Biochemical Engineering Journal 77 (2013) 161170

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Biochemical Engineering Journal

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Regular article

Structural characterization of thermostable, solvent tolerant, cytosafe tannase from Bacillus subtilis PAB2
Arijit Jana, Chiranjit Maity, Suman K. Halder, Arpan Das, Bikash R. Pati, Keshab C. Mondal, Pradeep K. Das Mohapatra
Department of Microbiology, Vidyasagar University, Midnapore 721102, West Bengal, India

a r t i c l e

i n f o

a b s t r a c t
Tannase production by Bacillus subtilis PAB2, was investigated under solid state fermentation using tamarind seed as sole carbon source and it was found as the highest titer (73.44 U/gds). The enzyme was puried to homogeneity, which showed the molecular mass around 52 kDa (Km = 0.445 mM, Vmax = 125.8 mM/mg/min and Kcat = 2.88 min1 ). The enzyme was found stable in a range of pH (3.08.0) and temperature (3070 C) with an optimal activity at pH 5.0, pI of 4.4 and at 40 C temperature. It exhibited half-life (t1/2 ) of 4.5 h at 60 C. The enzyme comprised a typical secondary structure containing -helix (9.3%), -pleated sheet (33.6%) and -turn (17.2%). The native conformation of the enzyme was alike a 44 nm spherical nanoparticle upon aggregation. Thermodynamic parameters of tannase revealed that it was stable at 40 C and showed Q10 , Gd and Sd values of 2.08, 99.37 KJ/mol and 252.38 J mol1 K1 , respectively. Organic solvents were stimulatory with regard to enzyme activity. Moreover, the altered enzyme activity was determined to be correlated with the changes in structural conformation in presence of inducer and inhibitor. Tannase was explored to have no cytotoxicity on Vero cell line as well as rat model study. 2013 Elsevier B.V. All rights reserved.

Article history: Received 5 February 2013 Received in revised form 4 May 2013 Accepted 4 June 2013 Available online xxx Keywords: Tannase Bacillus subtilis PAB2 Solid-state fermentation Optimization Downstream processing Kinetic parameters

1. Introduction Microorganisms along with their enzymes play an important role in improving several industrial processes. The global market has a big need for industrial enzymes that is estimated to be worth about 1.6 billion $US, split among food enzymes (29%), feed enzymes (15%) and general technical enzymes (56%) [1]. Production of feed-grade enzymes such as amylase, cellulase, tannase and phytases by solid-state fermentation (SSF) and their applications in the feed have been examined in many studies [2]. But, the major obstacle against the comprehensive application of these enzymes in industry is their high cost of production as 3040% of the production cost is accounted for growth substrate [3]. Among the feed enzymes, tannase has attracted much more attention due to its multifaceted potential applications in beverage, food processing and pharmaceutical industries. The enzyme tannase (EC 3.1.1.20), is an extracellular/intracellular and inducible bio-molecule that increases the bioavailability of nutrients by the hydrolysis of the phenolic (tannin), a potent anti-nutritional factor in plant materials [4]. Tannase is used in wine-making, beer

Corresponding author. Tel.: +91 3222 276554x477; fax: +91 3222 275329. E-mail addresses: arijitjana.mic@rediffmail.com (A. Jana), pkdmvu@gmail.com (P.K. Das Mohapatra). 1369-703X/$ see front matter 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.06.002

chillproong, instant tea production and in the pre-treatment of animal feed [5,6]. In addition, it is used as a sensitive analytical probe for detecting cancerous cells [7]. Gallic acid, the product of tannin hydrolysis, also nds many applications including dye-making, pharmaceutical, leather and chemical industries [5]. However, due to its high production cost it is currently used in a few circumstances and effort should be taken to reduce production cost. Therefore, the use of tannin-rich agro-industrial by products as cheap and readily available substrates, can offer an alternative way of cost effective enzyme production and waste management that can couple with the production of valuable products. Apart from these, there is a substantial gap that still remains in our understanding of catalytic functions and biochemical information about this novel enzyme. Physico-chemical characterization of fungal tannase has been extensively studied [8], but information about bacterial tannase is very sparse. Recently, Banerjee et al. [9] have reported the physico-chemical characterization, functional motif and evolutionary relationship among fungal and bacterial tannase. It has been widely accepted that the catalytic functions of enzymes are related to protein structure . However, information concerning the tannase structure is scanty. So far, little is known about the tanninolytic machinery of bacteria for tannase production. In this study, an attempt had been made for production of tannase in solid state fermentation using tamarind seed as substrate

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and biochemical and structural characterization of puried tannase from Bacillus subtilis toward better understanding the mechanism of tannin degradation.

