Enzymes and cofactors interactions play a significant role in enzymatic function. Particularly, the covalent bonds between proteins and flavin cofactors are important for enzymatic activity and redox potential in covalent flavoproteins. For example, in pyranose 2-oxidase from the basidiomycete Phanerochaete chrysosporium (PcPOx), the flavin adenine dinucleotide (FAD) cofactor forms a covalent bond with histidine (His158), while FAD in other flavoproteins can form a covalent bond with other amino acid residues, such as cysteine, tyrosine, and aspartic acid. Considering the mechanism of forming a covalent bond with FAD, new covalent FAD patterns in PcPOx were expected. Here, we explored the potential for amino acids other than histidine to covalently bind FAD in PcPOx by conducting comprehensive site-directed mutagenesis at His158, and evaluated 19 mutants for covalent-bond-forming ability with FAD, as well as for oxidase and dehydrogenase activities towards D-glucose. All the mutants failed to form a covalent bond with FAD, though they could bind FAD noncovalently to various extents, except for H158D and H158P, which lost not only the covalent bonds with FAD but also the whole of FAD cofactors. The His158 variants showed markedly reduced both the oxidase and dehydrogenase activity toward D-glucose compared with the wild-type enzyme. Moreover, the apo-enzymes H158D and H158P were inactive. Our findings are expected to be helpful in the design of artificial cofactors for flavoproteins.

Here, we aimed to construct a biological saccharification process that combines the steps of enzyme production and enzymatic saccharification using an aerobic fungus Trichoderma reesei, an excellent cellulase producer. Sugar production consists of the growth phase at 28 °C and the saccharification phase at 50 °C. Final sugar yields from alkali-treated rice straw and microcrystalline cellulose using the T. reesei M2-1 strain were greatly affected by mycelial inoculum size and growth phase periods. The optimization of these factors yielded 74.5 % and 60.6 % of sugar from the alkali-treated rice straw and microcrystalline cellulose, respectively, at 120 h of the biological saccharification process. In comparison with the process employing anaerobic microorganisms, a relatively higher yield of sugars was achieved within a shorter period and the use of non-GM fungal strain. However, large variability in sugar yields based on feedstocks suggests imperceptible differences in initial conditions.

Cyanobacteria generally accumulate glycogen in their cells as a photosynthetic product. Interestingly, several unicellular diazotrophic species accumulate insoluble branched polysaccharide called cyanobacterial starch. Branching enzymes (BEs) belonging to glycoside hydrolase family 13 are universally found in the phylum cyanobacteria and are key enzymes in determining the branching pattern of polysaccharides. Many of the glycogen-producing cyanobacteria possess a single BE isozyme (BE1), while multiple BE isozymes (BE1, BE2, and BE3) are present in cyanobacterial starch-producing strains. A previous study suggested that the coexistence of three BE isozymes is essential for the trait of cyanobacterial starch-production. In this study, to obtain clues regarding the significance of the coexistence of the multiple isozymes, biochemical characterization using 11 purified recombinant BEs from both glycogen- and cyanobacterial starch-producing strains was performed. The BE1 and BE2 isozymes produced glucan chains with degree of polymerization (DP) 6 and 7 specifically, while BE3 isozymes produced short (DP 5-12) and long chains (DP 30-40) slightly. The BE1 and BE2 isozymes showed high activity, but those of BE3 isozymes were significantly low. The BE1 isozyme from cyanobacterial starch-producing Cyanobacterium sp. CLg1 showed markedly low activity. The BE1 and BE2 isozymes form cyanobacterial starch-producing Rippkaea orientalis PCC 8802 lacking BE3 isozyme shared similar reaction product specificity. These results suggested that the presence of the three isozymes is not essential and the roles of BE isozymes may vary depending on cyanobacterial species. These findings should deepen our understanding of the significance of BE isozymes in the biosynthesis of cyanobacterial starch.

