An enzyme hydrolyzing L-theanine in tea leaves.

Theanine hydrolase activity in tea leaves was assayed by measuring enzymatically released ethylamine from L-theanine.The o-phthalaldehyde derivative of ethylamine was measured by reverse phase HPLCrecorded with a spectrofluorometric detector.Theanine' hydrolase activity was purified about 4.6-fold by DEAE-cellulose column chromatography.Although this active fraction also had glutaminase activity, the yield of the glutaminase activity was about 50%of that of theanine hydrolytic activity.The theanine hydrolytic activity was inhibited by acidic amino acid and L-alanine, and stimulated by L-malic acid.The purified enzyme solution hydrolyzed not only theanine but also y-glutamylmethylamide, y-glutamyl-w-propylamide, y-glutamyl-«-butylamide, y-glutamyl-wo-butylamide, and y-glutamyl-«-amylamide, which were synthesized from L-pyroglutamic acid and corresponding alkylamines.However, Nmethylpropionamide and N-ethylpropionamide were not hydrolyzed.The theanine hydrolase activity and glutaminase in tea leaves showed the same pHoptimum (8.5).The activity of theanine hydrolase in tea leaves increased during the first lO hr after plucking but then decreased gradually, while that of glutaminase decreased constantly and was almost lost 48 hr after plucking.