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Therefore. 1992).elsevier.ibiod. present in various foods. 1983). 1998. 2007). doi:10. followed by DEAEcellulose and Sephadex G-150. All rights reserved. resist microbial attack and remains as recalcitrant in the environment (Field and Lettinga. Department of Biotechnology. The tannase catalyse the hydrolysis of ester and depside bonds presents in the hydrolysable tannins and gallic acid esters (Lekha and Lonsane. E.. with a few evidences showing degradation of phenol ring of CTs (Bhat et al. feeds and forages. The present investigation was carried out to study the degradation profile of tannins by Enterococcus faecalis and to purify tannase. The culture bottles were sealed under CO2 and incubated at 37  C for 72 h. Karnal 132001.. 2005b). India c Department of Biotechnology.1016/ Degradation of tannic acid and purification and characterization of tannase from Enterococcus faecalis Gunjan Goela. aerobic bacterial degraders and fungal species have been reviewed elsewhere (Bhat et al. fax: þ91 1731274375.: þ91 1731304148. Goel et al. 2001). with both beneficial and adverse effects in ruminants (Getachew et al. 1986.0% in minimal media) to gallic acid.. faecalis was observed to degrade tannic acid (1. Anil K. presence of tannase in microorganisms inhabiting rumen and gastrointestinal tract of animals results in detoxification of these tannins. 1. Though destructive in nature. Bhat et al.25 mM methyl gallate. Radaur 135133.. providing a general idea on the biodegradation of these polyphenols (William et al. 1998).7%..Author's personal copy International Biodeterioration & Biodegradation 65 (2011) 1061e1065 Contents lists available at SciVerse ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.1. JMIT Institute of Engineering and Technology. Introduction Tannins are the most common plant secondary metabolites that are poorly understood with respect to feeding habits. India b a r t i c l e i n f o Article history: Received 3 May 2011 Received in revised form 19 July 2011 Accepted 12 August 2011 Available online 15 September 2011 Keywords: Tannins Tannase Enterococcus faecalis Pyrogallol a b s t r a c t Tannins. being antimicrobial. Among tannin degrading microbes. Goel). Mullana. the key enzyme.2011. Materials and methods 2. These microorganisms have developed protective mechanisms such as secretion of binding polymers and synthesis of tannin resistant enzymes for their biodegradation (Goel et al. 1997). 1969.. the biological systems often have difficulty in removing toxic polyphenols to consistently low levels. 2010). Puniyaa. faecalis. 2.. bacterial tannase can degrade and hydrolyze natural tannins and tannic acid very efficiently (Lewis and Starkey. Ó 2011 Elsevier Ltd. The * Corresponding author. India. have anti-nutritional activity. Ambala 133201.7 folds. Vikas Beniwalb. Tannins (hydrolysable (HT) and condensed (CT)). Mamta Raghava. *. pyrogallol and resorcinol. Maharishi Markandeshwar University.. 0964-8305/$ e see front matter Ó 2011 Elsevier Ltd. India Department of Biotechnology. b. however. Biodegradation of tannic acid Tannic acid degrading E. Kishan Singha a Dairy Microbiology Division. 1998). faecalis was purified up to 18. Tannase from E. the present investigation was undertaken to study the degradation profile of an anaerobic Enterococcus faecalis isolated from faeces of goat and to characterise tannase from this bacterium. A number of reviews on tannin biodegradation have appeared in the past.. a major problem in the utilization of fungal strains for industrial applications is that degradation by fungi is relatively slow (Beniwal et al. Ashwani Kumarc.006 . is involved in the hydrolysis of tannins (Aguilar and Gutierrez-Sanchez. Ambala 133201. Mullana. The fungi have a better activity in degrading hydrolyzable tannins however. 2005a). using ammonium sulphate precipitation. Maharishi Markandeshwar University.08. 2000). National Dairy Research Institute.. Tel. It is reported that (G. The 45 kDa protein had an optimum activity at 40  C and pH 6. The overnight culture was centrifuged and cell pellet was collected and mixed (at the rate 106 cfu/ml) to the minimal media supplemented with 1% filter sterilized tannic acid (Nelson et al. However. E-mail address: gunjanmicro@gmail. with a recovery of 41. used in the present investigation was isolated from goat faeces (Goel et al. some rumen microorganisms are shown to possess the ability to grow on tannins and degrade HTs. Deschamp et al. 1995).. On the other end of the spectrum. All rights reserved.0 at substrate concentration of 0.

