Kim S W, Kim C Y, Benisek W F, Choi K Y
Department of Life Sciences, Pohang University of Science and Technology, Kyungbuk, Korea.
J Bacteriol. 1994 Nov;176(21):6672-6. doi: 10.1128/jb.176.21.6672-6676.1994.
The structural gene coding for the delta 5-3-ketosteroid isomerase (KSI) of Pseudomonas putida biotype B has been cloned, and its entire nucleotide sequence has been determined by a dideoxynucleotide chain termination method. A 2.1-kb DNA fragment containing the ksi gene was cloned from a P. putida biotype B genomic library in lambda gt11. The open reading frame of ksi encodes 393 nucleotides, and the amino acid sequence deduced from the nucleotide sequence agrees with the directly determined amino acid sequence (K. Linden and W. F. Benisek, J. Biol. Chem. 261:6454-6460, 1986). A putative purine-rich ribosome binding site was found 8 bp upstream of the ATG start codon. Escherichia coli BL21(DE3) transformed with the pKK-KSI plasmid containing the ksi gene expressed a high level of isomerase activity when induced by isopropyl-beta-D-thiogalactopyranoside. KSI was purified to homogeneity by a simple and rapid procedure utilizing fractional precipitation and an affinity column of deoxycholate-ethylenediamine-agarose as a major chromatographic step. The molecular weight of KSI was 14,535 (calculated, 14,536) as determined by electrospray mass spectrometry. The purified KSI showed a specific activity (39,807 mumol min-1 mg-1) and a Km (60 microM) which are close to those of KSI originally obtained from P. putida biotype B.
恶臭假单胞菌B型编码δ5-3-酮类固醇异构酶（KSI）的结构基因已被克隆，其完整的核苷酸序列通过双脱氧核苷酸链终止法确定。从λgt11中的恶臭假单胞菌B型基因组文库中克隆了一个包含ksi基因的2.1 kb DNA片段。ksi的开放阅读框编码393个核苷酸，从核苷酸序列推导的氨基酸序列与直接测定的氨基酸序列一致（K. Linden和W. F. Benisek，《生物化学杂志》261:6454 - 6460，1986）。在ATG起始密码子上游8 bp处发现了一个假定的富含嘌呤的核糖体结合位点。用含有ksi基因的pKK - KSI质粒转化的大肠杆菌BL21(DE3)在异丙基 - β - D - 硫代半乳糖苷诱导下表达高水平的异构酶活性。通过利用分级沉淀和以脱氧胆酸盐 - 乙二胺 - 琼脂糖亲和柱作为主要色谱步骤的简单快速程序，将KSI纯化至同质。通过电喷雾质谱法测定，KSI的分子量为14,535（计算值为14,536）。纯化的KSI显示出与最初从恶臭假单胞菌B型获得的KSI相近的比活性（39,807 μmol min-1 mg-1）和Km（60 μM）。
