固定化γ-谷氨酰转肽酶的催化特性研究

Catalytic properties of immobilized γ-glutamyltranspeptidase from B.subtilis NX-2

B.subtilisNX-2产γ-谷氨酰转肽酶(GGT)经纯化后,以Eupergit C250 L为载体进行了共价固定化,固定化酶活回收率为17.59%,蛋白回收率为32.74%;活性位点滴定的结果表明固定化后,GGT活性位点浓度为游离酶的79.8%。固定化酶的最适pH和温度分别为9.5和50℃,且温度和pH适用范围均有所扩大。测定得到了固定化GGT对供体(γGPNA)的米氏常数为Km=2.7463 mmol.L-1,谷氨酰基化反应催化常数为Kcat=2 670.01 min-1,较游离酶有所降低。固定化酶的温度及pH稳定性均较游离酶有大幅度提高,经50 d贮藏(4℃)和25个批次转化后,固定化GGT的残余活力仍可达初始值的79%。

γ-Glutamyltranspeptidase from B.subtilis NX-2 is purified and immobilized onto Eupergit C250 L.The yield of activity and recovery of protein are 17.59% and 32.74%,respectively.The active site concentration of immobilized GGT is titrated as 79.8% of the free enzyme by the Woodward's Reagent K(WRK).The optimal pH and temperature of immobilized GGT are 9.5 and 50℃,and the application ranges of pH and temperature are extended.The kinetic parameters of glutamylation catalyzed by immobilized GGT are determined as Km=2.7463 mmol·L-1 and Kcat=2670.01 min-1.The pH and temperature stability of GGT are increased after immobilization.During a 50 days of storage period at 4℃ and repeated usage for 25 batches,the immobilized enzyme still can keep over 79% of its initial activity.