Publications (16)64.63 Total impact
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Article: Applying Pairwise Combinations of Amino Acid Mutations for Sorting out Highly Efficient Glucosylation Tools for Chemo-Enzymatic Synthesis of Bacterial Oligosaccharides.
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ABSTRACT: Iterative saturation mutagenesis and combinatorial active site saturation focused on vicinal amino acids were used to alter the acceptor specificity of amylosucrase from Neisseria polysaccharea, a sucrose-utilizing α-transglucosidase, and sort out improved variants. From the screening of three semi-rational sub-libraries accounting in total for 20,000 variants, we report here the isolation of three double-mutants of N. polysaccharea amylosucrase displaying a spectacular specificity enhancement towards both sucrose, the donor substrate, and the allyl 2-acetamido-2-deoxy-α-D-glucopyranoside acceptor compared to the wild-type enzyme. Such levels of activity improvement have never been reported before for this class of carbohydrate-active enzymes. X-ray structure of the best performing enzymes supported by molecular dynamics simulations showed local rigidity of the -1 subsite as well as flexibility of loops involved in active site topology, which both account for the enhanced catalytic performances of the mutants. The study well illustrates the importance of taking into account the local conformation of catalytic residues as well as protein dynamics during the catalytic process, when designing enzyme libraries.Journal of the American Chemical Society 10/2012; · 9.91 Impact Factor -
Article: Characterization of a novel dextransucrase from Weissella confusa isolated from sourdough.
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ABSTRACT: Weissella confusa and Weissella cibaria isolated from wheat sourdoughs produce, from sucrose, linear dextrans due to a single soluble dextransucrase. In this study, the first complete gene sequence encoding dextransucrase from a W. confusa strain (LBAE C39-2) along with the one from a W. cibaria strain (LBAE K39) were reported. Corresponding gene cloning was achieved using specific primers designed on the basis of the draft genome sequence of these species. Deduced amino acid sequence of W. confusa and W. cibaria dextransucrase revealed common structural features of the glycoside hydrolase family 70. Notably, the regions located in the vicinity of the catalytic triad (D, E, D) are highly conserved. However, comparison analysis also revealed that Weissella dextransucrases form a distinct phylogenetic group within glucansucrases of other lactic acid bacteria. We then cloned the W. confusa C39-2 dextransucrase gene and successfully expressed the mature corresponding enzyme in Escherichia coli. The purified recombinant enzyme rDSRC39-2 catalyzed dextran synthesis from sucrose with a K (m) of 8.6 mM and a V (max) of 20 μmol/mg/min. According to (1)H and (13)C NMR analysis, the polymer is a linear class 1 dextran with 97.2 % α-(1→6) linkages and 2.8 % α-(1→3) branch linkages, similar to the one produced by W. confusa C39-2 strain. The enzyme exhibited optimum catalytic activity for temperatures ranging from 35 to 40 °C and a pH of 5.4 in 20 mM sodium acetate buffer. This novel dextransucrase is responsible for production of dextran with predominant α-(1→6) linkages that could find applications as food hydrocolloids.Applied Microbiology and Biotechnology 10/2012; · 3.42 Impact Factor -
Article: A novel dextransucrase is produced by Leuconostoccitreum strain B/110-1-2: an isolate used for the industrial production of dextran and dextran derivatives
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ABSTRACT: The industrial Leuconostoc strain B/110-1-2 producing dextran and dextran derivatives was taxonomically identified by 16S rRNA as L. citreum. Its dextransucrase enzymes were characterized according to their cellular location and reaction specificity. In the presence of sucrose, the strain B/110-1-2 produced two cell-associated dextransucrases (31.54% of the total glucosyltransferase activity) with molecular weights of 160 and 240kDa and a soluble dextransucrase (68.46%) at 160–180kDa. Two open reading frames (ORF) coding for L. citreum strain B/110-1-2 dextransucrases were identified. One of them shared a 52% identity with the alternansucrase ASR of L. citreum NRRL B-1355 and with a putative annotated alternansucrase sequence found in the genome of L. citreum KM20. The structural analysis (HPAEC-PAD, HPSEC, and 13C-NMR) of the polymer and oligodextrans produced by the B/110-1-2 dextransucrases suggest this novel glucansucrase has specificity similar to a dextransucrase but not to an alternansucrase, producing a soluble linear dextran with glucose molecules linked mainly in α-1,6 and α-1,3 with α-1,4 branches. These results enhance the understanding of this industrially significant strain and will aid in distinguishing between physiologically similar Leuconostoc spp. strains. KeywordsDextransucrases–Dextran–Gluco-oligosaccharides– LeuconostocJournal of Industrial Microbiology and Biotechnology 04/2012; 38(9):1499-1506. · 2.73 Impact Factor -
Article: Genome sequences of three Leuconostoc citreum strains, LBAE C10, LBAE C11, and LBAE E16, isolated from wheat sourdoughs.
