Alteration Of Substrate Specificity Of Lipase And Its Application In Hydrolysis Of Oils And Fats

Fatty acids are used widely in industries,and unsaturated fatty acids play an important role in human health,especially.Natural oils and fats contain a large amount of long-chain fatty acids,and enzymatic hydrolysis becomes the main method for the preparation of fatty acids due to the advantages of environmental friendness,and specificity.However,it is difficult to hydrolyze natural oils enriched with unsaturated fatty acids by Rhizopus chinensis lipase?RCL?.This lipase preferes medium-chain fatty acids,thus high-level production of fatty acids could not be achieved.At present,there are few studies on alteration of unsaturated fatty acids specificity of enzymes.In this research,RCL with enhanced specificity towards unsaturated long-chain fatty acids was obtained by rational design.The ability of this mutant enzyme in catalytic hydrolysis of soybean oil was correspondingly improved.Finally,these lipases was immobilized by mesoporous silica for reusability in hydrolysis of soybean oil.The major results are as follows:?1?Six residues located in binding pocket of R.chinensis lipase were mutated by site-directed mutagenesis and the mutant enzymes L285Q,T286Q,HQL,A116W,I281F,and A116W/I281F were obtained through whole-plasmid PCR,and expressed successfully in Pichia pastoris.The growth trend of recombined strain GS115/pPIC9K-RCLHQL was similar to that of the parent.?2?Properties of the variants were investigated.Compared to the parent,HQL showed enhanced specificity towards unsaturated fatty acids and increased hydrolytic activity for pNPC16 by 2.72 fold.The Km value remained the same as the parent,and the k_cat and k_cat/K_m values were 1.60 fold than that of RCL.Inserting a hydrophilic glutamine between His 284and Leu 285 increased the catalytic activity and promoted the interaction between the lipase and substrate,while activity of other mutant enzymes was not improved.The results showed that the optimum reaction temperature of the variants was 40°C,and the optimum reaction pH of the variants was 8.0,which were consistent with the parent.?3?The reaction conditions on hydrolysis of soybean oil by the lipase were optimized,which were the ratio of water to oil 1:1?w/w?,500 U·g^-1?oil mass?of lipase,pH 8.0,temperature 40°C and incubated for 24 h.Under the optimal reaction conditions,the hydrolysis rate of soybean oil by the variant HQL was increased to 98%due to the increased specificity towards unsaturated long-chain fatty acids,while that by the parentwas only 80%.?4?Mesoporous silica-immobilized lipases were prepared.The optimum reaction temperatures of RCL-HMMS and HQL-HMMS were 40°C and 50°C,respectively.The other optimum conditions for the hydrolysis of soybean oil by mesoporous silica immobilized lipase were determined:the ratio of water to oil 1:1?w/w?,500 U·g^-1?oil mass?of lipase,pH 8.0.Under the optimum reaction conditions,the hydrolysis rate of soybean oil by RCL-HMMS was 70%,while that by HQL-HMMS was increased to 95%.Reusability of the two immobilized lipases in hydrolysis of soybean oil were initially investigated.