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: 4.16). 08/2010; 106(5):707-20. DOI: 10.1002/bit.22741
Source: PubMed

ABSTRACT 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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    • "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 · 4.16 Impact Factor
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    • "Several different strategies involving many enzyme classes are used in natural degradation of recalcitrant biomass (Dashtban et al. 2009). However, industrial use of cellulases has mainly been focused on endo-1,4-β-D- glucanase and two types of cellobiohydrolases acting respectively from the reducing and from the non-reducing end of the polymer (Banerjee et al 2010). Additional industrially used cellulose-degrading enzymes are βglucosidases , which degrade β-D-glucose oligomers to glucose and the GH61 proteins, which boost cellulose decomposition by oxidative degradation of the glucose polymer (Harris et al. 2010; Langston et al. 2011; Quinlan et al. 2011; Westereng et al. 2011). "
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    ABSTRACT: Elucidation of fungal biomass degradation is important for understanding the turnover of biological materials in nature and has important implications for industrial biomass conversion. In recent years there has been an increasing interest in elucidating the biological role of thermophilic fungi and in characterization of their industrially useful enzymes. In the present study we investigated the cellulolytic potential of 16 thermophilic fungi from the three ascomycete orders Sordariales, Eurotiales and Onygenales and from the zygomycete order Mucorales thus covering all fungal orders that include thermophiles. Thermophilic fungi are the only described eukaryotes that can grow at temperatures above 45 [degree sign]C. All 16 fungi were able to grow on crystalline cellulose but their secreted enzymes showed widely different cellulolytic activities, pH optima and thermostabilities. Interestingly, in contrast to previous reports, we found that some fungi such as Melanocarpus albomyces readily grew on crystalline cellulose and produced cellulases. These results indicate that there are large differences in the cellulolytic potential of different isolates of the same species. Furthermore, all the selected species were able to degrade cellulose but the differences in cellulolytic potential and thermostability of the secretome did not correlate to the taxonomic position. PCR amplification and sequencing of 22 cellulase genes from the fungi showed that the level of thermostability of the cellulose-degrading activity could not be inferred from the phylogenetic relationship of the cellulases.
    08/2013; 3(1):47. DOI:10.1186/2191-0855-3-47
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    • "Reconstitution of defined cellulase components (cocktail) is another strategy for reducing enzyme cost. A study of a minimal fungal or bacterial cellulase mixtures suggested that the biomass can be hydrolyzed well by a careful combination of several enzymes [6] [7] [8]. However, the reconstitution cellulase cocktail is conducted with several organisms (each harboring a single enzyme), which increases the cost of organism fermentation. "
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