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ABSTRACT: Leuconostoc citreum is a key microorganism in fermented foods of plant origin. Here we report the draft genome sequence for three strains of Leuconostoc citreum, LBAE C10, LBAE C11, and LBAE E16, which have been isolated from traditional French wheat sourdoughs.Journal of bacteriology 03/2012; 194(6):1610-1. · 3.94 Impact Factor -
Article: Genome sequence of Weissella confusa LBAE C39-2, isolated from a wheat sourdough.
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ABSTRACT: Weissella confusa is a rod-shaped heterofermentative lactic acid bacterium from the family of Leuconostocaceae. Here we report the draft genome sequence of the strain W. confusa LBAE C39-2 isolated from a traditional French wheat sourdough.Journal of bacteriology 03/2012; 194(6):1608-9. · 3.94 Impact Factor -
Article: Structure and property engineering of α-D-glucans synthesized by dextransucrase mutants.
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ABSTRACT: Seven dextran types, displaying from 3 to 20% α(1→3) glycosidic linkages, were synthesized in vitro from sucrose by mutants of dextransucrase DSR-S from Leuconostoc mesenteroides NRRL B-512F, obtained by combinatorial engineering. The structural and physicochemical properties of these original biopolymers were characterized. When asymmetrical flow field flow fractionation coupled with multiangle laser light scattering was used, it was determined that weight average molar masses and radii of gyration ranged from 0.76 to 6.02 × 10(8) g·mol(-1) and from 55 to 206 nm, respectively. The ν(G) values reveal that dextrans Gcn6 and Gcn7, which contain 15 and 20% α(1→3) linkages, are highly branched and contain long ramifications, while Gcn1 is rather linear with only 3% α(1→3) linkages. Others display intermediate molecular structures. Rheological investigation shows that all of these polymers present a classical non-Newtonian pseudoplastic behavior. However, Gcn_DvΔ4N, Gcn2, Gcn3, and Gcn7 form weak gels, while others display a viscoelastic behavior that is typical of entangled polymer solutions. Finally, glass transition temperature T(g) was measured by differential scanning calorimetry. Interestingly, the T(g) of Gcn1 and Gcn5 are equal to 19.0 and 29.8 °C, respectively. Because of this low T(g), these two original dextrans are able to form rubber and flexible films at ambient temperature without any plasticizer addition. The mechanical parameters determined for Gcn1 films from tensile tests are very promising in comparison to the films obtained with other polysaccharides extracted from plants, algae or microbial fermentation. These results lead the way to using these dextrans as innovative biosourced materials.Biomacromolecules 11/2011; 13(1):187-95. · 5.48 Impact Factor -
Article: NMR-based structural glycomics for high-throughput screening of carbohydrate-active enzyme specificity.
