(19)
(11)EP 3 130 671 A1

(12)EUROPEAN PATENT APPLICATION
published in accordance with Art. 153(4) EPC

(43)Date of publication:
15.02.2017 Bulletin 2017/07

(21)Application number: 15758003.6

(22)Date of filing:  05.03.2015
(51)Int. Cl.: 
C12N 15/09  (2006.01)
A23C 9/13  (2006.01)
A23L 9/00  (2016.01)
C12N 1/15  (2006.01)
C12N 1/21  (2006.01)
C12N 9/78  (2006.01)
A21D 2/26  (2006.01)
A23C 13/16  (2006.01)
A23L 27/60  (2016.01)
C12N 1/19  (2006.01)
C12N 5/10  (2006.01)
C12P 21/02  (2006.01)
(86)International application number:
PCT/JP2015/056569
(87)International publication number:
WO 2015/133590 (11.09.2015 Gazette  2015/36)
(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
Designated Extension States:
BA ME
Designated Validation States:
MA

(30)Priority: 07.03.2014 JP 2014045531

(71)Applicant: Ajinomoto Co., Inc.
Tokyo 104-8315 (JP)

(72)Inventors:
  • YOKOYAMA, Noriko
    Kawasaki-shi Kanagawa 210-8681 (JP)
  • NAKANO, Mayu
    Kawasaki-shi Kanagawa 210-8681 (JP)
  • HASHIRO, Shuhei
    Kawasaki-shi Kanagawa 210-8681 (JP)
  • HIRAGA, Masako
    Kawasaki-shi Kanagawa 210-8681 (JP)
  • SHIMBO, Kazutaka
    Kawasaki-shi Kanagawa 210-8681 (JP)
  • TOKUNAGA, Ayaka
    Kawasaki-shi Kanagawa 210-8681 (JP)

(74)Representative: Strehl Schübel-Hopf & Partner 
Maximilianstrasse 54
80538 München
80538 München (DE)

  


(54)NEW PROTEIN DEAMIDASE


(57) A novel protein deamidase having an activity of directly acting on a side chain amide group of an asparagine residue in a protein to form a side chain carboxyl group and release ammonia, a microorganism that produces the same, a gene encoding the same, a method for producing the same, and use of the same are provided. A bacterium classified into the class Actinobacteria is cultured to generate protein deamidase, and the enzyme is collected from culture.




Description

Technical Field



[0001] The present invention relates to a novel protein deamidase, a polynucleotide encoding the enzyme, a recombinant vector containing the polynucleotide, a transformant introduced with the vector, a method for producing the enzyme, and a method for deamidating a protein using the enzyme.

Background Art



[0002] Deamidation of a protein improves various functional properties of the protein (Non-patent document 1). Therefore, it is expected that deamidation of a protein expands use of the protein. Among the amino acids that constitute proteins, glutamine and asparagine have an amide group. These are converted into glutamic acid and aspartic acid by deamidation, respectively. By deamidation, the negative charge of a protein is increased, and the isoelectric point of the protein is lowered. The solubility and water dispersibility of the protein are thereby markedly increased. Further, along with an increase of the electrostatic repulsive force, the interaction of proteins, i.e., association property of the proteins, is reduced. It is also known that the tertiary structure of a protein is loosen by deamidation to provide a change of the higher-order structure, so that a hydrophobic region that has been buried in the inside of the protein molecule is exposed on the molecular surface, and therefore the deamidated protein comes to have amphipathy, which results in improvement of the emulsification power, emulsification stability, foamability, and foam stability of the protein.

[0003] Techniques for deamidation of proteins are classified into chemical techniques and enzymatic techniques. As the chemical deamidation techniques, many methods based on a mild acid or alkaline treatment have been reported. However, all of these have problems, such as problems that they are based on nonspecific reactions, peptide bonds are also cleaved under the acidic or alkaline condition, unexpected by-products are produced. Further, they also have problems that they require facilities for using chemical substances, and impose significant environmental loads. The enzymatic techniques can overcome such problems of the chemical techniques. As the enzymatic deamidation techniques, there have been reported, for example, methods of using protein glutaminase (Patent documents 1 and 2, Non-patent document 2), methods of using protease (Non-patent documents 3 and 4), a method of using transglutaminase (Non-patent document 5), methods of using peptide glutaminase (Non-patent documents 6 and 7), and so forth.

[0004] Among these enzymes, protein glutaminase is the only enzyme that can catalyze deamidation reaction of a high molecular protein without accompanying any side reaction. Use of protease, transglutaminase, and peptide glutaminase has problems since the main reaction catalyzed by protease is cleavage of peptide bonds, the main reaction catalyzed by transglutaminase is crosslinking reaction based on formation of isopeptide bonds between glutamine and lysine, and peptide glutaminase is an enzyme that mainly catalyzes deamidation of decomposed low molecular weight peptides. It is considered that protein glutaminase has high practicality as an enzyme having a high deamidation ability for a high molecular weight protein. There have already been reported findings concerning improvement in functions of wheat proteins, milk proteins (casein and whey proteins), and soybean proteins provided by protein glutaminase (Non-patent documents 8 to 11). In patent documents, there have also been reported findings concerning improvement of qualities of actual foods, for example, yogurt, ice cream, coffee whitener, noodles, meat, and so forth provided by protein glutaminase (Patent documents 3 to 7). However, it is definitely described that protein glutaminase uses a glutamine residue in a protein as a substrate, and does not act on asparagine residue at all (Non-patent documents 2 and 12), and hence, effect of the treatment with this enzyme is limited. In fact, amino acids constituting plant and animal proteins contain a large amount of asparagine, and hence, it is preferable to deamidate not only glutamine, but also asparagine, for obtaining further functional reforming effect by deamidation.

[0005] Asparaginase (EC 3.5.1.1) is widely known as an enzyme that catalyzes hydrolysis of asparagine to generate aspartic acid. However, asparaginase is an enzyme that specifically acts on asparagine of the free form, and cannot deamidate an asparagine residue in a peptide or high molecular weight protein. There is also known an enzyme that catalyzes the reaction of deamidating an N-terminus asparagine residue having a free α-amino group is known (Non-patent document 13). However, this enzyme cannot deamidate an asparagine residue in a protein other than N-terminus asparagine residue.

[0006] As described above, any enzyme that deamidates an asparagine residue in a protein (except for N-terminus asparagine residue having a free α-amino group) is not known.

Prior art references


Patent documents



[0007] 

Patent document 1: Japanese Patent Laid-open (Kokai) No. 2001-218590

Patent document 2: Japanese Patent Laid-open (Kokai) No. 2005-052158

Patent document 3: WO2011/024994

Patent document 4: US2013-22710

Patent document 5: WO2011/108633

Patent document 6: Japanese Patent Laid-open (Kokai) No. 2009-219419

Patent document 7: WO2006/075771


Non-patent documents



[0008] 

Non-patent document 1: Hamada J.S., 1994, Critical Reviews in Food Science and Nutrition, 34, 283

Non-patent document 2: Yamaguchi S, Jeenes DJ, and Archer DB, 2001, Eur. J. Biochem., 268(5), 1410

Non-patent document 3: Kato A., Tanaka A., Matsudomi N., Kobayashi K., 1987, J. Agric. Food Chem., 35, 224

Non-patent document 4: Kato, A., Tanaka, A., Lee, Y., Matsudomi, N. & Kobayashi, K., 1987, J. Agric. Food Chem. 35, 285

Non-patent document 5: Motoki M., Seguro K., Nio N., Takinami K., 1986, Agric. Biol. Chem., 50, 3025-3030

Non-patent document 6: Hamada J.S., Marshall W.E., 1989, J. Food. Sci., 54, 598-601, 635

Non-patent document 7: Hamada J.S., Marshall W.E., 1988, J. Food. Sci., 53, 1132-1134, 1149

Non-patent document 8: Suppavorasatit I., De Mejia E.G., Cadwallader K.R., 2011, J. Agric. Food Chem., 59, 11621

Non-patent document 9: Yong Y.H., Yamaguchi S., Matsumura Y., 2006, J. Agric. Food Chem., 54, 6034

Non-patent document 10: Miwa, N., Yokoyama, K., Noriki Nio, N., Sonomoto, K., 2013, J. Agric. Food Chem., 61, 2205

Non-patent document 11: Miwa, N., Yokoyama, K., Wakabayashi, H., Nio, N., 2010, Int. Dairy J., 20, 393

Non-patent document 12: The Latest Technology and Application of Food Enzymatic Chemistry - Prospects to Food Proteomics, 2004, ISBN 978-4-7813-0127-3, Chapter 3, p.147

Non-patent document 13: Stewart AE, Arfin SM, Bradshaw RA, 1994, J. Biol. Chem., 1994 Sep. 23; 269(38): 23509-17


Summary of the Invention


Object to be Achieved by the Invention



[0009] An object of the present invention is to provide a novel protein deamidase (protein asparaginase) that catalyzes the reaction of deamidating an asparagine residue in a protein.

Means for Achieving the Object



[0010] The inventors of the present invention conducted various researches in order to achieve the aforementioned object. As a result, they found microorganisms that produce an enzyme that catalyzes the reaction of specifically deamidating an asparagine residue in a protein without causing decomposition and crosslinking of the protein by using a screening system using a peptide having an asparagine residue as a sole nitrogen source, and accomplished the present invention.

[0011] That is, the present invention can be embodied, for example, as follows.
  1. [1] A protein having an activity for catalyzing a reaction of deamidating an asparagine residue in a protein.
  2. [2] The protein according to [1], which does not substantially have an activity for catalyzing a reaction of hydrolyzing a peptide bond in a protein.
  3. [3] The protein according to [1] or [2], which is a protein defined in (A), (B), or (C) mentioned below:
    1. (A) a protein comprising the amino acid sequence of SEQ ID NO: 10 or 11, the amino acid sequence of positions 63 to 1260, 245 to 1378, 245 to 1260, or 131 to 1260 of SEQ ID NO: 10, or the amino acid sequence of positions 47 to 1257, 59 to 1258, 96 to 1101, 241 to 1378, 244 to 1258, 244 to 1257, 244 to 1101, 129 to 1258, 120 to 1257, or 120 to 1101 of SEQ ID NO: 11;
    2. (B) a protein comprising the amino acid sequence of SEQ ID NO: 10 or 11, the amino acid sequence of positions 63 to 1260, 245 to 1378, 245 to 1260, or 131 to 1260 of SEQ ID NO: 10, or the amino acid sequence of positions 47 to 1257, 59 to 1258, 96 to 1101, 241 to 1378, 244 to 1258, 244 to 1257, 244 to 1101, 129 to 1258, 120 to 1257, or 120 to 1101 of SEQ ID NO: 11 but including substitution, deletion, insertion, or addition of 1 to 10 amino acid residues, and having an activity for catalyzing a reaction of deamidating an asparagine residue in a protein;
    3. (C) a protein comprising an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 10 or 11, the amino acid sequence of positions 63 to 1260, 245 to 1378, 245 to 1260, or 131 to 1260 of SEQ ID NO: 10, or the amino acid sequence of positions 47 to 1257, 59 to 1258, 96 to 1101, 241 to 1378, 244 to 1258, 244 to 1257, 244 to 1101, 129 to 1258, 120 to 1257, or 120 to 1101 of SEQ ID NO: 11, and having an activity for catalyzing a reaction of deamidating an asparagine residue in a protein.
  4. [4] The protein according to any one of [1] to [3], which is a protein defined in (a), (b), or (c) mentioned below:
    1. (a) a protein comprising the amino acid sequence of SEQ ID NO: 2, 5, 7, 8, or 9, the amino acid sequence of positions 240 to 1355 of SEQ ID NO: 2, the amino acid sequence of positions 181 to 1180 or 67 to 1180 of SEQ ID NO: 5, the amino acid sequence of positions 193 to 1172 or 70 to 1172 of SEQ ID NO: 7, or the amino acid sequence of positions 146 to 989 or 21 to 989 of SEQ ID NO: 8;
    2. (b) a protein comprising the amino acid sequence of SEQ ID NO: 2, 5, 7, 8, or 9, the amino acid sequence of positions 240 to 1355 of SEQ ID NO: 2, the amino acid sequence of positions 181 to 1180 or 67 to 1180 of SEQ ID NO: 5, the amino acid sequence of positions 193 to 1172 or 70 to 1172 of SEQ ID NO: 7, or the amino acid sequence of positions 146 to 989 or 21 to 989 of SEQ ID NO: 8 but including substitution, deletion, insertion, or addition of one or several amino acid residues, and having an activity for catalyzing a reaction of deamidating an asparagine residue in a protein;
    3. (c) a protein comprising an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 2, 5, 7, 8, or 9, the amino acid sequence of positions 240 to 1355 of SEQ ID NO: 2, the amino acid sequence of positions 181 to 1180 or 67 to 1180 of SEQ ID NO: 5, the amino acid sequence of positions 193 to 1172 or 70 to 1172 of SEQ ID NO: 7, or the amino acid sequence of positions 146 to 989 or 21 to 989 of SEQ ID NO: 8, and having an activity for catalyzing a reaction of deamidating an asparagine residue in a protein.
  5. [5] A polynucleotide encoding the protein according to any one of [1] to [4].
  6. [6] A recombinant vector containing the polynucleotide according to [5].
  7. [7] A transformant introduced with the recombinant vector according to [6].
  8. [8] A method for producing a protein having an activity for catalyzing a reaction of deamidating an asparagine residue in a protein, the method comprising:

    culturing the transformant according to [7] in a medium to generate the protein according to any one of [1] to [4]; and

    collecting the protein from the culture broth.

  9. [9] A method for producing a protein having an activity for catalyzing a reaction of deamidating an asparagine residue in a protein, the method comprising:

    culturing a microorganism having an ability to produce the protein according to any one of [1] to [4] in a medium to generate the protein; and

    collecting the protein from the culture broth.

  10. [10] The method according to [9], wherein the microorganism is a bacterium belonging to the class Actinobacteria.
  11. [11] The method according to [10], wherein the bacterium is a bacterium belonging to the genus Luteimicrobium, Agromyces, Microbacterium, or Leifsonia.
  12. [12] The method according to [11], wherein the bacterium is Luteimicrobium album, Agromyces sp., Microbacterium testaceum, Leifsonia xyli, or Leifsonia aquatica.
  13. [13] The method according to any one of [8] to [12], which comprises treating the protein with a processing enzyme.
  14. [14] The method according to [13], wherein the processing enzyme is protease.
  15. [15] A method for producing a protein and/or peptide of which an asparagine residue has been deamidated, the method comprising:

    allowing the protein according to any one of [1] to [4] to act on a protein and/or peptide.

  16. [16] The method according to [15], wherein the protein and/or peptide is contained in a food or drink or raw material thereof.
  17. [17] The method according to [15] or [16], which further comprises allowing transglutaminase and/or protein glutaminase to act on the protein and/or peptide.
  18. [18] A method for reforming a food or drink or raw material thereof, the method comprising:

    allowing the protein according to any one of [1] to [4] to act on a food or drink or raw material thereof containing a protein and/or peptide.

  19. [19] A method for producing a reformed food or drink or raw material thereof, the method comprising:

    allowing the protein according to any one of [1] to [4] to act on a food or drink or raw material thereof containing a protein and/or peptide.

  20. [20] The method according to [18] or [19], which further comprises allowing transglutaminase and/or protein glutaminase to act on the food or drink or raw material thereof containing a protein and/or peptide.
  21. [21] The method according to any one of [16] to [20], wherein the food or drink is selected from mayonnaise, dressing, cream, yogurt, meat product, and bread.
  22. [22] The method according to any one of [15] to [21], wherein 0.001 to 500 U of the protein according to any one of [1] to [4] is used for 1 g of the protein and/or peptide.

Brief Description of the Drawings



[0012] 

[Fig. 1] A photograph showing the result of SDS-PAGE of protein asparaginase derived from Luteimicrobium album (Example 2).

[Fig. 2] A diagram showing the result of HPLC analysis of the insulin B chain treated with protein asparaginase derived from Luteimicrobium album (Example 5).

[Fig. 3] A photograph showing the results of isoelectric focusing of casein treated with protein asparaginase derived from Luteimicrobium album (Example 6). Control group is a group that used a treatment with water, Test group 1 is a group that used a treatment with protein asparaginase (0.68 U/ml), and Test group 2 is a group that used a treatment with protein asparaginase (2.7 U/ml).

[Fig. 4] A photograph showing the results of SDS-PAGE of casein treated with various enzymes (Example 6). Control group is a group that used a treatment with water, Test group 2 is a group that used a treatment with protein asparaginase (2.7 U/ml), and Comparative group is a group that used a treatment with protein glutaminase (10 U/ml). "-TG" means that transglutaminase was not used in combination, and "+TG" means that transglutaminase was used in combination.

[Fig. 5] Diagrams showing the results of HPLC analysis of the insulin B chain treated with protein asparaginase derived from Leifsonia xyli (Example 7).

[Fig. 6] A photograph showing the results of SDS-PAGE of protein asparaginase derived from Agromyces sp. (Example 9). Lane 1 is for a chromatography-purified fraction, and lanes 2 to 6 are for anion exchange chromatography-purified fractions.

[Fig. 7] A diagram showing the result of alignment of the sequences of SEQ ID NOS: 2 and 5.

[Fig. 8] A diagram showing the result of alignment of the sequences of SEQ ID NOS: 2, 5, 7, and 8.

[Fig. 9] A diagram showing the result of alignment of the sequences of SEQ ID NOS: 2, 5, 7, 8, and 9.

[Fig. 10] A design drawing of a pro-sequence-fused protein asparaginase.

[Fig. 11] A photograph showing the results of SDS-PAGE of a pro-sequence-fused protein asparaginase derived from Agromyces sp., which was expressed by secretory expression from Corynebacterium glutamicum (Example 12, <1>). The arrow indicates the position of the target protein.

