Production Of Fungi Laccase And Its Application In Oxidation Of Tea Polyphenol

Laccase-producing strains were screened by liquid fermentation using a-Chennai phenol and guaiacol as indicators, and enzyme activity was measured with ABTS. One strain of fungi(Pleurotus eryngii) was obtained with a moderate fermentation period and a relatively higher ability of enzyme production. The culture medium and conditions were optimized using single factor test and orthogonal design methods. The results showed that the optimal medium composition was (1000mL): potato200g, glucose5g, soybean meal8g, Cu2SO40.033g, MgSO4·7H2O1.5g, KH2PO43g. The optimal fermentation conditions for laccase production were:liquid volume120ml/250ml, inoculum size9%and pH value5.0. Under the optimum condition, the highest laccase activity reached5.207U/mL, which was about4.1times higher than that in original fermentation condition, and the fermentation time was decreased by four days.Enzymatic properties of the laccase produced by Pleurotus eryngii were studied. The optimum reaction temperature was60℃, and the optimum reaction pH value for oxidation of ABTS was4.0. The enzyme was stable at lower temperature (25℃-45℃) and a slightly acidic pH environment (5.0-7.0). Under standard assay conditions, Km and Vmax values of the enzyme were229.42μM and18.21μM/min, respectively。 The metal ion of Pb2+(1mM) had obvious inducing effect, and the laccase activity increased to126.8%of that of the blank control. The ion of Fe2+could inhibit the enzyme activity, which was only3.10%of that of the control.The fundamental studies on oxidation of tea polyphenols by fungi laccase were carried out. The results showed that laccase of Pleurotus eryngii could effectively convert tea polyphenols to tea pigments, and the contents of theaflavin, thearubigin and theabrownin all increased significantly after the reaction. The suitable conditions to theaflavins synthesis were:temperature55℃, pH value4.5, crude enzyme dosage0.2U/mL, substrate concentration4g/L, and reaction time90minutes. Under those conditions, the content of theaflavin was about5.4times that in the original tea solution. The results showed a good application prospect in quality improving of black tea.