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ABSTRACT: We report here the development of a straightforward, sensitive, and quantitative NMR-based method for high-throughput characterization of carbohydrate structure and screening of carbohydrate active enzyme (CAZyme) specificity. Automated assays starting from gene library expression to carbohydrate structure determination directly from crude reaction media have been established and successfully used to screen a library of 4032 CAZymes obtained by combinatorial engineering, at a rate of 480 enzyme variants per day. This allowed one to accurately discriminate 303 enzyme variants with altered specificity. The results demonstrate the potential of high-throughput NMR technology in glycomics, to mine artificial and natural enzyme diversity for novel biocatalysts.Analytical Chemistry 02/2011; 83(4):1202-6. · 5.86 Impact Factor -
Article: Isolation of a gene from Leuconostoc citreum B/110-1-2 encoding a novel dextransucrase enzyme.
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ABSTRACT: The amplicon encoding dextransucrase DSR-F from Leuconostoc citreum B/110-1-2, a novel sucrose glucosyltransferase (GTF)-specific for α-1,6 and α-1,3 glucosidic bond synthesis, with α-1,4 branching was cloned, sequenced, and expressed into Escherichia coli JM109. Recombinant enzyme catalyzed oligosaccharides synthesis from sucrose as donor and maltose acceptor. The dsrF gene encodes for a protein (DSR-F) of 1,528 amino acids, with a theoretical molecular mass of 170447.72 Da (~170 kDa). From amino acid sequence comparison, it appears that DSR-F possesses the same domains as those described for GTFs. However, the variable region is longer than in other GTFs (by 100 amino acids) and two APY repeats (a 79 residue long motif with a high number of conserved glycine and aromatic residues, characterized by the presence of the three consecutive residues Ala, Pro, and Tyr) were identified in the glucan binding domain. The DSR-F catalytic domain possesses the catalytic triad involved in the glucosyl enzyme formation. The amino acid sequence of this domain shares a 56% identity with catalytic domain of the alternansucrase ASR from L. citreum NRRL B-1355 and with the catalytic domain of a putative alternansucrase sequence found in the genome of L. citreum KM20. A truncated active variant DSR-F-∆SP-∆GBD of 1,251 amino acids, with a molecular mass of 145 544 Da (~145 kDa), was obtained.Current Microbiology 01/2011; 62(4):1260-6. · 1.82 Impact Factor -
Article: A novel dextransucrase is produced by Leuconostoc citreum strain B/110-1-2: an isolate used for the industrial production of dextran and dextran derivatives.
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ABSTRACT: The industrial Leuconostoc strain B/110-1-2 producing dextran and dextran derivatives was taxonomically identified by 16S rRNA as L. citreum. Its dextransucrase enzymes were characterized according to their cellular location and reaction specificity. In the presence of sucrose, the strain B/110-1-2 produced two cell-associated dextransucrases (31.54% of the total glucosyltransferase activity) with molecular weights of 160 and 240 kDa and a soluble dextransucrase (68.46%) at 160-180 kDa. Two open reading frames (ORF) coding for L. citreum strain B/110-1-2 dextransucrases were identified. One of them shared a 52% identity with the alternansucrase ASR of L. citreum NRRL B-1355 and with a putative annotated alternansucrase sequence found in the genome of L. citreum KM20. The structural analysis (HPAEC-PAD, HPSEC, and (13)C-NMR) of the polymer and oligodextrans produced by the B/110-1-2 dextransucrases suggest this novel glucansucrase has specificity similar to a dextransucrase but not to an alternansucrase, producing a soluble linear dextran with glucose molecules linked mainly in α-1,6 and α-1,3 with α-1,4 branches. These results enhance the understanding of this industrially significant strain and will aid in distinguishing between physiologically similar Leuconostoc spp. strains.Journal of Industrial Microbiology 01/2011; 38(9):1499-506. · 1.80 Impact Factor -
Article: Synthesis of dextrans with controlled amounts of alpha-1,2 linkages using the transglucosidase GBD-CD2.
