(19)
(11)EP 3 569 703 A1

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

(43)Date of publication:
20.11.2019 Bulletin 2019/47

(21)Application number: 18738844.2

(22)Date of filing:  15.01.2018
(51)Int. Cl.: 
C12N 9/16  (2006.01)
A23K 20/189  (2016.01)
C12N 15/55  (2006.01)
(86)International application number:
PCT/CN2018/072540
(87)International publication number:
WO 2018/130211 (19.07.2018 Gazette  2018/29)
(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 MD TN

(30)Priority: 15.01.2017 CN 201710027074

(71)Applicant: Feed Research Institute Chinese Academy of Agricultural Sciences
Beijing 100081 (CN)

(72)Inventors:
  • NIU, Canfang
    Beijing 100081 (CN)
  • YANG, Peilong
    Beijing 100081 (CN)
  • YAO, Bin
    Beijing 100081 (CN)
  • LI, Yangyang
    Beijing 100081 (CN)
  • DU, Yongkai
    Beijing 100081 (CN)
  • LUO, Huiying
    Beijing 100081 (CN)
  • HUANG, Huoqing
    Beijing 100081 (CN)
  • WANG, Yaru
    Beijing 100081 (CN)

(74)Representative: Teall, Charlotte 
Forresters IP LLP Skygarden Erika-Mann-Strasse 11
80636 München
80636 München (DE)

  


(54)PHYTASE YEAPPA MUTANTS HAVING IMPROVED GASTRIC PROTEIN RESISTANCE AND ACID RESISTANCE AND INCREASED CATALYTIC EFFICIENCY


(57) The present invention relates to the field of genetic engineering, particularly to phytase variant YeAPPA having improved pepsin resistance and acid resistance, and increased catalytic efficiency, by substituting Leucine at the 162th site of the sequence set forth in SEQ ID NO.1 with glycine or proline or substituting glutamic acid at the 230th site of the sequence set forth in SEQ ID NO.1 with glycine, proline or arginine, in the benefit of the development of economical feed enzyme industry.




Description

FTELD OF THE INVENTION



[0001] The present invention relates to the field of genetic engineering, particularly to phytase variants YeAPPA having improved pepsin resistance and acid resistance, and increased catalytic efficiency.

BACKGROUND OF THE INVENTION



[0002] Phytase can hydrolyze phytic acid into phosphoric acid residues to destroy the binding of phytic acid to mineral elements, and thus improve the utilization rate of nutrients. Therefore, phytase with high catalytic efficiency and protease resistance can produce good economic and ecological benefits, and will have a broad market in feed industry.

[0003] The catalytic function of phytase is directly related to its molecular structure. The study of the crystal structure of the different phytases will be able to help us to deepen understanding of the structure and function of phytases. At present, the crystal structures of several phytases with distinct structures have been reported. The phytases molecule consists of some structural components necessary for catalysis, and some unnecessary components which can be modified to adapt to hydrolyze the different substrates.

Order of the invention



[0004] One order of the present invention is to provide phytase variants having improved pepsin resistance and acid resistance, and increased catalytic efficiency by a method of site-directed mutagenesis.

[0005] Another order of the present invention is to provide a gene encoding the above phytase variants having improved pepsin resistance and acid resistance, and increased catalytic efficiency.

[0006] Another order of the present invention is to provide a recombinant vector comprising the above gene encoding the above phytase variants having improved pepsin resistance and acid resistance, and increased catalytic efficiency.

[0007] Another order of the present invention is to provide a recombinant cell comprising the above gene encoding the above phytase variants having improved pepsin resistance and acid resistance, and increased catalytic efficiency.

SUMMARY OF THE INVENTION



[0008] One aspect of the present invention is to provide a site-directed mutation variants of phytase of which the mature protein has amino acid sequence as set forth in SEQ ID NO.1, encoded by SEQ ID NO.2.

[0009] According to the present invention, said phytase variants YeAPPA having improved pepsin resistance and acid resistance, and increased catalytic efficiency are obtained by mutation at the 162th site of Leucine into glycine or alanine, or the 230th site of glutamic acid into glycine, proline or arginine for phytase with amino acid as set forth in SEQ ID NO.1.

[0010] According to the present invention, five phytase variants with having improved pepsin resistance and acid resistance named as YeAPPA-L162G, YeAPPA-L162A, YeAPPA-E230G, YeAPPA-E230P, and YeAPPA-E230R are obtained by site-directed mutation of the 162th site of Leucine into glycine or alanine, or the 230th site of glutamic acid into glycine, proline or arginine for phytase with amino acid as set forth in SEQ ID NO.1.

[0011] According to embodiment of the present invention, the phytase variant YeAPPA-L162G with amino acid sequence as set forth in SEQ ID NO.3 is obtained by mutation at the 162th site of Leucine into glycine for phytase with amino acid as set forth in SEQ ID NO.1.

