Swati Sucharita Dash

Catabolic pathways and biotechnological applications of microbial caffeine degradation

Catabolism of caffeine (1,3,7-trimethylxanthine) in microorganisms commences via two possible mechanisms: demethylation and oxidation. Through the demethylation route, the major metabolite formed in fungi is theophylline (1,3-dimethylxanthine), whereas theobromine (3,7-dimethylxanthine) is the major metabolite in bacteria. In certain bacterial species, caffeine has also been oxidized directly to trimethyl uric acid in a single step. The conversion of caffeine to its metabolites is primarily brought about by N-demethylases (such as caffeine demethylase, theobromine demethylase and heteroxanthinedemethylase), caffeine oxidase and xanthine oxidase that are produced by several caffeine-degrading bacterial species such as Pseudomonasputida and species within the genera Alcaligenes, Rhodococcus and Klebsiella. Development of biodecaffeination techniques using these enzymes or using whole cells offers an attractive alternative to the present existing chemical and physical methods removal of caffeine, which are costly, toxic and non-specific to caffeine. This review mainly focuses on the biochemistry of microbial caffeine degradation, presenting recent advances and the potential biotechnological application of caffeine-degrading enzymes.

Inducible nature of the enzymes involved in catabolism of caffeine and related methylxanthines

Previously isolated strain of Pseudomonas sp. has the capability of utilizing caffeine as the sole source of carbon and nitrogen and degrading caffeine at higher concentrations (>10 g l–1). In this study, an assay has been developed to study the enzymatic conversion of caffeine to subsequent methylxanthines by cell free extracts of Pseudomonas sp., the activity of which has been stabilized by use of stabilizers in the lysis buffer. Growth of the strain in various methylxanthines and later enzyme assay demonstrated that the enzyme(s) involved in degradation of caffeine and other methylxanthines were inducible in nature. The results also indicated that more than one enzyme are involved in degradation of caffeine to xanthine, which constitute the primary steps in bacterial caffeine catabolism. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Degradation Kinetics of Caffeine and Related Methylxanthines by Induced Cells of Pseudomonas sp.

In this study, the kinetics of degradation of caffeine and related methylxanthines by induced cells of Pseudomonas sp. was performed. The kinetics data showed that degradation of caffeine, theobromine, and 7-methylxanthine followed Michealis–Menten kinetics. The values of Km are low for caffeine and 7-methylxanthine and high for theobromine. Degradation of caffeine and theobromine was enhanced in the presence of NADH and NADPH, whereas the degradation of 7-methylxanthine was unaffected. Among the various metal ions tested, Fe2+ was found to enhance the rate of degradation for all three substrates, whereas Zn2+ and Cu2+ inhibited the degradation of caffeine and theobromine but not 7-methylxanthine. The differences in kinetic parameters and cofactor requirement suggest the possibility of the involvement of more than one N-demethylases in the caffeine catabolic pathway in Pseudomonas sp. The induced cells can serve as effective biocatalysts for the development of biodecaffeination techniques.

Chemotaxis of Pseudomonas sp. to caffeine and related methylxanthines

Pseudomonas sp. isolated from soil of coffee plantation area has been shown to degrade higher concentrations of caffeine (∼15 g l–1) by N‐demethylation at a rate higher than what has been reported for any strain so far. This strain exhibits positive chemotaxis towards caffeine (1,3,7‐trimethylxanthine) in swarm plate assay and modified capillary assay in a dose dependant manner. Related methylxanthines and xanthine also act as chemoattractants for the strain with the highest relative chemotactic response (RCR) seen for xanthine. Chemotaxis in Pseudomonas sp. is possibly plasmid mediated as indicated by positive chemotaxis of plasmid transformed E. coli DH5α. The chemotactic abilities of Pseudomonas sp. combined with higher rates of degradation of caffeine can be used in the development of strategies for biodecaffeination of caffeine containing wastes. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Industrial Applications of Caffeine Degradation by Pseudomonas sp.

Caffeine is a purine alkaloid naturally present in over 50 plant species with coffee beans, tea leaves and cocoa beans as its main sources. Caffeine majorly enters human system through tea and coffee which are used as the customary drinks throughout the world. High intake of caffeine causes a number of physiological effects on human body. Caffeine also has environmental consequences as effluents released from coffee and tea processing industries are rich in caffeine and pollute nearby water bodies and landmasses affecting natural eco-system. Therefore, decaffeination becomes important from health and environment perspective. Conventional methods of decaffeination include solvent extraction procedures which are toxic, expensive and non-specific. Microbial caffeine degradation can overcome these disadvantages as they are safe and eco-friendly. In this study, induced cells of Pseudomonas sp. were used to degrade caffeine in the effluent collected from Theni unit of Tata Coffee’s Instant Coffee Division and in commercially available tea samples from AVT. Caffeine (0.1 g/l) in the effluent was degraded completely within half an hour when pH of the effluent was adjusted to 7.8 and 8 g/l of induced Pseudomonas cells were used. Among various matrices used for immobilizing Pseudomonas sp., calcium alginate was found to be the best degrading 93 % of caffeine in 3 h when 18 % inoculum was used. Several studies were also done to show that caffeine in tea samples can be effectively removed without significant reduction in polyphenol content by sequential addition of induced Pseudomonas sp. This study has shown that Pseudomonas sp. is an efficient candidate for development of biological decaffeination techniques.