Cloning, expression and characterization of thermostable lipase from Geobacillus stearothermophilus DSMZ 1550

Hu Sheng; Huang Jun; Wu Hong; Mei Lehe

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Cloning, expression and characterization of thermostable lipase from Geobacillus stearothermophilus DSMZ 1550

Hu Sheng 1,Huang Jun 2,Wu Hong 3,Mei Lehe 3 *

1.School of Biotechnology and Chemical Engineering,Ningbo Institute of Technology, Zhejiang University, ZheJiang NingBo 315100

2.School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou, 310023, PR China

3.Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, PR China

*Correspondence author

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Funding: Doctoral Fund from Ministry of Education（No.200803350039）

Opened online: 1 April 2012

Accepted by: none

Citation: Hu Sheng,Huang Jun,Wu Hong.Cloning, expression and characterization of thermostable lipase from Geobacillus stearothermophilus DSMZ 1550[OL]. [ 1 April 2012] http://en.paper.edu.cn/en_releasepaper/content/4473260

A lipase gene of about 1.3 kb was cloned and sequenced from Geobacillus stearothermophilus strain DSMZ 1550. Nucleotide sequence homology analysis showed that the target gene sequence was completely identical to a published sequence (GenBank accession no. AF237623) from Bacillus stearothermophilus P1. The lipase gene was cloned into pET-28c (+) to construct an expression plasmid (pET-28c(+)-lipase), which was transformed into E. coli BL21 for expression. The cultivation parameters of the expression vector harboring strain were optimized to produce the lipase efficiently. The optimal conditions were determined as following: induction at middle stage of exponential growth (OD600 1.09) with 0.5 mM isopropylthio-d-galactoside (IPTG), induction temperature 28℃and post-induction expression for 6 h. Under the above conditions, the specific activity of lipase reached 1.89 IU/mg. Some characteristics of crude recombinant lipase were also investigated. The optimum pH and temperature of the crude lipase were 8.5 and 55℃. The enzyme activity was strongly inhibited by Zn2+, Fe2+, and phenylmethylsulfonyl fluoride (PMSF).

Keywords:Thermostable lipase; Geobacillus stearothermophilus; cloning; optimization; characterization

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