Identification and over-expression of a thermostable lipase from Geobacillus thermoleovorans Toshki in Escherichia coli.
A newly isolated thermophilic strain producing thermostable lipase was identified based on 16S rRNA sequencing, where phylogenetic analysis revealed its closeness to Geobacillus thermoleovorans. Thermostable lipase from this bacterium was cloned using consensus degenerate PCR primers. For over-expression in Escherichia coli, the lipase gene was sub-cloned in pET 15b vector with a strong T7 promotor. Lipase activity was approximately 4.5-fold higher than in the wild-type strain. The lipase enzyme was thermostable at 60 degrees C and pH 8, whereas a 30% residual activity was retained when incubated for 1h at 100 degrees C. Optimum lipase expression was obtained in 2 x YT medium after 70min of induction by IPTG.
Abdel-Fattah
Yasser R
YR
Bioprocess Development Department, Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific Research and Technology Applications, New Burg El-Arab City, Alexandria, Egypt. yasser1967@yahoo.com
Gaballa
Ahmed A
AA
D001406:Bacillaceae / Q000145:classification / Q000201:enzymology* / Q000302:isolation & purification; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D008049:Lipase; C428386:thermostable lipase