1. Tannin degraded compounds were eluted by a gradient of water/acetonitrile from 100 to 0% in 30 min.05 M citrate buffer (pH 5. Rf values were calculated and compared with the standards of gallic acid. ammonium sulphate was added to achieve 30e60% saturation and centrifuged at 16. faecalis. The retention time of 4. 60 and 70  C).Author's personal copy 1062 G.2. 0. The culture broth was centrifuged at 4000  g for 20 min and the supernatant was used for purification of enzyme.0625.05 M. pH 5. faecalis was able to degrade tannic acid to pyrogallol and further to resorcinol within 72 h of incubation. namely gallic acid and pyrogallol when compared with standards. The solvents were removed under vacuum and the extracted samples were reconstituted by adding 2 ml HPLC grade methanol. The tannic acid degraded products were analysed by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). 3. The samples were filtered through Millipore membrane filter (0. 4. The enzyme activity at each step was calculated as per Sharma et al. 0.5 cm  10 cm and was equilibrated with 0. slurry of silica (40 g in 80 ml of distilled water) was spread on glass plates (20  20 cm) to a thickness 0. RP-High Performance Liquid Chromatography Tannin degraded products were estimated using RP-HPLC by the modified method of Makkar (2000). 0. The enzyme activity and protein content (Lowry et al. 5 mm particle size.2. The gel suspension.05 min was observed for gallic acid. Characterization of tannase The molecular weight of the tannase was determined by SDSPAGE as per the protocol of Laemmli (1971).22 mm) and the filtrate was used for gradient HPLC analysis using C18 spherisorb column (ODS 2. Five.. / International Biodeterioration & Biodegradation 65 (2011) 1061e1065 samples for analysis were removed aseptically from the bottles after every 12 h interval. On TLC plate. 2. pyrogallol and resorcinol with an Rf value of 0. The enzyme preparation obtained after each ammonium sulphate fractionation step was dialysed using a 1000 MWCO (molecular weight cut-off) cellulose acetate membrane for about 18 h against citrate buffer (0. pH (pH 2.0).5 mm. Gallic acid and pyrogallol were observed as major degraded product of tannic acid after 72 h of incubation (Fig.0 mM). The fractions showing tannase activity were pooled and concentrated using PEG-6000.61. The enzyme activity was determined by the rhodanine method (Sharma et al. HPLC results indicated that E. The upper layer was collected and aliquots of 2 ml were made in duplicates. 2. 30. Results and discussion 3. The contents were mixed.25. tannic acid remained at the bottom line followed by gallic acid.05. Degradation of tannic acid Tannic acid degradation profile was observed using TLC and HPLC. The filtrate was clarified by centrifugation at 4000  g for 20 min at 4  C. To 950 ml of clarified culture supernatant.1.0) with 3e4 intermittent changes of citrate buffer (0.3 M citrate buffer. 0. 5 ml fractions for buffer of each molarity were collected and the fractions were monitored for the elution profile of protein and tannase activity as described above. (1999). The protein fractions were eluted from the column at a flow rate of 1 ml/min. DEAE-cellulose chromatography A column was packed with DEAE-Cellulose to a bed size of 2.0) overnight at room temperature and the fines were decanted off. Goel et al. 0. Clarification and ammonium sulphate precipitation The supernatant was treated with 1% activated charcoal to remove the colour and phenolic compounds (Mahendran et al. was packed into a 55 cm  2. Thin Layer Chromatography (TLC) Thin Layer Chromatography was done according to the protocol of Sharma et al. pH 5. the culture supernatant containing degraded tannic acid metabolites was filtered through Whatman filter paper. 1). 2.31 min.2.2. after deaerating for 10e15 min. The contents were then transferred to separating funnel and partitioning was allowed for 1 h.59 and 0. Enzyme purification E. 4.6  250 mm. TLC profile of degradation of tannic acid with E. 0. 