Purification and characterization of extracellular tannin acyl hydrolase ...

Biotechnology and Bioprocess Engineering 15: 793-799 (2010) DOI 10.1007/s12257-010-0058-3

RESEARCH PAPER

Purification and Characterization of Extracellular Tannin Acyl Hydrolase from Aspergillus heteromorphus MTCC 8818 Vinod Chhokar, Seema, Vikas Beniwal, Raj Kumar Salar, K. S. Nehra, Anil Kumar, and J. S. Rana

Received: 18 February 2010 / Accepted: 5 March 2010 © The Korean Society for Biotechnology and Bioengineering and Springer 2010

Abstract A tannase (E.C. 3.1.1.20) producing fungal 1. Introduction

strain was isolated from soil and identified as Aspergillus heteromorphus MTCC 8818. Maximum tannase production was achieved on Czapek Dox minimal medium containing 1% tannic acid at a pH of 4.5 and 30°C after 48 h incubation. The crude enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography. Diethylaminoethyl-cellulose column chromatography led to an overall purification of 39.74-fold with a yield of 19.29%. Optimum temperature and pH for tannase activity were 50°C and 5.5 respectively. Metal ions such as Ca , Fe , Cu , and Cu increased tannase activity, whereas Hg , Na , K , Zn , Ag , Mg , and Cd acted as enzyme inhibitors. Various organic solvents such as isopropanol, isoamyl alcohol, benzene, methanol, ethanol, toluene, and glycerol also inhibited enzyme activity. Among the surfactants and chelators studied, Tween 20, Tween 80, Triton X100, EDTA, and 1, 10-o-phenanthrolein inhibited tannase activity, whereas sodium lauryl sulfate enhanced tannase activity at 1% (w/v). 2+

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Keywords: Aspergillus heteromorphus, tannase, DEAEcellulose, tannic acid, metal ion, organic solvent

Vinod Chhokar*, Vikas Beniwal, Anil Kumar, J. S. Rana Department of Bio and Nano Technology, Guru Jambheshwar University of Science and Technology, Hisar 125-001, India Tel: +91-1662-263-355; Fax: +91-1662-276-240 E-mail: [email protected] Seema, Raj Kumar Salar Department of Biotechnology, Chaudhary Devi Lal University, Sirsa 125055, India K. S. Nehra Department of Biotechnology, F.G.M. Govt. College, Adampur125-052, India

Tannin acyl hydrolase (E.C. 3.1.1.20), commonly known as tannase, catalyses the hydrolysis of ester and depside bonds in hydrolysable tannins such as tannic acid to release glucose and gallic acid [1,2]. It is extensively used in the food, feed, pharmaceutical beverage, brewing, and chemical industries. The major uses of tannase are in the production of gallic acid, pyrogallol, propyl gallate, in wine-making, beer-chill proofing, production of instant tea by solubilization of tea cream, and in the manufacture of coffeeflavored soft drinks. In addition, the enzyme is also used as a sensitive analytical probe for determining the structures of gallic acid esters, in the pretreatment of animal feed additives, in the leather industry, and when cleaning up highly polluting tannery effluent [3,4]. Tannase cleaves polyphenolics such as dihydrodimer crosslinks present in plant cell walls, which are essential for plant cell wall digestibility [5]. Tannases are universally distributed throughout the animal [6], plant [7], and microbial kingdoms [8]. However, enzymes from microbial sources have dominated applications in industrial sectors [9]. Although several reports exist on the production of tannase from microbial sources, the search continues for a new tannase source with more desirable properties for industrial application. In the present study, we provide the first report of the purification and characterization of tannase from Aspergillus heteromorphus.

