Studies On Screening For Laccase-producing Endophytic Fungi From Tea Plants And The Separation And Characteristation Of Lacase

Laccase is a kind of copper polyphenol oxidase, it oxidates more than two hundred kinds of substrates. Laccase was widely used in food industry, wastewater treatment, paper industry and environmental monitoring. This paper presents the results of screening laccase -producing fungal strains, analyzing different nutritional factors and culture conditions on the influence of the fungi enzyme production, and optimizing the culture medium and culture conditions. Further, separation and purification of laccase and tea polyphenols bioconversion were also studied. A laccase-producing fungal strain CSN-4 was screened from 15 endophytic fungi isolated from health tea leaves by PDA medium amended with six colorimetric substrates. By the study of different nutritional factors and culture conditions, orthogonal tes of culture medium and culture conditions, it suggested that bran 30 g/L, peptone 2.5 g/L, CuSO4·5H2O 0.015 g/L, tea infusion 6 g/L, KH2PO4 2.0 g/L,MgSO4·7H2O 0.2 g/L and MnSO4·5H2O 0.5 g/L were the optimal liquid fermentation medium for laccase production by strain CSN-4. The best fermentation conditions for laccase enzyme production were:inoculating with six 6 mm fungal agar plugs, with liquid volume of 60 mL in 250 mL flask and rotate speed of 120 r/min, cultivating at pH 4.8 and 28 ℃. Laccase was purified by a three-step process:ammonium sulfate precipitation, DEAE-sepharose aion-exchange chromatography, Sephadex G-150 gel penetration chromatography. After the three-step purification, we obtained electrophoresis pure laccase, its purity and yield were 8.98 folds and 24.88% of the crude extract respectively. The molecular mass of the tannase was about 70 kDa. After studying the enzymology properties of purified laccase, its optimum temperature is 60 ℃ and the optimum pH is 4.0. When laccase were in 1 h below 35 ℃, it still had more than 95% of the enzyme activity. When laccase were in 1 h over 50 ℃, it only had below 60%% of the enzyme activity. The stability of the laccase is lost under the condition of high temperature. When laccase were in pH 4-6 for 4 h at 25 ℃, it had more than 96% of the enzyme activity. When laccase were in over pH8 for 4 h at 25 ℃, it only had below 26% % of the enzyme activity, the laccase is more stable in acidic conditions. Cu2+ low concentration have a promoting effect on laccase enzyme activity, more than 5 mmol/L of Cu2+ concentration has certain inhibitory effect on laccase.By using spectrophotometer and HPLC analysis, the effects of laccase on polyphenol transformation were studied. The results showed that transformation effect of laccase crude enzyme fluid is obvious, after the tea polyphenols transformation, the content of catechin EC, ECG fell by 17.28% and 28.6%, and the content of EGCG was the increase of 81.04%. The strain CSN-4 was a laccase-producing fungal strain by the plate screening, laccase enzyme activity raised about 24 times after medium and culture conditions optimization, laccase that was produced by liquid fermentation of strain CSN-4 can result in transformation of tea polyphenols.