[0012] According to embodiment of the present invention, the phytase variant YeAPPA-L162A with amino acid sequence as set forth in SEQ ID NO.4 is obtained by mutation at the 162th site of Leucine into alanine for phytase with amino acid as set forth in SEQ ID NO.1.

[0013] According to embodiment of the present invention, the phytase variant YeAPPA-E230G with amino acid sequence as set forth in SEQ ID NO.5 is obtained by mutation at the 230th site of glutamic acid into glycine for phytase with amino acid as set forth in SEQ ID NO.1.

[0014] According to embodiment of the present invention, the phytase variant YeAPPA-E230P with amino acid sequence as set forth in SEQ ID NO.6 is obtained by mutation at the 230th site of glutamic acid into proline for phytase with amino acid as set forth in SEQ ID NO.1.

[0015] According to embodiment of the present invention, the phytase variant YeAPPA-E230R with amino acid sequence as set forth in SEQ ID NO.7 is obtained by mutation at the 230th site of glutamic acid into arginine for phytase with amino acid as set forth in SEQ ID NO.1.

[0016] Another aspect of the invention is to provide a gene encoding the above phytase variants having improved pepsin resistance and acid resistance, and increased catalytic efficiency, with nucleotide sequence as set forth in SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12 respectively.

[0017] Another aspect of the invention is to provide a recombinant vector comprising polynucleotides encoding above phytase variants, preferably provide a recombinant Ecoli. expressing vector comprising the genes encoding phytase variants inserted between sites EcoRI and NotI as so to be controlled under the promoter T7-lac.

[0018] Yet another aspect of the invention is to provide a recombinant host cell comprising polynucleotides encoding above phytase variants, and preferably provide a recombinant Ecoli host, recombinant Ecoli BL21 (DE3).

[0019] Phytase variants of the present invention have the improved pepsin resistance and acid resistance, and the catalytic efficiency increased by 1.6 times and 2.4 times respectively compared with that of the wild phytase, in the benefit of the development of economical feed enzyme industry.

BRIEF DESCRIPTIONS OF THE DRAWINGS



[0020] 

FIG1 shows the comparison of effect of pepsin on activity of the modified phytase and the wild phytase.

FIG2 shows the comparison of the acid resistance of the modified phytase and the wild phytase.


Embodiment



[0021] The present invention is further illustrated with reference to the following Examples and the appended drawings, which should by no means be construed as limitations of the present invention.

Test materials and reagents



[0022] 
  1. 1. Strains and vectors: Expression vetor pET-22b (+) and host strain BL21 (DE3) (INovagen) .
  2. 2. Enzymes and other biochemical reagents: restriction endonucleases (TaKaRa), ligase (Invitrogen), and pepsin (p0685).
  3. 3. Medium:
    E.coli. LB medium: 1% of peptone, 0.5% of yeast extract, and 1% of NaCl, natural pH.


[0023] Suitable biology laboratory methods not particularly mentioned in the examples as below can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other kit laboratory manuals.

Example 1 Introduction of the mutant site to wild phytase



[0024] Gene encoding phytase YeAPPA having the nucleotide sequence as set in SEQ ID NO. 2 was performed with site-directed mutagenesis by Overlap PCR to obtain the genes enconding phytase variants YeAPPA-L162G, YeAPPA-L162A, YeAPPA-E230G, YeAPPA-E230P and YeAPPA-E230R, respectively. Overlap PCR was performed as being kept at 95°C for 5 min, followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30-90 sec, and keep 72°C for 10 min, with 12 mutation primers including the upper primer Ye-F and the reverse primer Ye-R for amplifying the full length of mutant gene, and the primers comprising the EcoRI and NotI sites marked in Italics or the mutant nucleotides marked in underlined for site-directed mutagenesis showed as below.

Ye-F: 5'-cgcgaattcgccccgattgctacaccgcc-3'

Ye-R: 5'-gatgcggccgcttaaatatggcaggctggctcga-3'

L162G-F: 5'-cgggggtctgtaaaggcgactcagcgaaaac-3'

L162G-R: 5'-gttttcgctgagtcgcctttacagacccccg-3'

L162A-F: 5'-cgggggtctgtaaagcggactcagcgaaaac-3'

L162A-R: 5'-gttttcgctgagtccgctttacagacccccg-3'

E230G-F: 5'-ttaaggtaaacgaaggcggtactaaagtttc-3'

E230G-R: 5'-gaaactttagtaccgccttcgtttaccttaa-3'

E230P-F: 5'-ttaaggtaaacgaaccggtactaaagtttc-3'

E230P-R: 5'-gaaactttagtacccggttcgtttaccttaa-3'

E230R-F: 5'-ttaaggtaaacgaacgtggtactaaagtttc-3'

E230R-R: 5'-gaaactttagtaccacgttcgtttaccttaa-3'



[0025] The modified gene is recovered, connected with the vector pEASY-T3, and sequenced.