2. Materials and Methods 2.1. Microorganism and culture maintenance

A tannase-producing fungus was isolated from a soil

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Biotechnology and Bioprocess Engineering 15: 793-799 (2010)

sample collected from Guru Jambheshwar University of Science and Technology, Hisar campus. The soil sample (1 g) was dissolved in 10 mL sterile distilled water. Of this, 1 mL was inoculated into potato dextrose broth containing 0.5% tannic acid and incubated at 30°C for 72 h. Aliquots of this inoculum were plated on agar plates containing 0.2% tannic acid. Fungal colonies capable of forming a clear zone around the mycelium due to tannic acid hydrolysis were selected and purified. The selected strain was then morphologically identified as A. heteromorphus MTCC 8818 by the Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India. The strain was maintained on potato dextrose agar slants with regular transfers in a refrigerator at 4°C.

reagent was added to each tube and was maintained for 15 min at room temperature for color stabilization. The absorbance was read at 530 nm using a T80 uv/vis spectrophotometer. One unit of enzyme activity was defined as the amount of enzyme required to hydrolyze 1 mM of tannic acid in 1 min under assay conditions and was expressed as U/mL.

2.2. Preparation of the spore inoculum

2.6.1. Ammonium sulfate fractionation and dialysis A volume of 100 mL of crude tannase was taken, and the required quantity of ammonium sulfate was added slowly under constant stirring at 4°C to obtain various saturation levels (50 ~ 90%). The precipitated proteins were separated by centrifugation at 3,000 × g for 20 min at 4°C and dissolved in a minimum amount of 0.02 M acetate buffer (pH 5.5). The separated proteins were dialyzed against acetate buffer (0.02 M, pH 5.5) for 24 h at 4°C.

2.3. Fermentation medium

2.6.2. DEAE-cellulose column chromatography A chromatography column was packed with DEAE-cellulose to a bed size of 2.5 × 10 cm and was equilibrated with 0.02 M acetate buffer (pH 5.5). The enzyme preparation obtained after dialysis was loaded on to the column and eluted first with 0.02 M acetate buffer (pH 5.5) followed by a linear gradient of 0 ~ 0.5 N NaCl in the same buffer (30 mL) at a flow rate of 5 mL/h. Two mL fractions were collected and analyzed for enzyme activity. Fractions with high enzyme activity were pooled together and used for further experiments.

A fungal spore inoculum was prepared by adding 2.5 mL of sterile distilled water containing 0.1% Tween 80 to a fully sporulated culture. The spores were dislodged using a sterile inoculation loop under strict aseptic conditions, and the number of spores in the suspension was counted using a Neubauer chamber. Finally, 1 mL of the prepared spore suspension was used as the inoculum, with a concentration of 5 × 109 spores. For the fermentation process, a 250 mL Erlenmeyer flask with 50 mL of Czapek Dox minimal medium [10] containing (g/L): NaNO3, 6; KH2PO4, 1.52; KCl, 0.52; MgSO4·7H2O, 0.52; FeSO4·7H2O, 0.01; and ZnSO4·7H2O, 0.01; was employed. The pH was adjusted to 5.0 and the medium was sterilized at 121°C for 15 min. A tannic acid solution was prepared separately, and its pH was adjusted to 4.5 with 0.1 M NaOH. The solution was then sterilized by filtering it through a sterile membrane (pore size 0.2 mm) and added to the medium resulting in a final tannic acid concentration of 1%. Flasks were incubated at 30°C in an incubator shaker at 150 rpm for 48 h. After the desired incubation period, the biomass was harvested by filtration through Whatman no.1 filter paper, and the cell-free culture broth was assayed for extracellular tannase activity.

2.4. Tannase assay

Tannase activity was determined colorimetrically using the method of Mondal et al. [11] A reaction mixture containing 0.3 mL of tannic acid (0.5% in 0.2 M sodium acetate buffer, pH 5.5) and 0.1 mL of enzyme was incubated at 50°C for 20 min. The enzymatic reaction was stopped by adding 3 mL BSA solution, which precipitated the remaining tannic acid. The mixture was centrifuged (5000 × g, 10 min), and the resulting precipitate was dissolved in 3 mL SDS-triethanolamine solution. One mL of FeCl3

2.5. Protein concentration assay

The protein concentration was determined by the Bradford method [12] using bovine serum albumin as the standard.

