Screening For Polyphenol Oxidase-producing Fungi From Tea Plants And Enzyme Purification

Polyphenol Oxidase is a kind of copper protein that can effectively catalyze polyphenolic compounds to form corresponding quinones materials by oxidation, and has been well investigated and applied in the phenolic wastewater treatment, lignin degradation, dye decolorization and other fields. It has wide application prospects.Tea is rich in polyphenolic compounds and polyphenol oxidase, which play an important role in the biosynthesis of tea and material conversion in the process of making tea and lay the material foundation for the formation of the flavor and special character of red tea and black tea. In this thesis, the endophytic fungi isolated from healthy tea were chosen as materials. The polyphenol oxidase producing strains were screened. The culture conditions of bacterial strains were investigated.Polyphenol Oxidase was isolated, purified and applied to the biological transformation of tea polyphenols, which laid the foundation for the use of endophytic fungi and polyphenol oxidase. With the approach of color screening and shake flask re-screenin, the relatively high-yielding strain of polyphenol oxidase, i.e. CSN-13, was selected from 14 strains. The change of tea polyphenols solution was tested by HPLC and Polyphenol oxidase production ability of strain CSN-13 was further confirmed. By morphological identification and fungi ITS sequence analysis, the strain CSN-13 was identified as Alternaria abutilonis. With the strain CSN-13 as starting strain, the liquid fermentation medium and fermentation conditions were optimized respectively by means of single factor and the response surface method. The optimized enzyme activity was increased 8.5 times and 10 times than before optimization. The the liquid fermentation of strain CSN-13 was conducted by use of the optimized conditions. The separation and purification of polyphenol oxidase were conducted by the methods of ammonium sulfate precipitation, DEAE- anion exchange chromatography, glucan G-150 gel chromatography. After the SDS-PAGE gel electrophoresis of received samples and Coomassie brilliant blue R- 250 staining, it indicated that two clear protein bands were found, whose relative molecular weights are 50 kDa and 70 kDa respectively. It was purified up to 3.50 times with a recovery of 3.15%. A research was made on the enzymatic property of purified polyphenol oxidase. It had an optimum temperature of 30 ℃ and an optimum pH of 5.5. The solid state fermentation medium of tea polyphenol oxidase was determined after optimization. Then the products in the solid fermentation and purified polyphenol oxidase were added into the tea polyphenol solution with certain concentration to react to each other. The HPLC test found that there was a big change of polyphenol content and existed multiple unknown peaks. It also indicated that both solid fermentation and liquid fermentation to produce enzymes had biological transformation effect.