Cloning of a beta-glucosidase gene from Ruminococcus albus and its expression in Escherichia coli.
A HindIII fragment of R. albus DNA encoding beta-glucosidase was cloned into E. coli. The DNA sequence (3158 bp) was determined, and the longest potential encoding sequence consisted of 2,841 bp (947 amino acids with the calculated molecular weight of 104,276. The deduced NH2-terminal amino acid sequence from the first (methionine) to the twentieth (glycine) was identical to that of the purified enzyme, suggesting that the gene for beta-glucosidase does not encode a signal peptide. The enzyme purified from the culture supernatant of the transformant had a molecular weight of 120,000 and its maximum activity was revealed at pH 6.5 and 30 degrees C. Reducing reagents activated the enzyme, whereas the sulfhydryl group-blocking reagents and reaction products (glucose) inhibited the activity. Hydrolyzates of celloorigomers contained glucose as a major product, indicating that the enzyme acts as beta-glucosidase. The enzyme from the transformant revealed similar properties to that from R. albus, and both enzyme proteins were immunologically the same to each other, indicating that the cloned gene encodes beta-glucosidase from R. albus.
Ohmiya
K
K
Faculty of Bioresources, Mie University, Tsu, Japan.
Takano
M
M
Shimizu
S
S
D000595:Amino Acid Sequence; D001438:Bacteroidaceae / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D013696:Temperature; D014170:Transformation, Genetic; D001617:beta-Glucosidase / Q000235:genetics*
D004269:DNA, Bacterial; D001617:beta-Glucosidase