Study Of The Transformation Isoeugenol To Vanillin By Isoeugenol Monooxygenasea

Vanillin is one of the most important flavours with the aroma of vanillaand widely used in foods, sweets, beverages, spices and so on. The supply of natural vanillin is excacted from vanilla beans which is less than 1% of the market requirement as 12,000 tons of vanillin every year. In 21 st century, the increasing concern for health and nutrition stimulates more and more people advocating "green" and "natural". In recent years, the United States and the European Union strictly limit the dosage of food additives and increase the demand for natural vanillin. Vanillin produced by biological conversion is equal to natural. Isoeugenol is one of the ideal precursors for vanillin production.The unique key enzyme of microbial transformation of isoeugenol into vanillin is isoeugenol monooxygenase. The technical problem for the bioconversion of isoeugenol into vanillin is that the isoeugenol monooxygenase activity is inhibited by product vanillin. This thesis aims to solve the product inhibition problem by chitosan membrane addition which can be connected with vanillin through schiff's base reaction. Semi-rational design was used for ISO gene sequence to predict mutation sites. The structure was simulated to predict key mutation sites and the production inhibition was removedor reduced by the saturation mutagenesis. The ISO gene was conducted with pPIC9 K and transferred into Pichia pastoris GS115 for its high expression of isoeugenol monooxygenase.The results were shown as following.(1) The bioconversion of isoeugenol to vanillin was conducted with the addition of chitosan film tothe recombinant E.coli BL21(DE3)-pET21a-ISO. The results showed that the chitosan membrane can connect with vanillin and reduce the product inhibition. Investigation of chitosan adsorbing capacity results showed that 0.7 g chitosan film could adsorpt 1.33 g vanillin at 25 oC and 48 h. Optimal biotransformation conditions were isoeugenol 40 g/L, wet cells 0.5 g, chitosan film0.2 g, pH10.5 glycine sodium hydroxide buffer solution 50 mmol/L, 37 oC, 200 rpm for 48 h and the highest concentration of vanillin reached 2.60 g/L. Compareing withthe condition without adding chitosan membrane, this way can make the concentration of vanillin up by 62.5%.(2) Site-saturation engineering of Asn120(Thr121, Phe281, Met298, Leu470) in isoeugenol monooxygenase was performed with E.coli BL21(DE3)-pET30a-ISO as the template and designed five pairs of special primers. The results were shown as following. 1. The influenceof F281 Q amino acid mutations is the highest which activity increased 76.9% than the wild type ISO. And five of mutation sites ordering:F281Q>M298K>L470S>T121P>N120I. 2. Thesmall space occupied of R- of amino acids has advantages. 3. Polarity neutral amino acid of Thr121 mutation into acidic amino acid is better than basic amino acid combined with the substrate and the mutation sites of Phe281 showed different results with Thr121. 4. The polar amino acid of Met298 are mutated into basic amino acid and its conversion efficiency is M298 K > M298 R > M298 H > ISO. It shows that basic amino acid could improve enzyme catalytic activity because of the enzyme and substrate was combined with hydrogen bond by- NH2 of the basic amino acid.(3) Construction of pPIC9K-ISO recombinant was conducted and the isoeugenol monooxygenase(ISO) was transformed into Pichia pastoris GS115 for expression.Optimal conditions were isoeugenol 60 g/L, isoeugenol mooxygenase 50 g/L, glycine sodium hydroxide buffer 50 mmol/L pH10.5, 20 oC, 200 rpm for 48 h and the highest concentration ofvanillin reached 2.45 g/L.