[Fig. 12] A photograph showing the results of SDS-PAGE of a pro-sequence-fused protein asparaginase, which was intracellularly expressed in Escherichia coli (Example 12, <2>). The arrows indicate the positions of the target protein.


Modes for Carrying out the Invention


<1> Protein deamidase



[0013] The present invention provides protein deamidase.

[0014] In the present invention, the term "protein deamidase" refers to a protein having an activity for catalyzing a reaction of deamidating an asparagine residue in a protein. This activity is also referred to as "protein asparaginase activity". In the present invention, protein deamidase is also referred to as "protein asparaginase".

[0015] The term "asparagine residue in a protein" means an asparagine residue existing in a protein, except for N-terminus asparagine residue having a free α-amino group. Protein deamidase may or may not have an activity for catalyzing a reaction of deamidating an N-terminus asparagine residue having a free α-amino group, so long as it has an activity for catalyzing a reaction of deamidating such an "asparagine residue in a protein". Protein deamidase may or may not have an activity for catalyzing a reaction of deamidating monomer asparagine.

[0016] In the present invention, a protein to be deamidated by protein deamidase is also referred to as "substrate protein". The length of the substrate protein is not particularly limited so long as the length is 2 residues (dipeptide) or longer. The length of the substrate protein may be, for example, 2 residues (dipeptide) or longer, 3 residues (tripeptide) or longer, 10 residues or longer, 50 residues or longer, or 100 residues or longer. That is, unless otherwise stated, the term "substrate protein" also includes a substance called peptide, such as oligopeptide and polypeptide.

[0017] An asparagine residue is hydrolyzed into an aspartic acid residue and ammonia by deamidation. Therefore, the protein asparaginase activity can be measured on the basis of, for example, generation of ammonia accompanying the deamidation of an asparagine residue. For example, protein deamidase can be allowed to act on a peptide having a length of two or more residues, containing asparagine residue, and not containing glutamine residue (such as Cbz-Asn-Gly) as a substrate, and then the protein asparaginase activity can be calculated on the basis of the amount of released ammonia. Specifically, the protein asparaginase activity can be calculated with, for example, the conditions described in the Examples section. That is, the protein asparaginase activity can be measured by adding 25 µL of an enzyme solution of an appropriate concentration to 125 µL of a 0.2 mol/L phosphate buffer (pH 6.5) containing 30 mmol/L of Cbz-Asn-Gly, incubating the mixture at 37°C for 60 minutes, then adding 150 µL of a 12% trichloroacetic acid solution to terminate the reaction, and measuring the ammonia concentration in the supernatant. In the present invention, the enzymatic activity for generating 1 µmol of ammonia in 1 minute under these conditions is defined as 1 unit (U) of the protein asparaginase activity.

[0018] It is preferred that protein deamidase does not substantially have an activity for catalyzing the reaction of deamidating a glutamine residue in a protein. This activity is also referred to as "protein glutaminase activity". A glutamine residue is hydrolyzed into a glutamic acid residue and ammonia by deamidation. Therefore, the protein glutaminase activity can be measured on the basis of, for example, generation of ammonia accompanying deamidation of a glutamine residue. For example, protein deamidase can be allowed to act on a peptide having a length of two or more residues, containing glutamine residue, and not containing asparagine residue (such as Cbz-Gln-Gly) as a substrate, and then the protein glutaminase activity can be calculated on the basis of the amount of released ammonia. Specifically, the protein glutaminase activity can be calculated with, for example, the conditions for measuring the protein asparaginase activity described in the Examples section, provided that the enzymatic reaction is performed by using Cbz-Gln-Gly instead of Cbz-Asn-Gly. That is, the protein glutaminase activity can be measured by adding 25 µL of an enzyme solution of an appropriate concentration to 125 µL of a 0.2 mol/L phosphate buffer (pH 6.5) containing 30 mmol/L of Cbz-Gln-Gly, incubating the mixture at 37°C for 60 minutes, adding 150 µL of a 12% trichloroacetic acid solution to terminate the reaction, and measuring the ammonia concentration in the supernatant. In the present invention, the enzymatic activity for generating 1 µmol of ammonia in 1 minute under these conditions is defined as 1 unit (U) of the protein glutaminase activity. The expression "protein deamidase does not substantially have the protein glutaminase activity" may mean that, for example, the ratio of the protein glutaminase activity to the protein asparaginase activity (namely, specific activity of the protein glutaminase activity/specific activity of the protein asparaginase activity) of protein deamidase is 1/100 or smaller, 1/1000 or smaller, 1/10000 or smaller, or 0 (zero). The ratio of the specific activities can be calculated by measuring the protein asparaginase activity and protein glutaminase activity.

[0019] It is preferred that protein deamidase does not substantially have an activity for catalyzing the reaction of hydrolyzing a peptide bond in a protein. This activity is also referred to as "protease activity". The protease activity can be measured by, for example, a known technique. Specifically, the protease activity can be calculated, for example, by using azocasein as a substrate under the following conditions. That is, 0.5 mL of an enzyme solution of an appropriate concentration is added to 1.0 mL of a 50 mM Tris-hydrochloric acid buffer (pH 8.0) containing 1% azocasein, the mixture is incubated at 37°C for 30 minutes, and then 2.0 mL of a 12% trichloroacetic acid solution is added to terminate the reaction. The reaction mixture is centrifuged (15,000 rpm, 4°C, 5 minutes), and then A405 of the supernatant is measured. As controls, the same procedure is performed for a test group in which 0.5 mL of water is added instead of the enzyme solution, and a test group for compensating influence of color of ingredients that may be contained in the enzyme solution, in which 1.0 mL of a 50 mM Tris-HCl buffer (pH 8.0) not containing azocasein is used instead of the 50 mM Tris-HCl buffer (pH 8.0) containing azocasein, and A405 of the supernatant is measured for each of the groups. On the basis of these measured values of A405, an increase of A405 provided by the enzyme is calculated. In the present invention, the enzymatic activity for increasing A405 by 0.01 in 1 minute under these conditions is defined as 1 unit (U) of the protease activity. The expression "protein deamidase does not substantially have the protease activity" may mean that, for example, when a protein is treated with protein deamidase so that deamidation of asparagine residues takes place in a desired degree, the protein is not significantly decomposed into lower molecular weight molecules due to cleavage of peptide bonds by the treatment. Also, the expression "protein deamidase does not substantially have the protease activity" may mean that, for example, the ratio of the protease activity to the protein asparaginase activity (namely, specific activity of the protease activity /specific activity of the protein asparaginase activity) of protein deamidase is 1/100 or smaller, 1/1000 or smaller, 1/10000 or smaller, or 0 (zero). The ratio of the specific activities can be calculated by measuring the protein asparaginase activity and protease activity.

[0020] It is preferred that protein deamidase does not substantially have an activity for catalyzing the reaction of crosslinking proteins. This activity is also referred to as "protein crosslinking activity". The protein crosslinking activity can be measured by, for example, a known technique. The expression "protein deamidase does not substantially have the protein crosslinking activity" may mean that, for example, when proteins are treated with protein deamidase so that deamidation of asparagine residues takes place in a desired degree, the proteins are not significantly crosslinked into higher molecular weight molecules due to crosslinking of the proteins caused by the treatment. The expression "protein deamidase does not substantially have the protein crosslinking activity" may specifically mean that, for example, when 25 µL of an enzyme solution adjusted to have 2.7 U/ml of the protein asparaginase activity is added to 500 µL of a 20 mM sodium phosphate buffer (pH 7.0) containing 2% w/v casein sodium, the reaction is performed at 37°C for 1 hour, and the enzyme is inactivated by a treatment at 100°C for 5 minutes, the amount of uncrosslinked casein corresponds to 90% or more, or 95% or more of the same of a control group (sample obtained by performing the reaction in the same manner as described above provided that 25 µL of water is added instead of the enzyme solution). The "amount of uncrosslinked casein" can be measured and compared by known techniques. For example, measurement and comparison of the "amount of uncrosslinked casein" may be performed by measuring molecular weight distribution using gel filtration chromatography, or by confirming position and concentration of an objective band in SDS-PAGE.

[0021] Examples of protein deamidase include, for example, protein deamidases of bacteria belonging to the class Actinobacteria. Examples of the bacteria belonging to the class Actinobacteria include bacteria belonging to the family Microbacteriaceae such as Leifsonia bacteria, Microbacterium bacteria, and Agromyces bacteria, and bacteria belonging to an unclassified family such as Luteimicrobium bacteria. Examples of the Leifsonia bacteria include Leifsonia xyli and Leifsonia aquatica. Examples of the Microbacterium bacteria include Microbacterium testaceum. Examples of the Agromyces bacteria include Agromyces sp. obtained in the Examples section mentioned later. Examples of the Luteimicrobium bacteria include Luteimicrobium album. That is, protein deamidase may be, for example, a protein derived from such bacteria.

[0022] The amino acid sequence of protein deamidase of Luteimicrobium album AJ111072 (NITE P-01650) and the nucleotide sequence of the gene encoding it are shown as SEQ ID NOS: 2 and 3, respectively. The amino acid sequence of protein deamidase of Agromyces sp. AJ111073 (NITE BP-01782) and the nucleotide sequence of the gene encoding it are shown as SEQ ID NOS: 5 and 6, respectively. The amino acid sequence of protein deamidase of Microbacterium testaceum is shown as SEQ ID NO: 7. The amino acid sequence of protein deamidase of Leifsonia xyli AJ111071 (NITE P-01649) is shown as SEQ ID NO: 8. The amino acid sequence of protein deamidase of Leifsonia aquatica is shown as SEQ ID NO: 9. That is, protein deamidase may be, for example, a protein having the amino acid sequence of SEQ ID NO: 2, 5, 7, 8, or 9. Protein deamidase may also be, for example, a protein encoded by a gene having the nucleotide sequence shown as SEQ ID NO: 3 or 6. The expression of "having an (amino acid or nucleotide) sequence" includes both cases of "comprising the (amino acid or nucleotide) sequence" and "consisting of the (amino acid or nucleotide) sequence".

[0023] Luteimicrobium album AJ111072 (NITE P-01650) was deposited at the independent administrative agency, National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (#122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on July 5, 2013, and assigned an accession number of NITE P-01650.

[0024] Agromyces sp. AJ111073 (NITE BP-01782) was deposited at the independent administrative agency, National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (#122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on December 11, 2013. Then, the deposit was converted to an international deposit under the provisions of the Budapest Treaty on March 4, 2015, and assigned an accession number of NITE BP-01782 (receipt number NITE ABP-01782).

[0025] Leifsonia xyli AJ111071 (NITE P-01649) was deposited at the independent administrative agency, National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (#122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on July 5, 2013, and assigned an accession number of NITE P-01649.

[0026] The amino acid sequence shown as SEQ ID NO: 2, 5, 7, 8, or 9 may contain a pre-pro-region (pre-sequence and pro-sequence). A protein containing a pre-pro-region is also referred to as "pre-pro-protein". A protein containing a pro-sequence, but not containing pre-sequence is also referred to as "pro-protein". A protein not containing pre-pro-region is also referred to as "mature protein". Protein deamidase may be, for example, a protein having the amino acid sequence of SEQ ID NO: 2, 5, 7, 8, or 9 except for a pre-pro-region (namely, amino acid sequence of mature protein), or a protein having the amino acid sequence of SEQ ID NO: 2, 5, 7, 8, or 9 except for a pre-region (namely, amino acid sequence of pro-protein). The amino acid sequence of the mature protein of protein deamidase of Luteimicrobium album corresponds to positions 240 to 1355 of SEQ ID NO: 2. The amino acid sequence of the mature protein of protein deamidase of Agromyces sp. corresponds to positions 181 to 1180 of SEQ ID NO: 5. The amino acid sequence of the mature protein of protein deamidase of Microbacterium testaceum corresponds to positions 193 to 1172 of SEQ ID NO: 7. The amino acid sequence of the mature protein of protein deamidase of Leifsonia xyli corresponds to positions 146 to 989 of SEQ ID NO: 8. The amino acid sequence of the pro-protein of protein deamidase of Agromyces sp. corresponds to positions 67 to 1180 of SEQ ID NO: 5. The amino acid sequence of the pro-protein of protein deamidase of Microbacterium testaceum corresponds to positions 70 to 1172 of SEQ ID NO: 7. The amino acid sequence of the pro-protein of protein deamidase of Leifsonia xyli corresponds to positions 21 to 989 of SEQ ID NO: 8. Protein deamidase may also be, for example, a protein encoded by a gene having a part of the nucleotide sequence of SEQ ID NO: 3 or 6, the part encoding the amino acid sequence other than the pre-pro-region (namely, nucleotide sequence encoding the amino acid sequence of the mature protein), or a protein encoded by a gene having a part of the nucleotide sequence of SEQ ID NO: 3 or 6, the part encoding the amino acid sequence other than the pre-region (namely, nucleotide sequence encoding the amino acid sequence of pro-protein). The nucleotide sequence encoding the amino acid sequence of the mature protein of protein deamidase of Luteimicrobium album corresponds to positions 718 to 4068 of SEQ ID NO: 3. The nucleotide sequence encoding the amino acid sequence of the mature protein of protein deamidase of Agromyces sp. corresponds to positions 541 to 3543 of SEQ ID NO: 6. The nucleotide sequence encoding the amino acid sequence of the pro-protein of protein deamidase of Agromyces sp. corresponds to positions 199 to 3543 of SEQ ID NO: 6. The position of the N-terminus residue of the mature protein may shift forward or backward by several residues depending on various conditions such as type of processing enzyme. The term "several residues" referred to here may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. That is, for example, the amino acid sequence of the mature protein of protein deamidase of Agromyces sp. may correspond to positions 181 ± several residues to 1180 of SEQ ID NO: 5. In an embodiment, the amino acid sequence of the mature protein of protein deamidase of Agromyces sp. may corresponds to positions 177 to 1180 of SEQ ID NO: 5.

[0027] Protein deamidase may be, for example, a protein having an amino acid sequence common to the amino acid sequences of two or more kinds of protein deamidases. The common amino acid sequence can be determined by aligning the amino acid sequences of two or more kinds of protein deamidases. The result of alignment of SEQ ID NOS: 2 and 5 is shown in Fig. 7, and the amino acid sequence common to them is shown as SEQ ID NO: 10. The result of alignment of SEQ ID NOS: 2, 5, 7, and 8 is shown in Fig. 8, and the amino acid sequence common to them is shown as SEQ ID NO: 11. The result of alignment of SEQ ID NOS: 2, 5, 7, 8, and 8 is shown in Fig. 9. That is, protein deamidase may be, for example, a protein having the amino acid sequence shown as SEQ ID NO: 10 or 11. Protein deamidase may also be, for example, a protein having a part of an amino acid sequence common to the amino acid sequences of two or more kinds of protein deamidases, the part corresponding to any of these protein deamidases. Examples of such a part of an amino acid sequence corresponding to protein deamidase include the amino acid sequence of positions 63 to 1260 of SEQ ID NO: 10 (corresponding to protein deamidase of Agromyces sp.), the amino acid sequence of positions 47 to 1257 of SEQ ID NO: 11 (corresponding to protein deamidase of Microbacterium testaceum), the amino acid sequence of positions 59 to 1258 of SEQ ID NO: 11 (corresponding to protein deamidase of Agromyces sp.), and the amino acid sequence of positions 96 to 1101 of SEQ ID NO: 11 (corresponding to protein deamidase of Leifsonia xyli). Protein deamidase may also be, for example, a protein having a part of an amino acid sequence common to the amino acid sequences of two or more kinds of protein deamidases, the part corresponding to the mature protein of any of those protein deamidases, or a protein having a part of an amino acid sequence common to the amino acid sequences of two or more kinds of protein deamidases, the part corresponding to the pro-protein of any of those protein deamidases. Examples of such a part of an amino acid sequence corresponding to a mature protein of protein deamidase include the amino acid sequence of positions 245 to 1378 of SEQ ID NO: 10 (corresponding to the mature protein of protein deamidase of Luteimicrobium album), the amino acid sequence of positions 245 to 1260 of SEQ ID NO: 10 (corresponding to the mature protein of protein deamidase of Agromyces sp.), the amino acid sequence of positions 241 to 1378 of SEQ ID NO: 11 (corresponding to the mature protein of protein deamidase of Luteimicrobium album), the amino acid sequence of positions 244 to 1258 of SEQ ID NO: 11 (corresponding to the mature protein of protein deamidase of Agromyces sp.), the amino acid sequence of positions 244 to 1257 of SEQ ID NO: 11 (corresponding to the mature protein of protein deamidase of Microbacterium testaceum), and the amino acid sequence of positions 244 to 1101 of SEQ ID NO: 11 (corresponding to the mature protein of protein deamidase of Leifsonia xyli). Examples of such a part of an amino acid sequence corresponding to a pro-protein of protein deamidase include the amino acid sequence of positions 131 to 1260 of SEQ ID NO: 10 (corresponding to the pro-protein of protein deamidase of Agromyces sp.), the amino acid sequence of positions 129 to 1258 of SEQ ID NO: 11 (corresponding to the pro-protein of protein deamidase of Agromyces sp.), the amino acid sequence of positions 120 to 1257 of SEQ ID NO: 11 (corresponding to the pro-protein of protein deamidase of Microbacterium testaceum), and the amino acid sequence of positions 120 to 1101 of SEQ ID NO: 11 (corresponding to the pro-protein of protein deamidase of Leifsonia xyli).

[0028] Protein deamidase may be a variant of any of the protein deamidases exemplified above (for example, a protein having the amino acid sequence of SEQ ID NO: 2, 5, 7, 8, 9, 10, or 11, or a protein having a part of any of those amino acid sequences), so long as the original function is maintained. Similarly, the gene encoding protein deamidase (also referred to as "protein deamidase gene") may be a variant of any of the protein deamidase genes exemplified above (for example, a gene having the nucleotide sequence of SEQ ID NO: 3 or 6, or a gene having a part of any of those nucleotide sequences), so long as the original function is maintained. Such a variant that maintains the original function is also referred to as "conservative variant". Examples of the conservative variant include, for example, a homologue and artificially modified version of the protein deamidases exemplified above and genes encoding them.

