Microbial ß-glucanases: production, properties, and engineering.

Lignocellulosic biomass, which mainly consists of cellulose and hemicellulose, is the most abundant renewable biopolymer on earth. ß-Glucanases are glycoside hydrolases (GHs) that hydrolyze ß-glucan, one of the dominant components of the plant cell wall, into cello-oligosaccharides and glucose. Among them, endo-ß-1,4-glucanase (EC 3.2.1.4), exo-glucanase/cellobiohydrolase (EC 3.2.1.91), and ß-glucosidase (EC 3.2.1.21) play critical roles in the digestion of glucan-like substrates. ß-Glucanases have attracted considerable interest within the scientific community due to their applications in the feed, food, and textile industries. In the past decade, there has been considerable progress in the discovery, production, and characterization of novel ß-glucanases. Advances in the development of next-generation sequencing techniques, including metagenomics and metatranscriptomics, have unveiled novel ß-glucanases isolated from the gastrointestinal microbiota. The study of ß-glucanases is beneficial for research and development of commercial products. In this study, we review the classification, properties, and engineering of ß-glucanases.

BsLPMO10A from Bacillus subtilis boosts the depolymerization of diverse polysaccharides linked via ß-1,4-glycosidic bonds.

Lytic polysaccharide monooxygenase (LPMO) is known as an oxidatively cleaving enzyme in recalcitrant polysaccharide deconstruction. Herein, we report a novel AA10 LPMO derived from Bacillus subtilis (BsLPMO10A). A substrate specificity study revealed that the enzyme exhibited an extensive active-substrate spectrum, particularly for polysaccharides linked via ß-1,4 glycosidic bonds, such as ß-(Man1 â†’ 4Man), ß-(Glc1 â†’ 4Glc) and ß-(Xyl1 â†’ 4Xyl). HPAEC-PAD and MALDI-TOF-MS analyses indicated that BsLPMO10A dominantly liberated native oligosaccharides with a degree of polymerization (DP) of 3-6 and C1-oxidized oligosaccharides ranging from DP3ox to DP6ox from mixed linkage glucans and beechwood xylan. Due to its synergistic action with a variety of glycoside hydrolases, including glucanase IDSGLUC5-38, xylanase TfXYN11-1, cellulase IDSGLUC5-11 and chitinase BtCHI18-1, BsLPMO10A dramatically accelerated glucan, xylan, cellulose and chitin saccharification. After co-reaction for 72 h, the reducing sugars in Icelandic moss lichenan, beechwood xylan, phosphoric acid swollen cellulose and chitin yielded 3176 ± 97, 7436 ± 165, 649 ± 44, and 2604 ± 130 µmol/L, which were 1.47-, 1.56-, 1.44- and 1.25-fold higher than those in the GHs alone groups, respectively (P < 0.001). In addition, the synergy of BsLPMO10A and GHs was further validated by the degradation of natural feedstuffs, the co-operation of BsLPMO10A and GHs released 3266 ± 182 and 1725 ± 107 µmol/L of reducing sugars from Oryza sativa L. and Arachis hypogaea L. straws, respectively, which were significantly higher than those produced by GHs alone (P < 0.001). Furthermore, BsLPMO10A also accelerated the liberation of reducing sugars from Celluclast® 1.5 L, a commercial cellulase cocktail, on filter paper, A. hypogaea L. and O. sativa L. straws by 49.58 % (P < 0.05), 72.19 % (P < 0.001) and 54.36 % (P < 0.05), respectively. This work has characterized BsLPMO10A with a broad active-substrate scope, providing a promising candidate for lignocellulosic biomass biorefinery.

A novel bifunctional glucanase exhibiting high production of glucose and cellobiose from rumen bacterium.

Herbivores gastrointestinal microbiota is of tremendous interest for mining novel lignocellulosic enzymes for bioprocessing. We previously reported a set of potential carbohydrate-active enzymes from the metatranscriptome of the Hu sheep rumen microbiome. In this study, we isolated and heterologously expressed two novel glucanase genes, Cel5A-h38 and Cel5A-h49, finding that both recombinant enzymes showed the optimum temperatures of 50 °C. Substrate-specificity determination revealed that Cel5A-h38 was exclusively active in the presence of mixed-linked glucans, such as barley ß-glucan and Icelandic moss lichenan, whereas Cel5A-h49 (EC 3.2.1.4) exhibited a wider substrate spectrum. Surprisingly, Cel5A-h38 initially released only cellotriose from lichenan and further converted it into an equivalent amount of glucose and cellobiose, suggesting a dual-function as both endo-ß-1,3-1,4-glucanase (EC 3.2.1.73) and exo-cellobiohydrolase (EC 3.2.1.91). Additionally, we performed enzymatic hydrolysis of sheepgrass (Leymus chinensis) and rice (Orysa sativa) straw using Cel5A-h38, revealing liberation of 1.91 ± 0.30 mmol/mL and 2.03 ± 0.09 mmol/mL reducing sugars, respectively, including high concentrations of glucose and cellobiose. These results provided new insights into glucanase activity and lay a foundation for bioconversion of lignocellulosic biomass.

Association of viral replication capacity with the pathogenicity of enterovirus 71.

Enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease, which is associated with fatal neurological disease. The mechanism of EV71 pathogenesis remains obscure. We compared the replication capacity of the severe and mild enterovirus 71 isolates. The replication kinetics of EV71 in RD cells and ICR mice was determined by qRT-PCR. The lung, muscular, brain, intestine tissues were used for histopathological and immunohistochemical assays. The growth curves of EV71 strains in RD cells showed that the severe EV71 strains (SDLY107 and SDLY52) replicated faster and generated more viral RNA than the mild EV71 strains (SDLY11 and SDLY1). The mice infected by the severe EV71 strains (SDLY107) showed more severe clinical symptoms, pathological changes and higher viral load than the mice infected by the mild EV71 strains (SDLY11). These results suggest that there was a difference in replication capacity between the severe and mild EV71 strains, which was possibly associated with EV71 pathogenesis.