Journal of applied glycoscience最新文献

Trehalose is known to protect enzymes from denaturation. In the present study, we observed promotion of apple polyphenol oxidase (PPO) inactivation in a trehalose solution with thermal treatment. Crude PPO from Fuji apple was mixed with either sucrose or trehalose solutions, then the samples treated at 25 or 65 °C. In the presence of trehalose, PPO activities were markedly decreased upon treatment at 65 °C with increasing trehalose concentration. Furthermore, the reduction in PPO activity in the presence of trehalose was proportional to storage time after thermal treatment and thermal treatment time. Comparing PPO activities between treatment time 0 and 90 min at 65 °C, activities decreased 89 % for trehalose concentration of 0.2 M. These results indicates that trehalose acts not only as inhibitor but as promoter of inactivation of PPO. The Lineweaver-Burk plot indicated that trehalose acts on PPO as a non-competitive inhibitor during the 65 °C treatment. Two mechanisms of PPO inactivation in the presence of trehalose were suggested; one is the suppression of PPO activation cause by a thermal treatment, and another is the conformational change to inactivation form of PPO in conjunction with trehalose and a thermal treatment. Additionally, apple juice including 0.2 or 0.5 M trehalose with 65 °C treatment indicated slow browning than the juice with 0.2 or 0.5 M sucrose or without sugars. This result demonstrates that the preventing of browning with trehalose is a viable industrial food process.

α-Mannosidase (ALMAN) extracted from onion (Allium cepa) was purified by column chromatography such as hydrophobic and gel filtration. ALMAN is an acidic α-mannosidase that exhibits maximum activity against pNP-α-Man at pH 4.0-5.0 at 50°C. Amino acid sequence analysis of ALMAN was consistent with α-mannosidase deduced from Allium cepa transcriptome analysis. The gene alman was amplified by PCR using mRNA extracted from onions, and a full-length gene of 3,054 bp encoding a protein of 1,018 amino acid residues was revealed. ALMAN is classified as Glycoside Hydrolase Family (GH) 38 and showed homology with other plant-derived α-mannosidases such as tomato and hot pepper.

Different forms of plant biomass have been utilised for various applications in daily life and have gained increasing attention as replacements for fossil fuel-based products in the pursuit of a sustainable society. Plant cell walls, the primary carbon sink of plant biomass, have a high-order polysaccharide architecture consisting of cellulose, hemicelluloses, pectins, lignin and some proteins. Hemicelluloses are a group of polysaccharides that interact with cellulose, which is fundamental to the different properties and functionality of the plant cell walls. However, for industrial applications, the complex polysaccharide architecture poses a barrier to their efficient use. Understanding the molecular basis of plant cell walls - especially cellulose-hemicellulose interactions - is therefore critical to improving the utilisation of plant biomass. Recent research has revealed that the detailed structures, modification patterns, and conformation of hemicelluloses play an influential role in their interaction with cellulose. In this review, we discuss the latest insights into hemicelluloses across different forms of plant biomass and how their structures affect cell wall assembly. Additionally, we explore recent findings on how alterations in hemicellulose structure and modification patterns affect the usability of plant biomass, including the extractability of polysaccharides and the digestibility of biomass by glycoside hydrolases for biofuel production. Furthermore, we address unsolved questions in the field and propose future strategies to maximize the potential of plant biomass.

Lytic polysaccharide monooxygenases (LPMO) are key enzymes for the efficient degradation of lignocellulose biomass with cellulases. A lignocellulose-degradative strain, Paenibacillus xylaniclasticus TW1, has LPMO-encoding PxAA10A gene. Neither the C1/C4-oxidizing selectivity nor the enzyme activity of PxAA10A has ever been characterized. In this study, the C1/C4-oxidizing selectivity of PxAA10A and the boosting effect for cellulose degradation with a cellulase cocktail were investigated. The full-length PxAA10A (rPxAA10A) and the catalytic domain (rPxAA10A-CD) were heterologously expressed in Escherichia coli and purified. To identify the C1/C4-oxidizing selectivity of PxAA10A, cellohexaose was used as a substrate with the use of rPxAA10A-CD, and the products were analyzed by MALDI-TOF/MS. As a result, aldonic acid cellotetraose and cellotetraose, the products from C1-oxidization and C4-oxidization, respectively, were detected. These results indicate that PxAA10A is a C1/C4-oxidizing LPMO. It was also found that the addition of rPxAA10A into a cellulase cocktail enhanced the cellulose-degradation efficiency.

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.