基于理性设计提高酿酒酵母烟酰胺核糖激酶1热稳定性  

Rational Design for Improving the Thermostability of Saccharomyces cerevisiae Nicotinamide Riboside Kinase 1

作　　者：王瑶 沈太松 李思晨 史红玲 姚伦广[1] 唐存多 WANG Yao;SHEN Taisong;LI Sichen;SHI Hongling;YAO Lunguang;TANG Cunduo(College of Life Science,Nanyang Normal University,Nanyang 473061,China;School of Bioengineering,Dalian University of Technology,Dalian 116024,China;Postdoctoral Innovation Practice Base,She Dian Lao Jiu Co.Ltd.,Nanyang 473300,China)

机构地区：[1]南阳师范学院生命科学学院,河南南阳473061 [2]大连理工大学生物工程学院,辽宁大连116024 [3]赊店老酒股份有限公司博士后创新实践基地,河南南阳473300

出　　处：《食品科学》2024年第24期92-99,共8页Food Science

基　　金：河南省青年科学家项目(225200810076)。

摘　　要：为了提高来源于酿酒酵母的烟酰胺核糖激酶1(nicotinamide riboside kinase 1 from Saccharomyces cerevisiae,Sc NRK1)的热稳定性,本研究基于计算机辅助技术虚拟筛选出Sc NRK1的6个单点突变体,通过定点突变技术完成6个单点突变的表达及酶学特性分析,然后挑选出3个优势突变体进行第2轮的组合突变。结果显示,经过两轮突变,获得了1个热稳定性和催化活性均显著提高的突变体T136P/S209A,其最适反应温度提高至45℃,45℃条件下的半衰期为48.98 min,是野生型Sc NRK1的4.2倍;其比活力为146.63 IU/mg,是野生型的1.98倍。本研究基于理性设计的手段,成功获得了热稳定性和催化活性显著增强的Sc NRK1突变体,有望为通过理性设计提高酶分子的热稳定性提供新思路,为烟酰胺单核苷酸的高效、低成本生产提供新酶源。In order to improve the thermal stability of nicotinamide riboside kinase 1 from Saccharomyces cerevisiae(ScNRK1),six single-point mutants of ScNRK1 were virtually designed using computer-aided technology,and their expression using site-directed mutagenesis and enzymatic characterization were carried out.Out of these,three superior mutants were selected for a second round of combined mutagenesis.The results showed that after two rounds of mutagenesis,a mutant named T136P/S209A with significantly improved thermal stability and catalytic activity was obtained.Its optimal reaction temperature was increased to 45℃,and its half-life at 45℃was 48.98 min,which was 4.2 times as high as that of the wild-type ScNRK1.The specific enzyme activity of the purified mutant enzyme was 146.63 U/mg,which was 1.98 times as high as that of the wild type.This study is expected to provide new ideas for improving the thermal stability of enzymes through rational design,and provide a new enzyme source for efficient and low-cost production of nicotinamide mononucleotide.

关 键 词：烟酰胺核糖激酶1 理性设计 定点突变 热稳定性 烟酰胺单核苷酸 

分 类 号：Q814[生物学—生物工程]