[0029] The expression "the original function is maintained" means that a variant of the gene or protein has a function (activity or property) corresponding to the function (activity or property) of the original gene or protein. That is, the expression "the original function is maintained" means that, in the case of protein deamidase, a variant of the protein has the protein asparaginase activity. Further, the expression "the original function is maintained" may also mean that, in the case of the protein deamidase gene, a variant of the gene encodes a protein that maintains the original function (namely, a protein having the protein asparaginase activity).

[0030] Examples of homologues of protein deamidase include, for example, protein deamidases produced by microorganisms obtained by the screening method mentioned later. Examples of homologues of protein deamidase also include, for example, proteins obtained from a public database by BLAST search and FASTA search using any of the aforementioned amino acid sequences as a query sequence. Also, homologues of the aforementioned protein deamidase genes can be obtained by, for example, PCR using a chromosome of any of various microorganisms as the template, and oligonucleotides prepared on the basis of any of those known gene sequences as the primers.

[0031] Protein deamidase may be a protein having any of the aforementioned amino acid sequences (for example, the amino acid sequence of SEQ ID NO: 2, 5, 7, 8, 9, 10, or 11, or a part (e.g. part corresponding to mature protein or part corresponding to pro-protein) of any of those amino acid sequences), but including substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions, so long as it maintains the original function. Although the number meant by the term "one or several" can differ depending on the positions of amino acid residues in the three-dimensional structure of the protein, or the types of amino acid residues, specifically, it is, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 to 3.

[0032] The aforementioned substitution, deletion, insertion, or addition of one or several amino acid residues is a conservative mutation that maintains normal function of the protein. Typical examples of the conservative mutation are conservative substitutions. The conservative substitution is a mutation wherein substitution takes place mutually among Phe, Trp, and Tyr, if the substitution site is an aromatic amino acid; among Leu, Ile, and Val, if it is a hydrophobic amino acid; between Gln and Asn, if it is a polar amino acid; among Lys, Arg, and His, if it is a basic amino acid; between Asp and Glu, if it is an acidic amino acid; and between Ser and Thr, if it is an amino acid having a hydroxyl group. Examples of substitutions considered as conservative substitutions include, specifically, substitution of Ser or Thr for Ala, substitution of Gln, His, or Lys for Arg, substitution of Glu, Gln, Lys, His, or Asp for Asn, substitution of Asn, Glu, or Gln for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp, or Arg for Gln, substitution of Gly, Asn, Gln, Lys, or Asp for Glu, substitution of Pro for Gly, substitution of Asn, Lys, Gln, Arg, or Tyr for His, substitution of Leu, Met, Val, or Phe for Ile, substitution of Ile, Met, Val, or Phe for Leu, substitution of Asn, Glu, Gln, His, or Arg for Lys, substitution of Ile, Leu, Val, or Phe for Met, substitution of Trp, Tyr, Met, Ile, or Leu for Phe, substitution of Thr or Ala for Ser, substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe, or Trp for Tyr, and substitution of Met, Ile, or Leu for Val. Further, such substitution, deletion, insertion, addition, inversion, or the like of amino acid residues as mentioned above includes a naturally occurring mutation (mutant or variant), such as those due to a difference of individuals or species of the organism from which the protein is derived.

[0033] Protein deamidase may be a protein having an amino acid sequence showing a homology of 80% or higher, preferably 90% or higher, more preferably 95% or higher, still more preferably 97% or higher, particularly preferably 99% or higher, to the whole of any of the aforementioned amino acid sequences, so long as the original function is maintained. In this description, "homology" can mean "identity".

[0034] When protein deamidase is a conservative variant of any of the aforementioned common amino acid sequences (for example, the amino acid sequence of SEQ ID NO: 10 or 11, or a part (e.g. part corresponding to mature protein or part corresponding to pro-protein) of any of those amino acid sequences), such a variant may be a protein, wherein a mutation has been introduced into a common portion and/or another portion in the common amino acid sequence, so long as the original function is maintained. It is preferred that such a variant is a protein, wherein the common part in the common amino acid sequence is conserved, and a mutation is introduced into another portion.

[0035] Protein deamidase may be a protein encoded by a DNA that hybridizes under stringent conditions with a probe that can be prepared from any of the aforementioned nucleotide sequences (for example, the nucleotide sequence of SEQ ID NO: 3 or 6, or a part (e.g. part encoding mature protein or part encoding pro-protein) of any of those nucleotide sequences), such as a sequence complementary to a part or the whole of any of the aforementioned nucleotide sequences, so long as the original function is maintained. Such a probe can be prepared by PCR using oligonucleotides produced on the basis of any of the aforementioned nucleotide sequences as primers, and a DNA fragment containing any of the aforementioned nucleotide sequences as the template. The "stringent conditions" refer to conditions under which a so-called specific hybrid is formed, and a non-specific hybrid is not formed. Examples of the stringent conditions include those under which highly homologous DNAs hybridize to each other, for example, DNAs not less than 80% homologous, preferably not less than 90% homologous, more preferably not less than 95% homologous, still more preferably not less than 97% homologous, particularly preferably not less than 99% homologous, hybridize to each other, and DNAs less homologous than the above do not hybridize to each other, or conditions of washing of typical Southern hybridization, i.e., conditions of washing once, preferably 2 or 3 times, at a salt concentration and temperature corresponding to 1 x SSC, 0.1% SDS at 60°C, preferably 0.1 x SSC, 0.1% SDS at 60°C, more preferably 0.1 x SSC, 0.1% SDS at 68°C. Further, for example, when a DNA fragment having a length of about 300 bp is used as the probe, the washing conditions of the hybridization can be, for example, 50°C, 2 x SSC, and 0.1% SDS.

[0036] Such protein deamidase as mentioned above may be, if the amino acid sequence thereof or the nucleotide sequence of the gene encoding it was known at the time of filling of this application, excluded from protein deamidase of the present invention.

[0037] Protein deamidase may be a fusion protein with another amino acid sequence. The "another peptide" is not particularly limited so long as the fusion protein has the protein asparaginase activity. The "another amino acid sequence" can be selected as required depending on various conditions such as purpose of use thereof. Examples of the "another amino acid sequence" include a peptide tag, signal sequence (pre-sequence), pro-sequence, and recognition sequence of a protease. The "another amino acid sequence" may be bound to, for example, either one or both of the N-terminus and C-terminus of protein deamidase. As the "another amino acid sequence", one kind of amino acid sequence may be used, or two or more kinds of amino acid sequences may be used in combination.

[0038] A peptide tag can be used for, for example, detection and purification of the expressed protein deamidase. Specific examples of the peptide tag include an His tag, FLAG tag, GST tag, Myc tag, MBP (maltose binding protein), CBP (cellulose binding protein), TRX (thioredoxin), GFP (green fluorescent protein), HRP (horseradish peroxidase), ALP (alkaline phosphatase), and Fc region of antibody. Examples of the His tag include 6xHis tag.

[0039] A signal sequence can be used for, for example, secretory production of protein deamidase. Examples of the signal sequence include a signal sequence recognized by the Sec system secretory pathway and a signal sequence recognized by the Tat system secretory pathway. Specific examples of the signal sequence recognized by the Sec system secretory pathway include a signal sequence of a cell surface protein of coryneform bacteria. Examples of the cell surface protein of coryneform bacteria include PS1 (CspA) and PS2 (CspB) of C. glutamicum (Japanese Patent Laid-open (Kohyo) No. 6-502548), and SlpA (CspA) of C. ammoniagenes (C. stationis) (Japanese Patent Laid-open (Kokai) No. 10-108675). Specific examples of the signal sequence recognized by the Tat system secretory pathway include the TorA signal sequence of E. coli, the SufI signal sequence of E. coli, the PhoD signal sequence of Bacillus subtilis, the LipA signal sequence of Bacillus subtilis, and the IMD signal sequence of Arthrobacter globiformis (WO2013/118544). As the signal sequence, a signal sequence of protein deamidase may be used. A signal sequence can be used by, for example, adding it to the N-terminus of the protein to be produced. Specifically, a signal sequence can be used by, for example, adding it to the N-terminus of a pro-protein or mature protein of protein deamidase. A signal sequence is generally digested with a signal peptidase, when a translation product is secreted out of a microbial cell. Therefore, if secretory production of protein deamidase is performed by using a signal sequence, protein deamidase not having the signal sequence may be secreted out of the microbial cell.

[0040] Specific examples of the pro-sequence include a pro-sequence of protein deamidase. Examples of the pro-sequence of protein deamidase include the sequence of positions 67 to 180 of SEQ ID NO: 5. A pro-sequence can be used by, for example, adding it to the N-terminus of the protein to be produced. Specifically, a pro-sequence can be used by, for example, adding it to the N-terminus of a mature protein of protein deamidase. When secretory production of protein deamidase is performed, protein deamidase may be constituted to contain a signal sequence, a pro-sequence, and a sequence of mature protein in this order from the N-terminus, and expressed. Expression of protein deamidase in the form of having a pro-sequence may contribute to structural stabilization of protein deamidase. By contrast, from the viewpoint of the protein asparaginase activity, it is preferred that protein deamidase eventually obtained does not have a pro-sequence.

[0041] A recognition sequence for a protease can be used for, for example, cleavage of the expressed protein deamidase. It is preferred that the recognition sequence for a protease is a recognition sequence for a protease showing high substrate specificity. Specific examples of the recognition sequence for a protease showing high substrate specificity include the recognition sequence for the factor Xa protease and the recognition sequence for the ProTEV protease. The factor Xa protease and the ProTEV protease recognize the amino acid sequence of Ile-Glu-Gly-Arg (= IEGR, SEQ ID NO: 14) and the amino acid sequence of Glu-Asn-Leu-Tyr-Phe-Gln (= ENLYFQ, SEQ ID NO: 15) in a protein, respectively, to digest the protein specifically at a position on the C-terminus side of the corresponding recognition sequence. For example, when protein deamidase is expressed as a fusion protein with another amino acid sequence such as a peptide tag or pro-sequence, if a recognition sequence for a protease is introduced between protein deamidase and such another amino acid sequence, such another amino acid sequence can be removed from the expressed protein deamidase by using the protease to obtain protein deamidase not having such another amino acid sequence.

[0042] The protein deamidase gene may be one having any of the nucleotide sequences of the protein deamidase genes exemplified above and conservative variants thereof, in which arbitrary codons are replaced with equivalent codons. For example, the protein deamidase gene may be modified so that it has codons optimized for codon usage observed in the host to be used.

[0043] In the present invention, the term "gene" is not limited to DNA, but may include an arbitrary polynucleotide, so long as it encodes a target protein. That is, the term "protein deamidase gene" may mean an arbitrary polynucleotide encoding protein deamidase. The protein deamidase gene may be DNA, RNA, or a combination thereof. The protein deamidase gene may be single-stranded or double-stranded. The protein deamidase gene may be a single-stranded DNA or a single-stranded RNA. The protein deamidase gene may be a double-stranded DNA, a double-stranded RNA, or a hybrid strand consisting of a DNA strand and an RNA strand. The protein deamidase gene may contain both a DNA residue and an RNA residue in a single polynucleotide chain. When the protein deamidase gene contains RNA, the aforementioned descriptions concerning DNA, such as those concerning nucleotide sequences exemplified above, may be applied to RNA with appropriately changing wordings to those for RNA as required. The mode of the protein deamidase gene can be chosen according to various conditions such as use thereof.

<2> Production of protein deamidase



[0044] Protein deamidase can be produced by using a host having an ability to produce the protein deamidase. That is, the present invention provides a method for producing protein deamidase, which method comprises culturing a host having an ability to produce protein deamidase in a medium to generate the protein deamidase, and collecting the protein deamidase from the culture broth. This method is also referred to as "the method for producing protein deamidase of the present invention". Protein deamidase can also be produced by expressing a protein deamidase gene in a cell-free protein synthesis system.

[0045] The host having an ability to produce protein deamidase may be one inherently having an ability to produce protein deamidase, or may be one modified so as to have an ability to produce protein deamidase.

[0046] Examples of the host having an ability to produce protein deamidase include such bacteria belonging to the class Actinobacteria as mentioned above, such as Luteimicrobium album AJ111072 (NITE P-01650), Agromyces sp. AJ111073 (NITE BP-01782), Microbacterium testaceum, and Leifsonia xyli AJ111071 (NITE P-01649).

[0047] Examples of the host having an ability to produce protein deamidase also include a microorganism obtained by the following screening technique.
  1. (1) A microorganism supply source such as soil is inoculated into a medium containing Cbz-Asn-Gly as a sole N source, and enrichment culture is performed.
  2. (2) The culture broth obtained in (1) is inoculated on an agar medium containing Cbz-Asn-Gly as a sole N source, and a grown strain is obtained.
  3. (3) The obtained strain is cultured in an appropriate liquid nutrition medium, and the activity for releasing ammonia from Cbz-Asn-Gly and casein contained in the culture broth is determined.


[0048] The composition of the medium used for the enrichment culture can be appropriately set according to the microorganism to be cultured, provided that Cbz-Asn-Gly is used as a sole N source. Culture conditions such as culture temperature can also be appropriately set according to the microorganism to be cultured. As for specific medium components and culture conditions, the descriptions concerning the culture of a host having an ability to produce protein deamidase described later can be referred to.

[0049] Examples of the host having an ability to produce protein deamidase also include a host introduced with a protein deamidase gene.

[0050] The host to be introduced with a protein deamidase gene is not particularly limited so long as it can express a functional protein deamidase. Examples of the host include, for example, bacteria, actinomycetes, yeast, fungi, plant cells, insect cells, and animal cells. Preferred examples of the host include microorganisms such as bacteria and yeast. More preferred examples of the host include bacteria. Examples of the bacteria include gram-negative bacteria and gram-positive bacteria. Examples of the gram-negative bacteria include, for example, bacteria belonging to the family Enterobacteriaceae, such as Escherichia bacteria, Enterobacter bacteria, and Pantoea bacteria. Examples of the gram-positive bacteria include Bacillus bacteria, and coryneform bacteria such as Corynebacterium bacteria. As the host, Escherichia coli can be especially preferably used. Also, when protein deamidase is produced by secretory production out of a microbial cell, particularly, coryneform bacteria such as Corynebacterium glutamicum and Corynebacterium stationis may preferably be used as the host (WO2013/065869, WO2013/065772, WO2013/118544, WO2013/062029).

[0051] A protein deamidase gene can be obtained by cloning from an organism having the protein deamidase gene. For the cloning, a nucleic acid containing the gene, such as a genomic DNA or cDNA, can be used. A protein deamidase gene can also be obtained by chemical synthesis (Gene, 60 (1), 115-127 (1987)).

[0052] Specifically, a protein deamidase gene can be cloned from, for example, such a microorganism having an ability to produce protein deamidase as mentioned above (for example, a bacterium belonging to the class Actinobacteria, or a microorganism obtained by the aforementioned screening technique) by such a method as described below.

[0053] First, protein deamidase is appropriately isolated and purified from a microorganism having an ability to produce the protein deamidase, and information on a partial amino acid sequence thereof is obtained. For determining the partial amino acid sequence, for example, a purified protein deamidase may be directly subjected to amino acid sequence analysis (protein sequencer PPSQ-21A (Shimadzu) etc.) according to the Edman degradation method [Journal of Biological Chemistry, 256, 7990-7997 (1981)] in a conventional manner, or protein deamidase may be subjected to limited hydrolysis by the action of a proteolytic enzyme, the generated peptide fragments may be isolated and purified, and the obtained purified peptide fragments may be subjected to amino acid sequence analysis. Then, the nucleotide sequence of the genomic DNA extracted from the microorganism is determined with a next-generation sequencer (MiSeq, Illumina, etc.), and the partial amino acid sequence obtained by the aforementioned method is searched for. That is, a contig sequence can be created by using CLC Genomics Workbench (CLC bio Japan) on the basis of the obtained nucleotide sequence of the genomic DNA, and the nucleotide sequence of the gene encoding the enzyme can be determined on the basis of the partial amino acid sequence of protein deamidase obtained beforehand. A protein deamidase gene can be cloned by a general method using PCR on the basis of the determined nucleotide sequence.

[0054] When a PCR method is used, such a method as mentioned below can be used. First, PCR is performed by using a genomic DNA of a microorganism having an ability to produce protein deamidase as the template, and synthetic oligonucleotide primers designed on the basis of the information on a partial amino acid sequence thereof to obtain a DNA fragment containing a part of the target protein deamidase gene. The PCR method is performed according to the method described in PCR Technology, Erlich, H.A. Eds., Stockton Press, 1989. Subsequently, if the nucleotide sequence of the amplified DNA fragment is determined by a method usually used such as the dideoxy chain terminator method, a sequence corresponding to the partial amino acid sequence of protein deamidase can be found in the determined sequence in addition to the sequences of the synthetic oligonucleotide primers, and that is, a part of the nucleotide sequence of the target protein deamidase gene can be determined. Further, by performing the hybridization method or the like using the obtained gene fragment as a probe, the protein deamidase gene can be cloned for the full length thereof.

[0055] Further, the thus-obtained protein deamidase gene can be modified as required to obtain a variant thereof. Modification of a gene can be performed by a known method. For example, by the site-specific mutagenesis method, an objective mutation can be introduced into a target site of a gene. That is, for example, a coding region of a gene can be modified by the site-specific mutagenesis method so that a specific site of the encoded protein include substitution, deletion, insertion, or addition of amino acid residues. Examples of the site-specific mutagenesis method include a method using PCR (Higuchi, R., 61, in PCR Technology, Erlich, H.A. Eds., Stockton Press, 1989; Carter P., Meth., in Enzymol., 154, 382, 1987), and a method of using a phage (Kramer, W. and Frits, H.J., Meth. in Enzymol., 154, 350, 1987; Kunkel, T.A. et al., Meth. in Enzymol., 154, 367, 1987). Further, a variant of the protein deamidase gene can also be obtained by, for example, a mutagenesis treatment. Examples of the mutagenesis treatment include methods such as treating a gene itself in vitro with hydroxylamine or the like, treating a microorganism such as a bacterium belonging to the class Actinobacteria and having a protein deamidase gene with X-ray, ultraviolet ray, or a mutation agent such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS), error prone PCR (Cadwell, R.C., PCR Meth. Appl., 2, 28 (1992)), DNA shuffling (Stemmer, W.P., Nature, 370, 389 (1994)), and StEP-PCR (Zhao, H., Nature Biotechnol., 16, 258 (1998)).

