Research Journal of Microbiology 2 (4): 327-336, 2007 ISSN 1816-4935 © 2007 Academie Journals ‘The Effect of Glucose on Growth and Degradation of Caffeine by Pyeudomonas sp. Sathyanarayana N. Gummadi, Anath C. Lionel, ‘Swati S, Dash and S. Gokulakrishinan Department of Biotechnology, Indian Institute of Technalogy-Madsas, ‘Chennai 600 036, India Abstract: The effect of glucose on eafleine degradation by Pseudomonas sp, which was previously isolated in cur laboratory, was investigated, Caffeine degradation was inhibited hen glucose vas preset in foe form inthe medium and ata concentration > I gL ‘Two-stage culture experiments indicated that glucose probatty interferes with the induetion of the enzymes invelved in the eaffeine degradation pathway. The addition of glucose at start and early stages of growth resulted in complete inhitition of eaffine degradation whereas addition at later stapes of growth has no inhibitory effect futher confirming the inhibitory effect of glucose is due suppression of caffeine catabolsing enzyme, which is ‘suggestive of catabolite repression. These findings are novel and have nce been reported in other Pseudomonas peptide strains degrading caffeine. Key words: Caffeine degradation, Pseudomonas, glucos, inhibition, two-stage culture, catabolite repression INTRODUCTION Microbial and enzymatic methods of caffeine degradation offer a bette alternative tothe present existing conventional techniques of decaifination (Gokulakrishnan et a, 2005) which is one of the ‘important steps in coffee processing Keeping in view the adverse effect of caffeine on health (Cooper ef a, 1992, James, 2004) andl environment (Bressani, 1987). However, the major constraint indevelopment of biodecaffeinaton techniques isthe lack of « microbial srain capable of degrading higher conoentrations of ean at an appreciable rte. In this comext, a calTeine degrading stain of Pseudomonas capable of uilizing caffeine as the sole souree of carbon and nitrogen fas been isolated ‘nur laboratory from the soil of eotee plantation area (Gokulakrshnan et af, 2006), which closely resembles Pseudomonas peptide as per 168 rRNA analysis (Dash andl Gummadi, 2006a). Kinetic studies on the strain showed that the isolate could tolerate high concentration of caffeine (-12.5- 15g1L~ calleine) and dhe minimum inhititory concentration of caffeine was 20 g L~'(Gokulakrishan anc Gommadi, 2000). Tis makes the strain an excellent candidate for calene biodegradation studies. In preliminary studies, the stain exhibited unique caffeine metabolism when different carbon. sources were supplied (Gokulaksishnan eral, 2006) Disscchardes (sucrose and acto) enhanced the ‘ate of eaffsine degradation by this stain without being uilized s carbon source, wheteas plucose completely inhibited the growth and caffeine degradation by this strain On the olher hand, monosaccharides like Fructose and galactose were used as carbon sources. Although there are reports ‘on the utilization of earbon sources by caffeine degrading Pseudomonas peptide (Blacher and Lingens, 1977), he phenomena of inhibition of caffeine degradation by glucose has not been reported + SalyararayanaN. Gira, Deprtnet of Bcteceleg, Indian tte of Teco Ceara 60086 nla Tek abDS7-ALLA Fee 91-25-0809 ‘27 Corresponding AtRes. J. Microbiol, 2 (4): 327-336, 2 inany other Pseudomonas pepide strains and appeats unique to the strain wed in this study. This will rove erucal in developing conditions for degradation of caffeine bythe strain when natural sources cof coffee and tea wastes, which contain different carbohydrates, are used. In this study an attempt has ‘been made to delve more into the matter and to elucidate the possible mechanism ofthis inhibitory phenomenon of glucose on caffeine degradation by Pseuiomonas sp MATERIALS AND METHODS. Microorganism Pseudomonas sp. previously isolated! was maintained on CAS agar medium which tid the following composition (gL): NaHPO, 0.12; KH,PO,, 1.3; CaCl, 03; MgSO,. 