Screening Method of Tyrosinase with Higher Ratio of Monophenolase/Diphenolase Activity for Phenolic Natural Compounds

Abstract

ortho-Monohydroxylation of daidzein, resveratrol and apigenin was attempted using tyrosinase. Tyrosinase proceeds two consecutive oxidation reactions, hydroxylation of monophenol compounds to diphenolic compounds (monophenolase reaction) and successive oxidation to make quinonic compounds (diphenolase reaction). Since the second step oxidation reaction consumes the first step diphenolic products such as piceatannol, 7,3',4'-trihydroxyisoflavone (3'-ODI), and tricetin, the yield of the first step products is quite low, limiting the use of tyrosinase for such monohydroxylation reaction. To overcome the drawback of tyrosinase, the changes in the ratio of monophenolase activity (k1) to diphenolase activity (k2) were attempted. For changing the ratio of monophenolase activity to diphenolase activity, our group developed a screening method and a method for obtaining kinetic constants and did a site-saturation mutagenesis with a tyrosinase from Streptomyces avermitilis MA4680 (SAV1137). According to the result, one mutant having higher ratio (1.4 fold higher than wild type) and productivity of piceatannol (1.4 fold higher than wild type) was found. However, the process was pretty labor-intensive, and the accuracy was poor. 5 mutants were screened from 300 mutants, but the only one mutants had meaningful value. Therefore, accuracy-improved and unexacting screening method was required. By changing pH and strength of buffer, concentration of iron and cell extract solution, and addition of 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH), we developed improved screening method which traces an accumulative quantity of diphenolic compounds in real time with UV spectrometer and an improved assay method for obtaining kinetic constants of mutants. For testifying screening method and finding mutants having higher ratio for natural phenolic compounds, site-saturation mutagenesis of tyrosinase from Bacillus megaterium was performed using the screening method based on x-ray crystal structure, homology modeling and enzyme-substrate docking simulation. Single mutations at V218S was effective to increase the yield of 3-ODI by 30% than by wild type and the yield of piceatannol by V218G was increased 440% than by wild type. R209A was also effective to increase the yield of tricetin by 40% comparing with wild type. Among these mutations, V218S showed 10.7 fold ratio change of k1 to k2 for daidzein and V218G showed 1.4 fold ratio change of k1 to k2 for resveratrol.