Current Research Journal of Biological Sciences 3(3): 246-253, 2011ISSN: 2041-0778© Maxwell Scientific Organization, 2011Received: March 24, 2011 Accepted: April 20, 2011 Published: May 05, 2011

Corresponding Author: Dr. J.S. Ghosh, Assistant Professor, Department of Microbiology, Shivaji University, Vidyanagar,Kolhapur 416004, India. Ph: +91 9850515620

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Production, Purification and Characterization of Tannase fromRhodococcus NCIM 2891

Naiem H. Nadaf and Jai S. GhoshDepartment of Microbiology, Shivaji University, Vidyanagar, Kolhapur 416004, India

Abstract: The study was done with the objective of tannic acid degradation by tannase from the RhodococcusNCIM 2891 which is actually a desulfurizing bacteria. Rhodococcus NCIM 2891cultivated in mediumcontaining 0.1% Tannic acid, produced tannase which showed maximum activity after 24 h. The enzyme waspurified by DEAE cellulose ion exchange chromatography and was shown to be dimeric in nature it has twosubunits as Tannase I and Tannase II, having specific activities of 0.23 U/mg of protein and 0.295 U/mg ofprotein, respectively. Both the enzyme showed optimum activity at pH 6 and 30ºC. The Km of both enzymeswas 0.034 and 0.040 mM respectively and Vmax of both enzymes were found to be 40 and 45 U/mL,respectively. SDS-PAGE analysis of purified proteins fraction revealed that their molecular weights are 60 and62 kDa. Tannase I was completely inhibited by 10 mM Hg2+, Cu2+, Fe3+, Co2+ and Tannase I and II both wascompletely inhibited by1 mM Hg2+ alone. The DNA damage- protecting activity of tannic acid and productobtained after tannic acid hydrolysis was assessed bringing about the DNA damage with H2O2 + UV exposurein absence of the hydrolyzed products of tannic acid. The experiment was carried out by using agarose gelelectrophoresis to analyze DNA damage in presence of H2O2 + UV. However, there was no damage of DNAby H2O2 + UV exposure in presence of the hydrolysis products.

Key words: Gallic acid, NCIM 2891, pollutants, Rhodococcus, tannase, tannin

INTRODUCTION

Tannins are naturally occurring water-solublepolyphenols which are often considered asrecalcitrant, that commonly exist in wastewater fromforestry, paper and leather industries and cause manyproblems associated with environmental pollution(Yagu et al., 2000). These are widespread in the plantkingdom, occurring mostly in leaves, fruits, bark andwood and are often considered nutritionally undesirable(Chung et al., 1998; Murugan and Saleh, 2010). Tanninsinhibit growth of various microorganisms, byprecipitating many enzymes and (Field andLettinga, 1992a).

Several conventional physiochemical techniques areavailable for tannin-containing wastewater treatment(Svitelska et al., 2004). However, the major drawbacks ofthese processes are high cost, tendency to form secondaryhazardous byproducts, and incomplete degradation. Thebiological treatment of tannin-containing wastewater isnow considered to be a better alternative, though these toohave certain limitations (Bhat et al., 1998; Ren, 2004;Field and Lettinga, 1992b).

Tannin acyl hydrolase (EC 3.1.1.20), commonlycalled tannase, is an enzyme that hydrolyzes the esterbonds of tannic acid, to produce gallic acid and glucoseand galloyl esters (Belur and Mugeraya, 2011). Besidesgallic acid production, the enzyme is extensively used inthe preparation of instant tea, wine, beer, and coffee-flavored soft drinks and also as additive fordetanniffication of food (Lekha and Lonsane, 1997). Inaddition, it is also used as a sensitive analytical probe fordetermining the structure of naturally occurring gallic acidester (Haslam and Stangroom, 1996). Although tannase ispresent in the plants, animals and microorganisms, it ismainly produced by the microorganisms (Ayed andHamdi, 2002). Filamentous fungi of the genusAspergillus, have been widely used for tannase production(Banerjee et al., 2001; Beniwal and Chhokar 2010).

