Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides

<p>Abstract</p> <p>Background</p> <p>High enzyme loading is a major economic bottleneck for the commercial processing of pretreated lignocellulosic biomass to produce fermentable sugars. Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant reduction in enzyme loading without sacrificing hydrolysis yield. This is especially important for alkaline pretreatments such as Ammonia fiber expansion (AFEX) pretreated corn stover. Hence, a diverse set of hemicellulases supplemented along with cellulases is necessary for high recovery of monosaccharides.</p> <p>Results</p> <p>The core fungal cellulases in the optimal cocktail include cellobiohydrolase I [CBH I; glycoside hydrolase (GH) family 7A], cellobiohydrolase II (CBH II; GH family 6A), endoglucanase I (EG I; GH family 7B) and β-glucosidase (βG; GH family 3). Hemicellulases tested along with the core cellulases include xylanases (LX1, GH family 10; LX2, GH family 10; LX3, GH family 10; LX4, GH family 11; LX5, GH family 10; LX6, GH family 10), β-xylosidase (LβX; GH family 52), α-arabinofuranosidase (LArb, GH family 51) and α-glucuronidase (LαGl, GH family 67) that were cloned, expressed and/or purified from different bacterial sources. Different combinations of these enzymes were tested using a high-throughput microplate based 24 h hydrolysis assay. Both family 10 (LX3) and family 11 (LX4) xylanases were found to most efficiently hydrolyze AFEX pretreated corn stover in a synergistic manner. The optimal mass ratio of xylanases (LX3 and LX4) to cellulases (CBH I, CBH II and EG I) is 25:75. LβX (0.6 mg/g glucan) is crucial to obtaining monomeric xylose (54% xylose yield), while LArb (0.6 mg/g glucan) and LαGl (0.8 mg/g glucan) can both further increase xylose yield by an additional 20%. Compared with Accellerase 1000, a purified cocktail of cellulases supplemented with accessory hemicellulases will not only increase both glucose and xylose yields but will also decrease the total enzyme loading needed for equivalent yields.</p> <p>Conclusions</p> <p>A diverse set of accessory hemicellulases was found necessary to enhance the synergistic action of cellulases hydrolysing AFEX pretreated corn stover. High glucose (around 80%) and xylose (around 70%) yields were achieved with a moderate enzyme loading (~20 mg protein/g glucan) using an in-house developed cocktail compared to commercial enzymes.</p

Glucose Recovery from Different Corn Stover Fractions Using Dilute Acid and Alkaline Pretreatment Techniques

Background: Limited availability of corn stover due to the competing uses (organic manure, animal feed, bio-materials, and bioenergy) presents a major concern for its future in the bio-economy. Furthermore, biomass research has exhibited different results due to the differences in the supply of enzymes and dissimilar analytical methods. The effect of the two leading pretreatment techniques (dilute acid and alkaline) on glucose yield from three corn stover fractions (cob, stalk, and leaf) sourced from a single harvest in Uganda were studied at temperatures 100, 120, 140, and 160 °C over reaction times of 5, 10, 30, and 60 min. Results: From this study, the highest glucose concentrations obtained from the dilute acid (DA) pretreated cobs, stalks, and leaves were 18.4 g/L (66.8% glucose yield), 16.2 g/L (64.1% glucose yield), and 11.0 g/L (49.5% glucose yield), respectively. The optimal pretreatment settings needed to obtain these yields from the DA pretreated samples were at a temperature of 160 °C over an incubation time of 30 min. The highest glucose concentrations obtained from the alkaline (AL) pretreated cobs, stalks, and leaves were 24.7 g/L (81.73% glucose yield), 21.3 g/L (81.23% glucose yield), and 15.0 g/L (51.92% glucose yield), respectively. To be able to achieve these yields, the optimal pretreatment settings for the cobs and stalks were 140 °C and for a retention time of 30 min, while the leaves require optimal conditions of 140 °C and for a retention time of 60 min. Conclusions: The study recommends that the leaves could be left on the field during harvesting since the recovery of glucose from the pretreated cobs and stalks is higher

Shotgun Approach to Increasing Enzymatic Saccharification Yields of Ammonia Fiber Expansion Pretreated Cellulosic Biomass

Most cellulolytic enzyme blends, either procured from a commercial vendor or isolated from a single cellulolytic microbial secretome, do not efficiently hydrolyze ammonia-pretreated (e.g., ammonia fiber expansion, AFEX) lignocellulosic agricultural crop residues like corn stover to fermentable sugars. Typically reported commercial enzyme loading (30–100 mg protein/g glucan) necessary to achieve &gt;90% total hydrolysis yield (to monosaccharides) for AFEX-treated biomass, within a short saccharification time frame (24–48 h), is economically unviable. Unlike acid-based pretreatments, AFEX retains most of the hemicelluloses in the biomass and therefore requires a more complex suite of enzymes for efficient hydrolysis of cellulose and hemicellulose at industrially relevant high solids loadings. One strategy to reduce enzyme dosage while improving cocktail effectiveness for AFEX-treated biomass has been to use individually purified enzymes to determine optimal enzyme combinations to maximize hydrolysis yields. However, this approach is limited by the selection of heterologous enzymes available or the labor required for isolating low-abundance enzymes directly from the microbial secretomes. Here, we show that directly blending crude cellulolytic and hemicellulolytic enzymes-rich microbial secretomes can maximize specific activity on AFEX-treated biomass without having to isolate individual enzymes. Fourteen commercially available cellulolytic and hemicellulolytic enzymes were procured from leading enzyme companies (Novozymes®, Genencor®, and Biocatalysts®) and were mixed together to generate several hundred unique cocktail combinations. The mixtures were assayed for activity on AFEX-treated corn stover (AFEX-CS) using a previously established high-throughput methodology. The optimal enzyme blend combinations identified from these screening assays were enriched in various low-abundance hemicellulases and accessory enzymes typically absent in most commercial cellulases cocktails. Our simple approach of blending crude commercially available enzyme cocktails allowed a drastic fourfold reduction in total enzyme requirements (from 30 to 7.5 mg enzyme/g glucan loading) to achieve near-theoretical cellulose and hemicellulose saccharification yields for AFEX-CS