Chapter 5. Purification and characterization of an alkaline cellulase from
5.2 Materials and Methods
5.2.5. Purification
5.2.5.1 Ammonium sulphate precipitation
All purification steps were performed at 4°C. After 48 h of cultivation at 39°C with shaking (121 rpm) the culture broth was centrifuged at 10,000g for 15 min.
Enzyme in the cell-free supernatant portion of the culture was precipitated by addition of ammonium sulphate ((NH4)2SO4) with constant stirring. Ammonium sulphate
fractions of 0-40% and 40-80% (w/v) were collected by centrifugation at 10,000g for 30 min and the enzyme pellet obtained was dissolved in minimal volume of 50 mM Tris-HCl (pH 8.0) buffer. The dissolved pellet was dialyzed against the same buffer with three changes. Dialysis was performed using molecular weight cut off membrane of 5 kDa (HiMedia, India).
5.2.5.2 Ion exchange chromatography
The enzyme extract (dialysate) was further purified using Fast Protein Liquid Chromatography, FPLC (GE Healthcare). 20 mL of the dialysate (0.5 mg/mL, specific activity 1.16 U/mg) was loaded on to diethylaminoethyl (DEAE)-Sepharose column (1.5 x 20 cm). Before loading the sample, the column was pre-equilibrated with 50 mM Tris-HCl buffer pH 8.0. The column was washed with two column volume of the same buffer and the adsorbed protein was eluted with a linear gradient of 0-0.8 M NaCl in equilibration buffer at a flow rate of 1 mL/min. Each fraction of 3 mL was collected for estimation of protein concentration (absorbance 280 nm) and CMCase activity (U/mL). The active fractions containing cellulase activities were pooled and stored at 4ºC for further analysis. SDS-PAGE of the active fractions were carried out to check the homogeneity of the enzyme and to determine its molecular weight. All the purification steps were performed at 4ºC.
5.2.5.3 Analysis of purification by SDS-PAGE
To check the purity of enzyme, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed following the method of Laemmli (1970). 10% (w/v) acrylamide for resolving gel and 4% (w/v) for stacking gel were
(w/v) SDS, 10% (w/v) glycerol, 5% (w/v) β-mercaptothanol and 0.05% (w/v) bromophenol blue. The purified enzyme sample was mixed with 5x loading dye buffer in the ratio of 4:1. The sample mixture was subjected to heat denaturation for 5 min and centrifuged at 12, 000 rpm for 1 min. The crude and column purified cellulase from different steps of purification were loaded on three identical 10% acrylamide gel and the electrophoresis was carried out using 1x running buffer (200 mM glycine, 0.1% SDS, 50 mM Tris-HCl pH 8.3) with a current of 2.5 mA per lane. The first two gels were loaded with same samples of crude supernatant and ammonium sulphate purified cellulase but stained with silver staining (Hames and Rickwood, 1996) and 0.25% (w/v) Coomassie brilliant blue (CBB) R-250, respectively. The third gel was loaded with column purified cellulase fractions and visualized by silver staining protocol. Molecular mass marker proteins used were phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa) and lysozyme (14.3 kDa) purchased from Bangalore Genei, India, was used as standard for SDS-PAGE.
5.2.5.4 Zymogram analysis
Zymogram analysis was performed according to the method of van Dyk et al.
(2010). It was carried out by using 0.2% CMC (w/v) polymerized within 12% SDS- PAGE gel. After electrophoresis, the lane containing the marker proteins was excised from rest of the gel and visualized by silver staining. Rest of the gel was immersed in 50 mM glycine-NaOH buffer (pH 9.2) containing 2.5% (v/v) Triton X-100 at shaking condition for 30 min with three changes to remove SDS. Subsequently, Triton X-100 was removed by washing the gel three times with 50 mM glycine-NaOH buffer (pH 9.2). The gel was then incubated in the same buffer at 45°C for 12 h. After incubation,
the gel was stained with 0.3% (w/v) Congo Red for 30 min and then destained with 1M NaCl until zone of clearance was visualized within the gel which confirmed cellulase activity. Gels were then counterstained with 1N HCl for better resolution.
