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Bioresource Technology 99 (2008) 2544–2551

Purification, immobilization and characterizationof tannase from Penicillium variable

Shashi Sharma, Lata Agarwal, Rajendra Kumar Saxena *

Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India

Received 29 December 2006; received in revised form 22 April 2007; accepted 22 April 2007Available online 13 June 2007

Abstract

Tannase from Penicillium variable IARI 2031 was purified by a two-step purification strategy comprising of ultra-filtration using100 kDa molecular weight cutoff and gel-filtration using Sephadex G-200. A purification fold of 135 with 91% yield of tannase wasobtained. The enzyme has temperature and pH optima of 50 �C and 5 �C, respectively. However, the functional temperature range is from25 to 80 �C and functional pH range is from 3.0 to 8.0. This tannase could successfully be immobilized on Amberlite IR where it retainsabout 85% of the initial catalytic activity even after ninth cycle of its use. Based on the Michaelis–Menten constant (Km) of tannase, tannicacid is the best substrate with Km of 32 mM and Vmax of 1.11 lmol ml�1 min�1. Tannase is inhibited by phenyl methyl sulphonyl fluoride(PMSF) and N-ethylmaleimide retaining only 28.1% and 19% residual activity indicating that this enzyme belongs to the class of serinehydrolases. Tannase in both crude and crude lyophilized forms is stable for one year retaining more than 60% residual activity.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Tannase; Chebulina myrobalan; Purification; Penicillium variable

1. Introduction

The enzyme tannase (E.C.3.1.1.20), also known as tan-nin acyl hydrolase, is a hydrolytic enzyme that acts on tan-nins. Tannase is an inducible enzyme that catalyses thebreakdown of ester linkages in hydrolysable tannins suchas tannic acid resulting in the production of gallic acidand glucose (Saxena and Saxena, 2004; Zhong et al.,2004; Batra and Saxena, 2005).

Tannase is extensively used in food, feed, beverage,brewing, pharmaceutical and chemical industries. Themajor applications of this enzyme are in the productionof gallic acid, which is used for the manufacture of an anti-malarial drug, trimethoprim and in the synthesis of estersas propyl gallate used as antioxidants in the food industry(Aguilar and Gutierrez-Sanchez, 2001). The enzyme is alsoused in the manufacture of instant tea, in the prevention of

0960-8524/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2007.04.035

* Corresponding author. Tel.: +91 11 24116559; fax: +91 11 24116427/5270.

E-mail addresses: rksmicro@hotmail.com, rksmicro@yahoo.co.in(R.K. Saxena).

phenol-induced mediarization in wine, manufacture of cof-fee-flavoured soft drinks, clarification of beer and fruitjuices, stabilization of malt polyphenol and as a sensitiveanalytical probe for determining the structure of naturallyoccurring gallic acid esters (Haslam and Tanner, 1970;Koichi and Tokuji, 1972; Suzuki, 1973; Massechelin andBatum, 1981; Canteralli et al., 1989; Giovanelli, 1989).Tannases have also been used for the cleavage of polyphen-olics such as dehydrodimer crosslinks present in cell wall ofplants which is essential for plant cell wall digestibility(Conesa et al., 2001).

Here, we report a fast and efficient protocol to purify thetannase produced from Penicillium variable along with itscharacterization in free and immobilized forms.

2. Methods

2.1. Growth and maintenance of Penicillium variable

Penicillium variable IARI 2031, procured from IndianAgricultural Research Institute (IARI), New Delhi, India,

S. Sharma et al. / Bioresource Technology 99 (2008) 2544–2551 2545

was selected as a potent tannase producer after exten-sive screening of a large number of fungi. The organismwas grown on potato dextrose agar (PDA) slants sup-plemented with 0.1% tannic acid at 30 �C for six days.The sporulated cultures were then maintained at 8 �C ina BOD incubator and were subcultured after every 15days.

2.2. Enzyme production

Conidia were harvested from 6-day-old culture by add-ing 10 ml of sterilized normal saline containing 0.01% ster-ile Tween-80. Approximately, 5 · 107 conidia wereinoculated in sterilized 50 ml of modified Czapek’s Doxmedium (pH 5.0) with the composition (g l�1): NaNO3

(6 g), KCl (0.52 g); MgSO4 Æ 7H2O (0.52 g); KH2PO4

(1.52 g); glucose (2.0 g) and Cu(NO3)2 Æ 3H2O; FeSO4 Æ7H2O and ZnSO4 Æ 7H2O were added as traces. Powderedsource of tannins from Chebulina myrobalan containing32% tannin was used (5.8 g/50 ml of medium). Flasks wereincubated at 30 ± 1 �C in an incubator shaker (Model G-25KC, New Brunswick Scientific, NJ, USA) at 150 rpm for72 h. After the desired incubation period, biomass was har-vested and the culture filtrate was analyzed for tannaseactivity.

