Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Protein adsorption wikipedia , lookup
Protein moonlighting wikipedia , lookup
Biochemistry wikipedia , lookup
Bottromycin wikipedia , lookup
Gaseous signaling molecules wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Biosynthesis wikipedia , lookup
List of types of proteins wikipedia , lookup
Western blot wikipedia , lookup
Metalloprotein wikipedia , lookup
Oxidative phosphorylation wikipedia , lookup
Evolution of metal ions in biological systems wikipedia , lookup
Journal of Biotechnology 125 (2006) 319–327 Purification and characterization of a novel caffeine oxidase from Alcaligenes species B.R. Mohapatra ∗ , N. Harris, R. Nordin, A. Mazumder Water and Watershed Research Program, Department of Biology, University of Victoria, Petch Building 116, 3800 Finnerty Road, Victoria, BC, Canada, V8P5C2 Received 13 November 2005; received in revised form 15 February 2006; accepted 13 March 2006 Abstract Alcaligenes species CF8 isolated from surface water of a lake produced a novel serine type metallo-caffeine oxidase. The optimal medium for caffeine oxidase production by this strain was (w/v) NaNO3 , 0.4%; KH2 PO4 , 0.15%; Na2 HPO4 , 0.05%; FeCl3 ·6H2 O, 0.0005%; CaCl2 ·2H2 O, 0.001%; MgSO4 ·7H2 O, 0.02%; glucose, 0.2%; caffeine, 0.05%, pH 7.5. The enzyme was purified to 63-fold by using ammonium sulfate precipitation, dialysis, ion exchange (diethylaminoethyl-cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified caffeine oxidase was monomeric with a molecular mass of 65 kDa. The purified caffeine oxidase with a half-life of 20 min at 50 ◦ C had maximal activity at pH 7.5 and 35 ◦ C. The purified caffeine oxidase had strict substrate specificity towards caffeine (Km 8.94 M and Vmax 47.62 U mg protein−1 ) and was not able to oxidize xanthine and hypoxanthine. The enzyme activity was not inhibited by para-chloromercuribenzoic acid, iodoacetamide, n-methylmaleimide, salicylic acid and sodium arsenite indicating the enzyme did not belong to xanthine oxidase family. The enzyme was not affected by Ca+2 , Mg+2 and Na+ , but was completely inhibited by Co+2 , Cu+2 and Mn+2 at 1 mM level. The novel caffeine oxidase isolated here from Alcaligenes species CF8 may be useful in biotechnological processes including waste treatment and biosensor development. © 2006 Elsevier B.V. All rights reserved. Keywords: Alcaligenes sp.; Caffeine; Caffeine oxidase; Kinetics; Purification 1. Introduction Sustaining the quality of freshwater for drinking, recreation and irrigation has been emerging as a challenge due to unprecedented release of anthropogenic ∗ Corresponding author. Tel.: +1 250 472 4833; fax: +1 250 721 7120. E-mail address: [email protected] (B.R. Mohapatra). 0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.03.018 pollutants, such as discharge of domestic wastes, leaching from septic tanks, and improper management of agro-industrial wastes (Vitousek et al., 1997). Appropriate counter measures against water pollution necessitate developing novel and efficient techniques to detect, manage and mitigate these pollutants. Caffeine (1,3,7-trimethylxanthine), a purine alkaloid occurs in more than 60 plant species including in the seeds of coffee, cacao and cola tree and in the leaves 320 B.R. Mohapatra et al. / Journal of Biotechnology 125 (2006) 319–327 of tea (Steffen, 2000). Caffeine is considered as one of the major agro-industrial wastes generated from coffee and tea processing plants. It is also an important constituent of a variety of beverages such as coffee, tea, caffeinated cola and soda, and cocoa-derived food products (chocolate, desserts and pastries). Coffee, tea, cola and cacao contain about 100, 50, 40 and 10 mg of caffeine per serving, respectively (Buerge et al., 2003). Caffeine is also an important pharmaceutical ingredient. It accelerates the analgesic actions in anticold and antipyretic drugs. Caffeine is used as a cardiac, neurological and respiratory stimulant and as a diuretic (Mazzafera, 2002). Considering