Molecular mechanism for endo-type action of glycoside hydrolase family 55 endo-ß-1,3-glucanase on ß1-3/1-6-glucan.

The glycoside hydrolase family 55 (GH55) includes inverting exo-ß-1,3-glucosidases and endo-ß-1,3-glucanases, acting on laminarin, which is a ß1-3/1-6-glucan consisting of a ß1-3/1-6-linked main chain and ß1-6-linked branches. Despite their different modes of action toward laminarin, endo-ß-1,3-glucanases share with exo-ß-1,3-glucosidases conserved residues that form the dead-end structure of subsite -1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-ß-1,3-glucanase MnLam55A, identified from Microdochium nivale. MnLam55A, like other endo-ß-1,3-glucanases, degraded internal ß-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. ß1-3-Glucans lacking ß1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal ß1-3-linkage of the laminarioligosaccharide moiety at the reducing end side of the main chain ß1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but kcat/Km values to laminarioligosaccharides (≤4.21 s-1 mM-1) were much lower than to laminarin (5920 s-1 mM-1). These results indicate that ß-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites -1 and -2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-ß-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-ß-1,3-glucanases.

Function and Structure of Lacticaseibacillus casei GH35 ß-Galactosidase LBCZ_0230 with High Hydrolytic Activity to Lacto-N-biose I and Galacto-N-biose.

ß-Galactosidase (EC 3.2.1.23) hydrolyzes ß-D-galactosidic linkages at the non-reducing end of substrates to produce ß-D-galactose. Lacticaseibacillus casei is one of the most widely utilized probiotic species of lactobacilli. It possesses a putative ß-galactosidase belonging to glycoside hydrolase family 35 (GH35). This enzyme is encoded by the gene included in the gene cluster for utilization of lacto-N-biose I (LNB; Galß1-3GlcNAc) and galacto-N-biose (GNB; Galß1-3GalNAc) via the phosphoenolpyruvate: sugar phosphotransferase system. The GH35 protein (GnbG) from L. casei BL23 is predicted to be 6-phospho-ß-galactosidase (EC 3.2.1.85). However, its 6-phospho-ß-galactosidase activity has not yet been examined, whereas its hydrolytic activity against LNB and GNB has been demonstrated. In this study, L. casei JCM1134 LBCZ_0230, homologous to GnbG, was characterized enzymatically and structurally. A recombinant LBCZ_0230, produced in Escherichia coli, exhibited high hydrolytic activity toward o-nitrophenyl ß-D-galactopyranoside, p-nitrophenyl ß-D-galactopyranoside, LNB, and GNB, but not toward o-nitrophenyl 6-phospho-ß-D-galactopyranoside. Crystal structure analysis indicates that the structure of subsite -1 of LBCZ_0230 is very similar to that of Streptococcus pneumoniae ß-galactosidase BgaC and not suitable for binding to 6-phospho-ß-D-galactopyranoside. These biochemical and structural analyses indicate that LBCZ_0230 is a ß-galactosidase. According to the prediction of LNB's binding mode, aromatic residues, Trp190, Trp240, Trp243, Phe244, and Tyr458, form hydrophobic interactions with N-acetyl-D-glucosamine residue of LNB at subsite +1.

Chemical synthesis of oligosaccharide derivatives with partial structure of ß1-3/1-6 glucan, using monomeric units for the formation of ß1-3 and ß1-6 glucosidic linkages.

ß1-3/1-6 Glucans, known for their diverse structures, comprise a ß1-3-linked main chain and ß1-6-linked short branches. Laminarin, a ß1-3/1-6 glucan extracted from brown seaweed, for instance, includes ß1-6 linkages even in the main chain. The diverse structures provide various beneficial functions for the glucan. To investigate the relationship between structure and functionality, and to enable the characterization of ß1-3/1-6 glucan-metabolizing enzymes, oligosaccharides containing the exact structures of ß1-3/1-6 glucans are required. We synthesized the monomeric units for the synthesis of ß1-3/1-6 mixed-linked glucooligosaccharides. 2-(Trimethylsilyl)ethyl 2-O-benzoyl-4,6-O-benzylidene-ß-d-glucopyranoside served as an acceptor in the formation of ß1-3 linkages. Phenyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(tert-butyldiphenylsilyl)-1-thio-ß-d-glucopyranoside and phenyl 2,3-di-O-benzoyl-4,6-di-O-levulinyl-1-thio-ß-d-glucopyranoside acted as donors, synthesizing acceptors suitable for the formation of ß1-3- and ß1-6-linkages, respectively. These were used to synthesize a derivative of Glcß1-6Glcß1-3Glcß1-3Glc, demonstrating that the proposed route can be applied to synthesize the main chain of ß-glucan, with the inclusion of both ß1-3 and ß1-6 linkages.

