Expression and mutation of soybean β‐amylase in Escherichia coli

Publisher Website
Google Scholar
Abstract
The cDNA clones corresponding to soybean β‐amylase mRNA were isolated and sequenced. The cDNA contained an open‐reading frame composed of 496 amino acids. The comparison of the amino acid sequence deduced from the cDNA with the N‐terminal peptide sequence from mature enzyme proved that β‐amylase had no leader sequence. Employing the cDNA, the β‐amylase was directly synthesized in Escherichia coli by the expression vector pKK233‐2 controlled by the tac promoter. The enzyme activity detected in E. coli lysate drastically increased with a lower cultivation temperature, and the total activity and specific activity of the enzyme in E. coli lysate cultured at 13°C was 130‐fold and 280‐fold, respectively, the value at 37°C. The enzyme produced in E. coli was purified by the affinity column chromatography of cyclomaltohexaose‐immobilized Sepharose 6B. Employing the established expression and purification system of the enzyme, the functional ionizable groups in the active site were searched. His93, involving an imidazole, and Asp348, involving a carboxylate, in the highly conserved regions within the β‐amylases were replaced by Arg (H93R) and Asn (D348N) by site‐directed mutagenesis, respectively. All β‐amylases, including the non‐mutant and mutant β‐amylases, produced in E. coli exhibited lower Vmax values than that of β‐amylase isolated conventionally from soybean seeds. Especially the Vmax value of [H93R]β‐amylase was reduced drastically compared to that of the non‐mutant; however, none of them lost their enzyme activities completely. Therefore, neither His93 nor Asp348 may participate in the catalytic reaction directly.