Assistant Professor

Biosynthesis of secondary metabolites, caffeine and vanillin, and enzymatic deodorization with polyphenolic compound (PP) and polyphenol oxidase (PPO) are being studied.

The pathway for caffeine biosynthesis is xanthosine Æ 7-methylxanthosine Æ 7-methylxanthineÆ theobromine (3,7-dimethylxanthine) Æ caffeine (1,3,7-trimethylxanthine) via nucleotides (AMP (or GMP) Æ IMP Æ XMP) and GMP Æ guanosine. We have demonstrated that the first methylation in caffeine biosynthesis is carried out during conversion of xanthosine to 7-methylxanthosine. Three novel enzymes, N-methyltransferase, N-methylnucleosidase, and guanosine deaminase, were partially purified from tea leaves and characterized. N-Methyltransferase, which is the key enzyme catalyzing the first methylation, is considered to be useful for production of naturally caffeine-free coffee using biotechnology. Blocking of caffeine synthesis in tea and coffee plants and production of caffeine in other plants will be investigated.

Though vanillin is one of the most important flavors, its biosynthetic pathway is not yet resolved. We have postulated a new pathway leading to vanillin formation from ferulic acid via ferulic acid CoA ester in Yanilla planifolia and have tried to demonstrate the pathway enzymologically. Production of vanillin may be controlled in the same way as caffeine in the near future.

The effect of PPs such as catechins and chlorogenic acid on deodorization has been known. We have been able to improve the deodorization effect by the addition of PPO to the reaction system. Furthermore, it was demonstrated that bad breath can be easily removed by eating raw fruits, vegetables and mushrooms, containing a large amount of both PP and PPO. The mechanism of enzymatic deodorization is considered to be that the radicals produced by the oxidation of PP with PPO add to methyl mercaptan to form the conjugate. Applications of PP-PPO reactions to other chemical and physiological functions are in progress.

Selected Publications. Negishi, O., et al.; Conversion of Xanthosine into Caffeine in Tea Plants, Agric. Biol. Chem., 49, 251-253 (1985) :Negishi, O., et al.; Methylation of Xanthosine by Tea-leaf Extracts and Caffeine Biosynthesis, Agric. Biol. Chem., 49, 887-890 (1985) :Negishi, O., et al.; The Role of Xanthosine in the Biosynthesis of Caffeine in Coffee Plants, Agric. Biol. Chem., 49, 2221-2222 (1985) :Negishi, O., et al.; N-Methyl Nucleosidase from Tea Leaves, Agric. Biol. Chem., 52, 169-175 (1988) :Negishi, O., et al.; Biosynthesis of Caffeine from Purine Nucleotides in Tea Plant, Biosci. Biotech. Biochem., 56, 499-503 (1992) :Negishi, O., et al.; Guanosine Deaminase and Guanine Deaminase from Tea Leaves, Biosci. Biotech. Biochem., 58, 1277-1281 (1994) :Negishi, O., and Ozawa, T.; Determination of hydroxycinnamic acids, hydroxybenzoic acids, hydroxybenzaldehydes, hydroxybenzyl alcohols and their glucosides by high-performance liquid chromatography, J. Chromatogr. A, 756, 129-136 (1996) :Negishi, O., and Ozawa, T.; Effect of Polyphenol Oxidase on Deodorization, Biosci. Biotech. Biochem., 61, 2080-2084 (1997).