1. No category

Biocatalysis for Carbohydrate Conversion
Andreas S. Bommarius
Georgia Institute of Technology
ChBE, also CHEM
Developing and Advancing Opportunities in the Bioeconomy
Atlanta, GA; March 10-11, 2015
Paradigm shift in biotechnology
process development
Conventional
Development
Ideal Development
Non-Ideal
Biocatalyst
Reaction
Constraints
Compromised
“Ideal” Process
Process
Reaction
Constraints
Create the Ideal
Biocatalyst
S.G. Burton, D.A. Cowan, and J.M. Woodley, "The Search for the Ideal
Biocatalyst", Nature Biotechnol. 2002, 20, 37-45
Perspective
Pathway 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.
Major Components
In Lignocellulosic
Biomass
H3CO
HO
OCH3OCH3
OCH3
OH
OH
O
O
OH
OH O
HO
O HO
O
OH

O
O HO
OH
O
O
OH
HO
O HO
O
OH
O
OH
OH O
OH O
O
HO
HO
O
OH
O
OH
HO
OH O
HO
OH O
O
OH
O HO
O
OH
O
OH
HO
OH O
OH
O HO
OH
OH
O
O
OH
OH O
O HO
OH
OH
O
OH
O
HO
O HO
OH O
HO
O
OH
OH O
OH
OH
O
OH
O
O HO
OH
OH
O
O HO
OH
OH O
O HO
O
OH
OH
O HO
OH
OH
O
O
O
O
OH
OH
O
HO
HO
OH
HO
O
OH
OH
O
OH O
O
HO
OH
OH
HO
OH
H3CO
OCH3
O HO
HO
O
O
O
HO
O
OH
OH
OH
O
OH
O
OH
O
OCH3OCH3
Hemicellulose: 23-32%
Long chains of beta-linked glucose
 Semicrystalline structure
O
OH
O
OCH3
OH
O
OCH3
OH
HO
OH
H3CO
OCH3
O
HO
O
HO
Complex network of
aromatic compounds
 High energy content
 Treasure trove of novel
chemistry

OH
OCH3

Cellulose: 38-50%
O
O
Lignin: 15-25%
A collection of 5- and 6-carbon
sugars linked together in long,
substituted chains- branched
 Xylose, arabinose, glucose,
mannose and galactose
H3CO
O
OCH3
O
OH
OH
O
OH
OH O
HO
HO
O HO
OH
O
OH
HO
OH O
OH
O
O
OH
OH O
O HO
OH
O HO
OH
O
OH
OH
O
OH
OH O
HO
O HO
OH
O
OH
OH O
OH
J.D. McMillan, NREL
MI Pretreatment on Lignocelluloses
Mechanical mixture of
Avicel and lignin (1:1 w/w)
MI
Ctrl
100
Temp: 25°C
Time: 5 min
MI: 100%
1-methylimidazole
m.p.
b.p.
MI
- 6oC
198oC
Percentage lignin dissolved
90
80
70
60
50
40
30
20
SBL
SEB
10
• MI – efficient delignifier
• Extract lignin without dissolving, degrading
or altering cellulose crystal structure
Y. Kang et al., Biotechnol. Progr. 2015, 31, 25-34
SEWS
SELP
0
0
20
40
60
80
100
120
Solid loading (g/L)
Bagasse (SEB) and Wheat straw (SEWS) provided by Dr. G. Zacchi
New PSE fellowship
recipient: Thomas Kwok
Cellobiohydrolase is a molecular machine
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)
U.S. Department of Energy Genome Programs (http://genomics.energy.gov)
Catalytic domain
Cellulase attack on cellulose
Cellobiohydrolase I acting on
cellulose
• Heterogeneous biocatalysis
Source: NREL (www.nrel.gov)
1. Adsorption
2. Find
chainend
end
Find
chain
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]*k
dX/dt = k*[Eadsorbed,active]*S
dX/dt = k*[Eadsorbed,active]*So(1-X)
If no rate hindrances, dX/dt ~(1-X)
• Crystallinity influences the order of the reaction
0
-2.5
-2
-1.5
-1
-0.5
-2
-4
-6
0
ln(dX/dt)
-3
-8
-10
-12
ln(1-X)
Apparent first order reaction with
amorphous cellulose
10
No apparent rate order with
crystalline cellulose (Avicel®)
Experimental results
3.5
Normalized parameter value
Rate (glucose in 10 min., mg/mL)
• 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
3
2.5
Due to clogging
Restart
2
1.5
Uninterrupted
1
0.5
0
0
20
40
60
80
100
Conversion (%)
Hydrolysability
1.4
Kad
[E]ads,max
1.2
Reactivity (k)
1
0.8
0.6
0.4
0.2
0
0
20
40
60
80
Conversion (%)
11
P. Bansal, et al., Bioresour Technol., 2012, 107, 243-250
Kinetic studies: summary

Factors involved in determining the rate
rate = k*[Eads,active]*So(1-X)
(So – initial substrate conc.)
rate = k*[Eads]*f*So(1-X)



Total cellulose
Accessible
Hydrolysable
Accessible/total ≡ [E]ads,max
Hydrolysable/Accessible = α
Rate/([E]ads,active) = k
[E]ads,active/[E]ads = f