[0056] The method for introducing a protein deamidase gene into a host is not particularly limited. In a host, a protein deamidase gene may be harbored in such a manner that it can be expressed under control of a promoter that functions in the host. In the host, the protein deamidase gene may exist on a vector autonomously replicable out of the chromosome such as plasmid, or may be introduced into the chromosome. The host may have only one copy of a protein deamidase gene, or may have two or more copies of a protein deamidase gene. The host may have only one kind of protein deamidase gene, or may have two or more kinds of protein deamidase genes.

[0057] The promoter for expressing a protein deamidase gene is not particularly limited so long as it is a promoter that functions in the host. The "promoter that functions in a host" refers to a promoter that shows a promoter activity in the host. The promoter may be a promoter derived from the host, or a heterologous promoter. The promoter may be a native promoter of the protein deamidase gene, or may be a promoter of another gene. The promoter may be a strong promoter so that a high expression amount of the gene can be attained. Specific examples of strong promoters that function in Enterobacteriaceae bacteria, such as Escherichia coli, include, for example, T7 promoter, trp promoter, trc promoter, lac promoter, tac promoter, tet promoter, araBAD promoter, rpoH promoter, PR promoter, and PL promoter. Examples of strong promoters that function in coryneform bacteria include the artificially modified P54-6 promoter (Appl. Microbiol. Biotechnol., 53, 674-679 (2000)), pta, aceA, aceB, adh, and amyE promoters inducible in coryneform bacteria with acetic acid, ethanol, pyruvic acid, or the like, cspB, SOD, and tuf (EF-Tu) promoters, which are potent promoters capable of providing a large expression amount in coryneform bacteria (Journal of Biotechnology, 104 (2003) 311-323; Appl. Environ. Microbiol., 2005 Dec; 71 (12):8587-96), as well as lac promoter, tac promoter, and trc promoter. Further, as the stronger promoter, a highly-active type of an existing promoter may also be obtained by using various reporter genes. For example, by making the -35 and -10 regions in a promoter region closer to the consensus sequence, the activity of the promoter can be enhanced (WO00/18935). Examples of highly active-type promoter include various tac-like promoters (Katashkina JI et al., Russian Federation Patent Application No. 2006134574) and pnlp8 promoter (WO2010/027045). Methods for evaluating the strength of promoters and examples of strong promoters are described in the paper of Goldstein et al. (Prokaryotic Promoters in Biotechnology, Biotechnol. Annu. Rev., 1, 105-128 (1995)), and so forth.

[0058]  Also, a terminator for termination of gene transcription may be located downstream of the protein deamidase gene. The terminator is not particularly limited so long as it functions in the bacterium of the present invention. The terminator may be a terminator derived from the host, or a heterogenous terminator. The terminator may be the native terminator of the protein deamidase gene, or a terminator of another gene. Specific examples of the terminator include, for example, T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

[0059] A protein deamidase gene can be introduced into a host, for example, by using a vector containing the gene. A vector containing a protein deamidase gene is also referred to as expression vector or recombinant vector for a protein deamidase gene. The expression vector for a protein deamidase gene can be constructed by, for example, ligating a DNA fragment containing the protein deamidase gene with a vector that functions in the host. By transforming the host with the expression vector for a protein deamidase gene, a transformant into which the vector has been introduced is obtained, i.e. the gene can be introduced into the host. As the vector, a vector autonomously replicable in the cell of the host can be used. The vector is preferably a multi-copy vector. Further, the vector preferably has a marker such as an antibiotic resistance gene for selection of transformant. Further, the vector may have a promoter and/or terminator for expressing the introduced gene. The vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, cosmid, phagemid, or the like. Specific examples of vector autonomously replicable in Enterobacteriaceae bacteria such as Escherichia coli include, for example, pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all of these are available from Takara Bio), pACYC184, pMW219 (NIPPON GENE), pTrc99A (Pharmacia), pPROK series vectors (Clontech), pKK233-2 (Clontech), pET series vectors (Novagen), pQE series vectors (QIAGEN), pACYC, and the broad host spectrum vector RSF1010. Specific examples of vector autonomously replicable in coryneform bacteria include, for example, pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)); plasmids obtained by improving these and having a drug resistance gene; plasmid pCRY30 described in Japanese Patent Laid-open (Kokai) No. 3-210184; plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX described in Japanese Patent Laid-open (Kokai) No. 2-72876 and U.S. Patent No. 5,185,262; plasmids pCRY2 and pCRY3 described in Japanese Patent Laid-open (Kokai) No. 1-191686; pAJ655, pAJ611, and pAJ1844 described in Japanese Patent Laid-open (Kokai) No. 58-192900; pCG1 described in Japanese Patent Laid-open (Kokai) No. 57-134500; pCG2 described in Japanese Patent Laid-open (Kokai) No. 58-35197; and pCG4 and pCG11 described in Japanese Patent Laid-open (Kokai) No. 57-183799. When the expression vector is constructed, for example, a protein deamidase gene having a native promoter region as it is may be incorporated into a vector, a coding region of protein deamidase ligated downstream from such a promoter as mentioned above may be incorporated into a vector, or a coding region of protein deamidase may be incorporated into a vector downstream from a promoter originally existing in the vector.

[0060]  Vectors, promoters, and terminators available in various microorganisms are disclosed in detail in "Fundamental Microbiology Vol. 8, Genetic Engineering, KYORITSU SHUPPAN CO., LTD, 1987", and those can be used.

[0061] A protein deamidase gene can also be introduced into, for example, a chromosome of a host. A gene can be introduced into a chromosome by, for example, using homologous recombination (Miller, J.H., Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of the gene transfer method utilizing homologous recombination include, for example, the Red-driven integration method (WO2005/010175), a transduction method using a phage such as P1 phage, a method of using a conjugative transfer vector, and a method of using a suicide vector without a replication origin that functions in a host. Only one copy, or two or more copies of a gene may be introduced. For example, by performing homologous recombination using a sequence which is present in multiple copies on a chromosome as a target, multiple copies of a gene can be introduced into the chromosome. Examples of such a sequence which is present in multiple copies on a chromosome include, for example, repetitive DNAs, and inverted repeats located at the both ends of a transposon. Further, a gene can also be randomly introduced into a chromosome by a method using a transposon or Mini-Mu (Japanese Patent Laid-open (Kokai) No. 2-109985, U.S. Patent No. 5,882,888, EP 805867 B1). When the gene is introduced into a chromosome, for example, a protein deamidase gene having a native promoter region as it is may be incorporated into a chromosome, a coding region for protein deamidase ligated downstream from such a promoter as mentioned above may be incorporated into a chromosome, or a coding region for protein deamidase may be incorporated into a chromosome downstream from a promoter originally contained in the chromosome.

[0062] Introduction of a gene into a chromosome can be confirmed by, for example, Southern hybridization using a probe having a sequence complementary to a part or the whole of the gene, or PCR using primers prepared on the basis of the nucleotide sequence of the gene.

[0063] The method for the transformation is not particularly limited, and conventionally known methods can be used. Examples of transformation method include, for example, a method of treating recipient cells with calcium chloride so as to increase the permeability thereof for DNA, which has been reported for the Escherichia coli K-12 strain (Mandel, M. and Higa, A., J. Mol. Biol., 1970, 53, 159-162), a method of preparing competent cells from cells which are in the growth phase, followed by transformation with DNA, which has been reported for Bacillus subtilis (Duncan, C.H., Wilson, G.A. and Young, F.E., Gene, 1977, 1:153-167), and so forth. Further, as the transformation method, there can also be used a method of making DNA-recipient cells into protoplasts or spheroplasts, which can easily take up recombinant DNA, followed by introducing a recombinant DNA into the DNA-recipient cells, which is known to be applicable to Bacillus subtilis, actinomycetes, and yeasts (Chang, S. and Choen, S.N., 1979, Mol. Gen. Genet., 168:111-115; Bibb, M.J., Ward, J.M. and Hopwood, O.A., 1978, Nature, 274:398-400; Hinnen, A., Hicks, J.B. and Fink, G.R., 1978, Proc. Natl. Acad. Sci. USA, 75:1929-1933). Further, as the transformation method, the electric pulse method reported for coryneform bacteria (Japanese Patent Laid-open (Kokai) No. 2-207791) can also be used.

[0064] A host inherently having a protein deamidase gene may have been modified so that the expression of the protein deamidase gene is increased. Examples of the means for increasing the expression of a protein deamidase gene include increasing the copy number of the protein deamidase gene, and improving the transcription efficiency of the protein deamidase gene. The copy number of a protein deamidase gene can be increased by introducing the protein deamidase gene into a host. Introduction of a protein deamidase gene can be performed as described above. The protein deamidase gene to be introduced may be a gene derived from the host, or heterogenous gene. The transcription efficiency of a protein deamidase gene can be improved by replacing the promoter of the protein deamidase gene with a stronger promoter. As such stronger promoter, the strong promoters mentioned above can be used.

[0065] By culturing such a host having an ability to produce protein deamidase as described above, protein deamidase can be expressed. During the culture, induction of gene expression may be performed, if necessary. Conditions for culture of the host and induction of gene expression may be chosen as required depending on various conditions such as type of marker, type of promoter, and type of the host. The medium used for the culture is not be particularly limited, so long as the host can proliferate in the medium and express a protein deamidase. As the medium, for example, a usual medium that contains a carbon source, nitrogen source, sulfur source, inorganic ions, and other organic components as required can be used.

[0066]  Examples of the carbon source include saccharides such as glucose, fructose, sucrose, molasses, and starch hydrolysate, alcohols such as glycerol and ethanol, and organic acids such as fumaric acid, citric acid, and succinic acid.

[0067] Examples of the nitrogen source include inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, and aqueous ammonia.

[0068] Examples of the sulfur source include inorganic sulfur compounds, such as sulfates, sulfites, sulfides, hyposulfites, and thiosulfates.

[0069] Examples of the inorganic ions include calcium ion, magnesium ion, manganese ion, potassium ion, iron ion, and phosphoric acid ion.

[0070] Examples of the other organic components include organic trace amount nutrients. Examples of the organic trace amount nutrients include required substances such as vitamin B1, yeast extract containing such substances, and so forth.

[0071] Although the culture may be performed as liquid culture or solid culture, liquid culture is preferred. The culture is preferably performed as aerobic culture. Examples of method for aerobic culture include shaking culture method, and aerobic deep culture method using a jar fermenter. In the aeration culture, oxygen concentration may be adjusted to, for example, 5 to 50%, preferably about 10%, with respect to the saturated concentration. Culture temperature may be, for example, 10 to 50°C, preferably 20 to 45°C, more preferably 25 to 40°C. pH of the medium may be adjusted to 3 to 9, preferably 5 to 8. For adjusting pH, inorganic or organic acidic or alkaline substances such as calcium carbonate, ammonia gas, and aqueous ammonia can be used. Culture period may be, for example, 12 hours to 20 days, preferably 1 to 7 days.

[0072] By performing the culture under such conditions as mentioned above, a culture broth containing protein deamidase is obtained. Protein deamidase is accumulated in, for example, microbial cells of the host and/or the medium. The term "microbial cell" may be appropriately read as "cell" depending on type of the host. Depending on the host to be used and design of the protein deamidase gene, it is also possible to accumulate protein deamidase in the periplasm, or to produce protein deamidase out of the cells by secretory production.

[0073] Protein deamidase may be used in a state that it is contained in microbial cells or the like, or may be separated and purified from microbial cells or the like to be used as a crude enzyme fraction or a purified enzyme, as required.

[0074] That is, for example, when protein deamidase is accumulated in microbial cells of the host, by subjecting the cells to disruption, lysis, extraction, etc. as required, the protein deamidase can be collected. The microbial cells can be collected from the culture broth by centrifugation or the like. Disruption, lysis, extraction, etc. of the cells can be performed by known methods. Examples of such methods include, for example, disruption by ultrasonication, disruption in Dyno-Mill, disruption in bead mill, disruption with French press, and lysozyme treatment. These methods may be independently used, or may be used in an appropriate combination. Also, for example, when protein deamidase is accumulated in the medium, a culture supernatant can be obtained by centrifugation or the like, and the protein deamidase can be collected from the culture supernatant.

[0075] Protein deamidase can be purified by known methods used for purification of enzymes. Examples of such methods include, for example, ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric precipitation. These methods may be independently used, or may be used in an appropriate combination. Protein deamidase may be purified to a desired extent.

[0076] The purified protein deamidase can be used as "protein deamidase" used in the method of the present invention. Protein deamidase may be used in a free form, or may be used as an immobilized enzyme immobilized on a solid phase of resin etc.

[0077] Not only the purified protein deamidase, but also an arbitrary fraction containing protein deamidase may be used as "protein deamidase" for deamidation of a protein. Such a fraction containing protein deamidase is not particularly limited, so long as it contains a protein deamidase so that the protein deamidase can act on a substrate protein. Examples of such a fraction include, for example, a culture broth of a host having an ability to produce protein deamidase, microbial cells collected from such a culture broth (cultured microbial cells), processed products of such microbial cells such as disruption product of the cells, lysate of the cells, extract of the cells (cell-free extract), and immobilized cells obtained by immobilizing such cells as mentioned above on a carrier such as acrylamide and carrageenan, culture supernatant collected from such a culture broth, partially purified products of these (roughly purified products), and combinations of these. These fractions each may be used alone, or may be used together with a purified protein deamidase.

[0078] The collected protein deamidase may be made into a formulation (preparation) as required. The dosage form of the formulation is not particularly limited, and can be appropriately determined according to various conditions such as purpose of use of protein deamidase. Examples of the dosage form include, for example, solution, suspension, powder, tablet, pill, and capsule. For preparing such a formulation, pharmaceutically acceptable additives such as excipients, binders, disintegrating agents, lubricants, stabilizers, corrigents, odor-masking agents, perfumes, diluents, and surfactants can be used.

[0079] When protein deamidase has a pro-sequence, the protein asparaginase activity may be improved by removal of the pro-sequence. Therefore, the method for producing protein deamidase of the present invention may further comprise removing a pro-sequence from protein deamidase having the pro-sequence. A pro-sequence can be removed by, for example, treating protein deamidase with a processing enzyme. Examples of the processing enzyme include protease. Specific examples of protease include serine proteases such as subtilisin, chymotrypsin, and trypsin; cysteine proteases such as papain, bromelain, caspase, and calpain; acid proteases such as pepsin and cathepsin; and metalloproteases such as thermolysin. Further, if protein deamidase is expressed with a recognition sequence for a specific protease inserted between sequences of a pro-sequence and a mature protein, the pro-sequence can be specifically removed by using the specific protease. Origin of protease is not particularly limited, and any of those derived from microorganisms, animals, plants, and so forth may be used. As protease, a homologue of a known protease or an artificially modified version of a known protease may be used. As protease, there can be used any of those in the form of, for example, a culture broth of a microorganism that produces protease, culture supernatant separated from such a culture broth, microbial cells separated from such a culture broth, processed product of such cells, agricultural, aquatic, or livestock product containing protease, processed product of such agricultural, aquatic, or livestock product, protease separated from any of these, commercial protease preparation, and so forth. Protease may be purified to a desired degree. Examples of the microorganism that produces protease include Bacillus bacteria and Aspergillus fungi. Examples of the commercial protease preparation include those mentioned in Table 5.

[0080] A specific example of the manufacturing procedure of protein deamidase will be explained below. For example, when Luteimicrobium album AJ111072 (NITE P-01650) is used as a host having an ability to produce protein deamidase, glycerol stock of the strain is inoculated in an amount of 1% to the tryptic soy medium (Difco), and shaking culture is performed at 30°C for 24 hours as preculture. Then, shaking culture is performed at 30°C for 24 hours in a medium containing Yeast Carbon Base (Difco) and polypeptone as main culture to obtain a culture broth containing protein deamidase. The protein deamidase can be purified by centrifuging the culture broth (12,000 rpm, 4°C, 20 minutes) to obtain a supernatant as a crude enzyme solution, and subjecting the crude enzyme solution to UF concentration (Hydrosart membrane, Sartorius, fractionation molecular weight 10,000), hydrophobic chromatography, ion exchange chromatography, gel filtration chromatography, or the like. Also when other hosts are used, the procedures for the case of using Luteimicrobium album AJ111072 (NITE P-01650) can be referred to.

<3> Use of protein deamidase



[0081] According to the present invention, an asparagine residue in a protein can be deamidated by using protein deamidase. That is, the present invention provides a method for deamidating an asparagine residue in a protein, which method comprises allowing protein deamidase to act on a protein. This method is also referred to as "deamidation method of the present invention". An embodiment of this method is a method for producing a protein containing a deamidated asparagine residue, which method comprises allowing protein deamidase to act on a protein.

[0082] The protein to be subjected to deamidation with protein deamidase (substrate protein) is not particularly limited so long as it is a protein containing an asparagine residue. As described above, the length of the substrate protein is not particularly limited so long as the length is 2 residues (dipeptide) or longer, and the term "substrate protein" also includes a substance called peptide, such as oligopeptide and polypeptide, unless otherwise stated. That is, specifically, the term "protein (substrate protein)" may mean a protein and/or a peptide. The substrate protein may be a natural substance or artificial product.

