Hiroyuki Nakai

Purification and Characterization of α-Glucosidase I from Japanese Honeybee (Apis cerana japonica) and Molecular Cloning of Its cDNA

α-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 °C and in a pH-range of 4.5–10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl α-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930 bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of α-amylase family enzymes. Western honeybees (Apis mellifera) had three α-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I.

Glucoamylase Originating from Schwanniomyces occidentalis Is a Typical α-Glucosidase

A starch-hydrolyzing enzyme from Schwanniomyces occidentalis has been reported to be a novel glucoamylase, but there is no conclusive proof that it is glucoamylase. An enzyme having the hydrolytic activity toward soluble starch was purified from a strain of S. occidentalis. The enzyme showed high catalytic efficiency (k cat⁄K m) for maltooligosaccharides, compared with that for soluble starch. The product anomer was α-glucose, differing from glucoamylase as a β-glucose producing enzyme. These findings are striking characteristics of α-glucosidase. The DNA encoding the enzyme was cloned and sequenced. The primary structure deduced from the nucleotide sequence was highly similar to mold, plant, and mammalian α-glucosidases of α-glucosidase family II and other glucoside hydrolase family 31 enzymes, and the two regions involved in the catalytic reaction of α-glucosidases were conserved. These were no similarities to the so-called glucoamylases. It was concluded that the enzyme and also S. occidentalis glucoamylase, had been already reported, were typical α-glucosidases, and not glucoamylase.

Bacteroides thetaiotaomicron VPI‐5482 glycoside hydrolase family 66 homolog catalyzes dextranolytic and cyclization reactions

Bacteroides thetaiotaomicron VPI‐5482 harbors a gene encoding a putative cycloisomaltooligosaccharide glucanotransferase (BT3087) belonging to glycoside hydrolase family 66. The goal of the present study was to characterize the catalytic properties of this enzyme. Therefore, we expressed BT3087 (recombinant endo‐dextranase from Bacteroides thetaiotaomicron VPI‐5482) in Escherichia coli and determined that recombinant endo‐dextranase from Bacteroides thetaiotaomicron VPI‐5482 preferentially synthesized isomaltotetraose and isomaltooligosaccharides (degree of polymerization > 4) from dextran. The enzyme also generated large cyclic isomaltooligosaccharides early in the reaction. We conclude that members of the glycoside hydrolase 66 family may be classified into three types: (a) endo‐dextranases, (b) dextranases possessing weak cycloisomaltooligosaccharide glucanotransferase activity, and (c) cycloisomaltooligosaccharide glucanotransferases.

Amino Acids in Conserved Region II Are Crucial to Substrate Specificity, Reaction Velocity, and Regioselectivity in the Transglucosylation of Honeybee GH-13 α-Glucosidases

Honeybees, Apis mellifera, possess three α-glucosidase isozymes, HBG-I, HBG-II, and HBG-III, which belong to glycoside hydrolase family 13. They show high sequence similarity, but clearly different enzymatic properties. HBG-III preferred sucrose to maltose as substrate and formed only α-1,4-glucosidic linkages by transglucosylation, while HBG-II preferred maltose and formed the α-1,6-linkage. Mutation analysis of five amino acids in conserved region II revealed that Pro226-Tyr227 of HBG-III and the corresponding Asn226-His227 of HBG-II were crucial to the discriminating properties. By replacing these two amino acids, the substrate specificities and regioselectivity in transglucosylation were changed drastically toward the other. The HBG-III mutant, Y227H, and the HBG-II mutant, N226P, which harbor HBG-I-type Pro-His at the crucial positions, resembled HBG-I in enzymatic properties with marked increases in reaction velocities on maltose and transglucosylation ratios. These findings indicate that the two residues are determinants of the enzymatic properties of glycoside hydrolase family 13 (GH-13) α-glucosidases and related enzymes.