7 H,0, 03. caffeine, 1.2; suotese, 5 and agar, 25. The strain was sub-cultred every altemate day fo maintain caffeine degrading ability Chemicals Caffzine (299% pity) was obtained ftom Merck, India. Theobromine, theophylline, T-methybsanthine and xanthine were obtsined ftom Sigma, All other chemicals were procured from Hi-Media, India. Media [Natsient Broth used fr seed cite lad the fallowing compenition (g L~ hoof extract 1s yeast ‘extract 25 peptone, 5; NaCl, 5, The composition of basal medium (BM) was as follows (g L~; Na,HPO,, 0.12; KH,PO,, 1.3; CaCh, 0.3; MgSO, 7 H,O, 03, CAS medium consisted of BM supplemented with 5 g L~' sucrose and 1.2g L~ caffeine. Carbon sourees added to the median were sterilized separately and then mixed with the medium under aseptic conditions, The initial ‘concentration of glucese and sucrose in the medium Was 5 g L~. Steck solution of 825 g L~ glucose vas prepared and autoclaved separately for intermittent additions. For an altemate nitrogen source caffaine vias replaced by 2.2 g Lt ammonium sulphate in CAS medium as per experimental ‘equitement. The intial pH of the medium was adjusted to 6.0 befone autoclaving. Flask Culture Experiments Foc flask calture experiments, three loops of cultare from CAS agar plat cultured at 30°C for 36 h was transfered to 25 mil. sterile Nutrient Broth and incubated in a rotary shaker (Ortitex, Scigenics Biotech, India) at 30°C and at 180 spm. After the seed OD, reached ~1.3, 0% (Wi) ofthe culture was transfered to 25 ml, of CAS medium in 100 ml. Erlenmeyer flasks and incubated on a ‘rotary shaker at 180 1pm and at 30°C, AL regutar intervals samples were collected and analyzed for cal grovith, caffeine degradation and sugar utilization. Three sets of experiments were performed as described below: Effect of Addition of Glucose with Other Sugars and at Different Concentrations “The medium for this experiment consisted of Basal Medium (BM) supplemented with plucose and other sugars at 5 gL concentation, separately or in combination. Whe two sugars were used, ‘composition of both the sugars was adjusted soeh thatthe total eatbon content was § pL. The carbon sourocs were autoclaved separatcly anl added just before inoculation. 6% (vi) of the seed culture at ODjq~1.3 was transferred to 25 ml, of medium in 100 ml. Enlenmeyer flask ar incubated ‘ona rotary shaker at 180 rpm and at 30°C. Samples were collected at gular intervals and analyzed for cal growth caffeine degradation end suger uflzaion. All experiments were perfonmed in triplicates ‘under identical conditions arn all results had a standard deviation of +1 to 3% about the mean, 28Res. J. Microbiol, 2 (8): 32 ‘Two Stage Culture Experiments “Two stage culture experiments were performed in order to determine the mechanism of inhitition ‘of caffeine degradation by glucose Cells wer nitially grown in glocose-ammonium sulphate medium and CAS medium and were laevested by centrifugation at 10000 x g (Centsfiage 5810 R, Eppendorf, (Germany) atthe mid log phase ~ 10), followed by twice washing with $0 mM potassium phesphate Duties (PH 70). Equal amoust of cells 0.016 g L~ dry weight) were transfered in the second stage inthe Fllowing manner: (from glucese-ammonium sulphate meunn to CAS medium, (i) fiom CAS ‘medium to glucose-caffeine medium and (i) from CAS medi to fresh CAS medium as contra, (Caffeine degradation and sugar consumption were monitored at diferent time intervals. In all second stage experiments, 0.016 g dry cell weight L~! of medium was maintained. Experiments were performed ‘in eiplicates anal yells had a stand deviation of +1 105% about the mean. Intermittent Addition of Glucose In this set of experiments, 6% (vv) of the seed culture at OD. ~1.3 was transferred t0 25 mL, ‘of CAS medium in 100 mL Erlenmeyer flasks and incubated ona rotary shaker at 180 spm and at 30°C, One mililter of stile glucose solution (625 g L~* ) was added to the culture at 0, 2,3, 5,7 and 8 hof grovith The contol consisted of CAS medium without addition of glucose, Samples were collected atthe shove