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International Biodeterioration & Biodegradation 65 (2011) 1061e1065

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International Biodeterioration & Biodegradation

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Degradation of tannic acid and purication and characterization of tannase from Enterococcus faecalis
Gunjan Goela, b, *, Ashwani Kumarc, Vikas Beniwalb, Mamta Raghava, Anil K. Puniyaa, Kishan Singha
a

Dairy Microbiology Division, National Dairy Research Institute, Karnal 132001, India Department of Biotechnology, Maharishi Markandeshwar University, Mullana, Ambala 133201, India c Department of Biotechnology, JMIT Institute of Engineering and Technology, Radaur 135133, 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, present in various foods, feeds and forages, have anti-nutritional activity; however, presence of tannase in microorganisms inhabiting rumen and gastrointestinal tract of animals results in detoxication of these tannins. The present investigation was carried out to study the degradation prole of tannins by Enterococcus faecalis and to purify tannase. E. faecalis was observed to degrade tannic acid (1.0% in minimal media) to gallic acid, pyrogallol and resorcinol. Tannase from E. faecalis was puried up to 18.7 folds, with a recovery of 41.7%, using ammonium sulphate precipitation, followed by DEAEcellulose and Sephadex G-150. The 45 kDa protein had an optimum activity at 40  C and pH 6.0 at substrate concentration of 0.25 mM methyl gallate. 2011 Elsevier Ltd. All rights reserved.

1. Introduction Tannins are the most common plant secondary metabolites that are poorly understood with respect to feeding habits, with both benecial and adverse effects in ruminants (Getachew et al., 2000). Tannins (hydrolysable (HT) and condensed (CT)), being antimicrobial; resist microbial attack and remains as recalcitrant in the environment (Field and Lettinga, 1992). Though destructive in nature, the biological systems often have difculty in removing toxic polyphenols to consistently low levels. However, some rumen microorganisms are shown to possess the ability to grow on tannins and degrade HTs, with a few evidences showing degradation of phenol ring of CTs (Bhat et al., 1998). These microorganisms have developed protective mechanisms such as secretion of binding polymers and synthesis of tannin resistant enzymes for their biodegradation (Goel et al., 2005a). A number of reviews on tannin biodegradation have appeared in the past, providing a general idea on the biodegradation of these polyphenols (William et al., 1986; Bhat et al., 1998). It is reported that tannase, the key enzyme, is involved in the hydrolysis of tannins (Aguilar and

Gutierrez-Sanchez, 2001). The tannase catalyse the hydrolysis of ester and depside bonds presents in the hydrolysable tannins and gallic acid esters (Lekha and Lonsane, 1997). Among tannin degrading microbes, aerobic bacterial degraders and fungal species have been reviewed elsewhere (Bhat et al., 1998; Goel et al., 2005b). The fungi have a better activity in degrading hydrolyzable tannins however, a major problem in the utilization of fungal strains for industrial applications is that degradation by fungi is relatively slow (Beniwal et al., 2010). On the other end of the spectrum, bacterial tannase can degrade and hydrolyze natural tannins and tannic acid very efciently (Lewis and Starkey, 1969; Deschamp et al., 1983). Therefore, the present investigation was undertaken to study the degradation prole of an anaerobic Enterococcus faecalis isolated from faeces of goat and to characterise tannase from this bacterium.

2. Materials and methods 2.1. Biodegradation of tannic acid Tannic acid degrading E. faecalis, used in the present investigation was isolated from goat faeces (Goel et al., 2007). 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% lter sterilized tannic acid (Nelson et al., 1995). The culture bottles were sealed under CO2 and incubated at 37  C for 72 h. The

* Corresponding author. Department of Biotechnology, Maharishi Markandeshwar University, Mullana, Ambala 133201, India. Tel.: 91 1731304148; fax: 91 1731274375. E-mail address: gunjanmicro@gmail.com (G. Goel). 0964-8305/$ e see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2011.08.006

