(19)
(11)EP 3 103 874 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
06.05.2020 Bulletin 2020/19

(21)Application number: 16162913.4

(22)Date of filing:  08.02.2007
(27)Previously filed application:
 08.02.2007 PCT/IB2007/000951
(51)Int. Cl.: 
C12N 15/54  (2006.01)
C12P 19/04  (2006.01)
C07K 19/00  (2006.01)
C12N 9/10  (2006.01)
C12P 19/18  (2006.01)

(54)

CONSTRUCTION OF NEW VARIANTS OF DEXTRANSUCRASE DSR-S BY GENETIC ENGINEERING

KONSTRUKTION NEUER VARIANTEN VON DEXTRANSUCRASE DSR-S MITTELS GENTECHNOLOGIE

CONSTRUCTION DE NOUVELLES VARIANTES DE LA DEXTRANSUCRASE DSR-S PAR GENIE GENETIQUE


(84)Designated Contracting States:
AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

(30)Priority: 08.02.2006 FR 0601117

(43)Date of publication of application:
14.12.2016 Bulletin 2016/50

(62)Application number of the earlier application in accordance with Art. 76 EPC:
07734270.7 / 1981914

(73)Proprietors:
  • Centre National de la Recherche Scientifique
    75794 Paris Cedex 16 (FR)
  • Institut national de recherche pour l'agriculture, l'alimentation et l'environnement
    75007 Paris (FR)
  • Institut National des Sciences Appliquées de Toulouse
    31077 Toulouse Cedex 4 (FR)

(72)Inventors:
  • MONSAN, Pierre
    31700 Mondonville (FR)
  • REMAUD-SIMEON, Magali
    31520 Ramonville (FR)
  • POTOCKI-VERONESE, Gabrielle
    31370 Lautignac (FR)
  • MOULIS, Claire
    31380 Garidech (FR)

(74)Representative: Desaix, Anne et al
Ernest Gutmann - Yves Plasseraud S.A.S. 66, rue de la Chaussée d'Antin
75009 Paris
75009 Paris (FR)


(56)References cited: : 
EP-A- 1 201 131
WO-A-2006/063862
US-A- 4 861 381
WO-A-89/12386
DE-A1- 10 225 380
  
  • ARGÜELLO-MORALES MARTHA ET AL: "Proteolytic modification of Leuconostoc mesenteroides B-512F dextransucrase.", ANTONIE VAN LEEUWENHOEK. FEB 2005, vol. 87, no. 2, February 2005 (2005-02), pages 131-141, XP002406199, ISSN: 0003-6072
  • DATABASE UniProt From Leuconostoc mesenteroides BHATNAGAR R.: "Dextransucrase", XP002406204, retrieved from EBI Database accession no. Q9ZAR4
  • MONCHOIS V ET AL: "Effect of Leuconostoc mesenteroides NRRL B-512F dextransucrase carboxy-terminal deletions on dextran and oligosaccharide synthesis.", APPLIED AND ENVIRONMENTAL MICROBIOLOGY. MAY 1998, vol. 64, no. 5, May 1998 (1998-05), pages 1644-1649, XP002406200, ISSN: 0099-2240
  • MONCHOIS V ET AL: "Glucansucrases: mechanism of action and structure-function relationships.", FEMS MICROBIOLOGY REVIEWS. APR 1999, vol. 23, no. 2, April 1999 (1999-04), pages 131-151, XP002406201, ISSN: 0168-6445
  • MONCHOIS VINCENT ET AL: "Cloning and sequencing of a gene coding for an extracellular dextransucrase (DSRB) from Leuconostoc mesenteroides NRRL B-1299 synthesizing only a alpha(1-6) glucan", FEMS MICROBIOLOGY LETTERS, AMSTERDAM, NL, vol. 159, no. 2, 15 February 1998 (1998-02-15), pages 307-315, XP002198584, ISSN: 0378-1097
  • MONCHOIS V ET AL: "CHARACTERIZATION OF LEUCONOSTOC MESENTEROIDES NRRL B-512F DEXTRANSUCRASE (DSRS) AND IDENTIFICATION OF AMINO-ACID RESIDUES PLAYING A KEY ROLE IN ENZYME ACTIVITY", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER VERLAG, BERLIN, DE, vol. 48, no. 4, October 1997 (1997-10), pages 465-472, XP008025068, ISSN: 0175-7598
  • RYU HWA-JA ET AL: "Cloning of a dextransucrase gene (fmcmds) from a constitutive dextransucrase hyper-producing Leuconostoc mesenteroides B-512FMCM developed using VUV", BIOTECHNOLOGY LETTERS, vol. 22, no. 5, March 2000 (2000-03), pages 421-425, XP002406202, ISSN: 0141-5492
  • FUNANE ET AL: "Changes in linkage pattern of glucan products induced by substitution of Lys residues in the dextransucrase", FEBS LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 579, no. 21, 29 August 2005 (2005-08-29), pages 4739-4745, XP005044268, ISSN: 0014-5793
  • ENDO H ET AL: "ON-LINE MONITORING OF THE VISCOSITY IN DEXTRAN FERMENTATION USING PIEZOELECTRIC QUARTZ CRYSTAL", BIOTECHNOLOGY AND BIOENGINEERING, vol. 36, no. 6, 1990, pages 636-641, XP008082755, ISSN: 0006-3592
  • VELJKOVIC V B ET AL: "STUDIES ON DEXTRAN FERMENTATION BROTH RHEOLOGY", ENZYME AND MICROBIAL TECHNOLOGY, vol. 10, no. 11, 1988, pages 686-688, XP008082754, ISSN: 0141-0229
  • KUBIK CELINA ET AL: "Immobilization of dextransucrase and its use with soluble dextranase for glucooligosaccharides synthesis", ENZYME AND MICROBIAL TECHNOLOGY, vol. 34, no. 6, May 2004 (2004-05), pages 555-560, XP002455538, ISSN: 0141-0229
  • ROBYT J F ET AL: "RELATIVE, QUANTITATIVE EFFECTS OF ACCEPTORS IN THE REACTION OF LEUCONOSTOC MESENTEROIDES B-512F DEXTRANSUCRASE", CARBOHYDRATE RESEARCH, ELSEVIER SCIENTIFIC PUBLISHING COMPANY. AMSTERDAM, NL, vol. 121, 1983, pages 279-286, XP001149187, ISSN: 0008-6215
  • MOULIS CLAIRE ET AL: "High-level production and purification of a fully active recombinant dextransucrase from Leuconostoc mesenteroides NRRL B-512F.", FEMS MICROBIOLOGY LETTERS. AUG 2006, vol. 261, no. 2, August 2006 (2006-08), pages 203-210, XP002406211, ISSN: 0378-1097
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description


[0001] The disclosure describes a recombinant process for the production of truncated and/or mutated dextransucrases while conserving their enzymatic activity and/or conserving their specificity for synthesizing α-1, 6 bonds. More precisely, the disclosure describes nucleic acid sequences of truncated or mutated dextransucrases, vectors containing said nucleic acid sequences and host cells transformed by sequences encoding truncated or mutated dextransucrases. The disclosure also describes a method for producing, in a recombinant manner, truncated and/or mutated dextransucrases which conserve their enzymatic activity and/or conserve their specificity for synthesizing α-1, 6 bonds in the final product and methods for producing dextrans or isomalto-oligosaccharides, in a single step, with a controlled molar mass and dextrans with modified rheological properties, especially compared with the properties of dextrans obtained with the native enzyme. The present invention relates to a dextransucrase consisting of a sequence having 90%, 95% or 98% sequence similarity to an amino acid sequence of SEQ ID NO: 22, 23, 24, 25 or 26, wherein the enzymatic activity of forming dextrans is maintained and wherein the dextransucrase conserves its specificity for synthesizing alpha-1,6 bonds. The present invention also relates to fusion proteins comprising a protein tag fused to said dextransucrase.

Field of the invention



[0002] Dextrans are α-D-glucans with various structures, comprising contiguous glycosyl units more than 50% of which have α-1,6 bonds in the principal chain and α-1,2, α-1,3 and/or α-1,4 branches [1]. The enzymes which produce such dextrans from sucrose are termed dextransucrases and belong to glycoside hydrolase family 70 [2]. During the reaction, fructose derived from the sucrose is released and may be upgraded elsewhere. Dextransucrases are produced by lactic bacteria from genera Leuconostoc, Streptococcus and Lactobacillus [1].

[0003] Dextransucrase (DSR-S) from Leuconostoc mesenteroides NRRL B-512F contains 1,527 amino acids [3]. This enzyme catalyzes the synthesis of glucose homopolymers with more than 95% α-1,6 bonds. The production of dextran may be redirected towards that of oligosaccharides or glucosylated conjugates by adding a suitable acceptor to the reaction mixture [4].

[0004] The number of industrial applications for dextrans and dextran derivatives is increasing, in particular for dextrans with a specific size. Dextrans with a size in the range 70,000 to 100,000 Da are, for example, used as a plasma substitute [5, 31]. Further, dextran of 40,000 Da is used to improve blood flow, most probably by reducing the viscosity of the blood and inhibiting erythrocytary aggregation [6,8]. After sulphation, smaller dextrans of about 10,000 daltons, for example, are used as transporters for iron [7] or anticoagulants [8]. Those compounds may have antiviral properties [9, 10].

[0005] Further, cross-linked dextran derivatives have long been used in the field of molecular separation; chromatography supports under the trade name Sephadex® have been sold since 1961 [6].

[0006] Moreover, the European Union has recently approved the use of dextran as a food ingredient in bakery products when these contain more than 95% of α-1,6 bonds and have a molar mass of more than 2 x 106 Da [15].

[0007] Dextransucrase may also produce isomalto-oligosaccharides (IMO) via an acceptor reaction. Acceptor reactions carried out by glucansucrases consist of a transfer of glucosyl residues from sucrose to other molecules added to the reaction medium. It is of increasing commercial interest, particularly in Japan, where the demand for isomalto-oligosaccharides represents about fifteen thousand tons per year [11]. Such small IMOs (DP 2 to 6) are used in bakery items, for drinks, in saké, in seasonings, in confectionery and as anticariogenic sweeteners. It has also been shown that said IMOs have prebiotic properties which are useful with respect to the intestinal and/or vaginal flora [12, 13]. These properties appear to vary with the size of the IMOs and are favored by high degrees of polymerization [14].

[0008] The only commercial and usual source of dextrans consists of cultivating L. mesenteroides NRRL B-512F with sucrose, leading to the formation of high molar mass polymers of about 108 Da. The direct synthesis of smaller dextrans of 10000 to 100000 Da is currently impossible. Dextrans are currently produced conventionally by acid hydrolysis of high molar mass native polymers followed by fractionation using organic solvents. This second step is, however, renowned for its low yields [19].

[0009] From a commercial viewpoint, IMOs of DP 2 to 6 are not produced by an acceptor reaction with dextransucrase DSR-S and glucose due to the low reaction yields, but from starch hydrolysates and a mixture of α-amylases and glucosidases [11].

[0010] Monchois et al [16] describe carboxy-terminal deletions from the dextransucrase of Leuconostoc mesenteroides NRRL B-512F and conclude that the role of the C-terminal domain is to facilitate transfer of dextran and oligosaccharides beyond the active site.

[0011] United States patent US-A-5,229,277 describes a process for producing dextran polymers having a homogeneous low molar mass using Leuconostoc mesenteroides and a mutant microorganism of Lipomyces starkeyi ATCC 74054, which is a yeast having dextranase activity, a specific enzyme for the hydrolysis of α-1,6 bonds of dextran. That method necessitates particular culture conditions and a precisely regulated duration and temperature so that the dextranase activity reduces the molar mass of the dextrans. Dextran polymers produced by that method have a molar mass in the range of 40,000 and 150,000 Da.

