Chapter 3. Biochemical and functional characterization of catalytic
3.2 Materials and Methods
3.2.3 Enzyme activity assay
The enzyme assay were initially performed in 100 µl of reaction mixture containing 20 mM sodium phosphate buffer pH 7.0 and 1% (w/v) substrate using 10 µl of purified enzyme (CtXynGH30 and CtXyn30A, 1.7 mg/ml each). The released reducing sugar was determined by the Nelson and Somogyi method (Nelson, 1944;
Somogyi, 1945). The reaction was carried out at 50°C for 15 min. After the analysis of the un-optimized assays, the results showed that the enzymes preferably hydrolyzed xylan based substrates. Afterwards, the assays were carried out using beechwood xylan under optimized conditions of pH 6.0 and temperature, 70°C.
3.2.3.1 Preparation of reagents for reducing sugar estimation Reagent A
Sodium carbonate anhydrous 6.25 g Sodium potassium tatarate 6.25 g Sodium bicarbonate 5.00 g Sodium sulphate anhydrous 50.0 g
The above mentioned components were dissolved in 100 ml of deionized water and the final volume was adjusted to 250 ml. The solution was filtered (Whatman No. 1) and stored at a temperature between 30-37°C.
Reagent B
Reagent B was prepared by dissolving 15 g of copper sulfate (CuSO4) in 50 ml deionized water and one or two drops of concentrated sulphuric acid was added to it.
The final volume was made up to 100 ml with deionized water and the solution was filtered (Whatman No. 1) and stored at room temperature.
Reagent C
Reagent C was prepared in two steps by under dark condition as it is light sensitive. First, 2.5 g of ammonium molybdate was dissolved in 45 ml of deionized water in 100 ml beaker and 2.1 ml of concentrated sulphuric acid was added to it. In another beaker 0.3 g of sodium arsenate was dissolved in 2.5 ml of deionized water.
Now, this solution was added to ammonium molybdate solution and the contents were mixed (total volume was around 50 ml). The solution was filtered (Whatman No. 1) under dark conditions and stored at 37°C. The solution was used after 24h incubation.
Reagent D
Reagent D was prepared by mixing reagent A and reagent B in the ratio 25:1.
Reagent D was always prepared freshly for use in the assay.
3.2.3.2 Generation of standard plot of D-xylose
The standard plot was prepared by varying the concentration of D-xylose from 10-250 µg/ml. The reaction volume was kept 100 µl with 20 mM sodium phosphate buffer pH 7.0. The reaction mixture containing buffer and D-xylose was incubated (in a 1.5 ml micro-centrifuge tube) at 70°C for 15 min and then 100 µl of solution D (Section 3.2.2.2) was added to it. The reaction mixture was then boiled for 20 min and cooled. 100 µl of solution C (Section 3.2.2.2) was added and the contents were mixed.
The colour development was rapid and completed after the evolution of carbon dioxide stopped. Then 700 µl of deionized water was added to make the final volume 1 ml. The absorbance at 500 nm wavelength (A500) was measured using UV-Visible spectrophotometer (Varian, Carry 100 Bio) against a buffer as blank. A standard plot of A500 versus D-xylose concentration (µg/ml) was generated and one absorbance unit
equivalent of D-xylose (µg/ml) was calculated. The one absorbance unit equivalent of
D-xylose (µg/ml) was converted in mg/ml for calculation of enzyme activity.
3.2.3.3 Calculation of enzyme activity
The activity of the enzyme was expressed as U/ml and the specific activity as U/mg of protein. One unit (U) of enzyme activity is defined as the amount of enzyme that liberates 1 µmole of reducing sugar per min. The enzyme activities of CtXynGH30 and CtXyn30A were calculated as described below,
∆A500 x C x V
Enzyme activity (U/ml) = = (µ mole/min/ml) 150 x t x v
where,
∆A500 = change in absorbance of the sample at 500 nm
C = 1 OD equivalent D-xylose concentration from standard plot V = volume of the reaction mixture (ml)
t = time of reaction (min)
150 = molecular weight of D-xylose
v = volume of the enzyme taken in assay (ml) for reducing sugar estimation.
3.2.3.4 Assay of CtXynGH30 and CtXyn30A with natural and synthetic p-nitrophenylglycoside substrates
CtXynGH30 and CtXyn30A were assayed against several natural polysaccharides, in order to determine the substrate specificity. The assays were performed in 100 µl reaction mixture using (1%, w/v) beechwood xylan and 10 µl of purified enzyme (CtXynGH30 or CtXyn30A, 1.7 mg/ml each) in 20 mM sodium phosphate buffer (pH 6.0) at 70°C for 10 minute following the procedure described in 3.2.1.3. The enzyme activity was determined by measuring the released reducing
sugar by the Nelson and Somogyi method (Nelson, 1944; Somogyi, 1945). The concentration of reducing sugar was estimated using a standard curve of xylose as CtXynGH30 and CtXyn30A showed maximum activity against the substrates with xylose residues in the main chain. One unit of activity was defined as the amount of enzyme which produced 1 µmole of xylose per min under the optimized condition of temperature and pH. The assay with synthetic p-nitrophenylglycoside substrates was performed in 1.0 ml 20 mM sodium phosphate buffer (pH 6.0) containing 50 µM of p- nitrophenylglycoside (pNP glycoside) and 10 µl of purified enzymes (CtXynGH30 or CtXyn30A, 1.7 mg/ml each) and incubated at 70°C for 10 min. The enzyme activity was determined by measuring the release of 4-nitrophenol (pNP) by monitoring the absorbance at 405 nm (A405) using a UV-Visible spectrophotometer (Varian, Cary 100 Bio) as described earlier (Ahmed et al., 2013). One unit of enzyme activity was defined as the amount of enzyme liberating 1 µmole of p-nitrophenol per min.
3.2.3.5 Substrate specificity of CtXynGH30 and CtXyn30A against a range of substrates
The activity of recombinant enzymes CtXynGH30 and CtXyn30A was analyzed against several natural polysaccharides. The polysaccharides were chosen on the basis of substrates reported so far for the family 30 and GH family 5 such as different xylans, celluloses, pectins and other hemicelluloses substrates. The assay was performed as mentioned in the section 3.2.3.4.