The Molecular Directed Evolution And Immobilization For The Lipase Lip906

Lipase(EC3.1.1.3)is a kind of enzyme which could specifically catalyze the hydrolysis of triacylglycerol.It widely exists in microorganisms,plants and animals.However,natural lipases derived from microorganisms are often limited in their application because their activity and stability can not meet the needs of industrial production.So a lot of protein transformation technology came into being.In our previous studies,a new lipase gene with a total length of 906 bp which was named Lip906 was found by metagenomic library screening.Due to its thermal stability cannot meet the needs of industrial production,we conducted a bioinformatics analysis,and transformed its molecular structure transformation through the error-prone PCR technology,and improved its stability further through the immobilization of the enzyme.We built random mutations library in use of error-prone PCR technology,and the mutant lipase Lip5-D whose activity was improved was selected from the library successfully.Only two base mutated,one was the synonymous mutation: G831A;the other was the missense mutation: A155 G,the corresponding amino acid changed to Q52 R.Compared with the wild type lipase Lip906,the activity of the mutant enzyme Lip5-D was increased by 4 times,the optimum reaction temperature was 60?,the optimum reaction pH was 7.5.The enzyme activity of Lip5-D kept at 70% in 65 ° C for 2 hours,at 60% in different pH(3 ~ 10)for 2 hours.The results show that it is feasible to reconstruct the molecular structure of the enzyme by using the error-prone PCR technique.The free enzyme is sensitive in outside environment and difficult to control.Moreover it can not be recycled.So,we conduct immobilization on the lipase Lip906.The results showed that the immobilization effect of mutant enzyme Lip5-D was the best with chitosan as carrier.,and the recovery rate of enzyme activity was 98%.Compared with the free enzyme,the optimum reaction temperature was 63?,the optimum reaction pH was 7.5;the activity of immobilized enzyme kept at about 65% in 70? for 2h,80% in different pH(3 ~ 10)for 2 hours.And its thermal stability and PH stable improved significantly.The immobilized enzyme was stored at 4? and room temperature for 20 days,the residual enzyme activity was 95.5% and 75.7%,respectively.However,The stability of the operation is not very rational,can only be repeated 6 to 8 times,and the tolerance of immobilized enzyme on the metal ions and organic solvents are significantly reduced.