High Density Cultivation Of Genetic Engineering Strain Of Lipase And Its Induced Production

In this thesis, the production conditions of Rhizopus oryzae lipase in Pichia pastoris were optimized and its high density cultivation was investigated. The inoculum concentration, glycerol and methanol feeding strategy of R. oryzae lipase in Pichia pastoris were studied. Various strategies of reducing recombinant lipase proteolytic degradation were investigated. R. oryzae lipase gene was successfully mutated and expressed in Pichia pastoris. The main results are as follows:1. The optimal production conditions for R. oryzae lipase in Pichia pastoris are shown as follows:initial culture pH6.0, additive amount of methanol 1%, induction time 96h, induction tempereture 30℃. Compared with yeast extract and peptone as nitrogen source,2% corn steep powder is more efficient to higher expression of the lipase. The lipase activity could reach 295U/mL in corn steep powder medium, which increased by 20% of that in yeast extract and peptone as nitrogen source.2.7.5% inoculum concentration is fit for growth and expression of R. oryzae lipase in Pichia pastoris. The DO and gradient-control glycerol feeding strategy need 6 h to increase the cell biomass and shorten 24h the glycerol feeding time. The methanol has been added on-line methanol electrode-based on-off control method during induction phase, the lipase activity and protein concentration were 692U/mL and 1.6g/L, which is two times as much as the expression in DO-stat method.3. The results indicate that the condition of pH5.0, adding 0.5% casein,22℃induction tempereture is a proper support to increase lipase activity and protein expression. SDS-PAGE analysis revealed that the degradation of lipase protein was reduced.4. On the basis of the above, high density cultivation of R. oryzae lipase in Pichia pastoris was expored in 10 L fermentor. The R. oryzae lipase in Pichia pastoris was optimized under the circumstance of 7.5% inoculating amount,30℃in growth phase, pH 5.0, DO and gradient-control glycerol feeding strategy,22℃in methanol feeding phase,0.5% casein, 0.3%-0.5% methenol added on-line methanol electrode-based on-off control method, the wet cell weight of broth reached 419.0g/L, the dry cell weight of broth reached 128.3g/L, the concentration of lipase protein was 3.0 g/L. The activity of lipase could reach 1302U/mL, which is 4.4 times of the expression in the shake flask.5. The R. oryzae lipase gene was mutated successfully and expressed in Pichia pastoris. The lipase activity and protein concentration were 244U/mL and 0.38g/L respectively in shake flask, and its lipase activity and protein concentration were 503U/mL and 0.91g/L of high density cultivation respectively.