4.3.1. Effect of optimal temperature on cellulase activity
Temperature is one of the most important factors that effects to enzyme activity. The higher temperature, the lower cellulose activity was When the temperature rose, kinetic energy increased (collision frequency develops) the frequency of enzyme-substrate complexes grew per unit of time so the reaction rate and the product increased. In response to the appropriate temperature controlled enzyme, the corresponding reaction rate was higher than the appropriate temperature (response rate decreased rapidly because of high temperature causing the enzyme). Operation centers lost their standard configuration and were no longer suitable for the substrate, thus the enzyme lost its catalytic role.
To investigate the optimal temperature of the cellulase enzyme from the Bacillus pumilus B6.4, the reaction was carried out at the temperatures ranging from 40 to 80 °C.
Figure 4.6. Effect of optimal temperature on enzyme activity
The results shown that the cellulase activity of the Bacillus pumilus B6.4 increased from 50 to 60 ºC and reached a maximum at 55 oC (5.157 U/ml). At 40 ºC the enzyme activity decreased to 66 % of the maximum activity, to 75 % at 60
oC to 20 % at 80 ºC compared to the maximum activity.
66
73 78
100
75
55
43
33
20 0
10 20 30 40 50 60 70 80 90 100
40 45 50 55 60 65 70 75 80
Relativie activity (%)
Temperature
Thus, the enzyme worked well in the range of 50 to 60˚C, the optimal reaction temperature of cellulase enzyme was 55˚C.
The previous studies have shown that the appropriate reaction temperature for Bacillus pumilus S124A was optimally at 50oC (Natesan and Nelson, 2014), and Bacillus pumilus EB3 was optimized at 60 oC (Ariffin et al., 2006). On the other hand, in Bacillus mycoides S122C was found at 50 oC (Balansubramanian et al., 2012), Bacillus subtilis YJ1 cellulase was found at 60 ˚C (Li et al., 2010).
4.3.2. Effect of optimal pH on cellulase activity
To determine the optimal pH for cellulase enzyme activity from the Bacillus pumilus B6.4, the reaction was performed at a substrate concentration of 1 % CMC; The substrate solution was mixed in a 50 mM acetate buffer pH (5 to 8) at the determined temperature (40 °C) using water bath. The results are shown in Figure 4.7.
Figure 4.7. Effect of optimal temperature on enzyme activity
The results showed that the cellulase activity increased during the pH range of 6.0 to 7.0, the activity was maximized at pH 6.5 (5.458 U/ml). At pH 7, cellulase activity of the Bacillus pumilus B6.4 decreased to 87 % and 36 % at pH 8.0.
The pH range suitable for cellulase activity from Bacillus circulans was found at 4.5 to 7.0 (Kim, 1995); for other Bacillus strains was at 5.0 to 6.5 (Mawadza et al., 2000); in Bacillus subtilis YJ1 was 6.0 to 6.5 (Li et al., 2010).
50
79
91
100
87
80
36
0 10 20 30 40 50 60 70 80 90 100
5 5.5 6 6.5 7 7.5 8
Relativie activity (%)
pH
Optimum pH values of 4.5 to 8.0 have been reported for different microbial cellulase (Immanuel et al., 2007; Dutta et al., 2008). Each enzyme has its own optimum pH and if the pH increases or decreases beyond the optimum, the ionization groups at the active site may change slowing or preventing the formation of an enzyme substrate complex (Eijsink et al., 2005).
4.3.3. Thermal stability on cellulase activity
Thermal stability of pre-purified cellulase was determined by measuring cellulolytic activity and residual activity at various temperatures ranging from 45 to 85 oC at different times ranging from 30 to 240 min at pH 5.0, shown in table 4.3 and figure 4.8.
