Factorial optimization of a six-cellulase mixture

Kim, Eunki; Irwin, Diana C.; Walker, Larry P.; Wilson, David B.

doi:10.1002/(sici)1097-0290(19980605)58:5<494::aid-bit5>3.0.co;2-8

Cited by 43 publications

(21 citation statements)

References 22 publications

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“…The E4 enzyme also contains a family III CBM that assists the enzyme in processivity (303). Factorial optimization of the T. fusca cellulase system was undertaken, and the highest synergistic effect was shown with the addition of CBHI from T. reesei (335).…”

Section: Cellulase Enzyme Systemsmentioning

confidence: 99%

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Lynd

Weimer

Zyl³

et al. 2002

Microbiol Mol Biol Rev

4,005

2,868

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Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for “consolidated bioprocessing” (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts

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Section: Cellulase Enzyme Systemsmentioning

confidence: 99%

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Lynd

Weimer

Zyl³

et al. 2002

Microbiol Mol Biol Rev

4,005

2,868

View full text Add to dashboard Cite

show abstract

“…A mixture of all six T. fusca cellulases was equivalent to a crude T. fusca mixture, but addition of T. reesei CBH1 increased activity by another 67%. Kim et al [30] worked with the same enzymes, but used factorial experimental design to optimize the ratios. Rosgaard et al [50] optimized the ratios of four T. reesei enzymes (two CBHs and two EGs).…”

Section: The Future Of Enzyme Research: Defined Enzyme Mixturesmentioning

confidence: 99%

Improving Enzymes for Biomass Conversion: A Basic Research Perspective

2010

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The cost of enzymes for converting plant biomass materials to fermentable sugars is a major impediment to the development of a practical lignocellulosic ethanol industry. Research on enzyme optimization with the goal of reducing the cost of converting biomass materials such as corn stover into glucose, xylose, and other sugars is being actively pursued in private industry, academia, and government laboratories. Under the auspices of the Department of Energy Great Lakes Bioenergy Research Center, we are taking several approaches to address this problem, including "bioprospecting" for superior key enzymes, protein engineering, and high-level expression in plants. A particular focus is the development of synthetic enzyme mixtures, in order to learn which of the hundreds of known enzymes are important and in what ratios. A core set comprises cellobiohydrolase, endoglucanase, β-glucosidase, endoxylanase, and β-glucosidase. Accessory enzymes include esterases, proteases, nonhydrolytic proteins, and glycosyl hydrolases that cleave the less frequent chemical linkages found in plant cell walls.

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“…In order to accelerate the development of better enzyme cocktails, several laboratories have developed high-throughput platforms for enzyme discovery and characterization. Efforts in this area have incorporated one or more of the following properties also found in GENPLAT: robotic dispensing of enzymes and biomass slurries, statistical design of experiment, and/or automated determination of Glc and Xyl (Berlin et al, 2007;Decker et al, 2009;Kim et al, 1998;King et al, 2009). GENPLAT extends these earlier efforts, most significantly in the complexity of the enzyme mixtures that can be analyzed from at most 6 components in the earlier studies to more than 16 in our latest work (Banerjee et al, 2010c).…”

Section: Discussionmentioning

confidence: 99%

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

Walton¹,

Banerjee²,

Car³

2011

JoVE

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The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.

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Factorial optimization of a six-cellulase mixture

Cited by 43 publications

References 22 publications

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Improving Enzymes for Biomass Conversion: A Basic Research Perspective

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

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