2.3. Enzyme assay

Tannase activity was estimated by the procedure of Des-champs et al. (1983). To 4 ml of the reaction mixture, 1 mlof 1% tannic acid (in 0.5 M citrate-phosphate buffer, pH5.0), 2 ml of 0.5 M citrate-phosphate buffer (pH 5.0) and1 ml of suitably diluted culture filtrate were added. Thismixture was incubated at 50 �C for 30 min in a water bathand the reaction was terminated by adding 4 ml of 2%bovine serum-albumin (BSA) solution. In control, BSAwas added in the reaction mixture prior to incubation.The tubes were left for 20 min at room temperature to pre-cipitate the residual tannins and were then centrifuged at3000g for 20 min. Tannase activity was estimated by dilut-ing 20 ll of the supernatant 500-fold with double distilledwater, and the absorbance was read at 260 nm (this wave-length corresponds to the optimal absorption of gallic acid)against double distilled water as blank in a UV spectropho-tometer (Model no. 1601, Shimadzu, Japan). The amountof gallic acid produced in the reaction mixture was esti-mated from the standard curve of gallic acid prepared inthe range of 10–100 lg ml�1.

Enzyme unit: One unit of tannase is defined as theamount of enzyme required to release one lmol of gallicacid per ml per minute of culture filtrate under the standardassay conditions.

Protein estimation: The protein content in the culture fil-trate was estimated according to the procedure of Bradford(1976).

2.4. Enzyme purification

2.4.1. Ultrafiltration

Culture filtrate (1000 ml) containing tannase was sub-jected to ultrafiltration using Spin Trex (New BrunswickScientific Co. Inc., NJ, USA) having membrane cartridgesof different molecular weight cutoffs (10, 50 and 100 kDa).The retentate and permeate collected after each run wereanalyzed for tannase activity and total protein contents.Observations were recorded in terms of volume concentra-tion, tannase yields and specific activities.

2.4.2. Gel-filtration chromatography

Fifty milliliter of the retentate obtained after ultra-filtra-tion was further concentrated to 10 ml using Speed Vac(Speed-Vac Plus, USA). Five milliliter of the concentratedsample was then loaded on to Sephadex G-200 column to abed size of 1.5 · 55 cm (bed volume 97.2 ml). Column wasequilibrated with 0.05 M citrate-phosphate buffer, pH 5.0and was eluted with the same equilibrating buffer at a flowrate of 1 ml min�1. Hundred fractions of one milliliter sizewere collected in fraction collector (Pharmacia Biotech,USA). The protein content in each sample was estimatedat 280 nm using spectrophotometer. Tannase activity inthese samples was also estimated. The fractions corre-sponding to high tannase activity were pooled, concen-trated and examined for purity by high performanceliquid chromatography (HPLC) (Shimadzu SPD-10A,LC-10AD pump, CTO-10AS column oven, Tokyo, Japan)and by sodium dodecyl sulphate–polyacrylamide gel elec-trophoresis (SDS–PAGE).

2.4.3. HPLC analysis of the purified P. variable tannaseAnalysis of purified P. variable tannase was carried out

on HPLC equipped with Shim-pack Diol – 150 column(size 7.9 mm u · 50 cm) using 50 mM Tris buffer contain-ing 0.2 M potassium chloride as a mobile phase with a flowrate of 0.6 ml min�1. UV–Visible detector was used at awavelength of 280 nm.

2.4.4. SDS–PAGE and molecular weight (Mr)

determination

The comparative mobility of purified tannase was car-ried out using 4 M urea in 5% SDS–PAGE with denaturedmolecular weight markers. Crude, partially purified andpurified tannase (40 lg) were loaded. After the run, thegel was stained with silver nitrate solution.

