Hakamada Y, Hatada Y, Ozawa T, Ozaki K, Kobayashi T, Ito S
Tochigi Research Laboratories of Kao Corporation, 2606 Akabane, Ichikai, Haga, 321-3497, Tochigi, Japan.
FEMS Microbiol Lett. 2001 Feb 5;195(1):67-72. doi: 10.1111/j.1574-6968.2001.tb10499.x.
An alkaliphilic Bacillus sp. strain, KSM-64, produces a mesophilic alkaline endo-1,4-beta-glucanase that is suitable for use in detergents. The deduced amino acid sequence of the enzyme showed very high homology to that of a thermostable alkaline enzyme from alkaliphilic Bacillus sp. strain KSM-S237. Analysis of chimeric enzymes produced from the genes encoding the mesophilic and thermostable enzymes suggested that the lysine residues at positions 137, 179, and 194 are responsible for their thermal stabilization. Replacing the corresponding Glu137, Asn179, and/or Asp194 with lysine by site-directed mutagenesis made the mesophilic enzyme more thermostable. Analyses of the hydrophilicity of deduced amino acid sequences and isoelectric focusing of the modified enzymes suggested that these three specific lysine residues and their replacements are all located on the surface of the enzyme molecule. This fact further suggested that specific ionic interaction is involved in the thermal stabilization of the enzyme.
嗜碱芽孢杆菌属菌株KSM - 64产生一种适用于洗涤剂的嗜温碱性内切-1,4-β-葡聚糖酶。该酶推导的氨基酸序列与嗜碱芽孢杆菌属菌株KSM - S237的一种耐热碱性酶的氨基酸序列具有很高的同源性。对编码嗜温和耐热酶的基因产生的嵌合酶的分析表明，第137、179和194位的赖氨酸残基负责它们的热稳定性。通过定点诱变将相应的Glu137、Asn179和/或Asp194替换为赖氨酸，使嗜温酶更耐热。对推导的氨基酸序列的亲水性分析和修饰酶的等电聚焦表明，这三个特定的赖氨酸残基及其替换均位于酶分子表面。这一事实进一步表明，特定的离子相互作用参与了酶的热稳定性过程。