2.5. Protein quantication, electrophoresis and mass spectrometry Protein concentration was determined by the method of Bradford [11] using bovine serum albumin as a standard. Homogeneity and probable molecular mass of the puried tannase were conrmed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using standard marker proteins. Zymogram analysis was performed according to the method of Maity et al. [12]. The molecular mass of the puried tannase was also analyzed in the linear mode by matrix assisted laser desorption ionization-time of ight (MALDI-TOF) mass spectrometry (MS) using a Voyager spectrometer (Applied Biosystems, USA). MALDITOF-MS analyses of the puried tannase fraction were carried out on aliquots containing about 5 pmol of protein samples, mixed with 1 l of matrix [10 mg/ml, -cyano-4-hydroxycinnamic acid in 50% acetonitrile], spotted onto MALDI sample plate and dried under ambient conditions. 2.6. Iso-electric focusing (IEF) Iso-electric point of the puried enzyme was determined by IEF (BioRad, Hercules, CA, USA) using the passive dehydration method (16 h of rehydration at 20 C) according to the manufacturers instruction. The strips were then transferred to an isoelectric focusing (IEF) cell (BioRad, Hercules, CA, USA). IEF was performed by applying a voltage of 250 V for 30 min, linear ramping to 8000 V over 2 h, and holding to 8000 V until 40 KVh was reached. Then the gel strips were equilibrated for 15 min twice in equilibration buffers. The second dimension was performed using 12% SDS-PAGE at 80 V. The gels were stained using the CBB staining and scanned with a BioRad GS-800 scanner. 2.7. Circular dichroism (CD) spectroscopy Secondary structure of tannase which is responsive to conformational changes was studied using a CD spectrometer (Jasco, J-810, USA) with a Peltier type cell holder for temperature control. A sample of 10 L (1 mg/ml) tannase solution was added into a quartz cuvette with a 1 mm path-length at room temperature under constant nitrogen spurging at the far UV region (200360 nm). The protein far-UV spectra were recorded by signal averaging of ve spectra, with a step resolution of 0.1 nm and a speed of 100 nm/min. The protein signal was obtained by subtracting buffer spectrum from the sample spectrum. CD spectra were recorded in milidegree (CD mdeg) and applied to calculate the secondary structure of the enzyme. The effect of temperature and pH on enzyme conformation was also measured after incubating tannase at different pH (3.08.0) and temperature (4070 C) for 1 h. 2.8. Scanning probe microscopy (SPM)

2. Methods 2.1. Microorganism A newly isolated tannase producing bacterial isolate, B. subtilis PAB2 (GenBank accession number-HM853662) was employed in this study.

2.2. Solid state fermentation and culture condition For tannase production, solid state fermentation (SSF) was carried out in 250 ml Erlenmeyer asks containing 5 g of broken tamarind (Tamarindus indica L.) seed (particle size 1 mm 1 mm) initially moistened with 5 ml of basal medium having a composition of (% w/v): NH4 Cl (4.0), mannose (0.01) and MgSO4 (0.01). The sterilized media was inoculated with 1 ml of 24 h fresh bacterial inoculum (2 103 cfu/ml) and incubated at 35 C in a programmable environmental chamber (REMI-CHM-6S) having 75% humidity, for 72 h. The inuencing parameters of SSF like various nitrogen sources, the secondary carbon sources and the metal salt were optimized at a nal concentration of (% w/v) 3, 0.1 and 0.1 respectively. The quasi-optimum protocol (single-variable approach) was initially used to evaluate the effect of an individual parameter and to incorporate it at the optimized level on the experiment before optimizing the next parameter. The enzyme was extracted by using water as extracting solvent by mixing in 1:6 ratio (w/v) and agitated through a rotary shaker at 150 rpm for 1 h at room temperature (35 C). The slurry was then squeezed through cheesecloth followed by centrifugation at 5000 g for 10 min at 4 C to remove the insoluble matter. The clear supernatant was used as crude tannase.

2.3. Tannase assay Tannase activity in the fermented medium was determined by the colorimetric method of Mondal et al. [10]. One unit of the tannase was dened as the amount of enzyme, which is able to hydrolyse 1 mol of the ester linkage of tannic acid in 1 min at specic condition.

2.4. Tannase purication The extracted crude preparation was used for purication of tannase. Ammonium sulfate was added slowly to the crude enzyme upto a nal concentration of 80% saturation. The precipitated proteins were separated by centrifugation at 10,000 g and resuspended in a minimum amount of acetate buffer (0.2 M, pH 5.0). Residual ammonium sulfate was removed by exhaustive dialysis against the same buffer. Concentrated 5 ml sample was then loaded onto Sephadex G-75 column with a bed size of 2.5 cm 70 cm. The column was equilibrated with 0.2 M acetate buffer (pH 5.0) and was eluted with the same equilibrating buffer at a ow rate of 1 ml/min. All the purication steps were carried out at 4 C. The protein content in each sample was estimated at 280 nm using a spectrophotometer. The fractions corresponding to high tannase activity were pooled, lyophilized and stored at -20 C for further characterization.