2.4.5. Molecular weight of native tannase

The molecular weight of native tannase was determinedby gel-filtration chromatography using Sephadex G-200.The mixture of high non-denaturing molecular weightmarkers (2 mg) were loaded on Sephadex G-200 and elutedwith 0.05 M citrate-phosphate buffer (pH 5.0) at a flow rateof 1 ml min�1. The void volume (Vo) of 25 ml was deter-mined using Blue Dextran 2000. The sample containing

2546 S. Sharma et al. / Bioresource Technology 99 (2008) 2544–2551

proteins were collected at their elution volume (Ve) and thegraph of V eV �1

o against log of molecular weight (Mw) wasplotted for the estimation of native molecular weight oftannase.

2.4.6. ImmobilizationDifferent supports such as Amberlite IR 1204, Amberlite

XAD 7, DEAE Cellulose, Celite and Silica were used toimmobilize P. variable tannase. 500 mg of each supportwas mixed with 10.0 ml of 0.1 M citrate-phosphate buffer,pH 5.0 and kept at room temperature for 2.0 h. To this,10 ml of partially purified enzyme was added and the sus-pension was agitated at 100 rpm and 4 ± 2 �C for 12 h.The support was filtered and washed thrice with buffer toremove the unbound protein. The filtrate was assayed forleft over tannase activity and protein contents. Efficiencyof immobilization was determined in terms of total proteinadsorbed per gram of the support. The reusability of theenzyme was also examined.

2.5. Characterization of tannase

2.5.1. Temperature tolerance

The optimum temperature for crude, purified andimmobilized tannases was studied by incubating the reac-tion mixtures at different temperatures ranging from 25to 100 �C for 30 min. Suitable dilutions of crude and puri-fied enzymes were made in 0.5 M citrate-phosphate buffer.Immobilized enzyme (100 mg) was suspended in 0.5 ml cit-rate-phosphate buffer (0.01 M, pH 5.0).

2.5.2. Temperature stability

The thermostability of all the three tannase forms wasdetermined by incubating the enzyme in 0.5 M citrate-phosphate buffer at temperatures ranging from 25 to100 �C for different time intervals (1/2, 1, 6, 12 and 24 h).After desired incubation periods, enzyme aliquots werewithdrawn and assayed at optimal assay conditions todetermine the residual tannase activities.

2.5.3. Activation energy (Ea)

The thermal inactivation of the purified enzyme wasexamined over a specified temperature range. Arrheniusplot of inactivation of the purified enzyme was plottedfrom the regression of natural logarithm ln (K) vs. inverseof temperature (T), where T was expressed in Kelvin andthe activation energy was calculated.

2.5.4. pH tolerance

The pH activity profile of all the three forms of tannasewas studied by incubating samples with tannic acid in buf-fers of different pH ranging from 3.0 to 11.0 at optimaltemperature of 50 �C. The buffers used were 0.5 M cit-rate-phosphate buffer (pH 3.0–5.0), 0.5 M phosphate buffer(pH 6.0–7.0), 0.5 M Tris buffer (pH 8.0–9.0) and 0.5 M gly-cine NaOH buffer (pH 10.0–11.0).

2.5.5. pH stability

The pH stability of all enzyme forms was studied byincubating the enzyme in different buffers of pH valuesranging from 3.0 to 10.0 for 1/2, 1, 6, 12 and 24 h at37 �C. After the desired incubation period, the residual tan-nase activity was estimated.

2.5.6. Substrate specificity

The ability of P. variable tannase to hydrolyse differentsubstrates, i.e. tannic acid, methyl gallate and propyl gal-late was examined at different concentrations (0.01–0.06 g ml�1). From the results obtained from the hydrolysisof the substrate, Line–Weaver plots were drawn and Km

and Vmax for each substrate was calculated.

2.5.7. Effect of surfactantsThe effect of surfactants (sodium choleate, sodium

taurocholeate, sodium dodecyl suphate, Tween-60,Tween-80 and Triton X-100) on tannase activity was exam-ined by incubating the lyophilized tannase (10 mg contain-ing 14.0 U) in 1.0 ml of surfactants for 5, 30 and 60 min.The residual activity was then determined.

2.5.8. Effect of organic solvents

The effect of different organic solvents on tannase activ-ity (acetone, carbon tetrachloride, formaldehyde, heptane,ethanol, petroleum ether, toluene and tetrahydrofuran)was examined by incubating 10 mg of the lyophilizedenzyme with 20% and 60% (v/v) organic solvents for 5and 60 min.

