Cloning, Modifying And Expression In Pichia Pastrois Of A Lipase Gene From Pseudomonas Fluorescens

Lipases are widespread hydrolyses, which catalyze the hydrolysis of ester bonds of triacylglycerols at the interface between the aqueous and non-aqueous phase. Lipases are a versatile group of enzymes and often show other activities. Because of their versatility and superiority as biocatalyst, lipases are widely used within many fields.In the body of animals, lipases are secreted by pancreas and digest the fat in the food. Lipases have an expansive prospect as feed additive. The reasons are lipases can remove the lipid anti-nutrition factors in the plant feed resources and reparation the deficiency of lipase in young domestic animals and birds. As regards the commercial lipases product, their pH stability is weak alkali and can't tolerate acid. Thus there are few fit for used as feed additive. The expression of acid tolerate lipases are very few. In this thesis, we study the expression in Pichia pastoris of lipase which is propitious to feed. The research includes the items as follows.1. Primers were designed based on the conserved nucleic acid sequences of lipase genes from Pseudomonas fluorescens. A gene fragment was amplified with these degenerate primers. Reverse PCR strategy was used to get the upstream and downstream sequences of this fragment. Thus the whole gene sequence had been gotten and registered in Genbank, whose accession number was DQ305493.2. According to bias in codon choice of Pichia pastoris and factors affecting gene expression and regulation in eukaryote, the gene encoding the lipase from Pseudomonas fluorescens was modified with the same amino acid sequence. Through DNA polymerase reaction, a modified lipase gene was obtained.3. The original and modified genes of P. fluorescens lipase were expressed in Pichia pastoris strain GS115 respectively. Both of them were secreted expression and their enzyme activities in the shake flask medium were 41.5U/mL and 91U/mL, which meaned through modification of the lipase gene the production was increased 2 times.4. The recombinant lipase had excellent characteristics. Its optimal activity pH was 8.5. Within the range of pH 4-10, its relative activity was beyond 60%. Having been treated in pH 2.2 to 12, the residual activity was more than 70%, which showed this lipase was acid-resistant and alkali- resistant. Its optimal activity temperature was 50℃. In the range from 35℃ to 60℃, the residual activity was beyond 60%. It possessed distinct thermal stability. Having been kept in 60℃ for 30 minutes, the residual activity was 70%. Metallic ion had very little influence on the recombinant lipase activity. The recombinantlipase could tolerate the action of pepsin and trypsin. It could tolerate acid treatment mimicing pH of the stomach and hydrolyzation of the pepsin. In the temperature of digestive tract, the recombinant lipase relative activity was about 80%. The recombinant lipase could tolerate the action of trypsin. According to all these experiments results, the lipase from P. fluorescens was fit for being used as feed additive.