Enzymatic saccharification is the most crucial and challenging step in lignocellulosic biorefineries for bioethanol production. The enzymatic saccharification requires a cocktail of hydrolytic enzymes, cellulases and hemicellulases for effective degradation of biomass. However, the complete bioconversion of biomass is governed by the proportion and synergistic action of hydrolytic enzymes present in the cocktail. Thus, this study aims to formulate an enzyme cocktail consisting of recombinant cellulases (bifunctional cellulolytic chimeric enzyme, CtGH1-L1-CtGH5-F194A and cellobiohydrolase, CtCBH5A) and xylanases (endo-1,4-β-xylanase, CtGH11A, and β-xylosidase, BoGH43), all in crude form (unpurified enzyme present in the total cellular proteins) for producing high yield of total reducing sugars using choline chloride:acetic acid pretreated rice-straw (CApRS). D-optimal mixture design approach was used for statistically designing the optimal proportion of crude recombinant enzyme cocktail for hydrolysis of delignified rice straw. The developed enzyme cocktail comprising chimera, CtCBH5A, CtGH11 and BoGH43 in ratio 35.1:41.8:10.0:13.1 resulted in cellulolytic enzyme activity, 5.6 FPU/mL and xylanase activity, 102 U/mL. Enzymatic saccharification of 3% (w/v) delignified CApRS using developed enzyme cocktail at 187 FPU/gCApRS, pH 5.8 and 35 °C, yielded total reducing sugar of 498 mg/gbiomass, glucose yield (410 mg/g) and xylose yield (71.3 mg/g) resulting in 72% saccharification efficiency. Whereas, significantly low total reducing sugar yield of 80 mg/g and 218 mg/g were released from raw RS and partially pretreated RS, respectively, by the developed cocktail under the same hydrolytic conditions, thereby inferring the effectiveness of the enzyme cocktail against CApRS biomass. The developed enzyme cocktail was compared with a commercial enzyme cocktail and prospects its applicability in lignocellulosic biorefineries.