Example 2 Preparing the phytase variants and measuring their activity



[0026] The modified gene encoding the phytase variants were inserted into expression vector pET-22b (+), and transformed into Ecoli. Strain BL21 (DE3), which was induced by IPTG in ImM, cultivated for 5h at 24°C to express the phytase, followed by being purified by columns Ni-NTA and DEAE to obtain the mutant protein with the same molecular weight as that of the wild.

Example 3 Measuring effect of pepsin on the enzyme activity of the phytase variants



[0027] Pepsin resistance of the phytase variants was measured by the remained activity and the amount of protein after being treated with different concentrations of pepsin.

Determining effect of pepsin on activity of the phytase variants



[0028] The effect of pepsin on the activity of the purified mutant phytase was determined by detecting the remained activity after being treated in pH 2 for 2 hours with the different concentrations of pepsin in a mass ratio to phytase ranging from 1/1000 to 1/1. The activity of phytase was detected by ferric molybdenum sulfate blue method by adding 50 ul of phytase solution to 950 ul of sodium phytate substrate in 1.5 mmol/L to react for 30min at 37 °C, followed by adding 1 mL of 10% (m/v) TCA to stop the reaction, and 2mL of developing color reagent. After developing, OD is measured at 700 nm to calculate the phytase activity. 1 unit of phytase activity is determined to be the enzyme amount releasing 1 µmol of phosphate for 1 minute. The absolute value of the measured phytase activity may be calculated based on the standard curve of inorganic phosphate in dilution. As showed by "A" and "B" of FIG 1, the phytase variants remained more enzyme activity after being treated for 2 h with different concentration of pepsin, than that of the wild phytase, wherein the retained activity of the phytase variants YeAPPA-E230G, YeAPPA-E230P, YeAPPA-E230R, YeAPPA-L162G and YeAPPA-L162A were 30%, 22%, 17%, 17% and 10% in order, but the wild phytase almost lost activity, demonstrating that pepsin resistance of phytase variants were improved.

Determining effect of pepsin on stability of the phytase variants



[0029] The effect of pepsin on the activity of the purified phytase variants was determined by detecting the retained phytase proteins by PAGE after being treated in pH 2 for 2 hours with the different concentrations of pepsin, and calculating the gray value of phytase protein bands. The amount of the retained phytase proteins after being treated with pepsin was represented by the ratio of the gray value of the retained phytase protein bands to that of the untreated phytase bands. As showed in "A" and "B" of FIG 2, the amounts of the retained protein of the phytase variants YeAPPA-E230G, YeAPPA-E230P, YeAPPA-E230R, YeAPPA-L162G and YeAPPA-L162A were more than that of the wild phytase with the ratio of 0.1, in case of being treated by pepsin in a ratio of 1/1000, and , the ratios of the gray value for the phytase variants YeAPPA-E230G, YeAPPA-E230P, YeAPPA-E230R, YeAPPA-L162G and YeAPPA-L162A were 0.54, 0.43, 0.33, 0.35, 0.25 in order, in case of being treated by pepsin in a ratio of 1/100. Therefore, the phytase variants of the present invention showed more retaining protein than that of the wild phytase after being treated with the different concentrations of pepsin, demonstrating that the phytase variants had the improve stability compare with the wild phytase.

Example 4 Determining stability of the phytase variants


(1) pH stability



[0030] The purified phytase variant was performed the enzymatic reactions in the substrate solutions with the different pHs using 0.1mol/L of Glycine-HCl buffer (pH1.0∼3.0), 0.1mol/L of acetic acid-sodium acetate buffer (pH3∼6), 0.1mol/L of Tris-Hcl buffer (pH6∼8) and 0.1mol/L of glycine-sodium hydroxide buffer (pH8∼12.0) at 37°C to deterimine the optimal pH. As showed in Table 1, the optimal pH values of the phytase variants YeAPPA-E230G, YeAPPA-E230P, YeAPPA-L162G and YeAPPA-L162A were pH 5.0 being similar to that of the wild enzyme, other than the phytase variant YeAPPA-E230R decreased one pH unit in optimal pH value. And, the phytase variants YeAPPA-E230G, YeAPPA-E230P, YeAPPA-E230R, YeAPPA-L162G and YeAPPA-L162A retaining more than 18-32% of enzyme activity were more stable than the wild phytase retaining 12% of enzyme activity after being treated in pH 1.0 to 2.0 for 1hour.

