Synthetic Enzyme Mixtures for Biomass Deconstruction: Production and Optimization of a Core Set

Department of Energy, Great Lakes Bioenergy Research Center, Michigan State University, E. Lansing, Michigan 48824, USA.
Biotechnology and Bioengineering (Impact Factor: 4.13). 08/2010; 106(5):707-20. DOI: 10.1002/bit.22741
Source: PubMed


The high cost of enzymes is a major bottleneck preventing the development of an economically viable lignocellulosic ethanol industry. Commercial enzyme cocktails for the conversion of plant biomass to fermentable sugars are complex mixtures containing more than 80 proteins of suboptimal activities and relative proportions. As a step toward the development of a more efficient enzyme cocktail for biomass conversion, we have developed a platform, called GENPLAT, that uses robotic liquid handling and statistically valid experimental design to analyze synthetic enzyme mixtures. Commercial enzymes (Accellerase 1000 +/- Multifect Xylanase, and Spezyme CP +/- Novozyme 188) were used to test the system and serve as comparative benchmarks. Using ammonia-fiber expansion (AFEX) pretreated corn stover ground to 0.5 mm and a glucan loading of 0.2%, an enzyme loading of 15 mg protein/g glucan, and 48 h digestion at 50 degrees C, commercial enzymes released 53% and 41% of the available glucose and xylose, respectively. Mixtures of three, five, and six pure enzymes of Trichoderma species, expressed in Pichia pastoris, were systematically optimized. Statistical models were developed for the optimization of glucose alone, xylose alone, and the average of glucose + xylose for two digestion durations, 24 and 48 h. The resulting models were statistically significant (P < 0.0001) and indicated an optimum composition for glucose release (values for optimized xylose release are in parentheses) of 29% (5%) cellobiohydrolase 1, 5% (14%) cellobiohydrolase 2, 25% (25%) endo-beta1,4-glucanase 1, 14% (5%) beta-glucosidase, 22% (34%) endo-beta1,4-xylanase 3, and 5% (17%) beta-xylosidase in 48 h at a protein loading of 15 mg/g glucan. Comparison of two AFEX-treated corn stover preparations ground to different particle sizes indicated that particle size (100 vs. 500 microm) makes a large difference in total digestibility. The assay platform and the optimized "core" set together provide a starting point for the rapid testing and optimization of alternate core enzymes from other microbial and recombinant sources as well as for the testing of "accessory" proteins for development of superior enzyme mixtures for biomass conversion.