its uptake with beverages, foods, and medicines, caffeine is probably the most widely consumed drugs in the world (Ogunseitan, 1996). It is added on the United States Environmental Protection Agency list of high production volume of chemical (USEPA, 2002). The global average consumption of caffeine is between 80 and 400 mg per person per day (Gokulakrishnan et al., 2005). In humans, dietary caffeine has been partially catabolized by hepatic cytochrome P450 1A2 (EC 1.14.14.1) through oxidative N-demethylation and/or ring oxidation to produce paraxanthine, theophylline, 1,3,7-trimethyluric acid and other byproducts those are excreted with unmetabolized caffeine in urine (Berthou et al., 1992; Ogunseitan, 2002). Because of its extensive use in food, beverages and medicines, caffeine has been detected in surface water, groundwater and wastewater effluents world wide (Buerge et al., 2003; Weigel et al., 2004; Glassmeyer et al., 2005). Recently, caffeine has also been proposed as a potential chemical marker for tracking human fecal waste input to source waters (Scott et al., 2002; Buerge et al., 2003). Decaffeination is being recommended for food, beverages and wastewaters because of potential chronic ingestion of caffeine and/or its byproduct 8chlorocaffeine (produced during chlorination) can have adverse effects on the physiological systems (Gould and Hay, 1982; White and Rasmussen, 1998). Decaffeination is usually performed by physico-chemical treatments, such as water diffusion (Feldman and Katz, 1977), solvent extraction (Smith, 1999) and super critical carbon dioxide oxidation (Udayasankar et al., 1986). However, these methods have high operational costs and usually produce waste products that require further treatment and/or disposal. In order to overcome these problems, a specific decaffeination process is necessary. The microbial bioprocessing is increasingly being considered to be an efficient technique for the caffeine abatement in environments (Gokulakrishnan et al., 2005). It has been reported that some bacteria belonging to the genera Pseudomonas and Serratia, and fungi belonging to the genera Aspergillus, Penicillium, Rhizopus and Stemphyllium have the ability to metabolize caffeine by enzymatic conversion (reviewed by Mazzafera, 2002). However, the enzymatic pathways for caffeine metabolisms in microorganisms are poorly understood (Madyastha and Sridhar, 1998). The enzymatic catabolism of caffeine in microbes is performed by N-demethylation and oxidation with the enzymes demethylases and oxidases, respectively (Gokulakrishnan et al., 2005). Caffeine oxidase, the key enzyme responsible for direct oxidation of caffeine to 1,3,7-trimethyluric acid has been purified to homogeneity only from mixed cultures of Klebsiella and Rhodococcus species isolated from wastewaters (Madyastha et al., 1999). Caffeine-oxidizing enzyme can be useful in the treatment of caffeine in the agroindustrial wastes of coffee pulps and husks (Mazzafera, 2002), groundwaters and wastewaters (Ogunseitan, 1996), and also as a biosensing element similar to other oxido-reductase enzymes (Eggins, 1996) for real-time monitoring of caffeine in natural waters. In view of the potential biotechnological application of caffeine oxidase, it is essential to study this enzyme from various microbial sources. This paper presents results on the purification and partial biochemical characterization of a novel caffeine oxidase from Alcaligenes species isolated from the surface waters. 