摘要
为提高肌酸酶(Creatinase,EC 3.5.3.3,CRE)的热稳定性,通过序列比对的方法确定了Arthrobacter nicotianae 23710 CRE热稳定性相关位点K195,并对其进行饱和突变。与野生酶相比,突变体K195V、K195T、K195C和K195L在50℃下的半衰期分别提高260%、230%、60%和20%;其中,K195V和K195C比酶活分别提高80.7%和88.2%,其他突变体比酶活无明显变化;突变体K195V、K195T、K195C和K195L的催化效率(kcat/Km)分别提高131%、218%、83%和100%。结构分析发现,突变体K195V、K195T、K195L较野生酶分别增加了7、12和13个氢键。结果表明:对Lys195的突变可有效改变CRE的热稳定性及催化效率,且分子内部氢键的增加可能是CRE热稳定性提高的重要原因之一。
To improve the thermal stability of creatinase(EC 3.5.3.3,CRE),the"hot-spot"Lys195 in Arthrobacter nicotianae 23710 CRE is identified by sequence alignment,and saturation mutagenesis is conducted at this site.In contrast to wild-type enzyme,the mutants K195V,K195T,K195C,and K195L exhibited 260%,230%,60%,and 20%increase in half-life at 50℃,respectively;the specific activity of K195V and K195C are respectively increased by 80.7%and 88.2%.Furthermore,the catalytic efficiency(kcat/Km)of mutants K195V,K195T,K195C,and K195L are increased by 131%,218%,83%and 100%,respectively.As indicated by structure analysis,the number of hydrogen bonds of the K195V,K195T,and K195L are increased by 7,12 and 13 in comparison with the wild-type enzyme,respectively.The results indicated mutations at Lys195 could affect the thermal stability and catalytic efficiency of CRE,and the increase in hydrogen bonds may account for the improved thermal stability of CRE.
作者
阮洁
刘松
李江华
堵国成
陈坚
RUAN Jie;LIU Song;LI Jianghua*;DU Guocheng;CHEN Jian(Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2019年第10期8-14,共7页
Journal of Food Science and Biotechnology
基金
国家自然科学基金项目(31401638)
江苏省重点研发计划社会发展项目(BE2016629)
关键词
肌酸酶
热稳定性
饱和突变
分子改造
氢键
creatinase
thermal stability
saturation mutagenesis
molecular modification
hydrogen bond
