Expression in Escherichia coli of a gene encoding a thermostable chitinase from Streptomyces thermoviolaceus OPC-520.
An expression plasmid for a thermostable chitinase gene from S. thermoviolaceus OPC-520 in E. coli was constructed. A cloned chitinase (Chi40) was purified from the periplasmic space of E. coli harboring the expression plasmid. The N-terminal sequence of Chi40 was 11 amino acids longer than that of chitinase from S. thermovilaceus OPC-520 (ST chitinase), however, a loss or addition of the amino acid residues did not affect the enzymatic properties. The mutations of Asp-145 and Glu-147 drastically decreased the specific activity of chitinase from the wild type, indicating that both amino acid residues are the best candidates for the essential catalytic residues of Chi40.
Tsujibo
H
H
Osaka University of Pharmaceutical Sciences, Japan.
Endo
H
H
Miyamoto
K
K
Inamori
Y
Y
D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D002688:Chitinases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005973:Glutamine / Q000737:chemistry / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012015:Reference Standards; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D005973:Glutamine; D001216:Asparagine; D002688:Chitinases