2. Materials and methods 2.1. Bacteria and culture conditions Caffeine oxidase-producing Alcaligenes species CF8, was isolated from the surface waters of Round lake, Smithers, British Columbia, Canada by spread plate technique with caffeine supplemented mineral medium (CMM) agar composed of (g l−1 ) NaNO3 , 4.0; KH2 PO4 , 1.5; Na2 HPO4 , 0.5; FeCl3 ·6H2 O, 0.005; CaCl2 ·2H2 O, 0.01; MgSO4 ·7H2 O, 0.2; glucose, 2.0; B.R. Mohapatra et al. / Journal of Biotechnology 125 (2006) 319–327 caffeine, 0.5 and agar powder 15 in distilled water. The isolated Alcaligenes sp. CF8 was routinely maintained on CMM agar plates. For production of caffeine oxidase, Alcaligenes species CF8 was cultivated aerobically in 3-L batch culture with CMM. Fermentation was carried out at 25 ◦ C for 72 h with agitation of 100 rpm on a rotary shaker. The bacterial cells were harvested by centrifugation at 5500 × g for 30 min at 4 ◦ C. The resulting cell pellets were resuspended in 0.2 M phosphate buffer (pH 7.5), and disrupted by sonication at 160 W for 20 min with an ultrasonicator. The cell debris were removed by centrifugation at 5500 × g for 30 min at 4 ◦ C. The cell-free supernatant was designated as crude caffeine oxidase. 2.2. Assay of caffeine oxidase Caffeine oxidase activity was assayed by the method of Madyastha et al. (1999) with some modifications. The standard assay mixture containing 0.5 ml of 0.2 M phosphate buffer (pH 7.5), 0.2 ml of 0.055 mM caffeine in the same buffer, 0.1 ml of 1.1 mM 2,6-dichlorophenolindophenol (DCIP) and 0.2 ml of 0.55 mM phenazine methosulfate (PMS). The reaction was started by the addition of 0.1 ml of enzyme solution in a total volume of 1.1 ml, and the rate of decrease in the absorbance at 600 nm was followed against a blank in which 0.22 m filtered distilled water was added in place of enzyme. One unit (U) of caffeine oxidase activity was defined as the mol of DCIP reduced per minute per milliliter of enzyme solution. The caffeine oxidase was also assayed with molecular oxygen (dissolved O2 ) as electron acceptor instead of PMS coupled DCIP by measuring the absorbance of the assay mixture at 300 nm according to a spectrophotometric method previously reported (Woolfolk, 1975). Protein concentration was quantified by using the method of Lowry et al. (1951) with bovine serum albumin as a standard. 2.3. Culture conditions optimization To study the effect of different carbon (fructose, dglucose, lactose, d-mannose, maltose, soluble starch and sucrose), and nitrogen sources (ammonium chloride, ammonium sulfate, casein, sodium nitrate, sodium nitrite, peptone, tryptone and yeast extract) on the caf- 321 feine oxidase production, 0.22 m filter sterilized individual carbon and nitrogen compounds were added to the 24 h old culture of Alcaligenes sp. CF8 growing in a 500 ml Erlenmeyer flask contained 100 ml of CMM without glucose and sodium nitrate, respectively. Incubation was carried out at 25 ◦ C with agitation of 100 rpm in an orbital shaker for another 48 h, and the enzyme activity and protein concentration was determined as mentioned above. 2.4. Purification of caffeine oxidase The crude caffeine oxidase was treated with 60% (w/v) (NH4 )2 SO4 . The precipitate was collected by centrifugation at 5500 × g for 30 min at 4 ◦ C. The precipitated enzyme was dissolved in 0.2 M phosphate buffer (pH 7.5), followed by dialysis (10 kDa molecular weight cut off) against the same buffer. The dialyzates were passed through a DEAE (diethylaminoethyl)cellulose column pre-equilibrated with 0.2 M phosphate buffer (pH 7.5). The elution of enzyme was performed with a linear gradient from 0.1 to 1 M KCl. The active enzyme fractions were pooled and further dialyzed with a 25 kDa dialysis membrane against 0.2 M phosphate buffer (pH 7.5). The dialyzates were passed through a Sephadex G-100 column previously equilibrated with 0.2 M phosphate buffer containing 0.15 M NaCl (pH 7.5). Fractions of a milliliter were collected per minute and estimated for protein concentration as well as caffeine oxidase activity. The active caffeine oxidase fractions were combined and dialyzed with 10 kDa cut off dialysis membrane against 0.2 M phosphate buffer (pH 7.5). The dialyzates were used for further studies. 