Journal of applied glycoscience最新文献

Dextran α-1,2-debranching enzyme (DDE) releases glucose with hydrolyzing α-(1→2)-glucosidic linkages in α-glucans, which are made up of dextran with α-(1→2)-branches and are generated by Leuconostoc bacteria. DDE was isolated from Microbacterium dextranolyticum (formerly known as Flavobacterium sp. M-73) 40 years ago, although the amino acid sequence of the enzyme has not been determined. Herein, we found a gene for this enzyme based on the partial amino acid sequences from native DDE and characterized the recombinant enzyme. DDE had a signal peptide, a glycoside hydrolase family 65 domain, a carbohydrate-binding module family 35 domain, a domain (D-domain) similar to the C-terminal domain of Arthrobacter globiformis glucodextranase, and a transmembrane region at the C-terminus. Recombinant DDE released glucose from α-(1→2)-branched α-glucans produced by Leuconostoc citreum strains B-1299, S-32, and S-64 and showed weak hydrolytic activity with kojibiose and kojitriose. No activity was detected for commercial dextran and Leuconostoc citreum B-1355 α-glucan, which do not contain α-(1→2)-linkages. The removal of the D-domain decreased the affinity for α-(1→2)-branched α-glucans but not for kojioligosaccharides, suggesting that D-domain plays a role in α-glucan binding. Genes for putative dextranases, oligo-1,6-glucosidase, sugar-binding protein, and permease were present in the vicinity of the DDE gene, and as a result these gene products may be necessary for the use of α-(1→2)-branched glucans. Our findings shed new light on how actinobacteria utilize polysaccharides produced by lactic acid bacteria.

β-Galactosidase (EC 3.2.1.23) hydrolyzes β-D-galactosidic linkages at the non-reducing end of substrates to produce β-D-galactose. Lacticaseibacillus casei is one of the most widely utilized probiotic species of lactobacilli. It possesses a putative β-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; Galβ1-3GlcNAc) and galacto-N-biose (GNB; Galβ1-3GalNAc) via the phosphoenolpyruvate: sugar phosphotransferase system. The GH35 protein (GnbG) from L. casei BL23 is predicted to be 6-phospho-β-galactosidase (EC 3.2.1.85). However, its 6-phospho-β-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 β-D-galactopyranoside, p-nitrophenyl β-D-galactopyranoside, LNB, and GNB, but not toward o-nitrophenyl 6-phospho-β-D-galactopyranoside. Crystal structure analysis indicates that the structure of subsite -1 of LBCZ_0230 is very similar to that of Streptococcus pneumoniae β-galactosidase BgaC and not suitable for binding to 6-phospho-β-D-galactopyranoside. These biochemical and structural analyses indicate that LBCZ_0230 is a β-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.

The purpose of this study was to understand the effect of relative humidity (RH) on amylose-lipid complex (ALC) formation in amylose-lauric acid blend powder held at 50 °C (temperature slightly higher than the melting point of lauric acid) using differential scanning calorimetry (DSC) and X-ray diffraction. From DSC curves, the melting of crystalized lauric acid and two melting peaks of ALC were observed depending on RH. ALC formation was confirmed by X-ray diffraction pattern. The melting enthalpy (∆H_m) of lauric acid in the sample held at RH 0 % was lower than that of lauric acid only though there was no ALC formation. This suggests that crystallization of lauric acid was prevented by amylose. The ∆H_m of lauric acid increased with an increase in RH up to 79.0 % because liquid lauric acid would have fused as the result of enhanced repulsive force between liquid lauric acid and hydrated amylose. The ∆H_m of ALC increased with an increase in RH between 79.0 and 95.0 %. For ALC formation, amylose has to be mobile in the system, but dehydrated amylose is in a glassy (immobilize) state. According to the glass to rubber transition behavior of amorphous polymer, amylose held at 50 °C is suggested to become rubbery (mobile) state at RH 76.0 %. This interpretation will explain the reason why ALC formation began to be observed at the RH range between 72.4 and 79.0 %.

