| Known as the "King of food flavors",vallinin is an excellent flavour and food additives which has relatively stable performance,pure and unique flavors and long lasting fragrance.It is one of the most widely used flavor compounds in food,beverages,pharmaceuticals,perfumes,and cosmeti industries.Vanillin produced by microbial or enzymatic conversion is equivalent to natural extracted vanillin.Compared with chemical synthesis,the reaction conditions of microbial conversion were mild and it could meet requirements from EU and FDA for safety of the natural flavors.Isoeugenol monooxygenase is the key enzyme which could efficiently convert isoeugenol to vanillin.However there are some problems such as substrate inhibition,product inhibition and difficult enzyme preparation.These have limited the wide application of isoeugenol monooxygenase.In this thesis,modified isoeugenol monooxygenase was constructed with association of an amphiphilic self-assembling peptide and the self-cleavable Mxe intein and then applied to the expression and purification of recombinant protein.The kinetic properties of recombinant protein were analyzed.of the immobilization of isoeugenol moonoxygenase with cross-linked enzyme aggregates(CLEAs)was studied.The methods and results of this thesis are shown as the following:(1)A recombinant plasmid pET(30a)-IEM-Mxe-ELK16 was successfully constructed associated with an amphiphilic self-assembling peptide and a self-cleavable intein and then expressed in E.coli BL21(DE3).The molecular weight of fusion protein was about 75 kDa from SDS-PAGE.Compared with the initial IEM,the Vmax,Km and Kcat of recombinant enzyme IEM-Mxe-ELK16 was 11.3,11.7 and 7.2 times,respectively.The results showed that the substrate-affinity to the modified enzyme IEM-Mxe-ELK16 was lower,so that the substrate inhibition was reduced and the maximum reaction rate was higher.The culture and reaction conditions were optimized.Isoeugenol monooxygenase was induced with 0.2 mg/L IPTG at 30? for 8 h with 200 rpm when OD600 reached 0.7.After centrifugation,9 mL 0.05 mol/L pH 10.5 glycine-sodium hydroxide buffer was used to resuspend cells.The highest concentration of vanillin was 1.80 g/L at the optimum reaction condition of 100 g/L crude enzyme,1 m L DMSO,150 mmol/L isoeugenol,25? for 48 h with shaking at 200 rpm.(2)Isoeugenol moonoxygenase was fused with the amphiphilic self-assembling peptideELK16 and the self-cleavable Mxe intein.The optimization on the self-cleavable system mediated by Mxe intein was carried out.The optimal reaction conditions were as following: 10 OD/m L lysed cells were suspended with cleavage buffer(20 mmol/L Tris-HCl,0.5 mol/L NaCl,1 mmol/L EDTA-2Na,pH 8.5)at 4? and cleaved for 24 h.(3)The isoeugenol moonoxygenase fusion protein was immobilized by CLEAs technology.It was showed that the optimal cross-linked enzyme IEM-Mxe-ELK16 was obtained after crossing linking with 100 mmol/L glutaraldehyde for 2 hours.The cross-linked enzyme has good stability in the range of 50?-100?,and it could be reused for 7 times.The highest concentration of vanillin could reach 0.96 g/L at the optimum conversion condition of 0.025 mol/L sodium carbonate-sodium hydroxide buffer(pH 10.5),10%(v/v)DMSO,100 mmol/L isoeugenol,30?,200 rpm for 60 h. |
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