Makino Y, Negoro S, Urabe I, Okada H
Department of Fermentation Technology, Faculty of Engineering, Osaka University, Japan.
J Biol Chem. 1989 Apr 15;264(11):6381-5.
A glucose dehydrogenase gene was isolated from Bacillus megaterium IWG3, and its nucleotide sequence was identified. The amino acid sequence of the enzyme deduced from the nucleotide sequence is very similar to the protein sequence of the enzyme from B. megaterium M1286 reported by Jany et al. (Jany, K.-D., Ulmer, W., Froschle, M., and Pfleiderer, G. (1984) FEBS Lett. 165, 6-10). The isolated gene was mutagenized with hydrazine, formic acid, or sodium nitrite, and 12 clones (H35, H39, F18, F20, F191, F192, N1, N13, N14, N28, N71, and N72) containing mutant genes for thermostable glucose dehydrogenase were obtained. The nucleotide sequences of the 12 genes show that they include 8 kinds of mutants having the following amino acid substitutions: H35 and H39, Glu-96 to Gly; F18 and F191, Glu-96 to Ala; F20, Gln-252 to Leu; F192, Gln-252 to Leu and Ala-258 to Gly; N1, Glu-96 to Lys and Val-183 to Ile; N13 and N14, Glu-96 to Lys, Val-112 to Ala, Glu-133 to Lys, and Tyr-217 to His; N28, Glu-96 to Lys, Asp-108 to Asn, Pro-194 to Gln, and Glu-210 to Lys; and N71 and N72, Tyr-253 to Cys. These mutant enzymes have higher stability at 60 degrees C than the wild-type enzyme. The results of this study indicate that the tetrameric structure of glucose dehydrogenase is stabilized by several kinds of mutation, and at least one of the following amino acid substitutions stabilizes the enzyme: Glu-96 to Gly, Glu-96 to Ala, Gln-252 to Leu, and Tyr-253 to Cys.
从巨大芽孢杆菌IWG3中分离出一个葡萄糖脱氢酶基因，并确定了其核苷酸序列。从核苷酸序列推导的该酶的氨基酸序列与Jany等人报道的巨大芽孢杆菌M1286中该酶的蛋白质序列非常相似（Jany, K.-D., Ulmer, W., Froschle, M., and Pfleiderer, G. (1984) FEBS Lett. 165, 6 - 10）。用肼、甲酸或亚硝酸对分离出的基因进行诱变，获得了12个含有热稳定葡萄糖脱氢酶突变基因的克隆（H35、H39、F18、F20、F191、F192、N1、N13、N14、N28、N71和N72）。这12个基因的核苷酸序列表明，它们包含8种具有以下氨基酸替换的突变体：H35和H39，Glu - 96突变为Gly；F18和F191，Glu - 96突变为Ala；F20，Gln - 252突变为Leu；F192，Gln - 252突变为Leu且Ala - 258突变为Gly；N1，Glu - 96突变为Lys且Val - 183突变为Ile；N13和N14，Glu - 96突变为Lys，Val - 112突变为Ala，Glu - 133突变为Lys，Tyr - 217突变为His；N28，Glu - 96突变为Lys，Asp - 108突变为Asn，Pro - 194突变为Gln，Glu - 210突变为Lys；N71和N72，Tyr - 253突变为Cys。这些突变酶在60℃时比野生型酶具有更高的稳定性。本研究结果表明，葡萄糖脱氢酶的四聚体结构通过几种突变得以稳定，并且以下氨基酸替换中的至少一种可使酶稳定：Glu - 96突变为Gly、Glu - 96突变为Ala、Gln - 252突变为Leu以及Tyr - 253突变为Cys。