The aggregation pattern of puried tannase from B. subtilis PAB2 was studied by SPM. Puried tannase was diluted with HPLC grade water and 5 L of the sample was spotted onto a cover slip and left for 10 min at room temperature. Cover slip was laid in the air and scanned at room temperature with an SPM instrument (Multiview1000TM , Nanonics Imaging Ltd., Israel) under the scanning mode equipped with image processing software (WSxM 5.0). 2.9. Determination of catalytic constants The kinetic parameters of tannase activity were determined using various concentrations (0.531.29 mM) of tannic acid (Merck, India) as substrates at standard assay condition. Accordance with a Lineweaver double reciprocal plot, MichaelisMenten plot

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was drawn using the Sigma plot (enzyme kinetics software). The Michaelis constant (Km ), maximum reaction velocity (Vmax ), catalytic efciency (kcat /Km ), turnover number (kcat ) and the second-order constant (Vmax /Km ) were estimated. 2.10. Temperature, pH optima and stability The optimum pH for tannase activity was determined at various pH levels between pH 3.0 and 8.0 using acetate (pH 3.06.0) and phosphate (6.58.0) buffers. For pH stability, residual tannase activity was measured after 1 h of incubation at 40 C in different pH buffers. Tannase activity was tested at different temperatures ranging from 30 to 70 C in 0.2 M acetate buffer (pH 5.0). The enzyme solution was incubated for 1 h at the tested temperature and residual activity was measured at optimum temperature and pH. 2.11. Salt tolerance test The enzyme was incubated in 0.2 M acetate buffer (pH-5.0) containing various concentrations of NaCl (15 M) for 24 h at 4 C, and then activities were measured. 2.12. Temperature quotient (Q10 ), thermal inactivation and kinetic modeling Temperature quotient or Q10 , the rate of an enzymatic catalytic reaction changes for every 10 C rise in temperature was calculated by the following equation: Q10 = R2 R1
10/(T2 T1 )

acetone] and then, residual activities were measured at standard assay condition. 2.14. Denaturation and renaturation pattern of tannase Denaturation and renaturation of tannase structure were studied in presence of Mg2+ and Hg2+ using F-7000 uorescence spectrophotometer (Hitachi, Japan). Preincubated (with 1 mM of Mg2+ and Hg2+ ion) puried tannase was loaded in 1 cm quartz cells. Both the excitation and emission slits were set at 2.5 nm. The excitation wavelengths were set at 278 and 295 nm, and the uorescence intensity of emission uorescence spectra was measured in the range of 310410 nm, with a scan speed of 1200 nm/min. 2.15. Cytotoxicity study 2.15.1. In vitro cytotoxicity assay The cytotoxicity of puried tannase was tested through MTT assay [13]. Vero cells (ATCC, Manassas, VA, USA) and isolated mice peritoneal macrophages were seeded into 96 well culture plates with 1.5 105 cells/ml concentration. Puried tannase was added to each culture well at a nal concentration of 1000 g/ml and incubated at 37 C with 5% CO2 for 48 h. Then, per well 10 l of MTT reagent (2.5 mg/mL 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazoliumbromid in PBS) was added. After 4 h of incubation, the formazan was solubilized by adding diluted HCl (0.04 N) in isopropanol and the absorbances were measured at 492 nm (reference 650 nm ) in an ELISA microplate reader. Cell viability (%) was calculated using the following equation Cell viability(%) = sample absorbance cell free sample blank mean media control absorbance 100 The 50% cytotoxic concentration (CC50 ) was represented as visible morphological changes in 50% of Vero cells with respect to cell control. 2.15.2. In vivo cytotoxicity testing In vivo cytotoxicity was tested according to method of Lane et al. [14]. For this experiment, male albino Wister rats (Rattus norvegicus), each 3 months old weighing 100 10 g, were used as model animal and housed according to the recommendations of Vidyasagar University Guide for the Care and Use of Laboratory Animals (Recommendation No.-VU/R/776/12 dt. 26.07.12). Animals were divided into three groups, each group having three rats. The group I did not receive any treatment (Control). Group II and III were regularly administered (oral) with tannase at a dose of 200 and 1000 mg respectively for 6 weeks at the rate of 10 ml/kg/day. At the end of the treatment, the rats were sacriced and liver, kidney and spleen were collected and xed in 10% (v/v) formalin. Tissue sections (45 m thickness) were stained with hematoxylin and eosin, and observed using a Nikon Eclipse LV100POL light microscope with digital camera (Tokyo, Japan) and the images were analyzed by Software Package NIS-Element F3.0. 2.16. Statistics All experiments were conducted in triplicate, and the values were represented as mean standard deviation (SD). 3. Results and discussion Optimization of nutritional parameters for tannase production in SSF was done by using quasi-optimum (one variable at

