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Understanding Free and Complexed Enzyme Mechanisms and Factors Contributing to
Cell Wall Recalcitrance
Michael G. Resch SIMB SBFC Clearwater, FL 4/29/2014
NREL/PR-2700-60927 NREL is a national laboratory of the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, operated by the Alliance for Sustainable Energy, LLC.
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Acknowledgements
Task Leaders Gregg Beckham Michael Himmel Steve Decker TEM and SEM Imaging Bryon Donohoe Peter Ciesielski Intern Jennifer Nill
Clean Fractionation Rui Katahira Kellene McKinney Cellulose Allomorphs and XRD Ashutosh Mittal BAT Team Techno Economic Analysis Mary Biddy
Funding
Biomass Technologies Office (BETO) Office of Science
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Economic Impact of Reducing Enzyme Loading
Humbird et al. 2011
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Gilbert and Fontes, Ann. Rev. Biochem. 2010
Lynd et al. MMR 2002
“free” cellulases
“Free” vs. Complexed Enzymes
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Different physical mechanisms of denconstruction
Cellulosomes “Free” Cellulases
Resch, M.G. et al. (2013) Energy and Environmental Science
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Do Free and complexed enzymes degrade cellulose synergistically?
Avicel conversion at 10 mg/g
Resch, M.G. et al. (2013) Energy and Environmental Science
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Enzyme Accessibility is Different
“Free” Enzymes Cellulosomes
• Cellulases are evenly distributed
• Cellulosomes are near the cell walls
Possible factors limiting cellulosome activity on DA pretreated biomass • Accessibility • Low koff compared to free cellulases • Binding to lignin
Resch, M.G. et al. (2013) Energy and Environmental Science
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Clean Fractionation Pretreatment (Organosolve)
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CEF
Solvent Temp (°C) Acid (mol/L) Lignin (%) Glucan (%) Xylan (%)
MIBK/Acetone/H2O 140 0.025 10.63 63.31 17.47 MIBK/Acetone/H2O 140 0.05 3.67 82.28 6.56 MIBK/Acetone/H2O 140 0.1 3.33 87.19 3.08
CEF Chemical Composition
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How does Clean Fractionation affect biomass morphology?
140°C 0.1M
Katahira, R. et al (in review)
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How does Clean Fractionation affect enzyme saccharification?
Resch, M. et al (in review)
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Affect of PT Severity on Enzymatic Saccharification
10 mg/g enzyme loading, 72 hours
Resch, M. et al (in review)
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Synergistic Combination of CTec2 and Cellulosomes
Resch, M. et al (in review)
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Technoeconomic Results from CF Work
Mary Biddy (NREL)
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SEM of Digested Cellulose Enriched Fraction
Resch, M. et al (in review)
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TEM of Digested Cellulose Enriched Fraction
Resch, M. et al (in review)
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Model of Enzymatic Deconstruction Mechanisms
Ciesielski, P., Resch, M. et al (in review)
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Cellulose Allomorphs
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Allomorph EH Study Objectives
Isolate cellulose allomorphs with identical crystallinity. How do complexed, free and a combination
perform on cellulose I, II and III? Does the crystallinity change over the course
of digestion? ? How does cellulose type effect the hydrolysis
mechanism with free, complexed and combination?
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Crystal Structure Models
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Cotton linters Allomorphs - XRD
10 20 30 402θ
Cellulose I
Cel I; CI - 62
PASC CL; CI - 44
PASC CL; CI - 34
Amorphous - Ball Milled - 6h
A
10 15 20 25 30 35 402θ
Cellulose II
Cel II @ 145C; CI - 57
Cel II @ 70C; CI - 49
Cel II @ 25C; CI - 40
Amorphous - Ball milled 6h
B
10 15 20 25 30 35 402θ
Cellulose III
Cel III @ 130C; CI - 60
Cel III @ 25C; CI - 49
Cel III @ -33C; CI - 32
Amorphous - Ball milled 6h
C
Allomorph Crystallinity Index (CrI)
Cellulose I 62 44 34
Cellulose II 57 49 40
Cellulose III 60 49 32 Ashutosh Mittal
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Enzymatic Hydrolysis of Cellulose Allomorphs
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XRD – 50% EH with Ctec2
10 15 20 25 30 35 40
Cellulose III - Ctec2
Cellulose III
Cel III Ctec2
10 15 20 25 30 35 40
Cellulose I - Ctec2
Cellulose I
Cel I Ctec2
10 15 20 25 30 35 40
Cellulose II - Ctec2
Cellulose II
Cel II Ctec2
No change in XRD of cellulose samples after 50% digestion with Ctec2
Ashutosh Mittal
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XRD – 50% EH with Cellulosomes
10 15 20 25 30 35 40
Cellulose III - Cellulosomes
Cellulose III Cellulosomes
Cellulose III
10 15 20 25 30 35 40
Cellulose I - Cellulosomes
Cellulose I
Cellulose I Cellulosomes
10 15 20 25 30 35 40
Cellulose II - Cellulosomes
Cellulose II
Cellulose II Cellulosomes
XRD of cellulose II and III allomorphs change after 50% digestion with Cellulosomes • Change in the intensity of XRD from various crystal planes • Emergence of a new peak at 2θ = 24° Do cellulosomes attack crystalline regions selectively? Currently looking at the digestions of cellulose allomorphs with combination of Ctec2 and Cellulosomes
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I
Free Complexed
II ? ?
III ? ?
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Conclusions
• “Free” enzymes and cellulosomes have distinct mechanisms in cellulose and biomass deconstruction
• “Free” enzymes and cellulosomes are synergistic • Biomass composition and enzyme accessibility to cellulose ends affect
cellulosomes more than “free” enzymes • Combining properties of “free” and multi-domain enzymes in
commercial enzyme cocktails could lead to enzyme cost reductions
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Acknowledgements
Task Leaders Gregg Beckham Michael Himmel Steve Decker TEM and SEM Imaging Bryon Donohoe Peter Ciesielski Intern Jennifer Nill
Clean Fractionation Rui Katahira Kellene McKinney Cellulose Allomorphs and XRD Ashutosh Mittal BAT Team Techno Economic Analysis Mary Biddy
Funding
Biomass Technologies Office (BETO) Office of Science
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Clean Fractionation – Xylan Conversion
Resch, M. et al (in review)