Cloning, Site-directed Mutation And Expression Of Alkaline Lipase Gene From Penicillium Cyclopium Mutant

Total RNA was isolated from Penicillium Cyclopium BD26 with TRIzol reagent by one step method.A 774 bp Lip PD gene was amplified by RT-PCR. Analysis of nucleotide sequence of the fragment suggested that Lip PD gene shared high similarity with the gene sequences of Penicillium Cyclopium(GenBank:AF274320)and Penicillium Expansum (GenBank:AAG22769)up to 99% identity.The cDNA encoding the mature peptide was cloned into expression plasmid pET-28a(+),pTrc-99a and pET-25b respectively, and expressed in Escherichia coli BL21(DE3) by IPTG induction respectively. A specific protein band of 27 kDa was detected by SDS–PAGE. With the halos appeared on solid agar plate,the activity of the recombinant lipase protein was detected. The further study on optimizing induction of BL21/pET-28a-Lip PD showed that the expression level of lipase was up to the highest specific activities of 29.30 U/mg with induction temperature 32℃, induction time 1h, the concentration of IPTG 1.0 mmol/L, strain density OD600 0.5. The further study on optimizing induction of BL21/ pTrc99a-Lip PD showed that the expression level of lipase exceeded the highest specific activity of 11.74U/mg with induction temperature 32℃, induction time 2h, the concentration of IPTG 0.5mmol/L, strain density OD600 0.8. The recombinant lipase protein of BL21/pET-25a-Lip PD was expressed in E. coli periplasm and the specific activity of the recombinant lipase was up to 24.19U/mg.The active site triad residues of Lip PD included Ser132,Asp188 and His241and they were identified by site-directed mutagenesis LipS132A,LipD188A and LipH241 ,respectively.The specific activity of LipG24C was 20.73U/mg at 25℃, which was 85.71% of the wild type at the same conditions. The optimum temperature of mutant was the same to the wild type . The optimum temperature for LipA252C was higher by 5℃than that of the wild type Lip PD. The specific activity of LipA252C was 34.59U/mg , which was 143% of the wild type at the same conditions.The gene of mature peptide was inserted into Pichia pastoris expression vector pPIC9K, resulting in the recombinant plasmid pPIC9K-LipPD. The pPIC9K-Lip PD was linearized by Sal I ,and then transformed into P. pastoris GS115 by electroporation. The intracellular activity of the recombinant lipase was up to 10.53U/mg. A specific protein band of 27 kDa was only detected by SDS–PAGE and the protein level was very low.