Enzymatic Synthesis And Purification Of Theaflavins

Theaflavins is an orange and yellow substance that can dissolve in ethyl acetate, from the oxidation of polyphenols and their derivatives from condensation. Theaflavins great medicinal value and economic value of making the request to its high purity, natural substances theaflavins were very low, and the extraction is difficult, using enzymatic oxidation of polyphenol oxidase synthesis of theaflavins, with its isolation and purification can greatly enhance the purification of theaflavins and propose the amount of material quality.Use pear, potato, apple and fresh tea leave as enzyme source material, extracted polyphenol oxidase, measured the protein content using Coomassie brilliant blue, The results were110.03μg/ml,2.137mg/ml,318.8μg/ml and200.8μg/ml, Use catechol as the substrate, select the temperature, pH, substrate concentration and the insoluble polyvinylpyrrolidone(PVPP) to do single factor and orthogonal tests, the optimal temperature of pear polyphenol oxidase conditions is25℃, pH6.0, the substrate concentration0.12mol/ml, PVPP0.75g, the optimal temperature of potato polyphenol oxidase conditions is30℃, pH7.0, substrate concentration0.12mol/ml, PVPP0.45g, the optimal temperature of apple polyphenol oxidase conditions is25℃, pH5.0, substrate concentration0.12mol/ml, PVPP0.75g, the optimal temperature of fresh tea leave polyphenol oxidase is30℃, pH6.0, substrate concentration0.14mol/ml, PVPP0.75g. The relative activity is pear> potato> Apple> fresh tea leave. In the optimal oxidation conditions, use the tea catechin as substrate, through the enzymatic oxidation synthesis and comparison the generate volume, results showed that the maximum conversion rate is from pear polyphenol oxidase.Selected pear polyphenol oxidase as the enzyme source, synthetic enzymatic oxidation of theaflavins, messured theaflavins purity by high performance liquid chromatography, and selection of Sephadex LH-20and polyamide resin to isolate and purify the crude theaflavins. The results showed that crude theaflavins in the four major theaflavins levels were TF0.106mg/mg, TF-3-G0.045mg/mg, TF-3’-G0.181mg/mg, TFDG0.058mg/mg, TFs39%, the system rate is72.8%. Crude theaflavins through Sephadex LH-20chromatography column has separated TF, TF-3-G+TF-3’-G and TFDG, their relative peak area is9.274%,67.442%and65.116%, respectively. Through polyamide resin, its relative peak area up to52.688%.