[0012] Argüello-Morales et al., Antonie Van Leeuwenhoek, vol 87 no. 2 (February 2005), 131-141 discloses two active forms of dextransucrase from Leuconostoc mesenteroides B-512 which have molecular weights of 155 and 129 kDa. These dextransucrases arise from the proteolytic cleavage of a 170 kDA precursor and behave in solutions as Newtonian fluids.

[0013] The foregoing shows that there is a need for the production of dextrans with a molar mass of about 10,000 to 100,000 Da using a faster method with a better yield, which in particular requires neither acid hydrolysis nor fractionation.

[0014] The disclosure describes dextransucrases produced in a recombinant manner, which are truncated and/or mutated, while conserving their enzymatic activity and/or conserving their specificity for synthesizing α-1, 6 bonds. The present invention concerns truncated variants of dextransucrase which produce dextrans with a controlled molar mass. More precisely, they conserve the binding specificity of native DSR-S and/or conserve their specificity for synthesizing α-1, 6 bonds and, starting from sucrose, produce high molar mass dextrans with interesting texturing properties and/or dextrans and IMOs with a controlled molar mass.

[0015] The disclosure also describes providing nucleic acid sequences of truncated and/or mutated dextransucrase, vectors and host cells transformed by said vectors, and amino acid sequences of truncated and/or mutated dextransucrases.

[0016] In particular, as will become apparent from the Examples, certain dextransucrases produce polymers with interesting texturing properties, i.e., substantially superior to those of the polymer produced by the native enzyme; others produce dextrans and isomalto-oligosaccharides with a controlled molar mass. Isomaltose is produced by at least one truncated and mutated dextransucrase.

[0017] Further aspects of the present invention will become apparent from the following description and Examples or preferred implementations.

Summary of the invention



[0018] The disclosure describes a nucleotide sequence consisting essentially of or consisting of a nucleotide sequence according to Figure 1 (SEQ ID NO: 1), a nucleotide sequence according to Figure 2 (SEQ ID NO: 2), a nucleotide sequence according to Figure 3 (SEQ ID NO: 3), a nucleotide sequence according to Figure 4 (SEQ ID NO: 4), a nucleotide sequence according to Figure 5 (SEQ ID NO: 5), a complementary sequence of one of the sequences with SEQ ID NO: 1, 2, 3, 4 or 5 or a sequence which hybridizes with a sequence with SEQ ID NO: 1, 2, 3, 4 or 5 under stringent hybridization conditions, provided that it conserves dextransucrase enzymatic activity.

[0019] The disclosure also describes nucleotide sequences of dextransucrase consisting essentially of or consisting of a nucleotide sequence selected from the fragment of SEQ ID NO: 1 from position 373 to position 4269 (SEQ ID NO: 17), the fragment of sequence SEQ ID NO: 2 from position 373 to position 4005 (SEQ ID NO:18), the fragment of sequence SEQ ID NO: 3 from position 373 to position 3408 (SEQ ID NO:19), the fragment of sequence SEQ ID NO: 4 from position 373 to position 3018 (SEQ ID NO:20), and the fragment of sequence SEQ ID NO: 5 from position 373 to position 4269 (SEQ ID NO:21).

[0020] It also describes nucleotide sequences consisting essentially of or consisiting of a nucleotide sequence selected from a complementary nucleotide sequence of the fragment of SEQ ID NO: 1 from the nucleotide at position 373 to that at position 4269, a complementary nucleotide sequence of the fragment of SEQ ID NO: 2 from the nucleotide at position 373 to that at position 4005, a complementary nucleotide sequence of the fragment of SEQ ID NO: 3 from the nucleotide at position 373 to that at position 3408, a complementary nucleotide sequence of the fragment of SEQ ID NO: 4 from the nucleotide at position 373 to that at position 3018 and a complementary nucleotide sequence to the fragment of SEQ ID NO: 5 from the nucleotide at position 373 to that at position 4269.

[0021] It also describes nucleotide sequences which hybridize under stringent conditions with a nucleotide sequence selected from the fragment of sequence SEQ ID NO: 1 from position 373 to position 4269, the fragment of sequence SEQ ID NO: 2 from position 373 to position 4005, the fragment of sequence SEQ ID NO: 3 from position 373 to position 3408, the fragment of sequence SEQ ID NO: 4 from position 373 to position 3018 and the fragment of sequence SEQ ID NO: 5 from position 373 to position 4269, provided that it conserves dextransucrase enzymatic activity and said nucleotide sequences that hybridizes thereto has the same number of nucleotides and hybridizes over the full length of the fragment.

[0022] The disclosure also describes nucleotide sequences encoding a protein consisting essentially of or consisting of consecutive amino acid sequences of any one of SEQ ID NOs:6 to 10 or 22 to 26.

[0023] The disclosure also describes vectors, for example plasmids, and host cells transformed by said vectors and containing said sequence of nucleic acids from truncated and/or mutated dextransucrase, in particular the variants of the Examples.

[0024] The disclosure describes a protein encoded by said truncated and/or mutated dextransucrase nucleotide sequence selected from the fragment of SEQ ID NO: 6 from the amino acid at position 125 to the amino acid at position 1423 (SEQ ID NO: 22), the fragment of SEQ ID NO: 7 from the amino acid at position 125 to the amino acid at position 1335 (SEQ ID NO: 23), the fragment of SEQ ID NO: 8 from the amino acid at position 125 to the amino acid at position 1136 (SEQ ID NO: 24), the fragment of SEQ ID NO: 9 from the amino acid at position 125 to the amino acid at position 1006 (SEQ ID NO: 25), and the fragment of SEQ ID NO: 10 from the amino acid at position 125 to the amino acid at position 1423 (SEQ ID NO: 26).

[0025] Further, the present invention concerns a truncated and/or mutated dextransucrase consisting of a sequence having 90%, 95% or 98% sequence similarity to an amino acid sequence selected from the fragment of SEQ ID NO: 6 from the amino acid at position 125 to the amino acid at position 1423 (SEQ ID NO:22), the fragment of SEQ ID NO: 7 from the amino acid at position 125 to the amino acid at position 1335 (SEQ ID NO:23), the fragment of SEQ ID NO: 8 from the amino acid at position 125 to the amino acid in position 1136 (SEQ ID NO:24), the fragment of SEQ ID NO: 9 from the amino acid at position 125 to the amino acid at position 1006 (SEQ ID NO:25), and the fragment of SEQ ID NO: 10 from the amino acid at position 125 to the amino acid at position 1423 (SEQ ID NO:26).

[0026] The disclosure describes the preparation of a mutated and/or truncated dextransucrase by culture of host cells containing a truncated and/or mutated dextransucrase under conditions allowing the expression of a dextransucrase, and isolating said dextransucrase from the culture medium.

[0027] The disclosure also describes a method for producing dextrans and/or isomalto-oligosaccharides (IMO) with a controlled molar mass controlled by reacting a mutated and/or truncated dextransucrase described herein with sucrose and optionally an acceptor, to obtain said dextrans or IMO with a controlled molar mass, including isomaltose.

[0028] A method for the direct production of IMOs essentially from sucrose is also described herein. The term "essentially" as used here means that it is not necessary for the acceptor to be employed in the reaction.

[0029] The high molar mass dextrans of the invention have modified rheological properties compared with those of dextran synthesized by a native enzyme, in particular a non-Newtonian, stringy and/or gelling nature.

[0030] Finally, the disclosure describes compositions comprising dextrans obtained by using said dextransucrases and the use of said dextransucrases for the production of dextrans and isomalto-oligosaccharides with a controlled molar mass in the range of 342 and 109 Da. More precisely, it can produce (i) isomaltose (342 Da), (ii) isomalto-oligosaccharides of 342 to 5,000 Da, (iii) dextrans with a controlled size of 1,300 to 52,000 Da, more precisely 5,000 to 22,000 Da, and centered around 10,000 Da, (iv) dextrans with a controlled size of 7,000 to 1.7 x 105 Da, more precisely between 22,000 and 70,000 Da, centered around 40,000 Da.

Brief description of the Figures



[0031] 

Figure 1 shows the amino acid and nucleotide sequence of a truncated DSR-S vardel Δ4N dextransucrase with a thioredoxin tag in the 5' terminal position of the sequence and 6 histidine tags in the 3' terminal position of the sequence as well as spacer arms between the protein tags and the sequence coding for dextransucrase.

Figure 2 shows the amino acid and nucleotide sequence for a truncated DSR-S vardel Δ3 with a thioredoxin tag in the 5' terminal position of the sequence and 6 histidine tags in the 3' terminal position of the sequence and spacer arms between the protein tags and the sequence coding for dextransucrase.

Figure 3 shows the amino acid and nucleotide sequence for a truncated DSR-S vardel Core with a thioredoxin tag in the 5' terminal position of the sequence and 6 histidine tags in the 3' terminal position of the sequence and spacer arms between the protein tags and the sequence coding for dextransucrase.

Figure 4 shows the amino acid and nucleotide sequence for a truncated DSR-S Core ΔA with a thioredoxin tag in the 5' terminal position of the sequence and 6 histidine tags in the 3' terminal position of the sequence and spacer arms between the protein tags and the sequence coding for dextransucrase.

Figure 5 shows the sequence of amino acid and nucleotides for a mutant DSR-S vardel Δ4N SEV663YDA with a thioredoxin tag in the 5' terminal position of the sequence and 6 histidine tags in the 3' terminal position of the sequence and spacer arms between the protein tags and the sequence coding for dextransucrase.

Figure 6 is a diagrammatic representation of the truncated variants of DSR-S and their relative activity. The four different domains (i) to (iv) of DSR-S correspond to: (i) signal peptide, (ii) variable region; (iii) catalytic domain and (iv) C-terminal domain as well as the repeat units A, C and N (in the shaded boxes) located in accordance with Monchois et al, 1998 [16].

Figure 7 (A, B) shows anti-thioredoxin (A) and anti-6xHis (B) Western blots carried out on a DSR-S vardel Δ4N produced by E. coli TOP10 at 23°C.

Figure 8 shows an electrophoresis gel after staining the proteins with colloidal blue on DSR-S vardel Δ4N extracts during affinity purification on nickel resin (Probond, Invitrogen). Track 1 corresponds to the supernatant from sonication of E. coli TOP10 at the end of culture; track 2 corresponds to the effluent obtained after binding the tagged 6xHis proteins on the resin, track 3 corresponds to the elution fraction and track 4 corresponds to the elution fraction after eliminating aggregates.

Figure 9 shows the elution profiles obtained by HPSEC of dextrans produced by the preparation of a) native DSR-S from L. mesenteroides NRRL B-512F, b) entire recombinant DSR-S, c) DSR-S vardel Δ4N before purification and d) purified DSR-S vardel Δ4N. Peak 1 corresponds to the high molar mass polymer (HMW), peak 2 to fructose, glucose and oligosaccharides with a DP of less than 7, not separated by the system. Between those two peaks, perturbations of the base line reflect the presence of dextrans with an intermediate size (between 103 to 107 Da) in a very low concentration.

Figure 10A-E shows the spectra obtained by proton NMR on dextrans synthesized by A) native DSR-S from L. mesenteroides NRRL B-512F, B) the entire recombinant DSR-S, C) DSR-S vardel Δ4N before purification and D) DSR-S vardel Δ4N after purification. Spectrum E) is a carbon-13 spectrum of the dextran synthesized by purified DSR-S vardel Δ4N.

Figure 11 corresponds to the HPAEC-PAD chromatogram of the digestion products using endodextranase (dase) of the four dextrans synthesized by native DSR-S, entire recombinant DSR-S and DSR-S vardel Δ4N, before and after purification.

Figure 12 shows the rheological behavior of four dextrans synthesized by native DSR-S (1) before and (2) after shearing, entire recombinant DSR-S (3) before and (4) after application of a second series of shear stresses, DSR-S vardel Δ4N (6) before and (7) after application of a second series of shear stresses, purified DSR-S vardel Δ4N (5) where A) represents the measurement of the viscosity flow, B) dynamic mode viscosity measurements (oscillations between 0 and 10 Pa), before determining the conservation G' and energy dissipation G" moduli for the dextrans synthesized by the non-purified DSR-S vardel Δ4N preparations (° and ●; solution type behavior, G'< G"; at 5% deformation) and purified preparation (□ and ■; gel type behavior G' > G"; 0.4% deformation).