2.6. Purification and characterization

2.7. Effect of temperature

To determine the effect of temperature on tannase activity, the reaction was conducted at different temperatures ranging from 20 to 70°C. Purified enzyme and substrate were incubated at various temperatures, and the enzyme assay was performed as described earlier.

2.8. Effect of pH

To study the effect of pH, tannase activity was measured at pH ranging from 3.5 to 6 using acetate buffer for pH 3.5 ~ 5.5 and phosphate buffer for pH 6.0.

2.9. Effect of metal ions, organic solvents, inhibitors, surfactants, and chelators on tannase activity An enzyme solution containing different concentrations of

Purification and Characterization of Extracellular Tannin Acyl Hydrolase from Aspergillus heteromorphus MTCC 8818

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metal ions, organic solvents, inhibitors, surfactants, and chelators was incubated at 50ºC for 20 min and assayed for enzyme activity. 3. Results and Discussion

3.1. Effect of incubation pH on tannase production

The effect of pH on tannase production was studied by varying the pH of the medium from 3.0 to 6.0 (Fig. 1). Initially there was a gradual increase in enzyme production with the increase in pH. Maximum enzyme production was obtained at a pH of 4.5 (1.06 U/mL). After that, a constant decline in enzyme production was observed until pH 6.0. Kumar et al. [13] and Kar & Benerjee [14] reported that a pH of 4.5 is optimum for enzyme production by A. ruber and Rhizopus oryzae, respectively.

3.2. Effect of incubation time and temperature on tannase production

The fermentation was conducted at 25, 30, 35, and 40°C in a rotary incubator shaker, and the samples were withdrawn after 12, 24, 36, 48, 60, and 72 h of incubation (Fig. 2). Enzyme production was low at 25°C and maximum at 30°C (1.45 U/mL). Thereafter, enzyme production declined with increases in incubation temperature. At 30°C, tannase production was maximum after 48 h of incubation but

Fig. 1.

Effect of pH on tannase production in Aspergillus hetero-

morphus.

Effect of incubation time and temperature on tannase production in Aspergillus heteromorphus. Fig.

2.

constantly declined until 72 h. Thus, an incubation temperature of 30°C for 48 h was optimum for enzyme production. An optimum temperature of approximately 30°C was reported for tannase production in A. rubber [13], Paecilomyces variotii [15], A. japonicus [16], and A. niger GH1 [17].

3.3. Purification of tannase

The results of a representative purification experiment are summarized in Table 1. Protein content and enzyme activity were determined after each purification step, and crude tannase was concentrated by ammonium sulfate fractionation. Fractional precipitation with 80% ammonium sulfate removed some of the nonenzymatic proteins, and 25.41% of total tannase was recovered. A specific activity of 1.07 U/mg protein was obtained, which was a two-fold enhancement in activity when compared to the crude enzyme. The elution profile of tannase from the DEAE-cellulose column showed two peaks. Maximum tannase activity was found in the first peak. The active fractions were pooled together. DEAE-cellulose column chromatography led to an overall purification of 39.74 fold with a yield of 19.29% (Table 1). Tannase from P. variotii and A. awamori were purified using the same techniques with yields of 3 and 13.5% respectively [15,3]. Tannase from A. niger GH1 was also purified to apparent homogeneity by ultrafiltration, anion-exchange chromatography, and gel filtration, which led to a purified enzyme with a specific activity of 238.14 IU/mg protein with a final yield of 0.3% and a 46-fold

Purification table of tannase from Aspergillus heteromorphus Total activity Total protein (U) (mg) Crude 119 206.6 Ammonium sulphate purification 30.24 28.32 DEAE-cellulose chromatography 22.96 1.01 Table 1.

