Chapter 6. Simultaneous Saccharification and Fermentation in shake flask at higher concentration of pretreated wild grass and
1. Quest for alternative energy
2.2 Materials and Methods
2.2.12 Analytical methods
2.2.12.1 Recombinant GH5 cellulase activity assay
The recombinant GH5 cellulase assay was performed by incubating the enzyme (10 µL) in a 100 µL reaction mixture containing 1% (w v-1) CMC in 20 mM sodium acetate buffer (pH 4.3) at 50°C for 10 min. The mixture was assessed for the released reducing sugars as reported earlier (Nelson, 1944; Somogyi, 1945). The amount of released reducing sugar was used to determine the enzyme activity. To 100 µL of reaction mixture containing the released reducing sugar, 100 µL of reagent D (as described in Section 2.2.12.3) was added. The solutions were mixed and heated for 20 min in the boiling water bath. After 20 min of boiling, the solution was ice cooled and 100 µL of reagent C (Section 2.2.12.3) was added. The colour developed rapidly with the evolution of carbon dioxide. The mixture was diluted by adding 700 µL water to make up the volume to 1 mL and the optical density (OD) was measured at 500 nm on a UV-Visible spectrophotometer (Varian, Cary 100 Bio). D-glucose in the range of 50-500 µg mL-1 was used for generating the standard plot as described in section 2.2.12.4.
2.2.12.2 Recombinant GH43 hemicellulase (α-L-arabinofuranosidase) activity assay GH43 hemicellulase (α-L-arabinofuranosidase) activity was determined by incubating 10 µL of enzyme in a 100 µL reaction mixture containing 1% (w v-1)
rye arabinoxylan in 100 mM sodium acetate buffer (pH 5.4) at 50°C for 10 min. The mixture was assessed for the released reducing sugars as reported earlier (Nelson, 1944; Somogyi, 1945) and as described in Section 2.2.12.3. The amount of released reducing sugar was used to calculate the enzyme activity. To 100 µL of reaction mixture, 100 µL of reagent D was added, mixed and heated for 20 min in boiling water bath. After cooling it to room temperature 100 µL of reagent C (Section 2.2.12.3) was added. The colour developed rapidly with the evolution of carbon dioxide. The mixture was diluted by adding 700 µL water to make up the volume to 1 mL and the OD was measured at 500 nm on a UV-Visible spectrophotometer (Varian, Cary 100 Bio). L-arabinosein the range of 10-250 µg mL-1 was used for generating the standard plot as described in Section 2.2.12.4.
2.2.12.3 Preparation of reagents for reducing sugar estimation Reagent A
Table 2.2.8 Chemical composition of Reagent A.
Constituents Concentration (g 100 mL-1)
Sodium carbonate anhydrous 6.25
Sodium potassium tartarate 6.25
Sodium bicarbonate 5.0
Sodium sulphate anhydrous 50.0
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 sulphate (CuSO4) in
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 24 h 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.
2.2.12.4 Generation of standard plot of D-glucose and L-arabinose
The standard plot of D-glucose was prepared by varying the concentration of
D-glucose from 50-500 µg mL-1. The reaction volume was kept to 100 µL with 50 mM sodium acetate buffer pH 4.3. The reaction mixture containing buffer and D-
glucose was incubated (in a 1.5 mL microcentrifuge tube) at 50°C for 15 min and then 100 µL of reagent D (Section 2.2.12.3) was added to it. The reaction mixture was then boiled for 20 min and cooled. 100 µL of reagent C (Section 2.2.12.3) was added and the contents were mixed. Then 700 µL of deionized water was added to make the final volume to 1 mL. The OD at 500 nm wavelength was measured using UV-Visible
spectrophotometer (Varian, Cary 100 Bio) against a buffer blank. A standard plot of OD versus D-glucose concentration (µg mL-1) was generated and 1 OD equivalent of D-glucose (µg mL-1) was calculated. The 1 OD equivalent of D-glucose (µg mL-1) was converted into mg mL-1 terms for calculation of enzyme activity.
