Filamentous fungi use various enzymes to degrade cellulose, some of which contain cellulose-binding domains (CBDs), most of which belong to carbohydrate-binding module family 1 (CBM1). We recently identified the novel fungal CBD, CBM104, from Gloeophyllum trabeum. Reportedly, CBM104 specifically binds to native crystalline cellulose, not to amorphous or artificially modified crystalline cellulose, exhibiting a unique adsorption characteristic. To gain further insights into CBM104, the adsorption properties of six different CBM104s, each appended to a different catalytic domain, were investigated. The adsorption tests illustrated that all CBM104s predicted to possess a three-dimensional structure in which two α-helices were crosslinked by disulfide bonds specifically adsorbed onto cellulose I. Conversely, CBM104 lacking these disulfide bonds failed to adsorb onto any form of cellulose used in this study, suggesting the importance of the fixed pair of α-helices for specific binding to cellulose I. To identify CBM104 homologs in which the disulfide bonds are conserved, a homology search was performed against fungal genomes, resulting in 144 hits. These CBM104 homologs were primarily appended to auxiliary activities (AA) family 9 or to domains that work cooperatively with AA9 enzyme. CBM104s were found only in certain orders of Agaricomycetes, and the majority of these fungi are suggested to have the ability to degrade plant cell walls. These results suggest that some Agaricomycetes utilize plant cell wall degradation systems involving CBM104-attached proteins. This study provides detailed insights into the structural factors involved in the adsorption capacity of CBM104, as well as its phylogenetic distribution.

Glycoside hydrolase family 32 (GH32) enzymes play key roles in fructooligosaccharide metabolism in gut bacteria. In this study, a GH32 enzyme (GenBank code, GFO85652) containing carbohydrate binding module 66 (CBM66) from the gut bacterium Anaerostipes butyraticus (AbFEH) was heterologously expressed in Escherichia coli. We constructed an expression plasmid that does not contain sequences for the N-terminal signal peptide and the C-terminal region potentially involving cell-wall binding. The enzyme obtained (AbFEH∆C) was purified and characterized. Thin-layer chromatography and high-performance liquid chromatography analyses revealed that AbFEH∆C produced fructose from all the substrates, sucrose, 1-kestose, inulin, and levan, and intermediate oligosaccharide products were not observed. The ratio of activities towards sucrose, 1-kestose, nystose, inulin, and levan was 6:100:83:8:95 under the conditions of this study. A region containing M and CBM66 domains was further removed from AbFEH∆C, and the activities for both 1-kestose and levan of this mutant enzyme were about 400-fold lower than those of AbFEH∆C. Kinetic analysis indicated a low K_m value for levan, while requiring higher substrate concentrations for 1-kestose and sucrose. Comparison of the predicted structure of AbFEH with crystal structures of some GH32 enzymes indicated that residues at subsite −1 were almost completely conserved, while some key residues found in GH32 enzymes were not present at subsites +1 and +2 in AbFEH. These observations suggest that AbFEH functions as fructan exohydrolase that exhibits low sucrose-hydrolyzing activity.

Perilla frutescens is a popular aromatic edible plant. The amount of Perilla residues produced by food or pharmaceutical industry is increasing because of the high demand for this plant. At present, most Perilla residues are incinerated, but there is increasing interest in using these materials as a biomass resource. In this study, an alkaline pretreatment to remove lignin from Perilla residues was optimized, and the ash, lignin, and total sugar contents of the treated materials were determined to evaluate their biomass potential. The optimum alkaline pretreatment for Perilla residues was 0.25 M NaOH at 121 °C for 60 min. The lignin and total sugar contents of the alkaline-pretreated Perilla residues were comparable to those reported for grain straw. These results suggest that alkaline-pretreated Perilla residues have high potential as biomass. With dual aims to reduce the volume of Perilla residues and to effectively use this resource, bacteria capable of decomposing Perilla seed shells after alkaline pretreatment were isolated from environmental samples. A total of 66 strains of degraders were isolated, of which one strain (strain SW8) was identified as Klebsiella aerogenes or Raoultella ornithinolytica with both cellulase and xylanase activities. Strain SW8 grew well at 25-35 °C with Perilla seed shells as the sole carbon source. Strain SW8 was identified as a useful bacterium to reduce the volume of, and effectively utilize, Perilla residues.

Rice bread, a gluten-free alternative to wheat bread, often suffers from poor texture due to inadequate viscoelasticity during baking. This study aimed to propose a baking method for pure rice bread by investigating the effects of hydroxypropylated potato starches (HPPS) with varying degrees of substitution on the baking performance of rice batter. First, the particle size distribution and thermal properties of HPPS were analyzed to characterize their fundamental attributes. Rice bread was then prepared using each type of HPPS, and their foaming properties were assessed. Additionally, dynamic viscoelastic measurements were performed during heating to assess rheological changes during baking. Results showed that a higher degree of substitution in HPPS reduced in the gelatinization onset temperature. Moreover, HPPS addition improved the cross-sectional structure of the rice bread. Notably, highly substituted HPPS suppressed the formation of large internal voids caused by bubble coalescence. These findings suggest that HPPS with a high degree of substitution enhances the overall quality of rice bread.