mentioned time points and analyzed for call growth, eaeine degradation and sugar uilization, Experimental values reported are an average of three sets With £14 to 45% standaed deviation about the mean. Analytical Determinations Cell density in the medium was monitored by measuring the optical density at 600.nm in a spectrophotometer Biorad, India) (ODay of 0.5 corresponds to 0:379 g dry weight L~), Caffsne, ‘heobromine,7mellylxandhine and xaoshine were estimated by HPLC (Agilent 1100 series) equipment using a ZORBAX C-18 column with 10mM ammonium phosphate baller (pH 2.Syacetonitile (LL, viv) as mobile phase at flow rate of I mL min~* and at 28.5°C. Detection of caffeine was done a 254 nm, The concentrations of carbon source in the medium were estimated by 3, -initesalicylic acid method (Miller, 1959), Sucrose and lactose were hydrolyzed with 3N Hl by heating at 100°C for 15 min and the reducing sugars released were analyzed by 3, S-dnttosalicylie aid method. RESULTS AND DISCUSSION BBoctril stains belonging to the gems Pseudomonas ate well known for their capability to metabolize compound that are toxic to other microbes and this attribute has evolved as an adaptation ‘osubstate availability in the natural environment. However eatabolite repression is often associated withthe degradation of such compounds whereby the catabolism of the xenobiotic is suppressed in the presence of simple sugars like glucose (Collier er a, 1996), Preliminary studies pertaining to caffeine degradation have shown that earbon sources have a distinctive effect on the grow andl caffeine degrading ability of Pseudomonas sp. used in this study (Golulakrishnan er a, 2006) Partulaly, the inhbitey eff of glucose on efeine deradstion and growth of this strain has not ‘ee reported for any caffeine degrading bacterial strains so fat, which suggests the unique metabolic tributes that this strain might have in relation to caffeine catabolism and carbon source usilization, Results obtained indicate the possible role of glucose in suppression of eafeine degrading enzymes ‘which account forts inititory effect. Effect of Addition of Glucose with Other Sugars In the previous reported study (Gobulabuishnan et al, 2006), it was obseaved that Pseudcmonas sp. could effectively lilize monosaccturides like fructose and galactose as carbon. 29Res. J. Microbiol, 2 (4): 327-336, 2 sources along with caffeine. Glucose inhibited the degradation of caffeine whereas glucose containing disaccharides ike sucrose and lotose didnot, ther the rato of caffeine degradation was enhanced in their presence twas therefore necessary to investigate whether the inhibitory effect of glucose holds 200d when glucose is present in the fee foe or it is masked when glues is present along with any ‘other monosaccharide thatthe strain is capable of lizing. Also it was necessary to determine the concentration of glucose necessary to cause inhibition. For this purpose, the degradation of caffeine ‘was studied in meditam containing glucose with other monosaccharides Like fructose and galactose (where lacose was present in the fice form) and in presence of disaccharides like sucrose and lactose (were glucose was present asa one of the mnomers ofthe disaccharides). Caffeine degradation by Pseudomonas sp, was also stutied by varying the initial concentration of glucose in the medium fiom 0.05 to 5 gL“ Reaulls showed that complete inhibition of caffeine degradation was observed when gluose was. present in fee form along with another sugar, However, this inhibition effect was not seen when alucose was present as a monomer in the disaccharide (sucrose or lactose) (Fig. 1a). The effect of slucose concentration on caffeine degradation was studied by varying the initial concentration of salucose in the medium, Calne degradation was completely inhibited when the initial coneentration ‘of glucose was 5g L~! (Fig 1b) andi the range 1-5 g L~ of suetese (data not shown). The inition effet deereased when the medium contained <0.5g L~plucose (Fig, Ib), However, degradation in the presence of all concentrations of glucose was lower than control where the isolate was grown in the presence of sucrose, 100). if Portage callie Bos 2 ‘aus cues Guas "Soe Geese ge") Fig 1: Theinhibitory effect of ghucoseon the caine degradation by Pseudomonas sp, (a) effect of ferent sugars an in combination with glucose on caffeine degradation. (b) eect of glucose ‘concentration on caffeine degradation All experiments were performed in wiphicaes under ‘dentcal conditions and all rsults had a standard deviation of +1 to-=3% abou the mean 20Res. J. Microbiol, 2 (4): 327-336, 2 ‘These results are in contrast tothe findings of Asano et. af. where they reported that glucose cenkianced the degradation of caffeine to theobromine by Pseudomonas peptide (Asano eta, 1993), The ability ofthe strain to ulilize galactose but not lactose is also in contrast tothe caffeine degrading Pseudomonas stain isolated by Pletcher anil Lingens (Blecher and Lingens, 1977) that was capable of ulizing elucote fuctove and lactose but not galactose as carbon source. These differences make the strain unique from other caffaine degrading strain isolated so far and probably it has different pathway in metabolizing carbohydrate, which isnot surprising since hexoses, in Pseudomonas sp ‘can be catabolized in more than one route (Vieente and Canovas, 1973), Therefore, glucose in the fee form or some intermediate of glucose catabolism unique to this strain is possibly responsible fortis inhibition. Also fcen the results it is clear that since disaccharides were not utilized by the stain the situin lacks the enzymes needed for the conversion of disaccharides to fee glucose and therefore no inhibition of eaeine degradation was observed in presence of disaccharides. Tt was also noted that the inhibitory effect of glucose was concentration dependent since caffeine degradation was completely inhibited when th ial concentration of glucose in the medium vwas>l gL and decreased when the medium contained <0.5 g L~” glucose. Similar effet was seen in ease of 2-chlosphenol degradation by a strain of Pseudomonas peptide (Fakhnudin and Quilty, 2005) which farther supports stay Effect of Glucose on Induction of Caffeine Degrading Enzymes (One of the possible easons for the inkbitory effect of glucose isthe repression of synthesis of the enzymes involved in caffeine degradation. The inducibe nature of caffeine depraing enzymes has ‘boom established ftom earlier reports (Woolfolk, 1975, Ogunseitan, 2002) and also fre studies carried ‘out om tho strain used inthe present study (Gummadi and Santosh, 2006), Sine these enzymes are inducible in nature, the presence of glucose might suppress thsir induction dae fo which eaffeine degradation s inhibited as en outcome of catabolite repression. This hyposhesis is strengthened by the ‘ati ndings where lucote has been shown to epeess the induction of enzymes sesponsible forthe degradation of various compounds lke catehlol and chlrcatechol, methyl phenol and benzyl alcohol (Hotel er af, 1994; Muller eral, 1996; MeFall er a, 1997) ‘This hypothesis was tsted by two stage culture experiments an approach that has been used inprevious tues to establish enzyme induction (Naidu e al, 2001). According to this strategy, cells ‘were iiflly grown in a medium conisning glucose and ammonium sulphate and then transferred 10 CAS medium at mid log phase of growth, Since eafTeine was absent in the fist stage of growth, the calls were assumed not o be induced with caffeine degrading enzymes. The contol consisted of an equal amount of cells, initially row n CAS modium for 10h being tansfared to anew CAS medium. Equal amount of calls om CAS modu were also transferred to a mediuun containing phucese and caffeine, presuming that the oolls were induced atthe time of transfer to secon stage, ‘The cells transferred ftom CAS medium to glucose and ealfeine medium showed significant growth and caffeine degradation despite the presence of both eaeine and glucose