Preliminary Studies On Purification And Characterization Of Chlorophyllase In Camellia Sinensis (L)O.Kuntze

The enzyme chlorophyllase (chlorophyll-chlorophyllidehydrolase, chlase) [EC 3.1.1.14] is an intrinsic membrane protein that is found in chloroplast photosynthetic membranes and was discovered by WillstSter and Stoll in 1913. It catalyzes the first step of chlorophyll (Chl) breakdown by hydrolysis of Chl into chlorophyllide and phytol in higher plants and algae. Under the optimized conditions for the enzyme extracting and activity analysis, chlorophyllase was purified and its characterization was studied for the first time in China. This will establish theoretical foundation for further research on the mechanism of Chl and photosynthesis, tea cultivation and processing. The main results are as follows:1 Optimization of conditions for extracting and activity analysis of ChlaseUsing Chla as substrate, the conditions for extracting and activity of Chlase in tea plant leaves were studied. The results indicated that the amount of polivinpolypyrrolidone (PVP) had great impact on chlase activity; enzyme activity attained the apex when the amount of PVP was equal to that of fresh leaves. The results also showed that chlase activity was effected by different buffers and pH values, chlase activity increased to max as pH value rose to 7.5, and then decreased. At the same pH value, enzyme activity was the highest with sodium phosphate buffer. It was found that the enzyme reaction was stable at 40℃in 15min.2 Changes of Chlase activity of different tea cultivars, different growing zone, processing and so onThe changes of chlase activity among eight cultivars of Camellia sinesis L. were investigated in this paper. As a result, the sequence of chlase activity from high to blow is: Fu yunliu haocha> Fuding dahaocha> Pingyang tezaocha > Fuding dabaicha> Echa1hao> Wuniuzao> Longjing43>Yingshuang.The differences of chlase activity in different growing zones of tea plants are significant. As a result, the sequence of chlase activity from high to blow is: Bud> The first leaf > The second leaf > The third leaf > old leaf > young stem. The changes of chlase activity in the process of spreading time were determined. It was found that during 6 hours spreading, chlase activity increased at first then decreased, and then increased greatly.And the diurnal changes of chlase activity. We determined chlase activity at 7:00, 10:00, 13:00, 16:00 and 19:00. The result indicated that, chlase activity was decreased gradually from 7:00 to 16:00, and increased from 16:00 to 19:00.There are differences of tea chlase activity in different growing seasons, chlase activity was higher in new shoots of spring tea, and new shoots of summer tea took second place, chlase activity of new shoots of atumn tea was lowest.Tea planting sunshade also can effected chlase activity. Two light shading percentages was applied in tea garden. The results showed that the chlase activity all decreased after 60% and 80% light shading percentage treatment.3 Isolation and purification of chlaseUsing the methods of ammonium sulfate precipitation and column chromatography on Sephadex G-200, Sepharose CL-6B, chlase in the tea leaves was purified. After three steps of Isolation and purification, it was purified 55.96 fold and a yield of 9.45%. There is one chlase stripe by SDS-PAGE. The subunit molecular mass was 37.2kDa.4 Research on characterization of chlaseResearch on the characterization of the purified chlase proved that the enzyme showed optimal activity at pH 7.5 and 40℃. It was stable in pH range from 6.0 to 8.0. Chlase activity decreased in large amplitude in acidic conditions. The result indicated that it was suitable for enzyme preservation under alkalescent condition.After conserved in different temperature a period, enzyme activity decreased at different degree. Chlase complete inactivation after treated with temperament 70℃in 30min and 80℃in 10min.The survival rate of enzyme activity remained 45% when stored 4℃for a month.Adding metal ions Ca²⁺,Mg²⁺,Zn²⁺,Fe³⁺ and EDTA into chlase solution. The result suggested that appropriate amount of metal ions and EDTA promoted chlase activity.