Table 4.3. Thermal stability on enzyme activity (U/ml) Temperature (oC)
Time (min) 45 55 65 75 85
0 3.679 3.679 3.679 3.679 3.679
30 2.711 3.211 3.541 1.724 1.145
60 2.241 2.904 3.413 1.253 0.996
90 1.832 2.673 3.112 1.096 0.722
120 1.627 2.416 2.735 0.891 0.526
150 1.394 2.127 2.367 0.552 0.275
180 1.140 1.718 1.806 0.278 0.177
240 0.903 1.487 1.642 0.088 0.027
More than 58 % of the original cellulase activity of the purified enzyme was maintained at the temperatures ranging from 55 to 65 oC after 150 min incubation at pH 5.0 and then it still remained about more than 40 % the comparison to the original residual activity of cellulase after 240 min. About 38 % of the original activity was maintained at temperatures 45 oC after 150 min and then dramatically decreased to 25 % after 240 min whereas less than 15 % of the original activity was observed at 75 to 85 oC at 150 min and went down to 1 % after 240 min.
Figure 4.8. Thermal stability on enzyme activity
This result suggested that this purification of enzyme was stable between 55 to 65 oC after 150 min and then decreased to 40 % after incubation for 240 min.
On the other hand, Natesan and Nelson (2014), reported that thermal stability on cellulase activity was at 60 to 70 oC in Bacillus pumilus S124A. In Bacillus amyoliquefaciens DL-3 was most stable at the temperatures ranging from 50 to 70
oC (Lee et al., 2008), while purification of enzyme produced by Bacillus subtilis was stable enough at 40 to 60 oC (Rekha and Lakshmi, 2012). For cellulase producing bacteria isolates as Bacilus sp., Pseudomonas sp., and Serratia sp. was found almost stable up to 55 oC (Prabesh Khativada et al., 2016).
4.3.4. pH stability
The pH stability of the purified enzyme between pH 5.5 to 8.5 was determined by incubating in 50 mM sodium acetate buffer of different pH values at 40 oC for various times ranging from 30 to 240 min and then assaying the residual enzyme activity under standard testing condition, shown in table 4.4 and figure 4.9.
More than 56 % of the original cellulase activity of the purified enzyme was maintained at pH ranging from 5.5 to 6.5 after 120 min incubation at temperature 40oC and then it still remained about more than 21 % in comparison with the original residual activity of cellulase after 240 min. About 30 % of the original activity was remained at pH 7.5 to 8.5 after 120 min and then dramatically decreased to 7 % after 240 min.
0 10 20 30 40 50 60 70 80 90 100
0 30 60 90 120 150 180 210 240
45 55 65 75 85
Time (min)
Relative activity (%)
Table 4.4. pH stability in enzyme activity (U/ml) pH
Time (min) 5.5 6.5 7.5 8.5
0 2.221 3.030 2.010 0.768
30 1.984 2.808 1.782 0.579
60 1.771 2.431 1.435 0.465
90 1.511 2.118 0.926 0.345
120 1.233 1.724 0.608 0.292
150 0.865 1.253 0.356 0.225
180 0.576 0.836 0.216 0.190
240 0.458 0.611 0.138 0.117
Figure 4.9. pH stability on enzyme activity
The cellulase enzyme of Bacillus pumilus B6.4 strain was relatively stable at pH 5.5 to 6.5 after 120 min. Some previous studies have also reported that the cellulase enzymes produced by several Bacillus sp. were stable over a wide pH range (Mawadza et al., 2000; Lee et al., 2008). Furthermore, Bacillus subtilis BY-4 was found to be stable at pH ranging from 4.5 to 6.0 which was the most stable at pH 5.0 of bacteria producing cellulase (Lima et al., 2015). Other Bacillus amyloliquefaciens DL-3 was stable over a broad pH ranging from 4.0 to 9.0 (Lee et al.,2008), Bacillus halodurans IND18 shown that pH stability various from 6.0 to 9.0, which was the most stable at pH 9.0 (Gao et al.,2008).
0 10 20 30 40 50 60 70 80 90 100
0 30 60 90 120 150 180 210 240
5.5 6.5 7.5 8.5
Time (min)
Relative activity (%)