Partial purification of capsaicin synthetase from hot pepper (Capsicum annuum)

Abstract

Capsaicin and its vanillic acid derivatives - capsaicinoids are well-known as major heat compounds synthesized by hot pepper (Capsicum annuum) during its fruit-maturation. Capsaicinoid biosysnthetic pathway is one of branches of plant phenolic compound biosysnthesis such as lignin, anthocyanin, flavonoid, and tannins. The most likely pathway of capsaicin biosynthesis was proposed but not yet fully elucidated. Capsaicinoids are synthesized by condensation of phenolic ring derived from phenylalanine and iso-type fatty acids derived from acetyl CoA``s and amino acid, valine. In most cases of chemical capsaicinoid constructions, phenyl ring is constant but iso-type fatty acids chain is variable in their number of carbons, double bonds, and branching. The condensation step is performed by capsaicinoid synthesizing enzyme, if any, capsaicin synthetase (CS). So far properties of CS - optimal pH, optimal temperature, cofactor requirements, and substrate specificities was studied but no purification-concerned themes. As substrates included in assay system, isocapric acid (8-methylnonanoic acid, iso-type fatty acid chain of dihydrocapsacin) and vanillylamine (4-methoxy-3-methoxybenzylamine) was used. Capsaicin synthetase was enriched by ammonium sulfate mediated salting-out and DEAE-cellulose anion-exchange chromatography. At 30 to 70% of ammonium sulfate saturation most of activity was visualized. And at about 35% salt gradient during elution of anion-exchange column most of activity was found. Nonradioactive detection by TLC was performed successfully but after longer incubation than radioactive detection method. Optimum incubation time was estimated as 16 hours or so. After 16 hours of incubation there was decomposition of pre-synthesized dihydrocapsaicin.