关键词: 铜绿假单胞菌, 硫酸化胆汁酸水解酶, 胆汁酸检测, 原核表达, 立体异构反应

Abstract: Objective To clone a gene encoding BSS H1 from Pseudomonas aeruginosa and express the product. To study its enzymatic activities. Methods A target sequence named BSS H1 was identified in Pseudomonas aeruginosa genome through sequence alignment. The complete sequence of BSS H1 was amplified by PCR. The obtained segment was inserted into pET30b(+) vector. The recombinant expression vector was transformed into Escherichia Coli BL21(DE3) and was induced to express the proteion. A high expression yield was achieved by optimizing the conditions of induction. The BSS-HSD double enzyme-linked method was employed in the study of the enzymatic characters. Results The highest level expression of the enzyme was achieved when the engineered E. coli having grown for 3 hrs was induced by IPTG with a final concentration of 1 mM for 5hrs. The hydrolyzates of bile acid sulfate included both 3α-and 3β-hydroxy bile acid. At pH8.5 the activity of the cloned enzyme was (632±65) U/L for 3α- catalyzing and (52±4) U/L for 3β-catalyzing respectively. Conclusion An engineered strain of high BSS H1 expression was obtained. The unique characteristic of BSS H1 that it can transform the substrate into two types of stereoisomers has not been reported in the study of other BSS. The mechanism is unknown, which is worthy of further research.

Key words: Pseudomonas aeruginosa, bile acid sulfate sulfatase, bile acid testing, prokaryotic expression, steric isomer reaction