Primary Research For Menthofuran Bioconversion Method

Menthofuran is also called menthol furan, with the chemical name4,5,6,7-tetrahydro-3,6-dimethylbenzofuran and the CAS number494-90-6. The chemical structural formula is and the molecular formula is C10H14O, with the molecular weight150.22. Menthofuran exists in natural peppermint oil, possessing the aroma of nuts or coffee, which can be mixed with edible essence. Methofuran is widely used on behalf of a low safety dosage of only about10mg/kg.Natural methofuran is mainly extracted from mint leaves. The yields are less but the price is so high that it is difficult to meet the great needs of the market As a result, people have tried to synthesis methofuran through chemical strategies. One of the most efficient strategies is the chlorinated oxidation method. However, methofuran produced by chlorinated oxidation method contained organochlorine, which is difficult to be removed completely. Moreover, the content of residual organochlorine does not meet the relevant requirements of food safety.It is a critical step to oxidize the epoxy isopulegol with chlorine in the chlorinated oxidation strategy, which is also the root of the organochlorine residue. To find out a bioconversion method which is safety, green, environment friendly, and could alternate the key step, this article tried three aspects of commercial enzyme transformation, recombinant enzyme transformation and microbial transformation to solve the problem of chlorine residue in product.Firstly, according to the skeleton structure and the key functional groups of the substrates, as well as the basic properties and species of commercial enzymes, we selected alcohol dehydrogenase, lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase and polyphenol oxidase successively. The five enzymes were used to convert epoxy isopulegol and isopulegol respectively. We constructed reaction systems of five enzymes by reference to the literature, then optimized the reaction conditions. We selected the conversion system which had highest enzyme activity. The result was that the ethanol dehydrogenase could react with epoxy isopulegol and isopulegol respectively. The reaction systems had absorption in340nm. Total product was tested by chromatography-mass spectrometry (GC-MS), but there were no epoxy isopulegone or isopulegone in them.Secondly, methofuran synthase and P450epoxidase respectively were used to transform isopulegol. We synthetised methofuran synthase (MFS) gene from mint artificially and the gene was expressed in E. coli. Due to MFS was membrane protein and NADH was required for electron transport when MFS catalyzed a substrate, so the broken cell mixture of recombinant strains was used to convert isopulegol. We expressed P450epoxidase (epoF) gene from Sorangium cellulosum SoO157-2in E. coli. The epoF belongs to P450family the same as MFS and the substrate specificity is small. We optimized seven factors including reaction buffer, time, temperature, acidification of the product, mixed reaction with5kinds of commercial enzymes respectively, whether to add hydrogen peroxide, and whether to add NADPH when two kinds of recombinant enzymes reacted with isopulegol. We compared the conversion effects of two kinds of recombinant enzymes using thin layer chromatography and the methofuran was not detected.Finally, we isolated microorganism from specific environments that could tolerate or using isopulegol. What we have tested was that whether the strains could transform isopulegol by the cell broken mixture.(1) We isolated94bacteria from three samples such as the soil of mint field in xinjiang. These bacteria were identified to the Bacillus, Pseudomonas and21genera. We isolated78fungi from the samples such as mint leaves. We detected the tolerance of the above strains to isopulegol respectively. We found that22bacteria and34fungi had tolerance with0.5%(v/v) of isopulegol. We transformed isopulegol using the broken cell mixture of56strains which had tolerance with isopulegol. However, we did not detect methofuran by thin layer chromatography.(2)We obtained the mixed strains XJTA-F2by enrichment of xinjiang using isopulegol. We cultured XJTA-F2with adding isopulegol. Then we analyzed fermentation products of21and28days by high pressure liquid ehromatography. The results showed that the content of isopulegol in28days was little than21days, but methofuran was not detected in21days or28days by further GC-MS.In conclusion, we synthetised menthofuran in vitro using bilogicol transformation method for the first time, but the approaches attempted in this thesis were failed in the biosynthesis in vitro and biological transformation of methofuran. We need to try more enzymes and strains to biosynthetic methoforan in vitro resulting from less of them in this paper. In further study, we can try to build an enzyme system which contains all enzymes needed in a strain. We will obtain an engineering bacterium which can convert isopulegol to menthofuran. In addition, there is an enzyme system which can synthesize methofuran with limonene as precursor in mint cells. So we can try to product methofuran through plant cell genetic engineering.