Members of the genus Rhodococcus occur widely,and are aerobic, non-sporulating bacteria, with a high GCcontent. These organisms are of environmental andbiotechnological importance because of their broadcatabolic diversity and array of unique enzymaticcapabilities (Bell et al., 1998) and their exceptional ability

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to degrade hydrophobic natural compounds andxenobiotics. However, there are no reports of tannic aciddegradation or production of tannase by Rhodococcus.

This investigation focuses about potential uses ofRhodococcus in tannin waste recycling and production ofvaluable products, applicable for the various industrialprocesses.

MATERIALS AND METHODS

The study was conducted between August 2010 andFebruary 2011. The work was carried out at theDepartment of Microbiology, Shivaji University,Kolhapur, India.

Microorganism: Rhodococcus NCIM 2891 was obtainedfrom NCIM (National Collection of IndustrialMicroorganisms, NCL, Pune, India). The culture wasmaintained on GYEME ( Glucose Yeast extract Maltextract ) agar having the following composition 1%glucose, 0.3% yeast extract, 0.3% malt extract 0.5%peptone and 1.5 % agar-agar.

Growth of Rhodococcus on tannic acid: TheRhodococcus NCIM 2891 was grown in mediumcontaining 0.3% NaNO3, 0.1% K2HPO4, 0.05% MgSO4,0.05%KCL, 0.0001% FeSO4, and 0.1% tannic acid. ThepH was adjusted to 7.0.

Screening of Rhodococcus for the production oftannase: Tannase production by Rhodococcus NCIM2891 was checked by growing the microorganism onnutrient agar supplemented with 2% tannic acid whichwas filter sterilized. Addition of tannic acid to nutrientagar plate forms a complex of tannic acid and proteinsand after cleavage of this complex, organism producesclear zone around colony.

Tannic acid degradation:Enzyme extraction and purification: The enzymestannase was purified from cell free medium. In first stepcell free medium was saturated up to 40% withammonium sulphate and allowed to precipitate for 6 hrsat 4ºC. This precipitate was discarded and the supernatantwas saturated with ammonium sulphate up to 80%, whichwas kept overnight for residual enzyme precipitation. Thisprecipitate was dissolved in 20 mL of 1 mM citrate buffer(pH = 6.0) and was further purified by DEAE - Celluloseion exchange column chromatography. The elution wascarried out at a flow rate of 0.5 mL/min, using NaClconcentration gradient from 0 to 0.2 M in 50 mM citratebuffer (pH = 6.0). The fractions were analyzed for tannaseactivities, and later used for SDS-PAGE analysis.

Tannase assay: The reaction mixture consisted of 0.3mL of tannic acid (0.7% (w/v) in 0.2 M citrate buffer (pH6.0) and 0.1 mL of the enzyme extract. This wasincubated at 30ºC for 20 min. The enzymatic reaction wasparalyzed by the addition of 3 mL of bovine serumalbumin solution - BSA (1 mg/mL), leading to theprecipitation of the remaining tannic acid. This was thencentrifuged at 9000×g for 15 min at 4ºC and the residuewas dissolved in 2 mL of SDS-triethanolamine, followedby the addition of 1 mL of FeCl3 reagent and holding for15 min for colour stabilization. The absorbance wasmeasured at 530 nm. One unit of tannase activity wasdefined as the amount of tannic acid hydrolyzed by 1 mLof enzyme minute of reaction.

High performance thin layer chromatography(HPTLC): The gallic acid produced was analyzed byusing High performance thin layer chromatography(HPTLC) system (CAMAG, Switzerland). Stationaryphase HPTLC silica gel 60 F254 (Merck, Germany),predeveloped with methanol, dried at 120ºC for 20 min,cooled to room temperature, and equilibrated with therelative humidity of the laboratory. The plates weredeveloped using saturated chamber containing developingsolvent (butanol: acetic acid: water in 4:1:1 ratio). Thechamber was saturated for 20 min prior to platedevelopment. After development the plate was scanned at280 nm. The results were analyzed by using HPTLC WinCATS 1.4.4.6337 software.

Effect of pH and temperature: The effect of pH andtemperature was checked for both the enzymes. The effectof pH on enzyme activity was studied at varying pH rangefrom 4.0 to 10.0, using citrate- buffer (pH 3.0-5.0),sodium- phosphate buffer (pH 6.0-8.0) and glycine-NaOH buffer (pH 9.0-10.0). The effect of temperature onenzyme activity was studied between temperatures 10 to90ºC. In all cases the preincubation of enzyme was carriedout for 30 min with respective pH and temperatures andthen activity was measured by standard assay as describedabove.

