Determination of the reactivity of cellulosic substrates towards enzymatic hydrolysis
An ever-growing scientific interest in the development of effective methods for transformation of various cellulosic resources into fermentable sugars necessitates development of a universal procedure for determination of the reactivity of cellulosic substrates towards enzymatic hydrolysis. The practical significance consists in maximum accessibility of the procedure for the labs of pilot-production enterprises engaged in testing and scaling up the biotech processes. The developed procedure fully complies with modern requirements and relies on measuring the concentration of reducing sugars (spectrophotometry and HPLC) in the enzymatic hydrolyzates obtained from pre-prepared substrates, the biocatalysis being run by a cocktail composed of available CelluLuxe-A and BrewZyme-BGX. On top of that, the procedure implies gravimetric analysis of the solid residues after hydrolysis of substrates. Cellulosic biomasses can usually be fermented for control without any pretreatment, however, commercial celluloses can be used as well. The use of the developed procedure is shown to provide prompt and high-quality assessment of the reactivity of a series of chosen substrates to enzymatic hydrolysis. In contrast to the methods of enzymatic hydrolysis discussed in literature for evaluation of the enzyme efficiency, the developed procedure allows arranging of chosen cellulosic raw materials in a descending order of their reactivity to hydrolysis using the same multi-enzyme cocktail and, moreover can demonstrate dependence of the reactivity of substrates on the pretreatment method. The results can be presented as a dependence of the concentration (yield) of reducing sugars on the duration of enzymatic hydrolysis of the substrate, and also in the form of the calculated hydrolysis rates, final yields of reducing sugars including pentoses, content of glucose component of reducing substances and decrease in mass. The procedure was repeatedly tested on a wide range of cellulosic substrates and provided reliable results regarding evaluation of their reactivity and forecasting of the scale-up results of enzymatic hydrolysis, including that in aqueous medium when preparing nutrient broths for microbiological synthesis.