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.
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)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
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)。
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
Streptomyces mobaraensis
transglutaminase
thermostability
dynamic cross-correlation
folding free energy