Effects of pH and incubation duration on the stability of the endoglucanase activity of seventeen exogenous fibrolytic enzyme preparations

Abstract Text: This study examined effects of pH and incubation duration on the stability of the endoglucanase activity of various exogenous fibrolytic enzymes (EFE). Seventeen commercial EFE sourced from Trichoderma reesei or and Aspergillus spp. were assayed in triplicate for endoglucanase (EN) activity at pH 4.0, 5.0 and 6.0 after incubation for 0, 24, 48 and 168 hours at 40°C. Endoglucanase activity was assayed in a 15-ml tube containing 1.0 ml of 1.0% (wt/vol) carboxymethyl cellulose as substrate and 0.9 ml of citrate-phosphate buffer (pH 4.0, 5.0 or 6.0). For the 0-h incubation, after a 10-min preincubation period, 0.1 ml of diluted EFE was added to cellulose to initiate the reaction and the suspension was incubated for 5 min. The reaction was terminated with 3 ml of dinitrosalicylic acid. For the other incubation periods, tubes were kept in a water bath at 40°C degrees for the respective durations. The unit of EN activity was the amount of EFE required to release 1 µmol of reducing glucose equivalents min^-1 mg^-1. Treatments were arranged in a 17 (enzymes) x 3 (pH) x 4 (incubation duration) factorial layout and data were analyzed with a model including these terms and the interactions using the GLM procedure of SAS. Endoglucanase activity was greatest (P < 0.0001) for all EFE at pH 4.0 after 0 h of incubation except for one EFE, which exhibited the greatest activity at pH 6.0 after 0 h of incubation. For 13 of the 17 EFE, increasing the incubation duration or the pH quadratically (P < 0.0001) decreased EN activity.  However, simultaneously increasing the pH and incubation duration linearly (P < 0.0001) decreased EN activity. Within 24 h of incubation, between 97.9 and 99.6 % of the EN activity was lost from each EFE.  Therefore, EN activity decreased substantially as the incubation duration increased.  This study shows that the EN activities of the EFE decreased with increasing pH and or incubation time. Endoglucanase activities were much lower at the usual ruminal pH of lacting dairy cows than at those (pH 4 – 5) typically used to assay EFE activities in the laboratory.

Keywords: endoglucanase, exogenous enzyme, incubation duration, pH