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In this work, we reported production of glucansucrases with molecular weights of about 300 kDa by strains of fructophilic lactic acid bacteria (FLAB) Lactobacillus pentosus AG8, Lactobacillus kunkeei AG9, AG10 and AG11 isolated from the gut of honey bee Apis mellifera. From sucrose, these enzymes synthesize average to high-molecular-weight dextran-type glucans ranging from 8.5 × 105 to >4 × 106 Da and containing 16 to 22% α-(1→3)-linked glucose units, with 5 to 8% branching in their structures. Enzyme preparations from the strains were applied in the synthesis of glucooligosaccharides (GOSs) with degrees of polymerization (DP) from 3 to 7 using maltose/sucrose ratios (M/S) from 8 to 0.12. The yields and composition of the obtained oligosaccharide products were strongly influenced by the ratios of the acceptor and donor of glucose units in the reactions. The highest yields of GOSs (>50 g/L) were achieved at M/S = 1, and the lowest content of α-(1→6) linkages (71%) combined with increased proportion of the products with DP > 3 were determined at M/S = 0.5. GOSs preparations from these two ratios were able to maintain the growth and acids production of three probiotic strains of lactobacilli, suggesting potential application as prebiotics.

In the present work, we report synthesis of glucooligosaccharides (GOSs) by glucansucrase enzymes from fructophilic lactic acid bacteria Lactobacillus pentosus AG8, Lb. kunkeei AG9, AG10 and AG11 isolated from the gut of honey bees Apis mellifera. As an acceptor of glucose units from sucrose was used maltose and maltose to sucrose ratios (M/S) 1, 2, 4, 6, 8, 0.12, 0.16, 0.25 and 0.5. GOSs with degrees of polymerization (DP) ranging from 3 to 7 were obtained. The yields of GOSs and their composition according to DP were strongly influenced by the concentrations of the acceptor and sucrose. The highest yields of GOSs were obtained at М/S=1 – 45 to 53 g/L with predomination of DP3 fraction of oligosaccharides – 47 to 53%. The further increase of M/S ratios led to gradual decrease of the amounts of GOSs and also of their diversity according to DP. At М/S=8 were achieved yields of 13 to 15 g/L, composed only by DP=3 and DP=4 GOSs. In the opposite, the increase of sucrose concentrations at the expense of maltose ones led to more even distribution of the synthesized GOSs according to their DP up to 7, and yields of 40 to 42 g/L at M/S=0.5. Thus by varying the acceptor/donor ratio we were able to produce GOSs preparations with more controlled composition. In addition, GOSs preparations synthesized with glucansucrases from the four strains were subjected to dextranase hydrolysis in order to determine the content of α-(1→6) linkages. The results showed content of α-(1→6) linkages that decreases towards the lower maltose concentrations and is between 71 and 96%. Knowing that the enzymes from the four strains introduce 16 to 22% α-(1→3) linkages in the synthesized glucan polysaccharides such type of linkages could be expected also in the oligosaccharide products and this is promising for their prebiotic potential., This work was supported by Department for Scientific Research – PU, Bulgaria, grant МУ19-БФ-019/2019, and by operational program "Science and education for smart growth" 2014–2020, grant number BG05M2OP001-1.002-0005-C01, Personalized Innovative Medicine Competence Centre (PERIMED).

International audience; The factors that define the resulting polymer length of distributive polymerases are poorly understood. Here, starting from the crystal structure of the dextransucrase DSR-M in complex with an isomaltotetraose, we define different anchoring points for the incoming acceptor. Mutation of one of these, Trp624, decreases the catalytic rate of the enzyme but equally skews the size distribution of the resulting dextran chains toward shorter chains. Nuclear magnetic resonance analysis shows that this mutation influences both the dynamics of the active site and the water accessibility. Monte Carlo simulation of the elongation process allows interpretation of these results in terms of enhanced futile encounters, whereby the less effective binding increases the pool of effective seeds for the dextran chains and thereby directly determines the length distribution of the final polymers.

Lactic acid bacteria use glucansucrase enzymes for synthesis of gluco-oligosaccharides and polysaccharides (α-glucans) from sucrose. Depending on the glucansucrase enzyme, specific α-glucosidic linkages are introduced. GTFA-ΔN (N-terminally truncated glucosyltransferase A) is a glucansucrase enzyme of Lactobacillus reuteri 121 that synthesizes the reuteran polysaccharide with (α1 → 4) and (α1 → 6) glycosidic linkages. Glucansucrases also catalyze glucosylation of various alternative acceptor substrates. At present it is unclear whether the linkage specificity of these enzymes is the same in oligo/polysaccharide synthesis and in glucosylation of alternative acceptor substrates. Our results show that GTFA-ΔN glucosylates catechol into products with up to at least 5 glucosyl units attached. These catechol glucosides were isolated and structurally characterized using 1D/2D (1)H NMR spectroscopy. They contained 1 to 5 glucose units with different (α1 → 4) and (α1 → 6) glycosidic linkage combinations. Interestingly, a branched catechol glucoside was also formed along with a catechol glucoside with 2 successive (α1 → 6) glycosidic linkages, products that are absent when only sucrose is used as both glycosyl donor and acceptor substrate.

