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枯草芽孢杆菌168脂肪酶基因的克隆、核苷酸序列及在大肠杆菌中的表达

Cloning, nucleotide sequence and expression in Escherichia coli of a lipase gene from Bacillus subtilis 168.

作者信息

Dartois V, Baulard A, Schanck K, Colson C

机构信息

Unité de Génétique, Université Catholique de Louvain-la-Neuve, Belgium.

出版信息

Biochim Biophys Acta. 1992 Jul 15;1131(3):253-60. doi: 10.1016/0167-4781(92)90023-s.

DOI:10.1016/0167-4781(92)90023-s
PMID:1320940
Abstract

The gene coding for an extracellular lipase of Bacillus subtilis 168 was cloned and found to be expressed in Escherichia coli. Enzyme activity measurements showed no fatty acid chain length preference. A set of Tn5 insertions which inactivate the gene were localized and used to initiate its sequencing. The nucleotide sequence was determined on two independent clones expressed in E. coli. In one of these clones, the sequence revealed a frameshift, due to the presence of an additional adenine in the N-terminal region, which caused the interruption of the open reading frame, probably allowing translation to initiate at a second ATG codon. The sequence of the wild-type lip gene from B. subtilis was confirmed on the chromosomal fragment amplified by polymerase chain reaction (PCR). When compared to other lipases sequenced to date, the enzyme described here lacks the conserved pentapeptide Gly-X-Ser-X-Gly supposed to be essential for catalysis. However, alignments of several microbial lipase sequences suggest that the pentapeptide Ala-X-Ser-X-Gly present in the lipase B. subtilis may function as the catalytic site. Homologies were found in the N-terminal protein region with lipases from different Pseudomonas species. The predicted M(r) and isoelectric point for the mature protein are 19,348 and 9.7 respectively.

摘要

枯草芽孢杆菌168胞外脂肪酶的编码基因被克隆,并发现其在大肠杆菌中表达。酶活性测定表明该酶对脂肪酸链长度没有偏好。一组使该基因失活的Tn5插入片段被定位,并用于启动其测序。在两个在大肠杆菌中表达的独立克隆上测定了核苷酸序列。在其中一个克隆中,序列显示由于N端区域存在一个额外的腺嘌呤导致移码,这使得开放阅读框中断,可能使翻译在第二个ATG密码子处起始。通过聚合酶链反应(PCR)扩增的染色体片段上证实了枯草芽孢杆菌野生型脂肪酶基因的序列。与迄今测序的其他脂肪酶相比,本文所述的酶缺乏被认为对催化至关重要的保守五肽Gly-X-Ser-X-Gly。然而,几种微生物脂肪酶序列的比对表明,枯草芽孢杆菌脂肪酶中存在的五肽Ala-X-Ser-X-Gly可能起到催化位点的作用。在N端蛋白质区域发现与不同假单胞菌属的脂肪酶具有同源性。成熟蛋白的预测相对分子质量(Mr)和等电点分别为19348和9.7。

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