0. Five millilitres fractions were collected after draining 80% of the void volume and analysed for tannase activity.2. 1951) at each step of purification was determined to calculate the specific activity.0).025% phosphoric acid) and acetonitrile. 300 Å pore size) Waters32 HPLC system.0.05 M citrate buffer (pH 5. 1.1. A mixture of ethyl acetate: chloroform: water (50:50:1) was used as mobile phase and the detection was done using iodine vapours in a saturated chamber.0e9. The supernatant was subsequently adjusted to 80 and 100% saturation. Briefly. 0. The peaks were detected at two wavelengths of 250 nm and 280 nm. 2005) for 2 h with intermittent shaking. The samples were centrifuged at 2000  g and the supernatant was transferred in fresh tube. pH 5. 40. (1999). 2. The thin layer chromatogram showed two identification spots. The molecular weight of the protein was determined by comparing with the protein marker standard (Low range marker from Biorad). An aliquot of 10 ml was spotted on the TLC plate. 4.0). Gel filtration chromatography Sephadex G-150 was soaked in 0. The solvent system composed of degassed HPLC grade water (acidified with 0. 2. 1999). The elution was done in discontinuous gradient system with citrate buffer of increasing molarity (0.125. 1. . 2.. The samples were kept at À20  C till further analysis.2. 50.05 M.0). 3.75. pyrogallol and Fig.4.57 min and 6.1.5 cm glass column at the operating pressure head of 30 cm.0 and 5. vortexed and stirred for 1 h. Equal volume of ethanoleethyl acetate (95% EA þ 5% ethanol) was added to the filtered broth.0) and substrate concentration (methyl gallate at 0. 2.0. All the experiments were done in triplicates.05 M.. The pellet in each case was dissolved in citrate buffer (0. However. pH 5.2.2. respectively.1. The plates were allowed to dry and were activated by placing in an oven at 110  C for 1 h. Briefly.26. faecalis was grown in minimal media containing 1% filter sterilised tannic acid (Sigma) for 24 h at 37  C.000  g for 20 min at 4  C.1. The activity of partially purified tannase was studied at different temperatures (20. pyrogallol and resorcinol.

098 0.07 Æ 0. .8% of the total tannase was recovered by 70% saturation with activity of 975.24 Æ 0.) from the rumen microflora of sheep.1 6. Odenyo and Osuji (1998) have reported three strains of a tannintolerating bacterium (Selenomonas sp.034 0.098 0.602 Protein (mg/ml) 0. Pyrogallol was the major product after 48 h and 72 h of incubation. heteromorphus MTCC 8818 was also purified through ammonium sulphate precipitation and ion exchange chromatography leading to an Table 2 Summary of partial purification profile of tannase.020 0. Mahendran et al.2. The results are in agreement with reports from Tsai and Jones (1975) who showed that the isolated Streptococcus strains and three Coprococcus strains from the bovine rumen were able to degrade 80% of phloroglucinol.49 0.011 0.2.8 27. whereas the remaining two strains (ES3 and EG19) were able to hydrolyse tannic acid to gallic acid only.7 fold with a yield of 27.005 0. 3.101 0. Total protein ¼ Protein (mg) Â Total volume (ml).16 Æ 0.268 8980 13. Hamdy (2008) purified tannase from Fusarium subglutinans with 26. at 48 h of incubation period. Chhokar et al. (2010a) reported maximum recovery of tannase at 80% ammonium sulphate fractionation.40 Æ 0.89 51.0 min) declined sharply with time of incubation with simultaneous increase in the peak area of pyrogallol.29 0. (1998) also reported a combined activity of tannase and gallate decarboxylase activity in Pantoea agglomerans T71 which can be used for the bioconversion of tannic acid to pyrogallol.884 29.022 0.28 0.98 fold purification and 42. Zeida et al. About 89.014 Resorcinol (Res) 0.45 Specific activity (U/mg) 112.51 975.08 Æ Æ Æ Æ Æ Æ 0.31 0. (2005) obtained 78.2. An extracellular tannase from A. Rajkumar and Nandy (1983) obtained a 69% of total recovery from