| Catechins,the principal components of tea polyphenol,are not only the most important characteristic metabolizing components in tea plant,but also the main chemical components of tea quality.Some enzymes related to the procedure of catechins biosynthesis include cinnamate 4-hydroxylase(C4H),4-coumaroyl-CoA ligase(4CL),chalcone synthase(CHS),chalcone isomerase(CHI),(2S)-flavanone-3-hydroxylase(F3H),dihydroflavonol-4-reductase (DFR),leucoanthocyanidin reductase(LAR),anthocyanidin reductase(ANR) and so on. DFR/LAR is very important enzymes in flavone biosynthesis pathway and have significant regulating function in catechin biosynthesis.The determination of DFR/LAR activity was primarily established and the kinetics properties of DFR/LAR were investigated in this paper.The relationships between DFR/LAR activity and the content of catechin(C) or gallocatechin(GC) in tea callus tissuse and different parts of tea leaf were also studied.1.The products of DFR/LAR enzyme reaction including leucocyanidin,C and GC were identified by means of High performance Liquid Chromatography(HPLC),Thin Layer Chromatography(TLC),ultraviolet-visible scaning and vanillin-hydrochloric acid colorimetry.According to these results,it was confirmed that the vanillin-hydrochloric acid colorimetry was a simple method to detecting the activity of DFR/LAR.2.The another detecting method for the activity of DFR/LAR was also obtained according to the consumption of coenzyme NADPH in reacting system.3.The kinetics characteristics of DFR/LAR were investigated by using vanillin-hydrochloric acid colorimetry and NADPH consuming method.The results showed that optimum temperature was 65℃and optimum pH was 5.5 and 7.5～8.0 when DHQ as substrate,while 45℃of ptimum temperature and 7.0 of optimum pH when DHM as substrate.The relationships between substrate concentration and reaction rate of DFR/LAR enzyme was not a typicla Michaelis-Menton equation due to the substrate inhibition.Four apparent Km with two substrates by using linearity graphical were obtained as 0.888mmol/L of KNADPH-DHQ,0.925mmol/L of KNADPH-DHM,0.337mmol/L of KDHQ-NADPH,1.145 mmol/L of KOHM-NADPH.The results also revealed that 4mmol/L of Mg2+ and 0.1mol/L of SDS could improve enzyme activity,but 0～1 mmol/L of EDTA could strongly restrain enzyme activity and Fe3+ had some inhibition on enzyme activity within range from 0 to 1 mmol/L. 4.The contents of total catechins,catechin,gallocatechin and the activity of DFR/LAR were measured in different parts of tea leaf and tea callus tissuse of "yunjing 63".The results showed that the activity of DFR/LARDHM was higher than that of DFR/LARDHQ no matter in tea leaf or tea callus tissuse.The activity of DFR/LAR declined gradually along with the maturity of tea leaf,and GC content presented a positive correlation with the activity of DFR/LARDHM. |
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