Multiple Seasonal Modifications in Enzymatic Flavour Formation in Tea

Carotenoid cleavage enzymes isolated from Japanese Camellia sinensis leaves(cultivar Yabukita) were used for investigating the structural patterns of carotenoid cleavage enzymes.Fresh tea leaves were used for the isolation of active enzymes and purified to single band stage in SDS PAGE gels after isoelectric focusing.The specific activity of the carotenoid cleavage enzymes was tested and the active fractions selected for further analysis.The sugar content and the amount of phosphate present in the purified enzymes were elucidated by the following methods:Phosphates were detected by phosphatase assays,fluorescence marker kits and ammoniumheptamolybdate complex measurements after incineration of the samples.Sugars were detected in gels using PAS reagent(periodide acid /Schiff reagent) staining and by GC-MS after hydrolysation of the proteins with trifluoric acid.Phosphorylations as well as glycosylations of the samples could be detected in all cases,thus giving evidence for an increasing phosphorylation level of proteins in Camellia sinensis from spring(1.84 g/mg) to autumn(2.39 g/mg) as well as the presence of at least four different sugars(arabinose,xylose,galactose and ribose).These secondary modifications of the carotenoid cleavage enzymes and their dependency on the harvesting season may well correspond to the changes on the functional level which were detected between spring(Michaelis Constant(K m) =9.45 mol/l) and autumn(K m= 17.16 mol/l) harvests.

Carotenoid cleavage enzymes isolated from Japanese Camellia sinensis leaves （cultivar Yabukita） were used for investigating the structural patterns of carotenoid cleavage enzymes.Fresh tea leaves were used for the isolation of active enzymes and purified to single band stage in SDS PAGE gels after isoelectric focusing.The specific activity of the carotenoid cleavage enzymes was tested and the active fractions selected for further analysis.The sugar content and the amount of phosphate present in the purified enzymes were elucidated by the following methods：Phosphates were detected by phosphatase assays,fluorescence marker kits and ammoniumheptamolybdate complex measurements after incineration of the samples.Sugars were detected in gels using PAS reagent （periodide acid/ Schiff reagent） staining and by GC-MS after hydrolysation of the proteins with trifluoric acid.Phosphorylations as well as glycosylations of the samples could be detected in all cases,thus giving evidence for an increasing phosphorylation level of proteins in Camellia sinensis from spring （1.84 g/mg） to autumn （2.39 g/rg） as well as the presence of at least four different sugars （arabinose,xylose,galactose and ribose）.These secondary modifications of the carotenoid cleavage enzymes and their dependency on the harvesting season may well correspond to the changes on the functional level which were detected between spring （Michaelis Constant （Km） =9.45 mol/1） and autumn （K =17.16 mol/l） harvests.