Starch is comprised of very large α-glucan molecules composed primarily of linear amylose and highly branched amylopectin. Most methods for analyses of starch structure use hydrolytic enzymes to cleave starch. When undegraded, whole starch structures can be analyzed by gel-permeation chromatography (GPC), but this typically yields a single peak each for amylopectin and amylose. The objective of this study was to stably separate amylopectins in whole starch based on their molecular weight using GPC, and to determine the structure of each peak. When alkali-gelatinized whole starch was applied to GPC columns (Toyopearl HW75S × 2, HW65S, and HW55S), it was separated into three peaks. Iodine staining and chain length distribution analyses of debranched samples showed that peaks were mainly composed of high-molecular weight (MW) amylopectin consisting of many clusters, low-MW amylopectin consisting of a small number of clusters, and amylose.

Algal sulfated polysaccharides are known to be effective hyaluronidase inhibitors. We evaluated hyaluronidase inhibitory activity of sulfated polysaccharide (SP) from Caulerpa lentillifera. Results showed that SP with IC₅₀ of 163 µg/mL appears to allosterically inhibit the hyaluronidase activity. Main sugar composition and sulfate content of SP was estimated to be Gal, Glc, Xyl, Man, uronic acids, and sulfate in the weight percent of 27.7: 28.9: 14.6: 22.5: 3.4: 21.7. We modified the SP by desulfation and partial hydrolysis with trifluoroacetic acid (TFA) to investigate the effect of sulfate content and molecular weight on inhibition. Hyaluronidase inhibitory activity of desulfated SP, 0.1 M TFA-hydrolyzed SP and 0.5 M TFA-hydrolyzed SP were significantly lower than that of native SP, revealing that sulfate content or molecular weight is important for hyaluronidase inhibition.

We created a Microsoft Excel file, Enzyme_Kinetics_Calculator, which includes macro programs that automatically calculates kinetic parameters for typical kinetic equations of enzymatic reactions, accompanied by their standard errors, by minimizing the residual sum of squares thereof. The [S]-v plot is automatically drawn with the theoretical lines and, similarly, the 1/[S]-1/v plot in the case of linear theoretical lines. Enzyme_Kinetics_Calculator is available as a supplementary file for this paper (see J. Appl. Glycosci. Web site).

Woody biomass is anticipated to be a resource for a decarbonized society, but the difficulty of isolating woody components is a significant challenge. Brown-rot fungi, a type of wood rotting fungi, decompose hemicellulose particularly efficiently. However, there are few reports on the hemicellulases from brown-rot fungi. An α-L-arabinofuranosidase belonging to glycoside hydrolase family 51 (GH51) from the brown-rot fungus Gloeophyllum trabeum (GtAbf51A) was cloned and characterized in the present study. Analyses of the phylogeny of GH51 enzymes in wood rotting fungi revealed the existence of two groups, intercellular and extracellular enzymes. After deglycosylation, the recombinant GtAbf51A produced by Pichia pastoris appeared on SDS-PAGE as approximately 71,777 daltons, which is the expected molecular weight based on the amino acid sequence of GtAbf51A. Maximum enzyme activity occurred between pH 2.2 and 4.0 and at 50 °C, while it was stable between pH 2.2 and 10.0 and up to 40 °C. Due to the presence of a signal peptide, GtAbf51A was thought to hydrolyze polysaccharide containing arabinose. However, the hydrolysis rate of arabinosyl linkages in polysaccharides was only 3-5 % for arabinoxylan and 18 % for arabinan. GtAbf51A, in contrast, efficiently hydrolyzed arabinoxylooligosaccharides, particularly O-α-L-arabinofuranosyl-(1→3)-O-β-D-xylopyranosyl-(1→4)-β-D-xylopyranose, which is the principal product of GH10 β-xylanase. These data suggest that GtAbf51A cooperates with other xylan-degrading enzymes, such as β-xylanase, to degrade xylan in nature.

