Effects Of Inhibitors On Mushroom Tyrosinase And Their Antimicrobial Activity

Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme, which is of the activity of monophenolase and diphenolase, is widely distributed in microorganisms, animals and plants. Its inhibitors can be used widely in many fields including whitening agents, keeping fruits and vegetables fresh, insecticides. Our research in the present paper is composed of two parts. In the first part, we chose cinnamic acid and its derivants, cinnamaldehyde and its derivants, methyl cinnamate, kojic, 4-cyanophenol and 3,4-dihydroxybenzonitrile as research objects, studied the inhibitory mechanism of analogs of them on the activity of monophenolase and diphenolase, and their kinetic constants were determined. In the second part, we studied the antimicrobials effect of these inhibitors.The effects of cinnamic acid, 4-hydroxy cinnamic acid and 4-methoxy cinnamic acid on mushroom tyrosinase were studied. Their IC50 for monophenolase were listed as: 0.58,0.27 and 0.50 mmol/L, IC50 for diphenolase were listed as 2.1,0.5 and 0.42 mmol/L. Through the study of inhibitory mechanism, the results showed, cinnamic acid and 4-methoxy cinnamic acid were non-competitive inhibitors of diphenolase, and 4-hydroxy cinnamic acid was competitive inhibitors of diphenolase. And the results of the antimicrobials experiment showed, the minimum inhibitory concentration (MIC) of cinnamic acid, 4-hydroxy cinnamic acid and 4-methoxy cinnamic acid to E. coli, B. subtilis, St. aureus was 500μg/mL, the minimum bactericidal concentration (MBC) was 1000μg/mL. To C. albicans, the MIC of cinnamic acid and 4-hydroxy cinnamic acid was 500μg/mL, the he minimum fungal concentration (MFC) was 1000μg/mL, and the MIC of 4-methoxy cinnamic acid was 250μg/mL, the MFC was 500μg/mL.The IC50 of cinnamaldehyde and 4-methoxy cinnamaldehyde for monophenolase were listed as: 0.64 and 0.39 mmol/L, for diphenolase were listed as 0.581and 0.700 mmol/L. And cinnamaldehyde and 4-methoxy cinnamaldehyde were non-competitive inhibitors of diphenolase, the inhibition constants (KI) were determined to be 0.581 and 0.700 mmol/L. The results of the antimicrobials MIC and MBC to E. coli, B. subtilis, St. aureus showed: to cinnamaldehyde, they were 250 and 500μg/mL, to 4-methoxy cinnamaldehyde, they were 125 and 250μg/mL. The MIC and MFC which of cinnamaldehyde to C. albicans were 1000 and 2000μg/mL, of 4-methoxy cinnamaldehyde were 62.5 and 125μg/mL.The effects of methyl cinnamate on mushroom tyrosinase were studied. The results showed, the IC50 of methyl cinnamate for monophenolase and diphenolase were 0.92 mmol/L and 1.65 mmol/L, and it was a non-competitive inhibitor. The MIC and MBC to E. coli were 500μg/mL and 1000μg/mL, to B. subtilis and St. aureus were 250μg/mL and 500μg/mL, The MIC and MFC to C. albicans were 1000μg/mL and 2000μg/mL.The IC50 value of kojic for monophenolase and diphenolase were estimated as 32μmol/L and 20μmol/L. It was a mixed type inhibitor, and the inhibition constant KI and KIS were determined to be 13.0 and 100.0μmol/L. The results of the antimicrobials experiment showed, the MIC to E. coli, B. subtilis, St. aureus was 1000μg/mL, and it couldn't kill them when its concentration was 2000μg/mL. The MIC and MFC to C. albicans were 1000μg/mL and 2000μg/mL.The IC50 value of 4-cyanophenol and 3,4-dihydroxybenzonitrile on the monophenolase were determined to be 0.22 mmol/L and 9.2μmol/L, on the diphenolase, which were determined to be 0.80 mmol/L and 13.5μmol/L. Through the study of the inhibitory mechanism of them, the results showed 4-cyanophenol was a competitive inhibitor, 3,4-dihydroxybenzonitrile was a non-competitive inhibitor. The MIC and MBC of 4-cyanophenol to E. coli, B. subtilis, St. aureus were estimated as 500 and 1000μg/mL, that of 3,4-dihydroxybenzonitrile were 1000 and 2000μg/mL. The MIC and MFC to C. albicans of 4-cyanophenol were 500μg/mL and 1000μg/mL, and that of 3,4-dihydroxybenzonitrile were 1000 and 2000μg/mL.