摘要
环糊精葡萄糖基转移酶(cyclodextrin glucosyltransferase,CGTase)能够以淀粉为底物发生环化反应生成环糊精,包括α-环糊精(α-cyclodextrin,α-CD)、β-环糊精及γ-环糊精,其中α-环糊精产量低且易降解。该研究选择Bacillus stearothermophilus NO2来源的CGTase,考察其突变体E142P、L277M、L277F、E142P/L277M、E142P/L277F、L277M/N353A及L277F/N353A以可溶性淀粉为底物生产α-CD的能力。与野生型相比,最优突变体为E142P/L277M,α-环糊精产量提高1.7 g/L,且在产物中占比提高14.3%。分子动力学(molecular dynamics,MD)模拟结果表明,E142P/L277M突变体中2个突变位点相互影响较小。此外,L277M/N353A催化效果较差,MD结果表明该突变体整体结构变化较大且溶剂可及表面积增大,不利于转糖苷反应的发生。该研究为CGTase的分子改造提高环化反应活性以及CGTase在α-环糊精工业化生产中提升产量提供了新思路。
Cyclodextrin glucosyltransferase(CGTase)can convert starch to cyclodextrins,includingα-cyclodextrin(α-CD),β-cyclodextrin(β-CD)andγ-cyclodextrins(γ-CD).Among these cyclodextrins,α-CD has characteristics of low yield and easy degradation.In this study,a CGTase derived from Bacillus stearothermophilus NO2 was selected and theα-CD producing ability of its mutants,including E142P,L277M,L277F,E142P/L277M,E142P/L277F,L277M/N353A,and L277F/N353A,were measured using soluble starch as substrate.Compared with the wild-type,the optimal mutant E142P/L277M increased the yield ofα-CD by 1.7 g/L and the proportion ofα-CD in the product increased by 14.3%.Molecular dynamics(MD)simulation results showed that the influence between the sites of E142 and L277 in E142P/L277M mutant was slight.In addition,the catalytic effect of L277M/N353A was poor,and MD results showed that the overall structure of L277M/N353A was changed greatly and its SASA was increased,which were not conducive to the occurrence of transglycoside reaction.This paper provides a new idea for the molecular modification of CGTase to improve the activity of cyclization reaction and increase the yield of CGTase in the industrial production ofα-CD.
作者
孔德民
左方圆
吴敬
王蕾
KONG Demin;ZUO Fangyuan;WU Jing;WANG Lei(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,China;Key Laboratory of Industrial Biotechnology Ministry of Education,Jiangnan University,Wuxi 214122,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2023年第15期1-7,共7页
Food and Fermentation Industries
基金
国家自然科学基金重点项目(31730067)
江苏省自然科学基金面上项目(BK20221536)
江苏省研究生科研与实践创新计划项目(KYCX21-2025)。
关键词
环糊精葡萄糖基转移酶
可溶性淀粉
Α-环糊精
定点突变
环化反应
cyclodextrin glucosyltransferase
soluble starch
α-cyclodextrin
site-directed mutation
cyclization reaction
