Development of Lignocellulase Enzyme Cocktail—A Logical Use of Statistical Experimental Design Techniques

The derivation of reduced sugars from lignocellulosic biomass requires an optimum blend of cellulolytic enzymes and reaction conditions that favour high sugar yield. In this respect, statistical design of experiment strategies become useful, but the technique is often misguided such that enzyme redundancy becomes overlooked. Herein, we demonstrate a systematic approach that involves simplex lattice mixture design and central composite design for optimizing enzyme cocktails for the saccharification of lignocellulosic biomass. The simplex lattice mixture design yielded 0.3333: 0.3333: 0.3333 of Celluclast (5%), Hemicellulase (5%) and Laccase (2%), respectively, as the optimum enzyme blend (volume) ratio for the saccharification of hydrothermally pretreated empty palm fruit bunch (EPFB, 10% solid loading). A subsequent application of central composite design resulted in 40 oC, pH 6 and 24 hrs as the optimum saccharification conditions. The individual Celluclast (5%) and Hemicellulase (5%) yielded a reduced sugar equivalence (RSE) of 1.77 mg/mL and 1.67 mg/mL, respectively. However, the blended enzyme cocktail upon subjection to simplex lattice mixture design and subsequent central composite design yielded an RSE of 2.215 mg/mL and 2.431 mg/mL, respectively. The overall results exemplify the significance of enzyme synergism in lignocellulosic biomass saccharification. The approach herein is intended as an easy-to-copy plan for optimizing enzyme cocktails.