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 (&gt;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 &amp; Co. KGaA.

Sathyanarayana Naidu Gummadi

Department Of Biotechnology

Bacteria (microorganisms)

Pseudomonas

Pseudomonas sp.

bacterial protein

Caffeine

enzyme

METHYLXANTHINE

XANTHINE DERIVATIVE

article

drug mixture

enzyme induction

enzymology

growth, development and aging

metabolism

Bacterial Proteins

COMPLEX MIXTURES

Enzymes

Metabolic Networks and Pathways

XANTHINES

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It currently offers undergraduate, postgraduate and research degrees across 16 disciplines in Engineering, Sciences, Humanities and Management. About 596 faculty belonging to science and engineering departments and centres of the Institute are engaged in teaching, research and industrial consultancy.

IIT Madras

Journal of Basic Microbiology

Pseudomonas sp. NCIM 5235 is a caffeine-degrading bacterial strain that metabolizes caffeine by sequential demethylation using methylxanthine demethylases. These enzymes belong to the class of two-component Rieske oxygenases and require an oxidoreductase, NdmD, for efficient catalysis. NdmD in Pseudomonas sp. has a unique domain fusion in its N-terminal that is not observed in any other Rieske oxygenase reductases reported so far. In this report, a ~ 1.7 kb ndmD gene from the gDNA of Pseudomonas sp. has been isolated and has been cloned in a pET28a expression vector. Soluble NdmD was over-expressed in Escherichia coli BL21 cells and purified by Ni2+ NTA chromatography. Monomeric molecular mass of the protein was found to be ~ 65 kDa and optimal activity was observed at 35 °C and pH 8.0. It showed broad substrate specificity with highest Kcat/km of 490.8 ± 17.7 towards cytochrome c. To determine the role of N-terminal Rieske domain in its reductase activity, two deletion constructs Δ114NdmD and Δ250NdmD were made. Cytochrome c reductase (ccr) activity of the NdmD constructs and demethylase activity of NdmA in the presence of NdmD constructs showed that there is no significant difference in the catalytic activity of NdmD upon deletion of its N-terminal Rieske domain. However, there might be some functional and evolutionary significance for the fusion of Rieske domain to NdmD and we hypothesize that this domain fusion is an intermediate phase of evolution towards the development of a more efficient enzyme system for xenobiotic degradation. © 2018, Springer-Verlag GmbH Germany, part of Springer Nature.

Applied Microbiology and Biotechnology

Identification and characterization of oxidoreductase component (NdmD) of methylxanthine oxygenase system in Pseudomonas sp. NCIM 5235

The growth of Pseudomonas sp. was studied in fed-batch process with an aim to improve the caffeine degradation rate and caffeine demethylase activity. The effects of varying initial caffeine concentrations in the batch mode, increase in the number of feeds, varying feed flow rates, and added nutrients to the feed on the fed-batch process were investigated. A maximum caffeine degradation rate of 0.82 g/L h and maximum caffeine demethylase activity of 2.6 U/mg were achieved using manual intermittent pulse feeds of caffeine with substrate concentration as feedback parameter for the fed batch started with an initial caffeine concentration of 3 g/L. A slight increase in the caffeine degradation rate (0.85 g/L h) and caffeine demethylase activity (3.4 U/mg) was observed when the additional nutrients were added along with caffeine in the feed. This is the first report showing complete degradation of large magnitudes of caffeine amounting to 237 g in 75 h. These results show that the fed-batch conditions achieved in this study using Pseudomonas sp. facilitate the development of a sustainable bioprocess to degrade the high concentrations of caffeine in industrial effluents. © 2011 Springer-Verlag.

Enhanced degradation of caffeine and caffeine demethylase production by Pseudomonas sp. in bioreactors under fed-batch mode

The effect of various initial caffeine concentrations on growth and caffeine demethylase production by Pseudomonas sp. was studied in bioreactor. At initial concentration of 6.5 g l-1 caffeine, Pseudomonas sp. showed a maximum specific growth rate of 0.2 h-1, maximum degradation rate of 1.1 g h-1, and caffeine demethylase activity of 18,762 U g CDW-1 (CDW: cell dry weight). Caffeine degradation rate was 25 times higher in bioreactor than in shake flask. For the first time, we show highest degradation of 75 g caffeine (initial concentration 20 g l-1) in 120 h, suggesting that the tested strain has potential for successful bioprocess for caffeine degradation. Growth kinetics showed substrate inhibition phenomenon. Various substrate inhibition models were fitted to the kinetic data, amongst which the double-exponential (R 2 = 0.94), Luong (R 2 = 0.92), and Yano and Koga 2 (R 2 = 0.94) models were found to be the best. The Luedeking-Piret model showed that caffeine demethylase production kinetics was growth related. This is the first report on production of high levels of caffeine demethylase in batch bioreactor with faster degradation rate and high tolerance to caffeine, hence clearly suggesting that Pseudomonas sp. used in this study is a potential biocatalyst for industrial decaffeination. © 2010 Society for Industrial Microbiology.

Journal of Industrial Microbiology and Biotechnology

Kinetics of growth and caffeine demethylase production of Pseudomonas sp. in bioreactor

Previously, we isolated caffeine degrading Pseudomonas strain from soil of coffee plantation area, which could utilize caffeine as sole carbon and nitrogen source and could tolerate caffeine up to 20 g/L. In this study, caffeine degradation by immobilized cells of this strain was investigated. Various matrices were considered and agar-agar was chosen based on degradation rate (0.08 g/(L h)), bead stability and reusability. Further, immobilization parameters, viz., bead size (mm), agar-agar concentration % (w/v) and cell concentration (g/L) were optimized using central composite design. The optimal conditions of cell concentration, agar-agar concentration and bead size were 7.8 g/L, 5% (w/v) and 6.2 mm. Under optimal conditions, caffeine degradation rate was found to 0.15 g/(L h), which closely agrees with the model predicted values. This is the first report on caffeine degradation at high concentrations (10 g/L) by immobilized cells of Pseudomonas sp. Immobilization efficiency was 80%. Damköhler number is very much higher than 1, suggesting that mass transfer is the rate limiting process. © 2008 Elsevier B.V. All rights reserved.

Biochemical Engineering Journal

Enhanced degradation of caffeine by immobilized cells of Pseudomonas sp. in agar-agar matrix using statistical approach

The effect of pH, aeration rate, and agitation rate on specific productivity of caffeine demethylase from Pseudomonas sp. was studied in a bioreactor. Maximum specific productivity of caffeine demethylase of 2,214 U g cell dry weight-1 h-1 was obtained at 0.27 vvm, 700 rpm, and pH 7.0. Under these conditions, volumetric oxygen transfer coefficient was 74.2 h-1, indicating that caffeine demethylase production by Pseudomonas sp. was highly oxygen-dependent. Different metabolite formation at different agitation and aeration rates can be used as a strategy for recovery of pharmaceutically important metabolites from caffeine by manipulation of conditions in a bacterial culture. This is the first report on production of high levels of caffeine demethylase in bioreactors. © 2009 Society for Industrial Microbiology.

Optimization of production of caffeine demethylase by Pseudomonas sp. in a bioreactor

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

Journal	Journal of Basic Microbiology
ISSN	0233111X
Open Access	No