Gene Cloning And Expression: Alkaline Lipase From Penicillium Expansum PF898

Penicillium expansum PF898, which is a mutant strain by many generation mutagenesis and breeding from the original strain isolated from soil in China, produced an alkaline lipase at a high level. The research on the gene and protein structure of the lipase from Penicillium expansum PF898 (designated PEL) that we have done is listed as follows:1.Purification and N-terminal amino acid sequence of PELThe lipase from P.expansum PF898 was purified 79.3-fold by ammonium-sulfated precipitation, DE52 ion exchange chromato-graph and Sephadex G 100 gel filtration to a final specific activity of 76.9U/mg. The molecular weight of the enzyme was 2 8-29 kDa determined by SDSPAGE and gel filtration. Purified protein was electrophoretically transfered to a PVDF membrane to analysis N-terminal amino acid sequence. The 12 aa of N-terminal were A-T-A-D-A-A-A-F-P-D-L-H and 3 of them are different from the N-terminal aa sequence of P.expansumDSM1994:V-A-A-S-A-A-F-P-D-L-X. The homologous is about 75% between the two N-terminal sequences.2.Gene cloning and sequence of PELThe cDNA and genomic DNA encoding PEL were amplified by PCR, RT-PCR, RACE. The amplified products were ligated to plasmid pBluescript H SK(+) and the sequences of them were analyzed. The whole cDNA sequence and genomic DNA sequence were obtained and submitted to GenBank and the accession numbers were AF284064 and AF330635 respectively. The whole cDNA of the enzyme consists of 855 bp and the genomic DNA of PEL is composed of 1135 bp and has six exons and five short introns (58 bp, 47 bp, 50 bp, 56 bp, and 69 bp). The homology is about 40% between the genomic DNA sequence of PEL and that of other lipases.3.Amino acid sequence analysis and model constraction of three-dimensional structure of PELThe whole amino acid sequence of PEL (GenBank protein_id AAG22769.1) was deduced according to the cDNA sequence which encodes a protein of 285 aa including a presumptive signal peptide or signal and prepro-sequence composed of 27 aa and a mature peptide composed of 258 aa. The homology of the mature peptide between PEL and several lipases of other fungi is 33-39%. The mature peptide of PEL has 4 Cys residues and has no N梘lycosylation site, and contains a pentapeptide G-X-S-X-G highly preserved among lipases. The threedimensional structure of PEL is similar with that of other lipases and the catalytic triad is composed of Ser條32~ Asp?88. His?41. The mature peptide has a molecular weight of 27.3 kDa which is larger than that of P.expansum DSM 1994 lipase (25 kDa).4.Gene expression of PELThe mature peptide gene of PEL was cloned to construct the expression vectors pET-22b(+)Iipase and pET-41b(+)lipase and expressed in E.coli BL21(DE3) using the T7 RNA polymerase proteins as a promotor. The products were accumulated at 25% and 18% of the soluble cell protein when expression strain BL-pET-22b(?lipase and BL-pET-41b(+)lipase induced by IPTG for 4 hours, but activities of the products were not detected.The expression vectors pPIC9-lipase and pPIC9k-lipase were constructed and transformed into methylotrophic yeast Pichia pastoris (GS 115) by electroporation. Transformant GS-pPIC9-lipase and GSpPIC9K-lipase can functionally expressed and secreted 20 U/mL lipase when 0.5% methanol were fed during the cultivition. 10-30 mM (the optimum 10 mM) Ca2~ was needed for the expression protein to hydrolyze olive oil on the olive oil plate. PEL need Ca2~ for enzyme activity.