2.5.9. Effect of inhibitors

The effect of different inhibitors on purified tannase wasexamined by incubating the purified enzyme with differentinhibitors (1 mM) as sodium choleate, phenyl boronic acid,propanolol chloride, b-mercaptoethanol, bromoacetic acid,dithiothrietol, cyanamide, mercuric benzoic acid, iodoace-tic acid, 1,10-phenanthrolein, phenyl methyl sulphonylfluoride, N-ethylmaleimide, 8-hydroxyquinone at roomtemperature for 30 min. Residual activity was calculatedagainst control.

2.5.10. Shelf-lifeShelf-life of crude and crude lyophilized P. variable tan-

nase at 4 �C and 30 �C was studied for a period of one yearwith time intervals at 0, 7, 15, 30, 60, 120, 180, 240 and 365days.

3. Results and discussion

A two-step efficient and reliable procedure for purifica-tion of P. variable tannase was carried out. In the first step,the culture filtrate containing the enzyme was efficientlyconcentrated to one-tenth the original volume by ultra-fil-tration using 100 kDa membrane cartridge, resulting in97% yield and 5.0-fold purification. This was followed by

Fig. 1. Elution profile of P. variable tannase on Sephadex G-200.

Fig. 2. SDS–PAGE of P. variable tannase. Lane A: molecular weightmarkers; Lane B: culture filtrate having the enzyme tannase; Lane C:partially purified tannase obtained after ultra filtration using 100 kDamembrane; lane D: purified tannase after gel filtration chromatography(Sephadex G-200).

S. Sharma et al. / Bioresource Technology 99 (2008) 2544–2551 2547

a second step, where the concentrated enzyme was purifiedto homogeneity by gel-filtration chromatography usingSephadex G-200, which resulted in 91% enzyme yield witha purification fold of 135 (Table 1, Fig. 1). A similar strat-egy of ultra-filtration for partial purification and concen-tration of A. niger LCF 8 tannase was reported usingfirst a 200 kDa membrane. The permeate obtained wasthen filtered through 100 kDa. This resulted in 80% recov-ery with a 14.9 purification fold (Barthomeuf et al., 1994).

HPLC analysis of the purified tannase showed that theenzyme eluted as a single peak with retention time at6.31 min.

P. variable tannase on Urea SDS–PAGE showed a sin-gle band with a molecular weight of 158 ± 2 kDa(Fig. 2). The native molecular weight estimated by gel-fil-tration chromatography is 310 kDa, indicating that P. var-

iable tannase probably may be a dimer of two subunits of158 kDa (Fig. 3). Tannases are generally high molecularweight and have mutimeric forms. Cryophonectrica parasi-

tica tannase had molecular weight of 240 kDa as deter-mined by gel-filtration and it was suggested that theenzyme was a tetramer comprising of four subunits of58 kDa (Farias et al., 1994). The molecular weight of A.

niger MTCC 2425 tannase has been reported as 185 kDawith two polypeptide chains of apparent molecular weightsof 102 and 83 kDa (Bhardwaj et al., 2003). Similarly, themolecular weight of tannase from Paecilomyces variotii

has been reported as 149.8 kDa through native PAGE witha monomeric unit of molecular mass 45 kDa as revealed bySDS–PAGE (Mahendran et al., 2005).

In the present investigation, results of immobilizationstudies showed that Amberlite IR 1204 is the best supportto immobilize P. variable tannase with 69% immobiliza-tion. However, DEAE-cellulose and Amberlite XAD-7supported only 46% and 25% immobilization (Table 2). Itwas observed that immobilized P. variable tannase couldbe reused at least six times with no appreciable loss inenzyme activity. However, in the ninth cycle about 85%residual activity was observed. Similarly, use of tannasefrom Paecilomyces variotii immobilized on alginate beadsfor hydrolyzing tannic acid has been reported, but afterthe third cycle, a gradual decline in activity was observed(Mahendran et al., 2005).

Table 1Steps for purification of Penicillium variable tannase

Steps Volume (ml) Total tannase (U) Total pr

Culture filtrate 1000 33,000 2160100 kDa (retentate) 185 32,142 404

Fifty ml of the retentate was concentrated to five ml volume using speed vac.

