假单胞菌ND6菌株水杨酸羟化酶基因(nahG)的核苷酸序列分析和酶鉴定

水杨酸羟化酶是细菌萘降解途径中的关键酶，它能催化水杨酸脱羟和羟化，生成儿茶酚．假单胞菌(Pseudomonas)DN6菌株的萘降解基因位于102kb的质粒pND6-1上，以pUC18质粒为载体，制备了含有1～3kb pND6-1 DNA随机片段的基因库，通过DNA测序和DNA序列同源性分析，从基因库中筛选出含有水杨酸羟化酶基因nahG的克隆．nahG基因的大小为1305bp，编码的水杨酸羟化酶(NahG)由434个氨基酸组成，与Pseudomonas putida NCIB 9816-4菌株的水杨酸羟化酶基因相比，核苷酸序列的同源性为100％，与其它10种细菌的水杨酸羟化酶基因相比，核苷酸序列的同源性为32％～99％．酶学实验表明，ND6菌株的水杨酸羟化酶具有广泛的底物特异性，它不仅能代谢水杨酸，还能代谢多种水杨酸的衍生物，如乙酰水杨酸、黄基水杨酸、3-甲基水杨酸、5-甲基水杨酸和5-氯水杨酸等．

NUCLEOTIDE SEQUENCE ANALYSIS AND ENZYME CHARACTERIZATION OF THE PSEUDOMONAS SP.ND6 SALICYLATE HYDROXYLASE GENE (nahG)

The salicylate hydroxylase, a important enzyme in bacterial naphthalene degradation pathway, catalyzes the decarboxylative hydroxylation of salicylate to form catechol. The naphthalene degradation genes of Pseudomonas sp. strain ND6 was localized to the 102 kb plasmid, pND6-1. Using pUC18 as a vector, a gene library containing 1-3 kb of DNA fragments from pND6-1 was constructed. By means of sequencing and analogous analysis of DNA fragments cloned, a clone containing salicylate hydroxylase gene, nahG, was selected. The size of the nahG gene is 1305 bp that corresponds to a protein of 434 amino acid residues (NahG). Pseudomonas sp. ND6 nahG gene possesses 100% identity to the salicylate hydroxylase gene of Pseudomonas putida NCIB 9816-4, and 32% to 99% identity to other ten bacterial salicylate hydroxylase genes. The enzyme assays indicated that Pseudomonas sp. ND6 NahG exhibits broad substrate specificities and metabolizes salicylate, acetylsalicylate, sulfosalicylate, 3-methylsalicylate, 5-methylsalicylate and 5-chlorosalicylate.$$$$