Improvement Of Isoeugenol Monooxygenase Activity By Semi-Rational Design And Directed Evolution

Vanillin is the third world largest flavor,having used in food,medicine,cosmetics,tobacco and other fields.Natural vanillin is extracted from vanilla pods,but due to it low yield and very expensive;some alternative methods is urgently needed.Therefore nowadays,vanillin production via microbial or enzymatic conversion method can be considered as a most promising alternative method due to high yield and low cost.Isoeugenol monooxygenase（IEM）is the only key enzyme for the conversion of isoeugenol to vanillin;but IEM is no so efficient because of product inhibition therefore it is an urgent needed to prepared a highly efficiently isoeugenol.At present,directed evolution is an effective method to improve enzyme activity,it requires a suitable high-throughput screening method and a mutation library.Therefore nowadays an urgent needs to develop alternative screening methods via optimizing the TBA and HPLC methods.By using this method a random mutation library of IEM was established and the mutant of V50D was obtaining with enhanced enzyme activity.In addition,by site-directed mutation T52P to mutant strain was obtained which in turn improved 35%enzyme activity.Also the present research project studies a new immobilization method（Ca-alginate/PAAm immobilization）and the method of fixing E.coli BL21（DE3）pET21a-IEM cells.The main research contents are as follows.Based on the sulfur-barbiturate（TBA）method,this study explores the detection limits of TBA,the relationship between TBA and vanillin concentration,effect of absolute ethanol,and the effect of substrate isoeugenol on TBA method.The introduction of a absolute ethanol improvement detection system,the optimized TBA detection system is 66.7%hydrochloric acid solution:1%thiobarbituric acid solution:anhydrous ethanol:sample to be tested=100μL:40μL:100μL:10μL.The standard deviation of the optimized detection system is 4.1%,which is according to the requirements of high-throughput screening.The addition amount of iseugenol should be10^-4-10^-55 mol/L.In addition,the optimal detection condition for HPLC re-screening method is gradient elution procedure 2（0 min:100%methanol/0.1%glacial acetic acid=35:65;7 min:100%methanol/0.1%glacial acetic acid）=80/20;13 min:100%methanol/0.1%glacial acetic acid=35/65).After that we combined cepPCR with Megawhop PCR to construct a library of isoeugenol monooxygenase gene mutation library.The IEM gene sequence was divided into three fragments of around 500 bp which was randomly mutated by cepPCR,and then with Megawhop PCR,to construct a series of mutant strains.Our results show that the concentration of 0.5 M manganese ion in the cepPCR mutation system is suitable.under the concentration of 0.5 M manganese ion,we obtain a plant of mutants with 1-3 mutation sites,which meets the requirements of high-throughput screening libraries.From the library,obtained eight IEM-Mutants with significant differences in enzyme activity compare to the IEM.They are F41D,V50D,F98T,V159G/D312H,G186G,K242,A348Q,and D361P.Among them,the positive mutant strains are V50D,F98T,G186G,K242E,which increase the relative IEM activity by 23%,13%,9%,15%,Using a method based on sequence conservative analysis and structural Gibbs free energy variability analysis,24 mutation sites were designed,and the corresponding IEM-mutant was successfully cloned.The enzyme activity of the mutant strain was tested and compared with the wild type（IEM）,the results show that about 50%of the mutants have higher activity than IEM.Furthermore,the IEM-T52P mutant has the highest activity.Under the above transformation system,0.63 g/L vanillin is obtained when catalyzed by the mutant IEM-T52P,which is about 35%higher than the wild-type strain IEM（0.46 g/L）.In addition,the mutants IEM-W279F/F281Q,IEM-S122N,and IEM-F216N all show light activity improvements.The yield of vanillin increased to 0.5 g/L,0.54 g/L,and 0.51 g/L,respectively.Ca-alginate/PAAm combined with trivalent aluminum ions was used to preliminary immobilize the cells,and the five conditions were studied:cell immersion time,glycerin immersion time,aluminum ion cross-linking concentration and cross-linking time.The optimal conditions for the immobilized cells were initially obtained by soaking the gel in 32g/L cells at 200 rpm for 10 h,washing the cells on the surface with pH 10.5 glycine-sodium hydroxide buffer at 100%.Then soak in glycerin for 1 h,cross-link with 0.01 M AlCl3solution for 90 min,and use glycine-sodium hydroxide solution to remove the excess AlCl₃solution on the surface of the gel to obtain immobilized cells.