Ultra-filtration (100 kDa) 50 8300 110Speed vac. sample 10 8200 94

Five ml of the concentrated tannase sample was loaded on Sephadex G-200 colu

Gel-filtration chromatography

Control 5 4100 47G-200 20 3740 1.82

Tannase of P. variable was found to be active in therange of 25–90 �C (Fig. 4). All the three enzyme forms

otein (mg) Specific activity (U/mg) Yield (%) Fold purification

15.27 100 1.079.56 97.41 5.2

75.45 97.41 5.087.23

mn and eluted with citrate-phosphate buffer

87.232055 91.0 135

Fig. 3. Estimation of native molecular weight of P. variable tannase bygel-filtration using Sephadex G-200. (1) a-Lactalbumin, 14 kDa; (2)carbonic anhydrase, 29 kDa; (3) ovalbumin, 45 kDa; (4) bovine serumalbumin, 66 kDa; (5) c-globulin (human), 132 kDa; (6) catalase (beefliver), 240 kDa; (7) glutamate dehydrogenase, 290 kDa; (8) P. variable

tannase, 310 kDa.

Fig. 4. Temperature tolerance of P. variable tannase.

2548 S. Sharma et al. / Bioresource Technology 99 (2008) 2544–2551

showed temperature optima at 50 �C. At 25 �C, crude andimmobilized enzyme showed 78% and 81% relative activitywhile purified tannase had a relative activity of 70% at thistemperature. At 80 �C, more than 50% relative activity wasobserved in crude and immobilized tannase while 45% inpurified form. At 90 �C, crude and immobilized enzymeretained 23% and 31% relative activity while purified tan-nase showed a relative activity of only 11%.

In many fungi, temperature optima for tannase activityhave been reported to be in the range of 30–40 �C (Fariaset al., 1994; Yu et al., 2004; Sabu et al., 2005). However,tannases from A. niger van Tieghem (Sharma et al., 1999)and Bacillus cereus KBR 9 (Mondal et al., 2001) have beenreported to have a temperature optima between 45 and60 �C. Temperature optimum of 60–70 �C has beenreported for A. niger tannase (Ramirez-Coronel et al.,2003). No shift in the temperature optima was observedwhen tannase of P. variable was immobilized on Amberliteas against the findings where temperature optima of immo-bilized A. niger tannase has been reported at 40 �C and offree enzyme at 30 �C (Yu et al., 2004).

In the present study, all the three forms of P. variable

tannase showed stability in the range of 25–70 �C for24 h retaining more than 60% residual activity at 25 �C,80% at 50 �C and more than 40% at 70 �C (Table 3). At

Table 2Immobilization of P. variable tannase

Carrier Enzyme added(A)

Unbound enzyme(B)

Immobilized(I)

Amberlite IR 17,000 5270 8094Amberlite XAD-7 17,000 4250 3188DEAE Cellulose 17,000 7820 4223Celite 17,000 14,790 287Silica 17,000 16,150 42.5

80 �C, very low residual activity of 7% and 3% in crude andpurified forms of P. variable was observed after 24 h. How-ever, in immobilized tannase of P. variable, 15% of theresidual activity was retained even after 24 h at this temper-ature. This result is supported by the findings where ther-mal stability of A. niger tannase is significantly improvedby the immobilization process (Abdel-Naby et al., 1999).

Based on the results of Arrhenius plot, activation energyof the tannase calculated in the temperature range 60–70 �C was 219 K cal. The half-life of the enzyme activityat 70 �C and 80 �C was 24 and 12 h, respectively. However,it has been reported that P. chysogenum tannase was stableonly up to 30 �C with 95% stability at 45 �C (Rajakumarand Nandy, 1983). Cryophonectrica parasitica tannase hasbeen reported to be stable at temperature below 30 �C,while at 50 �C and 60 �C the activity was lost in 20 minand 10 min (Farias et al., 1994).

Tannases from P. variable were functional in a broadpH range of 3.0–11.0 (Fig. 5). In all the three forms, morethan 80% relative activity at pH 3.0 was observed. At pH10.0, crude and purified tannases retained 28.6% and23.2% relative activity while in immobilized tannase,10.8% relative activity was observed. In many reports, thepH optimum has been reported in the range of 3.0–6.0(Banerjee et al., 2001; Ramirez-Coronel et al., 2003; Yuet al., 2004; Sabu et al., 2005). Two pH optima peaks at6.0 and 4.0 have been reported for A. niger van Tieghem

tannase (Sharma et al., 1999).

enzyme Specific activity of immobilizedenzyme

Immobilization yield =I/(A � B) (%)

