Expression Of The Yarrowia Lipolytica Lipase Lip2 In Mut~ S Recombinant Pichia Pastoris

The extracellular lipase Lip2 from Yarrowia lipolytic (YlLip2) with high activities of hydrolysis, esterification and transesterification, has been widely used as a catalyst in industry for biodiesel production, food process and optical resolution of chiral compounds. In this work, we constructed a Mut~s recombinant Pichia pastoris strain and selected a highest lipase production clone. Then, the fermentation conditions in shaking flask and 10L bioreactor were studied. The main work and the results were as follows:1. Recombinant plasmid pPICZα-lip2 previously constructed in our laboratory was transformed into P. pastoris KM71H, resulting in a series of Mut~s strains.2. After transformation, the transformed cell was spread for growth and selection on YPDS plates.Then, the transformants were picked and inoculated on rhodamine B plates to qualitatively screen high-level lipase expression clones. High-level expression clones were subsequently subjected to flask fermentation by quantitative screening. As a result, a high production of YlLip2 transformant No.25 was obtained, accumulating lipase activity in the supernatant to the level of 1,080 U/mL after induction for 72 h. The No.25 strain were transferred into shaking flasks for further determination of fermentation conditions with the test of methanol concentration, methanol inducement period, pH, liquid volume, inoculation quantity and the mixed substrate strategy.3. Fermentation was also performed in a 10 L bioreactor. The fermentation conditions of Mut~s strain were compared with those of Mut+ strain. When only adding methanol, the highest extracellular lipase activity and the highest protein content were 12,000 U/mL and 2.551 g/L. The effect of different carbon sources (glycerol, sorbitol and lactic acid) mixed with methanol at a ration of 1: 2 on YlLip2 production was also investigated. It was indicated that sorbitol was the best mixed substrate, which resulted in a maximum YlLip2 activity of 19,800 U/mL. Then, the effect of different mixed ratios of sorbitol and methanol on YlLip2 production was studied in order to further improve the expression level of YlLip2. The results showed that the highest extracellular lipase activity and the highest protein content were 30,400 U/mL and 4.758 g/L, respectively, under the optimum mixed substrates (methanol: sorbitol = 1: 1). The highest extracellular lipase activity and biomass under the optimum mixed substrates were approximately 2.53-and 1.21-fold higher than those by feeding the methanol alone. In addition, the pigment content was markedly lowered than other induction strategies, which is conducive to purification of the enzyme.