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
P. Bansal, et al., Bioresour Technol., 2012, 107, 243-250
12
Top Value Added Chemicals from
Biomass (2004 DOE report )
Bio Process for Soronatm: an example of both
Metabolic Engineering and Biocatalysis
OH
HO
HO
O
OPO3=
Gene 1
OH
HO
O
OH
HO
OH
OPO3=
Gene 2
HO
Glucose
OH
Gene 3
O
HO
O
O
Gene 4
HO
O
n
O
3GT
OH
3G
“T”
OH
Competition between chemical and
biological route on novel polymer
component: 1,3-propanediol
acrolein
ethylene oxide
H2O, [H2]
CO, H2
Shell
process
Degussa/Dupont
process
1,3-propanediol
E. coli
glucose
Dupont/Genencor
process
Glucose
Glycerol
Yeast
PDO
Bacterium
DuPont Soronatm
Insert Genes
in Microbe
Grow “Bugs”
O
(
--0-CH
2-CH2-CH2-0-C-1,3 Propanediol
3G
Process microbes,
separate product,
purify, polymerize
and form into end-use
O
)
---C--
Terephthalate
T
Propanediol (PDO) from Cornstarch
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
Poly(ethylene terephthalate) vs. poly(ethylene furanoate)
O
O
O
O
O
O
O
O
O
n
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.
n
PEF Plastic
• Bio-based
• From Hydroxymethylfurfural
(HMF)
• Avantium (YXY), Bird
Engineering (Netherlands)
• Material properties superior to
those of PET
Burgess, et al. Macromolecules 2014. dx.doi.org/10.1021/ma5000199
Survey of routes to FDCA
Glucose ↔
Harrison B. Rose
Georgia
Institute of Technology
Hydroxymethylfurfural 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
Example of a Sub-optimal Process
NaOH
Starch
α-amylase
breaks starch into
10-13 sugar units
HCl
Slurry
(40% solid)
Liquefaction
pH 3.5-4.2
Glucoamylase
Breaks into
glucose
monomers
Requires:
pH 6.0-6.2, Ca++
Saccharification
Thermo-tolerant:
105°C Short Step
95 °C 1-2 hr
Requires:
pH 4.2-4.5
NaOH
Glucose
Isomerase
Converts glucose to
fructose
Isomerization
Chrom.
Requires:
pH 7.8
Enrichment
$$$$
42%
Fructose
90% Fructose
Crabb and Shetty. (1999). Curr Opinion Micro 2:252-256
Final Product:
55% Fructose
A More Efficient Process
NaOH
X
Starch
α-amylase
breaks starch into
10-13 sugar units
Slurry
(40% solid)
HCl
X
Liquefaction
pH 3.5-4.2
Glucoamylase
Breaks into
glucose
monomers
Requires:
pH 6.0-6.2, Ca++
Saccharification
Thermo-tolerant:
105°C Short Step
95 °C 1-2 hr
Requires:
pH 4.2-4.5
NaOH
X
Glucose
Isomerase
Converts glucose to
fructose
Isomerization
Requires:
pH 7.8
X
Chrom.
______________________________
Enrichment
$$$$
42%
Fructose
90% Fructose
Final Product:
55% Fructose
Fine chemicals, vitamins:
Process routes to ascorbic acid
Reichstein-Grüssner
synthesis
Sorbitol Fermentation
Glucose Fermentation
D-Glucose
Hydrogenation
D-Sorbitol
Fermentation
L-Sorbose
Acetonization
Diacetone-L-sorbose
Oxidation/Hydrolysis
Fermentation
L-Sorbose
Fermentation
One step
Process
Fermentation
2-keto-L-gulonic Acid
Esterification
Methyl 2-keto-L-gulonic Acid
Lactonization
Ascorbic Acid
Chemical
Processing
Technology
Ascorbic Acid
Chotani, G. et al. , Biochimica et Biophysica Acta 1543 (2000) 434-455
Process routes to ascorbic acid
Table 20.3:
Step
Features of the steps in the Reichstein-Grüssner synthesis
Yield cycle time (h); T,p,
Work-up steps
(%) cat, solvent
Hydrogenation 95
2; 140°C, 80-125 bar; hot filtration, ion
Ra-Ni, H2O/MeOH
exchange,
filtration
Sorbitol
90
24; 30°C, 2 atm pH 5- centrifugation,
oxidation
deionization,
6 → 2; H2O
crystallization
24; 30°C,135°C /3
Acetonization 85
2x distillation,
Torr; acetone
filtration, vac.
/H2SO4, ether
Distillation
Oxidation
90
6; 50°C; pH H2O,
Precipitation,
acid, acetone; Pd/C
filtration, drying
Hydrolysis/
85
2; 100°C; pH 2:
distillation, rerearrangement
HCl/MeOH, ClHC,
crystallization,
EtOH
evaporation
ClHC: chlorinated hydrocarbon
[S]
Biggest
(g/L) challenges
200
Sterility & 2 atm
O2 requ.; Ni tank
material toxic
50
N2/CO2-atm
required
Current state of affairs : One-step biological
production of 2-keto-L-gulonic acid (2-KLG)
E1
D-Glucose
E2
Gluconic Acid
2-keto-D-Gulonic Acid
E1- glucose dehydrogenase
E2- gluconic acid dehydrogenase
E3- 2-keto-D-gluconic acid dehydrogenase
E4- 2,5-diketo-D-gluconic acid reductase
E3
2,5-diketo-D-Gulonic Acid
E4
2-keto-L-Gulonic Acid (2-KLG)
2-KLG Recovery
via Crystallization
Esterification/Lactonization
Recovery
Ascorbic Acid
Chotani, G., Biochimica et Biophysica Acta 1543 (2000) 434-455
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