Construction And Application Of Isoeugenol Monooxygenase Fusion Protein

As a flavor, vanillin(4-hydroxy-3-methoxybenxaldehyde) is of rich milk incense and widely applied in food, drink, cosmetics and medicine etc. Natural vanillin extracted from vanilla pods is limited and far away to meet people 's growing demand. Vanillin produced by microbial or enzymatic conversion is equal to natural exacted vanillin with short period and low cost. Bioconversion vanillin is one of the most promising alternatives. Isoeugenol is an ideal substrate of microbial transformation for vanillin due to its wide sources and low price. Isoeugenol monooxygenase is the key enzyme which efficiently catalyze isoeugenol to vanillin. O ne of the key problems of bioconversion vanillin is how to effenciently prepare the isoeugenol monooxygenase. The complex production process and high cost of enzyme have limited the widely application of enzyme. Some self-assembly amphiphilic short peptides(Self-assembly peptide, SAPs, 8-18 amino acid residue) can induce the formation of active protein aggregates when they are connected with the teminal of soluble enzyme. Immobilized enzyme with increased operational stability can be prepared by cross- linked enzyme aggregates(C LEAs). Besides, isoeugenol moonoxygenase expressed in Eschericha.coli is insoluble and easy to form inclusion bodies. To futher understanding of the structure and properties of the isoeugenol monooxygenase, MBP(Maltose-Binding Protein) tag is fused to increase its solubility.The rusults of this paper are shown as the followings:(1) In this paper, the active aggregates IEM-18 A was successfully constructed with the connection of amphiphilic short peptides 18A(EWLKAFYEKVLEK LKELF) and isoeugenol monooxygenase and efficiently expressed in Ecoli BL21(DE3). Recombinant bacteria Ecoli BL21(DE3) p ET30a-IEM-18 A was appiled in the catalyzation of isoeugenol. Condition optimization for biotransformation of E.coli BL21(DE3) IEM-18 A was carried out. The optimal culture and reaction conditions were that isoeugenol monooxygenase was induced with 0.8 mmol/L IPTG at 30? for 8 h with shaking at 200 rpm when OD600 was 0.8. And 9 mL 0.05 mol/L pH 10.5 glycine-sodium hydroxide buffer was used to suspend cells. The highest concentration of vanillin was 2.20 g/L at the optimum reaction condition of 150 g/L enzyme amount, 1 mL DMSO, 100 mmol/L isoeugenol, 25? for 36 h with shaking at 200 rpm in 50 mL conical flask.(2) When the active aggregates as described above were obtained, glutaraldehyde was adopted as cross- linking angent to prepare immobilized IEM-18 A. it was showed that cross linked enzyme could be abtained after crossing link with 100 mmol/L glutaraldehyde for 4 hours in 10 mL enzyme liquid at 4?. The highest activity was showed when the reaction was carried out at 25?, and the cross linked enzyme could be reused for 7 times.(3) Plasmid pRSET-MBP was adopted to construct fusion pritein IEM-MBP with the connection of isoeugenol monooxygenase. The optimal culture and reaction conditions were that the recombinant bacteria E.coli BL21(DE3) pRSET-IEM-MBP was induced with 0.8 mmol/L IPTG at 25? for 16 h with shaking at 200 rpm when OD600 was 0.8. And 10 mL 0.05 mol/L pH 10.5 glycine-sodium hydroxide buffer was used to suspend cells. The highest concentration of vanillin was 2.09 g/L at the optimum reaction condition of 100 g/L enzyme amount, 100 mmol/L isoeugenol, 25? for 48 h with shaking at 200 rpm in 50 mL conical flask.