Figure 13 shows a HPAEC-PAD chromatogram of products synthesized by mutant DSR-S vardel Δ4N SEV663YDA with 100 g/l of sucrose alone (A) or by acceptor reaction with 100 g/l of sucrose and 50 g/l of glucose (B). The symbol G signifies glucose, F: fructose, I2: isomaltose, I3: isomaltotriose, N/M: nigerose or maltose (not separated by the HPAEC-pad system) and the symbol "?" corresponds to products with an unknown structure.

Figure 14 shows the HPSEC chromatogram of dextrans synthesized by DSR-S vardel Δ3 at 20°C and 10°C. The arrows correspond to the retention times of commercial dextrans of 2 x 106 Da, 70,000 and 10,000 Da which served as references.

Figure 15 shows the HPSEC chromatogram of dextrans synthesized at 20°C with 100 g/l of sucrose and with 1 U/ml of (1) DSR-S vardel Δ4N, (2) DSR-S vardel Δ3, (3) DSR-S vardel Core and (4) DSR-S Core ΔA and the elution profile (5) of a commercial dextran of 10,000 Da (Sigma).

Figure 16 shows the HPAEC-PAD profile of dextrans synthesized at 20°C with 100 g/l of sucrose and with 1 U/ml of DSR-S vardel Δ4N (1), DSR-S vardel Δ3 (2), DSR-S vardel Core (3) and DSR-S vardel Core ΔA (4).

Figure 17 shows the HPAEC-PAD profile (A) and distribution (B) of IMOs produced by an acceptor reaction at 20°C with the variants DSR-S vardel Δ4N (1), DSR-S vardel Δ3 (2), DSR-S vardel Core (3) and DSR-S vardel Core ΔA (4). G: glucose; F: fructose; L: leucrose; T: trehalulose; I2 to I20: isomalto-oligosaccharides with DP 2 to DP 20. The insert of Figure B corresponds to an enlargement of the IMOs from DP of 15 to 27.


Detailed Description of the Preferred Embodiments



[0032] The term "enzyme having dextransucrase enzymatic activity" as used here means an enzyme which catalyzes the conversion of sucrose into oligosides and polyosides comprising more than 50% glucosyl units bound by α-1,6 bonds with a size in the range 342 and 109 Da, and more particularly dextrans and isomalto-oligosaccharides comprising more than 95% α-1,6 bonds. This conversion may take place in the presence of absence of external acceptors such as maltose, glucose, isomaltose or fructose or isomalto-oligosaccharides. Maltose, isomaltose and glucose are the preferred acceptors described herein. The enzymatic activity of the dextransucrases of the present invention may be measured as described in the Examples.

[0033] The terms "nucleotides", "polynucleotides" "nucleic acids" and "oligonucleotides" as used here are interchangeable and include, without being limited thereto, RNA, DNA, DNA/RNA sequences comprising more than one nucleotide in a single chain or in the form of a double chain. The polynucleotide sequences described herein may be prepared by any known method including, without being limited thereto, any recombinant synthesis method and any ex vivo generation method, as well as combinations of those methods.

[0034] The term "truncated" as used here means that at least one of the Nor C-terminal ends of the amino acid or nucleic acid sequence has been shortened. That shortening may be carried out using restriction enzymes, proteolytic enzymes or synthetically, including by specific amplification of nucleotide sequences, in particular by PCR.

[0035] The term "purified dextransucrase" as used here means a dextransucrase which has only one active form of dextransucrase in the preparations, which has a degree of protein purity of at least 70% or 85% or 95%.

[0036] The term "interesting original texturizing property" as used here means the rheological properties of the dextrans of the invention which, compared with dextrans synthesized by native enzyme under the same conditions, for example, exhibit non-Newtonian behavior, especially a gel or stringy type behavior. A "gel type polymer" is characterized here by dynamic mode rheological measurements, detecting the energy conservation (G') and energy dissipation (G") moduli. For a gel, G' is higher than G" over the entire frequency range studied, as will become apparent in Example 5. The stringy character can be identified with the naked eye. The stringy dextrans of the invention change from solution type behavior to gel type behavior after application of a second series of shear stresses, as will also be seen in Example 5.

[0037] The following abbreviations used here have the following meanings: DSR-S for dextransucrase from L. mesenteroides NRRL B-512F; DP for degree of polymerization; HMW for "high molar mass", IMW for "intermediate molar mass", IMW polymers being highly polydispersed polymers with sizes in the range 1,000 to 107 Da, where separation by HPSEC is difficult because of their low concentration. LMW polymers (low molar mass) are, according to the invention, a population which is much higher and easily detected between 750 and 70,000 Da, centered around 10,000 Da or in the range 2,000 to 1.7 x 105 Da and centered around 40,000 Da.

[0038] The term "10,000 Da dextran" as used here means a population of dextran with a size in the range 1,300 to 52,000 Da, more precisely between 5,000 and 22,000 Da, and centered at the height of the peak at about 10,000 Da. During characterization, the base of the elution peak obtained by gel permeation was in the range 1,300 to 52,000 Da, the range of molar mass estimated at the elution peak half height was in the range from 5,000 to 22,000 Da and the peak was centered at the height of the peak at about a mass of 10,000 Da. When the molar mass was expressed at the peak half height, at least 50% of the dextran population fell within the indicated range.

[0039] The term "40,000 Da dextran" as used here means a population of dextran with a size in the range 7000 to 1.7 x 105 Da, more precisely between 22,000 and 70,000 Da, and centered at the height of the peak at about 40,000 Da. During characterization, the base of the elution peak obtained by gel permeation was in the range 7,000 to 1.7 x 105 Da, the range of molar mass estimated at the elution peak half height was in the range 22,000 to 70,000 Da and the peak was centered at a mass of about 40,000 Da. When the molar mass was expressed at the peak half height, at least 50% of the dextran population fell within the indicated range.

[0040] IMO means isomalto-oligosaccharides.

[0041] The term "consisting essentially of" when used in connection with nucleic acids or amino acids as used here means that other minor ingredients or molecules may be present with the amino acid or nucleic acid sequences. The nucleic acid sequence has the exact same length as indicated in the sequence identification number, but may have 3 to 12 extra nucleotides at the N- and C- terminals. Like wise, the amino acid sequence has the exact same length as indicated in the sequence identification number but from 1 to 4 extra amino acids may be added at the N- or C-terminals. These extra amino acids have no effect on the enzyme activity.

[0042] The disclosure describes nucleic acids which encode a truncated dextransucrase or a mutated dextransucrase, a sequence complementary to all or part of those sequences or a sequence which hybridizes under stringent conditions with one of the above sequences provided that dextransucrase enzymatic activity is maintained. It should be appreciated that the nucleotide sequences that hybridizes thereto has the same number of nucleotides and hybridizes over the full length of the fragment.

[0043] The term "stringent hybridization conditions" as used here means conditions as described by Sambrook et al, Molecular Cloning Manual, 3rd edition (2001), i.e., as an example, the following conditions: hybridization buffers: 2 X SSC, 10 X Denhardts solution (Ficoll 400 & PEG & BSA, ratio 1:1:1), 0.1% SDS, 5 mM EDTA, 50 mM Na2HPO4, 250 µg/ml herring sperm DNA, 50 µg/ml of t-RNA or 0.25 M of sodium phosphate buffer with a pH of 7.2, 1 mM EDTA, 7% SDS;
Hybridization temperature: 60°C;
Washing buffer: 2 X SSC, 0.1% SDS;
Washing temperature: 60°C.

[0044] The nucleic acid molecules which hybridize under stringent conditions with the nucleic acids described herein may in principle encode dextransucrases from any microorganism such as bacteria, gram positive bacteria and, bacteria from the genera Leuconostoc, Streptococcus or Lactobacillus.

[0045] The disclosure describes nucleic acids which encode dextransucrase proteins having at least 70% or 80% or 90% sequence identity with those of sequences SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NOs 17 to 21, provided that the protein encoded by said sequences has dextransucrase enzymatic activity.

[0046] The disclosure describes nucleotide sequences encoding a protein consisting essentially of or consisting of consecutive amino acid sequences of any one of SEQ ID NOs:6 to 10 or 22 to 26.

[0047] As described herein, the sequences complementary to the sequences described herein or sequences which hybridize with said sequences under stringent conditions, provided that dextransucrase enzymatic activity is maintained, are also described herein.

[0048] Derivations from the basic nucleotide sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5), where the sequences are selected from the fragment of sequence SEQ ID NO: 1 from position 373 to position 4269, the fragment of sequence SEQ ID NO: 2 from position 373 to position 4005, the fragment of sequence SEQ ID NO: 3 from position 373 to position 3408, the fragment of sequence SEQ ID NO: 4 from position 373 to position 3018 and the fragment of sequence SEQ ID NO: 5 of a nucleotide in position 373 to position 4269, the sequences complementary to said sequences or sequences which hybridize with said sequences under stringent conditions provided that the dextransucrase enzymatic activity is maintained, may be produced by deletion, substitution, insertion or recombination, for example; the methods for carrying out said steps and transformations being well known in the art and described, for example, by Sambrook et al, supra.

[0049] It should be understood here that if any deletions, substitutions, insertions or recombinations of any of the sequences cited above take place, the proteins encoded by the sequences must maintain their dextransucrase enzymatic activity. Thus, 1 to 132, preferably 2 to 60 nucleotides, more preferably 15 to 36 nucleotides and still more preferably 12 to 27 nucleotides may be modified, for example, by deletion, substitution, insertion or recombination. As described herein, 90%, preferably 95% of the nucleotides remain unchanged.

[0050] The dextransucrase enzymatic activity can be measured, as described in the method section and in the Examples of the present application.

[0051] The oligonucleotides which may be used as a probe or primer are, for example, SEQ ID NO: 1 to SEQ ID NO: 5 or nucleotide sequences selected from the fragment of sequence SEQ ID NO: 1 from position 373 to position 4269, the fragment of sequence SEQ ID NO: 2 from position 373 to position 4005, the fragment of sequence SEQ ID NO: 3 from position 373 to position 3408, the fragment of sequence SEQ ID NO: 4 from position 373 to position 3018 and the fragment of sequence SEQ ID NO: 5 from a nucleotide in position 373 to position 4269.

[0052] The length of the probes and primers can vary depending on their applications. In general, they must have at least 25 nucleotides and may comprise all of the dextransucrase sequences described, such as 3,896 nucleotides. The length can also vary to be in the range of 25 to 150 nucleotides, 25 and 800 nucleotides or 25 and 3000 nucleotides, for example.

[0053] The primers generally comprise 18 to 25 nucleotides in length, but may also be longer, depending on the envisaged application. Examples of primers which may be used are:

GGC TTC TCT GGT GTG ATT (SEQ ID NO:11)

GAT CTG TCA GAA ACT GGC (SEQ ID NO:12)

ACA CAA CAA GTT AGC GGC (SEQ ID NO: 13)

CCA GAT ACT AAC TTG AGT (SEQ ID NO: 14)

TTC ATT GAT GCA GAC GGG (SEQ ID NO:15)

CAC GAC TAC GAC GCG CAA (SEQ ID NO :16)



[0054] It should be noted that the primers in the 5' and 3' terminal positions of the nucleotides encode the dextransucrase (SEQ ID NOs: 11 to 15) and the 5' and 3' side of the mutant sequence (SEQ ID NO: 16). However, a skilled person can use each of these sequences to produce primers or probes using consecutive nucleotides. Furthermore, these nucleotide sequences which are used as a probe may be tagged with radioactivity, enzymatic tagging, fluorescent tagging, in particular.

