文献类型：期刊文献

英文题名：Exploiting loop-grafting strategy resorting on computer-aided design to improve the thermostability of alpha-amylase from Geobacillus stearothermophilus

作者：Zhu, Mengyu[1];Zhai, Wenxin[1];Jiang, Haolin[2];Lin, Lin[3,4];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]Nanjing Univ Informat Sci & Technol, Sch Elect & Informat Engn, Nanjing 210044, Peoples R China;[3]Shanghai Inst Technol, Sch Chem & Environm Engn, Shanghai 201418, Peoples R China;[4]Natl Engn Res Ctr Nanotechnol, Res Lab Funct Nanomat, Shanghai 200241, Peoples R China

年份：2023

卷号：133

起止页码：28

外文期刊名：PROCESS BIOCHEMISTRY

收录：;EI(收录号：20233314567872);WOS:【SCI-EXPANDED(收录号:WOS:001065875100001)】；

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 Shanghai Institute of Technology's collaborative innovation fundation (10120K228051) , and the Shanghai Institute of Technology's scientific research start funds (10120K226107) .

语种：英文

外文关键词：Alpha-amylase; Loop-grafting; Molecular dynamic simulation; Thermostability; Corn starch liquefaction

摘要：Starch-based industries require a robust alpha-amylase capable of withstanding elevated temperatures and long incubation periods. In this study, a loop-grafting strategy based on a computer-aided design was proposed to engineer a thermostable alpha-amylase from Geobacillus stearothermophilus. Molecular dynamics (MD) simulations and trajectory analyses were employed to target the weak spots responsible for thermal inactivation, and three loop regions were identified as weak spots requiring improvement. Subsequently, substitute candidates from the thermophilic orthologous library were selected to refine these weak spots and the most promising mutant was screened using thermal unfolding and MD simulations. Expectedly, an alpha-amylase variant was constructed efficiently and specifically, which can withstand high-temperatures up to 100 degrees C without any stability-activity trade-off, giving an 8.0-fold longer half-life at 100 degrees C. Notably, the variant exhibited excellent thermotolerance during corn starch liquefaction at 100 degrees C, giving a 3.3-fold increased product concentration. Furthermore, the origin of the enhanced thermostability based on the dynamic trajectory of the variant was revealed. Successful attempts to tailor the thermal resistance and catalytic activity of alpha-amylase can further fuel the food industry, particularly high-temperature starch-based industries.