Selection of stabilized 3-isopropylmalate dehydrogenase of Saccharomyces cerevisiae using the host-vector system of an extreme thermophile, Thermus thermophilus
Selection of stabilized 3-isopropylmalate dehydrogenase of Saccharomyces cerevisiae using the host-vector system of an extreme thermophile, Thermus thermophilus
复制标题
使用极端嗜热菌、嗜热栖热菌的宿主载体系统选择酿酒酵母的稳定 3-异丙基苹果酸脱氢酶
作者：
M. Tamakoshi;Y. Nakano;S. Kakizawa;A. Yamagishi;T. Oshima
用质粒载体转化嗜热栖热菌TTY 1的leuB菌株，所述质粒载体指导由LEU 2基因编码的酿酒酵母3-异丙基苹果酸脱氢酶（IPMDH）的表达。原始菌株不能在50°C无亮氨酸的条件下生长，可能是因为S.酿酒酵母IPMDH。在50° C、60° C、62° C、65° C、67° C和70°C下逐步选择可以在没有亮氨酸的情况下生长的突变体。除了在50°C下分离的一种之外，所有突变菌株都积累了突变。突变连续累积：在每个步骤分别为Glu 255 Val、Asn 43 Tyr、Ala 62 Thr、Asn 110 Lys和Ala 112 Val。热处理后的残留活性和通过圆二色性监测的变性曲线的分析表明，热稳定性随着突变的积累而增加。大多数突变酶的动力学参数与野生型相似。然而，一些突变酶的稳定性和活性之间表现出反向相关性：酶的热稳定性大幅增加，表现出较低的活性。虽然野生型酶是不稳定的，在甘油的情况下，甘油的稳定效果没有观察到所有的突变体酶含有的Glu 255 Val取代，这是假设位于两个亚基之间的疏水界面。
A leuB strain of Thermus thermophilus, TTY1, was transformed with a plasmid vector that directed expression of 3-isopropylmalate dehydrogenase (IPMDH) of Saccharomyces cerevisiae encoded by the LEU2 gene. The original strain could not grow at 50°C without leucine, probably because of the low stability of S. cerevisiae IPMDH. The mutants that could grow without leucine were selected at 50°, 60°, 62°, 65°, 67°, and 70°C, step by step. All the mutant strains except for one isolated at 50°C accumulated mutations. Mutations were serially accumulated: Glu255Val, Asn43Tyr, Ala62Thr, Asn110Lys, and Ala112Val, respectively, at each step. The analyses of residual activity after heat treatment and the denaturation profile as monitored by circular dichroism showed that thermal stability was increased with accumulation of the mutations. The kinetic parameters of most mutant enzymes were similar to those of the wild type. However, some mutant enzymes showed a reverse correlation between stability and activity: the enzymes with a large increase in thermal stability showed lower activity. Although the wild-type enzyme is unstable in the absence of glycerol, the stabilizing effect of glycerol was not observed for all the mutant enzymes containing the Glu255Val substitution, which is assumed to be located at the hydrophobic interface between two subunits.
影响因子：
5.6
作者：
Gerlind Wallon;G. Kryger;Susan T. Lovett;Tairo Oshima;Dagmar Ringe;G. Petsko
通讯作者：
Gerlind Wallon;G. Kryger;Susan T. Lovett;Tairo Oshima;Dagmar Ringe;G. Petsko