Key roles of ß-glucosidase BglA for the catabolism of both laminaribiose and cellobiose in the lignocellulolytic bacterium Clostridium thermocellum.

The thermophilic bacterium Clostridium thermocellum efficiently degrades polysaccharides into oligosaccharides. The metabolism of ß-1,4-linked cello-oligosaccharides is initiated by three enzymes, i.e., the cellodextrin phosphorylase (Cdp), the cellobiose phosphorylase (Cbp), and the ß-glucosidase A (BglA), in C. thermocellum. In comparison, how the oligosaccharides containing other kinds of linkage are utilized is rarely understood. In this study, we found that BglA could hydrolyze the ß-1,3-disaccharide laminaribiose with much higher activity than that against the ß-1,4-disaccharide cellobiose. The structural basis of the substrate specificity was analyzed by crystal structure determination and molecular docking. Genetic deletions of BglA and Cbp, respectively, and enzymatic analysis of cell extracts demonstrated that BglA is the key enzyme responsible for laminaribiose metabolism. Furthermore, the deletion of BglA can suppress the expression of Cbp and the deletion of Cbp can up-regulate the expression of BglA, indicating that BglA and Cbp have cross-regulation and BglA is also critical for cellobiose metabolism. These insights pave the way for both a fundamental understanding of metabolism and regulation in C. thermocellum and emphasize the importance of the degradation and utilization of polysaccharides containing ß-1,3-linked glycosidic bonds in lignocellulose biorefinery.

Identification of the maturity of acacia honey by an endogenous oligosaccharide: A preliminary study.

Mature honeys that brew naturally in the hive develop distinct bioactive components, and thus carry a higher premium due to their superior quality. However, how to identify mature honeys remains difficult. Trace oligosaccharides are a likely source of biomarkers to indicate maturity. Here, we profiled trace oligosaccharides in acacia honey by GC-MS and used a metabolomics strategy to screen oligosaccharides that distinguish honeys with different maturities. Turanose content increased gradually in acacia honey samples and was closely related to the days stored in the hive (p < 0.05). To accurately quantify turanose, a UPLC-ELSD method was developed. Using the established method, honeys with ≥1.20 g/100 g of turanose could be classified as mature acacia honey. Based on the preliminary study, 500 commercial acacia honeys were analyzed, and only 77.2 % of these samples had a satisfactory level of turanose. This work offers a potential method to evaluate the quality of honeys.

Crystal structure and functional characterization of an oligosaccharide dehydrogenase from Pycnoporus cinnabarinus provides insights into fungal breakdown of lignocellulose.

BACKGROUND: Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the "Auxiliary Activity" family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoenzymes aryl-alcohol oxidase and glucose 1-oxidase. Based on sequence similarity to known fungal GDHs, an AA3_2 enzyme active on glucose was identified in the genome of Pycnoporus cinnabarinus, a model Basidiomycete able to completely degrade lignin. RESULTS: In our work, substrate screening and functional characterization showed an unexpected preferential activity of this enzyme toward oligosaccharides containing a ß(1→3) glycosidic bond, with the highest efficiency observed for the disaccharide laminaribiose. Despite its sequence similarity to GDHs, we defined a novel enzymatic activity, namely oligosaccharide dehydrogenase (ODH), for this enzyme. The crystallographic structures of ODH in the sugar-free form and in complex with glucose and laminaribiose unveiled a peculiar saccharide recognition mechanism which is not shared with previously characterized AA3 oxidoreductases and accounts for ODH preferential activity toward oligosaccharides. The sugar molecules in the active site of ODH are mainly stabilized through CH-π interactions with aromatic residues rather than through hydrogen bonds with highly conserved residues, as observed instead for the fungal glucose dehydrogenases and oxidases characterized to date. Finally, three sugar-binding sites were identified on ODH external surface, which were not previously observed and might be of importance in the physiological scenario. CONCLUSIONS: Structure-function analysis of ODH is consistent with its role as an auxiliary enzyme in lignocellulose degradation and unveils yet another enzymatic function within the AA3 family of the Carbohydrate-Active enZymes database. Our findings allow deciphering the molecular determinants of substrate binding and provide insight into the physiological role of ODH, opening new perspectives to exploit biodiversity for lignocellulose transformation into fuels and chemicals.