65 69.148 2527.8 4616.5 134.7 5.0

Table 3Temperature stability of P. variable tannase

Temperature (�C) 30 min 1 h 6 h 12 h 24 h

CT PT IT CT PT IT CT PT IT CT PT IT CT PT IT

25 EA 28.88 33.78 25.85 26.53 32.57 25.17 25.91 30.55 23.28 23.69 26.90 22.63 22.21 22.95 20.66RA 87.45 88.90 90.70 80.38 85.7 88.30 78.46 80.35 81.69 71.78 70.80 79.40 67.30 60.40 72.46

37 EA 32.14 34.43 27.99 31.85 34.85 27.27 31.12 32.41 25.74 26.63 27.47 24.40 24.92 25.04 22.40RA 97.40 90.60 98.20 96.50 91.70 95.70 94.30 85.30 90.34 80.70 72.30 85.60 75.49 65.90 78.59

45 EA 33.0 38.0 28.5 33.0 38.0 28.5 31.55 35.07 28.19 29.73 33.71 26.42 27.52 32.83 26.13RA 100 100 100 100 100 100 95.60 92.30 98.90 90.10 88.70 92.70 83.40 86.42 91.52

50 EA 33.0 38.0 28.5 33.0 38.0 28.5 33.0 38.0 28.5 30.20 34.05 25.91 29.44 32.19 24.85RA 100 100 100 100 100 100 100 100 100 91.50 89.60 90.09 89.17 84.70 87.19

60 EA 33.0 38.0 28.5 33.0 38.0 28.5 33.0 38.0 28.5 27.16 30.17 23.85 24.19 26.11 22.45RA 100 100 100 100 100 100 100 100 100 82.34 79.40 83.73 73.27 68.66 78.89

70 EA 29.27 30.25 25.76 26.50 27.17 24.62 26.68 24.24 22.37 19.34 19.53 19.04 14.45 15.28 14.93RA 88.74 79.60 90.38 80.26 71.50 86.40 74.76 63.80 78.52 58.64 51.30 66.80 43.84 40.23 52.35

80 EA 22.74 23.07 20.63 17.92 18.54 17.50 10.86 10.75 11.46 5.21 3.19 7.67 2.54 1.22 4.33RA 68.97 60.70 72.37 54.32 48.78 61.40 32.93 28.27 40.20 15.75 8.42 26.90 7.70 3.24 15.20

90 EA 7.69 4.33 8.84 – – – – – – – – – – – –RA 25.30 11.37 30.7 – – – – – – – – – – – –

100 EA – – – – – – – – – – – – – – –RA – – – – – – – – – – – – – – –

CT: crude tannase; PT: purified tannase; IT: immobilized tannase; EA: enzyme activity; RA: residual activity 100% residual activity (at 0 min) correspondsto 33.0 IU/ml for CT; 38.0 IU/ml for PT and 28.50 IU/ml for IT.

Fig. 5. pH tolerance of P. variable tannase.

S. Sharma et al. / Bioresource Technology 99 (2008) 2544–2551 2549

Tannase from P. variable is stable in pH range of 3.0–8.0for 24 h. It showed 60% residual activity at pH 3.0, almost100% stability in pH range of 4.0–6.0 for 24 h. At pH 10.0,crude and purified tannase retained 19.7% and 15.3% resid-ual activity for 24 h (Table 4). However, immobilized tan-nase retained 24.6% residual activity at this pH even after24 h. Stability of C. parasitica tannase has been reportedover a pH range of 4.0–7.5 for 12 h (Farias et al., 1994).A. niger PKL 104 tannase is stable only in narrow pHrange from 4.5 to 5.5 (Lekha et al., 1994).

P. variable tannase showed broad substrate specificitywith more affinity for tannic acid having a Km of 32 mMfollowed by methyl gallate (14 mM) and propyl gallate(12 mM). The Vmax/Km ratio for tannic acid is almost fourtimes higher than that for methyl gallate and 1.2 times forpropyl gallate. Other reports of Km for tannic acid are of0.28 mM for A. niger MTCC 2425 (Bhardwaj et al.,

2003) and 0.11 lM for A. niger tannase (Yu et al., 2004).Commercial grade tannase from A. oryzae (Sigma, USA)has Km of 0.42 lM.