[0055] In order to genetically engineer the prokaryotic or eukaryotic cell, the nucleic acids described herein or a portion of the nucleic acids described herein may be introduced into plasmids that allow mutagenesis or modification of sequences by recombination of nucleotide sequences. Standard methods using these techniques are known to the skilled person and have been described by Sambrook et al, supra, in particular. The DNA fragments can also be connected to each other by adapters or links and suitable restriction enzymes can be used to remove certain DNA sequences. Methods such as mutagenesis, restriction after the restoration of primers or ligatures can be used to obtain the desired sequence with the appropriate insertions, deletions or necessary or desirable substitutions.

[0056] Furthermore, well defined tags coding for nucleic acids may be attached to the N- or C-terminal ends of the nucleic acid sequences. They may be peptides such as poly-His, c-myc epitope or HA-tag or small proteins such as bacterial GST, MBP (maltose binding protein), thioredoxin, β-galactosidase, VSV-glycoprotein and the like.

[0057] Particular nucleic acids coding for other protein tags are His-tag, T7tag, S-tag, a "flag" peptide, trpE, avidin/streptavidin, staphylococcal A or G protein, dihydrofolate reductase, cellulose binding domains, polycysteine, polyphenylalanine and the like, which may also be used.

[0058] A nucleic acid coding for a thioredoxin may be fused to the N-terminal nucleic acid sequence. A nucleic acid coding for a 6xHis tag is fused to the 3' end of the nucleic acid sequences.

[0059] The nucleic acids may be linked to a transcription unit comprising (1) gene expression regulation elements such as promoters and amplifiers and (2) a coding or structural sequence which is transcribed into a mRNA and translated into the corresponding protein, and (3) appropriate initiation and termination signals.

[0060] A number of suitable expression control sequences are known in the art. General methods for expressing the recombinant protein are also known and exemplified in the document by R Kaufman, Methods in Enzymology 185, 537-566 (1990) [17].

[0061] The promoter regions which can be used in the vectors described herein include lacL, lacZ, T3, T7, gpt, lambda PR, tre and ara.

[0062] The disclosure also describes vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors which are known in the genetic engineering field and which comprise the nucleic acid sequences described herein, said vectors being plasmids and selected from DSR-S vardel Δ4N, DSR-S vardel Δ3, DSR-S vardel Core, DSR-S Core ΔA and DSR-S vardel Δ4N SEV663YDA.

[0063] The nucleic acids described herein may be expressed in prokaryotic or eukaryotic cells. Non-limiting examples of such cells which may be cited are VERO cells, HELA cells such as ATCC No CCL3, CHO cell lines such as ATCC CCL61, COS cells such as COS-7 and ATCC No CR cells: 1650, W138, BHK, HepG2, 3T3 such as ATCC No CRL6361, A549, PC12, K562, 293 cells, Sf9 cells such as ATCC No CRL 1711, Cv1 cells such as ATCC No CCL70 and JRKAT cells such as ATCC Tib152.

[0064] Non-limiting cells which can be used herein include strains of the prokaryotic host cells such as Eschierichia coli, Bacillus subtilis, Salmonella typhimurium or strains of the genus Pseudomonas, Streptomyces and Staphylococcus or strains of eukaryotic host cells such as the parasites Apicomplexan (Plasmodia, Toxoplasma, Cryptosporidia), Leishmania or Trypanosoma.

[0065] Other appropriate cells may be used herein and in particular include yeast cells such as Saccharomyces, for example Saccharomyces cerevisiae or pombe, Pichia pastoris and eukaryotic cells (plant cells, CHO cells and the like).

[0066] The cells used for expressing nucleic acids described herein are Escherichia coli and strains selected, for example, from JM109, BL21 (DE3)pLysS, TOP10 or Pir1. The INVsc strain of Saccharomyces cerevisiae may also be used.

[0067] The disclosure describes host cells transformed with the nucleic acid sequences described above or with a vector as described above and cells derived from transformed cells and containing the vector or the nucleic acid sequences described herein.

[0068] Examples of such host cells which may be cited are Escherichia coli, in which the truncated and/or mutated dextransucrase may be produced. The preparation of such host cells is known in the art.

[0069] Proteins and biologically active fragments of such proteins as well as mutated proteins which are encoded by the nucleic acid molecules described herein and their preparation methods are also described herein.

[0070] Thus, the disclosure also describes a method for preparing mutated and/or truncated dextransucrase, comprising the following steps:
  1. (a) culturing host cells transformed with the nucleic acid sequences described above or with a vector as described above under conditions allowing the expression of a dextransucrase; and
  2. (b) isolating said dextransucrase from the culture medium.


[0071] More specifically, the nucleic acid sequences may be selected from SEQ ID NO: 1 from position 373 to position 4269, the fragment of sequence SEQ ID NO: 2 from position 373 to position 4005, the fragment of sequence SEQ ID NO: 3 from position 373 to position 3408, the fragment of sequence SEQ ID NO: 4 from precursor 373 to position 3018, and the fragment of sequence SEQ ID NO: 5 from position 373 to position 4269, complementary sequences of said sequences and sequences which hybridize with said sequences under stringent conditions, provided that dextransucrase enzymatic activity is maintained.

[0072] After being isolated, the dextransucrases of the present invention may also be purified. In this respect, the usual purification methods may be used such as precipitation, ion exchange chromatography, affinity chromatography, hydrophobic exchange chromatography, gel filtration, reverse phase HPLC, phase demixing and the like. The mutated or truncated dextransucrases of the present invention may be purified using a resin charged with nickel, taking into account the existence of the thioredoxin and 6xHis tag.

[0073] The disclosure also describes dextransucrase proteins consisting essentially of or consisting of an amino acid sequence selected from SEQ ID NO: 6 to 10 or an amino acid sequence selected from the fragment of SEQ ID NO: 6 from the amino acid at position 125 to the amino acid at position 1423, the fragment of SEQ ID NO: 7 from the amino acid at position 125 to the amino acid at position 1335, the fragment of SEQ ID NO: 8 from the amino acid at position 125 to the amino acid at position 1136, the fragment of SEQ ID NO: 9 from the amino acid at position 125 to the amino acid at position 1006, and the fragment of SEQ ID NO: 10 from the amino acid at position 125 to the amino acid at position 1423.

[0074] A protein encoded by one of nucleotide sequences SEQ ID NO: 1 to SEQ ID NO: 5 or fragments of said sequences, as set forth above, is described herein.

[0075] Homologous amino acid sequences, i.e., wherein the degree of similarity with the sequences defined above is sufficient for the enzymatic activity to be maintained, are also described herein. Thus, Blast and Fasta programs may be used to investigate similarity. Since it was demonstrated herein that it was possible to truncate the N- and C-terminal ends of dextransucrases, maintaining enzymatic activity, sequence similarity cannot be considered for just the single complete sequence, but also for the truncated sequences. The disclosure thus describes any sequence containing 80%, 90% or 98% sequence similarity with the complete sequence, but also those which would have 80%, 90% or 98% sequence similarity with one of the truncated sequences, provided that enzymatic activity is maintained.

[0076] More specifically, the present invention concerns sequences having 90%, 95% or 98% similarity with amino acid sequences selected from the fragment of SEQ ID NO: 6 from the amino acid at position 125 to the amino acid at position 1423, SEQ ID NO: 7 from the amino acid at position 125 to the amino acid at position 1335, SEQ ID NO: 8 from the amino acid at position 125 to the amino acid at position 1136, SEQ ID NO: 9 from the amino acid at position 125 to the amino acid at position 1006, and SEQ ID NO: 10 from the amino acid at position 125 to the amino acid at position 1423, provided that these proteins have the enzymatic activity of said dextransucrases. Clearly, the amino acid sequences with a specific identity defined above have a majority of conservative amino acid substitutions.

[0077] Conservative amino acid substitutions include amino acid substitutions of the same class. These classes comprise, for example, amino acids having uncharged polar side chains, such as Asn, Gln, Ser, Thr or Tyr; amino acids containing basic side chains, such as His, Lys or Arg; amino acids containing acidic side chains, such as Glu or Asp and amino acids containing non-polar side chains, such as Ala, Gly, Leu, Val, Ile, Phe, Cys or Trp.

[0078] Furthermore, concerning the enzymatic activity of dextransucrase with amino acid substitutions, this can be tested as set forth in the Examples, but the activity can also be evaluated by HPLC analyses or using the usual predictions concerning the way amino acid changes affect protein functions.

[0079] In a further aspect, since the amino acid sequences are indicated here, the protein may be synthesized using R B Merrifield's method, 1963 [20]. For this reason, the synthesized dextransucrase proteins constitute another aspect described herein.

[0080] Mutant dextransucrases can be mutant SEV663YDA of DSR-S vardel Δ4N in which the serine, glutamic acid and valine in positions 663, 664 and 665 have been modified to tyrosine, aspartic acid and alanine respectively.

[0081] This mutant may be used to synthesize isomaltose from sucrose, using sucrose as the only substrate in a yield which is equivalent to that obtained when an acceptor, such as glucose is added to the reaction medium.

[0082] The disclosure describes a method for producing isomaltose directly from sucrose, said method comprising reacting mutant dextransucrase with SEQ ID NO: 10 with sucrose, and producing isomaltose.

[0083] The fusion proteins containing a protein tag as described above also form part of the present invention. In this regard, the mutated and/or truncated proteins of the present invention may be fused with at least one protein tag.

[0084] Thus the present invention also relates to a fusion protein comprising at the N - terminal and/or C - terminal position, a protein tag selected from the group of a poly-His tag, a c- myc epitope tag, a HA- tag, a T7- tag, a S-tag, a trpE tag, an avidin/streptavadin tag, a staphylococcus A tag, a G protein tag, a dihydrofolate reductase tag, cellulose binding domain tags, a polycysteine tag, a phenylalanine tag, a bacterial GST tag, a maltose binding protein tag, a thioredoxin tag, a beta-galactosidase tag and a VSV - protein tag fused to the dextransucrase according to the invention, wherein the fusion protein conserves its specificity for synthesizing alpha-1,6 bonds.

[0085] The preparation of high molar mass dextrans (about 106 - 108 Da) and with modified rheological properties compared with dextran synthesized by native DSR-S of L. mesenteroides NRRL B-512F using the truncated dextransucrase of the present invention is another aspect of the invention.

[0086] More specifically, microorganisms secreting dextransucrase or cellular extracts of microorganisms producing dextransucrase in an intracellular manner may be cultivated or used in a medium comprising sucrose, resulting in the synthesis of isomaltose (342 Da), (ii) isomalto-oligosaccharides of 342 to 5,000 Da, (iii) dextrans with a controlled size of 1,300 to 5,200 Da centered around 10,000 Da, (iv) dextrans with a controlled size of 7,000 to 1.7 x 105 Da centered around 40,000 Da, and (v) dextrans with a high molar mass from 2 x 106 Da to 109 Da. These compounds may be isolated from the culture medium by conventional methods such as ultrafiltration, nanofiltration, alcoholic precipitation, liquid chromatography and the like.

[0087] Alternatively, the truncated and/or mutated dextransucrases described in the present invention may be purified and used in a method for producing dextrans with a controlled molar mass.

[0088] Thus, the disclosure describes a method for producing dextrans and/or isomalto-oligosaccharides with a controlled molar mass, comprising reacting a mutated and/or truncated dextransucrase consisting essentially of or consisting of a sequence selected from nucleotide sequences SEQ ID NO: 6 to SEQ ID NO: 10 defined above with at least sucrose and optionally an acceptor.

[0089] The disclosure also describes a method for producing isomaltose, the method comprising reacting a mutated and/or truncated dextransucrase with sequence SEQ ID NO: 10 essentially with sucrose. The disclosure also concerns a method for producing dextrans with interesting textural properties, the method comprising reacting a mutated and/or truncated dextransucrase with the sequence of SEQ ID NO: 6.