Structural insight into a GH1 ß-glucosidase from the oleaginous microalga, Nannochloropsis oceanica.

Marine microalgae are promising sources of novel glycoside hydrolases (GHs), which have great value in biotechnical and industrial applications. Although many GH1 family ß-glucosidases have been extensively studied, studies on ß-glucosidases from microalgae are rare, and no structure of algal GH1 ß-glucosidase has been reported. Here, we report the biochemical and structural study of a GH1 ß-glucosidase BGLN1 from Nannochloropsis oceanica, an oleaginous microalga. Phylogenetic analysis of BGLN1, together with the known structures of GH1 ß-glucosidases, has indicated that BGLN1 is branched at the root of the eukaryotic part of the phylogenetic tree. BGLN1 showed higher activity against laminaribiose compared to cello-oligosaccharides. Unlike most of the other GH1 ß-glucosidases, BGLN1 is partially inhibited by metal ions. The crystal structure of BGLN1 revealed that BGLN1 adopts a typical (α/ß)8-barrel fold with variations in loops and N-terminal regions. BGLN1 contains extra residues at the N-terminus, which are essential for maintaining protein stability. BGLN1 has a more acidic substrate-binding pocket than other ß-glucosidases, and the variations beyond the conserved -1 site determine the substrate specificity. These results indicate that GH enzymes from microalgae may have unique structural and functional features, which will provide new insight into carbohydrate synthesis and metabolism in marine microalgae.

Continuous enzymatic production and adsorption of laminaribiose in packed bed reactors.

Bienzymatic production of laminaribiose from sucrose and glucose was combined with adsorption on zeolite BEA to introduce a first capture and purification step. Downstream processing including washing and desorption steps was characterized and optimized on a milliliter scale in batch mode. Results were then transferred to a packed bed system for enzymatic production and adsorption where the influence of adsorbent particle diameter on purity and productivity was evaluated. Finally, a continuous enzymatic production of laminaribiose was conducted over 10 days. The subsequent downstream processing of the loaded zeolites led to purities of over 0.5 gLaminaribiose gsugar -1 in the desorbate with a total productivity of 5.6 mgLaminaribiose Lenzyme bed -1 h-1 without the use of recycles.

The Construction of an In Vitro Synthetic Enzymatic Biosystem that Facilitates Laminaribiose Biosynthesis from Maltodextrin and Glucose.

Laminaribiose is a reducing disaccharide linked by a ß-1,3 glycosidic bond; it is also a precursor for building blocks in the pharmaceutical industry, a powerful germinating agent and antiseptic, as well as a potential prebiotic. In this study, an in vitro enzymatic biosystem composed of α-glucan phosphorylase, laminaribiose phosphorylase, isoamylase, and 4-glucanotransferase is designed for the one-pot synthesis of laminaribiose from low-cost maltodextrin and glucose. Through condition optimization, 51 mM laminaribiose is produced from 10 g L-1 maltodextrin (55.5 mM glucose equivalent) and 90 mM glucose. The product yield based on maltodextrin is 91.9%. To investigate the industrial potential of this in vitro enzymatic biosystem, the production of laminaribiose from high concentrations of substrates is also examined, and 179 mM laminaribiose is produced from 50 g L-1 of maltodextrin and 450 mM glucose. This in vitro enzymatic biosystem comprised of thermophilic enzymes can drastically decrease the manufacturing cost of laminaribiose and provide a green method for the production of other disaccharides using phosphorylases.

Continuous Laminaribiose Production Using an Immobilized Bienzymatic System in a Packed Bed Reactor.

The first continuous production system of laminaribiose from sucrose and glucose in a bienzymatic reaction is reported in this study. Immobilized laminaribiose phosphorylase and sucrose phosphorylase were used in a packed bed reactor system comprising of a 3-cm glass column at 35 °C with a steady feeding flow rate of 0.1 ml/min. Factors affecting product formation including enzyme ratio, peal concept (both enzymes in one pearl or in separate pearls), and pearl size were studied. An enzyme ratio of 2:1 of laminaribiose phosphorylase (LP) to sucrose phosphorylase (SP) when encapsulated separately in bigger size peals resulted in higher concentration of product. Laminaribiose (0.4 g/(L h)) is produced in the optimized system at steady state. The reaction system proved to be operationally stable throughout 10 days of continuous processing. A half-life time of more than 9 days was observed for both biocatalysts.