Branching enzymes (BEs) are essential for defining the branching patterns of glycogen and starch by catalyzing the formation of α-1,6-glucosidic linkages. While most cyanobacteria accumulate glycogen, some species, such as Crocosphaera subtropica ATCC 51142, produce an insoluble branched α-glucan known as cyanobacterial starch. This strain possesses three BE isozymes: cceBE1, cceBE2, and cceBE3. Our previous studies demonstrated that cceBE1 and cceBE2 share similar enzymatic properties and that a “stopper structure” contributes to their preferential production of short chains with a degree of polymerization (DP) of 6 and 7. In contrast, cceBE3 produces small amounts of short (DP5-12) and long (DP30-40) chains and lacks the amino acid sequence corresponding to the stopper structure. To investigate the role of the stopper structure, we constructed a deletion mutant of cceBE1 lacking the stopper structure and characterized its enzymatic properties. The mutant retained catalytic activity but lost the ability to selectively produce glucan chains with DP6 and 7 (transferred chains), providing direct evidence for the stopper structure's role in regulating product chain length. Furthermore, we determined the crystal structure of cceBE3, confirming the absence of the stopper structure. We also identified a unique structural feature in cceBE3, termed subdomain B, located within the predicted substrate-binding site. Deletion of subdomain B led to increased production of short chains (DP3-7), suggesting its involvement in substrate binding and the determination of product specificity. These findings reveal structural determinants of product specificity in cyanobacterial BEs and offer a strategy for engineering BEs to produce novel starch-based materials.

Alginate, a heteropolysaccharide composed of α-L-guluronic acid (G) and β-D-mannuronic acid (M), comprises poly-G, poly-M, and mixed poly-MG regions. Alginate lyases, classified within the polysaccharide lyase (PL) family, degrade alginate into unsaturated saccharides via β-elimination. Due to the abundance of alginate in brown algae, various marine bacteria produce alginate lyases for its assimilation. Recently, alginate lyases have also been identified in gut bacteria such as those of the genus Bacteroides. In this study, we purified an alginate lyase from enrichment culture supernatants containing alginate, using a human fecal sample, and isolated B. xylanisolvens strain MK6803, which can grow on alginate as a sole carbon source-unlike the type strain B. xylanisolvens XB1A. Draft genome sequencing of strain MK6803 revealed an alginate-metabolizing gene cluster encoding three alginate lyases belonging to PL6_1, PL17_2, and PL38, along with a putative oxidoreductase. This gene cluster was shared with B. ovatus CP926 and B. xylanisolvens CL11T00C41, but not with the type strain XB1A. Bacteroides species lacking this gene cluster exhibited no alginate assimilation, even if they possessed genes encoding one or more of the three alginate lyases. This suggests that the presence of the putative oxidoreductase, alongside the lyases, is essential for alginate assimilation in Bacteroides species. Phylogenetic analysis indicated horizontal gene transfer within the genus Bacteroides. These findings highlight the role of alginate metabolism in the adaptation of human gut microbiota.

Metagenomics can be used to obtain sequence information on putative genes in a microbial community. However, it is difficult to identify genes with specific functions among the numerous predicted genes. In this study, we attempted to identify genes induced in cultured microbes by the addition of saccharides using metagenomic and metatranscriptomic analyses. A mixture of arabinoxylan and its derived oligosaccharides was used as the inducer in this study. Some genes were highly induced in the presence of additive saccharides and formed gene clusters for the utilization of additive saccharides, suggesting that metatranscriptomic and metagenomic analyses are useful for analyzing carbohydrate-responsive genes in microbial communities and screening novel carbohydrate-active enzymes.

Short linear maltodextrin (SLMD) is a novel maltodextrin synthesized from starch using the combined enzymatic actions. SLMD exhibits unique aggregating and solidifying properties. In this study, we prepared SLMD aggregates, solidified materials under various conditions, and investigated their crystallinity. Aggregates formed in the 50 % SLMD solution at 4 °C (AGG-4), 25 °C (AGG-25), and 50 °C (AGG-50) showed clear X-ray diffraction peaks. A B-type crystal diffraction pattern was observed for AGG-4, whereas an A-type pattern was observed for AGG-25 and AGG-50. Kneading SLMD with a limited quantity of water produced solidified slurries at 4 °C (SS-4) and 25 °C (SS-25). SS-4 exhibited a C-type structure with low crystallinity, whereas SS-25 showed an A-type structure with high crystallinity. In addition, B-type crystals were detected in the aggregates in the emulsions solidified with vegetable oil. Therefore, SLMD crystals occurred in different forms in the aggregates or solidified bodies under various conditions.