Metabolism of Ferulic Acid Sucrose Esters in Anthers of Tulipa cv. Apeldoorn: II. Highly Specific Degradation of the Esters by Different Esterase Activities

Abstract

Abstract Protein extracts from anthers of Tulipa cv. Apeldoorn catalyze the degradation of ferulic acid sucrose esters. Different products are generated when triferuloyl sucrose (TFS) and diferuloyl sucrose (DFS) were applied as substrates. By the aid of reversed-phase HPLC , TLC and spectroscopy the products could be identified as free ferulic acid, monoferuloyl sucrose ester [feruloylsucrose(mono)] and two different diesters of ferulic acid and sucrose [feruloylsucrose(di) and the endogenously occurring diferuloylsucrose (DFS)]. By means of protein fractionation (chromatofocusing, anion exchange HPLC and molecular sieving HPLC) , four different enzyme activities involved in the degradation process could be separated. According to their catalytic properties, they were characterized as esterases (= EA). The partially purified esterase activity I (EAI) obtained after fractionation by chromatofocusing catalyzes the formation of feruloylsucrose(di) and ferulic acid when TFS is used as substrate. Incubations with EA la or EA Ib isolated in smaller portions lead to the same product pattern. The esterase activity II (EA II) degrades TFS to ferulic acid and DFS . DFS as substrate is only accepted by the EA I activities, in all three cases ferulic acid and feruloyl sucrose(mono) are formed as products. The kinds of different degradation reactions clearly indicate that one enzyme (= the EA II activity) catalyzes exclusively the formation of DFS from TFS. Both enzymes, EA I and EA II, exhibit a high specificity towards ferulic acid sucrose esters. Hydroxycinnamic sucrose esters with only sinapic acid moieties do not function as substrates. When enzymatically formed sucrose esters like feruloylsucrose(di), feruloylsucrose(mono) and monosinapoylsucrose were used as substrates, no product formation could be observed. Applying SFS as substrate, only the ferulic acid moiety was released by EA I. Further, naturally occurring esters (glucose- and CoA-esters of p-coumaric, caffeic, ferulic and sinapic acid; chlorogenic acid; BGM ) tested so far were not degraded by EA I and EA II. It is assumed that these esterase activities play a specific role in the ferulic acid metabolism in Tulipa anthers.