Liu J Q, Odani M, Yasuoka T, Dairi T, Itoh N, Kataoka M, Shimizu S, Yamada H
Laboratory of Biocatalytic Chemistry, Biotechnology Research Center, Toyama Prefectural University, Kosugi, Japan.
Appl Microbiol Biotechnol. 2000 Jul;54(1):44-51. doi: 10.1007/s002539900301.
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 Arthrobacter 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 Escherichia 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.
从木糖氧化产碱杆菌IFO 12669的染色体DNA中克隆出编码吡哆醛5'-磷酸（吡哆醛-P）依赖性低特异性D-苏氨酸醛缩酶的dtaAX基因。它包含一个由1134个核苷酸组成的开放阅读框，对应于377个氨基酸残基。预测的氨基酸序列与革兰氏阳性细菌节杆菌属DK-38的D-苏氨酸醛缩酶的氨基酸序列有54%的同一性，但与其他已知的吡哆醛-P酶没有显著相似性。这种革兰氏阴性细菌酶在重组大肠杆菌细胞中大量过量表达，细胞提取物中该酶的比活性高达18 U/mg（纯化酶为38.6 U/mg），比野生型产碱杆菌细胞提取物中的比活性高6000倍。因此，通过硫酸铵分级分离和DEAE- Toyopearl色谱步骤可将重组酶有效地纯化至同质。重组低特异性D-苏氨酸醛缩酶被证明是一种有效的生物催化剂，可用于拆分L-β-3,4-亚甲二氧基苯基丝氨酸，后者是帕金森病治疗药物生产中的一种中间体。
