MECHANISTIC INVESTIGATION IN ULTRASOUND INDUCED ENHANCEMENT OF ENZYMATIC
3.2 Materials and methods .1 Materials
Following chemicals have been procured from Fisher Scientific, India: citric acid, sodium mono-phosphate, sodium azide, sulphuric acid and Sodium hydroxide.
Commercial enzyme cellulase (6 U/mg) produced by Trichoderma reesei (ATCC 26921) and cellobiase (or β–1,4-glucosidase, 250 U/g) produced by Aspergillus niger was procured from Sigma Aldrich, USA.
96 3.2.2 Biomass collection and processing
The lignocellulosic biomass of all eight weeds: Arundo donax (AD), Chromolena odorata (CO), Eichhornia crassipes (EC), Ipomea carnea (IC), Lantana camara (LC), Mikania micrantha (MM), Parthenium hysterophorus (PH) and Saccharum spontaneum (SS) were collected from IIT Guwahati campus and nearby areas. The entire process followed was same as described in Chapter 2 (Subsection 2.2.1).
3.2.3 Pretreatment of raw biomass and hydrolysis of pretreated biomass
As noted earlier, all eight biomasses have been pretreated under constant conditions, viz. 1% (v/v) H2SO4 (equivalent to 0.36 N), 10% w/v biomass, autoclaving at 121°C and 15 psi for 30 min followed by rapid steam release. These conditions have been optimized for acid hydrolysis of the invasive weed of P. hysterophorus in our earlier study (Singh et al., 2014). The solid residue obtained after pretreatment was subjected to delignification by ultrasound assisted alkaline treatment. The conditions for delignification were as follows: 1.5% w/v NaOH, 2% w/v biomass, 30°C with treatment time of 10 min. These conditions have also been optimized for the invasive weed of P.
hysterophorus in our previous study (Bharadwaja et al., 2015). The residue obtained after both acid hydrolyzed and delignification was washed several times till neutral pH was obtained. The residual biomass was dried in a hot air oven at 60 ± 3 °C for 12 h. This residue was subjected to enzymatic hydrolysis using commercial cellulase and cellobiase (β– 1,4-glucosidase) enzymes under two protocols, viz. mechanical shaking and sonication. The lignocellulosic composition of the eight invasive weeds in native and post-treatment form is given in Table 2.1 (B) of chapter 2.
3.2.4 Protocol of enzymatic hydrolysis of biomass with mechanical shaking
Enzymatic hydrolysis with mechanical shaking (henceforth called as control experiments) was carried out in 150 mL Erlenmeyer flask with total reaction volume of 20 mL using an incubator–shaker (Scigenics, India). Mechanical shaking was applied at 150 rpm and temperature of 50 °C. pH of the reaction mixture was maintained 5 using citrate–phosphate buffer in all experiments. Other parameters of enzymatic hydrolysis (or saccharification) of delignified biomasses of all eight weed species were maintained at the optimum values reported by Bharadwaja et al. (2015) as follows: (1) concentration of pretreated biomass in reaction mixture = 4.2% w/v, and (2) concentrations of cellulase and cellobiase as 135 and 75 FPU/g biomass, respectively. Total time of enzymatic hydrolysis was 120 h. 0.005% w/v Sodium azide solution was added to the mixture to avoid external microbial contamination (Singh et al., 2015). The progress of the enzymatic hydrolysis was monitored by withdrawing 0.1 mL samples of reaction mixture periodically and analyzing for release of sugar.
