Myxococcus sp.V11海藻糖合酶TreSⅡ分子改造

Site-directed Mutagenesis Improves the Thermostability of Trehalose Synthase TreSⅡfrom Myxococcus sp.V11

来源于黏细菌Myxococcus sp.V11的海藻糖合酶(trehalose synthase,EC 2.4.1.245)TreSⅡ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显示出一定的应用潜力,但TreSⅡ对热敏感,在60℃保温3h,酶活性丧失,限制了其应用范围。目的:拟探索TreSⅡ影响热稳定性的氨基酸残基构成,通过对可能的氨基酸位点进行定点突变,以期获得耐热性的突变子,扩大TreSⅡ应用范围。方法:通过PCR介导的方法对TreSⅡ可能影响到热稳定性的氨基酸Q3、A283、W374、R449和Y537进行定点突变,以野生型重组酶为对照,比较突变型与野生型的最适反应温度和最适反应pH,通过测定不同温度下保存不同时间后的残留酶活,检测突变子的耐热效果。结果:研究表明突变子Q3D、A283R、W374D、R449Q和Y537H的比酶活与野生型无显著差异,且最适pH和最适反应温度也未发生改变;A283R、Y537H在60℃条件下,3h后活性剩余68%;Q3D、W374D、R449Q在温度60℃时,3h后活性剩余35%。结论:TreSⅡ分子结构中与金属离子结合的几个氨基酸残基的改变对蛋白质分子的耐热性具有显著影响。

The trehalose synthase(EC 2.4.1.245)from Myxococcus sp.V11(TreSⅡ)catalyzes the reversible interconversion of maltose and trehalose.The high catalytic activity and high conversion rate of maltose into trehalose of TreSⅡindicate that it has potential application in industrial production of trehalose.However,the thermal instability of TreSⅡlimits its wide application in trehalose production.Objective:The effects of amino acid residues mutations on the thermal stability,optima of pH and temperature,and specific activity of TreSⅡwere studied by site-directed mutagenesis.Methods:Site-directed mutation experiment of the two possible metal ion-binding sites(A283 and Y537)and the three sites(Q3,W374 and R449)in two regions which may correlate with thermostability by using overlapping PCR were performed.Mutants of A283 R,Y537 H,Q3 D,W374 D and R449 Q were heterogeneous expressed in E.coli BL21(DE3).At the same time the specific activity,the optimum reaction temperature,the optimum pH and the thermal stability of mutants were compared with wildtype strain.Results:Mutation of Q3 D,W374 D,R449 Q,A283 R and Y537 H enhanced the thermal stability,but did not affect the pH and temperature optima.Only the mutant R449 Q reduced the specific activity.The modified enzymes A283 R and Y537 H showed 68%and the mutants Q3 D,R449 Q,W374 D showed 35%of maximal activity after incubating in maltose substrate for 3 h at 60℃compared to only 20%activity for wild-type enzyme.Conclusion:These factors may render TreSⅡrelatively more thermostable among mesophilic trehalose synthases.The thermophilic amino acid residues provided herein may provide guidance for further protein engineering in the design of stabilized enzymes.