[0083] The substrate protein may be a protein itself or a material containing a protein. In other words, the substrate protein itself may be subjected to the deamidation reaction alone (namely, in an isolated state), or the substrate protein in a state of being contained in an arbitrary material may be subjected to the deamidation reaction. Examples of the substrate protein include, for example, agricultural, aquatic, or livestock products containing a protein, processed products thereof, and proteins separated therefrom. Examples of materials containing a vegetable protein include, for example, grains such as soybean, wheat, barley, corn, rice, and processed products thereof. Examples of materials containing an animal protein include, for example, meats such as beef, pork, and chicken, fish meat, milk, eggs, and processed products thereof. Examples of vegetable protein include soybean proteins such as glycinin, wheat proteins such as gluten, glutenin, and gliadin, and corn proteins such as cone gluten meal. Examples of animal protein include milk proteins such as casein, lactalbumin, and β-lactoglobulin, egg proteins such as ovalbumin, meat proteins such as myosin and actin, blood proteins such as serum albumin, and tendon proteins such as gelatin and collagen. Examples of the substrate protein also include proteins partially decomposed chemically with an acid, alkaline, or the like, or enzymatically with protease or the like, proteins chemically modified with various reagents, recombinant proteins produced in appropriate hosts, and synthetic peptides. The substrate protein may be one subjected to such a treatment as heating, steaming, pulverization, freezing, thawing, and drying, as required. The material containing a protein may contain one kind of protein or two or more kinds of proteins. As the substrate protein, one kind of protein may be used, or two or more kinds of proteins may be used.

[0084] The substrate protein may be, for example, a food or drink containing a protein, or may be a raw material of a food or drink containing a protein. In other words, the substrate protein may be subjected to the deamidation reaction in a state of being contained in, for example, a food or drink or raw material thereof. The type and form of the food or drink or raw material thereof are not particularly limited, so long as the food or drink or raw material thereof contains a protein. For example, each of such agricultural, aquatic, or livestock products containing a protein, processed products thereof, and proteins separated therefrom as mentioned above may be used as a food or drink or raw material thereof alone, an arbitrary combination of two or more of them may be used as a food or drink or raw material thereof, or an arbitrary combination of any one or more of them and another ingredient may be used as a food or drink or raw material thereof. By subjecting a food or drink or raw material thereof containing a protein to a deamidation with protein deamidase, a food or drink or raw material thereof containing a protein having a deamidated asparagine residue can be obtained. Further, a food or drink containing a protein having a deamidated asparagine residue can be produced by using a raw material of the food or drink containing a protein having a deamidated asparagine residue. That is, an embodiment of the deamidation method of the present invention may be a method for producing a food or drink or raw material thereof containing a protein having a deamidated asparagine residue, which method comprises allowing protein deamidase to act on a food or drink or raw material thereof containing a protein. The food or drink containing a protein having a deamidated asparagine residue obtained by an embodiment of the deamidation method of the present invention is also referred to as "food or drink of the present invention". The food or drink of the present invention can be produced by the same method using the same raw material as those for usual foods or drinks, except that the food or drink is obtained via a treatment with protein deamidase. A food or drink also includes a seasoning. Specific examples of the food or drink include, for example, mayonnaise, dressing, cream, yogurt, meat product, and bread.

[0085] The substrate protein may be subjected to the deamidation reaction in a state of, for example, solution, suspension, slurry, paste, or the like. The concentration of the substrate protein in a solution etc. is not particularly limited so long as a desired degree of deamidation is attained. The concentration of the substrate protein in a solution etc. can be appropriately determined according to various conditions, such as type and property of the substrate protein, and desired deamidation ratio. The solution etc. containing the substrate protein is not limited to an aqueous solution, and may be an emulsion in oil or fat. The solution etc. containing the substrate protein may consist of the substrate protein and a solvent, or may contain other ingredients. Examples of the other ingredients include, for example, salts, saccharides, proteins, perfumes, moisturizers, and coloring agents.

[0086] Reaction conditions (e.g. amount of enzyme, reaction time, reaction temperature, and reaction pH) are not particularly limited so long as a desired degree of deamidation is attained. The reaction conditions can be appropriately determined according to various conditions, such as type and purity of protein deamidase, type and purity of protein, and desired degree of deamidation. The amount of the enzyme may be, for example, 0.001 to 500 U, preferably 0.01 to 100 U, more preferably 0.1 to 10 U, with respect to 1 g of the substrate protein. Reaction temperature may be, for example, 5 to 80°C, preferably 5 to 40°C. pH of the reaction solution may be, for example, 2 to 10, preferably 4 to 8. Reaction time may be, for example, 10 seconds to 48 hours, preferably 10 minutes to 24 hours.

[0087] The deamidation can be carried out to a desired degree. The deamidation ratio ((Number of deamidated asparagine residues in substrate protein)/(Number of asparagine residues in substrate protein before deamidation)) may be, for example, 0.1% or higher, 1% or higher, 5% or higher, 10% or higher, or 20% or higher, or 100% or lower, 70% or lower, or 50% or lower.

[0088] The negative charge of a protein is increased by deamidation of the protein. With an increase of the negative charge, there may be provided such effects as a fall of the isoelectric point (pI), an increase of the hydration power, and an increase of the electrostatic repulsive force. Further, the higher-order structure of a protein may be changed by deamidation of the protein, and this change may provide such effects as an increase of the surface hydrophobicity. By these effects, there may be provided effects of improving the functional properties of a protein, such as improvement in the solubility and dispersibility, improvement in the foamability and foam stability, and improvement in the emulsifiability and emulsion stability. Such improvement in the functional properties of a protein provides an expanded use of the protein in the field of, for example, food industry. For example, since many vegetable proteins show poor functional properties such as low solubility, low dispersibility, and low emulsifiability, especially under weakly acidic conditions, which correspond to pH range of usual foods, use of them has been limited in many kinds of foods, for example, coffee whitener, acidic beverages such as fruit juice, dressing, mayonnaise, cream, and so forth. However, if such proteins are deamidated with protein deamidase, and the functional properties thereof such as solubility, dispersibility, and emulsifiability are thereby improved, it becomes possible to preferably use such proteins for many kinds of foods.

[0089] By deamidating a protein with protein deamidase, the mineral sensitivity of the protein may also be reduced, and the contained amount of a soluble mineral in a solution containing the protein and a mineral may be increased. It is generally known that the absorbability of calcium contained in foods into human bodies is improved by solubilizing calcium using an organic acid or casein phosphopeptide. Therefore, if the contained amount of a soluble mineral in a food or drink is increased by deamidating a protein with protein deamidase, the absorbability of a mineral such as calcium into human bodies can be improved. That is, protein deamidase can also be used as, for example, an active ingredient of a calcium absorption-promoting agent.

[0090] In the production of seasonings produced from proteins as raw materials, such as hydrolysates of animal proteins (HAP), hydrolysates of vegetable proteins (HVP), bean paste (miso), and soy sauce, deamidation of a protein with protein deamidase may provide such effects as a reduction of bitter taste and improvement in the ratio of proteolysis by protease. It is generally known that hydrophobic peptides serve as origins of bitter taste. However, by deamidation, the hydrophilic property of such peptides can be improved, and bitter taste thereof can be reduced. Further, deamidation of a protein may change the higher-order structure of the protein, and thereby increase protease sensitivity of the protein. That is, for example, the low decomposition ratio of a protein, which is one of the problems observed in the enzymatic production of HAP and HVP, can also be improved by deamidation.

[0091] As described above, the physical properties and functions of a protein or a material containing a protein can be modified by deamidation. Such modifications of physical properties or functions are also generically referred to as "reforming". That is, the deamidation method of the present invention may be, is other wards, a method for reforming a protein, which method comprises allowing protein deamidase to act on a protein, or may be a method for producing a reformed protein, which method comprises allowing protein deamidase to act on a protein. Also, an embodiment of this method may be, for example, a method for reforming a food or drink or raw material thereof, which method comprises allowing protein deamidase to act on a food or drink or raw material thereof containing a protein, or a method for producing a reformed food or drink or raw material thereof, which method comprises allowing protein deamidase to act on a food or drink or raw material thereof containing a protein.

[0092] Protein deamidase may also be used in combination with protein glutaminase. Protein glutaminase is an enzyme that catalyzes a reaction of deamidating a glutamine residue in a protein. If protein deamidase and protein glutaminase are used in combination, both asparagine residues and glutamine residues of a protein can be deamidated without causing decomposition of the protein into lower molecular weight molecules. Namely, since combinatory use of both the enzymes may further increase the deamidation ratio of the protein, and shift the isoelectric point (pI) of the protein to more acidic side, there may be provided further effects of improving the functional properties of the protein, such as improvement in the solubility even in a pH region more acidic than the pH region where proteins are originally easily insolubilized.

[0093] Protein deamidase may also be used in combination with transglutaminase. Transglutaminase is an enzyme that catalyzes the reaction of binding a glutamine residue and a lysine residue in proteins to crosslink the proteins. By the crosslinking, the protein can be gelled, or the functional properties of the protein can be improved. Therefore, transglutaminase is industrially used in a wide range of fields including the field of food industry as a protein reforming agent. In cases of performing both crosslinking and deamidation of a protein, if deamidation of the protein is performed with protein glutaminase, a glutamine residue as the substrate of transglutaminase is converted into a glutamate residue, and therefore the crosslinking reaction by transglutaminase is inhibited. By contrast, since the substrate of protein deamidase is an asparagine residue, it does not compete with transglutaminase for the substrate, and both crosslinking and deamidation of a protein can be efficiently performed by combinatory use with transglutaminase.

[0094] The type of an enzyme used in combination with protein deamidase can be appropriately chosen according to various conditions such as properties of protein deamidase. When protein deamidase is used in combination with another enzyme, timing or order of addition of the enzymes are not particularly limited. Both enzymes may be simultaneously added, or may be added at different timings. When protein deamidase and another enzyme are used in combination, reaction conditions (e.g. amounts of enzymes, reaction time, reaction temperature, and reaction pH) are not particularly limited so long as the desired effects are obtained. The reaction conditions can be appropriately determined according to various conditions such as type of such another enzyme. For example, when protein deamidase is used in combination with protein glutaminase, the amount of protein glutaminase to be used may be preferably 0.001 to 100 U with respect to 1 g of the substrate protein. Also, for example, when protein deamidase is used in combination with transglutaminase, the amount of transglutaminase to be used may be preferably 0.001 to 100 U with respect to 1 g of the substrate protein.

[0095] Protein deamidase can also be used as a reagent for protein engineering for modifying the function of a protein. When the substrate protein is an enzyme, the enzyme-chemical properties and physicochemical properties of the enzyme can be modified. For example, by deamidation of an enzyme protein with protein deamidase, the isoelectric point of the enzyme protein can be reduced, and the pH stability thereof can be thereby modified. Further, by changing the structure and electric environment of an active site of an enzyme protein, the properties of the enzyme protein, such as affinity to substrate, substrate specificity, reaction rate, pH dependency, temperature dependency, and thermal stability, can be modified.

[0096] Protein deamidase can also be used as a reagent for analysis or research of a protein, such as a reagent for determining the amide concentration of a protein, and a reagent for solubilizing a protein.

[0097] Protein deamidase can also be used for improving extraction efficiency and concentration efficiency for proteins of cereals or legumes. In general, many of proteins of cereals or legumes such as wheat and soybean are water-insoluble, and it is not easy to extract those proteins. However, for example, by treating a suspension of wheat flour or soybean flour with protein deamidase to increase the solubility of proteins, the proteins can be easily extracted, and an isolate having a high protein concentration can be obtained.

[0098] Protein deamidase can also be used for improving extraction efficiency of animal proteins. For example, gelatin is industrially produced by using mainly cow bone, oxhide, and pig skin as raw materials. In order to efficiently extract high quality gelatin, a pretreatment with an inorganic acid such as hydrochloric acid and sulfuric acid (acid treatment), or a pretreatment with lime (alkali treatment) is performed, but these pretreatments each impose high environmental impact, and require a long treatment time. However, if protein deamidase is used, proteins can be easily extracted, and since it is an enzymatic technique, environmental impact can be reduced.

[0099] A protein obtained as described above and showing improved functional properties exhibits superior effects when it is used in various kinds of foods, such as meat or fish meat products, and noodles, and may enable manufacture of a food having a novel mouthfeel and function.

Examples



[0100] Hereafter, the present invention will be more specifically explained with reference to examples. However, the present invention is not limited by these.

Example 1: Screening for protein asparaginase-producing bacterium based on enrichment culture



[0101] A soil sample was inoculated to a medium A (described below) containing Cbz-Asn-Gly (Peptide Institute) as a sole nitrogen source, and culture was performed for 5 days with shaking. The culture broth was applied to an agar plate of tryptic soy medium (Difco), and the grown colonies were chosen and collected. The obtained colonies each were applied to two kinds of agar media (containing 0.3% Cbz-Asn-Gly and not containing Cbz-Asn-Gly) similar to the medium A, and strains that showed significant difference in growth depending on the presence or absence of Cbz-Asn-Gly were chosen. These strains each were again cultured in the medium A, the culture supernatant was analyzed by high speed liquid chromatography (HPLC), and strains for which generation of the deamidation product (Cbz-Asp-Gly) was confirmed were regarded as candidate strains of protein asparaginase-producing bacteria. For these candidate strains, genus and species were identified by homology search based on 16S rDNA sequence analysis. The results are shown in Table 1.

[0102] Medium A: Yeast Carbon Base (1.17%, Difco) and Cbz-Asn-Gly (0.3%) were dissolved in distilled water, and the solution was subjected to filtration sterilization. pH of the medium was adjusted to 7.2.
[Table 1]
Table 1
Genus or speciesNumber of strainCbz-Asp-Gly (mM)
Leifsonia xyli 57 7.0
Leifsonia (other than xyli) 14 6.1
Microbacterium 11 4.7
Agromyces sp. 2 3.4
Paenibacillus 10 1.7
Luteimicrobium album 1 1.7
Raoultella ornithinolytica 1 0.7
Agrobacterium tumefaciens 1 0.6
Enterobacter asburiae 1 0.6
Citrobacter freundii 2 0.5
Arthrobacter aurescens 1 0.3
Rahnella sp. 1 0.2

Example 2: Purification of protein asparaginase derived from Luteimicrobium album



[0103] From the microorganisms obtained in Example 1, Luteimicrobium album AJ111072 (NITE P-01650) was chosen, and used to perform the following experiments.

[0104] Luteimicrobium album AJ111072 (NITE P-01650) was inoculated into a medium B (described below), and shaking culture was carried out at 30°C for 24 hours to obtain a culture broth.

[0105] Medium B: The same volumes of a solution obtained by dissolving 2.34% of Yeast Carbon Base in distilled water and subjecting the solution to filtration sterilization, and a 2% polypeptone (NIHON PHARMACEUTICAL) solution subjected to autoclaving (121°C, 20 minutes) were mixed. pH of the medium was adjusted to 7.2.

[0106] The aforementioned culture broth was centrifuged at 4°C and 8000 rpm for 15 minutes to remove the cells, and the obtained centrifugal supernatant was concentrated about 25 times with an ultrafiltration membrane (Sartorius), and filtered through Stericup 0.22 µm (Millipore). The filtrate was applied to a hydrophobic chromatography column, Hiprep Octyl FF 10/16, equilibrated with a 20 mM sodium phosphate buffer (pH 7.0) containing 1.0 M sodium sulfate (GE Healthcare), and the adsorbed proteins were eluted with a sodium sulfate linear density gradient of 1.0 to 0 M. Active fractions were collected, the buffer was exchanged with a 20 mM sodium phosphate buffer (pH 7.0), the resultant was applied to an anion exchange chromatography column, Hiprep DEAE FF 10/16 (GE Healthcare), equilibrated with the same buffer, and the adsorbed proteins were eluted with a sodium chloride linear density gradient of 0 to 0.5 M. Active fractions were collected again, the buffer was similarly exchanged with a 20 mM sodium phosphate buffer (pH 6.0), the resultant was applied to an anion exchange chromatography column, Hiprep DEAE FF 10/16 (GE Healthcare), equilibrated with the same buffer, and the adsorbed proteins were eluted with a sodium chloride linear density gradient of 0 to 0.5 M. Active fractions were concentrated with an ultrafiltration membrane, applied to a gel filtration chromatography column, Superdex™ 200 10/300, equilibrated with a 20 mM sodium phosphate buffer (pH 7.0) containing 0.1 M sodium chloride, and eluted with the same buffer. A purification table is shown as Table 2. Active fractions were mixed with a sample buffer for SDS-polyacrylamide gel electrophoreses (SDS-PAGE) containing a reducing agent, heat-treated, and subjected to electrophoresis on a 7.5% uniform polyacrylamide gel (e-PAGEL, E-T7.5L, Atto), and the gel after the electrophoresis was stained with Coomassie Brilliant Blue. The results are shown in Fig. 1. Judging from the magnitude of the activity and the density of the band, it became clear that the molecular weight of protein asparaginase of Luteimicrobium album AJ111072 (NITE P-01650) is about 110,000. In the active fractions obtained by the final purification process, the asparaginase activity for deamidating free asparagine or the protease activity for decomposing proteins was not detected.

[0107] The protein asparaginase activity was measured by the following procedures using Cbz-Asn-Gly as a substrate. Proteins were quantified by the Bradford method using bovine serum albumin as a standard protein.

