非天然D-型氨基酸生物转化中酶的纯化及其基因序列

D-海因酶 (D- Hydantoinase,HDTase )和 N -氨甲酰基 - D-氨基酸酰胺水解酶 (D- Carbam oylase,DCase )是医药和食品工业上用于生物转化 D-型氨基酸的酶。为了用人工酶替代现用的天然酶转化工艺 ,我们用热变性、硫酸铵分级及 Q - Sepharose、Phenyl- Sepharose、Superose12等多步柱层析 ,从现用菌株 No.2 2 6 2中纯化了 HDTase和 DCase,二酶均达到 SDS- PAGE纯 ,经 N -端序列分析 ,HDTase和 DCase的 N -末端氨基酸序列分别为 :MDKL IKNGTI和 MTRQKIL AF。首先 ,根据酶的 N -末端氨基酸顺序推论并合成了正向多核苷酸简并引物 ,然后按蛋白质数据库资料 ,在比较不同菌株氨基酸残基同源性基础上 ,化学合成了数对反向多核苷酸引物 ,以菌株 No. 2 2 6 2基因组 DNA为模板 ,经 PCR扩增获得了 HDTase的部分基因序列 (～ 70 0 bp )和 DCase的全部基因序列 (912 bp ) ,为使人工酶用于非天然 D-型氨基酸的生物转化奠定了基础。

Purification and Gene Sequence of Enzymes in Microbial Bioconversion of Nonnatural D-Amino Acids

Two enzymes, D-Hydantoinase(HDTase) and D-Carbamoylase(DCase), are commercially used in the process of microbial bioconversion for nonnatural D-amino acids. In order to construct engineered strain substituted for the strain which are presently used in the process of producing D-aminoacids, two enzymes mentioned above were purified to be SDS-PAGE pure from the strain No.2262 by some steps including thermal denaturation and ammonia sulfate fractionation as well as column chromatography such as Q-Sepharose, Phenyl-Sepharose, Superose-12 etc. By N-terminal amino acid residue analysis, the partial sequences of HDTase and DCase are MDKLIKNGTI and MTRQKILAF respectively. For the sake of cloning their genes, the forward degeneracy polynucleotide primers were deduced from N-terminal amino acid residues and chemically synthesized. Afterwards, according to the homologuous sequences of the two enzymes in the different strains obtained from protein data, several reverse primers were also synthesized. With the aid of PCR technology, the partial gene sequence of HDTase and the whole gene sequence of DCase were amplified with genome DNA as the template and sequenced. The results may play an important role in bioconversion of nonnatural D-aminoacids by engineered strains.