(2) Thermostability



[0031] The purified phytase variants were kept for 30 min at 30°C to 80°C, respectively to determine their optimal temperatures. As list in Table 1, the optimal temperatures of the phytase variant YeAPPA-E230P was 50°C , which was 5°C higher than those of the phytase variants YeAPPA-L162G, YeAPPA-L162A, YeAPPA-E230G, and YeAPPA-E230/R. And, phytase variants YeAPPA-E230P YeAPPA-E230G and YeAPPA-E230R retaining 12%, 21% and 9% of enzyme activity were more thermostable than phytase variants YeAPPA-L162G, YeAPPA-L162A and the wild phytase losing all of enzyme activity after being kept for 30 min at 60°C. Therefore, phytase variants YeAPPA-E230P, YeAPPA-E230G and YeAPPA-E230R were more thermostable than the wild phytase.
Table 1 Comparison of the effect temperature and pH on the activity and stability of the modified phytase and the wild phytase
VariantsOptimal pHOptimal temperaturepH stability of the phytaseafter being treated in different pHsThermostability of phytase kept for 30min at 60°C
YeAPPA 5 45°C pH 1-2,<12, pH 3-9,>89 0.6%
YeAPPA-L162G 5 45°C pH 1-2,>20, pH 3-9,>99 0.6%
YeAPPA-L162A 5 45°C pH 1-2,>18, pH 3-9,>96 0.6%
YeAPPA-E230G 5 45°C pH 1-2,>32, pH 3-9,>100 21%
YeAPPA-E230P 5 50°C pH 1-2,>24, pH 3-9,>99 12%
YeAPPA-E230R 4 45°C pH 1-2,>30, pH 3-9,>99 9%

Example 5 Measuring kinetic parameter of the phytase variants



[0032] The activity of phytase was measured with sodium phytate as substrate in different concentrations of 0.0625 mmol/L, 0.1 mmol/L, 0.125 mmol/L, 0.2 mmol/L, 0.25 mmol/L, 0.5 mmol/L, 1.0 mmol/L and 1.5mmol/L at the optimal temperature and pH, followed by calculating the values of km and Vmax by double reciprocal method for Michaelis equation, and Kcat according to the theoretical molecular weight. As showed in Table 2, the affinity to substrate (km) of each of phytase variants was similar to that of the wild phytase. Reaction rate Vmax and conversion rate Kcat of the phytase variant YeAPPA-E230G were greatly increased to 2.5 times of that of the wild phytase, and catalytic efficiency Kcatlkm was 2.5 times of that of the wild phytase. Reaction rate Vmax and conversion rate Kcat of the phytase variant YeAPPA-L162G were increased to 1.6 to 1.8 times of that of the wild phytase, and catalytic efficiency Kcatlkm was 1.7 times of that of the wild phytase. And, reaction rate, conversion rate and catalytic efficiency of the phytase variants YeAPPA-L162A, YeAPPA-E230P and YeAPPA-E230R were almost same as those of the wild phytase.
Table 2 Comparison of the enzymatic properties of the modified phytase and the wild phytase
PhytaseKm(mM)Vmax(U mg-1)Kcat(S-1)Kcat/Km(S-1 mM-1)
YeAPPA 0.19 6.4 4.9 26
YeAPPA-L162G 0.19 11 8.2 43
YeAPPA-L162A 0.19 6.5 5.0 27
YeAPPA-E230G 0.19 16 12 64
YeAPPA-E230P 0.19 6.7 5.1 26
YeAPPA-E230R 0.18 6.3 4.8 26























Claims

1. Phytase YeAPPA variant with the following characteristics of

having amino acid sequence substituting Leucine at the 162th site of the sequence set forth in SEQ ID NO.1 with glycine or proline, and

having improved pepsin resistance and acid resistance, and increased catalytic efficiency.


 
2. Phytase YeAPPA variant with the following characteristics of

having amino acid sequence substituting glutamic acid at the 230th site of the sequence set forth in SEQ ID NO.1 with glycine, proline or arginine, and

having improved pepsin resistance and acid resistance, and increased catalytic efficiency.


 
3. A polynucleotide encoding the phytase YeAPPA variant of claim 1 or 2.
 
4. Polynucleotide according to claim 3, is characterized of having the nucleotide sequence set in forth in SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO. 12, respectively.
 
5. DNA constructor comprising the polynucleotide of claim 3.
 
6. Recombinant cell comprising the polynucleotide of claim 3.
 
7. A method of producing a phytase variant comprising the steps of transforming host with DNA constructor of claim 5 to obtain recombinant host cell; cultivating the recombinant host cell to produce the supernatant containing phytase variant; and recovering the said phytase variant.
 
8. Use of the phytase variant of claim 1 or 2.
 




Drawing
















REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Non-patent literature cited in the description