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

Abstract

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

Comprehensive enzymatic analysis of the amylolytic system in the digestive fluid of the sea hare, Aplysia kurodai: Unique properties of two α‐amylases and two α‐glucosidases

“…To compare the substrate specificities of ApAGL74 and ApAGL86 precisely, the km and kcat values toward 4MU‐α‐glucoside, maltose, isomaltose, sucrose, dextran, glycogen, and starch for ApAGL74 and ApAGL86 were determined (Table 1 ). Although α‐glucosidase I from Japanese honeybee showed unusual kinetic features toward maltose, p‐nitrophenyl‐α‐glucoside, and sucrose [36], ApAGL74 and ApAGL86 exhibited normal Michaelis–Menten‐type kinetics for all substrates tested. Judging from the kcat / km , it is likely that ApAGL74 prefers short α‐1,4‐linked oligosaccharides.…”

“…Although a-glucosidase I from Japanese honeybee showed unusual kinetic features toward maltose, p-nitrophenyl-a-glucoside, and sucrose [36], ApAGL74 and ApAGL86 exhibited normal Michaelis-Menten-type kinetics for all substrates tested. Judging from the kcat/km, it is likely that ApAGL74 prefers short a-1,4-linked oligosaccharides.…”

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

“…One of them, JBG-I, displays non-MichaelisMenten type kinetic behavior, as observed for HBG-I. 10) In spite of the differences in enzymatic properties, honeybee -glucosidases belong to GH-13 and exhibit considerable sequence similarity to each other. 11,12) HBG-III shares 57% and 64% identical amino acids with HBG-I and HBG-II respectively.…”

Molecular Cloning of cDNAs and Genes for Three α-Glucosidases from European Honeybees,Apis melliferaL., and Heterologous Production of Recombinant Enzymes inPichia pastoris

“…The multiple alignment of -glucosidases of honeybee (Apis mellifera), Japanese honeybee (A. cerana), 31) mosquito (Aedes aegypti), 6) yeast (Saccharomyces cerevisiae), 2) and bacterium (B. cereus) 15) prepared using the Clustal W multiple alignment program 32) and the PHD prediction secondary structures program 33) indicated that the sequences for HBGases resembled each other, with 37 to 43% identities of the amino acid residues. Recently, an -glucosidase (JBGase I) was purified from Japanese honeybees, 31) and the enzymatic properties were found to be highly similar to those of HBGase I. Moreover, in the amino acid sequence alignment, the enzyme showed 78% identity towards HBGase I, compared with only 38 to 42% identities towards HBGases II and III.…”