Directed Evolution Of A Lipase From Cladosporium Sp.

Lipases can be used in a diverse array of areas including food industry, oleochemical industry, detergent industry and pharmaceutical industry. Lipases are added in detergents to remove grease stains, and used as catalysts in biodiesel production. Both surfactants in detergents and methanol as a substrate for biodiesel production, can inhibit the lipase activity. In this study, directed evolution of a lipase from Cladosporium sp. was carried out to improve the tolerance towards methanol or anionic surfactants.The mutant library of Cladosporium sp. lipase was constructed by error-prone PCR and mutant genes were expressed in Pichia pastoris. Firstly, positive colonies were collected in a pre-screening on Rhodamine B plates. Then lipases with higher tolerance towards methanol were screened on BMMY/glyceryl tributyrate double-layer plates. Finally, lipase solutions were obtained through shake flask fermentations and the tolerance towards methanol or surfactants was measured, by which better mutants were spotted.Mutant#467showed a stronger tolerance to methanol. The most significant results were observed in42.5%and45%methanol concentrations, which increased39.8%and68.7%compared to wild type, respectively. This mutant lipase has two amino acid substitutions: Ile110Thr and Glu355Lys.Mutant#496has a stronger tolerance to anionic surfactants. The residual activity of mutant#496showed about2.5-fold increase towards SDS (sodium dodecyl sulphate),2-fold towards AOS (a-olefin sulfonate), and1.5-fold towards LAS (linear alkylbenzene sulfonate) and AES (polyoxyethylene fatty alcohol sodium sulfate), respectively. Sequence analysis revealed that the mutant lipase has single amino acid substitution:Lys377Glu. Furthermore, a site-directed saturation mutagenesis was applied for the residue Lys-377.Mutant Lys377Asp showed a similar tolerance like mutant#496, but mutants Lys377Arg and Lys377His showed poor tolerance like the wild type. This result indicated that the tolerance towards anion surfactant was influenced by different electrical charge of residual side chain on this site.