where T1 and T2 are the initial and nal temperatures at which experiments were conducted. R1 and R2 are the yielding rates measured at T1 and T2 respectively. The kinetics of thermal inactivation was determined by incubating the puried enzyme at various temperatures (4070 C). The rst-order rate constants for denaturation (kd ) of the enzyme at different temperatures were determined from the slopes of semi-logarithmic plots according to ln(Ct /C0 ) = kd t (Ct = enzyme activity at the time t, C0 = initial enzyme activity, kd = rst order denaturation rate constant, t = time). The activation energy for denaturation (Ea,d ) was detected from the slope (Ea,d /R) of Arrhenius plot of ln kd versus 1/T. Free energy ( Gd ), enthalpy ( Hd ) and entropy ( Sd ) changes during enzymatic reactions were calculated using the following equation: Gd = RTln(Kd h/KB T) [h (Planck constant) = 6.626 1034 J s, KB (Boltzman constant) = 1.381 1023 J K1 , T = temperature], Hd = Ead RT [R (gas constant) = 8.314 J mol1 K1 ] and Sd = ( Hd Gd )/T. The half-life (t1/2 ) of the enzyme was obtained from t(1/2) = ln(2)/kd . The values of kd and Ea,d were estimated by regression analysis using the statistical package of Microsoft Excel . 2.13. Effect of additives on tannase activity To study the effect of various additives on tannase activity, puried tannase was incubated at room temperature for 1 h in presence of 1 mM of various metal ions [Ba2+ (barium chloride), Ca2+ (calcium chloride), Mg2+ (magnesium sulfate), Zn2+ (zinc chloride), Fe3+ (ferric chloride), Co2+ (cobalt chloride), Hg2+ (mercuric chloride), K+ (potassium chloride), Fe2+ (ferrous sulfate), Ag+ (silver nitrate)], 1% (v/v) of surfactants [tween 80, tween 60, SDS, triton X-100], 1 mM of inhibitor [dimethyl sulfoxide (DMSO), -mercaptoethanol, sodium azide, phenyl methyl sulfonyl uoride (PMSF), ethylene diamine tetra acetic acid (EDTA)] and 1% (v/v) organic solvents [isopropanol, methanol, ethanol, isoamyl alcohol, glycerol, butanol, acetic acid,

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A. Jana et al. / Biochemical Engineering Journal 77 (2013) 161170 29.85 1.19 64.51 1.93 Tannase (U/gds) 70.36 2.13 50.78 2.08 48.48 1.83 Each value represents the mean of three independent determinations SD. 56.06 2.48 41.28 1.57 55.77 2.17 60.48 2.93 73.44 2.36 63.6 1.78

a time/OVAT) protocol. Tamarind seed was successfully used as both carbon source as well as solid support. Kumar and Bhattacharya [15] reported that tamarind seed is a rich source of tannin (20.2%), protein, lipid, crude ber, minerals, amino acid etc. The bacterium, B. subtilis PAB2 produced a substantial amount of tannase in presence of all the tested nitrogen sources while highest enzyme biosynthesis of 63.6 U/gds occurred in presence of 3% (w/v) ammonium chloride (Table 1). Elemental nitrogen is very essential component for growth of microbes and production of secondary metabolites. The inducing effect of ammonium chloride on tannase production was also observed in Bacillus cereus KBR9 [16] and Bacillus licheniformis KBR6 [17]. Sabu et al. [18] reported maximum tannase production by Aspergillus niger after addition of KNO3 (1%, w/v) in tamarind seed containing media. Addition of the secondary carbon source like 0.1% (w/v) mannose stimulated tannase production (70.4 U/gds) 1.1 times more than the control (Table 1). Induction of tannase synthesis by additional carbon source like glucose was also reported in Klebsiella pneumonia [19] and Bacillus sphaericus [20]. Further, tannase production was enhanced to 73.44 U/gds when the production media was supplemented with 0.1% (w/v) magnesium sulfate (Table 1). Our nding is in agreement with the B. licheniformis KBR6 [17] and B. sphaericus [20]. But the actual role of metal ion in bacterial tannase production is still not clear. At optimized condition, maximum tannase production (73.44 U/gds) was achieved in presence of 3% (w/v) NH4 Cl, 0.1% (w/v) mannose and 0.1% MgSO4 and with incubation at 35 C for 72 h under humid condition. An overall 4.87 fold increase in tannase production was achieved after OVAT optimization. The level of tannase production by B. subtilis PAB2 was higher than previously reported tannase from any other bacterial origin [20]. Tannase was puried from the 72 h old solid state culture ltrate of B. subtilis PAB2 by two-step purication protocol consisting of ammonium sulfate precipitation (80%) followed by gel ltration chromatography (Sephadex G-75). In the nal step, enzyme was puried 24.18 fold with 5.04% recovery and specic activity of 2868.75 U/mg (Table 2). The purication of tannase from microbial source generally involves sequential chromatography techniques, mainly, ion exchange, gel ltration and HPLC [21,22]. In these steps, a considerable amount of enzyme is lost due to autolysis and some remain physically adsorbed on the matrix. To overcome these constraints, single step purication systems, like gel ltration chromatography has been employed. The homogeneity of the puried tannase was judged by the SDS and native PAGE. The electrophoregram showed only one band, and in-gel (zymogram) enzyme activity also conrmed its purity (Fig. 1A). The molecular mass of tannase from B. subtilis was found to be 52 kDa (Fig. 1A). Results of SDS and native PAGE indicate that the tannase from PAB2 is monomeric in its structure. Noteworthy, the MALDI-TOF-MS analysis (Fig. 1B) of puried tannase also indicated the presence of a single strong peak at m/z 52,000, thus denitely ruling out any possibility of multimeric protein being responsible for tannase activity. Previously, K. pneumonia and Lactobacillus plantarum tannase were also found to be monomer with molecular mass of 50 and 46.5 kDa, respectively [19,23]. To characterize the tannase further, isoelectric point of the enzyme was measured and it was found to be approximately 4.4 (Fig. 1A). The pI < 5.0 is most common for tannase of microbial origin imprinting its acidic character [24]. In this scenario, in silico study of Banerjee et al. [9] reported that the pI value of tannase ranged from 3.91 to 9.94 and most of the fungal tannases were in between 4.2 and 5.5. To determine the secondary structure composition of B. subtilis PAB2 tannase, CD spectra was studied. Native tannase was found to be composed of 9.3% -helix, 33.6% parallel -sheet, 17.2% -turn, and 39.9% random coil. This result recommended that conformation played a dominant role in tannase activity, implying that

