摘要
[目的]来源于黏细菌Myxococcus sp.V11的海藻糖合酶(trehalose synthase,EC 5.4.99.16)TreSI可通过转糖苷作用将麦芽糖转化成为海藻糖,在酶法生产海藻糖上有很高的应用前景,其热稳定性是制约该酶工业应用的关键。本文旨在通过定点突变对TreSI相关氨基酸残基进行改造,以期获得热稳定性强的突变子,提高其应用潜力。[方法]使用在线预测软件PoPMuSiC-2.1对TreSI每个氨基酸突变后的去折叠自由能变化(ΔΔG)进行初步预测,以SDM(site directed mutator)和mCSM(mutation cutoff scanning matrix)以及两者联合(Duet)进行预测。用重叠延伸PCR法构建11个海藻糖合酶突变体,经大肠杆菌表达和蛋白纯化后,对其酶学特性进行表征。[结果]成功筛选到2个热稳定性提高的突变子。突变子R149L、N193E的比酶活与野生型无显著差异,且最适pH值和最适反应温度也未发生改变;R149L、N193E在50℃处理1 h的残余酶活性为野生型的1.37和2.50倍;在60℃处理1 h的残余酶活性为野生型的2.41和4.32倍。[结论]本研究利用PoPMuSiC结合SDM、mCSM模拟的设计策略提高了TreSI的耐热性,为工业应用提供了新的酶资源,也为其他工业酶的定向改造提供了参考。
[Objectives]Trehalose synthase(EC 5.4.99.16)from Myxococcus sp. V11 can convert maltose into trehalose through transglycosidation, which has potential application value in the production of trehalose by enzyme catalysis. However, its heat resistance is key factor that restrict its utilization. The aim of this study was to modify the relevant amino acid residues of trehalose synthase TreSI by site-specific mutation in order to obtain mutants with higher thermostability and improve their application potential. [Methods]PoPMuSiC and SDM-mCSM duet were used to predict the amino acid sites that significantly affected the unfolding free energy change(ΔΔG). These strategies were combined to design the mutation sites for trehalose synthase(TreSI)from Myxococcus sp. V11. Site-directed mutagenesis was carried out by overlapping PCR,expression vectors were constructed for mutants. [Results]The mutant E.coli BL21(DE3)expression strains were constructed. After all mutants were expressed, the mutants R149L and N193E which had improved thermal stability was successfully screened. The specific enzyme activities of R149L and N193E mutants were not significantly different from that of the wild type, and the optimum pH and temperature of reaction were not changed. The R149L and N193E showed more residual activity than the wild type(1.37 and 2.50 times greater)after 1 h of treatment at 50 ℃. After treatment at 60 ℃ for 1 h, the residual activities of R149L and N193E were 2.41 and 4.32 times that of wild type, respectively. [Conclusions]A PoPMuSiC design strategy combined with SDM-mCSM duet were used to improve the thermostability of TreSI. The results provided new enzyme resources for industrial applications and a reference for the directional modification of other industrial enzymes.
作者
张丽霞
黄奕晴
吴嘉蕾
李元顺
王飞
ZHANG Lixia;HUANG Yiqing;WU Jialei;LI Yuanshun;WANG Fei(College of Bioscience and Bioengineering/Jiangxi Engineering Laboratory for the Development and Utilization of Agricultural Microbial Resources,Jiangxi Agricultural University,Nanchang 330045,China)
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2023年第1期55-62,共8页
Journal of Nanjing Agricultural University
基金
国家自然科学基金项目(31560031)
江西省教育厅科学技术研究项目(GJJ160387)。