Rice α-glucosidase isozymes and isoforms showing different starch granules-binding and -degrading ability

Insoluble starch granules stored in plant seeds have generally been considered to be degraded effectively by the combination of amylolytic enzymes following initial attack by de novo synthesized α-amylase at germination. We have shown that rice (Oryza sativa L., var Nipponbare) α-glucosidase isozymes (ONG1, ONG2, and ONG3) are also capable of binding to and degrading starch granules directly, indicating the direct liberation of glucose from starch granules by α-glucosidase at germination. ONG1 and ONG2 are encoded in a distinct locus of the rice genome, while ONG2 and ONG3 are generated by alternative splicing. Interestingly, each of the α-glucosidase isozymes showed different action toward starch granules. In addition, two ONG2 isoforms were found to be produced by post-translational proteolysis. The proteolysis induced changes in binding to and degradation of starch granules.

A Solanum torvum GH3 β-glucosidase expressed in Pichia pastoris catalyzes the hydrolysis of furostanol glycoside

Plant β-glucosidases are usually members of the glucosyl hydrolase 1 (GH1) or 3 (GH3) families. Previously, a β-glucosidase (torvosidase) was purified from Solanum torvum leaves that specifically catalyzed hydrolysis of two furostanol 26-O-β-glucosides, torvosides A and H. Furostanol glycoside 26-O-β-glucosides have been reported as natural substrates of some plant GH1 enzymes. However, torvosidase was classified as a GH3 β-glucosidase, but could not hydrolyze β-oligoglucosides, the natural substrates of GH3 enzymes. Here, the full-length cDNA encoding S. torvum β-glucosidase (SBgl3) was isolated by the rapid amplification of cDNA ends method. The 1887bp ORF encoded 629 amino acids and showed high homology to other plant GH3 β-glucosidases. Internal peptide sequences of purified native Sbgl3 determined by LC-MS/MS matched the deduced amino acid sequence of the Sbgl3 cDNA, suggesting that it encoded the natural enzyme. Recombinant SBgl3 with a polyhistidine tag (SBgl3His) was successfully expressed in Pichia pastoris. The purified SBgl3His showed the same substrate specificity as natural SBgl3, hydrolyzing torvoside A with much higher catalytic efficiency than other substrates. It also had similar biochemical properties and kinetic parameters to the natural enzyme, with slight differences, possibly attributable to post-translational glycosylation. Quantitative real-time PCR (qRT-PCR) showed that SBgl3 was highly expressed in leaves and germinated seeds, suggesting a role in leaf and seedling development. To our knowledge, a recombinant GH3 β-glucosidase that hydrolyzes furostanol 26-O-β-glucosides, has not been previously reported in contrast to substrates of GH1 enzymes.

Purification and characterization of a chloride ion-dependent α-glucosidase from the midgut gland of Japanese scallop (Patinopecten yessoensis)

Marine glycoside hydrolases hold enormous potential due to their habitat-related characteristics such as salt tolerance, barophilicity, and cold tolerance. We purified an α-glucosidase (PYG) from the midgut gland of the Japanese scallop (Patinopecten yessoensis) and found that this enzyme has unique characteristics. The use of acarbose affinity chromatography during the purification was particularly effective, increasing the specific activity 570-fold. PYG is an interesting chloride ion-dependent enzyme. Chloride ion causes distinctive changes in its enzymatic properties, increasing its hydrolysis rate, changing the pH profile of its enzyme activity, shifting the range of its pH stability to the alkaline region, and raising its optimal temperature from 37 to 55 °C. Furthermore, chloride ion altered PYG’s substrate specificity. PYG exhibited the highest Vmax/Km value toward maltooctaose in the absence of chloride ion and toward maltotriose in the presence of chloride ion. Graphical abstract An α-glucosidase was purified from the midgut gland of the scallop. The acarbose affinity chromatography was effective for the purification. The enzyme showed chloride ion dependency.