Detection And Preliminary Purification Of Enzymes Related To Catechins Biosynthesis In Tea Plant (Camellia Sinensis (L.) O. Kuntze)

Catechins, as the main secondary metabolites, are not only the main components of quality of tea, but also the major pharmacological features of tea. The health function of galloylated catechins is superior to that of nongalloylated catechins. Up to now, the biosynthesis of nongalloylated catechins in tea plant has been fully investigated, but the biosynthesis pathways and molecular mechanism of galloylated catechins are still not clear.In our previous work, we found that the biosynthesis way of galloylated catechins contained two-step reactions: In the first-step reaction, the galloylated acyl donor β-glucogallin(βG) was biosynthesized by (UDP-glucose: galloyl-1-O-β-D-glucosyltransferase, UGGT) from the substrates gallic acid (GA) and uridine diphosphate glucose (UDPG). In the second-step reaction, galloylated group was transferred to the3-postion of the C-ring in catechin and formed galloylated catechins by action of (epicatechin:1-O-galloyl-β-D-glucose O-galloyltransferase, ECGT). Besides, the galloylated catechins could be hydrolyzed to nongalloylated catechins and gallic acid with the (galloylated catechins hydrolase, GCH) action.Taking fresh tea leaves as materials, the effects of influence factors, including the optimal reaction time, the optimal temperature, the optimal pH, the optimal substrate concentration on the GCH, UGGT and ECGT active detection system were studied and a simple enzyme activity detection method for further purification enzyme protein was set up. GCH had been partially purified, which laied the foundation for further investigating the molecular mechanism of catechins.synthesis and metabolismThe main results were as follows.1. The enzymatic reaction products of GCH,UGGT and ECGT were quantificationally analyzed by HPLC.2. The optimal detecting systems of three enzymes GCH, UGGT and ECGT were established.(1) GCH reaction system: The2.5mL enzyme reaction assay included0.1mol/L phosphate buffer (pH6.5),0.2mmol/L EGCG(ECG or GCG),2.4mmol/L sodium ascorbate, crude enzyme extract (0.4mg protein), and then was incubated at30℃for30min.(2) UGGT reaction system: The1.5mL enzyme reaction assay included0.1mol/L phosphate buffer (pH6.0),1.4mmol/L GA,2.3mmol/L UDPG,4mmol/L sodium ascorbate,1.5mmol/L salicylic acid, crude enzyme extract (0.55mg protein), and then was incubated at30℃for90min.(3) ECGT reaction system: The1.5mL enzyme reaction assay included0.1mol/L phosphate buffer (pH6.0),0.8mmol/L βG,0.38mmol/L EGC(EC),4mmol/L sodium ascorbate,1.5mmol/L salicylic acid, crude enzyme extract (0.55mg protein), and then was incubated at30℃for60min.3. GCH was partially purified via ammonium sulfate fractionation, anion exchange chromatography on Q Sepharose FF and gel filtration chromatography on superdex200sequentially, which specific activity was1.71times higher than before and recovery was9.9%. Detection of the purified GCH by SDS-PAGE showed that the enzyme purity should be further improved.