Effect of metal ions: To study the effect of metal ions onenzyme activity, enzyme was pre-incubated in citratebuffer (pH 6.0) with metal ions Ca2+, Hg2+, Fe3+, Co2+,Mg2+, Cu2+ using their respective water soluble salts with1 and 10 mM concentration. The residual enzyme activitywas subsequently measured by standard assay asdescribed above.

Enzyme kinetics: The Km and Vmax of the Tannase I andII was determined by Lineweaver-Burk plots of reciprocalreaction velocities versus reciprocal substrate

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concentrations. Reactions were performed in citrate buffer(pH 6.0); with various concentrations of substrate 1%tannic acid by the addition of 0.1 to 1 mL. and enzymeactivity was monitored under the same conditions that arementioned above.

Protection against DNA damage induced by H2O2 +UV: DNA damage and DNA protecting activities onsample DNA according to protocol of Russo et al. (2000)modified by Skandrani et al. (2009). The test DNA usedin experiment was 8 phage DNA (Linear double strandedwith 48502 base pairs purchased from Genie, BangaloreIndia). The 8 phage DNA was oxidized with H2O2 + UVtreatment in presence and in absence of product obtainedafter hydrolysis of tannic acid.

The total volume of reaction mixture prepared was17µL containing 500 ng of 8 phage DNA, 10 µL tannic acid(0.1% w/v) and 10 µL incubated cell free medium (filtersterilized). H2O2 was added to this at a final concentrationof 100 mM. The whole system was exposed to UV light(wavelength of 312 nm) at ambient temperature for 5 min.After irradiation, the mixture was incubated at roomtemperature for 15 min. Untreated 8 phage DNA wasused as a control during the run of gel electrophoresisalong with UV and H2O2 treated sample of DNA.

Protein estimation: Protein estimation was done by theLowry method using bovine serum albumin as standard(Lowry et al., 1951).

Statistical analysis: Results obtained were the mean of 5replications. Analysis of the variants was carried out onall data at p<0.05 using Graph Pad software. (Graph PadInstat version 3.00, Graph Pad software, San Diego, CA,USA).

RESULTS

Growth of Rhodococcus NCIM 2891: It was observedthat the Rhodococcus NCIM 2891 was able to grow onTannic acid as a sole source of carbon. The maximumTannic acid degradation was observed in exponentialphase.

Screening of Rhodococcus for the production oftannase: The clear zone was observed around the growthof Rhodococcus NCIM 2891 after 48 h of incubationindicates that the microorganism producing tannaseenzyme cleave tannic acid protein complex byhydrolyzing tannic acid.

Fig. 1: The elution profile of DEAE-Cellulose Ion exchange chromatography of Tannase enzyme. Protein concentration of fractions(•) and activity of tannase enzymes (#)Results obtained are mean values±SD, n = 5

Table 1: Purification profile of tannase enzymesEnzyme Enzyme activity (units) Proteins (mg) Sp activity (Units/mg) Yield (%) Purification foldCrude 455 3500±0.05* 0.13±0.08* 100 1After ammonium 301 1005±0.06* 0.29±0.05* 66 2.23sulfate precipitationAfter ion exchange chromatographyFraction 1 60 268±0.03* 0.23±0.02* 13.18 1.76(Tannase - I)Fraction no 2 190 644±0.06* 0.295±0.05* 41.57 2.23(Tannase - II)Results obtained were the standard error of mean of 5 replications; *: p< 0.05

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1000.0[AU]800.0700.0600.0500.0400.0300.0200.0100.00.050.0

[AU]40.0

35.030.0

25.020.0

15.010.0

5.00.0

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0.000.00

0.400.20

0.000.20

1000.0[AU]800.0700.0600.0500.0400.0300.0200.0100.0

0.0

d

Fig. 2: SDS-PAGE of purified Tannase I (a) Tannase II (b),from Rhodococcus NCIM 2891 Size of the standardmarkers is at left. Markers (da) (m): phosphorylase b(97.4 kDa), Bovine Serum albumin (66 kDa), ovalbumin(43 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor(20.1 kDa), and lactabumin (14.3 kDa)

Purification of Tannase: The purification profile oftannase enzyme is given in Fig. 1 and the activity ofextracted tannase at different stages of purification isgiven in Table 1. It has been noted from Table 1, that theprecipitate at 80% saturation of (NH4)2SO4 was giving thebest yield of the enzyme. The same yield did not showany improvement after DEAE Cellulose ion-exchangechromatography, except that the 2 fractions seperated(Fig. 1).

