| As an important fine chemical product,n-butyl oleate has been extensively used in many fields.However,presently the commercial n-butyl oleate was chemically synthesized,which had many disadvantages,such as violent reaction conditions,environmental pollution and poor product quality.In order to overcome the shortcoming of traditional method in synthesizing n-butyl oleate,two lipases(Candida sp.and Aspergillus niger)were used as biocatalyst to investigate their feasibility and influence factors in synthesizing n-butyl oleate.Moreover,due to shortcoming of free lipase,two lipases were immobilized to investigate their feasibility in synthesizing n-butyl oleate.Also, the immobilization conditions and reaction conditions were optimized.The main results as followed:1.Two free lipases were used in synthesis of n-butyl oleate.The results indicated that both Candida sp.lipase and Aspergillus niger lipase can catalyze synthesis of n-butyl oleate,but Candida sp.lipase was more suitable to catalyze synthesis of n-butyl oleate than Aspergillus niger lipase. Among impact factors,polarity of reaction solvent had great effect on conversion rate,and solvents with logP＞2 were more suitable.In present study,hexane was used as reaction solvent.For Candida sp.lipase,the optimum conditions were:temperature 37℃,vibrating rate 200r·min-1, molar ratio of oleic acid to n-buanol 1,lipase dose 5000IU/25mL solvent, and the greatest conversion rate was 59.1%in 4h reaction.2.In order to overcome shortcoming of free lipase in application,lipase immobilization was carried out,and effects of support characterics and impact factors were also investigated.The results indicated that polysulfone(PS)membrane was the most suitable carrier among five kinds of membrane.Compared two lipases,immobilized Candida sp.lipase had higher protein loading and lipase activity than immobilized Aspergillus niger.The optimum conditions for immobilization of Candida sp.lipase were:adsorption time 10h,adsorption temperature 25℃,lipase solution pH 6.0,and the protein loading and lipase activity were 0.09mg·cm-2and 0.55U·cm-2,respectively.Investigation on characteristic of immobilized Candida sp.lipase showed that it was more stable than free lipase under high temperature,and it had good repeatability with remaining activity 39.0%after 5 cycles reuse.Moreover,immobilized Candida sp.lipase had good storage stability and the residual activity was 72.7%after 10 days under 4℃.3.Candida sp.lipase immobilized on PS membrane was used to catalyze synthesis of n-butyl oleate.The results indicated that,compared to free lipase,the optimum temperature of immobilized lipase didn't shift,but its ability to endure temperature fluctuation increased.Study on thermal stability of lipase showed that relative conversion rates of free and immobilized lipase were 29.9%and 65.3%of initial activity after 2h under 50℃,respectively.In addition,relative conversion rate was 65.6%after 4 cycles reuse,which indicated that immobilization increased lipase reusability.The immobilization was futher studied.Candida sp.lipase was immobilized on PTFE membrane which has non-woven fabric as support layer through filtering and crosslinking.Then the immobilized lipase was used to catalyze synthesis of n-butyl oleate,the result shows that reaction conversion rate was up to 82.0%in 4h,and there was no significant decrease after 9 cycles reuse.
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