[0090] The disclosure also describes dextrans and isomalto-oligosaccharides having the characteristics defined herein which may be obtained by the methods described here. These characteristic properties include the fact that high molar mass dextrans have non-Newtonian behavior and have the character of a gel or a stringy nature, and the property of changing form a solution type behavior to that of a gel after application of a second series of shear stresses.

[0091] As will become apparent in the Examples, advantageously, the different rheological properties may be obtained depending on whether the enzyme is purified or non-purified.

[0092] The enzymatically produced dextrans may be used as a support in the pharmaceutical industry, as a plasma substitute, additives in textiles or paints, in cosmetics and in the agroalimentary industry, as well as a texturing agent, for example as a substitute for gum Arabic or a gelling agent. The disclosure also describes compositions comprising the dextrans and IMOs described herein.

[0093] One important application of the dextrans and isomalto-oligosaccharides described herein is their use as prebiotics. These products are not completely metabolized and are selectively fermented in the colon by appropriate bacterial species such as Bifidobacteria and Lactobacilli.

[0094] Oligosaccharides have traditionally been used for human or animal foodstuffs, in the pharmaceutical industries and in the cosmetics industry or as a sweetener, stabilizer or filler [21]. During the last fifteen years, a new field of activity has developed for the prebiotic properties of certain non digestible molecules [23]. Oligosaccharides as prebiotics are interesting with respect to their capacity to resist attack by digestive enzymes and to accentuate the growth of "healthy" bacteria, primarily Bifidobacteria and Lactobacilli, in the intestine. This concept has been stimulated by the emergence of commercial prebiotic products which have rapidly gained popularity. Oligomers such as fructo-oligosaccharides, lactulose, galacto-oligosaccharides, xylo-oligosaccharides, oligosaccharides extracted from soya or isomalto-oligosaccharides which are usually obtained by biological processes or by extraction from plants, are also promising. Currently, research in this field has centered on the production of novel oligosaccharide structures termed second generation prebiotics which should have novel physico-chemical properties and more specific biological activities [18].

[0095] The disclosure describes a composition comprising a dextran obtained from a dextransucrase described herein, and a pharmaceutically acceptable vehicle or a food quality vehicle.

[0096] The acceptable vehicle may, for example, be selected from adjuvants, salts and the like and the adjuvants may be selected from muramyl peptides, alum, montanide and the like. The mutated and/or truncated dextransucrases may be a purified protein, a protein produced in a recombinant manner or a synthetically produced protein.

[0097] Regarding the method for producing the dextrans and/or IMOs described herein, preferred acceptors, when used, are glucose, isomaltose, maltose and isomalto-oligosaccharides.

[0098] Preferably, as described herein, the method for producing isomalto-oligosaccharides with a controlled molar mass comprises reacting a mutated and/or truncated dextransucrase consisting of sequences SEQ ID NO: 7, 8, 9 or 10 essentially with sucrose. The degree of polymerization thus may vary from 2 to 60 glucosyl units (DP2 to DP60).

[0099] The production reaction may take place at temperatures in the range 4 °C to 80 °C, preferably 4 °C to 40 °C.

[0100] Preferably, when the sequence is SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, the temperature may be in the range 4°C to 15°C, preferably 8°C to 12°C, and more preferably the temperature may be of the order of 10°C for the production of dextrans with a controlled size. Further, for such sequences, the temperature may be preferably in the range from about 8°C to 25 °C, more preferably on the order of 20 °C for IMO synthesis.

[0101] Furthermore, preferably when the sequence is SEQ ID NO: 6 or SEQ ID NO: 10, the temperature may be in the range 15°C to 45°C, preferably 17°C to 30 °C, and more preferably on the order of 20 °C to 25 °C.

[0102] Further, the sucrose concentration may be in the range 10 to 600 g/l, preferably 75 to 400 g/l, and more preferably 90 to 280 g/l.

[0103] When the sequence is SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, the concentration of sucrose in the medium may be preferably on the order of 250 g/l.

[0104] Further, when the sequence is SEQ ID NO: 6 or SEQ ID NO: 10, the concentration of sucrose may be on the order of 100 g/l.

[0105] Further, as appropriate, the sucrose/acceptor weight ratio may be on the order of 0.5 to 12, preferably 1 to 4, more preferably about 2.

[0106] In the method described herein, the dextransucrase is in the free form or immobilized on a support. Said immobilization may be effected by adsorption, inclusion or covalent binding, for example.

[0107] Finally, to carry out the method described herein, the pH is in the range 3.0 to 10.0, preferably 4.0 to 7.0, more preferably 4.5 to 6.0 and still more preferably about 5.2.

[0108] Other aspects of the invention may become apparent from a study of the Examples below.

Example 1: Construction of variants



[0109] The pBad/TOPO Thiofusion vector (Invitrogen) was used for cloning and expressing truncated and/or mutated dsrS genes under the control of the L-arabinose promoter. It allows fusion of the gene to the 6xHis tag at the C-terminal end, and to a thioredoxin tag at the N-terminal end.

[0110] For use as a matrix, genomic DNA from L. mesenteroides NRRL B-512F was extracted using the "Blood and Cell culture DNA maxi" kit (Qiagen). The strain is derived from the NCAUR collection, Peoria, IL, USA.

[0111] One Shot TOP10 cells (Invitrogen) were used for expression of the truncated and/or mutated dsrS genes. The restriction enzymes were purchased from New England Biolabs and used in accordance with the manufacturer's instructions. DNA was purified using "QIAquick" (purification by PCR and gel extraction) and "QlAprep" (plasmid purification) kits from Qiagen.

[0112] The variants were constructed by PCR amplification of the DSR-S gene from genomic DNA from L. mesenteroides NRRL B-512F using the "Expand High fidelity" polymerase (Roche) and the following primers (given in the 5'→ 3' direction):
  1. 1 DSR-S vardel Δ4N was constructed using the pBad and DSR-S vardel primers: 454-acacaacaagttagcggcaagtacgttgaaaaagac-490 and PBad Δ4N: 4350-actcaagttagtatctggatccacaatgatagc-4317. It contained amino acids T152 to S1450 of DSR-S.
  2. 2 DSR-S vardel Δ3 was constructed using the PBad and DSR-S vardel primers: 454- acacaacaagttagcggcaagtacgttgaaaaagac-490 and PBad Δ3: 4086-cccgtctgcatcaatgaattcacc-4062. It contained amino acids T152 to G1362 of DSR-S.
  3. 3 DSR-S vardel Core was constructed using the PBad and DSR-S vardel primers: 454- acacaacaagttagcggcaagtacgttgaaaaagac-490 and PBad Core: 3489-gccagtttctgacagatcattagttaactg-3459. It contained amino acids T152 to G1162 of DSR-S.
  4. 4 DSR-S Core ΔA was constructed using the PBad DSR-S cat primers: 843-ggcttctctggtgtgattgatggtcaa-870 and PBad Core: 3489-gccagtttctgacagatcattagttaactg-3459. It contains amino acids G282 to G1162 of DSR-S.
  5. 5 The mutant DSR-S vardel Δ4N SEV663YDA was constructed by directed mutagenesis using the "mega primer" technique [33, 21] and DNA polymerase Pfu (Strategene). A first PCR reaction was carried out using the DSR-S vardel Δ4N plasmid matrix and the SEV663YDA primer pair: 1965-agctttgtacgagctcacgactacgacgcgcaaacggtt-2004 and rev: 3447-gtcaccatcctcagtgttcgaaacg-3422, comprising the BstBI restriction site (underlined). This PCR product was then used as a reverse mega primer in a second PCR with the forw primer: 1329-caaccacagtggaatgaaactagtc-1354 comprising the SpeI restriction site. This second PCR product was then digested with the two restriction enzymes SpeI and BstBI in accordance with the manufacturer's conditions (New England Biolabs) and cloned into the pBad DSR-S vardel Δ4N vector previously digested with the same enzymes. The SEV663YDA primer was designed to introduce a single restriction site to select positive clones (in this case, the SacI site).


[0113] The primary structure of each of the variants DSR-S vardel Δ4N, DSR-S vardel Δ3, DSR-S vardel Core and DSR-S Core ΔA is diagrammatically shown in Figure 6.

Example 2: Production of variants in E coli



[0114] Cultures were carried out in a baffled Erlenmeyer flask on 2X YT medium buffered to a pH of 6.4 with 100 mN of Tris-HCl, DSR-S being known to be unstable under alkaline pH conditions [3].

Composition of medium 2X YT:



[0115] 
Bactotryptone 16 g/l
Yeast extract 10 g/l
NaCl 5 g/l
Tris 12.1 g/l


[0116] E. coli TOP10 cells carrying pBad DSR-S vardel Δ4N and pBad DSR-S vardel Δ4N SEV663YDA plasmids were cultivated at 23°C. L arabinose induction was carried out when cell growth reached OD600nm of 0.2 with 0.002% (w/v) of inducer. Culturing was stopped when cell growth reached a plateau (OD600nm of about 3-3.5) before starting the cell lysis phase.

[0117] E. coli TOP10 cells carrying pBad DSR-S vardel Δ3, pBad DSR-S vardel Core and pBad DSR-S Core ΔA plasmids were brought to 16°C. Induction was carried out when the cell growth reached OD600nm of 0.2 with 0.005% (w/v) of L arabinose in the case of DSR-S vardel Δ3 and 0.02% (w/v) in the case of DSR-S vardel Core and DSR-S Core ΔA. Culturing was halted when the cell growth reached a plateau (OD600nm of about 2.5) before starting the cell lysis phase.

[0118] Following culture, the cells were recovered by centrifuging (8,000 x g, 10 minutes, 4°C), re-suspended and concentrated to an OD600nm of equivalent to 80 in a sodium acetate buffer 50 mM, pH 5.2, supplemented with 0.05 g/l of 1 mM CaCl2 and phenylmethanesulfonyl fluoride (PMSF). Cell rupture was carried out by sonication. The preparations were then centrifuged once again (20,000 x g, 30 min, 4°C) to eliminate cellular debris and recover only the sonication supernatant.

[0119] The enzymatic activity of the extracts was measured using the dinitrosalicylic acid (DNS) method of Sumner and Howell, 1935 [22]. An enzymatic unit is defined as the quantity of enzyme which catalyses the formation of one µmole of fructose per minute at a given temperature (4°C to 40°C depending on the case, more precisely 20°C or 30°C) and in a sodium acetate buffer (50 mM), pH 5.2, containing 0.05 g/l of CaCl2 and 100 g/l of sucrose.

Example 3: Purification of DSR-S vardel Δ4N variant



[0120] Different enzymatic forms of DSR-S vardel Δ4N were produced during the culture of E. coli TOP10: a vastly major entire form and different degraded forms at the C-terminal end (Figure 7). The origin of these degradations remains unclear. Production in Example 2 reached about 5500 U/l of culture in the sonication supernatants (activity assayed at 30 °C).

[0121] To determine the number of active enzymatic forms in the extracts, electrophoresis gels were produced under native or denaturing conditions. After gel re-naturing, it was incubated overnight at 25°C in a sodium acetate buffer, 50 mM, pH 5.2 supplemented with 100 g/l of sucrose. The active enzymatic forms then synthesized polymer at the region to which they migrated in the gel. A reagent (Schiff's reagent) which specifically colored the polymers synthesized by active dextransucrases, after oxidation of primary alcohol functions of the periodic acid polymer was used and the gels were stained with this reagent. This type of gel is termed a zymogram. In the case of DSR-S vardel Δ4N, or its mutant SEV663YDA, only the two higher molar mass forms were detected as being active (results not shown). However, only the entire form had both the thioredoxin tag and the 6xHis tag.

[0122] The presence of the 6xHis tag only in the entire form of DSR-S vardel Δ4N was exploited to purify the enzyme by affinity chromatography on nickel resin (Probond Ni-NTA, Invitrogen).