2.5. Characterization of caffeine oxidase To evaluate the optimum temperature and pH for the purified caffeine oxidase activity, the enzyme assay was performed at different temperatures from 5 to 60 ◦ C with 5 ◦ C increments and with buffers of different pH, such as 0.2 M citrate (pH 3, 4 and 5), 0.2 M phosphate (pH 6.0, 7.0 and 7.5), 0.2 M Tris–HCl (pH 8), 0.2 M borate (pH 9–10) and 0.2 M borax–NaOH (pH 11–12). For the pH and thermal stability studies, the purified caffeine oxidase solution was pre-incubated for 2 h with the above-mentioned buffers of different pH (3–12) and 30 min with above-mentioned temper- 322 B.R. Mohapatra et al. / Journal of Biotechnology 125 (2006) 319–327 atures (from 5 to 60 ◦ C with 5 ◦ C increments), respectively and the residual enzyme activity was assayed as mentioned above. The extinction coefficients of DCIP at different pH were calculated from the data of Armstrong (1964). The effect of different metal ions and chemicals were performed by pre-incubating the purified enzyme solution with individual metal ions and chemicals for 15 min at 25 ◦ C. The residual caffeine oxidase activity was estimated. To determine the effect of different analogues of caffeine on the caffeine oxidase activity, the assay conditions were the same as mentioned above except different caffeine analogues were used as the substrate instead of caffeine. Separate blanks with individual metal ions, chemicals and caffeine analogues were also prepared. The kinetics parameters, such as Michaelis–Menten constant (Km ) and maximum reaction velocity (Vmax ) values against caffeine as the substrate were calculated using double reciprocal Lineweaver–Burk plot. 2.6. Molecular mass determination and purity checking The relative molecular mass and purity of the purified caffeine oxidase was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) by the methods of Laemmli (1970). Briefly, the purified caffeine oxidase and low molecular weight protein standard (Biorad Inc., US) were diluted with 1:4 and 1:20, respectively with Laemmli sample buffer (1.6 ml glycerol, 0.4 ml -mercaptoethanol, 1.6 ml 10% SDS, 1.0 ml 0.5 M Tris buffer pH 6.8, and 0.4 ml 0.5% bromophenol blue in a total volume of 8 ml). The purified caffeine oxidase solution and protein standard in Laemmli sample buffer were heated at 95 ◦ C for 4 min and loaded onto a 10% acrylamide gel. Electrophoresis was performed at 200 V constant voltages. After electrophoresis, the gel was stained with BioSafetyTM Coomassie blue (Biorad Inc., US) for 1 h, followed by destaining for 30 min in distilled water and photographed. 3. Results and discussion Table 1 Effect of different carbon source on caffeine oxidase production by Alcaligenes sp. CF8 Carbon source (2 g l−1 ) Caffeine oxidase activitya (U mg protein−1 ) Controlb Soluble starch Lactose Maltose Sucrose Fructose d-Glucose d-Mannose 0.318 ± 0.029 0 0.113 ± 0.011 0.129 ± 0.008 0.156 ± 0.006 0.322 ± 0.014 0.613 ± 0.011 0.099 ± 0.006 a All the measurements were performed thrice and expressed as mean ± standard deviation. b Control culture was grown in the CMM without glucose. eral medium supplemented with caffeine as the sole source of carbon and/or nitrogen. To improve the production of caffeine oxidase, the medium composition was optimized in shake flask. The maximum enzyme production was obtained at pH 7.5 and 25 ◦ C (data not shown). Various carbon sources were added at a concentration of 2 g l−1 to the caffeine supplemented cultivation medium (Table 1). Addition of glucose significantly increase the activity by almost two-fold. A low level of enzyme secretion was observed in the presence of disaccharides (lactose, maltose and sucrose). The synthesis of caffeine oxidase was ceased with soluble starch. Among all the nitrogen sources tested (4 g l−1 ) the caffeine oxidase production ranged between 0.011 U mg protein−1 and 0.631 U mg protein−1 (Table 2). The optimum activity Table 2 Effect of different nitrogen source on caffeine oxidase production by Alcaligenes sp. CF8 Nitrogen source (4 g l−1 ) Caffeine oxidase activitya (U mg protein−1 ) Controlb Ammonium chloride Ammonium sulfate Casein Sodium nitrate Sodium nitrite Peptone Tryptone Yeast extract 0.306 0.018 0.011 0.199 0.630 0.183 0.598 0.568 0.581 a Alcaligenes species CF8 produced a significant amount of caffeine oxidase when grown in