60.6 2.20 44.8 1.50 55.96 1.67 60.48 2.54 62.6 1.75 70.4 2.53 62.6 1.75 58.7 2.50 64.6 2.20 63.8 1.66 Tannase (U/gds)

Maltose

NH4 Cl

43.87 1.43

KNO3

Arabinose

38.49 1.84

Urea

Lactose

29.28 0.66

Peptone

Cellulose

Yeast extract

25.72 1.10

Mannitol

26.49 0.85

Beef extract

Mannose

30.14 .75

NaNO3

33.69 1.01

(NH4 )2 SO4

Fructose

Table 1 Effect of different nutritional components on tannase production.

16.22 0.49

Glucose

Starch

NH4 NO3

15.07 0.60

Control

Secondary carbon

Tannase (U/gds)

Nitrogen source

Control

Metal salt

Control

ZnCl2

ZnSO4

K2 SO4

CaCl2

MgCl2

NaCl

MnSO4

MgSO4

KCl

A. Jana et al. / Biochemical Engineering Journal 77 (2013) 161170 Table 2 Summary of purication of tannase from Bacillus subtilis PAB2. Purication step Crude enzyme Ammonium sulfate fractionation Sephadex gel-ltration Total activity (U) 45532.80 27689.20 2295.00 Total protein (mg) 383.78 60.69 0.80 Specic activity (U/mg) 118.64 456.24 2868.75 Purication fold 1 3.84 24.18

165

Recovery (%) 100 60.81 5.04

the tannase is a globular protein. The secondary structure composition of tannase of PAB2, is approximately similar to that of Penicillium herquei [21]. This also indicates that the folding pattern of the tannase is presumably similar in both bacteria and fungi. The changes of -helix, -sheet and -turn percentage in the tannase structure at different pH and temperature, were studied

and represented in a graphical manner (Fig. 2A and 2B). It has been revealed that -helical content decreased at far acidic and basic pH range as compared to mid pH 5.5. However, the overall change in helix content was not signicant at varied pH range. On the contrary, the -sheet content increased at lower (pH 3.05.0) and higher pH (7.08.0) values. The sheet content maximally decreased

Fig. 1. (A) SDS-PAGE, zymogram and isoelectric point analysis of puried tannase from B. subtilis PAB2 where M, Molecular mass marker; P, Puried tannase; Z, Zymogram analysis of tannase activity; I, Isoelectric point of puried tannase. (B) Molecular mass spectroscopic analysis of puried tannase.

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A
-helix, -sheet and turn (%)

50 40 30 20 10 0

-helix

-sheet

turn

3.5

4.5

5.5 pH

6.5

7.5

35 30

-helix

-sheet

turn

25 20 15 10 5 0

40

50

60

70

Temperature (OC)
Fig. 2. Circular dichroism analysis of puried tannase from B. subtilis PAB2 (A) at different acidic, neutral and basic pH range and (B) temperature (4070 C) on secondary structure of tannase.

