Expression and mutation of soybean beta-amylase in Escherichia coli.
The cDNA clones corresponding to soybean beta-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 beta-amylase had no leader sequence. Employing the cDNA, the beta-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 degrees C was 130-fold and 280-fold, respectively, the value at 37 degrees 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 beta-amylases were replaced by Arg (H93R) and Ash (D348N) by site-directed mutagenesis, respectively. All beta-amylases, including the non-mutant and mutant beta-amylases, produced in E. coli exhibited lower Vmax values than that of beta-amylase isolated conventionally from soybean seeds. Especially the Vmax value of [H93R]beta-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.
Totsuka
A
A
Genetic Engineering Laboratory, National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Ibaraki, Japan.
Fukazawa
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003505:Cyclodextrins; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics; D012685:Sepharose; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000235:genetics*; D047391:alpha-Cyclodextrins; D001614:beta-Amylase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D003505:Cyclodextrins; D012333:RNA, Messenger; D047391:alpha-Cyclodextrins; D012685:Sepharose; D001614:beta-Amylase; C032613:alpha-cyclodextrin