Effect of temperature and substrate concentration
The negative interactive coefficient (Eq. (4)) and non-significant
value (Prob > F) (Table 3) indicated lack of interaction among the
independent variables. Cellulase activity (model designed point)
9.48 U/ml at 65 ◦C and 90.0 mg/ml substrate concentration is less
then the cellulase activity in the optimum area in the vicinity of
62–63 ◦C (Fig. 5). No change in activity observed at higher substrate
concentrations suggest that the increase availability of substrate
did not outweigh the negative effect on the rate caused by the
increase in average time for the cellobiohydrobiolases to reach the
chain end. The clearly identifiable hydrolyzed cellulose filter paper lane A, B and C indicated the mobility and lateral diffusion of cellulose
enzyme, also the already adsorbed enzyme are less effected
by the higher temperature (Fig. 6). A total of 25l of lyophilized
and sonicated enzyme preparation were spotted and dried at ambient
temperature. The paper chromatography analysis assays were
carried out in 0.05M citrate buffer (pH 4.8) at 50, 60 and 70 ◦C of
the lane A, B and C respectively. The degradation products were
dried and sprayed with 50% sulphuric acid and dried at room temperature.
The intensity of lane colour increased with increased in
temperature but slight decrease in the lane width indicated that
the lateral diffused enzymes are deactivated at high temperature
(Fig. 6C). The loss of the substrate inhibition pattern at high temperature
suggested that the rate of lateral diffusion is enhanced by
increase in temperature to such an extent that, it is no longer a
prominent rate limiting factor