in the medium (ig. 2aand b), The glucose uilization in dhe second sage is ~90% (Fig. 2¢) It vas observed that CAS _grovin cells hen transferred to glucose medium exhiited 90% culfeine depradation in 12b, Sinoe the cells had been grown in CAS medium dung first stage in the absence ofphuose, the caffeine degrading ‘enzymes are already present when the cells are transferred to the second stage (caffeine ae glucose medium). On the other hand, the cells transferred from glucose-«mmenium sulphate medium 10 CAS medium shoved litle growth in CAS medium and showed very less eafeine degradation (Soin 18h) (Fig, 2a and b), This can be atributed tothe fact that glucose present inthe first stage ‘of gowth (ihe ghacese-«moniuan sulphate mean ibbited the induction and expression of caffeine degrading enzymes, ‘The two stage culture experiment suppets the hypothesis that inhibition of caffeine degradation, ‘by glucose is doe fo intial inhibition ofthe expression of he ealTeine depraing enzymes anid that the a1Res. J. Microbiol, 2 (4): 327-336, 2007 16) —e- cas w Gtacatine a cisAsto cas a= caste cas Bog colley welt (@> H cASta Glatetine Ta Guise cas SE cisteas atin emsning 6) 3 8 8 8 hocosesealnag *) 8 casa cnncattioe 3 10 15 Tins) Fig. 2: Two stage ete experiment to show the effet of glucose on induction of caffeine de enzymes by Pseudomonas sp. Equal amount of eels initially grown in glucose-ammonium sulphate medium and CAS medium were tansterted to CAS and elucese-armceium sulphate, respectively. Control consisted of cells transfered from CAS medium fo fresh CAS medium, (call growth (0) caffeine degradation profile and (e) glucose consumption pol second stage culture ll experiments were performed in triplicates under identical conditions and al results ad a standatdl deviation of to-£5¥% about the mean the phenomena of catabolite repression occurs in this stain also, Degradation of cafvine in Pseudomonas follows sequential demthyiation leading to formation of theobromine, 7-methybsanthin and xanthine by eaffene N-demethylases (Dash and Gurmmadi, 2006b) and it also happens soin the strain used in a2Res. J. Microbiol, 2 (4): 327-336, 2 tis study (Dash and Gummadi, 20068). Giveose nas been showin to repress the gene coding for & domethy aso in case of the fingus Nectria haematococca in an catir seport (Khan and Staney, 1999). Furthermore, along with glucose, intermediates of glacose metabolism like gluconic acid have been ‘eported to cause the repression of the enzymes involved in eatechol degradation at wanscriptional level (MoFall et al, 1997), Probably, such a mechanism also operates in Pseudomonas 3p. used inthis sty in relation to the metabolism of ealTine fect of Addition of Glucose at Various Time Points of Growth From the above experiment, it was established that inhibitory effect of glucese on caffeine degradation is due to inhibition of enzyme expression. Therefore further experiments were needed 10 find out whether this catabolite repression i effective ony atthe initial stazes of growth ofthe bacteria inane medium or does it occur at all stages. For this purpose, glucose was added to CAS grown culture at various time points such that final concentration of glucese was 5 g L~ ‘When glucose was added a early time points (0 and 2), there was no significant difference in call growth compared to the control (CAS medium without addition of glucose) (Fig. 3a). Also, ‘complete inhibition of ean degradation was cbserved when glucose was added at Oh, wheteas very litle degradation (-5-8%) was abserved when glucose was added at 2h (Fig. 3). The glucose lization was also observe to be negligible a the carly adkition (Fig. 3). This indicates clearly that ‘the bacterumis not able to metabolize eafeine when glucose is added dusing initial stages of growth and neither i uilized sines there is no grovcth of the stain, ‘The station is however differnt when glicose was added atthe time of acive growth of the strain in caffeine medium. Significant growth was observed when