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ABSTRACT: GBD-CD2 is an alpha-1,2 transglucosidase engineered from DSR-E, a glucansucrase naturally produced by Leuconostoc mesenteroides NRRL B-1299. This enzyme catalyses from sucrose, the alpha-1,2 transglucosylation of glucosyl moieties onto alpha-1,6 dextran chains. Steady-state kinetic studies showed that hydrolysis and transglucosylation reactions occurred at the early stage of the reaction in the presence of 70 kDa dextran as acceptor and sucrose. The transglucosylation reaction catalysed by GBD-CD2 follows a Ping Pong Bi Bi mechanism with a high kcat value of 970 s(-1). The amount of the synthesised alpha-1,2 side chains was found to be directly dependent on the initial molar ratio [Sucrose]/[Dextran]. Dextrans with controlled alpha-1,2 linkage contents ranging from 13% to 40% were synthesised. The procedure resulted in the production of dextrans with the highest content of alpha-1,2 linkages ever reported.Applied Microbiology and Biotechnology 10/2009; 86(2):545-54. · 3.42 Impact Factor -
Article: Glucansucrases of GH family 70: What are the determinants of their specifities?
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ABSTRACT: Glucansucrases from family 70 of glycoside-hydrolases catalyse the synthesis of α-glucans with various types of osidic linkages from sucrose. Among these enzymes, alternansucrase (ASR) and dextransucrase E (DSR-E) catalyse the formation of unusual α-glucans. ASR catalyses the synthesis of linear glucan with α-1,3 and α-1,6 alternating linkages and DSR-E synthesizes a glucan containing α-1,6 linkages in the linear chain and α-1,2 branches. The sequence analysis of these enzymes enabled the identification of structural elements suspected to be involved in the enzyme specificities. Biochemical characterization of ASR and DSR-E variants obtained from gene truncations or site-directed mutagenesis experiments showed that the specificity of these enzymes to form different types of osidic linkage is controlled by two different approaches. For ASR, the double specificity is controlled by only one catalytic domain where important amino acids involved in the enzyme specificity have been identified. In the case of DSR-E, the double specificity is controlled by two different catalytic domains both belonging to family 70, each domain being specific of one type of linkage.07/2009; 24(1-2):137-145. -
Article: Design of alpha-transglucosidases of controlled specificity for programmed chemoenzymatic synthesis of antigenic oligosaccharides.
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ABSTRACT: Combined with chemical synthesis, the use of biocatalysts holds great potential to open the way to novel molecular diversity. We report in vitro chemoenzymatic pathways that, for the first time, take advantage of enzyme engineering to produce complex microbial cell-surface oligosaccharides and circumvent the chemical boundaries of glycochemistry. Glycoenzymes were designed to act on nonnatural conveniently protected substrates to produce intermediates compatible with a programmed chemical elongation. The study was focused on the synthesis of oligosaccharides mimicking the O-antigen motif of Shigella flexneri serotypes 1b and 3a, which could be used for the development of multivalent carbohydrate-based vaccines. A semirational engineering approach was successfully applied to amylosucrase, a transglucosidase that uses a low cost sucrose substrate as a glucosyl donor. The main difficulty was to retain the enzyme specificity toward sucrose, while creating a new catalytic function to render the enzyme able to regiospecifically glucosylate protected nonnatural acceptors. A structurally guided library of 133 mutants was generated from which several mutants with either completely new specificity toward methyl alpha-l-rhamnopyranoside or a tremendously enhanced one toward allyl 2-acetamido-2-deoxy-alpha-d-glucopyranoside acceptors were isolated. The best variants were used to synthesize glucosylated building blocks. They were then converted into acceptors and potential donors compatible with chemical elongation toward oligosaccharide fragments of the O-antigens of the two targeted serotypes. This is the first report of a successful engineering of an alpha-transglycosidase acceptor binding site that led to new specificities. It demonstrates the potential of appropriate combinations of a planned chemoenzymatic pathway and enzyme engineering in glycochemistry.Journal of the American Chemical Society 06/2009; 131(21):7379-89. · 9.91 Impact Factor -
Article: Search for a dextransucrase minimal motif involved in dextran binding.