The specific activities of Tannase I and Tannase IIwas measured about 0.23 U/mg of protein and 0.295U/mg of protein using tannic acid as substrate.SDS/PAGE analysis revealed that the two enzymes hadmolecular weights of 60kDa and 62 kDa respectively asshown in Fig. 2.

HPTLC analysis: The HPTLC analysis of incubatedbroth indicated the production of gallic acid due tohydrolysis of tannic acid by Tannase, as shown in Fig. 3.The Rf value of standard gallic acid was 0.90 where as theproduct of hydrolysis gave Rf values 0.89 and 0.88. theseRf values linearly equals each other, indicating thattannase produced by the microorganism was able todegraded tannic acid in to its monomer, gallic acid.

Fig. 3: HPTLC analysis of standard gallic acid and incubated broth after 24 h. and 48 h,(a) standard gallic acid; (b) broth after 24h. incubation; (c) Broth after 48h. Incubation; (d) 3D peak profile of gallic acid; (e) developed HPTLC plate in that

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4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0

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Fig. 4: Effect of pH on enzyme activity tannase I (•) andTannase II (#).Results obtained are mean values±SD,n = 5

Fig. 5: Effect of Temperature on enzyme activity Tannase I (•)and Tannase II (#).Results obtained are meanvalues±SD, n = 5

Fig. 6: Effect of heavy metals on tannase I with 1mM and 10mM concentration. Results obtained are meanvalues±SD, n = 5

pH and temperature effect: The optimum pH of theTannase I and Tannase II were 6 whereas temperatureoptimum for Tannase I and Tannase II were 30ºC asshown in Fig. 4 and 5.

Effect of heavy metals: The Tannase I showed completeinhibition by Hg+2

in 1mM concentration and by 10 mMFe3+, Cu2+, Co2+ whereas the activity of tannase wasretained 100 %, by 1 mM Cu2+ as shown in Fig. 6.

Fig. 7: Effect of heavy metals on Tannase II with 1mM and 10mM concentration. Results obtained are meanvalues±SD, n = 5

Fig. 8: Km of Tannase I with Tannic acid as a substrate resultsobtained are mean values±SD, n = 5

Fig. 9: Km of Tannase II with Tannic acid as a substrateResults obtained are mean values±SD, n = 5

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a b c d

Fig. 10: Profile of agarose gel electrophoresis lane (a) DNA +H2O2, lane (b) Control DNA, lane (c) DNA+ H2O2+incubated cell free medium and lane (d) DNA+ H2O2+Tannic acid

The Tannase II showed complete inhibition by Hg+2

at 1 mM concentration and 25% of the activity wasinhibited by 1 mM Mn+2

, and 10mM Mg2+, Cu2+, Co2+.

Tannase I showed increase in activity by 37% aboveoptimum, in presence of 1 mM Cu2+

, 10 mM Ca2+, by 25%in presence of Co2+ and up to 12% in presence of Fe3+

(Fig. 7).

Enzyme kinetics: The Km of Tannase I is 0.034 mMwhereas the Km Tannase II 0.040mM. The Vmax of bothenzymes were found to be 40 and 45 U/mL, respectivelyas shown in (Fig. 8 and 9).

Protection against DNA damage induced by H2O2 +UV by obtained product after hydrolysis of tannicacid: Figure 10 shows that the DNA damage wasobserved in first sample (a) where the DNA was treatedwith 100 mM H2O2. Sample (b) which was unexposedcontrol, shows no damage of DNA. Sample(c), which wastreated with 100 mM H2O2 and 10 :L incubated cell freemedium containing gallic acid shows similar band as incontrol. Sample (d) which had tannic acid, showed somedamage of the DNA.