[0108] Activity measurement method: An enzyme solution (25 µL) was added to 125 µL of a 0.2 mol/L phosphate buffer (pH 6.5) containing 30 mmol/L of Cbz-Asn-Gly, the mixture was incubated at 37°C for 60 minutes, and then 150 µL of a 12% trichloroacetic acid solution was added to terminate the reaction. The reaction mixture was centrifuged (15,000 rpm, 4°C, 5 minutes), then the ammonia concentration in the supernatant was measured by using F-Kit ammonia (Boehringer Mannheim), and the protein asparaginase activity was calculated. The protein asparaginase activity that generates 1 µmol of ammonia in 1 minute was defined as 1 unit (U) of the protein asparaginase activity.
[Table 2]
Table 2: Purification table
 Total amount of proteinsTotal activitySpecific activityRecovery
 (mg)(U)(U/mg)(%)
Ultrafiltration concentration (molecular weight cut off, 10,000) 145.52 219.02 1.5 100
Hiprep Octyl FF 16/10 53.5 85.58 1.6 39.1
Hiprep DEAE FF 16/10 (pH 7.0) 2.59 49.26 19 22.5
Hiprep DEAE FF 16/10 (pH 6.0) 1.86 27.99 15 12.8
Superdex™ 200 10/300 0.53 13.65 25.9 6.2

Example 3: Determination of N-terminus amino acid sequence of protein asparaginase derived from Luteimicrobium album



[0109] The purified protein asparaginase obtained in Example 2 was analyzed with a protein sequencer (PPSQ-21A, Shimadzu) to determine the N-terminus amino acid sequence thereof for 5 residues. The N-terminus amino acid sequence of the protein asparaginase of Luteimicrobium album AJ111072 (NITE P-01650) was Ala-Val-Thr-Ala-Asp (SEQ ID NO: 1).

Example 4: Determination of full-length amino acid sequence of protein asparaginase derived from Luteimicrobium album



[0110] The full-length amino acid sequence of the protein asparaginase was determined by a technique of identifying a protein from LC-MS/MS data using a genome sequence obtained with a next-generation sequencer as a database. That is, Luteimicrobium album AJ111072 (NITE P-01650) was cultured at 30°C for 24 hours on the tryptic soy agar medium, the genomic DNA was extracted from grown cells, and the nucleotide sequence was obtained with Miseq (Illumina). Then, the purified protein asparaginase obtained in Example 2 was subjected to SDS-PAGE, and the objective band was excised and digested with trypsin. The digested fragments were subjected to LC-MS/MS analysis to obtain partial amino acid sequences of the enzyme. Further, from the genome sequence information, partial amino acid sequences, and N-terminus amino acid sequence (SEQ ID NO: 1), which were obtained by the aforementioned methods, the amino acid sequence of 1355 residues of the protein asparaginase of Luteimicrobium album AJ111072 (NITE P-01650) including the pre-pro-region (SEQ ID NO: 2) and the full-length nucleotide sequence of the gene encoding this enzyme (SEQ ID NO: 3) were obtained.

Example 5: Deamidation of protein with protein asparaginase derived from Luteimicrobium album



[0111] Insulin B chain (Sigma) was dissolved in a 0.1 M sodium phosphate buffer at a concentration of 2.5 mg/ml to prepare a substrate solution. To 45 µL of the substrate solution, 45 µL of a solution of the protein asparaginase of Luteimicrobium album AJ111072 (NITE P-01650) (about 0.3 U/ml) or 45 µL of water as a control was added, the reaction was performed at 37°C for 1 hour, and then 10 µL of 1 N hydrochloric acid was added to terminate the reaction. Then, the reaction mixture was centrifuged, the supernatant was filtered, and the filtrate was analyzed by HPLC. The results are shown in Fig. 2. Whereas the elution time of the insulin B chain observed for the control group (solid line) was 4.88 minutes, the elution time of the insulin B chain observed for the enzyme addition group (dashed line) was 4.96 minutes. Thus, a small difference of the elution time was observed. Therefore, the solutions after the reaction were analyzed with a protein sequencer (PPSQ-21A, Shimadzu), and it was confirmed that the N-terminus sequence of the insulin B chain of the enzyme addition group was Phe-Val-Asp-Gln-, while the N-terminus sequence of the insulin B chain of the control group was Phe-Val-Asn-Gln-. Therefore, it was verified that asparagine of the third residue from the N-terminus of the insulin B chain was converted into aspartic acid by deamidation by this enzyme. By contrast, glutamine as the fourth residue from the N-terminus did not change.

[0112] Then, in order to investigate the reactivity against various proteins, α-casein (Sigma), α-lactalbumin (Sigma), casein sodium ("MIPRODAN", Nippon Shinyaku), milk whey protein ("BiPro", Davisco), porcine-derived acidic gelatin (Sigma), bovine-derived alkaline gelatin (Sigma), fish-derived gelatin (Nippi), cornmeal gluten (Sigma), and ovalbumin (Sigma) were each dissolved in a sodium phosphate buffer (0.02 M, pH 6.5) at a concentration of 2% w/v. A 6% w/v solution of skim milk powder (Yotsuba Milk Products, low heat type) was also dissolved in the same buffer. To 100 µL of each substrate solution, 10 µL of a solution of the protein asparaginase of Luteimicrobium album AJ111072 (NITE P-01650) (about 5 U/ml) was added, the reaction was performed at 37°C for 1 hour, and then 100 µL of 12% trichloroacetic acid was added to terminate the reaction. Then, the reaction mixture was centrifuged, and ammonia contained in the centrifugal supernatant was quantified with F-Kit ammonia (Roche). As shown in Table 3, it was confirmed that this enzyme acts on various proteins. Further, a part of the reaction mixtures after completion of the reaction was subjected to SDS-PAGE, and the results were compared with those of the control. As a result, any increase or decrease of the molecular weight of the protein caused by this enzyme was not observed. That is, any activity for crosslinking proteins or protease activity was not detected for the protein asparaginase of Luteimicrobium album AJ111072 (NITE P-01650).
[Table 3]
Table 3
SubstrateAmount of generated ammonia (mM)
α-Casein 4.69
α-Lactalbumin 1.84
Skim milk powder 1.22
Casein sodium 2.98
Milk whey protein 1.17
Acidic gelatin (porcine) 2.23
Alkaline gelatin (bovine) 0.73
Fish gelatin 1.27
Soybean proteins 0.98
Rice proteins 0.04
Cornmeal 0.41
Ovalbumin 0.01

Example 6: Modification of properties of protein with protein asparaginase derived from Luteimicrobium album



[0113] Casein sodium (Nippon Shinyaku, trade name: MIPRODAN) was dissolved in a 20 mM sodium phosphate buffer (pH 7.0) at a concentration of 2% w/v to prepare a substrate solution. To 500 µL of the substrate solution, 25 µL of a solution of the protein asparaginase of Luteimicrobium album AJ111072 (NITE P-01650) (0.68 U/ml or 2.7 U/ml) was added, the reaction was performed at 37°C for 1 hour, and then the enzyme was inactivated by a treatment at 100°C for 5 minutes. The 0.68 U/ml enzyme addition group is referred to as test group 1, and the 2.7 U/ml enzyme addition group is referred to as test group 2. As a control, 25 µL of water was added instead of the enzyme solution, and the mixture was treated in the same manner (control group). The ammonia concentrations of the reaction mixtures were quantified, and the value of the control group was subtracted from those of the experimental groups. As a result, the released ammonia amounts obtained with addition of 0.68 U/ml and 2.7 U/ml of the enzyme solution were 1.0 mM and 1.3 mM, respectively. The results of isoelectric focusing (IEF) performed for these samples are shown in Fig. 3. In the samples added with the enzyme (test groups 1 and 2), the isoelectric point (pI) shifted to more acidic side compared with the control group, and thus a fall of pI of the protein due to deamidation was confirmed.

[0114] The solubility was investigated for the samples of the control group and the test group 2 by the following method. To 5 µL of each of the samples, 200 µL of each of buffers of various pH values (mentioned below) was added, the mixture was left standing at room temperature for 5 minute, and then centrifuged at 15,000 rpm for 5 minutes, and the protein concentration in the supernatant was determined by the Bradford method. The solubility was calculated as a relative value based on the protein concentration at pH 9.0 of the control group, which was taken as 100%. The results are shown in Fig. 4. In the test group 2, in which the enzyme was added, the solubility was increased compared with the control group, especially around pH 5.0, and thus improvement in the solubility of proteins provided by deamidation was confirmed.

[0115] The pH buffers: acetate buffer (pH 4 to 6.0), phosphate buffer (pH 6.0 to 7.5), and Tris-hydrochloric acid buffer (pH 7.5 to 9.0), of which the concentrations were 0.2 M.

<Effect of combinatory use with transglutaminase>



[0116] Transglutaminase is an enzyme that forms an isopeptide bond between a glutamine residue and lysine residue in proteins to crosslink the proteins. Transglutaminase and protein glutaminase have a problem that since they both use glutamine in a protein as a substrate, if these are used together, the reactions conflict to each other. By contrast, since protein asparaginase of the present invention uses asparagine in a protein as a substrate, it does not compete with transglutaminase, and therefore combinatory use of them provides both the effect of deamidation and the effect of crosslinking. Therefore, in this example, effects of combinatory use of transglutaminase with protein asparaginase and transglutaminase with protein glutaminase were compared. As transglutaminase, a product purified from Activa (registered trademark) TG bulk powder was used. As protein glutaminase, a purified product prepared by the method described in WO2006/075771 was used. For comparison, to 500 µL of 2% v/v solution of casein sodium, 25 µL of the protein glutaminase (10 U/ml) was added, the reaction was performed at 37°C for 1 hour, and then the enzyme was inactivated with a treatment at 100°C for 5 minutes (comparative group). To the casein solutions of the control group, test group 2, and the comparative group, 6.5 U of the transglutaminase was added per 1 g of casein, the reaction was performed at 37°C for 100 minutes, and then the enzyme was inactivated by a treatment at 95°C for 5 minutes. The reaction mixtures each were subjected to SDS-PAGE, and molecular weight change of casein was investigated. The results are shown in Fig. 4. Whereas crosslinking by transglutaminase was suppressed in the comparative group (protein glutaminase-treated casein), formation of crosslinking products was confirmed in the test group 2 (protein asparaginase-treated casein) almost similarly to the control group. Therefore, it was suggested that enhancement of physical properties due to crosslinking and improvement in functions such as solubility due to deamidation are expected to be simultaneously provided by combinatory use of transglutaminase and protein asparaginase.

Example 7: Analysis of protein asparaginase derived from Leifsonia xyli



[0117] From the microorganisms obtained in Example 1, Leifsonia xyli AJ111071 (NITE P-01649) was chosen, and used to perform the following experiments.

[0118] Leifsonia xyli AJ111071 (NITE P-01649) was inoculated into a medium C (described below), and shaking culture was carried out at 30°C for 24 hours to obtain a culture broth.

[0119] Medium C: The same volumes of a solution obtained by dissolving 1.17% of Yeast Carbon Base in distilled water and subjecting the solution to filtration sterilization, and a 1% solution of casein sodium (Wako Pure Chemical Industries) subjected to autoclaving (121°C, 20 minutes) were mixed. pH of the medium was adjusted to 7.2.

[0120] The culture broth was centrifuged at 15000 rpm for 15 minutes, and the protease activity in the obtained supernatant was measured. As a result, the protease activity was not detected. By contrast, the protein asparaginase activity in the supernatant was 0.029 U/ml.

[0121] A 2% casein sodium solution (100 µL) as a substrate was reacted with 100 µl of the supernatant for 3 hours, and then 200 µl of 12% TCA was added to terminate the reaction. As a control group, a mixture was also prepared by adding 12% TCA, and then adding the supernatant. Ammonia in each of the reaction mixtures was quantified, and from the ammonia amount of the reaction mixture observed after the reaction for 3 hours, the corresponding value of the control group was subtracted. As a result, 0.289 mM of ammonia was released, and it was confirmed that the enzyme produced by this bacterium deamidates casein.

[0122] In the same manner as that described in Example 5, the insulin B chain was used as the substrate, and reacted with the culture supernatant. The results of HPLC analysis of the reaction mixtures obtained after the reaction at 37°C for 2 hours and 8 hours are shown in Fig. 5. Peaks of both the unreacted substrate and the reaction product were observed for the reaction mixture obtained after the reaction for 2 hours, while the substrate was totally converted into the reaction product in the reaction mixture obtained after the reaction for 8 hours. When the reaction mixture obtained after the reaction for 8 hours was analyzed with a protein sequencer, it was confirmed that asparagine of the third residue from the N-terminus of the insulin B chain was converted into aspartic acid by deamidation. By contrast, glutamine of the fourth residue from the N-terminus did not change.

Example 8: Analysis of protein asparaginase derived from Agromyces sp. (1)



[0123] From the microorganisms obtained in Example 1, Agromyces sp. AJ111073 (NITE BP-01782) was chosen, and used to perform experiments similar to those of Examples 2 to 4. That is, protein asparaginase was purified from a culture broth of Agromyces sp. AJ111073 (NITE BP-01782), and subjected to SDS-PAGE. The objective band was excised, and digested with trypsin, and peptides were isolated from the trypsin digestion product of the objective enzyme, and analyzed with a protein sequencer (PPSQ-21A, Shimadzu). As a result, an internal amino acid sequence of 12 residues shown as SEQ ID NO: 4 (Ala-Arg-Gly-Gln-Leu-Ile-Leu-Asp-Thr-Leu-Thr-Met) was determined. A gene encoding the amino acid of SEQ ID NO: 4 was searched for by using the total genome sequence of Agromyces sp. AJ111073 (NITE BP-01782) obtained beforehand with a next-generation sequencer as a database. As a result, the amino acid sequence of 1180 residues of the protein asparaginase of Agromyces sp. AJ111073 (NITE BP-01782) including a pre-pro-region (SEQ ID NO: 5), and the full-length nucleotide sequence of the gene encoding this enzyme (SEQ ID NO: 6) were obtained.

Example 9: Analysis of protein asparaginase derived from Agromyces sp. (2)


<1> Purification of protein asparaginase derived from Agromyces sp.



[0124] From the microorganisms obtained in Example 1, Agromyces sp. AJ111073 (NITE BP-01782) was chosen, and used to perform the following experiments.

<1-1> Cultivation



[0125] Agromyces sp. AJ111073 (NITE BP-01782) was inoculated into the medium C (described below), and shaking culture was carried out at 37°C for 24 hours to obtain a culture broth.

[0126] Medium C: The same volumes of a solution obtained by dissolving 1.17% of Yeast Carbon Base in distilled water and subjecting the solution to filtration sterilization, and a 1% solution of casein sodium (Wako Pure Chemical Industries) subjected to autoclaving (121°C, 20 minutes) were mixed. pH of the medium was adjusted to 7.2.

<1-2> Treatment for activation of protein asparaginase



[0127] The obtained culture broth was mixed with 1/10 volume of a culture supernatant of Bacillus subtilis (described below), and the mixture was left standing overnight. By this operation, the protein asparaginase activity of the culture supernatant was improved from 0.005 U/ml to 1.06 U/ml.

[0128] Culture supernatant of Bacillus subtilis: Bacillus subtilis subsp. subtilisT JCM1465 was inoculated into the aforementioned medium C, and shaking culture was carried out at 37°C for 24 hours to obtain a culture broth. The obtained culture broth was centrifuged at 4°C and 8,000 rpm for 15 minutes to remove the cells, and the supernatant was filtered with Stericup 0.22 µm (Millipore) to obtain a culture supernatant.

<1-3> Purification and molecular weight determination of protein asparaginase



[0129] The culture broth after the activation treatment was centrifuged at 4°C and 40,000 rpm for 1 hour to remove the cells, and the obtained centrifugal supernatant was filtered with Stericup 0.22 µm (Millipore). NaCl was dissolved to the filtrate at a final concentration of 2.0 M, and the mixture was applied to a hydrophobic chromatography column, Hiprep Phenyl FF 10/16 (GE Healthcare), equilibrated with a 20 mM sodium phosphate buffer (pH 6.0) containing 2.0 M NaCl. The adsorbed proteins were eluted with a NaCl linear density gradient of 2.0 to 0 M to collect active fractions, and the buffer thereof was exchanged with a 20 mM sodium phosphate buffer (pH 6.0). The sample was applied to an anion exchange chromatography column, Hiprep DEAE FF 10/16 (GE Healthcare), equilibrated with the same buffer, and the adsorbed proteins were eluted with a sodium chloride linear density gradient of 0 to 0.5 M. A purification table is shown as Table 4. Active fractions were mixed with a sample buffer for SDS-polyacrylamide gel electrophoreses (SDS-PAGE) containing a reducing agent, heat-treated, and subjected to electrophoresis on a 4-12% Bis-Tris Gel (NuPAGE, Invitrogen), and the gel after the electrophoresis was stained with SimplyBlue SafeStain (Invitrogen). The results are shown in Fig. 6. Judging from the magnitude of the activity and the density of the band, it became clear that the molecular weight of protein asparaginase of Agromyces sp. AJ111073 (NITE BP-01782) is about 120,000. In the active fractions obtained by the final purification process, the asparaginase activity that acts on free asparagine or the protease activity for decomposing proteins was not detected.
[Table 4]
Table 4: Purification table of protein asparaginase
 Total activity (U)Recovery (%)
Culture supernatant 819.75 100
Hiprep Phenyl FF 16/10 415.56 50.7
Hiprep DEAE FF 16/10 (pH 6.0) 195.79 23.9

<2> Determination of internal amino acid sequence and N-terminus amino acid sequence of protein asparaginase derived from Agromyces sp.



[0130] The band corresponding to the protein asparaginase was excised from the gel after the electrophoresis, and digestion was performed in the gel. That is, the excised gel piece was washed, and treated with a Tris-hydrochloric acid buffer (pH 8.5) containing lysyl endopeptidase at 35°C for 20 hours. Then, the treated sample was subjected to reverse phase HPLC to separate fragmented peptides. Analyzable peptide peaks were isolated, and analyzed with a protein sequencer (Procise 494 HT Protein Sequencing System). As a result, a sequence containing Ala-Arg-Gly-Gln-Leu-Ile-Leu-Asp-Thr-Leu-Thr-Met (SEQ ID NO: 4) was confirmed. The purified protein asparaginase obtained above was also analyzed with a protein sequencer (PPSQ-21A, Shimadzu) to determine the N-terminus amino acid sequence for 5 residues. The N-terminus amino acid sequence of the protein asparaginase (mature protein) of Agromyces sp. AJ111073 (NITE BP-01782) was Ala-Ala-Thr-Glu-Asp (SEQ ID NO: 12).

