海洋氧化节杆菌KQ11右旋糖酐酶催化位点关键氨基酸研究

摘要/Abstract

摘要： 摘要：通过同源比对，对来自海洋氧化节杆菌（Arthrobacter oxydans KQ11）的右旋糖酐酶（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种突变型右旋糖酐酶原核表达载体：pColdⅢ-KQN-Q418G、pColdⅢ-KQN-D420G、pColdⅢ-KQN-E423G、pColdⅢ-KQN-D439G、pColdⅢ-KQN-D440G。（表达产物记为：Q418GDex、D420GDex、E423GDex、D439Dex、D440GDex）。经过发酵表达，Q418GDex、D420GDex、E423GDex、D439Dex几乎没有酶活。D440GDex酶活与AoDex一致；所不同的是，D440GDex在25℃到40℃时的酶活提高了2-3倍，最适pH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的功能改造奠定了基础。

关键词: 关键词：右旋糖酐酶, 定点突变, 催化域, 关键氨基酸, 结构预测

Abstract: Abstract: To investigate the structure and functionality of dextranase from Arthrobacter oxydans KQ11 (AoDex) . The catalytic domain and key residues were predicted by homology alignment. Five residues were chosen from the catalytic domains: 418 -QTDGIELYKGSTMKNTFFNANDD - 440. Site-directed mutagenesis was used and five vectors were obtained: pColdⅢ-KQN-Q418G, pColdⅢ-KQN-D420G, pColdⅢ-KQN-E423G, pColdⅢ-KQN-D439G, pColdⅢ-KQN-D440G. (the protein as Q418GDex, D420GDex, E423GDex, D439Dex, D440GDex). After fermentation, Q418GDex, D420GDex, E423GDex, D439Dex almost no enzyme activity. D440GDex enzyme activity was same as AoDex. The D440GDex also showed a significant increase at 25 ° C to 40 ° C; the optimum pH also changed from 5.5 to 6.5. The four residues: Q418, D420, E423 and D439 of AoDex are in the catalytic domain and the key residues. The D440 does not be general base of the AoDex catalytic domain. The enzyme activity will be affected dramatically when the D440 was mutated to glycine. The findings suggest that AoDex and GH family 49 have a similar catalytic mechanism.

Key words: Key words: dextranase, site-directed mutagenesis, catalytic domain, key residues, structure prediction

中图分类号:

Q812

刘乐丁一王紫玄房耀维王淑军吕明生. 海洋氧化节杆菌KQ11右旋糖酐酶催化位点关键氨基酸研究[J]. 食品科学, 0, (): 0-0.

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