基于动态互相关性和折叠自由能变分析的谷氨酰胺转氨酶热稳定性改造  

Thermostability modification of transglutaminase based on dynamic cross-correlation and folding free energy analysis

作　　者：阳鹏辉 邱文轩 叶佳才 堵国成 刘松 YANG Penghui;QIU Wenxuan;YE Jiacai;DU Guocheng;LIU Song(Science Center for Future Foods,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China;Jiaxing Institute of Future Food,Jiaxing 314031,China)

机构地区：[1]江南大学,未来食品科学中心,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122 [3]嘉兴未来食品研究院,浙江嘉兴314031

出　　处：《食品与发酵工业》2024年第24期1-8,共8页Food and Fermentation Industries

基　　金：国家重点研发计划(2021YFC2101400);国家自然科学基金(32071474,31771913)。

摘　　要：茂原链霉菌(Streptomyces mobaraensis)谷氨酰胺转氨酶(transglutaminase,TGase)能催化蛋白质交联,被广泛用于食品加工。前期研究构建了S.mobaraensis TGase耐热突变体FRAPD-TGm2,提高了其在高温条件下的应用性能。该研究基于动态互相关性鉴定得到热稳定性进一步提高的TGase突变体。首先,通过分子动力学模拟和动态互相关性分析确定了FRAPD-TGm2分子中与其底物口袋柔性区域具有动态互相关性的48个残基。其次,基于Rosetta Cartesian_ddg对上述残基进行虚拟饱和突变,对折叠自由能变化小于-1 kcal/mol的20个突变体进行表达、纯化和酶学性质表征。其中,突变体FRAPD-TGm2-Y34 W的60℃半衰期和65℃比酶活力分别达到100.5 min和134.2 U/mg,较FRAPD-TGm2提升89.1%和28.5%。此外,发现了远端位点His201和Asn32对FRAPD-TGm2比酶活力和热稳定性具有显著影响。结果表明,基于动态互相关性和折叠自由能变分析的酶分子改造能有效提高TGase的热稳定性。Streptomyces mobaraensis transglutaminase(TGase),with its ability to catalyze protein cross-linking,is widely applied in food processing.Previously,a thermostable variant FRAPD-TGm2 of S.mobaraensis TGase was constructed to improve its application performance under high temperatures.This study identified a TGase variant with further improved thermostability based on dynamic cross-correlation analysis.Firstly,based on molecular dynamics simulation and dynamic cross-correlation analysis,this study identified 48 residues exhibiting dynamic cross-correlation with the flexible region of the FRAPD-TGm2 substrate-binding pocket.Subsequently,virtual saturation mutagenesis on these residues employing Rosetta Cartesian_ddg resulted in 20 variants with a folding free energy change of less than-1 kcal/mol.These mutants were then expressed,purified,and subjected to enzymatic property characterization.Among them,the 60℃half-life and 65℃specific enzyme activity of the variant FRAPD-TGm2-Y34 W reached 100.5 min and 134.2 U/mg,respectively,which were 89.1%and 28.5%higher than that of FRAPD-TGm2.Furthermore,this study observed significant effects on enzyme activity and thermostability in FRAPD-TGm2 due to distal residues His201 and Asn32.The above results indicate that enzyme molecular redesign,based on dynamic cross-correlation and folding free energy analysis,can effectively enhance the thermostability of TGase.

关 键 词：茂原链霉菌 谷氨酰胺转氨酶 热稳定性 动态互相关性 折叠自由能 

分 类 号：TS201.25[轻工技术与工程—食品科学]