A novel lipase from Pseudomonas fluorescens HU380:: Gene cloning, overproduction, renaturation-activation, two-step purification, and characterization
A novel lipase from Pseudomonas fluorescens HU380:: Gene cloning, overproduction, renaturation-activation, two-step purification, and characterization
复制标题
DOI：
10.1016/s1389-1723(03)80188-3
复制
发表时间：
2003-09-01
影响因子：
2.8
通讯作者：
Shimizu, S
中科院分区：
文献类型：
--
作者：
Kojima, Y;Kobayashi, M;Shimizu, S
从pBluescriptSK+构建的基因组文库中克隆了荧光假单胞菌（PseudomonasfluorescensHU 380）胞外脂肪酶基因（利帕）。核苷酸序列分析表明，该脂肪酶的开放阅读框为1854 bp。推导的氨基酸序列包括该菌株脂肪酶的内部氨基酸序列。该脂肪酶与粘质沙雷氏菌和荧光假单胞菌的脂肪酶序列相似性较高。在大肠杆菌中，利帕以包涵体的形式表达，随后用尿素溶解，然后透析。复性后的蛋白质具有可溶性和生物活性。从E.经变性-复性和两步柱层析纯化的重组脂肪酶与从R fluorescens HU 380中纯化的脂肪酶具有相同的电泳迁移率，并且两种酶在比活等理化性质上非常相似，表明重组脂肪酶蛋白具有内在的体外折叠能力。本文还讨论了它的C-末端区的功能。
The extracellular lipase gene (lipA) from Pseudomonas fluorescens HU380 was cloned from a genomic library constructed in pBluescript SK+. Nucleotide sequence analysis revealed an open reading frame of 1854 bp encoding the lipase. Its deduced amino acid sequence included internal amino acid sequences of the lipase from this strain. The lipase showed significant sequence similarity to lipases of Serratia marcescens strains and P fluorescens strains. In Escherichia coli, lipA was expressed in the form of inclusion bodies, which were subsequently solubilized by urea followed by dialysis. The refolded protein was soluble and biologically active. The lipase purified from the E. coli transformant by this denaturation-renaturation procedure followed by only two steps of column chromatographs exhibited the same electrophoretic mobility as did the enzyme purified from R fluorescens HU380, and both enzymes were quite similar in physicochemical properties such as specific activity, suggesting that the recombinant lipase protein has an intrinsic folding capability in vitro. The function of its C-terminal region is also discussed.