Viscosimetric Assay of Endoglucanase Activity in Enzyme Fodder Additives

Abstract

Worldwide a large number of enzymes, including those with endo-β-glucanase activity are produced by different companies as fodder additives. In Russia, there is no uniform method for endo-β-glucanase activity analysis that causes difficulties in assessing quality of the enzyme preparations. The aim of the study was to develop such uniform method. In this, we optimized a viscometric determination based on change of the relative viscosity of a substrate mixed with enzyme preparation using Ostwald capillary viscometer. The following regime has been accepted: the diameter of the capillary is 0.73 mm; the temperature of hydrolysis is 30 C; the solvent is the acetate buffer (pH 4.7); time of incubation is 5, 10, 15 min in accordance with the kinetics of the enzymatic reaction; and βglucane (0.4 %) was used as substrate. The carboxymethyl cellulose is shown not to be relevant so far as it gives results incomparable with those obtained with β-glucane due to estimation of the cellulase but not the β-glucanase activity, that does not correspond to the objectives of forage production. The unit used in the developed method is the amount of enzyme which increases the relative substrate flow rate in 1 min-1 under standard conditions of hydrolysis. The measurement of accuracy of the method was carried out according to GOST R ISO 5725-2002 (the state standards). In this, 5 certified samples with different activity were prepared from a commercial sample of β-glucanase (Sigma-Aldrich, USA) with known activity (GLA/g). The activity of the standard was expressed in GLA/g as glucose equivalents corresponding to the amount of enzyme which acts on -glucane, releasing 1 µmol of sugars in terms of glucose. Certified samples were analyzed under intermediate precision conditions with three factors changed (i.e., operator, matrix, reagents). The matrices were mineral fillers, the calcium carbonate and zeolite. To calculate the accuracy of the method the viscosity of certified samples was expressed in GLA units using the calibration curves. Expanded uncertainty of measurements ranged within 10-22 % for the coverage factor k = 2.