A novel ß-glucosidase from Saccharophagus degradans 2-40T for the efficient hydrolysis of laminarin from brown macroalgae.

BACKGROUND: Laminarin is a potential biomass feedstock for the production of glucose, which is the most preferable fermentable sugar in many microorganisms by which it can be converted to biofuels and bio-based chemicals. Also, laminarin is a good resource as functional materials because it consists of ß-1,3-glucosidic linkages in its backbone and ß-1,6-glucosidic linkages in its branches so that its oligosaccharides driven from laminarin have a variety of biological activities. It is industrially important to be able to produce laminarioligosaccharides as well as glucose from laminarin by a single enzyme because the enzyme cost accounts for a large part of bio-based products. In this study, we investigated the industrial applicability of Bgl1B, a unique ß-glucosidase from Saccharophagus degradans 2-40T, belonging to the glycoside hydrolase family 1 (GH1) by characterizing its activity of hydrolyzing laminarin under various conditions. RESULTS: Bgl1B was cloned and overexpressed in Escherichia coli from S. degradans 2-40T, and its enzymatic activity was characterized. Similar to most of ß-glucosidases in GH1, Bgl1B was able to hydrolyze a variety of disaccharides having different ß-linkages, such as laminaribiose, cellobiose, gentiobiose, lactose, and agarobiose, by cleaving ß-1,3-, ß-1,4-, and ß-1,6-glycosidic linkages. However, Bgl1B showed the highest specific activity toward laminaribiose with a ß-1,3-glycosidic linkage. In addition, it was able to hydrolyze laminarin, one of the major polysaccharides in brown macroalgae, into glucose with a conversion yield of 75% of theoretical maximum. Bgl1B also showed transglycosylation activity by producing oligosaccharides from laminarin and laminaribiose under a high mass ratio of substrate to enzyme. Furthermore, Bgl1B was found to be psychrophilic, exhibiting relative activity of 59-85% in the low-temperature range of 2-20 °C. CONCLUSIONS: Bgl1B can directly hydrolyze laminarin into glucose with a high conversion yield without leaving any oligosaccharides. Bgl1B can exhibit high enzymatic activity in a broad range of low temperatures (2-20 °C), which is advantageous for establishing energy-efficient bioprocesses. In addition, under high substrate to enzyme ratios, Bgl1B can produce high-value laminarioligosaccharides via its transglycosylation activity. These results show that Bgl1B can be an industrially important enzyme for the production of biofuels and bio-based chemicals from brown macroalgae.

Chitosan-based hybrid immobilization in bienzymatic reactions and its application to the production of laminaribiose.

A hybrid-immobilization method was developed to improve the long-term stability of laminaribiose phosphorylase immobilized on epoxy supports Sepabeads EC-EP/S. Entrapment in chitosan retained all of the enzyme activity depending on the amount of entrapped solid materials and increased half-life by a factor of 10-94.4 h. No enzyme activity loss was determined during 12 times reuse. The immobilization method is also applicable to sucrose phosphorylase immobilized on Sepabeads EC-EP/S. Up to 31.9 g/L laminaribiose were produced in bienzymatic batch experiments with reaction-integrated product separation by adsorption on zeolites.

Immobilization and Characterization of E. gracilis Extract with Enriched Laminaribiose Phosphorylase Activity for Bienzymatic Production of Laminaribiose.

Immobilization methods and carriers were screened for immobilization of Euglena gracilis extract with laminaribiose phosphorylase activity. The extract was successfully immobilized on three different carriers via covalent linkage. Suitable immobilization carriers were Sepabeads EC-EP/S and ECR 8209M with epoxy groups and ECR 8309M with amino groups as functional units. Immobilization on Sepabeads EC-EP/S resulted in highest retained activity (65%). The immobilizates were characterized for pH, temperature, and buffer molarity preferences. The immobilized enzyme lost 48% of its activity when used seven times. Together with sucrose phosphorylase, laminaribiose phosphorylase was successfully applied for bienzymatic production of laminaribiose from sucrose and glucose with a final laminaribiose concentration of 14.3 ± 2.1 g/L (20% yield).

Facile preparation of highly crystalline lamellae of (1 → 3)-ß-D-glucan using an extract of Euglena gracilis.

