Glycoside hydrolase family 1 (GH1) includes enzymes with a wide range of specificities in terms of reactions, substrates and products, with plant GH1 enzymes covering a particularly wide range of hydrolases and transglycosylases. In plants, in addition to β-D-glucosidases, β-D-mannosidases, disaccharidases, thioglucosidases and hydroxyisourate hydrolase, GH1 has recently been found to include galactosyl and glucosyl transferases that utilize galactolipid and acyl glucose donors, respectively. The amino acids binding to the nonreducing monosaccharide residue of glycosides and oligosaccharides in subsite -1 are largely conserved in GH1 glycoside hydrolases, despite their different glycon specificities, and residues outside this subsite contribute to sugar specificity. The conserved subsite -1 residues form extensive hydrogen bonding and aromatic stacking interactions to the glycon to distort it toward the transition state, so they must make different interactions with different sugars. Aglycon specificity is largely determined by interactions with the cleft leading into the active site, but different enzymes appear to interact with their substrates via different residues. The most extended aglycon binding interactions that have been studied extensively are those for cellooligosaccharides. Rice Os3BGlu7 (BGlu1) β-D-glucosidase, which binds cellooligosaccharides residues in subsites +1 to +4 primarily by water-mediated hydrogen bonds and a few aromatic-sugar stacking interactions, appears to show remarkable plasticity in this binding. Although mutations that change the mechanism of the hydrolases, such as glycosynthases and thioglycoligases create transglycosylases, the structural basis for natural transglycosylase vs. glycoside hydrolase activities in GH1 enzymes remains to be determined.
Analyses of function of starch biosynthesis-related isozymes for a thorough understanding of starch biosynthesis were performed in rice (Oryza sativa L.). Starch synthase (SS) I, IIIa and pullulanase (PUL)-deficient mutant lines of rice were isolated using reverse genetics and the function of these isozymes was clarified. Artificial modified amylopectins exhibiting dramatically different gelatinization temperatures or crystallinity as compared to wild type were also produced in rice endosperm. This was accomplished by manipulating isoamylase1 (ISA1), a debranching enzyme gene and branching enzyme IIb (BEIIb). In addition to these in vivo analyses, purified isozymes were expressed in E. coli for direct study. The in vitro results supported the in vivo analyses using mutant and transgenic rice lines. Recently, the production of various combinations of double mutant lines from crosses involving single mutant lines was promoted. The endosperm starches found in these rice lines have a potential for industrial use in food applications and engineering fields. Additionally, these endosperm starches are good source materials for understanding the mechanisms of starch biosynthesis.
Original enzymes screened from nature are not necessarily perfect for industrial applications. Thermostable enzymes from hyperthermophiles often show broad reaction/substrate specificities. Although the enzymes from plants or mesophilic microorganisms exhibit relatively strict reaction/substrate specificities, their thermostabilities are not high enough for industrial purposes. Here we describe two practical approaches for overcoming this problem, introducing amylose production with α-glucan phosphorylases and cycloamylose production with amylomaltases.
The properties of rice flour and the qualities of rice bread made with vital wheat gluten were evaluated and varietal differences were compared by twenty-six rice cultivars of various amylose content and amylopectin structure to determine the influence of rice starch on rice bread. Rice bread made from high-amylose cultivars had a less concave shape, and there was a significant positive correlation between amylose content and specific loaf volume (p < 0.01). Amylose content was also positively correlated with bread hardness. Among the high-amylose cultivars, cultivars with a higher proportion of amylopectin long chains yielded breads of harder texture than the breads made from those with lower proportions of amylopectin long chains, regardless of the small difference in amylose contents. Rice breads made from cultivars that had high pasting temperatures (PT) had harder texture than those made from cultivars that had low PT. Using three near-isogenic lines (NILs) for the Wx and Alk locus on the cv. Nipponbare genetic background, we found that water absorption by the rice flour of NILs was lower than that of Nipponbare and that bread hardness values for all NILs were higher than the value for Nipponbare. These results indicate that amylose content and amylopectin structure affect dough and bread qualities, including water absorption, bread volume, shape and hardness, and that rice cultivars with intermediate amylose content and lower PT are suitable for making rice bread.
Isoprimeverose-producing oligoxyloglucan hydrolase (IPase; EC 22.214.171.124) is a unique β-glycosidase that cleaves xyloglucan oligosaccharides at the nonreducing end, producing isoprimeverose. Here, we report the first gene cloning and expression of IPase. Previously, we reported that IPase from an actinomycetes, Oerskovia sp. Y1, has a molecular mass of 105 kDa. In this study, the full-length DNA encoding IPase was cloned and sequenced, and shown to have a 3,054-bp open reading frame encoding a protein of 1,018 amino acid residues. Based on the amino acid sequence, the catalytic domain was classified as glycoside hydrolase family 3 enzyme, and it has a family 6 carbohydrate-binding module at its C-terminus. IPase was expressed in Escherichia coli and the recombinant protein was purified. Although the recombinant protein was expressed as an inclusion body, renaturation was successful, and enzymatically active recombinant IPase was obtained. An analysis of the substrate specificity revealed that IPase strictly recognizes the isoprimeverose unit at the nonreducing end of a xyloglucan oligosaccharide. In addition, the binding assay revealed that IPase had binding ability to xyloglucan.
A his7-tagged β-glucosidase from Magnaporthe oryzae (MoCel3A) was expressed under the control of a cellobiohydrolase I (cbh1) promoter in Trichoderma reesei. Accumulation of extracellular his7-tagged MoCel3A and glucose production were evaluated during growth on a variety of substrates. Enzyme preparations obtained from the recombinant T. reesei strain grown in the presence of cellulose produced more his7-tagged MoCel3A, exhibited greater hydrolytic activity towards pNPG and produced more glucose from PSC, cellulose, rice straw and laminarin than the wild type parent. These results indicate that expression of his7-tagged MoCel3A enhanced glucose production not only from β-1,4-glucan but also from β-1,3-glucan. More his7-tagged MoCel3A accumulated when the recombinant strain was grown in the presence of cellooligosaccharides with various degrees of polymerization. Interestingly, accumulation of his7-tagged MoCel3A was highly enhanced by growth on laminarioligosaccharides. An active enzyme preparation was obtained from the recombinant strain grown on rice straw, an inexpensive and renewable substrate.
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