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samples for analysis were removed aseptically from the bottles after every 12 h interval. The samples were kept at 20  C till further analysis. The tannic acid degraded products were analysed by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). All the experiments were done in triplicates. 2.1.1. Thin Layer Chromatography (TLC) Thin Layer Chromatography was done according to the protocol of Sharma et al. (1999). Briey, slurry of silica (40 g in 80 ml of distilled water) was spread on glass plates (20 20 cm) to a thickness 0.5 mm. The plates were allowed to dry and were activated by placing in an oven at 110  C for 1 h. The samples were centrifuged at 2000 g and the supernatant was transferred in fresh tube. An aliquot of 10 ml was spotted on the TLC plate. 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. Rf values were calculated and compared with the standards of gallic acid, pyrogallol and resorcinol. 2.1.2. RP-High Performance Liquid Chromatography Tannin degraded products were estimated using RP-HPLC by the modied method of Makkar (2000). Briey, the culture supernatant containing degraded tannic acid metabolites was ltered through Whatman lter paper. Equal volume of ethanoleethyl acetate (95% EA 5% ethanol) was added to the ltered broth. The contents were mixed, vortexed and stirred for 1 h. The contents were then transferred to separating funnel and partitioning was allowed for 1 h. The upper layer was collected and aliquots of 2 ml were made in duplicates. The solvents were removed under vacuum and the extracted samples were reconstituted by adding 2 ml HPLC grade methanol. The samples were ltered through Millipore membrane lter (0.22 mm) and the ltrate was used for gradient HPLC analysis using C18 spherisorb column (ODS 2, 4.6 250 mm, 5 mm particle size, 300 pore size) Waters32 HPLC system. The solvent system composed of degassed HPLC grade water (acidied with 0.025% phosphoric acid) and acetonitrile. Tannin degraded compounds were eluted by a gradient of water/acetonitrile from 100 to 0% in 30 min. The peaks were detected at two wavelengths of 250 nm and 280 nm. 2.2. Enzyme purication E. faecalis was grown in minimal media containing 1% lter sterilised tannic acid (Sigma) for 24 h at 37  C. The culture broth was centrifuged at 4000 g for 20 min and the supernatant was used for purication of enzyme. The enzyme activity at each step was calculated as per Sharma et al. (1999). The enzyme activity and protein content (Lowry et al., 1951) at each step of purication was determined to calculate the specic activity. 2.2.1. Clarication and ammonium sulphate precipitation The supernatant was treated with 1% activated charcoal to remove the colour and phenolic compounds (Mahendran et al., 2005) for 2 h with intermittent shaking. The ltrate was claried by centrifugation at 4000 g for 20 min at 4  C. To 950 ml of claried culture supernatant, ammonium sulphate was added to achieve 30e60% saturation and centrifuged at 16,000 g for 20 min at 4  C. The supernatant was subsequently adjusted to 80 and 100% saturation. The pellet in each case was dissolved in citrate buffer (0.05 M, pH 5.0). 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 (0.05 M, pH 5.0) with 3e4 intermittent changes of citrate buffer (0.05 M, pH 5.0).

2.2.2. DEAE-cellulose chromatography A column was packed with DEAE-Cellulose to a bed size of 2.5 cm 10 cm and was equilibrated with 0.05 M citrate buffer (pH 5.0). The elution was done in discontinuous gradient system with citrate buffer of increasing molarity (0.05, 0.1, 0.2, 0.3 M citrate buffer, pH 5.0). Five, 5 ml fractions for buffer of each molarity were collected and the fractions were monitored for the elution prole of protein and tannase activity as described above. 2.2.3. Gel ltration chromatography Sephadex G-150 was soaked in 0.05 M citrate buffer (pH 5.0) overnight at room temperature and the nes were decanted off. The gel suspension, after deaerating for 10e15 min, was packed into a 55 cm 2.5 cm glass column at the operating pressure head of 30 cm. The protein fractions were eluted from the column at a ow rate of 1 ml/min. Five millilitres fractions were collected after draining 80% of the void volume and analysed for tannase activity. The fractions showing tannase activity were pooled and concentrated using PEG-6000. 2.2.4. Characterization of tannase The molecular weight of the tannase was determined by SDSPAGE as per the protocol of Laemmli (1971). The molecular weight of the protein was determined by comparing with the protein marker standard (Low range marker from Biorad). The activity of partially puried tannase was studied at different temperatures (20, 30, 40, 50, 60 and 70  C), pH (pH 2.0e9.0) and substrate concentration (methyl gallate at 0.0625, 0.125, 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0 and 5.0 mM). The enzyme activity was determined by the rhodanine method (Sharma et al., 1999).

3. Results and discussion 3.1. Degradation of tannic acid Tannic acid degradation prole was observed using TLC and HPLC. The thin layer chromatogram showed two identication spots, namely gallic acid and pyrogallol when compared with standards. On TLC plate, tannic acid remained at the bottom line followed by gallic acid, pyrogallol and resorcinol with an Rf value of 0.26, 0.59 and 0.61, respectively. Gallic acid and pyrogallol were observed as major degraded product of tannic acid after 72 h of incubation (Fig. 1). However, HPLC results indicated that E. faecalis was able to degrade tannic acid to pyrogallol and further to resorcinol within 72 h of incubation. The retention time of 4.31 min, 4.57 min and 6.05 min was observed for gallic acid, pyrogallol and

Fig. 1. TLC prole of degradation of tannic acid with E. faecalis.