the min- ± ± ± ± ± ± ± ± ± 0.029 0.006 0.002 0.013 0.037 0.023 0.016 0.012 0.029 All the measurements were performed thrice and expressed as mean ± standard deviation. b Control culture was grown in the CMM without sodium nitrate. B.R. Mohapatra et al. / Journal of Biotechnology 125 (2006) 319–327 323 Table 3 A typical purification of caffeine oxidase isolated from Alcaligenes sp. CF8 Step Total protein (mg) Total activity (U) Specific activity (U mg−1 ) Purification fold Crude extract 60% (NH4 )2 SO4 (Dialysis Mr 12 kDa cut off) DEAE-cellulose (Dialysis Mr 25 kDa cut off) Sephadex G-100 (Dialysis Mr 12 kDa cut off) 3120 260 18.75 9.75 1965.6 985.4 580.88 388.34 0.630 3.79 30.98 39.83 1 6 49 63 (0.630 U mg protein−1 ) was achieved with the inorganic nitrogen compound sodium nitrate, followed by the organic nitrogen compounds peptone, yeast extract and tryptone. Enzyme production was substantially suppressed in the presence of ammonium sulfate and ammonium chloride. These results suggest that the external carbon and nitrogen sources have regulatory effect on caffeine oxidase biosynthesis by Alcaligenes species. Furthermore, the enzyme synthesis is demonstrated to be modulated by induction and repression mechanism. The potent inducers for caffeine oxidase production were glucose and sodium nitrate. The catabolite repressors for the enzyme synthesis were starch and ammonium salts (ammonium sulfate and ammonium chloride). Previous studies have also demonstrated that the enzymatic pathways of caffeine catabolism in bacteria are inducible. Glucose has been found to be the effective inducers for mixed cultures of Klebsiella and Rhodococcus species (Madyastha et al., 1999), and fructose and tryptone for Pseudomonas sp. No. 6 (Asano et al., 1993). A typical purification of caffeine oxidase isolated from Alcaligenes sp. CF8 is summarized in Table 3. The crude extract obtained after centrifugation was precipitated with 60% (w/v) ammonium sulfate. This step yielded a six-fold purification step with a specific activity of 3.79 U mg protein−1 . This fraction was chromatographed with DEAE-cellulose column and caffeine oxidase activity was eluted between 0.4 and 0.6 M KCl. The purification fold was increased to 49-fold with specific activity of 30.98 U mg protein−1 . Caffeine oxidase was purified further by gel filtration chromatography with Sephadex G-100 column. The major peak was eluted with 0.2 M phosphate buffer (pH 7.5) containing 0.15 M NaCl. By this method, the caffeine oxidase was purified 63-fold with a specific activity 39.83 U mg protein−1 . The homogeneity of caffeine oxidase was checked with SDS-PAGE. The relative molecular mass of the purified caffeine oxi- dase was 65 kDa (Fig. 1). The enzyme also displayed a molecular mass of 65 kDa when chromatographed with Sephadex G-100 suggesting the enzyme is monomeric. This molecular mass is found to be lower than the Fig. 1. Negative image of electrophoretogram (SDS-PAGE) of purified caffeine oxidase from Alcaligenes sp. CF8 on 10% polyacrylamide gel. Lane A: purified caffeine oxidase. Lane B: crude caffeine oxidase. Lane C: low molecular weight markers: phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa) and soybean trypsin inhibitor (21.5 kDa). 324 B.R. Mohapatra et al. / Journal of Biotechnology 125 (2006) 319–327 Fig. 2. Effect of pH on activity () and stability () of purified caffeine oxidase from Alcaligenes sp. CF8. value reported for the caffeine oxidase isolated from the mixed cultures of Klebsiella and Rhodococcus species (Madyastha et al., 1999) and xanthine oxidase isolated from human liver (Krenitsky et al., 1986) and Arthrobacter species (Woolfolk and Downard, 1978). The optimum pH for maximum caffeine oxidase activity was found at pH 7.5 (Fig. 2). The pH optimum of purified caffeine oxidase isolated from mixed consortium of Klebsiella and Rhodococcus species (Madyastha et al., 1999) was pH 7.5, and xanthine oxidase isolated from Pseudomonas putida L. was pH 7.0 (Yamaoka and Mazzafera, 1999). The enzyme lost