DISCUSSION

This is a first report of tannase production fromRhodococcus NCIM 2891 where this microorganismproduces 2 extracellular tannase namely Tannase I andTannase II. The study regarding the production ofTannase by other microorganisms reveals that the fungiare predominant tannase producers and extensivelystudied (Bradoo et al., 1996). Deschamps et al. (1983)reported that the optimum enzyme production by Bacilluspumilus, Bacillus polymyxa, Cornybacterium sp. andKlebsiella pneumoniae in presence of chestnut bark assole carbon source. The preliminary study to checktannase production from Rhodococcus NCIM 2891included plate assay. The production of gallic acid duringincubation was checked by HPTLC method.

Monika et al. (2007) reported the production of twoextracellular tannase but cold adapted from Verticillumsp. 9 each consisting of approximately 40 and 60 kDa subunits Rhodococcus NCIM 2891 also produces twoenzyme namely Tannase I and Tannase II havingapproximately molecular weight around 60 and 62 kDa.The other known fungal tannase are multimeric proteinsand molecular weight varying from 168 to over 310 kDa(Sabu et al., 2005; Hatamoto et al., 1996).

Both tannase were optimally active at pH 6 howeverthe optimum pH was 5-5.5 in case of fungal tannase(Sabu et al., 2005). The optimum temperature of theTannase I and and II is 30oC which is same as reported forthe Bacillus licheniformis KBR 6 (Mondal andPatil, 2000) as compared many reports regardingtemperature variability among tannase produced bydifferent type of fungi (Sabu et al. 2005).

The Km of Tannase I and II are 0.034mM and 0.040mM which is very low as compared to the Km value for asimilar enzyme produced by Cryphonectria parasitica,using tannic acid as substrate, have been found to be 0.94mM (Farias et al., 1994). The Km values for tannases fromSelenomonas ruminantium and Cryphonectria parasaticausing methyl gallate as substrate have been found to be1.6 mM (Skene and Brooker, 1995) and 7.49 mM(Farias et al., 1994). The Km values for tannic acid oftannases produced by other fungi ranged from 0.20 to1.03 mM (Mukherjee and Banerjee 2006;Sharma et al., 1999; Sabu et al., 2005).

The enzyme was completely inhibited by Hg2+, Cu2+,Fe2+, Co2+ ions the effect of metal ions are similar to thosereported previously but the only difference was thatactivity of other tannases are increased in presence ofMg2+ (Kar et al., 2003).

There are reports which states important applicationof tannase that is the production of gallic acid andpropylgallate, fine-chemicals employed as antioxidants infoods, cosmetics, hair products, adhesives, and lubricantindustry (Kar et al., 2002; Yu et al., 2004; Yu andLi, 2006). Min-Jer and Chinshuh, (2008) studiedenzymatic modification by tannase which increased theantioxidant activity of green tea. The antioxidantproperties of tannic acid degradation, was checked byusing DNA damage assay. The oxidative DNA damageinduced by tea catechins especially epigallocatechingallate (EGCG) in presence of metals complexes such asCu2+ and Fe3+ has been reported to decrease due to EGCGhydrolysis (Furukawa et al., 2003). These are similar withour results but the difference was only that there were nometals complexes.

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CONCLUSION

The tannase production by microorganism isextensively studied with respect to the food andpharmaceutical industries. However, it was mostly studiedin fungi and in some extent in other microbial species.The results of this investigation clearly indicate thattannase produced by Rhodococcus NCIM 2891 has higheraffinity for tannic acid as compared to those produced byother bacterial species. Tannic acid is water solublemetabolite of plant showing antagonistic activity againstbacteria and its high concentration in environment cansignificantly affect the biodiversity. The findings of thisstudy clearly indicates that tannase from RhodococcusNCIM 2891 can be used as a potent bioremediation toolfor industrial waste containing tannins or their derivatives.The antioxidant properties of tannic acid degradationproducts was another significant, novel finding whichcould be used to remove tannins from various substanceslike fodder for sheep and goats and thus improve thehealth of these animals.

ACKNOWLEDGMENT

The authors are very grateful to the Department ofMicrobiology, Shivaji University, Kolhapur, for extendingthe laboratory facilities to complete the investigation.

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