Bio-chemical Synthesis Of Melanin Precursor 5,6-dihydroxyindole

As a green dye,5,6-dihydroxyindole（5,6-dihydroxyindole,DHI）can effectively replace p-phenylenediamine,a carcinogenic component of traditional dyes,with the same mechanism of action and obvious dyeing.At present,most of the synthesis of DHI adopts chemical methods,the reaction conditions are harsh and the environmental pollution is serious.In this study,the biological oxidation step of L-DOPA to synthesize dopachrome（DC）catalyzed by Saccharomyces cerevisiae BY4741/p YX212-TYR and the chemical step of DC reductive synthesis of 5,6-dihydroxyindole（DHI）were coupled to construct the bio-chemical synthesis step of DHI.The culture conditions of the recombinant strains were optimized to maximize their vitality,the influencing factors in the biological oxidation reaction were investigated,and the effects of chemical environment and chemical additives on DC transformation were investigated.The anti-polymerization effect of the polymerization agent on DHI laid the foundation for the green preparation of DHI.The specific research contents are as follows:（1）The tyrosinase heterologous expression strain S.cerevisiae BY4741/p YX212-TYR was constructed by the optimized Aspergillus oryzae tyrosinase gene,host strain S.cerevisiae BY4741,and shuttle plasmid p YX212.The culture time has a great influence on the viability of the recombinant strains.By optimizing the culture conditions,the recombinant tyrosinase activity reached 8.02 U·mg^-1 cells.（2）The influencing factors of S.cerevisiae BY4741/p YX212-TYR catalyzed L-DOPA oxidation to synthesize DC,such as catalyst concentration,reaction temperature,reaction p H,substrate L-DOPA concentration and dissolved oxygen concentration,were investigated.The conditions were as follows:50 g·L^-1 S.cerevisiae BY4741/p YX212-TYR,35℃,p H 6.0,and the maximum conversion efficiency could be obtained by introducing oxygen at the substrate L-DOPA concentration of 3 mmol·L^-1.Among them,p H has a great influence on the reaction rate.Compared with before adjustment,the substrate conversion rate is increased by 2.3 times,and the yield is increased by 3 times.After optimization,the conversion rate of L-DOPA reached 94.75%,and the yield of DHI was 47.09%,which was 6.3 times higher than that before optimization,and the yield was 16 times higher.（3）The effects of chemical environment and chemical additives on the chemical reduction of DC to DHI were investigated,and DHI can be accumulated to the greatest extent at 20℃and p H 3.0;on this basis,0.5 mmol·L^-1 Al³⁺can increase the yield of DHI up to60.06%,50%ethanol can increase the yield of DHI to 75.3%,3.0%sodium ascorbate can increase the DHI yield to 79.4%,and the combined treatment of 50%ethanol and 3%sodium ascorbate can further increase the yield to 90.03%.（4）The LC/MS identification of the product showed that there were DHI-soluble oligomers in the reaction system,and the content of oligomers continued to increase,mainly dimers and tetramers.Polyvinyl alcohol（PVA）and dimethyl sulfoxide（DMSO）can effectively alleviate the polymerization of DHI.1.25%PVA and 15%DMSO had the best anti-polymerization effect,which increased the DHI yield in the reaction system from 90.03%to 93.41%and 91.98%,respectively.