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G. Goel et al. / International Biodeterioration & Biodegradation 65 (2011) 1061e1065 Table 1 Degradation prole of tannic acid by E. faecalis at different time intervals. Incubation Time (h) 12 24 36 48 60 72 Degraded products (mg/ml) Gallic acid (GA) 0.16 0.28 0.20 0.11 0.08 0.00 0.013 0.022 0.012 0.020 0.123 Pyrogallol (PYR) 0.00 0.11 0.16 0.21 0.24 0.35 0.011 0.023 0.034 0.077 0.014 Resorcinol (Res) 0.000 0.07 0.15 0.21 0.40 0.56 0.005 0.010 0.015 0.100 0.098 0.83 0.54 0.49 0.46 0.28 0.08 0.101 0.095 0.102 0.098 0.111 0.002 17.43 45.89 51.42 54.28 72.18 91.88 Tannic acid (TA)

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% TA degradation

Values presented are mean SD.

resorcinol, respectively. The peak area of the tannic acid at retention time (11.0 min) declined sharply with time of incubation with simultaneous increase in the peak area of pyrogallol. The degradation prole of tannic acid by E. faecalis (Table 1) shows that initially at 12 h of incubation period, only gallic acid was formed indicating a 17% degradation of tannic acid. Further increase in incubation period upto 24 h resulted in maximum gallic acid production followed by pyrogallol and traces of resorcinol. Pyrogallol was the major product after 48 h and 72 h of incubation. However, at 48 h of incubation period, all the metabolites i.e. gallic acid, pyrogallol and resorcinol were observed in signicant amount resulting in 55% degradation of tannic acid and nally after 72 h of incubation, 92% of tannic acid was mineralized into pyrogallol and resorcinol as the major end products. Several other ruminal microorganisms were reported to possess the ability to degrade phenolic monomers. 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. Odenyo and Osuji (1998) have reported three strains of a tannintolerating bacterium (Selenomonas sp.) from the rumen microora of sheep, goat and antelope that had either been fed or had browsed on tanniniferous forages. One of the strains (EAT2) of this ruminal bacterium could hydrolyse tannic acid to gallic acid and subsequently to pyrogallol, whereas the remaining two strains (ES3 and EG19) were able to hydrolyse tannic acid to gallic acid only. Zeida et al. (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. Eubacterium oxidoreducens, a strictly anaerobe was reported to degrade gallate, phloroglucinol and pyrogallol to acetate and butyrate in presence of hydrogen and formic acid (Krumholz and Bryant, 1986). Another anaerobe, Syntrophococcus sucromutans was reported to have ability to demethoxylate various phenolic monomers (Krumholz and Bryant, 1988). The degradation of tannic acid by our isolate indicates that the tannineprotein complexes, which were thought to be hydrolysed only under the acidic

condition of the abomasum, may also be subject to microbial degradation in rumen.

3.2. Enzyme purication The results pertaining stepwise purication of tannase are presented in Table 2. 3.2.1. Clarication and ammonium sulphate precipitation Culture ltrate treated with 1% activated carbon removed more than 80% of the colour according to the visual observation. The colour due to oxidation of gallic acid was removed by activated charcoal. The claried ltrate was having an activity of 32.51 U/ml with a yield of 94.7%. Fractional precipitation with 50% ammonium sulphate removed some of the nonenzymatic proteins. Tannase was precipitated at 70% saturation. About 89.8% of the total tannase was recovered by 70% saturation with activity of 975.64 U/ml. Rajkumar and Nandy (1983) obtained a 69% of total recovery from ammonium sulphate at 100% saturation. Mahendran et al. (2005) obtained 78.7% of the total tannase at 70% saturation with ammonium sulphate. Chhokar et al. (2010a) reported maximum recovery of tannase at 80% ammonium sulphate fractionation. 3.2.2. DEAE-cellulose chromatography The 70% saturated fraction was further puried through DEAEcellulose ion exchange chromatography. The elution prole yielded a single protein peak corresponding with the tannase activity. The tannase at this stage has total activity of 8980 U with a specic activity of 2896 U/mg. From DEAE-cellulose chromatography, the overall purication of 25.7 fold with a yield of 27.5% was obtained. Hamdy (2008) puried tannase from Fusarium subglutinans with 26.98 fold purication and 42.6% recovery by ammonium sulphate, ion exchange chromatography (DEAE-cellulose) and gel ltration (Sephedax G-150). An extracellular tannase from A. heteromorphus MTCC 8818 was also puried through ammonium sulphate precipitation and ion exchange chromatography leading to an

Table 2 Summary of partial purication prole of tannase. Purication step Culture ltrate Clarication Ammonium sulphate precipitation DEAE-cellulose Sephadex G-150 Volume (ml) 1000 950 30 10 15 Tannase activity (U/ml) 32.58 32.51 975.64 898 906.81 Total Activity (U) 32,580 30,884 29,268 8980 13,602 Protein (mg/ml) 0.29 0.29 0.89 0.31 0.43 Total Protein (mg) 290 275.5 26.7 3.1 6.45 Specic activity (U/mg) 112.3 112.1 1096 2896 2109 Purication fold 1 1 9.7 25.7 18.7 Yield (%) 100 94.7 89.8 27.5 41.7

Total activity Activity (U) Total volume (ml). Total protein Protein (mg) Total volume (ml). Specic activity Total activity (U)/Total protein (mg). Purication fold Specic activity of the sample/Initial specic activity. Yield [Total activity of the sample/Initial total activity] 100.