Example 10: Activation of protein asparaginase precursor by processing



[0131] From the microorganisms obtained in Example 1, Agromyces sp. AJ111073 (NITE BP-01782) was chosen, and used to perform investigation of processing enzymes for converting a protein asparaginase precursor in a culture broth into a mature enzyme.

[0132] Aqueous solutions of various commercial proteases at concentrations of 1% (100-fold diluted solutions in cases of liquid enzymes) were prepared, and each added in an amount of 5% v/v to the culture broth of Agromyces sp. AJ111073 (NITE BP-01782), and the mixtures each were left standing at room temperature for 30 minutes. The protein asparaginase activities of the culture broths treated with the proteases are shown in Table 5. In the table, "PA act." means the protein asparaginase activity. The protein asparaginase activity of the culture broth was 0.21 U/mL before the activation (before the protease treatment), but it was improved even to 4.6 to 5.7 U/mL by the protease treatment. Also, when a culture supernatant of Bacillus subtilis (described below) was added in a volume of 10% v/v to the culture broth of Agromyces sp. AJ111073 (NITE BP-01782), and the mixture was left standing at room temperature for 30 minutes, the same activation effect was obtained (Table 5).

[0133] Culture supernatant of Bacillus subtilis: Bacillus subtilis subsp. subtilisT JCM1465 was inoculated into the aforementioned medium C, and shaking culture was carried out at 37°C for 24 hours to obtain a culture broth. The obtained culture broth was centrifuged at 4°C and 8,000rpm for 15 minutes to remove the cells, and the supernatant was filtered with Stericup 0.22 µm (Millipore) to obtain a culture supernatant.
[Table 5]
Table 5: Protein asparaginase activation effect of various proteases
ProductManufacturerPA act. (U/mL)Origin
Protin SD-NY10 Amano Enzyme 5.62 Bacillus amyloliquefaciens
Sumizyme ACP-G Shinnihon Chemicals 5.69 Aspergillus oryzae
Protease P "Amano" 3SD Amano Enzyme 5.45 Aspergillus melleus
Protease S "Amano" G Amano Enzyme 5.18 Bacillus stearothermophilus
Corolase N Higuchi Inc. 5.31 Bacillus subtilis
Purified papain for foods Nagase ChemteX 4.56 Papaya
Papain W-40 Amano Enzyme 5.28 Carica papaya L.
Actinase AS Kaken Pharmaceutical 5.45 Streptomyces griseus
Sumizyme LP Shinnihon Chemicals 5.22 Aspergillus oryzae.
Pancreatic Trypsin Novo Novozymes 5.36 Porcine pancreatic trypsin
Nucleicin HBI Enzymes 5.59 Bacillus subtilis
Protin SD-AC10F Amano Enzyme 5.65 Bacillus licheniformis
Protin SD-AY10 Amano Enzyme 5.50 Bacillus licheniformis
DELVOLASE DSM 5.58 Bacillus licheniformis
Alcalase Novozymes 5.56 Bacillus licheniformis
Enzylon ALK-4 Rakuto Kasei Industrial 4.75 Bacillus licheniformis
Protease M "Amano" SD Amano Enzyme 5.56 Aspergillus oryzae
Culture supernatant of Bacillus subtilis - 5.26 -
Before activation - 0.21 -


[0134] The N-terminus amino acid sequence of the protein asparaginase precursor of Agromyces sp. AJ111073 (NITE BP-01782) was determined to be VPEHGVIASGD (SEQ ID NO: 13), and it was found that it located upstream from the N-terminus amino acid sequence of the mature enzyme (SEQ ID NO: 12) by 114 residues.

Example 11: Reforming of gelatin with protein asparaginase derived from Agromyces sp.



[0135] An aqueous solution of porcine acidic gelatin (Nitta Gelatin) at a concentration of 5% wt (adjusted to pH 7.0) was prepared. To the aqueous solution, the protein asparaginase of Agromyces sp. AJ111073 (NITE BP-01782) was added in an amount of 1, 2, or 10 U per 1 g of the raw material gelatin, and an enzymatic treatment was performed at 37°C for 2 hours. For comparison, a sample was prepared by adding water instead of the enzyme, and treated in the same manner. The samples after completion of the reaction each were diluted 50 times with water, and the isoelectric point (pI) thereof was determined with a zeta potential meter (Zetasizer Nano ZS, Malvern) equipped with an automatic titrator (MPT-2). The results are shown in Table 6. With an increase in the amount of the added enzyme, the isoelectric point of gelatin was reduced, and thereby gelatins having different surface charges were prepared. As described above, use of protein asparaginase of the present invention enables control of the surface charge of a gelatin protein, which is important for functional expression of the gelatin protein.
[Table 6]
Table 6: Change of isoelectric point of gelatin provided by protein asparaginase treatment
Enzyme (U/g (protein raw material))Isoelectric point (PI)
0 8.96
1 7.41
2 6.65
10 6.05

<Effect of combinatory use with protein glutaminase (1)>



[0136] Aqueous solutions of porcine acidic gelatin and bovine alkaline gelatin (both are products of Nitta Gelatin) at concentrations of 5% wt (adjusted to pH 7.0) were prepared. To each of the aqueous solutions, the protein asparaginase of Agromyces sp. AJ111073 (NITE BP-01782) was first added in an amount of 15 U per 1 g of the raw material gelatin. Immediately thereafter, protein glutaminase prepared by the method described in WO2006/075771 (purified enzyme) was added in an amount of 50 U per 1 g of the raw material gelatin, and the enzymatic treatment was performed at 37°C for 2 hours. The groups in which the acidic gelatin and alkaline gelatin were treated with the enzymes are referred to as test groups A and B, respectively. As controls, the above procedure was repeated with adding water instead of the enzymes, and these groups are referred to as control groups A and B, respectively. After completion of the reaction, the samples were diluted 50 times with water, and the isoelectric points (pI) thereof were determined in the same manner as described above. As a result, the pI values of the control groups A and B were 8.98 and 5.03, respectively, while pI values of the test groups A and B were 4.85 and 4.81, respectively, and thus it was confirmed that pI values shifted to more acidic side in both of the test groups. In particular, pI of the enzyme-treated acidic gelatin (test group A) markedly changed, and thus it was demonstrated that the treatment markedly changes the electric properties of the gelatin. A further fall of pI was also observed for the enzyme-treated alkaline gelatin (test group B).

<Effect of combinatory use with protein glutaminase (2)>



[0137] Aqueous solutions of fish acidic gelatin (Nippi) and bovine alkaline gelatin (Sigma) at concentrations of 1% wt were prepared with a 20 mM sodium phosphate buffer (pH 6.5), respectively. To each of the aqueous solutions, the protein asparaginase of Agromyces sp. AJ111073 (NITE BP-01782) was first added in an amount of 50 U per 1 g of the raw material gelatin, and the enzymatic treatment was performed at 37°C for 1 hour. Then, protein glutaminase prepared by the method described in WO2006/075771 (purified enzyme) was added in an amount of 80 U per 1 g of the raw material gelatin, and the enzymatic treatment was performed at 55°C for 1 hour. Then, the enzymes were inactivated by a treatment at 100°C for 5 minutes. The groups in which the acidic gelatin and alkaline gelatin were treated with the enzymes are referred to as test groups A and B, respectively. As controls, the above procedure was repeated with adding water instead of the enzymes, and these groups are referred to as control groups A and B, respectively. The isoelectric points (pI) of these samples were determined with a zeta potential meter (Zetasizer Nano ZS, Malvern). As a result, the pI values of the control groups A and B were 8.63 and 5.25, respectively, while pI values of the test groups A and B were 4.59 and 4.61, respectively, and thus it was confirmed that pI values shifted to more acidic side in both of the test groups. In particular, pI of the enzyme-treated acidic gelatin (test group A) markedly changed, and thus it was demonstrated that the treatment markedly changes the electric properties of the gelatin. A further fall of pI was also observed for the enzyme-treated alkaline gelatin (test group B).

[0138] It can be expected that gelatin, which is broadly utilized for food, medical, industrial uses etc., can be made to have higher quality and higher added value by use of protein asparaginase as described above.

Example 12: Heterogeneous expression of protein asparaginase


<1> Secretory expression of protein asparaginase derived from Agromyces sp. in Corynebacterium glutamicum


<1-1> Construction of plasmid for secretory expression of protein asparaginase



[0139] When protein asparaginase is heterogeneously expressed, expression of the protein asparaginase in the form of having the original pro-sequence on the N-terminus side may contribute to stabilization of the structure of the protein asparaginase. For expressing an objective protein in the form of a fusion protein with an amino acid sequence other than the objective protein, there is widely known a method of providing a recognition sequence for a specific protease showing high substrate specificity between the amino acid sequence of the objective protein and the fused amino acid sequence, so that the expressed fusion protein is cleaved with the specific protease to easily obtain the objective protein. Further, as the protease showing high substrate specificity, for example, there are known the factor Xa protease and the ProTEV protease, and they recognize the sequence of Ile-Glu-Gly-Arg (= IEGR) (SEQ ID NO: 14) and Glu-Asn-Leu-Tyr-Phe-Gln (= ENLYFQ) (SEQ ID NO: 15) in a protein, respectively, to specifically cleave the protein on the C-terminus side of the respective sequences. Therefore, for example, concerning a pro-sequence-fused protein asparaginase, if a pro-sequence-fused protein asparaginase gene is constructed so that a nucleotide sequence encoding the recognition sequence for the factor Xa protease (IEGR) or the recognition sequence for the ProTEV protease (ENLYFQ) is inserted between the nucleotide sequence encoding the pro-sequence amino acid residues of protein asparaginase and the nucleotide sequence encoding a mature protein asparaginase, and the pro-sequence-fused protein asparaginase is expressed from the gene, it becomes possible to easily remove the pro-sequence from the pro-sequence-fused protein asparaginase to obtain the mature protein asparaginase by using any of these proteases.

[0140] In consideration of the codon usage of C. glutamicum, there was designed a DNA sequence (SEQ ID NO: 16) encoding a fusion protein (SEQ ID NO: 17) in which, from the N-terminus, the signal sequence of the CspA protein derived from C. ammoniagenes (WO2013/06029), the pro-sequence of the protein asparaginase derived from Agromyces sp. AJ111073 (NITE BP-01782), the recognition sequence for the ProTEV protease (ENLYFQG), and the sequence of the mature protein asparaginase derived from Agromyces sp. AJ111073 (NITE BP-01782) were ligated in this order (Fig. 10). Further, a DNA sequence (SEQ ID NO: 18) comprising the above DNA sequence (SEQ ID NO: 16), the CspB promoter region provided immediately upstream of the above DNA sequence, and the recognition sequences for the restriction enzymes KpnI and BamHI on the 5' side and 3' side thereof, respectively, was synthesized by an artificial gene synthesis method, and cloned into the plasmid vector pPK4 (Corynebacterium-E. coli shuttle vector carrying the kanamycin resistance gene, Japanese Patent Laid-open (Kokai) No. 9-322774). Specifically, the synthesized DNA sequence (SEQ ID NO: 18) and pPK4 were simultaneously digested at two sites with the restriction enzymes KpnI and BamHI, then both DNA fragments were ligated, and used to transform competent cells of the Escherichia coli JM109 strain (TaKaRa), and the transformed cells were applied to the LB agar medium containing 50 µg/ml of kanamycin, and cultured overnight at 37°C. Then, single colonies were separated from the colonies that appeared to obtain transformants. Plasmid DNAs were extracted from the obtained transformants in a conventional manner, the objective plasmid was confirmed by DNA sequencing, and this plasmid was designated as pPK4-Pro-TEV-PA.

<1-2> Secretory expression of protein asparaginase in C. glutamicum



[0141] Then, the C. glutamicum YDK010 strain (WO2004/029254) was transformed with pPK4-Pro-TEV-PA in a conventional manner to obtain YDK010/pPK4-Pro-TEV-PA strain. The C. glutamicum YDK010 strain is a strain deficient in the cell surface protein PS2 (CspB), of C. glutamicum AJ12036 (FERM BP-734) (WO2004/029254). The AJ12036 strain was originally deposited at the Agency of Industrial Science and Technology, Fermentation Research Institute (presently, the independent administrative agency, National Institute of Technology and Evaluation, International Patent Organism Depositary, #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on March 26, 1984 as an international deposit, and given with an accession number of FERM BP-734. The YDK010/pPK4-Pro-TEV-PA strain was cultured at 30°C for 72 hours in the MM liquid medium (120 g of glucose, 3 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate, 1.5 g of potassium dihydrogenphosphate, 0.03 g of iron sulfate heptahydrate, 0.03 g of manganese sulfate tetrahydrate, 0.45 mg of thiamine hydrochloride, 0.45 mg of biotin, 0.15 g of DL-methionine, and 50 g of calcium carbonate in a volume of 1 L made with water, adjusted to pH 7.0) containing 25 mg/L of kanamycin. After the culture for 72 hours, the culture broth was centrifuged (13,800 x g, 2 minutes), 4 µL of the supernatant was subjected to reducing SDS-PAGE, and staining was performed with SimplyBlue SafeStain (Novex), to analyze the proteins secreted in the culture broth. As a result, a band was confirmed at position around 115 kDa, which is the expected molecular weight size of the pro-sequence-fused protein asparaginase (Fig. 11). Since the amino acid composition of the protein asparaginase contains many acidic amino acids, it was estimated that the molecular weight slightly shifted to the higher molecular weight side from 115 kDa.

[0142] Since this fusion protein has the recognition sequence for ProTEV in the connection part between the pro-sequence and the mature protein asparaginase, the mature protein asparaginase can be obtained by digesting this fusion protein with ProTEV. Therefore, this fusion protein contained in the crude enzyme solution (culture supernatant) was processed into the mature protein asparaginase by digestion with ProTEV, and it was investigated whether the protein asparaginase activity was observed for the resultant. The crude enzyme solution (culture supernatant) obtained as described above was concentrated about 9 times by using Vivaspin 10,000 MWCO (GE Healthcare), and subjected to the processing with ProTEV. The processing was performed by adding 4 µL of ProTEV Plus (Promega), 10 µL of 10 x buffer (1 M NaCl, 500 mM Tris-HCl, 50 mM CaCl2, pH 8.0), and 46 µL of Mili-Q water to 40 µL of the concentrated crude enzyme solution, and incubating the mixture at 25°C for 2 hours. The protein asparaginase activity of the concentrated crude enzyme solution treated with ProTEV was measured, and found to be 0.37 U/ml. As seen from the above result, secretory expression of protein asparaginase derived from Agromyces sp. was attained in C. glutamicum.

<2> Intracellular expression of protein asparaginase in E. coli


<2-1> Expression of protein asparaginase derived from Agromyces sp.



[0143] In consideration of the codon usage of E. coli, a DNA sequence (SEQ ID NO: 19) encoding a fusion protein (SEQ ID NO: 20) in which, from the N-terminus, the pro-sequence of the protein asparaginase derived from Agromyces sp. AJ111073 (NITE BP-01782), the recognition sequence for the ProTEV protease (ENLYFQG), and the sequence of the mature protein asparaginase (mature PA) derived from Agromyces sp. AJ111073 (NITE BP-01782) were ligated in this order was synthesized by an artificial gene synthesis method, and cloned into a plasmid vector pCold TF DNA (TaKaRa). pCold TF DNA is a cold shock expression vector, with which transcription for the objective protein is induced at a low temperature, designed so that an objective gene is expressed to provide the objective protein in the form of being fused with the trigger factors (TF), which is a kind of chaperon, as a soluble tag. The cloning was carried out according to the following procedures by using In-Fusion HD Cloning Kit (Clontech). A pCold TF DNA fragment was amplified by PCR using the pCold TF DNA as the template and the primers of SEQ ID NOS: 21 and 22. A DNA fragment for cloning encoding the fusion protein was amplified by PCR using the synthesized DNA sequence (SEQ ID NO: 19) as the template and the primers of SEQ ID NOS: 23 and 24. As the polymerase, Prime STAR GXL (TOYOBO) was used. In PCR, a cycle of reactions at 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 60 seconds was repeated 30 times. The amplified fragments were ligated by the In-Fusion reaction, and the E. coli JM109 strain was transformed with the reaction product, applied on the LB agar medium containing 50 µg/ml of ampicillin, and cultured overnight at 37°C. Then, single colonies were separated from the colonies that appeared to obtain transformants. From the obtained transformants, there was obtained a strain having an expression plasmid for the pro-sequence-fused protein asparaginase derived from Agromyces sp. (proPA_Agro), in which plasmid the DNA encoding the fusion protein of the pro-sequence, the recognition sequence for ProTEV, and the mature PA sequence was correctly ligated downstream from the DNA encoding TF, which plasmid was designated as pCTF-proPA_Agro.

[0144] pCTF-proPA_Agro was extracted from the obtained strain by using a plasmid extraction kit, QIAprep Spin Miniprep Kit (QIAGEN). The E. coli Rosetta 2 strain (Novagen) was transformed with pCTF-proPA_Agro to construct a proPA_Agro-expression strain, E. coli Rosetta2/pCTF-proPA_Agro. This expression strain was cultured overnight at 37°C on the LB agar medium (Difco) containing ampicillin at a final concentration of 100 µg/ml as seed culture. The cells were inoculated to fresh LB medium containing ampicillin at a final concentration of 100 µg/ml with a 1 µl inoculation loop, and shaking culture was performed at 37°C for about 3 hours. Then, isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM, and the culture temperature was lowered to 15°C to induce transcription from the cspA promoter of pCTF-proPA_Agro (low temperature induction). Culture was performed at 15°C for 24 hours, and then the cells were collected by centrifugation. As a control, the cells immediately before the low temperature induction were also collected in a similar manner. Then, the cells from 1 ml of the culture broth were suspended in 350 µl of 20 mM Tris-HCl (pH 8.0), and disrupted by repeating a cycle of sonication of being "ON for 30 seconds, and OFF for 30 seconds" 10 times under cooling conditions by using an ultrasonicator UCD-250 (Cosmobio). The obtained disrupted cell suspension was centrifuged (21,600 x g, 4°C, 10 minutes) to remove the insoluble fraction, and the supernatant fraction was regarded as a crude extract. Then, 5 µl of the NuPAGE LDS sample buffer (Novex) and 2 µl of the NuPAGE sample reducing reagent (Novex) were added to 13 µL of the crude extract, and the mixture was heat-treated at 70°C for 10 minutes, and then subjected to electrophoresis using NuPAGE 4-12% Bis-Tris Gel (Novex). As a result, as shown in Fig. 12, a band of a protein at position around the objective molecular weight of 176 kDa was more distinctly observed for the sample subjected to the low temperature induction of the expression plasmid, compared with that before the low temperature induction. Since the theoretical molecular weights of TF and proPA_Agro are 52 kDa and 124 kDa, respectively, the molecular weight of the objective protein of this expression experiment, TF-proPA_Agro fusion protein, is estimated to be about 176 kDa.