ammonium sulphate at 100% saturation. DEAE-cellulose chromatography The 70% saturated fraction was further purified through DEAEcellulose ion exchange chromatography.54 0.81 Total Activity (U) 32.58 32. The colour due to oxidation of gallic acid was removed by activated charcoal. Syntrophococcus sucromutans was reported to have ability to demethoxylate various phenolic monomers (Krumholz and Bryant. The tannase at this stage has total activity of 8980 U with a specific activity of 2896 U/mg.15 Æ 0.010 0.21 Æ 0. only gallic acid was formed indicating a 17% degradation of tannic acid.08 0.83 0.64 898 906. Specific activity ¼ Total activity (U)/Total protein (mg).43 45. Further increase in incubation period upto 24 h resulted in maximum gallic acid production followed by pyrogallol and traces of resorcinol.023 0.21 Æ 0.123 Pyrogallol (PYR) 0. Purification step Culture filtrate Clarification Ammonium sulphate precipitation DEAE-cellulose Sephadex G-150 Volume (ml) 1000 950 30 10 15 Tannase activity (U/ml) 32.00 Æ Æ Æ Æ Æ 0.35 Æ 0. The degradation of tannic acid by our isolate indicates that the tannineprotein complexes.000 0.11 0. 3.002 17.5% was obtained. gallic acid. 92% of tannic acid was mineralized into pyrogallol and resorcinol as the major end products. may also be subject to microbial degradation in rumen.580 30. The elution profile yielded a single protein peak corresponding with the tannase activity. From DEAE-cellulose chromatography.7 Yield (%) 100 94.28 0.16 0.51 U/ml with a yield of 94. Goel et al. / International Biodeterioration & Biodegradation 65 (2011) 1061e1065 Table 1 Degradation profile of tannic acid by E. the overall purification of 25. However. phloroglucinol and pyrogallol to acetate and butyrate in presence of hydrogen and formic acid (Krumholz and Bryant.7 18. resorcinol.7 25.7 Total activity ¼ Activity (U) Â Total volume (ml).3 112.015 0.42 54.2.7 89.Author's personal copy G.46 0. a strictly anaerobe was reported to degrade gallate.88 Tannic acid (TA) 1063 % TA degradation Values presented are mean Æ SD.89 0. which were thought to be hydrolysed only under the acidic condition of the abomasum. Purification fold ¼ Specific activity of the sample/Initial specific activity. 3. pyrogallol and resorcinol were observed in significant amount resulting in 55% degradation of tannic acid and finally after 72 h of incubation.00 0.6% recovery by ammonium sulphate.012 0.11 Æ 0. Clarification and ammonium sulphate precipitation Culture filtrate treated with 1% activated carbon removed more than 80% of the colour according to the visual observation. The clarified filtrate was having an activity of 32. faecalis (Table 1) shows that initially at 12 h of incubation period.18 91.20 0. Another anaerobe. Enzyme purification The results pertaining stepwise purification of tannase are presented in Table 2.7 3. 1988).077 0. respectively.100 0.1.5 26.1 1096 2896 2109 Purification fold 1 1 9.29 0. Incubation Time (h) 12 24 36 48 60 72 Degraded products (mg/ml) Gallic acid (GA) 0. The degradation profile of tannic acid by E. faecalis at different time intervals.7%. goat and antelope that had either been fed or had browsed on tanniniferous forages.102 0.56 Æ 0.64 U/ml.5 41.43 Total Protein (mg) 290 275. Tannase was precipitated at 70% saturation. ion exchange chromatography (DEAE-cellulose) and gel filtration (Sephedax G-150).095 0. all the metabolites i.111 0.28 72. Several other ruminal microorganisms were reported to possess the ability to degrade phenolic monomers. 1986).013 0. Yield ¼ [Total activity of the sample/Initial total activity] Â 100. Fractional precipitation with 50% ammonium sulphate removed some of the nonenzymatic proteins.7% of the total tannase at 70% saturation with ammonium sulphate. The peak area of the tannic acid at retention time (11. Eubacterium oxidoreducens. One of the strains (EAT2) of this ruminal bacterium could hydrolyse tannic acid to gallic acid and subsequently to pyrogallol.e.

