The Study On Synthesis Of Heliotropin By Co-expressing Trans-anthole Oxygenase And Formate Dehydrogenase

Heliotropin is an important member of flavor and fragrance,and has important application value in food,cosmetics and medicine industries.At present,heliotropin is mainly synthesized by chemical methods,but this process has problems such as cumbersome reactions,large energy consumption,and difficulty in the later treatment of waste liquid.Compared with the chemical method,bio-transformation has the advantages which are single reaction process,mild reaction conditions and environmental friendly.Our laboratory had developed a method for the synthesis of heliotropin using recombinant E.coli whole cells.In this paper,we studied the effects of different cofactors and coenzymes on the trans-anethole oxygenase?TAO?activity,and then co-expressed formate dehydrogenase?FDH?and TAO in Escherichia coli.Finally,the induction conditions and whole cell catalytic conditions were optimized.All the above are in order to achieve efficient synthesis of heliotropin.Details are listed as follows:?1?Cloning,Expression and Purification of TAO,enzymatic analysis and effects of different cofactors on TAO activity.The tao gene was cloned into plasmids pET-28a,pETDuet-1,pET-20b,and pGEX-6p-1 respectively,and the recombinant plasmids were expressed and induced in Escherichia coli.It was obtained that E.coli BL21DE?3?/pETDuet-1-tao had higher enzyme activity and played better in protein expression,and its crude enzyme had an enzyme activity of 0.71 U·OD^-1.TAO was purified by affinity chromatography and a single protein was obtained.Enzymatic analysis showed that the optimal reaction temperature and pH of TAO were 30oC and 8,respectively.When isolafrole taken as substrate,its K_m and K_cat were 20.01 mmol·L^-1 and 1.47 s^-1,respectively.Assaying for TAO enzyme activity,it was found that flavin adenine dinucleotide?FAD??nicotinamide adenine dinucleotide?NADH?and formate dehydrogenase can significantly increase the reaction rate.Thus,the strategy of coenzyme coupling for the catalytic reaction was determined.?2?Optimizing co-expression system of TAO and FDH.FDH was cloned from Candida boidinii and codon-optimized,and then different co-expression systems were constructed?E.coli BL21 DE?3?/pETDuet-1-tao&pCDFDuet-1-fdh,E.coli BL21 DE?3?/pETDuet-1-tao-fdh,E.coli BL21 DE?3?/pETDuet-1-fdh-tao,E.coli BL21 DE?3?/p ETDuet-1-fdh-tao&pCDFDuet-1-fdh?.It was obtained that E.coli BL21 DE?3?/pETDuet-1-fdh-tao had an enzyme activity of 3.37 U·OD^-1,which was 3 times higher than that of expression of TAO.The best system above was applied to the mutant TAO_3G2?V71L/G249C?obtaining E.coli BL21 DE?3?/pETDuet-1-fdh-tao_3g2 with an enzyme activity of 4.11 U·OD^-1.?3?Using E.coli BL21 DE?3?/pETDuet-1-fdh-tao_3g2 for whole cell synthesis of heliotropin.First,the fermentation conditions of bio-transformation using lactose as an inducer instead of traditional IPTG was optimized.It was found that the initial induction OD₆₀₀ was 0.95?2.20,the inducer lactose concentration was 4 g·L^-1,the induction temperature was 20?25oC,and the induction time was 10 h.Then,the conditions of whole-cell catalysis was optimized.The optimum concentration of biomass was 7.5?10%,substrate concentration added was 20?25 g·L^-1,and the molar ratio of substrate to auxiliary substrate was 1:2.Finally,fermentation and catalytic synthesis was carried out on a 3 L fermentor.It was found that the OD₆₀₀ obtained in the 3 L fermentor was 18.84.When substrate with a concentration of 25 g·L^-1 was added and transformation was lasted for 7 h,heliotropin with a concentration of 18.47 g·L^-1 was finally obtained,with a conversion rate of79.79%and a space-time yield of 2.64 g·?L·h?^-1.After preliminary extraction,the crystals of helitropin with a purity of 98.63%could be obtained.