near the range of iso-electric pH of the native tannase (4.4), which may be due to the forced precipitation. The variation of reaction temperature also affected the content of secondary structure as reected in Fig. 2B. This may be due to the pH and temperature

dependent destabilization of non-covalent bonds, particularly hydrogen bonding, as it is the main binding force for stabilization of secondary structure of tannase. Protein aggregation pattern of puried tannase was analyzed by SPM that enlightened overall morphological information about the protein particle. It was detected that tannase from B. subtilis PAB2 showed different degree of protein aggregation, approximately round to oval shaped structure of different sizes with an average diameter of 44 nm (Fig. 3). This nding is in agreement with tannase of P. herquei [21]. The topological details can edify assembly patterns, dynamic behavior or ability to interact with other molecules with this enzyme. The kinetic parameter of puried tannase was determined by LineweaverBurk reciprocal plot (Fig. S1). It is evident from the plot that puried tannase exhibited Km , Vmax , Kcat , Kcat /Km , Vmax /Km value of 0.445 mM, 125.8 mM/mg/min, 2.88/min, 6.47 mM/min and 282.7 U/mg/mM, respectively. The tannase produced by PAB2 showed quite lower afnity (Km ) and higher Vmax than recently reported P. herquei [21] and Emericella nidulans [22] conrming its higher applicability toward different industrial sectors. Temperature and pH are the key factors that affect the catalytic efcacy and stability of the enzyme. The puried tannase was optimally active at pH 5.0 and relatively stable in a range of pH (3.08.0) and completely stable (95100%) in a pH range of 4.06.5 (Fig. 4A). It may be due to the presence of the acidic nature of enzyme and abundance of acidic amino acid at the active site. Sabu et al. [25] reported the pH dependent enzyme activity being determined by the nature of the amino acids at the active site, which undergoes protonation and deprotonation by the inuence of environmental pH. Till date, most of the reported fungal tannases are active in the range of pH 2.08.0 whereas, the bacteria are in the range of pH 4.55.5 [8]. The optimal temperature of tannase activity was found at 40 C (Fig. 4B), which was similar to other tannases [16,19]. Tannase was stable at moderate temperature (3060 C) and even retained its activity (14%) at 70 C (Fig. 4B). Our results are comparable with reported tannase [25]. But tannase from L. plantarum showed temperature stability 45 C [23]. Stability of the enzyme is one of the important parameters which determine the economic feasibility of applying them in industrial processes. The high pH and

Fig. 3. Morphological view of protein aggregation pattern of puried B. subtilis PAB2 tannase. (A) Scan size 10 m 10 m, (B) three dimensional projection view of A, (C) magnied image of selected area of A.

helix, sheet and turn (%)

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Fig. 4. (A) Effect of pH on the tannase activity and stability. The optimal pH was determined by measuring the tanninolytic activity within a pH range of 3.08.0 at 40 C for 1 h. The pH stability was estimated by measuring the remaining activity after incubating the enzyme for 1 h at 40 C with different buffers: 0.2 M of acetate (pH 3.06.0) and phosphate (6.58.0) buffers. (B) Effect of temperature on the tannase activity and stability. The optimal temperature was determined by measuring the tanninolytic activity at different temperatures (2070 C) for 1 h. The thermal stability of tannase was evaluated by measuring the remaining tanninolytic activity after incubating the enzyme in various temperatures (3070 C) for 1 h at pH 5.0. (C) Effect of NaCl (salt) concentration on stability of the puried tannase. Relative activity was dened as the percentage of activity detected with respect to the maximum enzyme activity. For determining the stability, the activity of the enzyme without any treatment was taken as control. Each value represents the mean SD of three independent experiments.

thermostability of PAB2 tannase allows the performance of the biotechnological processes at minimal risk of microbial contamination and also favors reaction efciency. The effect of temperature on rate of reaction was measured in terms of Q10 . The usefulness of calculating a Q10 value is that it suggests whether or not the metabolic reactions are mainly controlled by temperature or by some other factors. For enzymatic reactions, the value of Q10 is between 1 and 2 and any deviation from this value indicates the inuence of other factors [26]. The Q10 value for tannic acid hydrolysis by tannase of B. subtilis PAB2 was found to be 2.08, reecting that some factor other than temperature is controlling the rate of reaction.