Surfactants are reported to play key role in the catalyticactivity of the enzymes. Tannase activity was enhanced inP. variable by sodium choleate and sodium taurocholeatewhile SDS, Tween-60, Tween-80 completely inactivatedthe enzyme. In Triton X-100, it retained 100% residualactivity (Table 5a). Inhibition by Tween-60 and sodiumdodecyl sulphate is in agreement with the results of Karet al. (2003). This inhibition may be the result of the com-bined effect of factors such as reduction in the hydrophobicinteractions that play a crucial role in holding the proteintertiary structure and the direct interactions with the pro-tein molecule. In Triton X-100, a non-ionic surfactant,P. variable tannase showed enhancement in tannase activ-ity with respect to time. However, a decrease in tannaseactivity in Triton X-100 was observed (Kar et al., 2003).

P. variable tannase showed different degrees of stabilityin various organic solvents with more than 60% residualactivity in 20% v/v of carbon tetrachloride, heptane, petro-leum ether and toluene after 60 min. In acetone, 49% activ-ity was retained while in formaldehdye only 12% activitywas retained after 60 min. The enzyme was stable morethan 50% in 60% v/v of carbon tetrachloride, heptane,petroleum ether and toluene for 5 min. In tetrahydrofuran,the enzyme is totally denatured (Table 5b).

Inhibition studies on P. variable tannase showed that theenzyme was inhibited by phenyl methyl sulphonyl fluoride(PMSF) and b-mercaptomethanol retaining 28% and39.6% residual activity suggesting it to be a serine hydro-lase. Moreover, N-ethylmaleimide showed strong inhibitionwith only 7% residual activity while 1,10-O-phenanthrolien

Tab

le4

pH

stab

ilit

yo

fP

.va

ria

ble

tan

nas

e

pH

30m

in1

h6

h12

h24

h

CT

PT

ITC

TP

TIT

CT

PT

ITC

TP

TIT

CT

PT

IT

3.0

EA

28.8

130

.51

25.4

826

.86

29.9

424

.34

25.1

827

.51

22.9

923

.66

24.8

120

.95

23.0

322

.84

19.7

2R

A87

.380

.30

89.4

081

.40

78.8

085

.40

76.3

072

.40

80.7

071

.70

65.3

073

.50

69.8

060

.10

69.2

04.

0E

A33

.038

.028

.50

33.0

38.0

28.5

033

.038

.028

.50

33.0

38.0

28.5

033

.038

.028

.50

RA

100.

010

0.0

100.

010

0.0

100.

010

0.0

100.

010

0.0

100.

010

0.0

100.

010

0.0

100.

010

0.0

100.

05.

0E

A33

.038

.028

.50

33.0

38.0

28.5

033

.038

.028

.50

33.0

38.0

28.5

033

.038

.028

.50

RA

100.

010

0.0

100.

010

0.0

100.

010

0.0

100.

010

0.0

100.

010

0.0

100.

010

0.0

100.

010

0.0

100.

06.

0E

A33

.038

.028

.50

33.0

38.0

28.5

033

.038

.028

.50

33.0

38.0

28.5

033

.038

.028

.50

RA

100.

010

0.0

100.

010

0.0

100.

010

0.0

100.

010

0.0

100.

010

0.0

100.

010

0.0

100.

010

0.0

100.

07.

0E

A29

.30

30.4

425

.42

23.6

729

.83

24.7

126

.17

28.6

123

.57

23.2

327

.21

22.1

720

.63

25.3

520

.06

RA

88.7

880

.12

89.2

080

.67

78.4

686

.68

79.3

075

.30

82.7

270

.40

71.6

077

.80

62.5

066

.70

70.4

08.

0E

A24

.52

26.0

022

.74

22.0

422

.08

20.8

920

.49

20.5

619

.64

18.2

817

.78

17.1

915

.94

15.4

716

.73

RA

74.3

268

.30

79.7

869

.78

62.1

473

.25

62.1

054

.10

68.9

055

.40

46.8

060

.30

48.3

040

.70

58.7

09.

0E

A16

.63

16.0

716

.22

15.6

814

.71

14.3

412

.74

13.0

312

.17

10.2

010

.49

11.4

98.

428.

5110

.20

RA

50.3

642

.30

56.9

047

.50

38.7

050

.30

38.6

034

.30

42.7

030

.90

27.6

040

.28

25.5

22.4

035

.80

10.0

EA

14.7

212

.65

14.3

913

.43

11.2

112

.34

10.7

68.