[0123] Purification was carried out at 4°C. All of the buffers had concentrations of 50 mM sodium acetate, 400 mM of NaCl, different concentrations of imidazole and were adjusted to a pH of 7.5. The resin was equilibrated with 8 volumes of buffer having a concentration of 40 mM of imidazole. Fixing was carried out for 2 hours with 7 volumes of enzymatic extract supplemented with 20 mM of imidazole and adjusted to a pH of 7.5. Next, the resin was washed with 40 volumes of 40 mM imidazole buffer, 8 volumes at 60 mM and 4 volumes at 100 mM. Finally, the proteins were eluted with 7 volumes of buffer having a concentration of 250 mM of imidazole.

[0124] The fractions containing the eluted fusion proteins were mixed and dialyzed overnight at 4°C against a buffer containing a concentration of 50 mM of sodium acetate, pH of 5.2, and 0.05 g/l of CaCl2. The protein concentration was determined by the microbradford method (Biorad Laboratories) with BSA (bovine serum albumin) as the standard.

[0125] The purity of the preparation at the end of the procedure was estimated at about 90% (Figure 8). The purified DSR-S vardel Δ4N proteins had a very strong tendency to aggregate, causing the formation of white precipitates and limiting the yields obtained at the end of the procedure (Table 1). However, the specific activity of the preparation was estimated at 584 U/mg of protein, which corresponded to the best described specific activity of a recombinant dextransucrase. By way of comparison, the specific activity of native DSR-S (expressed by L. mesenteroides NRRL B-512F) was estimated at about 170 U/mg [24].
Table 1: Purification of DSR-S vardel Δ4N by affinity chromatography on nickel resin
Purification stageVolume (ml)Activity (U/ml)Protein conc (mg/l)Specific activity (U/mg)Purification factorYield (%)
Sonication supernatants 150 149.2 9.46 15.7 1 100
Elution fraction after dialysis 150 67.6 0.25 270.5 17 45.3
Soluble fraction after eliminating aggregates 150 38.2 0.09 424.4 27 25.4

Example 4: Nucleotide sequences and amino acid sequences



[0126] The constructs were sequenced and the corresponding sequences are shown in Figures 1 to 5.

Example 5: Synthesis of dextran by DSR-S vardel Δ4N, comparison with DSR-S from L. mesenteroides NRRL B-512F



[0127] Dextran was synthesized from native DSR-S from L. mesenteroides NRRL B-512F, entire recombinant DSR-S (sonication supernatant) and DSR-S vardel Δ4N (sonication supernatant and purified enzyme).

Synthesis conditions and analysis of products formed



[0128] Entire recombinant DSR-S was constructed on the same principle as the variants described in Example 1, with primers which were suitable for amplification of the entire gene. E. coli TOP10 cells carrying the pBad DSR-S plasmid were cultivated using the protocol described for DSR-S vardel Δ4N (Example 2). The supernatant contained three enzymatic forms, including two with higher active molar mass.

[0129] The form with the greatest size contained DSR-S in its entirety; the two other forms were degraded at their N-terminal position (data not shown).

[0130] The activity of each enzymatic preparation was determined at 30°C.

[0131] Dextran syntheses were carried out at 25°C starting with a 100 g/l sucrose solution, in a 50 mM sodium acetate buffer containing 0.05 g/l of CaCl2 and with 1 unit per ml of enzyme. The progressive exhaustion of sucrose was monitored by HPAEC-PAD analyses (see below) and the reaction was stopped after its complete consumption, by heating for 5 min at 95°C (complete denaturing of cited dextransucrases).

[0132] The products formed were analyzed by HPAEC-PAD (high performance anion exchange chromatography with pulsed amperometric detection) with respect to the mono, di and oligosaccharides, and by HPSEC (high performance size exclusion chromatography) with respect to the polysaccharides.

[0133] The HPAEC-PAD system comprised a Dionex "Carbopack PA100" 4 x 250 mm column. A 6 to 300 mM sodium acetate gradient in 28 minutes in a 150 mM sodium hydroxide solution was applied at a flow rate of 1 ml/min. Detection was carried out by amperometry using a Dionex ED40 module with a gold electrode and an Ag/AgCl pH reference electrode.

[0134] The HPSEC system was constituted by two Shodex OH-Pack SB-805 and SB-802.5 columns in series, using 0.45 M sodium nitrate + 1% (v/v) ethylene glycol as the solvent, in an amount of 0.3 ml/min. The columns and pre-columns were kept at 70°C and the samples were filtered on 0.45 µm filters (Sartorius) prior to injection. Detection was of the refractometric type, coupled to a light diffusion detector (Wyatt) to determine the mass of the dextrans.

[0135] The concentrations by weight of glucose, fructose and leucrose (sucrose isomer) were determined by HPAEC-PAD analyses. The percentages of glucosyl residues from the sucrose incorporated into the free glucose and leucrose were calculated using the following formula:

and

where [glucosetf] and [leucrosetf] correspond to the final concentrations of glucose and leucrose at the end of the reaction and [sucroseto] corresponds to that of the initial substrate (g/l).

[0136] The percentage of glucosyl residues incorporated into the HMW polymer was determined by HPSEC analyses using the formula:

in which surface areadextran tf corresponds to the surface area of the dextran peak, determined using the HPSEC chromatogram at the end of the reaction, and surface areasucrose-t0 corresponds to that of the peak of the initial substrate. For a given concentration, the surface obtained by refractometry is identical regardless of the sugar.

[0137] The proportion of glucosyl units incorporated into the IMW polymers or oligosaccharides for which the concentration could not be directly quantified by HPAEC-PAD or HPSEC was determined using the formula:



[0138] The elution profiles of the four dextrans obtained by HPSEC are shown in Figure 9. Different populations can be distinguished: a first peak eluted at 38 minutes, corresponding to the high molar mass polymer (HMW), and a second peak at 75 minutes corresponding to fructose, glucose, leucrose (5-O-α-D glucosyl fructose) and other oligosaccharides with a degree of polymerization (DP) of less than 7, not separated by the system or in very low concentrations. Between these two principal peaks, as indicated by the base line perturbations, the products of intermediate size (IMW dextrans) were also present. These compounds, with very variable sizes, between 1000 and 107 Da, were highly polydispersed and in very low concentrations, which explains their low intensity on the chromatogram. HPAEC-PAD analyses confirmed their presence, however (results not shown).

[0139] The relative quantity of glucosyl units derived from sucrose and incorporated into the different products is listed below in Table 2. The synthesis yield for HMW dextran represents about 60% of the glucosyl units for each of the preparations. The transfer of glucosyl units to water (glucose) or fructose (leucrose) represents less than 8%, while the synthesis of intermediate size dextrans (IMW) accounted for 25% to 32% of the transferred glucosyl units. All of the recombinant forms of DSR-S tended to synthesize more intermediate size dextrans. The HPSEC analyses also showed that the native enzyme appeared to synthesize two different populations of dextran, as opposed to only one for the recombinant enzymes. The molar mass of HMW dextrans was determined by light diffusion and estimated to be over 107 g/mol for all of the samples (exclusion limit of the columns used).
Table 2: Percentage of glucosyl units incorporated into the various products derived from the synthesis of dextran at 25°C and 100 g/l of sucrose, for the four cited DSR-S preparations
 GlucoseLeucroseIMW dextransHMW dextrans
Rel %HMW (g/mol)
Native DSR-S 4.12 5.80 25.60 64.47 1.5 x 108 8.88 x 107
Entire DSR-S 2.32 5.39 29.32 62.96 1.86 x 108
DSR-S vardel Δ4N 2.43 5.90 31.03 60.64 4.87 x 107
Purified DSR-S vardel Δ4N 2.33 5.80 32.24 59.62 2.47 x 107

Structure of dextrans formed



[0140] The structure of the dextran produced by DSR-S vardel Δ4N (purified or otherwise) was compared with that of dextrans synthesized from entire recombinant DSR-S and native DSR-S. These structures were determined by nuclear magnetic resonance (1H NMR) using a Brücker AC 300, at 85°C and with an acquisition frequency of 300.13 MHz. The acquisition time was 3 s, with 32 to 64 passes. The dextrans were initially separated from the coproduced fructose by precipitating 3 times with 1 volume of absolute ethanol, recovered by centrifuging, washed with distilled water and freeze dried. The samples were dissolved in D2O to a concentration of 6 mg/ml.

[0141] The NMR spectra are shown in Figure 10. Only α-1,6 bonds were detected. Carbon-13 NMR analysis was also carried out on the dextran synthesized by purified DSR-S vardel Δ4N. The spectrum obtained was identical to those published for the dextran from L. mesenteroides NRRL B-512F and entire DSR-S [3].

[0142] These polymers were also digested with endodextranase from Chaetomium gracile carried out for 16 h at 37°C with 3 enzyme units per ml of synthesis medium. The digestion products were analyzed by HPAEC-PAD (Figure 11). The digestion profiles obtained were identical for the four analyzed dextrans, confirming that they all had at least 95% α-1,6 bonds.

[0143] The deletions made in the N and C-terminal positions of the DSR-S to construct the DSR-S vardel Δ4N variant thus have no significant influence on the initial activity of DSR-S or on the portion of glucosyl units derived from sucrose incorporated into the synthesis of the HMW dextran, the size or the structure of the polysaccharide.

Rheological behavior of dextrans formed



[0144] The rheological behavior of the four dextrans was analyzed using a cone-plane system (AR 1000, TA Instruments) provided with a 4 cm diameter cone at an angle of 3.59°, and covering speeds of 0.01 to 100 s-1. The measurements were carried out at 25°C. Dynamic experiments were carried out in the linear domain between 0 and 10 Pa, with a deformation of 8% for the dextran synthesized by native DSR-S from L. mesenteroides NRRL B-512F (control), 3% for that synthesized by the entire recombinant DSR-S, 5% for that synthesized by a non-purified extract of DSR-S vardel Δ4N and 0.4% for that synthesized by purified DSR-S vardel Δ4N. The complex stiffness modulus is defined by the relationship:



[0145] The energy conservation modulus G'(ω) is larger when the sample is predominantly elastic or highly structured. The loss modulus G" (ω) represents the energy dissipated during deformation. Predominantly viscous samples have a high G" (ω).

[0146] These rheological analyses produced entirely original results (Figure 12). As described in the literature, native DSR-S synthesized a dextran with Newtonian behavior [25].

[0147] The entire recombinant DSR-S extracts and non-purified DSR-S vardel Δ4N extracts produced viscous solutions with identical behavior (viscosity about 10 times higher than that of dextran produced by native enzyme). When observed with the naked eye, they also had a fairly pronounced stringy behavior. Further, after application of new shear stresses, the behavior of said polymers changed from a solution type to a gel type, which is a a novel property which has been identified for this type of biopolymer. The dextran produced by the native enzyme, in contrast, was not stringy, and its behavior was entirely reversible after application of a second series of stresses (Figure 12A).

[0148] The purified enzyme directly synthesized a polymer having the properties of a highly structured gel (Figure 12B, modulus G' much higher than G"), retaining its characteristics through a range of temperatures from 10°C to 70°C (results not shown). This behavior is completely different from that of the native enzyme.

[0149] Only the preparation of purified DSR-S vardel Δ4N contained only one active dextransucrase in the extract. Native DSR-S is known to be prone to problems of proteolytic degradation [26] and the purification techniques developed could not resolve that problem [27, 28, 29]. Entire recombinant DSR-S used in the test contained at least two active enzymatic forms, like the DSR-S vardel Δ4N preparation prior to purification. However, the degraded forms of native DSR-S, entire recombinant DSR-S and DSR-S vardel Δ4N are entirely different. It is currently assumed that cooperation between these different active enzymatic forms present in the medium could be the origin of modifications to the dextran chains, causing these differences in behavior.

Example 6: Synthesis of isomaltose from sucrose



[0150] The capacity of mutant DSR-S vardel Δ4N SEV663YDA to synthesize only isomaltose (IMO with DP 2) from sucrose to the detriment of high molar mass dextrans was studied.

[0151] The mutant was purified by affinity chromatography using the procedure described for DSR-S vardel Δ4N given in Example 3.