3.2.5 Protocol for enzymatic hydrolysis of biomass with sonication
The protocol for enzymatic hydrolysis with sonication (henceforth called test experiments) was very similar to that with mechanical shaking. Parameters such as reaction volume, biomass concentration, enzyme concentration were exactly same as for the mechanical shaking. Experiments were carried out in an ultrasound bath (Elma, Germany; model: Transonic T-460, volume: 2 L, dimensions: 25 cm x 15 cm x 10 cm) operating at a frequency of 35 kHz and theoretical power input of 35 W. This bath was filled with water, which acted as medium for propagation of ultrasound. This bath had facility of automatic amplitude compensation due to which the net acoustic power delivered to the bath remained constant irrespective of the changes occurring in the bath
during course of reaction. Prior to the experiments, the pressure amplitude of the ultrasound wave generated in the bath was determined using calorimetric technique as 1.5 bar (Sivasankar et al., 2007; Bhasarkar et al., 2013). The temperature of water in the bath increased during sonication. By periodic replacement of some quantity of water in the bath, the temperature of water was maintained at 30°C during the sonication period. A 100 mL conical flask made of borosilicate glass was used for the experiments with total reaction volume of 20 mL. The temperature of the reaction mixture inside the flask was same as that of water outside. The flask was dipped to about 1/4th of its height in water inside the bath. Due to significant spatial variation of the acoustic intensity in the bath, the position of reaction flask was carefully maintained constant in all experiments. Total time of sonication of the enzymatic hydrolysis reaction mixture was 10 h with 10% duty cycle (i.e. 1 min of sonication and 9 min of mechanical shaking for every 10 min of treatment). Similar to the control experiments, progress of the sugar release in enzymatic hydrolysis was monitored by periodic withdrawal of 0.1 mL aliquots of reaction mixture and analyzing them for concentration of reducing sugar.
3.2.6 Total reducing sugar estimation
Hexose-rich enzyme hydrolysates collected intermittently from both set of experiments were subjected to centrifugation for 10 min at 10,000 rpm (26,832g) at 4°C to remove particulate matter. Total reducing sugar in the hydrolyzate was estimated using method of Nelson (1944) and Somogyi (1945).
3.2.7 Analysis of enzyme structure
The morphological changes in the structure (i.e. secondary and tertiary structures) of cellulase and cellobiase enzyme induced by physical and chemical effects of
ultrasound/cavitation have been determined using intrinsic fluorescence and circular dichroism analysis. The procedures for the same have been described below.
18.104.22.168 Intrinsic fluorescence analysis of enzymes before and after exposure to sonication
Three samples of cellulase and cellobiase enzymes were used for measurement of intrinsic fluorescence, viz. (1) native enzyme (i.e. without any treatment), (2) enzyme after treatment with mechanical agitation, and (3) enzyme after ultrasound treatment at ambient pressure of 101.3 kPa. The total treatment time was 1 h with both mechanical agitation and sonication. A duty cycle of 10% was employed for enzyme treatment with ultrasound (as stated previously in the experimental section). Intrinsic fluorescence spectra of all three enzyme samples were measured at temperature of 25° ± 1°C with fluorescence spectrophotometer (Horiba Scientific, model: FluoroMax-4) at 280 nm excitation wavelength (slit = 10 nm), 300–500 nm emission wavelength (slit = 10 nm) and 10 nm/s of scanning speed. Citrate phosphate buffer (0.05 M, pH 5) used for dissolution of cellulase enzyme was used as a blank solution for these measurements.
22.214.171.124 Circular dichroism (CD) analysis of enzymes before and after exposure to sonication
Far UV circular dichroism (CD) spectra of the cellulase and cellobiase enzymes were recorded from the difference in absorption of left and right-circularly polarized light in a Jasco J-815 spectro-polarimeter using a suprasil quartz cuvette of 1 mm optical path length at temperature (25 ± 1°C). The spectra were recorded between 240 and 190 nm with 3 replicates at a scan rate of 100 nm/min with 1 nm bandwidth and a path length of the sample cell of 1 mm. The CD data were expressed in terms of mean residue ellipticity
[h] based on the average molecular weight range of (42–68 kDa) and concentration 50 mM of cellulase or cellobiase solution (Coral et al., 2002). The spectrum of phosphate buffer solution was used as blank and subtracted from the average of three spectra to obtain a corrected spectrum for each samples. The secondary structures of native enzyme and enzyme after treatment with mechanical agitation or sonication were analyzed using an online server DICHROWEB (Sreerama and Woody, 2000; Whitmore and Wallace, 2004 & 2008).