only 35% and 40% of the activity at pH 6 and 9, respectively. The enzyme was stable (>80%) in the pH range between pH 6 and 9 for at least 2 h incubation. Above pH 9, the stability sharply declined and retained only 60% of its maximum activity at pH 10. The effect of temperature on stability and activity of purified caffeine oxidase is shown in Fig. 3. Caffeine oxidase was quite stable at 35 ◦ C for almost 60 min incubation. The enzyme activity rapidly decreased above 40 ◦ C, and the enzyme revealed a half-life of 20 min at 50 ◦ C. The optimum temperature for maximum enzyme activity was found at 35 ◦ C. Nearly complete inactivation of enzyme occurred at 55 ◦ C. At 45 and 50 ◦ C, the enzyme exhibited 70% and 46% of its maximum enzyme activity, respectively. The activation and deactivation energies of the purified caffeine oxidase estimated from an Arrhenius plot were 68.69 and 122.06 kJ mol−1 , respectively (Fig. 4). This optimum temperature was found to be higher than the value reported for caffeine oxidase from mixed cultures of Klebsiella and Rhodococcus species (Madyastha et al., Fig. 3. Effect of temperature on activity () and stability () of purified caffeine oxidase from Alcaligenes sp. CF8. 1999) and xanthine oxidase isolated from Arthrobacter species (Woolfolk and Downard, 1978) and P. putida L. (Yamaoka and Mazzafera, 1999). Caffeine oxidase of Alcaligenes sp. CF8 utilizes molecular oxygen (O2 ) as electron acceptor for oxidation of caffeine with production of hydrogen peroxide in the assay mixture. Hydrogen peroxide was determined spectrophotometerically by using the peroxidase method (Ngo and Lenhoff, 1980). Specific activity (4.55 U mg protein−1 ) of caffeine oxidase with O2 was eight times lower than the value of specific activity (39.83 U mg protein−1 ) with DCIP as electron acceptor. Molecular oxygen was also found to be a poor electron acceptor compared to DCIP for caffeine oxidase isolated from Klebsiella and Rhodococcus species (Madyastha et al., 1999). The ultravioletvisible absorption spectrum of the purified caffeine oxidase of Alcaligenes sp. CF8 showed maxima at 278, Fig. 4. Arrhenius plot to estimate the activation and deactivation energies of purified caffeine oxidase from Alcaligenes sp. CF8. B.R. Mohapatra et al. / Journal of Biotechnology 125 (2006) 319–327 325 Table 4 Substrate specificity of purified caffeine oxidase from Alcaligenes sp. CF8 Substrate (0.055 mM) Relative activity (%) Caffeine Xanthine Hypoxanthine Theophylline Theobromine Ethyltheobromine 100 1.26 3.89 16.76 10.23 19.66 365, 450, 480 and 550 nm. The absorption spectrum of the enzyme was similar to that of xanthine oxidase from Arthrobacter sp. (Woolfolk and Downard, 1978). The relative oxidizing efficiencies of purified caffeine oxidase of Alcaligenes sp. CF8 were compared by measuring the reduction of DCIP with various substrates of caffeine analogues (Table 4). The enzyme showed strict specificity to caffeine. The enzyme did not catalyze xanthine and hypoxanthine. The DCIP reduction was only 10%, 16% and 19% in the presence of theobromine, theophylline and ethyltheobromine, respectively. Similarly the caffeine oxidase isolated from Klebsiella and Rhodococcus species did not show specificity towards xanthine and theophylline, however, the enzyme had an affinity towards theobromine and its derivatives ethyltheobromine (Madyastha et al., 1999). Kinetics parameters like the Michaelis–Menten constant (Km ) and maximum reaction velocity (Vmax ) were determined for purified caffeine oxidase with respect to caffeine and PMS at 35 ◦ C by Lineweaver–Burk plots. Under the optimal conditions (35 ◦ C, pH 7.5) the enzyme exhibited Michaelis–Menten type kinetics. The kinetic constant (Km ) measured for caffeine was 8.94 M and Vmax was 47.62 U mg protein−1 (Fig. 5). The Km value for phenazine methosulfate (PMS) was estimated to be 37.42 M (Fig. 6). The Km value with caffeine as substrate was lower than the value (11.4 M) reported for the mixed cultures of Klebsiella and Rhodococcus species