Aspergillus foetidus BY AQUEOUS TWO PHASE

Transcription

1 33 CHAPTER 3 PARTIAL PURIFICATION OF TANNASE FROM Aspergillus foetidus BY AQUEOUS TWO PHASE EXTRACTION AND ITS CHARACTERIZATION 3.1 INTRODUCTION Partial purification of proteins in general and tannase in particular is carried out by various techniques such as salt precipitation (Mahendran et al 2006), solvent precipitation (Gargi and Rintu 2006) and ultra filtration (Barthomeuf et al 1994). Partial purification by aqueous two phase extraction (ATPE) has many advantages and they include negligible mass transfer resistance, amenability to continuous processing, a high capacity for handling of solids and lower investment cost (Subhash and Prashant 2003). Tannase is utilized in a number of industrial applications including manufacture of instant tea, wine and gallic acid (Ascension et al 2003) and solubilization of tea cream in instant tea processing (Nagalakshmi et al 1985). One of the major commercial applications of tannase is the hydrolysis of tannins to gallic acid, a key intermediate required for the synthesis of an antibiotic drug, trimethoprim (Hadi et al 1994). As the range of application of this enzyme is wide, there is always a scope for novel tannase with better characteristics, which may be suitable for diverse applications. Studies on the purification and characterization of tannase greatly help in finding out suitable applications. Hence, the objective of this study

2 34 involves partial purification of tannase from A.foetidus employing ATPE and characterization of the partially purified tannase for its stability against ph, temperature, salts, surfactants and detergents. 3.2 MATERIALS AND METHODS Microorganism and Growth Conditions A new strain of Aspergillus foetidus MTCC 6322 isolated from tannery sludge (Common Effluent Treatment Plant, Chrompet, Chennai) was used in the study. The organism was identified at the Institute of Microbial Technology, Chandigarh, India and it was maintained at 4 o C in Czapek-Dox agar slants containing 0.5% tannic acid. 100 µl of the spore suspension containing 6.0x10 7 CFU/ml were used as the seed inoculum. Fermentation was carried out in 250 ml Erlenmeyer flasks containing 50.0 ml of production medium by batch mode with agitation of 150 rpm at 37 o C. The production medium had the following composition (g/l): Tannic acid ; Fructose - 7.5; NaNO ; NaH 2 PO 4 2H 2 O ; KH 2 PO 4-2.0; MgSO 4. 7H 2 O - 0.3; FeSO 4.7H 2 O ; MnSO 4.7H 2 O ; ZnSO 4.7H 2 O , CaCl 2.2H 2 O The culture medium except tannic acid was sterilized by autoclaving at 121 C for 20 min. After the culture medium was cooled, tannic acid (filtered through 0.22 µ filter) was added under sterile condition Tannase Assay The tannase activity was assayed according to Lekha and Lonsane (1994). To state briefly, 3.0 ml of tannic acid (0.004%), dissolved in 20 mm acetate buffer, ph 5.0 was taken in a 3.0 ml cuvette and the reaction was initiated by the addition of 0.1 ml of culture supernatant. The absorbance

3 35 change at 310 nm was followed continuously for 5.0 min. One unit (U) of tannase activity was defined as the amount of enzyme which gave a decrease of 0.01 absorbance per min at 310 nm. The tannase activity was expressed as U/ml/min Protein Content (Lowry et al 1951) Proteins react with copper at an alkaline ph to form copper-protein complex. This complex, when treated with phosphomolybdic-tungstic reagent (Folin-ciocalteau), forms blue colour that is measured at 660 nm in a Shimadzu UV-VIS 2401 spectrophotometer. Procedure: To 100µl of protein samples was made upto 1.0 ml with water. To this, 5 ml of freshly prepared alkaline copper sulphate solution (mixture of 50 ml of 2% sodium carbonate in 0.1N sodium hydroxide and 1.0 ml of 0.5% copper sulphate penta hydrate in 1% potassium sodium tartarate was added and kept at room temperature (28 ± 2 C) for 20 min. The resultant blue colour was read at 660 nm Partial Purification of Tannase Partial purification of tannase was carried out by ammonium sulphate precipitation as well as aqueous two phase extraction. To the crude broth, ammonium sulphate (80% saturation) was added and equilibrated for 30 min at 4 o C with stirring and the suspension was centrifuged at 10,000 rpm for 20 min at 4 º C. The resultant precipitate was suspended in 20mM acetate buffer, ph 6.0 and analyzed for tannase activity. For aqueous two phase extraction, culture filtrate was mixed with 30% polyethylene glycol (PEG 6000), 5% ammonium sulphate and 2.5% sodium chloride and then kept at 4 o C which resulted in tannase partitioning

