Totsuka A, Fukazawa C
Genetic Engineering Laboratory, National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Ibaraki, Japan.
Eur J Biochem. 1993 Jun 15;214(3):787-94. doi: 10.1111/j.1432-1033.1993.tb17981.x.
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.
分离并测序了与大豆β-淀粉酶mRNA对应的cDNA克隆。该cDNA包含一个由496个氨基酸组成的开放阅读框。将从cDNA推导的氨基酸序列与成熟酶的N端肽序列进行比较，证明β-淀粉酶没有前导序列。利用该cDNA，通过受tac启动子控制的表达载体pKK233-2在大肠杆菌中直接合成β-淀粉酶。在大肠杆菌裂解物中检测到的酶活性随着培养温度的降低而急剧增加，在13℃培养的大肠杆菌裂解物中该酶的总活性和比活性分别是37℃时的130倍和280倍。在大肠杆菌中产生的酶通过固定化环麦芽六糖的琼脂糖凝胶6B亲和柱色谱法进行纯化。利用已建立的该酶表达和纯化系统，搜索活性位点中的功能性可电离基团。通过定点诱变，分别将β-淀粉酶高度保守区域中涉及咪唑的His93和涉及羧酸盐的Asp348替换为Arg（H93R）和Ash（D348N）。在大肠杆菌中产生的所有β-淀粉酶，包括非突变型和突变型β-淀粉酶，其Vmax值均低于常规从大豆种子中分离的β-淀粉酶。特别是[H93R]β-淀粉酶的Vmax值与非突变型相比急剧降低；然而，它们都没有完全丧失酶活性。因此，His93和Asp348都可能不直接参与催化反应。
