Biocatalysis for Carbohydrate Conversion...J.K. Blum, and M.J. Abrahamson, Curr. Opin. Chem. Biol....
Transcript of Biocatalysis for Carbohydrate Conversion...J.K. Blum, and M.J. Abrahamson, Curr. Opin. Chem. Biol....
Biocatalysis for Carbohydrate Conversion
Andreas S. BommariusGeorgia Institute of Technology
ChBE, also CHEMDeveloping and Advancing Opportunities in the Bioeconomy
Atlanta, GA; March 10-11, 2015
Non-Ideal Biocatalyst
Reaction Constraints
Compromised Process
Conventional Development
Reaction Constraints
Create the Ideal Biocatalyst
“Ideal” Process
Ideal Development
Paradigm shift in biotechnology process development
S.G. Burton, D.A. Cowan, and J.M. Woodley, "The Search for the Ideal Biocatalyst", Nature Biotechnol. 2002, 20, 37-45
PerspectivePathway to developing a biocatalyst useful in synthesis has evolved
A.S. Bommarius, J.K. Blum, and M.J. Abrahamson, Curr. Opin. Chem. Biol. 2011, 15, 194-200
Contents
• Overview• Deconstruction of cellulose• Synthetic reactions with carbohydrates
– Biomonomers• 1,3-propanediol (PDO)• Furandicarboxylic acid (FDCA)
– Bulk Chemicals• Fructose from Glucose• Ascorbic acid
Summary and Perspective
• Process opportunities exist for cellulose, hemicellulose, and lignin
• To succeed, products from renewables have to feature superior properties w.r.t. products from non-renewables, not just feature “Greenness”
• Request: set the goal to RBI (i.e. the faculty) to develop process routes (incl. catalysts, solvents) – from defined raw materials to defined products, or– to products with defined properties.
O
OO
OH
OH
OH
HOHO
OHO
O
OO
OH
OH
OH
HOHO
OHO
O
OO
OH
OH
OH
HOHO
OHO
O
OO
OH
OH
OH
HOHO
OHO
O
OO
OH
OH
OH
HOHO
OHO
O
OO
OH
OH
OH
HOHO
OHO
O
OO
OH
OH
OH
HOHO
OHO
O
OO
OH
OH
OH
HOHO
OHO
OHO
HO
H3CO
OH
OCH3
OCH3
O
O
O
OH
OCH3
OCH3
H3CO
OO
HO
H3CO
HO
OCH3
OCH3
OHO
HO
H3CO
OH
OCH3
OCH3O
O
OH
OCH3
OCH3
OCH3
OO
O
OH
HO
O
OO
O
OH
HO
OH
OH
OO
O
OH
HO
OH
OH
OO
O
OH
HO
OH
OH
O
OO
OH
OH
OH
HOHO
OHO
O
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OH
OH
OH
HOHO
OHO
O
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OH
OH
OH
HOHO
OHO
O
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OH
OH
OH
HOHO
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O
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OH
OH
OH
HOHO
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O
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OH
OH
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Lignin: 15-25% Complex network of aromatic compounds High energy content Treasure trove of novel chemistry
Hemicellulose: 23-32% A collection of 5- and 6-carbon sugars linked together in long, substituted chains- branched Xylose, arabinose, glucose, mannose and galactose
Cellulose: 38-50% Long chains of beta-linked glucose Semicrystalline structure
Major ComponentsIn LignocellulosicBiomass
J.D. McMillan, NREL
MI Pretreatment on Lignocelluloses
0
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0 20 40 60 80 100 120Pe
rcen
tage
lign
in d
isso
lved
Solid loading (g/L)
SBL SEWSSEB SELP
MI Ctrl
Temp: 25°CTime: 5 min
MI: 100%
Mechanical mixture of Avicel and lignin (1:1 w/w)
• MI – efficient delignifier• Extract lignin without dissolving, degrading or altering cellulose crystal structure
Bagasse (SEB) and Wheat straw (SEWS) provided by Dr. G. Zacchi
1-methylimidazole m.p. b.p.