glucose was added at later points (G to 8h) (Fig, 3a), Cleary, the possibility of diausio growth is ruled out as no growth was noticed ‘when glucose was present in the initial stages slong with caffeine. Supplementing this, significant ‘increase in degradation was observed when glocese was added atthe later stages and was observed 10 ‘be was > 90% when glucose was added at 8h (Fig. 3b). Moreover, glucose was also metabolized by the strain wen added at later stages and at 8h of glucose ation, glucose consumption was around 100% (Fig. 3c). A masismum dry col Weight of 23 g L~* was obtained when glucose was added at 8 ‘ith a yield of 0.46 g call dry weight 2 gluoose suggesting that pluoese was being used as an ‘adtional carbon source Ihas also been observed tat suerose Wes not consumed (ata not shovin. ‘This proves thatthe inhibitory eff2xt oF glucose on caffeine degradation was effective only when slucose was presenti the inital tages of growth whercas degradation was enhanced when glucese was added at later stages of grovlh (Fig. 3). I father establishes thatthe addition of glucose at caly stages ‘of gronth inhibits the incuction and expression of caffeine degrading enzymes that are symihesized in ‘the lag phase, This effect ceases to exist when glucose was added at later stages of growth because already the genes necessary for expression of caffeine degrading ezymes would have been transcribed ‘in the cell and the strain is active growing in caffeine medium. Once sufficient biomass has been achieved, Pseudomonas sp. used in this study is also able to ublize glucose as carbon source, ‘The next sep was to investigate whether this phenomenon slmited to caffeine or does it extend to the other methylxanthines, which ate metabolites of caffeine degradation pathway. To check this, ‘the isolate was grown in CAS medium in which caffeine was replaced by theobromine,theaphi line, ‘-medhylsanthine and xanthine (tethylsanthines at |g L~ concentration) Tt vas obverved that isolate ‘could metabolize heobromin, 7-methylxanshine and xanthine but noe theophylline, Siniler experiment 1was performed inthe presence of glucose and found that isolate could not grow and degrade caffeine imctabolites. Thecphyine was not utilized in both the cases because itis not a metabolite in the degradation pathway ofthe bacteria, as shown earlier (Dash and Guramaci, 2006) It indicates that «enzymes bringing the conversion of caffeine to its metabolites ar repressed by glucose. More study ‘onthe molecular axpeets of regulation is required to arrive ata decisive thoory 33Fig. 3 Res. J Microbiol © (Cady wight) 4 IY 10 o w} © o wo aco reas) 2» 5 0 2 (9): 327-336, 2007 Tine) @ 7” Time @) cont on = 3h sh ar 3h cont on a “3h 5h en oon con on 2h 3h sh mh hn Elfect of addition of glucose at different growth points on calfeine degradation by Psendomenas sp. Glacose was sed from stock solution to achieve a concentration of Sg L-* in the medium at 0,2, 3, 5.7 and & h, respectively. Control consisted of growth of cells in CAS medium, (a) cell growth, (b) caffeine degradation and (c) sugar consumption were ‘monitored at regular intervals, Data presented is the average of triplicates with standard deviation about the meanRes. J. Microbiol, 2 (4): 327-336, 2 “These findings throw light cn the complex interaction between ghicose and caffeine metabolism, Either the pathways of metabolism of both these compounds are inerinked or glucose has effect on ‘he regulation of caffeine degrading enzymes. Pseudomonas sp. proves to be an attuetive model 10 ‘understand the degradation of caffeine on the molecular level, which in turn will prove beneficial in developing biological decaffeination techniques, ACKNOWLEDGMENT We are gratefil to Departinent of Science and Technology, India for their Financial support. REFERENCES Asano, ¥., 7. Komeda and #1, Yamada, 1993, Microbial production of theabromine fiom caffeine Biosci, Biotech. Biochem. 57: 1280-1289, Blecher, R. and. 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