[0152] The activity was assayed at 30 °C.

[0153] With a specific activity of only 9 U/mg, the SEV663YDA mutations induced severe effects on the activity of DSR-S (loss of 98% of the initial sucrose consumption rate). That specific activity, however, is equivalent to that of recombinant amylosucrase from N. polysaccharea [32], which has been widely studied for its application potential.

[0154] The characterizations which were carried out demonstrate the feasibility of producing isomaltose by this mutant DSR-S, while the wild enzyme produces only high molar mass dextrans. Syntheses were carried out at 25°C in a buffer containing a concentration of 50 mM of sodium acetate at a pH of 5.2 and 0.05 g/l of CaCl2, 1 U/ml of purified enzyme and using 100 g/l of sucrose as the only substrate, or by acceptor reaction starting with 100 g/l of sucrose and 50 g/l of glucose. Exhaustion of sucrose was monitored by HPAEC-PAD analyses (see Example 4 for analysis conditions) and the reactions were interrupted after complete consumption.

[0155] Isomaltose production thus reached a yield of 47% using sucrose as the only substrate (Table 3 and Figure 13), a yield which was equivalent to that obtained by the acceptor reaction. Adding an exogenous acceptor was thus not necessary. Traces of isomaltotriose, maltose or nigerose (not separated by the system) were also identified (Figure 13) as well as the presence of other oligosaccharides with a DP of less than 7 and of unknown structure.
Table 3: Synthesis of isomaltose by mutant DSR-S vardel Δ4N SEV663YDA from 100 g/l of sucrose alone, or by acceptor reaction with 50 g/l of glucose. Concentration of different products present at the end of the reaction.
 100 g/l sucrose100 g/l sucrose + 50 g/l glucose
Glucose 16.73 33.14
Fructose 45.95 42.31
isomaltose 23.99 47.17
Other oligosaccharides 13.33 27.38
% of glucose residues transferred to isomaltose 47.98% 47.17%2
2: calculated from glucosyl residues derived from exogenous glucose and sucrose added to medium.


[0156] Thus in this Example, the production of isomaltose attained a yield of 47%. Currently, this is the first method involving a single enzyme for synthesizing isomaltose from sucrose; all prior studies being linked to the degradation of starch by a cocktail of α-amylases and glycosidases [11], or to the joint action of dextransucrase and dextranase [30]. Further, sucrose is a cheap and widely available substrate and the fructose released during the syntheses constitute a co-product the value of which can be exploited separately.

Example 7: Synthesis of dextran by DSR-S vardel Δ3



[0157] Different enzymatic forms of DSR-S vardel Δ3 were produced during culture of E. coli TOP10. However, the entire form was vastly in the majority and the zymograms produced (see Example 3) showed that only the entire form was active.

[0158] The optimum activity temperature for this variant was 20°C. Thus, activity assays were carried out at this temperature. Production of DSR-S vardel Δ3 in accordance with Example 2 reached about 320 U/l of culture.

[0159] Dextran syntheses were carried out at 20°C in a buffer containing 50 mM of sodium acetate, pH of 5.2, and 0.05 g/l of CaCl2, 100 g/l of sucrose and 1 U/ml of non-purified DSR-S vardel Δ3 extract. The DSR-S vardel Δ3 extract could be purified by affinity chromatography on nickel resin using the protocol described for DSR-S vardel Δ4N in Example 3. However, since the sonication supernatant contained only a single enzymatic form of dextransucrase and E. coli did not produce another enzyme which could consume the sucrose, purification of the variant did not constitute a prerequisite for rigorous characterization of its properties. By way of comparison, dextran syntheses were carried out under the same conditions as with (non-purified) DSR-S vardel Δ4N. The disappearance of the sucrose was monitored by HPAEC-PAD analyses and the reactions were stopped (5 minutes, 95°C) after total exhaustion.

[0160] The synthesized products were analyzed and quantified by HPAEC-PAD and HPSEC using the conditions described in Example 4. For the HPSEC analyses, the size of the dextrans was estimated using commercially available dextrans with sizes of 2 x 106, 503 x 103, 70,000, 10,000 Da, maltoheptaose and glucose (Sigma).

[0161] As can be seen in Figure 13, at 20°C the DSR-S vardel Δ3 variant synthesized two populations of polymers; major population of HMW dextran with a size of 2 x 106 Da, representing about 39% of the glucosyl residues derived from sucrose (Table 4) and a second population of 1,300 to 52,000 Da, centered at the highest peak at around 10,000 Da (about 25% glucosyl residues). This is the first time that a second population of dextran which is clearly visibly on the HPSEC chromatogram has been observed for a DSR-S variant.

Effect of temperature on the profile of the products



[0162] Dextran syntheses were also carried out at a temperature of 10°C, still with a buffer containing 50 mM of sodium acetate, pH 5.2, 0.05 g/l of CaCl2 and 1 U/ml of enzyme (activity assayed at 20°C). Sucrose exhaustion was monitored by HPAEC-PAD analyses and the reactions were stopped (5 min, 95°C) after total consumption thereof.

[0163] As can be seen in Figure 14, at 10°C the DSR-S vardel Δ3 variant synthesized a population of dextran which was very different from that produced at 20°C. The major polymer (about 44%) formed at that temperature had a molar mass in the range 7,000 and 1.7 x 105 Da centered at the peak at around 40,000 Da.
Table 4: Percentage of glucosyl units incorporated into different products synthesized by DSR-S vardel Δ4N nd DSR-S vardel Δ3 at 10°C and 20°C starting from 100 g/l of sucrose
 DSR-S vardel Δ4NDSR-S vardel Δ3
 20 °C10°C20 °C10°C
HMW dextran > 2 x 106 Da 55.2 37.1 39.2 8.8
Dextran 40,000 Da nd1 nd nd 43.9
Dextran 10,000 Da 18.2 14.7 24.8 nd
Oligosaccharides with DP ≤ 82 16.2 39.1 27.3 36.7
Leucrose 9.2 7.6 5.3 9.3
Glucose 1.2 1.5 3.4 1.3
1: nd: not detected
2: degree of polymerization calculated from retention time estimated at lower limit of 10000 Da dextran peak.

Effect of sucrose concentration



[0164] Four increasing concentrations of sucrose were tested (100, 150, 200 and 250 g/l) for the dextran syntheses carried out at 20 °C and 10 °C with DSR-S vardel Δ3 (1 U/ml). The total consumption of sucrose was monitored by HPAEC-PAD analyses and the syntheses were stopped after its total consumption (less than 48 h).

[0165] For the two temperatures, the initial increase in the concentration of substrate encouraged the synthesis of low molar mass dextrans. At 20°C, the synthesis of 10,000 Da dextran thus changed from a yield of 25% to 48% on changing from 100 to 250 g/l of initial sucrose. At 10°C and from 250 g/l, HMW dextran synthesis was completely abolished, and that of dextran with the main population with a molar mass centered around 40,000 Da advantageously reached a yield of 69%.

[0166] For all of the dextrans synthesized by DSR-S vardel Δ3, at 10°C and 20°C, and from 100 to 250 g/l of sucrose, the endodextranase digestion profiles (see Example 5) carried out confirmed that the binding specificity of DSR-S was unchanged (same oligosaccharide profiles detected by HPAEC-PAD as with DSR-S vardel Δ4N, i.e., at least 95% α-1,6 bonds).

Example 8: Synthesis of dextran by DSR-S vardel Core and DSR-S Core ΔA



[0167] The DSR-S vardel Core and DSR-S Core ΔA variants were also slightly degraded during expression by E. coli TOP under the conditions described in Example 2. However, as was the case for the DSR-S vardel Δ3 variant, only the entire form, which was in the vast majority, was active according to the zymogram (results not shown).

[0168] The optimum activity temperature for these variants was also 20°C. Production thus reached 38 and 180 U/L of culture for DSR-S vardel Core and DSR-S Core ΔA respectively.

[0169] Dextran syntheses were carried out at 20°C and 10°C using 100 to 250 g/l of sucrose in a buffer containing 50 mM of sodium acetate, pH 5.2, 0.05 g/l of CaCl2 and 1 U/ml of enzymatic extract (non-purified). Sucrose consumption was monitored by HPAEC-PAD analyses and the syntheses were stopped (5 min, 95°C) after complete exhaustion (less than 48 h). The products formed were analyzed by HPAEC-PAD and HPSEC and their concentration was quantified as described in Example 5.

[0170] Figure 15 shows the profile of the products synthesized at 20°C by the two variants (HPSEC chromatogram). It can clearly be seen that with these variants, and in contrast to DSR-S vardel Δ4N and DSR-S vardel Δ3, the major population of dextran formed had a molar mass of close to 10,000 Da with the base of the peak between 1,300 and 52,000 (at half height between 5,000 and 22,000). With the DSR-S Core ΔA variant, the synthesis of HMW dextran was completely abolished (Table 5). A reduction in temperature to 10°C could increase the yields of dextran with ∼ 10,000 Da without a significant size difference, as was the case with DSR-S vardel Δ3 (Table 5). Dextran synthesis with the DSR-S Core ΔA variant thus reached a yield of 75%. An equivalent yield was obtained with the DSR-S vardel Core variant when the initial concentration of sucrose was 250 g/l (results not shown).
Table 5: Percentage of glucosyl units incorporated into different products synthesized by DSR-S vardel Core and DSR-S Core ΔA at 10°C and 20°C starting from 100 g/l of sucrose
 DSR-S vardel Δ4NDSR-S vardel CoreDSR-S Core ΔA
 20 °C10°C20 °C10°C20°C10°C
HMW dextran > 2 x 106 Da 55.2 37.1 9.9 2.4 nd Nd
Dextran 10,000 Da 18.2 14.7 57.5 62.5 64.4 74.5
Oligosaccharides with DP ≤ 82 16.2 39.1 19.6 14.8 19.8 10.0
Leucrose 9.2 7.6 6.5 10.2 12.7 12.8
Glucose 1.2 1.5 6.5 10.1 3.1 2.7
1: nd: not detected
2: degree of polymerization calculated from retention time estimated at lower limit of 10000 Da dextran peak.


[0171] HPAEC-PAD analysis of the dextran synthesized from 100 g/l of sucrose at 20°C by the different variants showed the very high polydispersibility of the product (Figure 16), containing isomalto-oligosaccharides with a DP of 2 to a DP of about 60 for DSR-S Core ΔA in particular.

[0172] For all of the dextrans synthesized by DSR-S vardel Core and DSR-S Core ΔA at 10°C and 20°C, and using 100 to 250 g/l of sucrose, the endodextranase digestion profiles (see Example 5) carried out confirmed that the binding specificity of DSR-S was unchanged (even the oligosaccharide profiles detected by HPAEC-PAD compared with DSR-S vardel Δ4N, thus at least 95% α-1,6 bonds).

Example 9: Acceptor reaction with glucose



[0173] Acceptor reactions were carried out at 20°C with a sucrose/glucose ratio of 2 (100 g/l of sucrose, 50 g/l of glucose), 1 U/ml of extract of DSR-S vardel Δ4N, DSR-S vardel Δ3, DSR-S vardel Core and DSR-S Core ΔA in a buffer containing 50 mM of sodium acetate at a pH of 5.2 and 0.05 g/l of CaCl2. The total consumption of sucrose was monitored by HPAEC-PAD and the reactions were stopped after it had been completely exhausted. All of the variants synthesized isomalto-oligosaccharides (IMO) with a DP of 2 to about 30, to the detriment of the synthesis of polymer with a higher DP.

[0174] However, the yields obtained were higher for the variants truncated of A units. Hence, IMO production reached 52% in the case of DSR-S vardel Δ3 and 58% for DSR-S vardel Core and DSR-S Core ΔA, as opposed to 47% in the case of DSR-S vardel Δ4N. The oligosaccharide distribution was also modified (Figure 17).

