海洋氧化节杆菌KQ11右旋糖苷酶催化位点关键氨基酸

Dextranase from Arthrobacter oxydans KQ11: Identi?cation of Key Residues in Catalysis

通过同源比对,对来自海洋氧化节杆菌(Arthrobacter oxydans KQ11)的右旋糖苷酶(记作AoDex)催化域及关键氨基酸进行预测,运用定点突变将AoDex催化域418-QTDGIELYKGSTMKNTFFNANDD-440中的5个氨基酸突变为甘氨酸,获得5种突变型右旋糖苷酶原核表达载体:pColdIII-KQN-Q418G、pColdIII-KQN-D420G、pColdIII-KQN-E423G、pColdIII-KQN-D439G、pColdIII-KQN-D440G,表达产物分别记为Q418GDex、D420GDex、E423GDex、D439GDex、D440GDex。经过发酵表达,Q418GDex、D420GDex、E423GDex、D439GDex几乎没有酶活力。D440GDex酶活力与AoDex一致;所不同的是,D440GDex在25～40℃时的酶活力提高了2～3倍,最适pH值也从AoDex的5.5变为6.5。数据表明,Q418、D420、E423、D439四个氨基酸残基是AoDex催化域中的关键氨基酸。D440突变为甘氨酸对该酶的性质有较大影响,也表明其不是催化域中的广义碱。本研究表明AoDex的催化机制与糖苷酶49家族是一致的,为AoDex的功能改造提供了理论支持。

The catalytic domain and key amino acid residues of dextranase from Arthrobacter oxydans KQ11(AoDex) were predicted by homologous sequence alignment. The mutants Gln418→Gly, Asp420→Gly, Glu423→Gly, Asp439→Gly, and Asp440→Gly were obtained from the catalytic domain 418-QTDGIELYKGSTMKNTFFNANDD-440 by site-directed mutagenesis. The mutant dextranases Q418 GDex, D420 GDex, E423 GDex, and D439 GDex had almost no enzymatic activity, while D440 GDex had equal enzymatic activity to AoDex. However, at temperatures between 25 and 40 ℃,D440 GDex activity increased by 2–3 folds compared to AoDex activity. The optimum pH for D440 GDex was 6.5 while that for AoDex was 5.5. Q418, D420, E423, D439 were the key amino acid residues in the catalytic domain of AoDex. Mutation of D440 had a great impact on the enzymatic properties, suggesting it to be not the general base of the AoDex catalytic domain. These findings suggest that AoDex and the GH family 49 share a similar catalytic mechanism, which will provide theoretical support for improving the enzymatic properties of AoDex.