Yamashita M, Kinoshita T, Ihara M, Mikawa T, Murooka Y
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University.
J Biochem. 1994 Dec;116(6):1233-40. doi: 10.1093/oxfordjournals.jbchem.a124669.
To study the structure and function of pullulanase from Klebsiella aerogenes, a method involving random mutagenesis of the entire gene for pullulanase was used. Out of 50,000 clones screened at high temperature, seven genes for mutant proteins were identified by DNA sequencing. The amino acid substitutions in the seven mutant proteins were clustered on the NH2-terminal side of the four conserved regions found in alpha-amylases. These mutant pullulanases were classified into two types: those whose catalytic activity was altered and those whose thermal stability was increased. The results presented here and in previous reports suggest that pullulanase from K. aerogenes has similar active sites to those of alpha-amylases with the four conserved regions, as well as another substrate-binding site closer to the NH2-terminus. The plate assay method used for isolation of thermostable variants may be applicable to the generation of useful variants of other enzymes.
为研究产气克雷伯菌普鲁兰酶的结构与功能，采用了一种对普鲁兰酶全基因进行随机诱变的方法。在高温筛选的50000个克隆中，通过DNA测序鉴定出7个突变蛋白基因。这7个突变蛋白中的氨基酸替换集中在α-淀粉酶中发现的4个保守区域的氨基末端一侧。这些突变普鲁兰酶分为两类：催化活性改变的和热稳定性增加的。本文及之前报道的结果表明，产气克雷伯菌的普鲁兰酶具有与具有4个保守区域的α-淀粉酶相似的活性位点，以及另一个更靠近氨基末端的底物结合位点。用于分离热稳定变体的平板检测方法可能适用于其他酶的有用变体的产生。