[0175] For DSR-S vardel Δ3, the proportion of IMO with a DP of 2 to DP of 15 was less than that of products synthesized by DSR-S vardel Δ4N. The situation was reversed for IMOs with a DP of more than 15.

[0176] Similarly, the DSR-S vardel Core and DSR-S Core ΔA mutants were shown to perform better for the synthesis of IMO with a high DP than DSR-S vardel Δ4N or native DSR-S (DP essentially 2 to 15): the production of IMO with a DP of 12 to a DP of 27 was two to five times higher with these two variants (according to the ratio of the surface areas obtained by HPAEC-PAD).

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  22. [22] Sumner, J., & Howell, S. 1935. A method for determination of invertase activity. Journal of Biological Chemistry 108, 51.
  23. [23] Gibson, G. R., Roberfroid, M. B., 1995 Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics J. Nutr., 125, 1401-12.
  24. [24] Paul, F., Auriol, D., Oriol, E., & Monsan, P. 1984. Production and purification of dextransucrase from Leuconostoc mesenteroides NRRL B-512F. Ann. N.Y. Acad. Sci. 434, 267-270 .
  25. [25] Carrasco, F., Chornet, E., Overend, R. P., & Costa, J. 1989 A generalized correlation for the viscosity of dextrans in aqueous solutions as a function of temperature, concentration, and molecular weight at low shear rate. J Appl Polymer Sci 37, 2087-98.
  26. [26] Arguello-Morales, M., Sanchez-Gonzalez. M., Canedo, M., Quirasco. M., Farres, A., & Lopez-Munguia, A. 2005. Proteolytic modification of Leuconostoc mesenteroides B-512F dextransucrase. Antonie Van Leeuwenhoek 87, 131-41.
  27. [27] Miller, A. W., Eklund, S. H., & Robyt, J. F. 1986. Milligram to gram scale purification and characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F. Carbohydr.Res. 147, 119.
  28. [28] Kobayashi, M., & Matsumada, K. 1986. Electrophoretic analysis of the multiple forms of dextransucrase from Leuconostoc mesenteroides. J.Biochem. (Tokyo) 100, 615
  29. [29] Kobayashi, M., Mihara, K., & Matsuda, K. 1986. Dextransucrase from Leuconostoc mesenteroides NRRL B-512F: characterization of the enzyme bound to Sephadex gel. Agric Biol Chem 50,551-556.
  30. [30] Paul, F., Monsan, P., Remaud, M., Pelenc, V. Process for preparing enzymatically a sugar mixture having a high content of isomaltose from sucrose, US Patent 4,861,381
  31. [31] Blood Products Committee 83rd Meeting - July 21, 2005
  32. [32] Potocki-de-Montalk, G., Remaud-Simeon, M., Willemot, R.M., Planchot, V., and Monsan, P. 1999. Sequence analysis of the gene encoding amylosucrase from Neisseria polysaccharea and characterization of the recombinant enzyme. J. Bacteriol. 181, 375- 381.
  33. [33] Barik, S. 1995. site-directed mutagenesis by double polymerase chain reaction. Mol Biotechno/3, 1-7.

SEQUENCE LISTING



[0178] 

<110> CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE INSTITUT NATIONAL DES SCIENCES APPLIQUEES DE TOULOUSE

<120> CONSTRUCTION OF NEW VARIANTS OF DEXTRANSUCRASE DSR-S BY GENETIC ENGINEERING

<130> B6624A-JAZ/JV

<140> New PCT patent application
<141> 2007-02-08

<150> FR 06/01117
<151> 2006-02-08

<160> 26

<170> PatentIn version 3.3

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Claims

1. A dextransucrase consisting of a sequence having 90%, 95% or 98% sequence similarity to an amino acid sequence selected from the fragment from the amino acid in position 125 to the amino acid in position 1423 of SEQ ID NO: 6 and as defined in SEQ ID NO: 22, the fragment from the amino acid in position 125 to the amino acid 1335 of SEQ ID NO: 7 and as defined in SEQ ID NO: 23, the fragment from the amino acid in position 125 to the amino acid in position 1136 of SEQ ID NO: 8 and as defined in SEQ ID NO: 24, the fragment from the amino acid in position 125 to the amino acid in position 1006 of SEQ ID NO: 9 and as defined in SEQ ID NO: 25, and the fragment from the amino acid in position 125 to the amino acid in position 1423 of SEQ ID NO: 10 and as defined in SEQ ID NO: 26, wherein the enzymatic activity of forming dextrans is maintained and wherein the dextransucrase conserves its specificity for synthesizing alpha-1,6 bonds.
 
2. The dextransucrase according to Claim 1, wherein the dextrans formed have a controlled size of 1,300 to 52,000 Da centered around 10,000 Da or a controlled size of 7,000 to 1.7 X 105 Da centered around 40,000 Da.
 
3. The dextransucrase according to Claim 1 or 2, wherein the dextrans formed have a high molar mass from 106 to 108 Da and have modified rheological properties.
 
4. The dextransucrase according to Claim 3, wherein the modified rheological properties are those that have the property of changing from a solution type behavior to a gel after application of a second series of shear stresses.
 
5. The dextransucrase according to Claim 4, wherein said property is non-Newtonion behavior.
 
6. A fusion protein comprising at the N - terminal and/or C - terminal position, a protein tag selected from the group of a poly-His tag, a c- myc epitope tag, a HA- tag, a T7- tag, a S-tag, a trpE tag, an avidin/streptavadin tag, a staphylococcus A tag, a G protein tag, a dihydrofolate reductase tag, cellulose binding domain tags, a polycysteine tag, a phenylalanine tag, a bacterial GST tag, a maltose binding protein tag, a thioredoxin tag, a beta-galactosidase tag and a VSV - protein tag fused to the dextransucrase according to any one of claims 1 to 5, wherein the fusion protein conserves its specificity for synthesizing alpha-1,6 bonds.
 


Ansprüche

1. Dextransukrase, bestehend aus einer Sequenz mit 90%, 95% oder 98% Sequenzähnlichkeit mit einer Aminosäure-Sequenz, die aus dem Fragment von der Aminosäure in der Position 125 ausgewählt ist, mit der Aminosäure in der Position 1423 der SEQ ID NR. 6 und gemäß Definition in SEQ ID NR. 22, aus dem Fragment von der Aminosäure in der Position 125 der Aminosäure 1335 der SEQ ID NR. 7 und gemäß Definition in der SEQ ID NR. 23, aus dem Fragment von der Aminosäure in der Position 125 der Aminosäure in der Position 1136 der SEQ ID NR. 8 gemäß Definition in der SEQ ID NR. 24, aus dem Fragment von der Aminosäure in der Position 125 der Aminosäure in der Position 1006 der SEQ ID NR. 9 und gemäß Definition in der SEQ ID NR. 25 und aus dem Fragment von der Aminosäure in der Position 125 der Aminosäure in der Position 1423 der SEQ ID NR. 10 und gemäß Definition der SEQ ID NR. 26, wobei die Enzymaktivität des Bildens von Dextranen aufrecht erhalten ist und wobei die Dextransukrase ihre Spezifizität beim Synthetisieren von Alpha-1,6-Verbindungen beibehält.
 
2. Dextransukrase gemäß Anspruch 1, wobei die gebildeten Dextrane eine kontrollierte Größe von 1.300 bis 52.000 Da, die um 10.000 Da zentriert ist, oder eine kontrollierte Größe von 7.000 bis 1,7 X 105 Da, die um 40.000 Da zentriert ist, aufweisen.
 
3. Dextransukrase gemäß Anspruch 1 oder 2, wobei die gebildeten Dextrane eine höhere Molmasse von 106 bis 108 Da aufweisen und modifizierte rheologische Eigenschaften aufweisen.
 
4. Dextransukrase gemäß Anspruch 3, wobei die modifizierten rheologischen Eigenschaften diejenigen sind, die die Eigenschaft aufweisen, nach Anwendung einer zweiten Serie von Scherbeanspruchungen von einem Verhalten vom Lösungstyp in ein Gel überzuwechseln.
 
5. Dextransukrase gemäß Anspruch 4, wobei die genannte Eigenschaft ein Nicht-Newtonsches Verhalten ist.
 
6. Fusionsprotein, umfassend an der N-Terminal und / oder C-Terminalposition ein Protein-Tag, das aus der Gruppe eines Poly-His-Tags, eines c-myc-Epitop-Tags, eines HA-Tags, eines T7-Tags, eines S-Tags, eines trpE-Tags, eines Avidin- / Straptavidin-Tags, eines Staphylococcus-A-Tags, eines G-Protein-Tags, eines Dihydrofolatreduktase-Tags, eines Zellulose bindenden Domänen-Tags, eines Polycystein-Tags, eines Phenylalanin-Tags, eines bakteriellen GST-Tags, eines Maltose bindenden Protein-Tags, eines Thioredoxin-Tags, eines Beta-Galaktosidase-Tags und eines VSV-Protein-Tags, der mit der Dextransukrase gemäß irgendeinem der Ansprüche 1 bis 5 verschmolzen ist, ausgewählt ist, wobei das Verschmelzungsprotein seine Spezifizität zum Synthetisieren von Alpha-1,6-Bonds beibehält.
 


Revendications

1. Dextransucrase consistant en une séquence ayant une similarité de séquence de 90%, 95% ou 98% avec une séquence d'acides aminés choisie parmi le fragment de l'acide aminé en position 125 à l'acide aminé en position 1423 de la SEQ ID NO: 6 et tel que défini dans la SEQ ID NO: 22, le fragment de l'acide aminé en position 125 à l'acide aminé 1335 de la SEQ ID NO: 7 et tel que défini dans la SEQ ID NO: 23, le fragment de l'acide aminé en position 125 à l'acide aminé en position 1136 de la SEQ ID NO: 8 et tel que défini dans la SEQ ID NO: 24, le fragment de l'acide aminé en position 125 à l'acide aminé en position 1006 de la SEQ ID NO: 9 et tel que défini dans la SEQ ID NO: 25, et le fragment de l'acide aminé en position 125 à l'acide aminé en position 1423 de la SEQ ID NO: 10 et tel que défini dans la SEQ ID NO: 26, dans laquelle l'activité enzymatique de formation de dextrans est maintenue et dans laquelle la dextransucrase conserve sa spécificité pour la synthèse de liaisons alpha-1,6.
 
2. Dextransucrase selon la revendication 1, dans laquelle les dextrans formés ont une taille contrôlée de 1300 à 52000 Da centrée autour de 10000 Da ou une taille contrôlée de 7000 à 1,7 X 105 Da centrée autour de 40000 Da.
 
3. Dextransucrase selon la revendication 1 ou 2, dans laquelle les dextrans formés ont une masse molaire élevée de 106 à 108 Da et ont des propriétés rhéologiques modifiées.
 
4. Dextransucrase selon la revendication 3, dans laquelle les propriétés rhéologiques modifiées sont celles qui ont la propriété de passer d'un comportement de type solution à un gel après application d'une deuxième série de contraintes de cisaillement.
 
5. Dextransucrase selon la revendication 4, dans laquelle ladite propriété est un comportement non Newtonien.
 
6. Protéine de fusion comprenant, en position N-terminale et/ou C-terminale, un marqueur de protéine choisi dans le groupe constitué par un marqueur poly-His, un marqueur d'épitope c-myc, un marqueur HA, un marqueur T7, un marqueur S, un marqueur trpE, un marqueur avidine/streptavadine, un marqueur de staphylocoque A, un marqueur de protéine G, un marqueur de dihydrofolate réductase, des marqueurs de domaine de liaison de cellulose, un marqueur de polycystéine, un marqueur de phénylalanine, un marqueur de GST bactérien, un marqueur de protéine de liaison de maltose, un marqueur de thiorédoxine, un marqueur de bêta-galactosidase et un marqueur de protéine VSV fusionnés à la dextransucrase selon l'une quelconque des revendications 1 à 5, dans laquelle la protéine hybride conserve sa spécificité pour la synthèse de liaisons alpha-1, 6.
 




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REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description