MI - 6oC 198oC
Y. Kang et al., Biotechnol. Progr. 2015, 31, 25-34
New PSE fellowship recipient: Thomas Kwok
Cellobiohydrolase is a molecular machine
U.S. Department of Energy Genome Programs (http://genomics.energy.gov)
Cellobiohydrolase (CBH, exo-glucanase, E.C. 3.2.1.x) consists of several domains that help to pre-organize the cellulose chain; some of the major issues are: - kinetics on a heterogeneous surface, and - processivity: number of glucose/cellobiose units cleaved during a binding event
Cellulose-binding domain (CBD)
Catalytic domain
• Heterogeneous biocatalysis
Cellulase attack on cellulose
Find chain end
Cellobiohydrolase I acting on cellulose
Source: NREL (www.nrel.gov)
1. Adsorption
2. Find chain end 3. E:S complex (thread into tunnel)
4. Hydrolysis(bond cleavage, expulsion, de-crystallization)
Beckham GT et al., J. Phys. Chem. B, 2011, 115, 4118–4127 Bansal P et al., Biotechnology Advances, 2009, 27, 833-848
Rate slowdown along conversion• Rate slow-down is not simply due to substrate depletion• Importance of rate-order
dX/dt = [Enzymes cleaving the β-glycosidic bond]*kdX/dt = k*[Eadsorbed,active]*SdX/dt = k*[Eadsorbed,active]*So(1-X)If no rate hindrances, dX/dt ~(1-X)
• Crystallinity influences the order of the reaction
Apparent first order reaction with amorphous cellulose
No apparent rate order with crystalline cellulose (Avicel®)
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0-3 -2.5 -2 -1.5 -1 -0.5 0
ln(1-X)
ln(d
X/dt
)
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Experimental results
• Restart rates account for most of the rate reduction, higher restart rates – the remainder is attributed to clogging
• Hydrolysability (α), Kad decrease with conversion, [E]ads,max does not vary strongly, reactivity (k) no noticeable trend
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alue
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HydrolysabilityKad[E]ads,maxReactivity (k)
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Rat
e (g
luco
se in
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g/m
L)
Conversion (%)
Restart
Uninterrupted
Due to clogging
11P. Bansal, et al., Bioresour Technol., 2012, 107, 243-250
Kinetic studies: summary
Total cellulose Accessible Hydrolysable
Accessible/total ≡ [E]ads,maxHydrolysable/Accessible = αRate/([E]ads,active) = k[E]ads,active/[E]ads = f
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P. Bansal, et al., Bioresour Technol., 2012, 107, 243-250
Factors involved in determining the raterate = k*[Eads,active]*So(1-X) (So – initial substrate conc.)rate = k*[Eads]*f*So(1-X)
f = 1 or α/y, where y = [E]ads/[E]ads,max
Ratio of accessible to total cellulose decreases (adsorption studies) Productive adsorption - fraction of hydrolyzable cellulose
Unproductive adsorption: obstacles, improper orientation of cellulose chain, exhaustion of reactive sites, etc. Cause cannot be explicitly measured or determined but productive adsorption related to hydrolyzability
Intrinsic reactivity decreases with conversion
Top Value Added Chemicals from Biomass (2004 DOE report )
Bio Process for Soronatm: an example of both Metabolic Engineering and Biocatalysis
OOH
OHHOHO
OH
HO OH
HO OHOH
HO OPO3=O
O
O O
O
n 3G
3GT
HO OPO3=OH
HO OGene 3
Gene 4
Gene 1
Gene 2Glucose
“T”
Competition between chemical and biological route on novel polymer
component: 1,3-propanediol
1,3-propanediol
acrolein ethylene oxideH2O, [H2]
Degussa/Dupontprocess
CO, H2Shell process
glucose
E. coli Dupont/Genencor process
Propanediol (PDO) from Cornstarch
Insert Genesin Microbe
Grow “Bugs”Process microbes,separate product,purify, polymerize
and form into end-use
Yeast Bacterium
Glucose Glycerol PDO
1,3 Propanediol3G
TerephthalateT
OO--0-CH2-CH2-CH2-0-C-- ---C--( )
DuPont Soronatm
credit: Ray Miller, Dupont
2,5-FDCA (2,5-furandicarboxylic acid): building block for biopolymers
2,5-FDCA is a building block for polyethylene furanoate (PEF) or polypropylene furanoate (PPF) polyesters from renewables
PET Plastic• Petroleum-based• Food packaging (e.g., soda
and water bottles)• Textiles (e.g., polyester)• 19.1 Megatons by 2017
Smithers Pira organization. 2012.
PEF Plastic• Bio-based
• From Hydroxymethylfurfural(HMF)
• Avantium (YXY), Bird Engineering (Netherlands)
• Material properties superior to those of PET
Poly(ethylene terephthalate) vs. poly(ethylene furanoate)
O
OO
O
n
OO
OO
O
n
Burgess, et al. Macromolecules 2014. dx.doi.org/10.1021/ma5000199
Survey of routes to FDCAGlucose ↔
Harrison B. Rose Georgia Institute of Technology
Hydroxymethyl-furfural oxidase
(HMFO)
HFMO is a very new enzyme- Flavoprotein- O2-dependent
W.P. Dijkman, ACS Catalysis 2015, 5, 1833-39
Food: glucose isomerase (GI), the commercially most important biocatalyst (!?)