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Available from: Melissa S Borrusch, Mar 19, 2014
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    • "It contained (by mass) 35% cellobiohydrolase 1 (CBH1), 7% cellobiohydrolase 2 (CBH2; GenBank P07987), 5% endo-β1,4-glucanase 1 (EG1, GenBank AAA34212), 4% β-glucosidase (BG, GenBank AAA18473), 36% AA9 (formerly GH61A, GenBank CAA71999), 2% endo-β1,4-xylanase 2 (EX2, GenBank AAB29346), 9% endo-β1,4-xylanase 3 (EX3; GenBank BAA89465), and 3% β-xylosidase (BX; GenBank CAA93248). CBH1 was obtained from Megazyme, Ltd. (Bray, Ireland) and the other proteins were expressed in Pichia pastoris, including T. reesei Cel5A (TrCel5A; GenBank ABA64553 or AAA34213), by the method described earlier [13]. Bulk quantities were produced by Lucigen, Inc. (Madison, WI). S. thermophile Cel5A (StCel5A) was produced by expression in A. niger [14]. "
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    ABSTRACT: Enzymatic conversion of lignocellulosic materials to fermentable sugars is a limiting step in the production of biofuels from biomass. We show here that combining enzymes from different microbial sources is one way to identify superior enzymes. Extracts of the thermophilic fungus Sporotrichum thermophile (synonym Myceliophthora thermophila) gave synergistic release of glucose (Glc) and xylose (Xyl) from pretreated corn stover when combined with an 8-component synthetic cocktail of enzymes from Trichoderma reesei. The S. thermophile extracts were fractionated and an enhancing factor identified as endo-β1,4- glucanase (StCel5A or EG2) of subfamily 5 of Glycosyl Hydrolase family 5 (GH5_5). In multi-component optimization experiments using a standard set of enzymes and either StCel5A or the ortholog from T. reesei (TrCel5A), reactions containing StCel5A yielded more Glc and Xyl. In a five-component optimization experiment (i.e., varying four core enzymes and the source of Cel5A), the optimal proportions for TrCel5A vs. StCel5A were similar for Glc yields, but markedly different for Xyl yields. Both enzymes were active on lichenan, glucomannan, and oat β-glucan; however, StCel5A but not TrCel5A was also active on β1,4-mannan, two types of galactomannan, and β1,4-xylan. Phylogenetically, fungal enzymes in GH5_5 sorted into two clades, with StCel5A and TrCel5A belonging to different clades. Structural differences with the potential to account for the differences in performance were deduced based on the known structure of TrCel5A and a homology-based model of StCel5A, including a loop near the active site of TrCel5A and the presence of four additional Trp residues in the active cleft of StCel5A. The results indicate that superior biomass-degrading enzymes can be identified by exploring taxonomic diversity combined with assays in the context of realistic enzyme combinations and realistic substrates. Substrate range may be a key factor contributing to superior performance within GH5_5.
    PLoS ONE 10/2014; 9(10):e109885. DOI:10.1371/journal.pone.0109885 · 3.23 Impact Factor
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    • "So the reason could be that BX itself may also have ability to hydrolyze xylan chains on solid substrate. This possible " Endo-acting " ability of BX can also be found from other experiments (Banerjee et al., 2010a,b,c) and need to be further studied. We further investigate the effect of substrate composition on hydrolysis by testing different initial percentages of cellulose and xylan with the same values of overall substrate accessibility and the initial amount of exposed D-xylose units. "
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    ABSTRACT: We develop a novel and general modeling framework for enzymatic hydrolysis of cellulose and hemicellulose simultaneously. Our mechanistic model, for the first time, takes into consideration explicitly the time evolution of morphologies of intertwining cellulose and hemicelluloses within substrate during enzymatic hydrolysis. This morphology evolution is driven by hydrolytic chain fragmentation and solubilization, which is, in return, profoundly affected by the substrate morphology. We represent the substrate morphology as a randomly distributed Smallest Accessible compartments (SACs) which are described by geometric functions to track total volume and exposed surface substrate materials, including both cellulose and hemicelluloses. Our morphology-plus-kinetics approach then couple the time-dependent morphology with chain fragmentation and solubilization resulted from enzymatic reactions between various bonds in cellulose and hemicelluloses and a mixture (i.e. endo-, exo- and oligomer- acting) of cellulases and hemicellulases. In addition, we propose an advanced and generalized site concentration formalism that considers different polysaccharide chain types and different monomer unit types on chains. The resulting ODE system has a substantially reduced size compared to conventional chain concentration formalism. We present numerical simulation results under real enzymatic hydrolysis experimental conditions from literature. The comparisons between the simulation results and the experiment measurements demonstrate effectiveness and wide applicability of the proposed mechanistic model. Biotechnol. Bioeng. © 2014 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 09/2014; 111(9). DOI:10.1002/bit.25244 · 4.13 Impact Factor
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    • "To test this, enzyme optimization experiments were performed. Synthetic mixtures of eight ’core’ enzymes (BG, EG1, CBH1, CBH2, GH61, BX, EX2 and EX3) were optimized [19,20]. A minimum proportion of 5% was set as a lower limit for all enzymes. "
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    ABSTRACT: Many different feedstocks are under consideration for the practical production of biofuels from lignocellulosic materials. The best choice under any particular combination of economic, agronomic, and environmental conditions depends on multiple factors. The use of old fields, restored prairie, or marginal lands to grow biofuel feedstocks offers several potential benefits including minimal agronomic inputs, reduced competition with food production, and high biodiversity. However, a major component of such landscapes is often herbaceous dicotyledonous plants, also known as forbs. The potential and obstacles of using forbs as biofuel feedstocks compared to the more frequently considered grasses and woody plants are poorly understood. The factors that contribute to the yield of fermentable sugars from four representative forbs were studied in comparison with corn stover. The forbs chosen for the study were lamb's quarters (Chenopodium album), goldenrod (Solidago canadensis), milkweed (Asclepias syriaca), and Queen Anne's lace (Daucus carota). These plants are taxonomically diverse, widely distributed in northern temperate regions including the continental United States, and are weedy but not invasive. All of the forbs had lower total glucose (Glc) content from all sources (cell walls, sucrose, starch, glucosides, and free Glc) compared to corn stover (range 16.2 to 23.0% on a dry weight basis compared to 39.2% for corn stover). When digested with commercial enzyme mixtures after alkaline pretreatment, yields of Glc as a percentage of total Glc were lower for the forbs compared to corn stover. Enzyme inhibition by water-extractable compounds was not a significant contributor to the lower yields. Based on experiments with optimized cocktails of pure glycosyl hydrolases, enzyme imbalance probably accounted for much of the lower yields. Addition of xyloglucanase and alpha-xylosidase, two enzymes targeting Glc-containing polysaccharides that are more abundant in dicotyledonous plants compared to grasses, enhanced Glc yields from lamb's quarters, but Glc yields were still lower than from corn stover. The potential utilization of forb-rich plant communities as biofuel feedstocks must take into account their lower Glc content compared to grasses such as corn stover. Furthermore, new enzyme mixtures tailored to the different cell wall composition of forbs will have to be developed.
    Biotechnology for Biofuels 04/2014; 7(1):52. DOI:10.1186/1754-6834-7-52 · 6.04 Impact Factor
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