Relationship between an increase in thermostability and amino acid substitutions in N-carbamyl-D-amino acid amidohydrolase

被引:19
作者
Ikenaka, Y [1 ]
Nanba, H [1 ]
Yajima, K [1 ]
Yamada, Y [1 ]
Takano, M [1 ]
Takahashi, S [1 ]
机构
[1] Kaneka Corp, Fine Chem Res Labs, Takasago, Hyogo 6768688, Japan
关键词
N-carbamyl-D-amino acid amidohydrolase; thermostabilized enzyme; D-amino acid;
D O I
10.1271/bbb.62.1672
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
For the production of D-amino acids using stable N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, the DCase gene of Agrobacterium sp. KNK712 was mutagenized to increase its enzymatic thermostability. In a search for thermostability-related amino acid sites besides the two known sites of DCase, i.e., the 57th and 203rd amino acids, the new mutant enzyme found, in which the 236th amino acid, valine, had been changed to alanine, showed a 10 degrees C increase in thermostability. These known three thermostability-related amino acids were changed to other amino acids by the PCR technique, and it was proved that the thermostability of the DCase increased when the 57th amino acid of DCase, histidine, was changed to leucine, the 203rd amino acid, proline, to asparagine, glutamate, alanine, isoleucine, histidine, or threonine, and the 236th amino acid, valine, to threonine or serine, in addition to the known mutations.
引用
收藏
页码:1672 / 1675
页数:4
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