Investigation of thermodynamic parameters like melting temperature, activation energy of denaturation (Ea,d ) and change in enthalpy ( Hd ), entropy ( Sd ), free energy ( Gd ) and half life (t1/2 ) of enzyme is necessary to understand the behavior of molecules in different conditions [27]. The kd values, activation energy, enthalpy, entropy, free energy and the half life of enzyme at different temperature are given in Table 3. The high stability of puried tannase at 40 C is also revealed by high t1/2 value suggesting its applicability in bioprocess industries. The low G* value for the heat labile enzyme corresponds to the large H* and S* contributions and conversely, the high G* corresponds to the low H* and S* for heat-stable enzymes. The value of S* > 0 implied increased randomness at the activated transition state reecting an increased disorder, which is the main driving force of heat denaturation. The half life of the puried tannase is in direct relationship with temperature. Similar results have been reported for the tannase of Aspergillus niger GH1 [28]. Puried tannase of PAB2 is salt tolerant upto 4 M NaCl (Fig. 4C), that retained 67% of its original activity after 24 h of incubation. This is the rst time observation that bacterial tannase also has a high salt tolerant property up to 4 M NaCl. Previously, the salt tolerant property upto 3 M has been reported from bacterial origin [16]. The enzyme with high salt tolerance capability is very important for its commercialization in bioprocess industry and treatment of tannin containing hard sewage water. Effect of metal ions on PAB2 tannase was studied and shown in Table 4. Metal ions act as salt or ion bridges to maintain the conformation of the enzyme or to stabilize the binding of the substrate and enzyme complex (as cofactor). Among the metal ions, Mg2+ at a concentration of 1 mM was able to enhance the activity of the puried tannase dramatically. Increased activity (>2 fold) in the presence of Mg2+ may be due to the stabilization of enzyme in its active conformation rather than it being involved in the catalytic reaction. It probably acts as a salt or an ion bridge via a cluster of carboxylic groups and thereby maintains the rigid conformation of the enzyme molecule. Inhibitory effect of metal ions on tannase activity have shown in decreasing order as Fe3+ > Hg2+ > Fe2+ > Ba2+ > Ca2+ > Co2+ > Zn2+ > Ag+ > K+ . Strong inhibitory effect of Fe3+ and Hg2+ may be the result of binding to thiol groups or interaction with tryptophan residue or the carboxy group of amino acid in the enzyme. Inhibition of tannase in presence of Hg2+ is indicative of its thiol hydrolase nature. Thiol hydrolase nature of tannase was also reported by many workers [2931]. The effect of the detergents on tannase activity was investigated. Among the detergents, SDS at 1% concentration showed stimulatory (13.2%) effect on tannase activity, while Triton X100 had the maximum inhibitory effect (Table 4). This action of the detergents on the protein can well be correlated to its resistance against hydrophobic/non-polar microenvironment [32]. The activation of tannase activity in presence of SDS is in agreement with earlier E. nidulans and Aspergillus heteromorphus tannases [22,31]. Inhibition studies primarily provide information about its structural pattern, its cofactor requirements, and the nature of the active site. Various inhibitors such as DMSO, -mercaptoethanol, sodium azide, PMSF and EDTA disodium salt were tested at 1 mM concentration each on tannase activity (Table 4). DMSO was the most potent inhibitor of tannase, causing a 75.1% loss in original tannase activity, followed by sodium azide, -mercaptoethanol, EDTA and PMSF. Moreover, DMSO showed strong inhibition with only 24.9% residual activity, while PMSF was a mild inhibitor. The inhibition of PAB2 tannase by -mercaptoethanol could be due to the reduction of disulde bonds in the active site. Our ndings are in agreement with Qiu et al. [21] and Chhokar et al. [31] who reported that the tannase from P. herquei and A. heteromorphus was also inhibited

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Table 3 Kinetic and thermodynamic parameters for thermal denaturation of puried enzyme. T (K) 313 K (40 C) 323 K (50 C) 333 K (60 C) 343 K (70 C)

kd (min1 ) 1.699 10 1.0335 103 0.002539 0.0459

4

t1/2 (min) 4077.58 670.68 273 15.1

Gd (kJ mol1 ) 99.377 97.787 98.411 93.196

Hd (kJ mol1 ) 178.373 178.289 178.207 178.123

Sd (J mol1 K1 ) 252.38 249.23 239.63 247.6

Table 4 Effect of additives on activity of tannase from B. subtilis PAB2. Additive Control Metal ion Ba2+ Ca2+ Mg2+ Zn2+ Fe3+ Co2+ Hg2+ K+ Fe2+ Ag+ Surfactant Tween 80 Tween 60 SDS Triton X-100 Relative activity (%) 100 8.28 0.40 24.48 1.10 205.31 6.57 56.63 2.01 5.21 0.26 30.41 1.27 2.25 0.11 92.31 4.70 6.14 0.26 86.28 3.27 78.18 2.25 80.18 1.84 113.2 3.40 58.18 2.51 Additive Inhibitor DMSO -Mercaptoethanol Sodium azide PMSF EDTA Organic solvent Isopropanol Methanol Ethanol Isoamyl alcohol Glycerol Butanol Acetic acid Acetone Relative activity (%) 24.9 65.56 60.07 93.22 82.05 110.8 102.3 107.14 103.4 121.7 60.43 27.47 42.67 1.19 2.68 2.32 2.42 2.83 3.32 4.30 3.49 5.17 4.98 1.81 1.01 1.86

Each value represents the mean of three independent determinations SD.