The molecular weight of the purified cellulase was estimated based on its mobility calculated with standard calibration proteins using Hendricks plot (Hendrick and Smith, 1968).
5.2.6 Optimization of reaction conditions for maximum cellulase activity 5.2.6.1 Effect of temperature on enzyme activity and stability
The optimum temperature for assay of the enzyme was determined by incubating the mixture of 20 µL purified enzyme (1.16 U/mg, 0.5 mg/mL) with 80 µL of 2% (w/v) CMC in 50 mM glycine–NaOH (pH 9.2) buffer at different temperatures ranging from 20°C to 80°C for 10 min. 100 µ L of reaction mixture was analyzed for enzyme activity as described in Section 5.2.2.
The thermal stability of the enzyme was determined by incubating 50 µ L purified enzyme (1.16 U/mg; 0.5 mg/mL) in 50 mM glycine–NaOH buffer (pH 9.2) at various temperatures ranging from 20°C to 80°C for 30 min and 60 min. Aliquots of 20 µ L were withdrawn at different time intervals and the residual activity was determined according to the method described in Section 5.2.2.
5.2.6.2 Effect of pH on enzyme activity
The optimum pH of the purified cellulase was determined by incubating 100 µ L reaction mixture containing 20 µ L purified enzyme (1.16 U/mg, 0.5 mg/mL) and 80 µ L of 2% CMC in the presence of appropriate buffers at 45°C for 10 min. The
and acetic acid in two different ratios to obtain pH of 4.0 and 5.0; 50 mM sodium phosphate buffer prepared by mixing two different ratios of sodium dihydrogen phosphate and disodium hydrogen phosphate to obtain pH of 6.0 and 7.0; 50 mM Tris- HCl buffer prepared by mixing two different volumes of HCl to 50 mM Tris to obtain pH of 8.0 and 8.8; 50 mM glycine-NaOH buffer prepared by mixing different volumes of NaOH to 50 mM glycine to obtain pH of 9.0, 9.2, 9.5, 9.8, 10.0 and 11.0 and 50 mM KCl –NaOH buffer (pH 12.0).
5.2.6.3 Substrate specificity of the enzyme
The substrate specificity of the enzyme was determined by incubation of 20 µ L of purified enzyme (1.16 U/mg, 0.5 mg/mL) with 80 µ L 2% (w/v) of the cellulosic substrates: CMC, hydroxyethyl cellulose, lichenan, laminarin, avicel, steam exploded bagasse and barley β-glucan in 50 mM glycine-NaOH buffer (pH 9.2) at 45°C for 10 min. The total reaction mixture was 100 µL. The amount of reducing sugar produced by the reaction was measured by assay method as mentioned in Section 5.2.2.
5.2.6.4 Kinetic parameters
The enzyme-substrate (CMC) reaction was characterized in terms of Michaelis–Menten kinetic constants (Km and Vmax) (Michaelis and Menten, 1913) using the Lineweaver–Burk plot (Lineweaver and Burk, 1934) by assaying the enzyme at CMC concentration ranging from 0.1 mg/mL to 7.0 mg/mL in 50 mM glycine- NaOH buffer (pH 9.2) at 45°C for 10 min. The data were analyzed using GraphPad Prism software (GraphPad Software, Inc. USA).
Purification step Vol (mL)
Enzyme activity (U/mL)
Total units (U)
Protein (mg/mL)
Total protein
(mg)
Specific activity (U/mg)
Overall Yield
(%)
Fold purification
Crude 250 0.6 150 1.8 450 0.34 - -
80% (NH4)2SO4 20 0.58 11.6 0.5 10 1.16 7.73 3.4
DEAE- Sepharose
chromatography 42 0.2 8.4 0.06 2.52 3.33 5.6 9.8
5.3 Results and Discussion