4 36 selectively to the lower aqueous phase. Initially, PEG of different molecular weights ( ) were tried and PEG 6000 was selected for further study. Subsequently, different concentrations of PEG 6000 (15%-30%) were tested for finding the optimum concentration for bioseparation of tannase Characterization of Partially Purified Tannase ph optimum and ph stability Tannase activity was determined with the use of tannic acid as substrate employing different buffers viz M acetate (ph ); 0.02M phosphate (ph 6-8); 0.02 M carbonate (ph 9-10). The activity was estimated as described in Tannase assay. 5.0 ml aliquots of partially purified enzyme were mixed with an equal amount of 0.02 M buffer of different ph values viz. ph and incubated at 30 o C for 1 h. The residual activity was then determined Optimum temperature and temperature stability 3.0 ml of 0.004% Tannic acid in 0.02 M acetate buffer, ph 6.0 was incubated with 100 µl of enzyme solution at various temperatures (10 o -70 o C) for 5 min. After the incubation period, the enzyme activity was estimated. 5.0 ml aliquots of partially purified enzyme were mixed with an equal amount of 0.02 M acetate buffer, ph 6.0 and incubated at different temperatures from 10 o -70 o C for 1 h. The residual activity was then assayed Salt stability Stability of tannase towards various salts was determined by incubating 5.0 ml of partially purified enzyme with equal volume of salt

5 37 solution (1mM) in 0.02M acetate buffer, ph 6.0 for 1 h at 30 o C and the residual activity for each salt was determined Detergent stability Detergent stability was evaluated by incubating 5.0 ml of the partially purified enzyme with equal volume of detergent solution (7 mg/ml of respective detergent) in 0.02 M acetate buffer, ph 6.0 for 1 h at 30 o C and the residual activity for each detergent was measured. 3.3 RESULTS AND DISCUSSION Partial Purification of Tannase The crude tannase when precipitated using ammonium sulphate 80% saturation showed 1.7 fold purification with 67% yield of tannase activity (Table 3.1). Tannase, when subjected to aqueous two phase extraction, selectively partitioned into the lower phase when 25% polyethylene glycol (molecular weight 6000) with 9.3% ammonium sulphate and 2.5% sodium chloride were used. When varying molecular weights of 30 % PEG ( ) were tried, PEG with molecular weight of 6000 was found to have a yield of 77.3% with 2.62 fold purification (Table 3.1). Subsequently, when different concentrations of PEG 6000 were tried, 25% PEG 6000 was found to show a yield of 82.0% with 2.7 fold purification (Table 3.1). A rapid procedure for partial purification of extracellular tannase using a combination of 0.1% tannic acid and 0.1% PEG-6000 was reported by Gupta et al (1997). The specific activity as well as yield of partially purified tannase obtained by ATPE were higher when compared to ammonium sulphate precipitated enzyme.

6 38 Table 3.1 Partial purification of tannase by ammonium sulphate precipitation and aqueous two phase extraction Fraction Vol (ml) Activity (U) Total Activity (U) Protein (mg) Total Protein (mg) Specific activity (U/mg) Yield (%) PF Crude extract (NH 4 ) 2 SO 4 Precipitation- 80% saturation Aqueous two phase Extraction - 30%PEG (MW 4000) % PEG(MW6000) % PEG (MW8000) % PEG (MW9000) Aqueous two phase Extraction - 15% PEG (MW6000) % PEG (MW6000) % PEG (MW6000) % PEG (MW6000) PEG- Polyethylene glycol; PF- Purification fold; % Yield = Total activity in the fraction / Total activity in the crude extract 100

7 39 Partial purification studies showed that the recovery of tannase was low when ammonium sulphate precipitation was resorted to. ATPE of tannase using polyethylene glycol resulted in an yield of 82.0% whereas tannase from Aspergillus japonicus, when precipitated with 0.1% tannic acid and 0.1% PEG, showed 50.3% yield which was reported to be ph dependent (Gupta et al 1997). It was shown earlier that when tannase from Paecilomyces variotii was precipitated using 50% saturation of ammonium sulphate, some of the nonenzymatic proteins were shown to be removed and at 70% saturation of ammonium sulphate, a yield of 78.7% recovery was reported (Mahendran et al 2006) Characterization of Partially Purified Tannase ph optimum and ph stability Tannase activity as a function of ph was studied using tannic acid as substrate (Figure 3.1). The tannase showed maximum activity at ph 6.0. The enzyme was active in the acidic range; but, its activity decreased with increase in ph. It was reported earlier that the tannase from Aspergillus awamori nakazawa exhibited ph optimum at 5.0 (Mahapatra et al 2005) and tannase from Bacillus cereus showed optimum activity at ph 4.5 (Keshab et al 2001). Similarly, the extra and intracellular tannase from Aspergillus aculeatus were noticed to have the same ph optimum at 5.0 (Banerjee et al 2001). The enzyme was stable in the range of whereas at ph 8.0, the stability was around 70% (Figure 3.1). Similarly, tannase from Bacillus cereus remained stable between ph (Keshab et al 2001), whereas for Aspergillus aculeatus tannase, it was reported to be between ph (Banerjee et al 2001). The observed ph stability of tannase from A.foetidus at

