Cloning and expression of thermostable beta-glycosidase gene from Thermus nonproteolyticus HG102 and characterization of recombinant enzyme.
The gene coding for beta-glycosidase (EC 3.2.1.21) from Thermus nonproteolyticus HG102 was cloned and expressed in Escherichia coli. The gene open reading frame was 1311 bp, and it codes for 437 amino acids. The deduced amino acid sequence of the enzyme showed identity with members of the glycosyl hydrolase family I. The enzyme had high content of Arg and Pro. The recombinant enzyme was purified to homogeneity with heat precipitation, ammonium sulfate precipitation, DEAE-cellulose (DE52) chromatography, and prepared slab polyacrylamide gel electrophoresis. The enzyme was monomeric and had a molecular mass of 48,900 Daltons and a pI of 5.2. The enzyme showed optimum activity at pH 5.6 and 90 degrees C. It catalyzed the hydrolysis of beta-D-glucoside, beta-D-galactoside, beta-D-fucoside, and beta-D-mannoside. Lineweaver-Burk plots showed that the kcat/Km ratio for beta-D-glucoside and beta-D-fucoside was higher than for beta-D-mannoside and beta-D-galactoside. The enzyme was extremely thermostable, with a half-life of 2.5 h at 90 degrees C, and was stable over a wide range of pH. It also had transglycosidic activity at high temperature.
Xiangyuan
H
H
Institute of Microbiology, Chinese Academy of Science, Beijing.
Shuzheng
Z
Z
Shoujun
Y
Y
D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis; D017422:Sequence Analysis, DNA / Q000379:methods; D013379:Substrate Specificity / Q000502:physiology; D013824:Thermus / Q000235:genetics* / Q000378:metabolism; D001617:beta-Glucosidase
D009844:Oligosaccharides; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D001617:beta-Glucosidase; C441633:beta-glycosidase, Thermus nonproteolyticus