Chitin is a cell wall structural component of many fungi and is important for mycelium growth. Therefore, enzymes like chitinase which break down chitin, are likely important in fungi during morphological changes. Irpex lacteus, a white-rot fungus isolated from wood-rotting fungi, produces several chitinases. Although it produces a range of chitinases, there are currently no characterization reports exploring them, despite the interest and body of published works evaluating carbohydrate degrading enzymes. In this study, IlChi18C was cloned and recombinantly produced using Pichia pastoris as a host. Properties of purified IlChi18C were determined, revealing an optimal pH of 5.0 and temperature of 50 °C when using pNP-N,N'-diacetyl-β-D-chitobioside (pNP-(GlcNAc)₂) as a substrate. It is activated in the presence of metal ions such as Mg²⁺, Ca²⁺, and Mn²⁺, but inhibited by DMSO, EtOH, and SDS. The Km and Vmax of IlChi18C for this substrate are 3.48 mM and 5.46 µM min⁻¹, respectively. Using pNP-(GlcNAc)₂ and chitin powder as substrates, IlChi18C predominantly exhibited exo-type chitinase activity, releasing chitobiose from the non-reducing ends of chitin chains. It was also observed that this enzyme acts on the fruiting body of Flammulina velutipes, releasing chitobiose as the main product.

Western honeybee (Apis mellifera) α-glucosidase III (HBG-III), which is secreted from the hypopharyngeal glands of honeybees, plays a role in converting nectar into honey. Consequently, hypothesizing that HBG-III is a suitable marker of honey authenticity, we developed an analytical method to determine the HBG-III content and investigated its applicability to various commercial products. Following extraction from honey using phosphate-buffered saline, HBG-III was concentrated using an ultrafiltration membrane and subsequently fragmented with trypsin and lysyl endopeptidase mixture. The specific peptide fragments were used for quantitation by liquid chromatography-tandem mass spectrometry. The established method was validated for linearity, accuracy, precision, and the limit of quantitation (LOQ). As a result, the calibration curve was linear in the range of 0.01-0.3 μM, the mean recovery ranged from 73.8 to 89.2 %, the within-laboratory reproducibility (RSD_wr) ranged from 3.9 to 6.5 %, and the LOQ was 1.9 mg/kg. An investigation of HBG-III concentrations in 65 honey products available on the Japanese market revealed that the HBG-III content of 15 low-priced honey products was below the LOQ. This suggested that these products may be adulterated with non-honey syrups. Therefore, this method can serve as an effective tool to verify the authenticity of honey products.

Lacto-N-biosidase hydrolyzes the β-GlcNAc or β-GalNAc bond of sugar chains to release lacto-N-biose I (Gal-β1,3-GlcNAc) or galacto-N-biose (Gal-β1,3-GalNAc) from the non-reducing end. Typical substrates for lacto-N-biosidase include type I oligosaccharides contained in human breast milk, such as lacto-N-tetraose. Lacto-N-biosidases have recently received significant attention because of their potential to synthesize milk oligosaccharides. Bifidobacterial lacto-N-biosidases belonging to glycoside hydrolase families 20 and 136 have been studied. The GH20 lacto-N-biosidases utilize a substrate-associated hydrolysis mechanism. LnbB from Bifidobacterium bifidum is the only lacto-N-biosidase with reported crystal structures in GH20. In this study, the crystal structure of the lacto-N-biosidase from Streptomyces sp. strain 142 (StrLNBase) was solved in a complex with lacto-N-biose and galacto-N-biose. The stabilizing residue, which recognizes the nitrogen atom of the N-acetyl group of the −1 subsite, and the catalytic acid/base residue, were determined to be D304 and E305, respectively. The structure of StrLNBase is similar to that of LnbB; however, in the complex with galacto-N-biose, there were two structures exhibiting different sugar conformations. A phylogenetic analysis revealed that lacto-N-biosidases discovered in the soil bacteria Streptomyces spp. and human gut bacteria Bifidobacterium spp. may be divided into two separate groups, which suggests that they evolved divergently.

Onions (Allium cepa L.) accumulate fructans, which are fructose polymers, in their bulbs as reserve carbohydrates and as a source of energy for sprouting. Onions with high fructan content and slow fructan degradation by hydrolysis are considered suitable for long-term storage. We have previously found that ‘Pole Star’ accumulates fewer fructans than ‘Kita-momiji 2000’ in their bulbs. In this study, we attempted to clarify the differences in storage characteristics, fructan content, and fructan metabolizing enzyme activities between the two cultivars during storage. Sprouting was not observed in the bulbs of ‘Kita-momiji 2000’ stored at 15 °C for up to 20 weeks, whereas it was observed in ‘Pole Star’ from around 14 weeks. The fructan content during storage showed a gradual decrease in the inner and outer scales of the bulbs in ‘Kita-momiji 2000’, whereas a rapid decrease was observed in ‘Pole Star’. In the basal plate, the fructan contents in ‘Kita-momiji 2000’ were higher than those in ‘Pole Star’ after 16 weeks of storage. Fructan metabolizing enzyme activities were low and constant in ‘Kita-momiji 2000’, whereas their activities increased in ‘Pole Star’ during storage. The low fructan content of ‘Pole Star’ was thought to be due to the high activity of fructan metabolizing enzymes, but the variation of fructan content was difficult to clearly explain using the balance of fructan hydrolase and synthase activities alone.