2810

.74

8.18

7.49

8.24

6.50

5.81

7.01

RA

44.6

333

.27

50.4

740

.70

29.5

043

.30

32.6

021

.80

37.7

024

.80

19.7

028

.919

.70

15.3

024

.60

CT

:cr

ud

eta

nn

ase;

PT

:p

uri

fied

tan

nas

e;IT

:im

mo

bil

ized

tan

nas

e;E

A:

enzy

me

acti

vity

;R

A:

resi

du

alac

tivi

ty10

0%re

sid

ual

acti

vity

(at

0m

in)

corr

esp

on

ds

to33

.0IU

/ml

for

CT

;38

.0IU

/ml

for

PT

and

28.5

0IU

/ml

for

IT.

Table 5aCharacterization of P. variable tannase with respect to surfactants

Surfactants RA (%)

5 min 30 min 60 min

Control 100 100 100Sodium choleate 90.5 105 244Sodium taurocholeate 100 136 105Sodium dodecyl sulphate – – –Tween-60 – – –Tween-80 – – –Triton X-100 76 87 100

RA, residual activity (%).

Table 5bCharacterization of P. variable tannase with respect to organic solvents

Organic solvents 20% v/v (RA %) 60% v/v (RA %)

5 min 60 min 5 min 60 min

Control 100 100 100 100Acetone 75 49 – –Carbon tetrachloride 68 68 57 –Formaldehyde 12 12 29 –Heptane 90 62 69 69Ethanol 70 61 47.1 35.7Petroleum ether 78.2 65 50 33Toulene 63 63 54 44Tetrahydrofuran – – – –

RA, residual activity (%).

Table 6Effect of inhibitors on tannase activity

Inhibitors (1 mM) IU (ml) Residual activity (%)

Control 38 100Sodium choleate 19.99 52.6Phenyl boronic acid 25.50 67.1Propanolol chloride 18.85 49.6b-Mercaptoethanol 15.04 39.6Bromoacetic acid 20.25 53.3Dithiothrietol (DTT) 20.56 54.1Cyanamide 23.45 61.7Mercuric benzoic acid 18.54 48.8Iodoacetic acid 18.24 48.01,10-Phenanthrolein 15.2 40.0Phenyl methyl sulphonyl fluoride 10.68 28.1N-ethylmaleimide 7.22 19.028-Hydroxyquinone 17.94 47.2

2550 S. Sharma et al. / Bioresource Technology 99 (2008) 2544–2551

was a mild inhibitor (Table 6). Inhibition of enzyme byPMSF is in accordance to the results of Suseela et al.(1985) who worked with P. chrysogenum tannase. R. ory-

zae tannase is inhibited by dimethyl sulphoxide (DMSO),b-mercaptoethanol and 1,10-O-phenanthrolien (Karet al., 2003). Similarly, A. niger LCF 8 tannase is inacti-vated by b-mercaptoethanol (Barthomeuf et al., 1994).

The shelf-life studies on tannase in crude and crudelyophilized form at 4 �C and 30 �C were carried out fora period of one year. The results (Table 7) showed thatmore than 90% residual activities retained till 15 days in

Table 7Shelf-life of crude and crude lyophilized P. variable tannase at 4 �C and at30 �C

Number of days % Residual activity (4 �C) % Residual activity(37 �C)

C CL C CL

0 100a 100 100 1007 99 99 99 98

15 99 99 95 9030 85 94 90 8760 80 90 88 80

120 72 80 79 76180 69 76 77 74240 67 73 75 73365 60 67 72 70

C: crude tannase; CL: crude lyophilized tannase.a 100% Residual activity of crude tannase – 33.0 IU/ml, 100% Residual

activity of crude lyophilized tannase – 14.0 U/ml/10 mg of lyophilizedpowder.

S. Sharma et al. / Bioresource Technology 99 (2008) 2544–2551 2551

crude and crude lyophilized enzyme forms both at 4 �C and30 �C. However after 120 days, more than 70% residualactivities were retained. About 60–70% residual enzymeactivities were retained even after one year.

4. Conclusions

Though there are many reports on tannases describingtheir properties, however, the present investigation is a firstof its kind where the enzyme possesses desirable propertiessuch as stability at extreme pH and temperature, broadsubstrate specificity, good shelf-life which permits its bio-technological potential to be exploited in food, feed, bever-age, brewing, pharmaceutical and chemical industries.

Acknowledgement

Shashi Sharma acknowledges with thanks to theCouncil of Scientific and Industrial Research (CSIR),New Delhi, Government of India, for the award of a SeniorResearch Fellowship.

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