Gene Cloning And Expression Of Lipases

Lipase genes, LipRs from Rhizopus stolonifer YF6 and Lip42 from metagenome of oil dirt on the hearth of the restaurant were cloned by using degenerate PCR. A nevol highthroughput Pichia pastoris cell-surface display expression vector was constructed based on Flo1 from Saccharomyces cerevisiae and the display expression of RSL was detected. At the same time, preparing biodesiel by cells of R. Stolonifer YF6 immobilized, overexpression and characterization of Lip42 in P. pastoris were conducted. Details as following:A novel lipase-encoding cDNA from R. stolonifer YF6 was cloned by using degenerate PCR, rapid amplification of cDNA ends (RACE) and RT-PCR, and its structural gene was amplified by PCR. No intron was found in this lipase gene by nucleotide sequences alignment between the structural gene and its corresponding cDNA. The cDNA of the putative lipase gene (LipRs) consisted of 1 173 bp, including an open reading frame encoding a 26-amino acid signal peptide at the N-terminal end and a 365-amino acid mature protein with a predicted molecular mass of 39 268, a pI value of 7.66 and 5 potential N-glycosylation sites (N-X-T/S). The 7 conserved amino acid residues, GHSLGGA, required to the active site of the lipase from Rhizopus sp. were all found in this lipase, RSL. The amino acid sequences alignment result showed that the RSL had a high degree of identity with other Rhizopus lipases, and the highest one from Rhizopus oryzae (AAF32408) was 83%. This lipase gene sequence has been submitted to GenBank and its accession number is DQ139862.A novel system based on Flo1 from S. cerevisiae was developed for cell-surface display of heterologous proteins in P. pastoris. In order to test the vector, R. oryzae lipase RSL was fused to the C-terminal of the truncated peptide of Flo1p. The expression of fusion protein Flo1p-RSL was detected throughout the P. pastoris cell surface by confocal laser scanning microscopy. And lipase specific activity was detected on the MM agar medium containing olive oil and Rodanmine B. The results indicated that a Flo1p-based system could express proteins on the surface of P. pastoris and that the fusion proteins did not affect the function of which expressed protein.And also, the methanolysis of plant oils was investigated by cells of R. Stolonifer YF6 immobilized within polyurethane material. In order to enhance the methanolysis activity of whole cell, substrate-related compounds, such as 0.5% rapeseed oil and 0.1 % soybean oil, were added to the culture medium. The lyophilized immobilized whole cell was used as biocatalyst to prepare biodiesel. The molar ratio of methanol/oil was 3:1. Methanol should be added 3 times stage by stage to avoid reducing the specific activity of the immobilized whole cells. At optimized condition, reaction yield reached 89%. Various plant oils could be used as stuff to prepare biodiese fuel, not only vegetable oil, such as rapeseed oils and soybean oils, but also high acidic value wasted oil.A lipase gene was cloned by degenerate PCR from metagenome of the oil dirt on the hearth of the restaurant and designated as Lip42. Its sequence was submitted to GenBank with the accession number DQ313172 obtained. The cDNA of the putative lipase gene (Lip42) consisted of 1 692 bp, including an open reading frame encoding a 19-amino acid signal peptide at the N-terminal end and a 544-amino acid mature protein with a predicted molecular mass of 61 541.51 Dal, a pI value of 5.482 and 2 potential N-glycosylation sites (N-X-T/S). The open reading frame was transformed into P. pastoris strain GS115 under control of the AOX1 promoter by using the vector pHBM906. Secretion expression of Lip42 was detected from P. pastoris recombinant strain [GS115(pAMB768)]. Furthermore the expression was carried out at three different mediums (MM\BMM\BMMY), four different methanol concentration (0.5%/1.0%/1.5%/2.0%) to optimize the expression for shake-flask cultures at 22℃. The maximum yield of lipase is about 147.93 mg/L of culture medium. The recombinant lipase was purified in a three-step procedure involving ammonium sulfate fractionation and ion exchange. Characterizations of the purified enzyme revealed a molecular mass of 68 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, maximum activity at 30°C and pH 8.0 for hydrolysis of olive oil. The highest recombinant lipase activity, 50.60 U/mL, was got under the optimum reaction conditions. The metal ions Mg2+ can activate the recombinant lipase, whereas Ca2+, K+, Fe2+, Cu2+, and Co2+ inhibited it, and Tween 20,Triton-X100, SDS inhibited it strongly.