Construction of a plasmid used for the expression of a sevenfold-mutant barley beta-amylase with increased thermostability in Escherichia coli and properties of the sevenfold-mutant beta-amylase.
To increase the thermostability of beta-amylase, seven kinds of single-mutant plasmids were constructed with an expression vector of barley beta-amylase by mutagenesis. The remaining activity versus temperature curves were used to determine the temperatures (T50) at which 50% of the initial activity was lost during a 30-min heating period. These mutations increased the T50 values by amounts ranging from 0.8 to 3.2 degrees C. To express the sevenfold-mutant beta-amylase in Escherichia coli, plasmid pB927 was constructed. E. coli harboring plasmid pB927 produced sevenfold-mutant beta- amylase. The T50 value of purified sevenfold-mutant beta-amylase (69.0 degrees C) was higher than that of not only the original recombinant beta-amylase (57.4 degrees C) by 11.6 degrees C but also soybean beta-amylase (63.2 degrees C) by 5.8 degrees C. The intragenic amino acid replacements were found to have simple additive effects on the thermostability of beta-amylase. The sevenfold-mutant beta-amylase was found to be stable at pHs up to 12.5, while the original recombinant beta-amylase was unstable at pHs above 9.5. The data obtained from kinetics studies suggested that the sevenfold-mutant beta-amylase acquired enhanced thermostability, but its function as a beta-amylase remained unchanged.
Yoshigi
N
N
Brewing Research Laboratories, Sapporo Breweries Ltd., Shizuoka.
Okada
Y
Y
Maeba
H
H
Sahara
H
H
Tamaki
T
T
D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005822:Genetic Vectors; D001467:Hordeum / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D013379:Substrate Specificity; D013696:Temperature; D001614:beta-Amylase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D008670:Metals; D010940:Plant Proteins; D001614:beta-Amylase