Slowly digestible carbohydrates are needed for nutritional support in diabetic patients with malnutrition. They are a good source of energy and have the advantage that their consumption produces a low postprandial peak in blood glucose levels because they are slowly and completely digested in the small intestine. A high-amount isomaltomegalosaccharide containing carbohydrate (H-IMS), made from starch by dextrin dextranase, is a mixture of glucose polymers which has a continuous linear structure of α-1,6-glucosidic bonds and a small number of α-1,4-glucosidic bonds at the reducing ends. It has a broad degree of polymerization (DP) distribution with glucans of DP 10－30 as the major component. In our previous study, H-IMS has been shown to exhibit slow digestibility in vitro and not to raise postprandial blood glucose to such levels as that raised by dextrin in vivo. This marks it out as a potentially useful slowly digestible carbohydrate, and this study aimed to evaluate its in vivo digestibility. The amount of breath hydrogen emitted following oral administration of H-IMS was measured to determine whether any indigestible fraction passed through to and was fermented in the large intestine. Total carbohydrate in the feces was also measured. H-IMS, like glucose and dextrin, did not result in breath hydrogen excretion. Carbohydrate excretion with dietary H-IMS was no different from that of glucose or water. These results show that the H-IMS is completely digested and absorbed in the small intestine, indicating its potential as a slowly digestible carbohydrate in the diet of diabetic patients.

Paenibacillus xylaniclasticus strain TW1, a gram-positive facultative anaerobic bacterium, was isolated as a xylanolytic microorganism from the wastes of a pineapple processing factory. A gene encoding one of its xylanolytic enzymes, a β-xylosidase, was cloned and sequenced. Sequence analysis revealed that this β-xylosidase, named PxXyl43A, was composed of a glycoside hydrolase (GH) family 43 subfamily 12 catalytic module and an unknown function module (UM). The full-length PxXyl43A (PxXyl43A) was heterologously expressed in Escherichia coli and purified. Recombinant PxXyl43A exhibited hydrolysis activity against both p-nitrophenyl-β-D-xylopyranoside (pNPX) and p-nitrophenyl-α-L-arabinofuranoside at specific activities of 250 and 310 mU/mg, respectively. The optimal reaction pH and temperature for pNPX hydrolysis were 7.1 and 54 ˚C, respectively. At pH 7.0 and 54 ˚C, the K _m and k _cat for pNPX were 1.2 mM and 2.8 ± 0.15 s^-1, respectively. It was also discovered that the recombinant unknown function module of PxXyl43A (PxXyl43A-UM) could bind to insoluble xylans like birchwood xylan and oat spelt xylan, whereas it did not bind to cellulosic substrates such as ball-milled cellulose, carboxymethyl cellulose or lichenan. The PxXyl43A-UM's binding constant value K _a for oat spelt xylan was 2.0 × 10^-5 M^-1. These results suggest that PxXyl43A possesses a novel carbohydrate-binding module, named as CBM91, specific for xylan-containing polysaccharides.

The GH-16 type β-1,3-glucanase (BgluC16MK) gene of Lysobacter sp. MK9-1 was cloned to study its antifungal activities. BgluC16MK displays amino acid sequence similarity with GluC from L. enzymogenes strain N4-7. BgluC16MK includes a signal sequence, a catalytic domain and carbohydrate-binding module family 6-type β-glucan binding domain (B-GBD). The expression of the BgluC16MK gene in Escherichia coli without the signal sequence resulted in antifungal activity at a dose of 0.6-0.8 nmol/disk. However, BgluC16MK displayed antifungal activity at a dose of 0.025 nmol/disk in combination with Chi19MK. Substrate-specific assay revealed that purified BgluC16MK hydrolyzed insoluble curdlan more readily than the soluble substrate. Furthermore, to explore the binding selectivity of B-GBD of BgluC16MK, we constructed a fusion protein (B-GBD-GFP) using the B-GBD and green fluorescent protein. The activity of the fusion protein against various substrates indicates that B-GBD was selective for glucans with β-1,3-linkages. An additional study demonstrated the binding ability of B-GBD-GFP to the cell-wall of living fungi, such as T. reesei and Aspergillus oryzae. These findings suggest that BgluC16MK can be utilized to generate antifungal enzyme preparations and that the fusion protein B-GBD-GFP can be used to identify the fungal cell surface structure using β-glucans.