Gene cloning and overproduction of low-specificity D-threonine aldolase from Alcaligenes xylosoxidans and its application for production of a key intermediate for parkinsonism drug

被引:58
作者
Liu, JQ
Odani, M
Yasuoka, T
Dairi, T
Itoh, N
Kataoka, M
Shimizu, S
Yamada, H
机构
[1] Toyama Prefectural Univ, Biotechnol Res Ctr, Lab Biocatalyt Chem, Kosugi, Toyama 9390398, Japan
[2] Kyoto Univ, Grad Sch Agr, Div Appl Life Sci, Kyoto 6068502, Japan
基金
日本学术振兴会;
关键词
D O I
10.1007/s002539900301
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
The dtaAX gene encoding a pyridoxal 5'-phosphate (pyridoxal-P)-dependent low-specificity D-threonine aldolase was cloned from the chromosomal DNA of Alcaligenes xylosoxidans IFO 12669. It contains an open reading frame consisting of 1,134 nucleotides corresponding to 377 amino acid residues. The predicted amino acid sequence displayed 54% identity with that of D-threonine aldolase from gram-positive bacteria Ar-throbacter sp. DK-38, but showed no significant similarity with those of other known pyridoxal-P enzymes. This gram-negative bacterial enzyme was highly overproduced in recombinant Escherickia coli cells, and the specific activity of the enzyme in the cell extract was as high as 18 U/mg (purified enzyme 38.6 U/mg), which was 6,000 times higher than that from the wild-type Alcaligenes cell extract. The recombinant enzyme was thus feasibly purified to homogeneity by ammonium sulfate fractionation and DEAE-Toyopearl chromatography steps. The recombinant low-specificity D-threonine aldolase was shown to be an efficient biocatalyst for resolution of L-beta-3,4-methylenedioxyphenylserine, an intermediate for production of a therapeutic drug for Parkinson's disease.
引用
收藏
页码:44 / 51
页数:8
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