Isolationi And Purification Of Polyphenol Oxidase Isoenzyme From Camellia Sinensis Var. Longjing 43, And Enzymatic Properties Of PPO Ⅲ-2 And PPO Ⅴ-3

Polyphenol oxidases(PPO) can effectively catalyse the polymerization and oxidation of polyphenols to the corresponding quinones, and play a very important role in the physiology and metabolism of Camellia sinensis, the processing of tea products and enzymatic preparation of theaflavins. It was known that the content of theaflavins is positively related to the quality and price of black tea, and the low activity of PPO in the leaves results in the low quality of Chinese black tea and broken black tea. Experiments showed that the activity of PPO isozymes is higher than the crude PPO from C. sinensis, but the domestic researches on tea PPO isozymes are only in the band levels of one-dimensional electrophoresis. If PPO isozymes with high activity were selected to improve the tea quality and economic benefits, enhance in vitro the efficiency of producing theaflavins, and reduce the waste of spring and summer fresh tea leaves which accounted for 60% of the annual total output in China, it is very important to the development of tea industry. In this study, the PPO isozymes, PPO Ⅲ-2, and PPO Ⅴ-3 were isolated from the fresh leaves of C. sinensis cv. Longjing NO.43, and their identification, enzymatic properties and the catalytic preparation of theaflavins were studied. The main results are as follows:From fresh leaves of Longjing NO.43, two PPO isozymes, PPO Ⅲ-2, and PPO Ⅴ-3, were isolated and purified sequentially by homogenate extraction, ammonium sulfate precipitation, and anion exchange chromatography. There were four kinds of obvious PPO isozymes in crude PPO, and PPO Ⅲ-2, and PPO Ⅴ-3 were the second, and the third PPO isozyme, respectively. Compared with crude PPO, the specific activity of PPO Ⅲ-2 was 1293.97 U/mg, and its purification fold was 3.39; the specific activity of PPO Ⅴ-3 was 684.81 U/mg, and its purification fold was 1.79. Electrophoretically pure PPO isozyme proteins were further purified by gel filtration chromatography, and the sizes of PPO subunits were 38, 45, 60, and 80 ku, respectively; their specific activities were respectively 3173.08, 715.94, 3942.59, and 4852.98 U/mL, and their purification folds were 7.62, 1.72, 9.46, and 11.65, respectively. The N-terminal amino acid of the highest purity of PPO isozyme(45 ku) was sequenced, and seven amino acids were failed to detect in 15 amino acids possibly for the PPO isozyme(45 ku) was methylated.It was found that PPO had the highest absorption at the wavelength of 410 nm, so the wavelength of 410 nm was chosen for PPO activity assay. Crude PPO, PPO Ⅲ-2, PPO Ⅴ-3 had higher activity within the temperature range of 30-50 ℃, optimum temperatures were 50 ℃, 30 ℃ and 40 ℃, respectively. PPO Ⅲ-2, PPO Ⅴ-3 were less thermostable than the crude PPO, but the activities of PPO Ⅲ-2 and PPO Ⅴ-3 both increased obviously after been incubated at 30 ℃. The pH optimums for crude PPO were 5.2 and 8.0, the results were 611.67 U/m L and 958.89 U/mL, respectively. At the same time, the activity of crude PPO was higher at alkaline pH than acidic pH. But PPO Ⅲ-2 and PPO Ⅴ-3 had higher activities at acidic pH(4.0-4.8), and the optimum pH was 4.4. The crude PPO, PPO Ⅲ-2 and PPO Ⅴ-3 all have catalytic effects against catechol and pyrogallic acid, but not against m-dihydroxybenzene and hydroquinone. Furthermore, pyrogallic acid had higher affinity to PPO comparing to the other inhibitors tested, the most effective inhibitors of PPO activity was ascorbic acid, followed by L-Cysteine, sodium metabisulfite, at the concentration of 0.5 mmol/L,they can completely inhibitthe catalytic activities of PPO. However, EDTA-Na2 and Citric acid showed slightly effects on the catalyst activities, the isoenzymes still had high activities at 1 mmol/L.It was discovered that metal ions of K+, Na+ inhibited the PPO activities slightly, while Mg2+ increased the activity. When the concentration of Mg2+ was 30 mmol/L, the relative activities of the crude PPO, PPO Ⅲ-2, PPO Ⅴ-3 were 136.30%、207.57% and 214.79%, respectively. Cu2+ increased the PPO activity at low concentration, but inhibited the activity at high concentration. The crude PPO obtained the highest activity at 5 mmol/L, while both PPO Ⅲ-2 and PPO Ⅴ-3 had the highest activities at 10 mmol/L. When the concentration of Cu2+ was higher than 25 mmol/L, Cu2+ showed strong inhibitive effects against the PPO activities. Fe3+ and Al3+ showed the same effects with Cu2+. The highest activities of the crude PPO were found at 15 mmol/L of Fe3+ and Al3+, while 10 mmol/L to PPOⅢ-2 and PPO Ⅴ-3. When the concentrations of Fe3+ and Al3+ reached 25 mmol/L, these irons showed strong inhibitive effects against the PPO activities of both the crude PPO and isoenzymes.The optimum conditions for making theaflavins with the crude PPO, PPO Ⅲ-2 and PPO Ⅴ-3 were as follows: The optimum substrate concentration was 3.0, 1.5, 1.0 mg/mL, respectively. The optimum reaction pH was 4.8, 4.8, 4.4, respectively. The optimal reaction temperature were 37, 37, 40 ℃, respectively.In addition, 1 h was considered the best reaction time for all of them. Comparing theproductions of theaflavins, PPO Ⅲ-2 is more suitable for the synthesis of theaflavins, followed by PPO Ⅴ-3 and the crude PPO.