Specific activity (U/mg) 0.575 1.07 22.85

Purification (fold) 1 1.85 39.74

Yield (%) 100 25.41 19.29

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activity, the temperature was varied from 25 to 80°C. With an increase in temperature, the tannase activity increased, and optimum activity (4.06 U/mL) was recorded at 50°C (Fig. 4). Further increases in temperature resulted in a decrease in activity. The optimum temperature for tannase activity was 50°C, which was similar to that obtained for P. variable tannase [20]. However, tannases from A. niger van Tieghem [23] and B. cereus KBR 9 [19] have a temperature optima between 45 and 60°C. A temperature optimum of 60 ~ 70°C has also been reported for A. niger tannase [24]. Fig. 3.

Effect of pH on tannase activity.

increase at purification [17].

3.4. Effect of pH on enzyme activity

Effect of pH (Fig. 3) on tannase activity showed that the activity was extremely low at pH 3.0 (1.74 U/mL). Enzyme activity increased gradually with the increase in pH, and the maximum was observed at pH 5.5 (3.89 U/mL). A further increase in pH resulted in a decrease in tannase activity. Most of the tannases are active in a pH range of 4.5 ~ 6.0. For example, the optimum pH for tannase activity in A. niger IIT25A5 [18] and Bacillus cereus KBR9 [19] is 4.5. Whereas its pH 5.0 for Penicillium variable [20], pH 5.5 for A. ruber [13], and a range from 5 to 7 in P. variotii [21]. The effect of pH on enzyme activity is determined by the nature of the amino acids at the active site, which undergoes protonation and deprotonation, and by the conformational changes induced by the ionization of other amino acids [22].

3.5. Effect of temperature on tannase activity

To evaluate the effect of temperature on purified tannase

3.6. Effect of organic solvents on enzyme activity

Enzyme activity is influenced by various organic solvents. Organic solvents can be advantageous in various industrial enzymatic processes, and the use of organic solvents can increase the solubility of non-polar substrates, increase the thermal stability of enzymes, decrease water-dependent side reactions, or eliminate microbial contamination [25]. To determine the effect of organic solvents on tannase from A. heteromorphus, various organic solvents (Table 2) were used at different concentrations (1 and 5%). Isopropanol, isoamyl alcohol, benzene, methanol, ethanol, toluene, and glycerol stimulated tannase activity at a concentration of 1%. At higher concentrations, these solvents resulted in inhibition. At lower concentrations, the solvents caused an increase in enzyme activity by making the buried disulfides accessible. At a higher concentration, organic solvents might have caused denaturation through a conformational change in the enzyme tertiary structure. Saborowski et al. [25] studied the effect of organic solvents on endopeptidases and found that the protease and chymotrypsin activity were slightly elevated at 5 and 10% concentrations of acetone, isopropanol, methanol, and ethanol respectively. In contrast, trypsin activity rose concomitantly by 8-fold at a concentration of 40% isopropanol. . Effect of organic solvents on tannase activity Relative activity (%) Organic solvent 1% 5% a 100 100 Control Isopropanol 106.6 96.3 Isoamyl alcohol 114.0 100.8 Benzene 116.7 98.0 Methanol 103.2 95.8 Ethanol 104.4 83.1 Glycerol 118.2 110.5 Butanol 103.4 115.5 DMSO 110.8 122.4 Toluene 119.4 109.8

Table 2

Fig. 4.

Effect of temperature on tannase activity.

a

pH 5.5, 50°C, and 20 min incubation.

Purification and Characterization of Extracellular Tannin Acyl Hydrolase from Aspergillus heteromorphus MTCC 8818