by the -mercaptoethanol and EDTA. Kar et al. [30] found that the tannase was inactivated by DMSO and -mercaptoethanol at 1 mM. The effect of organic solvents at 1% concentration on tannase activity has been investigated and summarized in Table 4. Polar and protic solvents like glycerol, isopropanol, ethanol, methanol and isoamyl alcohol were found to increase the enzyme activity, whereas, butanol, acetic acid and acetone were found to decrease the enzyme activity. The former group of solvents may facilitate substrate availability of the active site of the enzyme and enhance the rate of catalysis. On the contrary, Klibanov [33] reported that polar solvents reduce the catalytic efcacy by absorbing the essential water molecule from the enzyme. Enzymatic stability of PAB2 tannase in the presence of organic solvents makes it attractive for transesterication reactions for production of propyl gallate. The functional property of the tannase differed with the interaction of salts manipulated in its structure as well as its active site. In general, this effect is traced by the property of intrinsic uorescence of the present aromatic amino acid. Presence of aromatic amino acids like tryptophan (Trp), tyrosine (Tyr), phenyl alanine (Phe) in a protein molecule contributes to its intrinsic uorescence property. Intrinsic uorescence based structural refolding and unfolding of tannase was studied in presence of metallo activator (Mg2+ ) and inhibitor (Hg2+ ) (Fig. 5). Free Trp showed the excitation at 280 nm and emit maximum intensity at 350353 nm. But blue shift of the maximum emission spectra from 353 to 332 nm indicated that Trp concealed in the hydrophobic environment of the tannase. Addition of Mg2+ (1 mM) altered conformation of tannase that exposed the Trp to the hydrophilic domain (active site) as the relative intensity of the emission spectra increased. A decrease in relative uorescence intensity (at 278 nm) of tannase at maximum wavelength was observed after addition of Hg2+ . But, the excitation at 295 nm showed no evident shift of emission spectra. It was obvious from this experiment that tannase activity and active conformation depended upon Trp, which is very sensitive for salts interaction and the microenvironment of the enzyme. Banerjee et al. [9] reported that amino acid sequences of functional motif of tannase are representing its structural specicity and enzymatic function.

Although tannase is widely used in clarication of beer, fruit juices, wine and other food preparation but little is known about its safety to health. In the present investigation detailed toxicological studies for safety have been performed. The rst step in evaluating the cytotoxicity of the B. subtilis PAB2 tannase was to screen the minimum concentration with detectable cytotoxic effects. It was found that (Fig. S2) tannase having concentration upto 1000 g/ml has no demonstratable ill effect on Vero cell, indicating its non-toxicity to the cells. The CC50 value of puried tannase is 1720 g/ml. Till date, no such report is available on in vitro cytotoxic activity of tannase. In vivo cytotoxicity was studied on rats and no abnormal behavior was observed during the experimental period. Histological sections of the liver, kidney and spleen of tannase treated rats showed normal cellular architecture and no pathological damages. Histological observation could be a good indicator for safety and non-toxicity study [34]. Microscopic analysis of histological

Fig. 5. Intrinsic uorescence spectrum of B. subtilis PAB2 tannase in 0.2 M acetate buffer, pH 5.0, where, (a) puried tannase, (b) tannase + Mg2+ , (c) tannase + Hg2+ . Experiments were carried out at 25 C and corrected for buffer contribution.

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Fig. 6. Hematoxylin and eosin stained sections of kidneys, liver and spleen at 400 magnication. (A, B, and C) Kidney sections of control, group II and III rat showing normal histology (arrows depict normal glomerulus and renal tubules); (D, E, and F) liver sections of control, group II and group III rat showing normal histology (arrows depict normal hepatocytes, portal area and dotted arrow indicated koofer cell); (G, H, and I) spleen sections of control, group II and group III rat showing normal histology. The different capital letter indicated the different portion of the spleen (W, white pulp, R, red pulp, C, central arteriole).

sections revealed no abnormal signs and symptoms like degeneration of hepatocytes in liver, epithelial cell desquamation in kidney and hematopoiesis degeneration in spleen (Fig. 6). Among these, liver is an important organ for any toxicological study because it is the major site of detoxication in the body of all drugs/toxins [35]. The overall (in vitro and in vivo) toxicological data of B. subtilis PAB2 tannase provide a potential basis of biotechnological attention which, given its GRAS (Generally Regarded As Safe) status, can nd it further exploitation in food and feed sectors. Till date, this is the rst report on tannase, where its safety aspect has been dealt with. 4. Conclusion B. subtilis PAB2 has prospect for highest titer of tannase production (73.44 U/gds) using tamarind seed as low cost substrate. Tannase from PAB2 is an extracellular thiol hydrolase of lowmolecular mass. B. subtilis PAB2 derived tannase exhibits high activity and stability over a broad range of temperature. The properties of tannase suggest its potential relevance in various industrial applications where high salt concentrations and high temperatures are preferred. Furthermore, the cytosafe status of tannase may extend its application from developing eco-friendly industrial technologies to the food industry. Thus, this work sets the platform for more detailed structural and functional investigations of the bacterial tannase.

Acknowledgement The authors are greatly indebted to the University Grants Commission, New Delhi for nancial assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2013.06.002. References
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