low range marker Biorad). Cryphonectria. 2005). 1999) and Aspergillus awamori (Chhokar et al..2 mM (Sharma et al. faecalis tannase has interesting and attractive properties for industrial use for production of pyrogallol.1.86 mM (Yamada et al. variotii (Mahendran et al. 1994) and P. A. the tannase activity was found to decline sharply with no activity at 80  C as increase in temperature increases the rate of denaturation of the enzyme. Effect of pH on tannase activity. with molecular weight of 186 to over 300 kDa. Lekha and Lonsane. faecalis has higher affinity for methyl gallate. 1968). thus those fractions were pooled and concentrated.4. faecalis was 40  C (Fig. 1. with the loss of secondary and tertiary structures (Sabu et al. The tannase activity was not observed in other peaks.5 which were reported to be due to different depsidase and esterase activities (Barthomeuf et al.0 (Fig.. Rajkumar and Nandy. yielding about 17. Properties of tannase 3. 2009).. The results presented in this work shows that E. 3. flavus. 3. niger Van Tieghem using methyl gallate as substrate have been found to be 0. 4. respectively. 2010b). Srivastava and Kar.2. 2007). Tannase from other sources like A.. At gel filtration the enzyme was purified up to 18.7% recovery of the total tannase.6 mM (Skene and Brooker. 3. 1983.4. 3. Fig. Penicillium chrysogenum. 2). The same pH range was reported for other tannase obtained from A.49 mM (Farias et al. However.. Fig. Effect of substrate concentration The analysis of the graph of substrate concentration against tannase activity yielded Km values of 0. respectively. Effect of temperature on tannase activity..Author's personal copy 1064 G.0 and a secondary peak at 4.2. a considerable potential exists for the exploitation of such potential isolates using in vivo studies in order to use them for trans-inoculation into other animal species to improve the nutritional status and productivity of the livestock..0 and 7. 2. . S. 4). ruminantium. Fig. 2010a). 1997). Being grazers. 3. Aspergillus niger have been reported to have their optimal activity at 30e40  C (Iibuchi et al. Molecular weight (Mr) determination The purified protein resulted in a single band of 45 kDa (Fig. 7.74 fold with a yield of 19.4.4. Hence. However. 1994. Yamada et al. 3. 1997. The tannases with a molecular weight of 59 kDa from Selenomonas ruminantium (Skene and Brooker.6% of total tannase. / International Biodeterioration & Biodegradation 65 (2011) 1061e1065 overall purification of 39. (1968) and Hatamoto et al. With the further increase in temperature. (2003) reported that the total tannase activity was made up of nearly equal activity of esterase and depsidase with a molecular weight 102 and 83 kDa. The Km values for tannases from A. faecalis (M: Marker. (1999) also obtained four peaks of the protein content with a single peak of tannase activity where the purification was obtained upto 29 folds with 2. niger Van Tieghem MTCC 2425 (Sharma et al.3. Effect of temperature The temperature for the optimum activity of tannase from E. 2005) have already been reported.. goats are found to be more adapted to tannins in feeds as they harbour tannin degrading microflora in situ.5% recovery from the Sephadex G-200 column eluted with 0. One of the prominent peak between fractions from 14 to 28 coincides with the peak of tannase activity.02 M acetate buffer. Sabu et al. Cryphonectria (Farias et al.. Goel et al. Sharma et al. niger Van Tieghem MTCC 2425 as reported by Sharma et al. (1999) resulted in two different peaks of tannase activity at a pH of 6. SDS profile of tannase from E.. 2005. niger (Ramırez-Coronel et al.7 folds with a yield of 41. A lower temperature optima (30  C) has been reported for tannase from A.0 with an optimum activity at pH of 6. Mahendran et al.7%. 1994) and 0. 1968.. Bhardwaj et al. 1999). Effect of pH The tannase from E.3.. 1995) and 45 kDa from Paecilomyces variotii (Mahendran et al. Rajkumar and Nandy (1983) obtained 18.29% (Chhokar et al. 2005). The tannase at this stage has total activity of 13. oryzae. This implies that tannase of E. tannase from A. 3).. (1996) have reported that the multimeric tannases from Aspergillus flavus and Aspergillus oryzae.43 mmol..3. 2003. faecalis possess an activity between 5. it can be concluded that the potential tannin degrading microorganisms reside in the gut of small ruminants. Lekha and Lonsane.602 U with a specific activity of 2109 U/mg. (2005) obtained three protein peaks when tannase was eluted from Sephadex G-200. From the present investigation. 1995). Battestin and Macedo. Sephadex G-150 chromatography The elution profile of the enzyme from gel filtration resulted in four protein peaks of which two were prominent.

. M. Jones.. Rana. Microbiology 149.S. Nature 227. M. V. T. J. 449e461. Vienna. pyrogallol.. N. Kar. Yamada. Chhokar...07 .. S. 1998.A. V. M. Journal of Basic Microbiology 45. 2007.. Tannase production by Paecilomyces variotii. N. Getachew. H. Isolation and characterization of anaerobic ruminal bacterium capable of degrading hydrolyzable tannins. Protein measurement with the folin phenol reagent... Marcel Dekker Inc. Elkins. 215e221. (Eds.. R. A.K. K.A. gas production: fermentation kinetics for feed evaluation and to access microbial activity. B. 2001.K. J. Wageningen. Purification and characterization of tannin acyl hydrolase from A.. Beniwal. New Delhi. Journal of Scientific and Industrial Research 45. O. 321e323.P.. Sato. Rosebrough.. N. G. Microbial degradation of tannins: a current perspective. Production of tannase and degradation of chestnut tannins by bacteria. C.. Goel. Agricultural and Biological Chemistry 32.. 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