3.7. Effect of metal ions on enzyme activity

The effect of various metal ions such as Ba , Ca , Hg , Na , K , Mn , Fe , Fe , Zn , Ag , Co , Cu , Cu , Mg , and Cd on tannase activity was examined at different concentrations (Table 3). Among all the metal ions tested, Ca , Fe , Na , Cu , and Cu were enzyme activators. The most effective activator was Ca , which increased tannase activity by 12.8% at 4 mM (data not shown) but a further increase in the Ca concentration (5 mM) inhibited enzyme activity by 6%. Fe , Cu , and Cu also showed stimulating effects on tannase activity at 1 mM concentration, while further increase in ion concentration caused decrease in enzyme activity. Hg , K , Zn , Ag , Mg , and Cd acted as enzyme inhibitors and decreased tannase activity with increasing concentrations. Tannase activity was strongly inhibited (44%) by Hg (5 mM). The inhibition of enzyme activity by Hg ions may have been due to its interaction with sulfhydryl groups, suggesting that an important cysteine residue is in or close to the enzyme active site [26]. Ba had no significant effect on tannase activity up to 3 mM (data not shown), but a slight inhibitory effect was observed at higher concentrations. Co and Mn showed an inhibitory effect at 1 mM and slight variations were observed at higher concentrations. The effect of metal ions on tannase activity was studied by Kar et al. [27]. They observed that 1.0 mM Mg or Hg activated tannase activity. In contrast, Ba , Ca , Zn , Hg , and Ag inhibited tannase activity at 1.0 mM. Mukherjee and Banerjee [4] found that Mg at low concentrations increases tannase activity, whereas it is inhibited maximally by Hg followed by Fe , Zn , and Ba . Sabu 1+

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Effect of metal ions on tannase activity Relative activity (%) Salt 1 mM 5 mM a 100 100 Control 105.6 94.0 CaCl2 HgCl2 71.2 56.0 BaCl2 100.0 91.1 CdCl2 102.4 80.2 ZnSO4 88.4 80.0 FeSO4 107.6 94.0 106.2 74.1 CuSO4(Cu2+) CuSO4(Cu1+) 108.6 74.6 AgNO3 93.0 57.0 MnCl2 79.0 96.3 KCl 97.5 73.3 NaCl 100.7 89.1 CoCl2 87.6 90.6 MgCl2 89.0 77.5

Table 3.

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et al. [22] also studied the effect of metal ions on tannase from A. niger ATCC 16620 and found that adding metal

ions such as Zn , Mn , Cu , Ca , Mg , and Fe inhibited enzyme activity. Kasieczka-Burnecka et al. [28] have recently reported the inhibitory effect of Zn , Cu , K , Cd , Ag , Fe , Mn , Co , Hg , Pb , and Sn on tannase from Verticillium sp. Metals stabilize and activate enzymes. More than 75% of enzymes require the presence of metal ion activators to express their full catalytic activity. Enzyme activation by metal ions is important industrially for achieving maximal catalytic efficiency. At low concentrations, metal ions act as cofactors for many enzymes, thereby increasing enzyme catalytic activity, whereas at high concentrations catalytic activity is reduced. This may be due to partial enzyme denaturation in the presence of excessive free ions in the enzyme extract. Ca and Fe increased and stabilized tannase activity possibly due to metal ion activation. In contrast, Hg , Mn , Zn , Ag , Co , and Mg inhibited tannase activity. This may have been due to non specific binding or enzyme aggregation. Moreover, the disulfide bond could also be hydrolytically degraded by the action of Ag and Hg . 2+

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3.8. Effect of inhibitors on enzyme activity

Various inhibitors such as sodium azide, benzoic acid, sodium bisulfite, gallic acid, propyl gallate, pyrogallol, iodoacetamide, phenyl methyl sulfonyl fluoride (PMSF), sodium thioglycolate, n-bromosuccinic acid, 4-aminobenzoic acid, and 2-mercaptoethanol were tested at 1 mM concentration each for their effect on tannase activity (Table 4). 2mercaptoethanol was the most potent inhibitor of tannase, causing a 40.6% loss in original tannase activity, followed by n-bromosuccinic acid (26.1%), sodium bisulfate (18.7%), Effect of inhibitors on tannase activity Inhibitors Relative activity (%) a 100 Control Sodium azide 82.8 Benzoic acid 83.1 Sodium bisulfite 81.3 Gallic acid 82.1 Propyl gallate 85.1 Pyrogallol 95.0 Iodoacetamide 100.0 PMSF 86.1 Sodium thioglycolate 83.1 n-Bromosuccinic acid 73.9 4-aminobenzoic acid 81.9 B-mercaptoethanol 59.4

Table 4.

