Study On Microbial Transformation Of L-tyrosine And The Analysis And Identification Of Its Metabolites

L-tyrosine, an aromatic amino acid, has various metabolite, some of which are ofimportant physiological function and pharmacological properties, moreover,metabolites and their contents in fermented food may cast significant influence uponfood safety and physiological functions. Therefore, it is necessary to further study onmicrobial conversion products of L-tyrosine. Meanwhile, through microbialtransformation study, it is promising that a cleaner and more efficient microbialenzyme production method could be developed for L-tyrosine metabolites. Therefore,to explore metabolic products and metabolic pathways of L-tyrosine are of importantresearch value and practical significance in a wide range of fields including food,medication, forage, chemical industry, disease detection, etc.In this study,106strains were obtained from nature, and through preliminaryscreening, rescreening, strain ZL-20was finally selected, which could transformedinto eight products in different conditions. This strain is finally identified as Bacillusmegaterium ZL-20by16s rDNA molecule identification and phylogenetic evolutiontree (homologous rate reaches99.0%), as well as physiological and biochemical test.By optimizing factors like the culture medium and transformation conditions, therespective conditions for three main products were optimized, therefore, thetransformation rate and concentration was significantly improved, product (1)conversion rate reaches above65%after24h, with an increase of21%or so; Product(2) reaches75%after36h, with an increase of33%or so; Product (3) is as high as98%after48h, with an increase of25%or so.Based on the above，and through pretreatment of the conversion liquid, the targetproduct were separated and obtained by organic solvent extraction, ion exchangeresin separation, Prep-HPLC and so on. The products were further purified by coolingcrystallization, enrichment crystallization and recrystallization. Then, the crystalproducts obtained were tested by HPCE and HPLC. As the results showed, the crystalpurity of product (1) is more than98%, the product (2) is96%, and product(3) is95%. Finally, the three targeted products were then determined by NMR and MStechnology, combining with the result of16s rDNA molecule identification andL-tyrosine metabolism Pathway in KEGG Pathway Database. As is indicated by theresults that product (1) is L-Dopa(C9H11NO4), belonging to the melanin pathway,generated by polyphenol oxidase; and Product (2) is4-hydroxy-enol-benzene ketoacid(C9H8O4), belonging to the oxidation decomposition approach, generated byaspartate aminotransferase, phenylalanine tautomerism enzyme; Product (3) isTyramine(C8H11NO), a branch of oxidation decomposition approach, generated byL-tyrosine decarboxylase. It is noted that in NCBI Gene Database, phenylalaninetautomerism enzyme is not found in bacillus, which indicates that this enzyme maybe a new and unannotated enzyme in bacillus.In conclusion, Strain ZL-20screened in this study showed good stability andrich transformation products; and an effective method for the analysis of L-tyrosinemetabolites was established by successfully identifying three of the products whichwere extracted under the optimized conditions; It laid a solid foundation for furtherstudies on the discovery for more new products, development of enzyme resourceswith potential industrial application, and safety evaluation on fermented food.