The standard plot of L-arabinose was prepared by varying the concentration of L-arabinose from 10-250 µg mL-1. The reaction volume was kept to 100 µL with 50 mM sodium acetate buffer pH 5.4. The reaction mixture containing buffer and L- arabinose was incubated (in a 1.5 mL microcentrifuge tube) at 50°C for 15 min and then 100 µL of reagent D (Section 2.2.12.3) was added to it. The reaction mixture was then boiled for 20 min and cooled. 100 µL of reagent C (Section 2.2.12.3) was added and the contents were mixed. Then 700 µL of deionized water was added to make the final volume to 1 mL. The OD was measured at 500 nm wavelength using UV-Visible spectrophotometer (Varian, Cary 100 Bio) against a buffer blank. A standard plot of OD versus L-arabinose concentration (µg mL-1) was generated and 1 OD equivalent of L-arabinose (µg mL-1) was calculated. The 1 equivalent of L-arabinose (µg mL-1) was converted in mg mL-1 terms for calculation of enzyme activity.
2.2.12.5 Calculation of enzyme activity of recombinant GH5 cellulase and GH43 hemicellulase (α-L-arabinofuranosidase)
The activity of the enzyme was expressed as U mL-1 and the specific activity as U mg-1 of protein. One unit (U) of enzyme activity is defined as the amount of enzyme that liberates 1 µmole of reducing sugar (glucose or arabinose) per min. The enzyme activity of recombinant GH5 cellulase was calculated as described below,
∆A500 × C × V
Enzyme activity (U mL-1) = = (µ mole min-1 mL-1) 180 × t × v
where,
∆A500 = change in OD of the sample at 500 nm
C = 1 OD equivalent is D-glucose concentration from standard plot (µg mL-1) V = volume of the reaction mixture (mL)
t = time of reaction (min)
180 = molecular weight of D-glucose
v = volume of the enzyme taken in assay (mL) for reducing sugar estimation.
The enzyme activity of recombinant GH43 hemicellulase (α-L- arabinofuranosidase) was calculated as described below,
∆A500 × C × V
Enzyme activity (U mL-1) = = (µ mole min-1 mL-1) 150 × t × v
where,
∆A500 = change in OD of the sample at 500 nm
C = 1 OD equivalent is L-arabinose concentration from standard plot (µg mL-1) V = volume of the reaction mixture (mL)
t = time of reaction (min)
180 = molecular weight of L-arabinose
v = volume of the enzyme taken in assay (mL) for reducing sugar estimation.
2.2.12.6 Protein concentration estimation
The concentration of protein was detected by mixing the enzyme (10 L) with distilled water (90 L) in a total reaction volume (100 L) with final addition of 1 mL Bradford reagent (Bradford, 1976). The reaction mixture was kept at 25°C for 20 min and OD was determined using a UV-visible spectrophotometer (Perkin Elmer, Model lambda-45) at 595 nm. A Bovine serum albumin (BSA) standard curve was prepared by varying the concentration of BSA from 0.01-0.2 mg mL-1 to determine the protein concentration. The specific activity (U mg-1) of the cell free supernatant was calculated from enzyme activity and protein concentration.
2.2.12.7 Preparation of Bradford’s reagent
Bradford assay involves the spectral properties of Coomassie Brilliant Blue G- 250 to estimate the amount of protein in a solution (Bradford, 1976). 100 mg 0.01%
(w v-1) Coomassie Brilliant Blue G-250 was weighed and dissolved in 50 mL 95%
ethanol (in an amber colour bottle). 100 mL 85% (w v-1) phosphoric acid was added to it. A magnetic bead was placed inside the bottle and the contents were mixed properly by keeping on magnetic stirrer until the dye completely dissolved. The dye was finally diluted to 1 L with deionized water, filtered (Whatman, No. 1 paper) under dark conditions and stored at 4°C.
The concentration of protein was calculated as follows:
∆ A595 × C × V
Protein concentration (mg mL-1) = = (mg mL-1) v
where,
∆ A595 = change in OD of the sample
C = 1 OD equivalent of BSA from standard plot V = volume of the reaction mixture (mL)
v = volume of the enzyme used for assay (mL)