Alteration of Substrate Specificity and Transglucosylation Activity of GH13_31 α-Glucosidase from Bacillus sp. AHU2216 through Site-Directed Mutagenesis of Asn258 on ß→α Loop 5.

α-Glucosidase catalyzes the hydrolysis of α-d-glucosides and transglucosylation. Bacillus sp. AHU2216 α-glucosidase (BspAG13_31A), belonging to the glycoside hydrolase family 13 subfamily 31, specifically cleaves α-(1→4)-glucosidic linkages and shows high disaccharide specificity. We showed previously that the maltose moiety of maltotriose (G3) and maltotetraose (G4), covering subsites +1 and +2 of BspAG13_31A, adopts a less stable conformation than the global minimum energy conformation. This unstable d-glucosyl conformation likely arises from steric hindrance by Asn258 on ß→α loop 5 of the catalytic (ß/α)8-barrel. In this study, Asn258 mutants of BspAG13_31A were enzymatically and structurally analyzed. N258G/P mutations significantly enhanced trisaccharide specificity. The N258P mutation also enhanced the activity toward sucrose and produced erlose from sucrose through transglucosylation. N258G showed a higher specificity to transglucosylation with p-nitrophenyl α-d-glucopyranoside and maltose than the wild type. E256Q/N258G and E258Q/N258P structures in complex with G3 revealed that the maltose moiety of G3 bound at subsites +1 and +2 adopted a relaxed conformation, whereas a less stable conformation was taken in E256Q. This structural difference suggests that stabilizing the G3 conformation enhances trisaccharide specificity. The E256Q/N258G-G3 complex formed an additional hydrogen bond between Met229 and the d-glucose residue of G3 in subsite +2, and this interaction may enhance transglucosylation.

Identification and characterization of extracellular GH3 ß-glucosidase from the pink snow mold fungus, Microdochium nivale.

Glycoside hydrolase family 3 (GH3) ß-glucosidase exists in many filamentous fungi. In phytopathogenic fungi, it is involved in fungal growth and pathogenicity. Microdochium nivale is a severe phytopathogenic fungus of grasses and cereals and is the causal agent of pink snow mold, but its ß-glucosidase has not been identified. In this study, a GH3 ß-glucosidase of M. nivale (MnBG3A) was identified and characterized. Among various p-nitrophenyl ß-glycosides, MnBG3A showed activity on d-glucoside (pNP-Glc) and slight activity on d-xyloside. In the pNP-Glc hydrolysis, substrate inhibition occurred (Kis = 1.6 m m), and d-glucose caused competitive inhibition (Ki = 0.5 m m). MnBG3A acted on ß-glucobioses with ß1-3, -6, -4, and -2 linkages, in descending order of kcat/Km. In contrast, the regioselectivity for newly formed products was limited to ß1-6 linkage. MnBG3A has similar features to those of ß-glucosidases from Aspergillus spp., but higher sensitivity to inhibitory effects.

Extreme ultra-low lasing threshold of full-polymeric fundamental microdisk printed with room-temperature atmospheric ink-jet technique.

We experimentally demonstrated an extreme ultra-low lasing threshold from full-polymeric fundamental microdisk cavities fabricated by a novel fabrication method, the ink-jet printing method, which is much simpler and easier than previous methods such as lithography. The ink-jet printing method provides additive, room-temperature atmospheric, rapid fabrication with only two steps: (i) stacking cladding pedestal and waveguiding disk spots using the ink-jet technique, and (ii) partial etching of the cladding pedestal envelope. Two kinds of low-viscosity polymers successfully formed microdisks with high surface homogeneity, and one of the polymers doped with LDS798 dye yielded whispering-gallery-mode lasing. The fundamental disks exhibited an extremely ultra-low lasing threshold of 0.33 µJ/mm(2) at a wavelength of 817.3 nm. To the best of our knowledge, this lasing threshold is the lowest threshold obtained among both organic and inorganic fundamental microdisk cavity lasers with a highly confined structure.