| Lipase was a special ester bond hydrolase, which can act on the ester bond of triglyceride and cause triglyceride degrade into diacylglycerol, monoglyceride, glycerol and fatty acids. Lipase was an important industrial enzyme and widely used in various industrial fields, which was one of the earliest enzymes being studied. Since the microbial lipase was discovered, screening high-yield microbial strains of lipase has become a research focus at home and abroad because of its following advantages such as diversity, highly specificity of substrate, wide range action pH and temperature, convenient to realize industrialization, easy to obtain and so on.In this thesis, the protoplast fusion of Geotrichum candidum NS3 protoplast and Candida valida T2 protoplast was studied by chemical PEG fusion method. In the meanwhile, the resistance of drugs was also used to screen a fusion strain with high-yield of lipase and strong growth ability. Moreover, the solid-state fermentation conditions of fusant for producing lipase were studied preliminary. The specific methods and research results are as follows:(1) The resistant marker was selected from the parents stains produced lipase. Twelve kinds of drugs were chosen as the sign of resistance to antibiotics of Geotrichum candidum NS3 and Candida valida T2, respectively by the method of concentration gradient and principles of medical administration. The resistant markers of the two stains were obtained. One of the medical tags is Qumixi slave at a concentration of 2×103μg/ml when Geotrichum candidum NS3 is resistance and Candida valida T2 non-resistance. The other one is CuSO4 at the concentration of 2×103μg/ml when Candida valida T2 is resistance and Geotrichum candidum NS3 is non-resistance.(2) The parents of the strains protoplast preparation and regeneration conditions were studied. The various factors that affect the formation rate and regeneration rate of Geotrichum candidum NS3 protoplast and Candida valida T2 protoplast were investigated by means of single factor experiment. The results showed the optimum preparation conditions for protoplasts of Candida valida T2 and Geotrichum candidum NS3 were as following:using middle exponential phase cells, the action of 1.5% cellulase enzyme at 30℃for 1 h, taking 0.05% cysteine as the reagent of promoting cell wall degradation and using 0.7M NaCl as osmotic stabilizer.(3) The protoplast fusion of the parents and identification of fusant. The protoplast fusion of parents strains protoplasts was studied by chemical PEG fusion method. Fifty individual fusants have been got by replica platting method, and eight individual plants were isolated from the first generation, a steady fusant was selected from the tenth generation culture, then the fusant named CG16. By comparing the colonial morphology and microstructure of the parents and fusant CG16, and the full-cell solubility protein electrophoresis, the result showed that CG16 fusant was a new strain derived from the protoplast fusion of parents.(4) The invesitigetion and optimization of solid-fermentation conditions of fusant CG16 have been carry out by single factor comparison experiment. The results showed that the optimal medium composition for producing lipase by fusant CG16 was:12 g solid matter (wheat bran: soybean meal=3:2),8 ml nutrient solution (g/L: KH2PO4 7.07, Na2HPO4 22.06, MgSO4·7H2O 2.0, Na2CO3 2.0, NaNO3 7.07). The lipase activity produced by CG16 reached 82.22 U/g dry matter under the fermentation conditions with 30℃, initial pH 7.0 and culture 72 h. The lipase activity of CG16 was higher 32% and 171% than that of the parent strains produced, respectively.
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