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ABSTRACT: Fourteen truncated forms of Leuconostoc mesenteroides NRRL B512-F dextransucrase, involving N-, C- or N- plus C-terminal domain truncations were tested for their ability to bind dextrans. The shortest fragment (14kDa molecular weight) that still exhibited a strong interaction with dextran was localized between amino acids N1397 and A1527 of the C-terminal domain (GBD-7) and consists of six YG repeats. With a dissociation constant K(d) of 2.8x10(-9)M, this motif shows a very high affinity for isomaltohexaose and longer dextrans, supporting the proposed role of GBD in polymer formation. The potential application of GBD-7 as an affinity tag onto cheap resins like Sephacryl S300HR for rapid purification was evaluated and is discussed.FEBS Letters 11/2007; 581(24):4675-80. · 3.54 Impact Factor -
Article: Understanding the polymerization mechanism of glycoside-hydrolase family 70 glucansucrases.
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ABSTRACT: Glucan formation catalyzed by two GH-family 70 enzymes, Leuconostoc mesenteroides NRRL B-512F dextransucrase and L. mesenteroides NRRL B-1355 alternansucrase, was investigated by combining biochemical and kinetic characterization of the recombinant enzymes and their respective products. Using HPAEC analysis, we showed that two molecules act as initiator of polymerization: sucrose itself and glucose produced by hydrolysis, the latter being preferred when produced in sufficient amounts. Then, elongation occurs by transfer of the glucosyl residue coming from sucrose to the non-reducing end of initially formed products. Dextransucrase preferentially produces an isomaltooligosaccharide series, whose concentration is always low because of the high ability of these products to be elongated and form high molecular weight dextran. Compared with dextransucrase, alternansucrase has a broader specificity. It produces a myriad of oligosaccharides with various alpha-1,3 and/or alpha-1,6 links in early reaction stages. Only some of them are further elongated. Overall alternan polymer is smaller in size than dextran. In dextransucrase, the A repeats often found in C-terminal domain of GH family 70 were found to play a major role in efficient dextran elongation. Their truncation result in an enzyme much less efficient to catalyze high molecular weight polymer formation. It is thus proposed that, in dextransucrase, the A repeats define anchoring zones for the growing chains, favoring their elongation. Based on these results, a semi-processive mechanism involving only one active site and an elongation by the non-reducing end is proposed for the GH-family 70 glucansucrases.Journal of Biological Chemistry 11/2006; 281(42):31254-67. · 4.77 Impact Factor -
Article: High-level production and purification of a fully active recombinant dextransucrase from Leuconostoc mesenteroides NRRL B-512F.
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ABSTRACT: Recombinant expression of the dextransucrase dsrS gene by Escherichia coli was optimized to produce 5850 U L(-1) (culture) of DSR-S, corresponding to a 30-fold increase compared with previous studies. Rational deletions of the signal peptide, the beginning of the variable region and the last four repeats of the C-terminal end caused no loss of activity. This new variant successfully purified was remarkably stable. With a k(cat) of 584 s(-1), it is the most efficient recombinant glucansucrase described to date. The synthesized polymer possesses more than 95% of alpha-1,6 links, like the dextran produced by the native enzyme, and innovative gel properties were obtained.FEMS Microbiology Letters 09/2006; 261(2):203-10. · 2.04 Impact Factor -
Article: High‐level production and purification of a fully active recombinant dextransucrase from Leuconostoc mesenteroides NRRL B‐512F
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ABSTRACT: Recombinant expression of the dextransucrase dsrS gene by Escherichia coli was optimized to produce 5850 U L−1culture of DSR-S, corresponding to a 30-fold increase compared with previous studies. Rational deletions of the signal peptide, the beginning of the variable region and the last four repeats of the C-terminal end caused no loss of activity. This new variant successfully purified was remarkably stable. With a kcat of 584 s−1, it is the most efficient recombinant glucansucrase described to date. The synthesized polymer possesses more than 95% of -1,6 links, like the dextran produced by the native enzyme, and innovative gel properties were obtained.FEMS Microbiology Letters 07/2006; 261(2):203 - 210. · 2.04 Impact Factor
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Institutions
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2012
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Université Paul Sabatier - Toulouse 3
- Laboratoire de Biotechnologies Agroalimentaires et Environnementales - EA 4565 - LBAE
Toulouse, Midi-Pyrenees, France
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