Glucansucrases são enzimas que atuam em reações de síntese de polissacarídeos e oligossacarídeos. Para que esses biocatalisadores sejam aplicados em escala industrial, é desejável ótimas estabilidades térmica e operacional, o que pode ser alcançado com a imobilização de enzimas. Como alternativa aos suportes sólidos amplamente estudados, está a quitosana, polímero que não apresenta toxicidade e possui alta biocompatibilidade e alta afinidade com proteínas. Outra possibilidade promissora na imobilização de enzimas, é a síntese dos agregados enzimáticos entrecruzados (CLEAs), os quais apresentam alta atividade catalítica e alta estabilidade. Contudo, uma peculiaridade das glucansucrases quando produzidas em meio contendo sacarose é a camada de polímero que as envolve, e que bloqueia o acesso aos grupos reativos na superfície da proteína. No caso da expressão heteróloga das glucansucrases em Escherichia coli essa dificuldade pode ser contornada. Além disso, o uso da mutagênese sítio-dirigida pode proporcionar modificações de aminoácidos na superfície da enzima, tais como os resíduos Lys, Cys, His, com o intuito de que melhorias na imobilização sejam alcançadas. Sendo assim, na primeira etapa desse trabalho, uma extensa discussão é apresentada em relação às metodologias de imobilização de dextransucrase encontradas na literatura. A seguir, estudos referentes à imobilização da dextransucrase de Leuconostoc mesenteroides B-512 F em esferas de quitosana ativadas com glutaraldeído foram realizados. Esse imobilizado apresentou alta atividade catalítica (197 U/g) quando utilizada a carga de proteína de 400 mg/g de suporte. Além disso, observou-se que a imobilização covalente e os açúcares maltose e glicose promoveram proteção à enzima em temperaturas de 40 ºC e 50 ºC. Na etapa seguinte, a produção e a caracterização de CLEAs de dextransucrase de L. mesenteroides B-512 F foram investigados. Demonstrou-se que o tratamento com a dextranase foi essencial para a imobilização da glucansucrase e que o isopropanol foi o melhor agente precipitante. Os CLEAs apresentaram pH e temperatura ótimos de 3,0 e 60 ºC, respectivamente, enquanto que a dextransucrase imobilizada nas esferas de quitosana funcionalizada com glutaraldeído apresentaram os valores de 4,5 e 20 ºC. Ambas formas imobilizadas apresentaram boa estabilidade operacional na síntese de oligossacarídeos uma vez que após 10 ciclos, 40 % de atividade residual foi observada. Por fim, estão apresentados estudos sobre a modelagem das estruturas tridimensionais e a mutagênese sítio-dirigida das glucansucrases DSR-S vardel Δ4N and ASR C-APY del. Os modelos preditos demonstraram boa qualidade e a mutagênese sítio-dirigida não promoveu perdas significativas na atividade enzimática dos mutantes. Somente o mutante DSR_S326C mostrouse inativo. Os resultados obtidos sugerem que a imobilização da dextransucrase foi satisfatória e que cada técnica possibilita diferentes características ao imobilizado. Além disso, os imobilizados foram adequados para síntese de dextrana e oligossacarídeos. Glucansucrases are enzymes that catalyze the synthesis of polysaccharides and oligosaccharides. In order to assure continuous processing and reuse of the biocatalyst in industrial applications, enzyme immobilization techniques are required to promote good thermal and operational stabilities. Among the several solid supports for enzyme immobilization, chitosan shows interesting properties because it is non-toxic, it is biocompatible, and it has high protein affinity. Other possibility is the production of cross-linked enzyme aggregates (CLEAs), which presents high catalytic activity and good stability. However, glucansucrases have a particularity when produced in sucrose medium, since a polymer layer surrounds the protein, blocking the access to reactive groups on the enzyme surface. To overcome this problem, it is possible to make the heterologous production of glucansucrases in Escherichia coli. Likewise, the site-directed mutagenesis may promote changes in the amino acids located on the surface to improve immobilization parameters. Therefore, this work aimed to discuss the several techniques applied for dextransucrase immobilization, and to design new immobilized biocatalysts. In a first step, it is presented a review about the distinct immobilization methodologies for dextransucrase. In a second study, an investigation about dextransucrase from Leuconostoc mesenteroides B-512 F immobilized on glutaraldehyde-activated chitosan particles was carried out. The novel immobilized biocatalyst showed 197 U/g (400 mg/g dried support) of catalytic activity. The covalent immobilization promoted protection against enzyme damages at 40 ºC and 50 ºC, whereas maltose and glucose acted as stabilizers. Furthermore, it was studied the production and characterization of CLEAs dextransucrase from L. mesenteroides B-512 F. It was demonstrated that dextranase treatment was crucial for immobilization. Isopropanol was chosen as the best precipitant agent. CLEAs presented optimal pH and temperature of 3.0 and 60 ºC, respectively, whereas it was found values of 4.5 e 20 ºC for dextransucrase immobilized on glutaraldehyde-activated chitosan particles. Both immobilized biocatalysts showed good operational stability in the oligosaccharides synthesis, exhibiting 40 % of residual activity after 10 cycles. Finally, the study concerning the homology modeling and site-directed mutagenesis of glucansucrases DSR-S vardel Δ4N and ASR C-APY del is presented. The predicted models showed good quality and it has been demonstrated that the site-directed mutagenesis did not promote significant losses in the variant enzyme activities. Only one mutant (DSR_S326C) had shown no dextransucrase activity. The results obtained in this work suggest that the immobilization of dextransucrase was satisfactory, also showing that each technique promotes different characteristics to the immobilized biocatalyst. Besides, these immobilized enzymes were feasible for the synthesis of dextran and oligosaccharides.

