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J. Biochem, 1995, Vol. 118, No. 3 562-567
© 1995 Japanese Biochemical Society

other

Construction of a Plasmid Used for the Expression of a Sevenfold-Mutant Barley ß-Amylase with Increased Thermostability in Escherichia coli and Properties of the Sevenfold-Mutant ß-Amylase

Naohiro Yoshigi, Yukio Okada, Hideo Maeba, Hirohisa Sahara and Teruo Tamaki

Brewing Research Laboratories, Sapporo Breweries Ltd. Yaizu, Shizuoka 425

To increase the thermostability of ß-amylase, seven kinds of single-mutant plasmids were constructed with an expression vector of barley ß-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°C. To express the sevenfoldmutant ß-amylase in Escherichia coli, plasmid pB927 was constructed. E. coli harboring plasmid pB927 produced sevenfold-mutant ß-amylase. The T50 value of purified sevenfoldmutant ß-amylase (69.0°C) was higher than that of not only the original recombinant ß-amylase (57.4°C) by 11.6°C but also soybean ß-amylase (63.2°C) by 5.8°C. The intragenic amino acid replacements were found to have simple additive effects on the thermostability of ß-amylase. The sevenfold-mutant ß-amylase was found to be stable at pHs up to 12.5, while the original recombinant ß-amylase was unstable at pHs above 9.5. The data obtained from kinetics studies suggested that the sevenfold-mutant ß-amylase acquired enhanced thermostability, but its function as a ß-amylase remained unchanged.


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