The changes in moisture content over time were measured for commercially available dried spaghetti with five different diameters (1.17 mm to 1.90 mm) during the rehydration process at 30 °C to 80 °C. The change in moisture content over time at any temperature was modeled using a hyperbolic equation for all spaghetti diameters. The activation energy for the initial rehydration process did not depend on the diameter. The temperature dependence of the equilibrium moisture content differed at temperatures higher and lower than approximately 55 °C, which is close to the starch gelatinization temperature. The rehydration process of spaghetti with diameters of 1.17 mm and 1.42 mm differed from that of thicker spaghetti, with thinner spaghetti rehydrating faster at any temperature. A stochastic model was proposed to explain this phenomenon.

Super Ohtaka^®, a fermented beverage of plant extracts, is prepared from approximately 50 kinds of vegetables and fruits is a naturally fermented mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp.). In this study, we separated water-soluble polysaccharides from Super Ohtaka^® using dialysis and chromatography, yielding four polysaccharide fractions. The polysaccharide fraction designated as OEP3 exhibited hyaluronidase inhibitory activity. The half-maximal inhibitory concentration was 860 µg/mL. This polysaccharide not only stimulated macrophages but also inhibited hyaluronidase activity and showed weak 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity.

Glycoside hydrolase family 131 (GH131) proteins are found in oomycetes, ascomycetes, and basidiomycetes, and have been reported to hydrolyze various β-glucan polysaccharides. Coprinopsis cinerea, a model basidiomycete, contains two GH131 proteins, CcGH131A and CcGH131B. This study focuses on the structural and functional properties of CcGH131B, a protein that lacks the carbohydrate bonding module 1 (CBM1) domain present in CcGH131A. The crystal structure of CcGH131B was determined. The structure displayed a β-jelly roll fold with extra loops and α-helices, resulting in a deeper substrate-binding groove compared to CcGH131A and also PaGluc131A, a GH131 protein from Podospora anserina. A cellobiose-bound structure of the E161A mutant, in which the potential catalytic residue Glu161 was substituted with Ala, showed that the region of the minus subsites bind cellulose. In contrast, the region of the plus subsites mainly consists of hydrophobic amino acid residues and appeared to interact with hydrophobic molecules rather than with carbohydrates. Analysis using native affinity polyacrylamide gel electrophoresis showed that CcGH131B interacted with cellulosic polysaccharides such as methylcellulose and carboxymethylcellulose, while the protein exhibited no detectable enzymatic activity under the tested conditions. These results suggest that the substrate specificity of CcGH131B is likely to be different from those of CcGH131A and PaGluc131A.

Ice formation and growth during freezing in processed foods containing water can deteriorate food quality. Naturally derived antifreeze proteins and antifreeze polysaccharides are an attractive solution to this problem. Alkaline extracts from the basidiomycete Flammulina velutipes (enokitake) are known to inhibit ice crystal growth and are expected to maintain frozen food quality. In this study, polysaccharides/oligosaccharides (POS) were obtained from the readily available edible mushrooms F. velutipes, Hypsizygus marmoreus, Pleurotus eryngii, and Grifola frondosa. POS extracts were isolated by treatment of the fruiting mushroom body with the cell wall-lytic enzyme Uskizyme, then precipitated by ethanol addition. All POS showed antifreeze activity by suppressing ice crystal growth. The benefit of the POS isolated from enzyme-treated edible mushrooms towards frozen processed products quality and shelf-life for foods containing egg protein, fish protein, and rice starch was evaluated. POS derived from F. velutipes was effective in maintaining egg protein (chawanmushi) quality. For fish protein (surimi), the POS derived from F. velutipes and G. frondosa mushrooms suppressed freezing-induced increases in hardness and elasticity. However, for rice starch (shiratama), none of the POS had any effect in preventing retrogradation. This study is the first report to show that components obtained from mushroom cell walls by enzymatic treatment can be effectively used to improve the physical properties of foods. These results suggesting the possibility of new applications for mushrooms as potential cryoprotectants in the frozen food industry.