International audience; Certain enzymes of the GH70 family dextransucrases synthesize very high molar mass dextran polymers, whereas others produce a mixed population of very high and low molar mass products directly from sucrose substrate. Identifying the determinants dictating polymer elongation would allow the tight control of dextran size. To explore this central question, we focus on the recently discovered DSR-M enzyme from Leuconostoc citreum NRRL B-1299, which is the sole enzyme that naturally, exclusively, and very efficiently produces only low molar mass dextrans from sucrose. Extensive biochemical and structural characterization of a truncated form of DSR-M (DSR-MΔ2, displaying the same biochemical behavior as the parental enzyme) and X-ray structural analysis of complexes with sucrose and isomaltotetraose molecules together with accurate monitoring of the resulting polymer formation reveal that DSR-MΔ2 adopts a nonprocessive mechanism attributed to (i) a high propensity to recognize sucrose as a preferred acceptor at the initial stage of catalysis, (ii) an ability to elongate oligodextrans irrespective of their size, and (iii) the presence of a domain V showing a weak ability to bind to the growing dextran chains. In this study, we present the 3D structure with the largest defined domain V reported to date in the GH70 family and map sugar binding pockets on the basis of the structure of the complex obtained with isomaltotetraose. Altogether, these findings give insights into the interplay between the domain V and the catalytic site during polymerization. They open promising strategies for GH70 enzyme engineering aiming at modulating glucan size.

International audience; 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 alpha-1,6 and alpha-1,3 with alpha-1,4 branches. These results enhance the understanding of this industrially significant strain and will aid in distinguishing between physiologically similar Leuconostoc spp. strains.

Lactobacillus reuteri strain ATCC 55730 (LB BIO) was isolated as a pure culture from a Reuteri tablet purchased from the BioGaia company. This probiotic strain produces a soluble glucan (reuteran), in which the majority of the linkages are of the α-(1→4) glucosidic type (∼70%). This reuteran also contains α-(1→6)- linked glucosyl units and 4,6-disubstituted α-glucosyl units at the branching points. The LB BIO glucansucrase gene ( gtfO ) was cloned and expressed in Escherichia coli , and the GTFO enzyme was purified. The recombinant GTFO enzyme and the LB BIO culture supernatants synthesized identical glucan polymers with respect to linkage type and size distribution. GTFO thus is a reuteransucrase, responsible for synthesis of this reuteran polymer in LB BIO. The preference of GTFO for synthesizing α-(1→4) linkages is also evident from the oligosaccharides produced from sucrose with different acceptor substrates, e.g., isopanose from isomaltose. GTFO has a relatively high hydrolysis/transferase activity ratio. Complete conversion of 100 mM sucrose by GTFO nevertheless yielded large amounts of reuteran, although more than 50% of sucrose was converted into glucose. This is only the second example of the isolation and characterization of a reuteransucrase and its reuteran product, both found in different L. reuteri strains. GTFO synthesizes a reuteran with the highest amount of α-(1→4) linkages reported to date.

International audience; The amplicon encoding dextransucrase DSR-F from Leuconostoc citreum B/110-1-2, a novel sucrose glucosyltransferase (GTF)-specific for alpha-1,6 and alpha-1,3 glucosidic bond synthesis, with alpha-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 (similar to 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-Delta SP-Delta GBD of 1,251 amino acids, with a molecular mass of 145 544 Da (similar to 145 kDa), was obtained.