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overall purication of 39.74 fold with a yield of 19.29% (Chhokar et al., 2010b). 3.2.3. Sephadex G-150 chromatography The elution prole of the enzyme from gel ltration resulted in four protein peaks of which two were prominent. One of the prominent peak between fractions from 14 to 28 coincides with the peak of tannase activity, thus those fractions were pooled and concentrated. The tannase activity was not observed in other peaks. The tannase at this stage has total activity of 13,602 U with a specic activity of 2109 U/mg. At gel ltration the enzyme was puried up to 18.7 folds with a yield of 41.7%. Rajkumar and Nandy (1983) obtained 18.5% recovery from the Sephadex G-200 column eluted with 0.02 M acetate buffer. Sharma et al. (1999) also obtained four peaks of the protein content with a single peak of tannase activity where the purication was obtained upto 29 folds with 2.7% recovery of the total tannase. Mahendran et al. (2005) obtained three protein peaks when tannase was eluted from Sephadex G-200, yielding about 17.6% of total tannase. 3.3. Molecular weight (Mr) determination The puried protein resulted in a single band of 45 kDa (Fig. 2). The tannases with a molecular weight of 59 kDa from Selenomonas ruminantium (Skene and Brooker, 1995) and 45 kDa from Paecilomyces variotii (Mahendran et al., 2005) have already been reported. However, Yamada et al. (1968) and Hatamoto et al. (1996) have reported that the multimeric tannases from Aspergillus avus and Aspergillus oryzae, respectively, with molecular weight of 186 to over 300 kDa. Bhardwaj et al. (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. 3.4. Properties of tannase 3.4.1. Effect of temperature The temperature for the optimum activity of tannase from E. faecalis was 40  C (Fig. 3). With the further increase in temperature, 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, with the loss of secondary and tertiary structures (Sabu et al., 2005). Tannase from other sources like A. oryzae, Penicillium chrysogenum, Aspergillus niger have been reported to have their optimal activity at 30e40  C (Iibuchi et al., 1968; Rajkumar and Nandy, 1983; Lekha and Lonsane, 1997; Sabu et al., 2005). A lower temperature optima (30  C) has been reported for tannase from A. niger Van Tieghem MTCC 2425 (Sharma et al., 1999) and Aspergillus awamori (Chhokar et al., 2010a).

Fig. 3. Effect of temperature on tannase activity.

3.4.2. Effect of pH The tannase from E. faecalis possess an activity between 5.0 and 7.0 with an optimum activity at pH of 6.0 (Fig. 4). The same pH range was reported for other tannase obtained from A. niger (Ramrez-Coronel et al., 2003; Srivastava and Kar, 2009), Cryphonectria (Farias et al., 1994) and P. variotii (Mahendran et al., 2005; Battestin and Macedo, 2007). However, tannase from A. niger Van Tieghem MTCC 2425 as reported by Sharma et al. (1999) resulted in two different peaks of tannase activity at a pH of 6.0 and a secondary peak at 4.5 which were reported to be due to different depsidase and esterase activities (Barthomeuf et al., 1994; Lekha and Lonsane, 1997). 3.4.3. Effect of substrate concentration The analysis of the graph of substrate concentration against tannase activity yielded Km values of 0.43 mmol. This implies that tannase of E. faecalis has higher afnity for methyl gallate. The Km values for tannases from A. avus, S. ruminantium, Cryphonectria, A. niger Van Tieghem using methyl gallate as substrate have been found to be 0.86 mM (Yamada et al., 1968), 1.6 mM (Skene and Brooker, 1995), 7.49 mM (Farias et al., 1994) and 0.2 mM (Sharma et al., 1999), respectively. From the present investigation, it can be concluded that the potential tannin degrading microorganisms reside in the gut of small ruminants. Being grazers, goats are found to be more adapted to tannins in feeds as they harbour tannin degrading microora in situ. Hence, 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. The results presented in this work shows that E. faecalis tannase has interesting and attractive properties for industrial use for production of pyrogallol.

Fig. 2. SDS prole of tannase from E. faecalis (M: Marker, low range marker Biorad).

Fig. 4. Effect of pH on tannase activity.

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Acknowledgements The authors wish to thank Indian Council of Agriculture Research, New Delhi, and National Dairy Research Institute, Karnal, India, for providing nancial support and essential facilities required for the research work. References
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