Ikenaka Y, Nanba H, Yajima K, Yamada Y, Takano M, Takahashi S
Fine Chemicals Research Laboratories, Kaneka Corporation, Hyogo, Japan.
Biosci Biotechnol Biochem. 1998 Sep;62(9):1672-5. doi: 10.1271/bbb.62.1672.
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.
为了利用固定化形式的稳定N-氨甲酰-D-氨基酸酰胺水解酶（DCase）生产D-氨基酸，对土壤杆菌属菌株KNK712的DCase基因进行诱变以提高其酶的热稳定性。在寻找除DCase已知的两个位点（即第57位和第203位氨基酸）之外的与热稳定性相关的氨基酸位点时，发现的新突变酶中第236位氨基酸缬氨酸已变为丙氨酸，其热稳定性提高了10℃。通过PCR技术将这三个已知的与热稳定性相关的氨基酸替换为其他氨基酸，结果证明，除了已知的突变外，当DCase的第57位氨基酸组氨酸变为亮氨酸、第203位氨基酸脯氨酸变为天冬酰胺、谷氨酸、丙氨酸、异亮氨酸、组氨酸或苏氨酸以及第236位氨基酸缬氨酸变为苏氨酸或丝氨酸时，DCase的热稳定性会提高。