[0145] Since this fusion protein has the digestion recognition site (Glu-Asn-Leu-Tyr-Phe-Gln) for the ProTEV protease in the connection part between the pro-sequence and the mature PA, the mature PA can be obtained by digesting this fusion protein with ProTEV (Novagen). Therefore, PA contained in the crude extract obtained after the low temperature induction was processed into the mature PA by digestion with ProTEV, and it was investigated whether the PA activity was observed for the resultant. The crude enzyme solution obtained as described above was concentrated about 20 times by using Vivaspin 10,000 MWCO (GE Healthcare), and subjected to the processing with ProTEV. The PA activity of the concentrated crude enzyme solution treated with ProTEV was measured, and found to be 0.069 U/ml. As seen from the above result, intracellular expression of protein asparaginase derived from Agromyces sp. was attained in E. coli.

<2-2> Expression of protein asparaginase derived from Leifsonia xyli



[0146] In consideration of the codon usage of E. coli, a DNA sequence (SEQ ID NO: 25) encoding a fusion protein (SEQ ID NO: 26) in which, from the N-terminus, the pro-sequence of the protein asparaginase derived from Leifsonia xyli AJ111071 (NITEP-01649), the recognition sequence for the ProTEV protease (ENLYFQG), and the sequence of the mature protein asparaginase (mature PA) derived from Leifsonia xyli AJ111071 (NITEP-01649) were ligated in this order was synthesized by an artificial gene synthesis method, and cloned into the plasmid vector pCold TF DNA (TaKaRa). The procedures of the following experiments were the same as those of Example 12, <2-1>, unless otherwise stated. A pCold TF DNA fragment was amplified by PCR using the pCold TF DNA as the template and the primers of SEQ ID NOS: 21 and 22. A DNA fragment for cloning encoding the fusion protein was amplified by PCR using the synthesized DNA sequence (SEQ ID NO: 25) as the template and the primers of SEQ ID NOS: 27 and 28. The amplified fragments were ligated by the In-Fusion reaction, and the E. coli JM109 strain was transformed with the reaction product. From the obtained transformants, there was obtained a strain having an expression plasmid for the pro-sequence-fused protein asparaginase derived from Leifsonia xyli (proPA_Leif), in which plasmid the DNA encoding the fusion protein of the pro-sequence, the recognition sequence for ProTEV, and the mature PA sequence was correctly ligated downstream from the DNA encoding TF, which plasmid was designated as pCTF-proPA_Leif.

[0147] The plasmid was extracted from the obtained strain, and transformation of the E. coli Rosetta 2 strain (Novagen), culture of a proPA_Leif-expression strain, and preparation of a crude extract were performed. The crude extract was subjected to electrophoresis. As a result, as shown in Fig. 12, a band of a protein at position around the objective molecular weight of 159 kDa was more distinctly observed for the sample subjected to the low temperature induction of the expression plasmid, compared with that before the low temperature induction. Since the theoretical molecular weights of TF and proPA_Leif are 52 kDa and 107 kDa, respectively, the molecular weight of the objective protein of this expression experiment, TF-proPA_Leif fusion protein, is estimated to be about 159 kDa.

[0148] Since this fusion protein has the digestion recognition site for the ProTEV protease (Glu-Asn-Leu-Tyr-Phe-Gln) in the connection part between the pro-sequence and the mature PA, the mature PA can be obtained by digesting this fusion protein with ProTEV (Novagen). Therefore, PA contained in the crude extract obtained after the low temperature induction was processed into the mature PA by digestion with ProTEV, and it was investigated whether the PA activity was observed for the resultant. The crude enzyme solution was concentrated about 20 times by using Vivaspin 10,000 MWCO (GE Healthcare), and the pro-sequence was cleaved by a treatment with ProTEV for 2 hours. The PA activity of the concentrated crude enzyme solution treated with ProTEV was measured, and found to be 0.047 U/ml. As seen from the above result, intracellular expression of protein asparaginase derived from Leifsonia xyli was attained in E. coli.

<2-3> Expression of protein asparaginase derived from Microbacterium testaceum



[0149] In consideration of the codon usage of E. coli, a DNA sequence (SEQ ID NO: 29) encoding a fusion protein (SEQ ID NO: 30) in which, from the N-terminus, the pro-sequence of the protein asparaginase derived from Microbacterium testaceum, the recognition sequence for the ProTEV protease (ENLYFQG), and the sequence of the mature protein asparaginase (mature PA) derived from Microbacterium testaceum were ligated in this order was synthesized by an artificial gene synthesis method, and cloned into the plasmid vector pCold TF DNA (TaKaRa). The procedures of the following experiments were the same as those of Example 12, <2-1>, unless otherwise stated. A pCold TF DNA fragment was amplified by PCR using the pCold TF DNA as the template and the primers of SEQ ID NOS: 22 and 23. A DNA fragment encoding the fusion protein was amplified by PCR using the synthesized DNA sequence (SEQ ID NO: 29) as the template and the primers of SEQ ID NOS: 31 and 32. The amplified fragments were ligated by the In-Fusion reaction, and the E. coli JM109 strain was transformed with the reaction product. From the obtained transformants, there was obtained a strain having an expression plasmid for the pro-sequence-fused protein asparaginase derived from Microbacterium testaceum (proPA_Micro), in which plasmid the DNA encoding the fusion protein of the pro-sequence, the recognition sequence for ProTEV, and the mature PA sequence was correctly ligated downstream from DNA encoding TF, which plasmid was designated as pCTF-proPA_Micro.

[0150] The plasmid was extracted from the obtained strain, and transformation of E. coli Rosetta 2 strain (Novagen), culture of a proPA_Micro-expression strain, and preparation of a crude extract were performed. The crude extract was subjected to electrophoresis. As a result, as shown in Fig. 12, a band of a protein at position around the objective molecular weight of 174 kDa was more distinctly observed for the sample subjected to the low temperature induction of the expression plasmid, compared with that before the low temperature induction. Since the theoretical molecular weights of TF and proPA_Micro are 52 kDa and 122 kDa, respectively, the molecular weight of the objective protein of this expression experiment, TF-proPA_Micro fusion protein, is estimated to be about 174 kDa.

[0151] Since this fusion protein has the digestion recognition site for the ProTEV protease (Glu-Asn-Leu-Tyr-Phe-Gln) in the connection part between the pro-sequence and the mature PA, the mature PA can be obtained by digesting this fusion protein with ProTEV (Novagen). Therefore, PA contained in the crude extract obtained after the low temperature induction was processed into the mature PA by digestion with ProTEV, and it was investigated whether the PA activity was observed for the resultant. The crude enzyme solution was concentrated about 20 times by using Vivaspin 10,000 MWCO (GE Healthcare), and the pro-sequence was cleaved by a treatment with ProTEV for 2 hours. The PA activity of the concentrated crude enzyme solution treated with ProTEV was measured, and found to be 0.022 U/ml. As seen from the above result, intracellular expression of protein asparaginase derived from Microbacterium testaceum was attained in E. coli.

Industrial Applicability



[0152] According to the present invention, a novel protein deamidase (protein asparaginase) that catalyzes a reaction of deamidating an asparagine residue in a protein is provided. According to an embodiment thereof, by using this enzyme, an asparagine residue in a protein can be deamidated to improve the functional properties of the protein.

Explanation of Sequence Listing



[0153] 

SEQ ID NO: 1, N-Terminus amino acid sequence of protein asparaginase derived from Luteimicrobium album (5 residues)

SEQ ID NO: 2, Amino acid sequence of protein asparaginase derived from Luteimicrobium album (including pre-pro-region)

SEQ ID NO: 3, Full-length nucleotide sequence of protein asparaginase gene derived from Luteimicrobium album

SEQ ID NO: 4, Internal amino acid sequence of protein asparaginase derived from Agromyces sp. (12 residues)

SEQ ID NO: 5, Amino acid sequence of protein asparaginase derived from Agromyces sp. (including pre-pro-region)

SEQ ID NO: 6, Full-length nucleotide sequence of protein asparaginase gene derived from Agromyces sp.

SEQ ID NO: 7, Amino acid sequence of protein asparaginase derived from Microbacterium testaceum

SEQ ID NO: 8, Amino acid sequence of protein asparaginase derived from Leifsonia xyli

SEQ ID NO: 9, Amino acid sequence of protein asparaginase derived from Leifsonia aquatica

SEQ ID NO: 10, Common amino acid sequence for sequences of SEQ ID NOS: 2 and 5

SEQ ID NO: 11, Common amino acid sequence for sequences of SEQ ID NOS: 2, 5, 7, and 8

SEQ ID NO: 12, N-Terminus amino acid sequence of mature protein of protein asparaginase derived from Agromyces sp. (5 residues)

SEQ ID NO: 13, N-Terminus amino acid sequence of precursor (pro-protein) of protein asparaginase derived from Agromyces sp. (11 residues)

SEQ ID NO: 14, Recognition sequence for factor Xa protease

SEQ ID NO: 15, Recognition sequence for ProTEV protease

SEQ ID NO: 16, Nucleotide sequence of gene encoding pro-sequence-fused protein asparaginase derived from Agromyces sp. for secretory expression in C. glutamicum

SEQ ID NO: 17, Amino acid sequence of pro-sequence-fused protein asparaginase derived from Agromyces sp. for secretory expression in C. glutamicum

SEQ ID NO: 18, Nucleotide sequence of insertion fragment containing gene encoding pro-sequence-fused protein asparaginase derived from Agromyces sp. for secretory expression in C. glutamicum

SEQ ID NO: 19, Nucleotide sequence of gene encoding pro-sequence-fused protein asparaginase derived from Agromyces sp. for intracellular expression in E. coli

SEQ ID NO: 20: Amino acid sequence of pro-sequence-fused protein asparaginase derived from Agromyces sp. for intracellular expression in E. coli

SEQ ID NOS: 21 to 24, Primers

SEQ ID NO: 25, Nucleotide sequence of gene encoding pro-sequence-fused protein asparaginase derived from Leifsonia xyli for intracellular expression in E. coli

SEQ ID NO: 26, Amino acid sequence of pro-sequence-fused protein asparaginase derived from Leifsonia xyli for intracellular expression in E. coli

SEQ ID NOS: 27 and 28, Primers

SEQ ID NO: 29, Nucleotide sequence of gene encoding pro-sequence-fused protein asparaginase derived from Microbacterium testaceum for intracellular expression in E. coli

SEQ ID NO: 30, Amino acid sequence of pro-sequence-fused protein asparaginase derived from Microbacterium testaceum for intracellular expression in E. coli

SEQ ID NOS: 31 and 32, Primers




















































































































































































Claims

1. A protein having an activity for catalyzing a reaction of deamidating an asparagine residue in a protein.
 
2. The protein according to claim 1, which does not substantially have an activity for catalyzing a reaction of hydrolyzing a peptide bond in a protein.
 
3. The protein according to claim 1 or 2, which is a protein defined in (A), (B), or (C) mentioned below:

(A) a protein comprising the amino acid sequence of SEQ ID NO: 10 or 11, the amino acid sequence of positions 63 to 1260, 245 to 1378, 245 to 1260, or 131 to 1260 of SEQ ID NO: 10, or the amino acid sequence of positions 47 to 1257, 59 to 1258, 96 to 1101, 241 to 1378, 244 to 1258, 244 to 1257, 244 to 1101, 129 to 1258, 120 to 1257, or 120 to 1101 of SEQ ID NO: 11;

(B) a protein comprising the amino acid sequence of SEQ ID NO: 10 or 11, the amino acid sequence of positions 63 to 1260, 245 to 1378, 245 to 1260, or 131 to 1260 of SEQ ID NO: 10, or the amino acid sequence of positions 47 to 1257, 59 to 1258, 96 to 1101, 241 to 1378, 244 to 1258, 244 to 1257, 244 to 1101, 129 to 1258, 120 to 1257, or 120 to 1101 of SEQ ID NO: 11 but including substitution, deletion, insertion, or addition of 1 to 10 amino acid residues, and having an activity for catalyzing a reaction of deamidating an asparagine residue in a protein;

(C) a protein comprising an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 10 or 11, the amino acid sequence of positions 63 to 1260, 245 to 1378, 245 to 1260, or 131 to 1260 of SEQ ID NO: 10, or the amino acid sequence of positions 47 to 1257, 59 to 1258, 96 to 1101, 241 to 1378, 244 to 1258, 244 to 1257, 244 to 1101, 129 to 1258, 120 to 1257, or 120 to 1101 of SEQ ID NO: 11, and having an activity for catalyzing a reaction of deamidating an asparagine residue in a protein.


 
4. The protein according to any one of claims 1 to 3, which is a protein defined in (a), (b), or (c) mentioned below:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 2, 5, 7, 8, or 9, the amino acid sequence of positions 240 to 1355 of SEQ ID NO: 2, the amino acid sequence of positions 181 to 1180 or 67 to 1180 of SEQ ID NO: 5, the amino acid sequence of positions 193 to 1172 or 70 to 1172 of SEQ ID NO: 7, or the amino acid sequence of positions 146 to 989 or 21 to 989 of SEQ ID NO: 8;

(b) a protein comprising the amino acid sequence of SEQ ID NO: 2, 5, 7, 8, or 9, the amino acid sequence of positions 240 to 1355 of SEQ ID NO: 2, the amino acid sequence of positions 181 to 1180 or 67 to 1180 of SEQ ID NO: 5, the amino acid sequence of positions 193 to 1172 or 70 to 1172 of SEQ ID NO: 7, or the amino acid sequence of positions 146 to 989 or 21 to 989 of SEQ ID NO: 8 but including substitution, deletion, insertion, or addition of one or several amino acid residues, and having an activity for catalyzing a reaction of deamidating an asparagine residue in a protein;

(c) a protein comprising an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 2, 5, 7, 8, or 9, the amino acid sequence of positions 240 to 1355 of SEQ ID NO: 2, the amino acid sequence of positions 181 to 1180 or 67 to 1180 of SEQ ID NO: 5, the amino acid sequence of positions 193 to 1172 or 70 to 1172 of SEQ ID NO: 7, or the amino acid sequence of positions 146 to 989 or 21 to 989 of SEQ ID NO: 8, and having an activity for catalyzing a reaction of deamidating an asparagine residue in a protein.


 
5. A polynucleotide encoding the protein according to any one of claims 1 to 4.
 
6. A recombinant vector containing the polynucleotide according to claim 5.
 
7. A transformant introduced with the recombinant vector according to claim 6.
 
8. A method for producing a protein having an activity for catalyzing a reaction of deamidating an asparagine residue in a protein, the method comprising:

culturing the transformant according to claim 7 in a medium to generate the protein according to any one of claims 1 to 4; and

collecting the protein from the culture broth.


 
9. A method for producing a protein having an activity for catalyzing a reaction of deamidating an asparagine residue in a protein, the method comprising:

culturing a microorganism having an ability to produce the protein according to any one of claims 1 to 4 in a medium to generate the protein; and

collecting the protein from the culture broth.


 
10. The method according to claim 9, wherein the microorganism is a bacterium belonging to the class Actinobacteria.
 
11. The method according to claim 10, wherein the bacterium is a bacterium belonging to the genus Luteimicrobium, Agromyces, Microbacterium, or Leifsonia.
 
12. The method according to claim 11, wherein the bacterium is Luteimicrobium album, Agromyces sp., Microbacterium testaceum, Leifsonia xyli, or Leifsonia aquatica.
 
13. The method according to any one of claims 8 to 12, which comprises treating the protein with a processing enzyme.
 
14. The method according to claim 13, wherein the processing enzyme is protease.
 
15. A method for producing a protein and/or peptide of which an asparagine residue has been deamidated, the method comprising:

allowing the protein according to any one of claims 1 to 4 to act on a protein and/or peptide.


 
16. The method according to claim 15, wherein the protein and/or peptide is contained in a food or drink or raw material thereof.
 
17. The method according to claim 15 or 16, which further comprises allowing transglutaminase and/or protein glutaminase to act on the protein and/or peptide.
 
18. A method for reforming a food or drink or raw material thereof, the method comprising:

allowing the protein according to any one of claims 1 to 4 to act on a food or drink or raw material thereof containing a protein and/or peptide.


 
19. A method for producing a reformed food or drink or raw material thereof, the method comprising:

allowing the protein according to any one of claims 1 to 4 to act on a food or drink or raw material thereof containing a protein and/or peptide.


 
20. The method according to claim 18 or 19, which further comprises allowing transglutaminase and/or protein glutaminase to act on the food or drink or raw material thereof containing a protein and/or peptide.
 
21. The method according to any one of claims 16 to 20, wherein the food or drink is selected from mayonnaise, dressing, cream, yogurt, meat product, and bread.
 
22. The method according to any one of claims 15 to 21, wherein 0.001 to 500 U of the protein according to any one of claims 1 to 4 is used for 1 g of the protein and/or peptide.
 




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REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




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