In vitro synthesis of (1 → 3)-ß-D-glucan was performed using laminaribiose phosphorylase obtained by an extraction of Euglena gracilis with sucrose phosphorylase. The synthetic product was a linear (1 → 3)-ß-D-glucan with a narrow distribution of degree of polymerization (DP) centered on DP=30. X-ray diffraction and electron microscopy revealed that the glucan molecules obtained were self-organized as highly crystalline hexagonal lamellae. This synthetic product has quite high structural homogeneity at every level from primary to higher-order structure, which is a great advantage for the detailed analyses of physiological functions of (1 → 3)-ß-D-glucan.

A laminaribiose-hydrolyzing enzyme, AkLab, from the common sea hare Aplysia kurodai and its transglycosylation activity.

Endo-ß-1,3-glucanases (laminarinase, EC 3.2.1.6) from marine molluscs specifically degrades laminarin from brown algae producing laminaribiose and glucose, but hardly degrades laminaribiose. For the complete depolymerization of laminarin, other enzymes that can hydrolyze laminaribiose appear to be necessary. In the present study, we successfully isolated a laminaribiose-hydrolyzing enzyme from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by conventional column chromatographies. This enzyme, AkLab, named after the scientific name of this animal and substrate specificity toward laminaribiose, shows an approximate molecular mass of 110kDa on SDS-PAGE, and optimum pH and temperature at around pH5.5 and 50°C, respectively. AkLab rapidly hydrolyzes laminaribiose and p-nitrophenyl-ß-D-glucoside, and slowly cellobiose, gentiobiose and lactose, but not sucrose and maltose. AkLab shows high transglycosylation activity and can produce a series of laminarioligosaccharides larger than laminaritetraose from laminaribiose (a donor substrate) and laminaritriose (an acceptor substrate). This enzyme is suggested to be a member of glycosyl hydrolase family 1 by the analysis of partial amino-acid sequences.

Synthesis of O- and C-glycosides derived from ß-(1,3)-D-glucans.

A series of ß-(1,3)-d-glucans have been synthesized incorporating structural variations specifically on the reducing end of the oligomers. Both O- and C-glucosides derived from di- and trisaccharides have been obtained in good overall yields and with complete selectivity. Whereas the O-glycosides were obtained via a classical Koenigs-Knorr glycosylation, the corresponding C-glycosides were obtained through allylation of the anomeric carbon and further cross-metathesis reaction. Finally, the compounds were evaluated against two glycosidases and two endo-glucanases and no inhibitory activity was observed.

Modulation of acceptor specificity of Ruminococcus albus cellobiose phosphorylase through site-directed mutagenesis.

Cellobiose phosphorylase (EC 2.4.1.20, CBP) catalyzes the reversible phosphorolysis of cellobiose to α-D-glucose 1-phosphate (Glc1P) and d-glucose. Cys485, Tyr648, and Glu653 of CBP from Ruminococcus albus, situated at the +1 subsite, were mutated to modulate acceptor specificity. C485A, Y648F, and Y648V were active enough for analysis. Their acceptor specificities were compared with the wild type based on the apparent kinetic parameters determined in the presence of 10 mM Glc1P. C485A showed higher preference for D-glucosamine than the wild type. Apparent kcat/Km values of Y648F for D-mannose and 2-deoxy-D-glucose were 8.2- and 4.0-fold higher than those of the wild type, respectively. Y648V had synthetic activity toward N-acetyl-D-glucosamine, while the other variants did not. The oligosaccharide production in the presence of the same concentrations of wild type and each mutant was compared. C485A produced 4-O-ß-D-glucopyranosyl-D-glucosamine from 10 mM Glc1P and D-glucosamine at a rate similar to the wild type. Y648F and Y648V produced 4-O-ß-D-glucopyranosyl-D-mannose and 4-O-ß-D-glucopyranosyl-N-acetyl-D-glucosamine much more rapidly than the wild type when D-mannose and N-acetyl-D-glucosamine were used as acceptors, respectively. After a 4h reaction, the amounts of 4-O-ß-D-glucopyranosyl-D-mannose and 4-O-ß-D-glucopyranosyl-N-acetyl-D-glucosamine produced by Y648F and Y648V were 5.9- and 12-fold higher than the wild type, respectively.