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4-aminobenzoic acid (18.1%), gallic acid (17.9%), sodium azide (17.2%), benzoic acid (16.9%), sodium thioglycolate (16.9%), propyl gallate (14.9%), pyrogallol (5%), and PMSF (13.9%). Iodoacetamide did not inhibit tannase activity. Our results are in agreement with Battestin and Macedo [15], who reported that tannase from P. variotii was inhibited by sodium bisulfite, 2-mercaptoethanol, 4-aminobenzoic acid, sodium azide, n-bromosuccinimide, and cysteine. Strong inhibition of tannase by 2-mercaptoethanol was also reported by Kar et al. [27]. Inhibition studies on P. variable tannase [20] showed that the enzyme was inhibited by PMSF, and 2-mercaptomethanol and that it retained 28 and 39.6% of its residual activity, suggesting it is a serine hydrolase. Moreover, N-ethylmaleimide showed strong inhibition with only 7% residual activity, while 1, 10 ophenanthrolien was a mild inhibitor. Inhibition studies primarily provide an insight into the nature of an enzyme, its cofactor requirements, and the nature of the active site. Inhibition by n-bromosuccinic acid indicated that tryptophan residues play an important role in maintaining the active conformation of the enzyme. Inhibition of tannase by 2-mercaptoethanol suggests the presence of sulfur-containing amino acids at the enzyme active site. 3.9.

Effect

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Surfactants play a role in enzyme catalytic activity [27]. The data in Table 5 show that tannase activity was inhibited by Tween 20 (30.8%) and Tween 80 (27.4%). Inhibition by Tween 20 and Tween 80 agrees with the results of Battestin and Macedo [15]. This inhibition may be the result of a combined effect of factors such as a reduction in the hydrophobic interactions that play a crucial role in holding the tertiary protein structure together and a direct interaction with the protein molecule. It was also observed that Triton X-100 caused a decrease in tannase activity by 21.9 at concentrations of 1% (v/v). These results are in agreement with the findings of Kar et al. [27]. Surprisingly, sodium lauryl sulfate (SLS) increased tannase activity by Effect of surfactants and chelators on tannase activity Inhibitors Concentration Relative activity (%) 100 Control a Tween 20 1 % (v/v) 69.2 Tween 80 1 % (v/v) 72.6 Sodium lauryl sulfate 1 % (v/v) 145.0 Triton X-100 1 % (v/v) 78.1 EDTA 1 mM 65.0 1,10-o-phenanthrolein 1 mM 58.0 Table 5.

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45%. The possible reason is that SLS at higher concentrations apparently enhanced changes in the enzyme itself, resulting in greater catalytic activity [29]. However, an inhibitory effect of SLS on tannase was reported earlier [4,28,30]. EDTA and o-phenanthroline at 1.0 mM inhibited tannase activity by 35 and 42% respectively. Tannase from A. niger was reportedly inactivated by o-phenanthroline and EDTA [31], while the tannase from Verticillium sp. P9 is strongly inhibited [28]. Tannase from A. oryzae is completely inactivated by EDTA [32], whereas no inhibition was observed with the chelating agents EDTA and ophenanthroline in the case of tannase from A. flavus [33]. Yeast tannase is also not inhibited by EDTA [34]. 4. Conclusion

This is the first report on the production, purification, and characterization of tannase from A. heteromorphus. Maximum production of the enzyme was observed on Czapek Dox minimal medium containing 1% tannic acid with a pH of 4.5 at 30°C after 48 h incubation. The enzyme was purified to homogeneity by ammonium sulfate precipitation and DEAE-cellulose column chromatography. The optimum conditions of temperature and pH were investigated. Tannase from this new isolate exhibited optimum activity at pH 5.5 and 50°C. The effect of additives such as metal ions, surfactants, reducing agents, chelators, organic solvents, and inhibitors was also studied. References

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