Lipolytica Candida Lipase Phenylethanol Chiral Separation And Bacteria Production Fermentation Conditions, The Enzymatic Properties

Lipase(EC3.1.1.3, triacylglycerol acylhydrolase) is the enzyme that degrade the fat. It was a kind of special ester-hydrolysis enzyme, found in most animals, plants and microorganism, and could catalyze the following reaction: Triglyceride + H₂O = Glycerin + fatty acid. Lipase was widely used in the synthesis of esters, resolution of racemic componds, the selective protection of intermediate, and the synthesis of high polymers and peptides.In this study, the ability of resolvingα- Phenylethanol of different strains capable of producing lipase was compared. The fermentation conditions of Candida lipolytica, purification and the characterization of this lipase was studied. The results of the experiments were listed as follows:1. Screening of strains capable of resolvingα- Phenylethanol47 strains isolated from soil and water, together with 3 reserved strains in the lab were selected to construct a pool of strains producing lipase. Then, 16 strains with high productivity were selected for next round screening. According to the results, Candida lipolytica was selected as the initial strains for following study because of its high lipase activity and high ability of resolving.2. The study of resolution ofα-PhenylethanolThe resolution ofα-Phenylethanol by lipase from Candida lipolytica was studied. The optimum resolving conditions were: reaction system(4mL) consisting ofα-Phenylethanol (0.6mol/L),Vinyl acetate (1.2mol/L) and methylenechloride using as solvent, 220 r/min, 40℃, and 72h. The optimum amount of enzyme powder was 0.2g according to the standard reaction system. Through the optimization the conversion rate of total substrate was increased from 36.1% to 45.2%, R-substrate from 61.9% to 85.4%, while S-substrate decreased from 9.8% to 4.15%. The e.e. value of the substrate was increased from 39.6% to 70.2% and products from 73.3% to 95.1%. E value increased from 9.5 to 83.9. These results showed that the ability of the enzyme powder was greatly increased through this optimization.3. Optimization of the fermentation conditions of Candida lipolytica The fermentation conditions of Candida lipolytica was optimized through one-factor, Plackett-Burman, Response Surface Methodology designs. The optimum condition was: glucose 0.250%, peptone 0.600%, K₂HPO₄ 0.603%, KH₂PO₄ 0.100%, MgSO₄·7H₂O 0.050%, olive oil 0.500%, tween-80 0.500%, initial pH 7.85, rotate speed 180 r/min, incubate in 28℃for 48h. Lipase activity after optimization increased from 91.2 U /mL to 208.9 U /mL, increased by 129%.4. The characterization of lipase from Candida lipolyticaThe properties of enzyme solution were studied. The results showed that the optimum reaction temperature was 40℃and the optimum pH was 8.0. Enzyme solution was stable under 50℃, pH 6-8. Zn²⁺, Mg²⁺, K⁺ could increase enzyme activity while Cu²⁺, EDTA inhibited enzyme activity. The process of making enzyme powder was also studied. The main procedure was sedimentation through ammonium sulfate, condensing by Polyethylene Glycol 6000 and freeze drying. The final activity of lipase powder was 3740U/g.