Gene Cloning, Expression And Directed Evolution Of Lipase From Thermophilic Fungi

Lipases (EC3.1.1.3) which full name is triacylglycerol acylhydrolases widely exists in organisms. Lipase can catalyze the hydrolysis of triacylglycerols into fatty acids and glycerol at the water-lipid interface and its reverse reaction in non-aqueous solvents. Apart from their hydrolytic capacity in aqueous medium, lipases are efficient in various biological reactions such as esterification, transesterification, and aminolysis. Lipases are ubiquitous in plants, animals, and microorganisms. Of these, microbial lipases are presently receiving great attention for diverse catalytic activity, high yield, and low-cost production as well as relative ease of genetic manipulation. Thermophilic enzymes have significant potential for industry applications because of their high inherent stability and high reaction rates at high temperatures. Thus, great efforts have been made to select thermostable lipases from thermophilic fungi.T. aurantiacus var. aurantiacus is a soil-borne thermophilic fungus that grows well at50℃. It was grown in inducing olive oil PDA medium to express lipase. Degenerate primers were designed according to the conserved sequence of lipases deposited in GenBank and used to amplify partial sequence of target lipase gene from T. aurantiacus var. aurantiacus. Full-length cDNA of T. aurantiacus var. aurantiacus lipase was obtained via RT-PCR,3’-RACE and5’-TAIL PCR. A novel lipase gene, lg, was cloned from the thermophilic fungus T. aurantiacus var. aurantiacus. DNA sequencing revealed that the genomic DNA of Ig was1118bp, with three introns. cDNA had an open reading frame of894bp, encoded297amino acid residues. Amino acid sequence was used to search the NCBI database to identify the protein by the BLASTP. It revealed this lipase belonged to the Lipase3family of Lipase Superfamily. SignalP3.0Server program analysis revealed it contained a putative signal peptide of21amino acids which was identical to the registered lipase gene lg. Post-translational modifications program of CBS Prediction analysis revealed it contained three potential N-glycosylation sites and one O-glycosylation site. The obtained nucleotide sequences were deposited in GenBank under Accession No. KC920898and KC920899.The recombinant plasmid pPIC9K/lg was constructed and electroporated into P. pastoris cells for heterologous expression of lg under transcriptional control of the AOX1promoter. Hundreds of transformants were obtained by electroporation on MD/MM plates. Multicopy transformants were detected by PCR and screened out on YPD plate with the highest concentration of G418. The clone with the highest yield of lipase, designated GS-LG-6, was chosen for lipase purification and characterization. After methanol induction, lipase LG was secreted into the culture medium. The hydrolytic activity of recombinant lipase in the culture supernatant reached19.92U/mg after induced expressionfor144h. After purification by ion-exchange chromatography, the recombinant lipase displayed a single band in the electrophoresis gel image, corresponding to a molecular mass of58kDa. Under different temperature conditions, the recombinant lipase LG showed the highest activity at50℃. This results indicated that the lipase was thermostable. Under different pH conditions, the lipase LG was relatively stable at pH8.0-12.0and showed the highest activity at pH8.0. This result indicated that the lipase LG was an alkali lipase enzyme. The lipase activity was noticeably enhanced by Ca2+. The optimum substrate of the lipase was4-nitrophenol palmitate and had good hydrolysis activity of fatty acid ester with carbon chain length of more than10.The method of directed evolution and high throughput screening for higher activity were used to modify the lipase gene lg from T. aurantiacus var. aurantiacus to enhance the activity and stability. Error-prone PCR method was adopted to establish the mutant library, which contained9654transformations. After high troughput screening, nine transformants were selected. But only one strain showed1.24fold higher activities than that expressed from the wide lg gene, which didn’t reach the expected effect.The same method were used to modify the lipase gene LN from Thermomyces lanuginosus to enhance the activity and stability. The mutant library was establish and contained11736transformations. After high troughput screening, two transformants GS-LN4and GS-LN11showed3.4and2.2fold higher activities than that expressed from the wide In gene, respectively. GS-LN4had two mutant amino acids:A96V and Q132R. Meantime, E16D, D50V, L98F, R130K, F147Y, N231D and T236S were exhibited in GS-LN11. After the mutant and wild-type lipase were purified by using methods of the fractional ammonium sulphate precipitation and ion exchange chromatography on DEAE-Sepharose, their natures were compared. And we found that the optimal temperature, optimal pH and thermostablity of the mutations lipase had changed. To understand how mutant amino acids affect lipase property,3-dimentional structures of the wide-type and mutant lipase proteins from T. lanuginosus in the study were predicted by the method of homology modeling.