Formulation of enzyme blends to maximize the hydrolysis of alkaline peroxide pretreated alfalfa hay and barley straw by rumen enzymes and commercial cellulases

Abstract

BackgroundEfficient conversion of lignocellulosic biomass to fermentable sugars requires the synergistic action of multiple enzymes; consequently enzyme mixtures must be properly formulated for effective hydrolysis. The nature of an optimal enzyme blends depends on the type of pretreatment employed as well the characteristics of the substrate. In this study, statistical experimental design was used to develop mixtures of recombinant glycosyl hydrolases from thermophilic and anaerobic fungi that enhanced the digestion of alkaline peroxide treated alfalfa hay and barley straw by mixed rumen enzymes as well as commercial cellulases (Accelerase 1500, A1500; Accelerase XC, AXC).ResultsCombinations of feruloyl and acetyl xylan esterases (FAE1a; AXE16A_ASPNG), endoglucanase GH7 (EGL7A_THITE) and polygalacturonase (PGA28A_ASPNG) with rumen enzymes improved straw digestion. Inclusion of pectinase (PGA28A_ASPNG), endoxylanase (XYN11A_THITE), feruloyl esterase (FAE1a) and β-glucosidase (E-BGLUC) with A1500 or endoglucanase GH7 (EGL7A_THITE) and β-xylosidase (E-BXSRB) with AXC increased glucose release from alfalfa hay. Glucose yield from straw was improved when FAE1a and endoglucanase GH7 (EGL7A_THITE) were added to A1500, while FAE1a and AXE16A_ASPNG enhanced the activity of AXC on straw. Xylose release from alfalfa hay was augmented by supplementing A1500 with E-BGLUC, or AXC with EGL7A_THITE and XYN11A_THITE. Adding arabinofuranosidase (ABF54B_ASPNG) and esterases (AXE16A_ASPNG; AXE16B_ASPNG) to A1500, or FAE1a and AXE16A_ASPNG to AXC enhanced xylose release from barley straw, a response confirmed in a scaled up assay.ConclusionThe efficacy of commercial enzyme mixtures as well as mixed enzymes from the rumen was improved through formulation with synergetic recombinant enzymes. This approach reliably identified supplemental enzymes that enhanced sugar release from alkaline pretreated alfalfa hay and barley straw.

Production of cellulolytic enzymes from ascomycetes: Comparison of solid state and submerged fermentation

“…Such blends can be further optimized by tailoring them for specific biomasses. Tailored blends might also decrease costs pertaining to enzyme production by utilizing an optimal ratio between the enzymes and thereby lowering the enzyme usage [8]. By improving the method of enzyme production a higher yield may be achieved, which would also increase the feasibility of biorefineries.…”

An optimal blend of single autodisplayed cellulases for cellulose saccharification – a proof of concept

“…According to Khattak et al ., the utilization of sugars from feed during microbial conversion of cellulose is a crucial drawback. Also, feedback inhibition of cellulases by both intermediate and end products has been widely reported . Nevertheless, it should be observed that Tween 20 was able to maintain the performance and functionality (stability) of the cellulases better than CaCl 2 during EFB saccharification.…”

Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme

“…Cel7B might improve overall cell wall conversion through the hydrolysis of polysaccharides covering the cellulose microfibers and thus making them exposed to cellulase action. In addition, Cel7B are capable of generating more reducing and non-reducing ends, available to Cel7A and Cel7B attack (Badhan et al 2014). Based on ThCel7B biochemical characterization, we speculate that this enzyme might be equally important for biomass enzymatic hydrolysis, not only directly synergizing with exoglucanases in cellulose microfibers depolymerization but also making them available for cellobiohydrolase action through active hydrolysis of other polysaccharides of the plant cell wall matrix.…”

“…It was shown that enzymatic hydrolysis of alkaline peroxide pretreated alfalfa hay and barley straw by rumen enzymes and commercial cellulases was significantly enhanced by addition of endoglucanase from GH7 family (Badhan et al 2014). Augmented glucose release as a consequence of the endoglucanase addition was thought to be the result of its broad substrate specificity (Badhan et al 2014). Cel7B might improve overall cell wall conversion through the hydrolysis of polysaccharides covering the cellulose microfibers and thus making them exposed to cellulase action.…”