at pH 7.8 and 30 ◦ C (Madyastha et al., 1999). The effect of metal ions and chemicals on the purified caffeine oxidase is shown in Table 5. The enzyme was not affected by the metal ions Ca+2 , Mg+2 and Na+ at 1 mM, however, the enzyme was completely inhibited by Co+2 , Cu+2 and Mn+2 . The metal chelating agents, EDTA and o-phenanthroline also inhibited Fig. 5. Lineweaver–Burk plot of purified caffeine oxidase activity against caffeine concentration in the presence of 0.55 mM PMS. S: caffeine concentration; V: caffeine oxidase specific activity. Error bars are standard deviation of triplicate measurements. Error bars are shown when they exceed dimension of the symbol. the enzyme activity at 1 mM concentration, indicating the caffeine oxidase is a metalloenzyme, and requires metal ions for its activity. Diphenyliodonium chloride inhibited the caffeine oxidase activity, suggesting the enzyme to be a flavoprotein. Further inhibition of caffeine oxidase by phenylmethylsulfonyl fluoride, pointing towards the participation of serine at the active site of the enzyme. The non-inhibition of enzyme activity by iodoacetamide and para-chloromercuribenzoic acid precluding the involvement of sulfhydryl (–SH) group at the active sites. The caffeine oxidase activity was Fig. 6. Lineweaver–Burk plot of purified caffeine oxidase activity against PMS concentration in the presence of 0.055 mM caffeine. S: PMS concentration; V: caffeine oxidase specific activity. Error bars are standard deviation of triplicate measurements. Error bars are shown when they exceed dimension of the symbol. 326 B.R. Mohapatra et al. / Journal of Biotechnology 125 (2006) 319–327 Table 5 Effect of different metal ions and chemical agents on purified caffeine oxidase of Alcaligenes sp. CF8 Metal ions and chemicals Ca+2 Mg+2 Na+ Co+2 Cu+2 Mn+2 EDTA PCMB Iodoacetamide O-phenanthroline DPI PMSF Salicylic acid Sodium arsenite N-methylmaleimide Percent activity retained 0.1 mM 1 mM 100 100 100 2 15 12 8 100 98 11 3 18 97 96 94 100 100 100 0 3 0 0 93 96 3 0 1 95 92 91 PCMB: para-chloromercuribenzoic acid; PMSF: phenylmethylsulfonyl fluoride; DPI: diphenyliodonium chloride. not inhibited by the xanthine oxidase inhibitors such as n-methylmaleimide, salicylic acid and sodium arsenite indicating the enzyme is not belonging to xanthine oxidase family (Woolfolk and Downard, 1978). The novel caffeine oxidase isolated from the Alcaligenes species CF8 is thermostable, salt-tolerant and has broad pH optima for its activity. Further it has a lower Km value, and has strict substrate specificity towards caffeine. These results suggest the superior quality of this enzyme for its potential biotechnological utilization including waste treatment and biosensor development. Future studies should be undertaken to elucidate the structure of caffeine oxidase as well as immobilization of caffeine oxidase on various matrices to increase its biocatalytic efficacy. Acknowledgements This research work was supported by the Natural Sciences and Engineering Research Council of Canada Industrial Research Chair Program in Environmental Management of Drinking Water, and the Canadian Institute of Health Research Grant under Safe Food and Water Initiative to AM. We also acknowledge the support from British Columbia Ministry of Environment for collection of water samples. References Armstrong, J.M., 1964. The molar extinction coefficient of 2,6-dichlorophenol indophenol. Biochim. Biophys. Acta 86, 194–197. Asano, Y., Komeda, T., Yamada, H., 1993. Microbial production of theobromine from caffeine. Biosci. Biotech. Biochem. 57, 1286–1289. Berthou, F., Guillois, B., Riche, C., Dreano, Y., Jacqz-Aigrain, E., Beaune, P., 1992. Interspecies variations in caffeine metabolism related to cytochrome P4501A enzymes. Xenobiotica 22, 671–680. Buerge, I.J., Poiger, T., Müller, M.D., Buser, H.R., 2003. Caffeine, an anthropogenic marker for wastewater contamination of surface waters. Environ. Sci. Technol. 