Purification and characterization of alpha-glucosidase I from Japanese honeybee (Apis cerana japonica) and molecular cloning of its cDNA

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2006-12, Vol.70 (12), p.2889-2898
• Main Authors: Wongchawalit, J.(Hokkaido Univ., Sapporo (Japan)), Yamamoto, T, Nakai, H, Kim, Y.M, Sato, N, Nishimoto, M, Okuyama, M, Mori, H, Saji, O, Chanchao, C, Wongsiri, S, Surarit, R, Svasti, J, Chiba, S, Kimura, A
• Format: Article
• Language: English
• Subjects: ABEILLE DOMESTIQUE; ABEJA MELIFERA; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; allosteric enzyme; alpha-Glucosidases - genetics; alpha-Glucosidases - isolation & purification; alpha-Glucosidases - metabolism; Animals; Apis cerana; Apis mellifera; Base Sequence; Bees - enzymology; Biological and medical sciences; Carbohydrate Metabolism; CHROMATOGRAPHIE; CHROMATOGRAPHY; Chromatography, Gel; CLONACION MOLECULAR; CLONAGE MOLECULAIRE; Cloning, Molecular; CROMATOGRAFIA; DNA Primers; DNA, Complementary; ELECTROFORESIS; ELECTROPHORESE; ELECTROPHORESIS; Electrophoresis, Polyacrylamide Gel; ENZYME ACTIVITY; Enzyme Stability; FRACCIONAMIENTO; FRACTIONATION; FRACTIONNEMENT; Fundamental and applied biological sciences. Psychology; GLUCOSIDASA; GLUCOSIDASE; GLUCOSIDASES; HONEY BEES; honeybee isoenzymes; Hydrogen-Ion Concentration; Japanese honeybee α-glucosidase; MOLECULAR CLONING; PURIFICACION; PURIFICATION; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; TEMPERATURA; TEMPERATURE; western honeybee α-glucosidase
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.60302

Alpha-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 82000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 deg 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 alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1930 bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I.