文献类型：期刊文献

英文题名：A method to rationally increase protein stability based on the charge-charge interaction, with application to lipase LipK107

作者：Zhang, Lujia[1,2];Tang, Xiaomang[1];Cui, Dongbing[1];Yao, Zhiqiang[1];Gao, Bei[1];Jiang, Shuiqin[1];Yin, Bo[1,3];Yuan, Y. Adam[3,4];Wei, Dongzhi[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Nanjing Univ Technol, State Key Lab Mat Oriented Chem Engn, Nanjing 211800, Jiangsu, Peoples R China;[3]Natl Univ Singapore, Suzhou Res Inst, Suzhou 215123, Jiangsu, Peoples R China;[4]Natl Univ Singapore, Dept Biol Sci, Ctr Bioimaging Sci, Singapore 117543, Singapore

年份：2014

卷号：23

期号：1

起止页码：110

外文期刊名：PROTEIN SCIENCE

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000328566600010)】；

基金：Grant sponsor: The National Natural Science Foundation of China; Grant numbers: 20976053/B060804, 21002029/B020706; Grant sponsor: National High Technology Research and Development Program of China; Grant numbers: 2012AA020403; Grant sponsor: "973" Program; Grant number: 2012CB721003; Grant sponsor: National Major Science and Technology Projects of China; Grant number: 2012ZX09304009; Grant sponsor: National University of Singapore (Suzhou) Research Institute; Grant number: R-2012-N-007; Grant sponsor: Open Funding Project of the State Key Laboratory of Bioreactor Engineering; Grant sponsor: The funding of State Key Laboratory of Materials-Oriented Chemical Engineering; Grant number: KL10-09.

语种：英文

外文关键词：designed protein stabilization; electrostatic stabilization; lipase stabilization; surface charge-charge interaction; Lipk107

摘要：We report a suite of enzyme redesign protocol based on the surface charge-charge interaction calculation, which is potentially applied to improve the stability of an enzyme without compromising its catalytic activity. Together with the experimental validation, we have released a suite of enzyme redesign algorithm Enzyme Thermal Stability System, written based on our model, for open access to meet the needs in wet labs. Lipk107, a lipase of a versatile industrial use, was chosen to test our software. Our calculation determined that four residues, D113, D149, D213, and D253, located on the surface of LipK107 were critical to the stability of the enzyme. The model was validated with mutagenesis at these four residues followed by stability and activity tests. LipK107 mutants D113A and D149K were more resistant to thermal inactivation with approximate to 10 degrees C higher half-inactivation temperature than wild-type LipK107. Moreover, mutant D149K exhibited significant retention in residual activity under constant heat, showing a 14-fold increase in the half-inactivation time at 50 degrees C. Activity tests showed that these mutants retained the equal or higher specific activity, among which noteworthy was the mutant D253A with as much as 20% higher activity. We suggest that our protocol could be used as a general guideline to redesign protein enzymes with increased stabilities and enhanced activities.