37, 691–700. Eggins, B.R., 1996. Biosensors: An Introduction. Wiley-Teubner Inc., New York, p. 212. Feldman, J.R., Katz, S.N., 1977. Caffeine. In: Encyclopedia of chemical processing design. Marcel Dekker Inc, pp. 424–440. Glassmeyer, S.T., Furlong, E.T., Kolpin, D.W., Cahill, J.D., Zaugg, S.D., Werner, S.L., Meyer, M.T., Kryak, D.D., 2005. Transport of chemical and microbial compounds from known wastewater discharges: potential for use as indicators of human fecal contamination. Environ. Sci. Technol. 39, 5157–5169. Gokulakrishnan, S., Chandraraj, K., Gummadi, S.N., 2005. Microbial and enzymatic methods for the removal of caffeine. Enzyme Microb. Technol. 37, 225–232. Gould, J.P., Hay, T.R., 1982. The nature of the reactions between chlorine and purine and pyrimidine bases: products and kinetics. Water Sci. Technol. 14, 629–640. Krenitsky, T.A., Spector, T., Hall, W.W., 1986. Xanthine oxidase from human liver: purification and characterization. Arch. Biochem. Biophys. 247, 108–119. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227, 680–685. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265–275. Madyastha, K.M., Sridhar, G.R., 1998. A novel pathway for the metabolism of caffeine by a mixed culture consortium. Biochem. Biophys. Res. Commun. 249, 178–181. Madyastha, K.M., Sridhar, G.R., Vadiraja, B.B., Madhavi, Y.S., 1999. Purification and partial characterization of caffeine oxidase – a novel enzyme from a mixed culture consortium. Biochem. Biophys. Res. Commun. 263, 460–464. Mazzafera, P., 2002. Degradation of caffeine by microorganisms and potential use of decaffeinated coffee husk and pulp in animal feeding. Sci. Agric. 59, 815–821. Ngo, T., Lenhoff, H., 1980. A sensitive and versatile chromogenic assay for peroxidase and peroxidase-coupled reactions. Anal. Biochem. 105, 389–397. Ogunseitan, O.A., 1996. Removal of caffeine from wastewater by Pseudomonas putida Biotype A: implications for water pollution index. World J. Microbiol. Biotechnol. 12, 251–256. Ogunseitan, O.A., 2002. Caffeine-inducible enzyme activity in Pseudomonas putida ATCC 700097. World J. Microbiol. Biotechnol. 18, 423–428. B.R. Mohapatra et al. / Journal of Biotechnology 125 (2006) 319–327 Scott, T.M., Rose, J.B., Jenkins, T.M., Farrah, S.R., Lukasik, J., 2002. Microbial source tracking: current methodology and future directions. Appl. Environ. Microbiol. 68, 5796–5803. Smith, R.M., 1999. Supercritical fluids in separation science – the dreams, the reality and the future. J. Chromatogr. A 856, 83–115. Steffen, D.G., 2000. Chemistry and health benefits of caffeinated beverages: symposium overview. In: Parliament, T.H., Ho, C.T., Schieberle, P. (Eds.), Caffeinated Beverages, Health Benefits, Physiological Effects, and Chemistry, Ser. 754. ACS, Washington, DC, pp. 2–8. Udayasankar, K., Manaohar, B., Chokkalingam, A., 1986. A note on supercritical carbon dioxide decaffeination of coffee. J. Food Sci. Technol. 23, 326–328. U. S. Environmental Protection Agency, Office of Pollution Prevention and Toxics. OPPT High Production Volume Chemicals. http://www.epa.gov (September 2002). Vitousek, P.M., Mooney, H.A., Lubchenko, J., Mellilo, J.M., 1997. Human domination of Earth’s ecosystem. Science 277, 494–499. 327 Weigel, S., Berger, U., Jensen, E., Kallenborn, R., Thoresen, H., Hühnerfuss, H., 2004. Determination of selected pharmaceuticals and caffeine in sewage and seawater from Tromsø/Norway with emphasis on ibuprofen and its metabolites. Chemosphere 56, 583–592. White, P.A., Rasmussen, J.B., 1998. The genotoxic hazards of domestic wastes in surface waters. Mut. Res. 410, 223– 236. Woolfolk, C.A., 1975. Metabolism of N-methylpurines by a Pseudomonas putida strain isolated by enrichment on caffeine as the sole source of carbon and nitrogen. J. Bact. 123, 1088– 1106. Woolfolk, C.A., Downard, J.S., 1978. Bacterial xanthine oxidase from Arthrobacter S-2. J. Bact. 135, 422–428. Yamaoka, D.M., Mazzafera, P., 1999. Catabolism of caffeine and purification of a xanthine oxidase responsible for methyluric acids production in Pseudomonas putida L. Rev. Microbiol. 30, 62–70.