8 40 acidic ph could be exploited in various industrial applications such as tea cream solubilization Tannase activity (Units) % Residual activity ph 0 ph optima ph stability Figure 3.1 ph optima and ph stability of tannase from A. foetidus Optimum temperature and temperature stability To evaluate the effect of different temperatures on the activity of tannase, the temperature range was varied from 30 o to 55 o C. With a rise in temperature, the tannase activity was observed to increase and optimum activity was recorded at 50 o C (Figure 3.2). An optimum temperature of 35 o C was reported for tannase from Aspergillus awamori nakazawa (Mahapatra et al 2005) and in case of Penicillium variable (Sharma et al 2007), the optimum temperature was at 50 o C. In this study, with a further increase in temperature, there was a decrease in activity. This was in good agreement

9 41 with the results obtained earlier for tannase from Bacillus cereus (Keshab et al 2001) Tannase activity ( Units) % Residual activity Temperature ( º C) 0 Temperature optima Temperature stability Figure 3.2 Temperature optima and temperature stability of tannase from A. foetidus Though optimal stability of tannase was found to be around 30 o C, good stability was observed in the temperature range of 15 o - 40 o C. Above 40 ºC, there was a significant reduction in thermal stability (Figure 3.2). The observation of this study showing thermal stability in the range of 15 o - 40 o C concurred with an earlier report for tannase from Paecilomyces variotii (Mahendran et al 2006). It was also reported earlier that tannase from Aspergillus aculeatus DBF9 showed good stability at 50ºC (Banerjee et al 2001).

10 Salt stability The tannase of this study was highly stable in presence of salts such as CuSO 4, KH 2 PO 4, MnSO 4, CaCl 2, and NaCl. But, there was a slight reduction in activity in presence of MgSO 4 and ZnSO 4 (Figure 3.3). An earlier report showed that while MgCl 2 and HgCl 2 activated the tannase activity from Rhizopus oryzae, ZnCl 2, HgCl 2, BaCl 2, CaCl 2, and AgNO 3 were shown to inhibit the activity (Kar et al 2003). Tannase from Aspergillus aculeatus DBF9 showed good stability at high NaCl (Banerjee et al 2001). Activation of enzyme reactions by metal ions was important industrially to achieve maximal catalytic efficiency % Residual activity Control MgSO 4 CaCl 2 CuSO 4 ZnSO 4 MnSO 4 NaCl KH 2 PO 4 Salts Figure 3.3 Salt stability of tannase from A. foetidus

11 Detergent stability Tannase from A. foetidus exhibited considerable stability in the presence of surfactants such as sodium dedecyl sulphate (SDS) and commercial detergents such as Sunlight, Rinshakthi and Wheel and a considerable loss in activity was observed in presence of Triton X100 and Tween 60 (Figure 3.4). In contrast, tannase from Rhizopus oryzae was reported to have decreased stability in presence of sodium lauryl sulphate (Kar et al 2003) % Residual activity Control Tritonx100 Tween60 SDS Sunlight Rinshakthi Wheel Detergents Detergents Figure 3.4 Detergent stability of tannase from A. foetidus

12 44 Thus, this study showed that purification by ATPE was better than that of ammonium sulphate saturation. Tannase from A.foetidus exhibited good ph and thermal stability as well as stability towards salts and detergents. 3.4 CONCLUSION Tannase, produced by the fungal strain Aspergillus foetidus MTCC 6322 by submerged culture fermentation, was partially purified by Ammonium sulphate precipitation and Aqueous Two Phase Extraction (ATPE). ATPE extraction was found to be a better purification strategy and it resulted in a purification fold of 2.70 and yield of 82.0%. A concentration of 25% polyethylene glycol (Molecular weight, 6000) was found to be optimal for extraction of tannase by ATPE. This extracellular tannase exhibited good stability towards ph, temperature, salts, surfactants and commercial detergents indicating its suitability for commercial utilization.