文献类型：期刊文献

英文题名：Computer-Aided Protein Surface Modification Strategy to Improve the Thermostability of α-Amylase

作者：Zhai, Wenxin[1];Zhu, Mengyu[1];Lin, Lin[2,3];Wei, Wei[1];Wei, Dongzhi[1]

机构：[1]East China Univ Sci & Technol, Newworld Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Shanghai Inst Technol, Sch Chem & Environm Engn, Shanghai 201418, Peoples R China;[3]Natl Engn Res Ctr Nanotechnol, Res Lab Funct Nanomat, Shanghai 200241, Peoples R China

年份：2025

卷号：77

期号：1

外文期刊名：STARCH-STARKE

收录：;EI(收录号：20243116801032);WOS:【SCI-EXPANDED(收录号:WOS:001281052900001)】；

基金：This work was supported by the grant from the National Key Research and Development Program of China (2021YFC2100300), the Shanghai Natural Science Foundation (No. 20ZR1415400), the Scientific Research Foundation of Shanghai Institute of Technology (No. YJ2022-13), and the Collaborative Innovation Project of Shanghai Institute of Technology (No. XTCX2022-14).

语种：英文

外文关键词：alpha-amylase; protein surface engineering; thermostability

摘要：Stability under high-temperature environments is crucial for amylase to function in starch-based industries. This study develops a method of modifying protein surfaces by combining computer-aided tools including FoldX, PoPMuSiC, Discovery Studio, and I-Mutant 2.0 with conserved sequence analysis. A truncated alpha-amylase triangle Amy(PTG) from Parageobacillus thermoglucosidasius DSMZ 2542 is rationally designed through this method to improve its thermostability. Seven single-site variants are constructed and five of them displayed enhanced thermostability. Next, three double-site variants are constructed with one particularly successful double-site variant N31RT213R, exhibiting a 4.3-fold longer half-life at 80 degrees C. Notably, the specific activity of N31RT213R reaches 10 567.16 U mg(-1), higher than triangle Amy(PTG) (6645.43 U mg(-1)). When applied to the corn starch liquefaction reaction, the mutant N31RT213R gets a higher yield of product concentration of about 255.70 mu g mL(-1), compared to 190.72 mu g mL(-1) for triangle Amy(PTG). Intramolecular forces analysis and surface electrostatic charges analysis are conducted to determine possible causes for the improvement. Also, molecular dynamics simulation is used to analyze the flexibility shifts of the entire protein. This innovative rational engineering approach has proven to be a successful strategy for the selection of hot spots for protein thermostability evolution and has the potential to be applied to other enzymes.