E.C. 5.3.1.5.
Tetramer, composed of two dimers
Subunit: 43 kDa
Mg catalytically essential, also requirement for Co; Mg/Ca-ratio critical for proper activity
Found originally as a xylose isomerase
Starch
Liquefaction
Saccharification
Isomerization
Slurry(40% solid)
pH 3.5-4.2
Requires:pH 6.0-6.2, Ca++
Thermo-tolerant:105°C Short Step95 °C 1-2 hr
α-amylasebreaks starch into 10-13 sugar units
NaOH
Requires:pH 4.2-4.5
GlucoamylaseBreaks into glucosemonomers
HCl
Glucose IsomeraseConverts glucose to fructose
NaOH
42% Fructose
Requires:pH 7.8
Chrom
.
Enrichm
ent$$$$
Final Product:55% Fructose
90% Fructose
Example of a Sub-optimal Process
Crabb and Shetty. (1999). Curr Opinion Micro 2:252-256
A More Efficient Process
Starch
Liquefaction
Saccharification
Isomerization
Slurry(40% solid)
pH 3.5-4.2
Requires:pH 6.0-6.2, Ca++
Thermo-tolerant:105°C Short Step95 °C 1-2 hr
α-amylasebreaks starch into 10-13 sugar units
NaOH
Requires:pH 4.2-4.5
GlucoamylaseBreaks into glucosemonomers
HCl
Glucose IsomeraseConverts glucose to fructose
NaOH
42% Fructose
Requires:pH 7.8
Chrom
.
Enrichm
ent$$$$
Final Product:55% Fructose
90% Fructose
X
X
X
X______________________________
Fine chemicals, vitamins: Process routes to ascorbic acid
Reichstein-Grüssner Sorbitol Fermentation Glucose Fermentationsynthesis
Hydrogenation
Fermentation
Oxidation/Hydrolysis
Acetonization
Esterification
Lactonization
D-Glucose
Diacetone-L-sorbose
D-Sorbitol
2-keto-L-gulonic Acid
L-Sorbose
Methyl 2-keto-L-gulonic Acid
Ascorbic Acid
Fermentation
L-Sorbose
Fermentation
Fermentation
One step Process
ChemicalProcessingTechnology
Ascorbic AcidChotani, G. et al. , Biochimica et Biophysica Acta 1543 (2000) 434-455
Process routes to ascorbic acidTable 20.3: Features of the steps in the Reichstein-Grüssner synthesis Step Yield
(%) cycle time (h); T,p, cat, solvent
Work-up steps [S] (g/L)
Biggest challenges
Hydrogenation 95 2; 140°C, 80-125 bar; Ra-Ni, H2O/MeOH
hot filtration, ion exchange, filtration
Sorbitol oxidation
90 24; 30°C, 2 atm pH 5-6 → 2; H2O
centrifugation, deionization, crystallization
200 Sterility & 2 atm O2 requ.; Ni tank material toxic
Acetonization 85 24; 30°C,135°C /3 Torr; acetone /H2SO4, ether
2x distillation, filtration, vac. Distillation
50
Oxidation 90 6; 50°C; pH H2O, acid, acetone; Pd/C
Precipitation, filtration, drying
Hydrolysis/ rearrangement
85 2; 100°C; pH 2: HCl/MeOH, ClHC, EtOH
distillation, re-crystallization, evaporation
N2/CO2-atm required
ClHC: chlorinated hydrocarbon
Current state of affairs : One-step biological production of 2-keto-L-gulonic acid (2-KLG)
Gluconic Acid 2-keto-D-Gulonic Acid
2,5-diketo-D-Gulonic Acid
D-Glucose
Ascorbic Acid
2-KLG Recovery via Crystallization
Esterification/LactonizationRecovery
E1 E2
E4
E3
E1- glucose dehydrogenaseE2- gluconic acid dehydrogenaseE3- 2-keto-D-gluconic acid dehydrogenaseE4- 2,5-diketo-D-gluconic acid reductase
Chotani, G., Biochimica et Biophysica Acta 1543 (2000) 434-455
2-keto-L-Gulonic Acid (2-KLG)
Messages From This Presentation[Andreas Bommarius]
• Possible applications of the insights/techniques/ findings/opportunities in this presentation – Lowering cost of clean raw materials (glucose, xylose)– Combine use of chemo/bio/catalysis to innovative products
• Barriers and challenges to success– Cellulose hydrolysis to glucose still too expensive– Catalyses compartmentalized, unoptimized for $2-10/kg
products• Additional research opportunities
– (ligno)cellulose structure, cellulase kinetics – Catalyses in cascades and in benign, mostly aqueous solvents