Rapid optimization of enzyme mixtures for deconstruction of diverse pretreatment/biomass feedstock combinations.

Abstract

Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings.
When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-β1,4-xylanase (EX3) and a lower proportion of endo-β1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, β-mannanase, amyloglucosidase, α-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-β1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-β1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and β-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h.
The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.

Full text

Page 1

RESEARCHOpen Access
Rapid optimization of enzyme mixtures for
deconstruction of diverse pretreatment/biomass
feedstock combinations
Goutami Banerjee, Suzana Car, John S Scott-Craig, Melissa S Borrusch, Jonathan D Walton*
Abstract
Background: Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from
lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for
multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT,
which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays.
Proportions of six core fungal enzymes (CBH1, CBH2, EG1, b-glucosidase, a GH10 endo-b1,4-xylanase, and b-
xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all
combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers’ grains plus solubles
[DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline
peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for
three pretreatment/substrate combinations. Results were compared to the performance of two commercial
enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings.
Results: When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and
with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl
yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers
pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and
digested with the core set required a higher proportion of endo-b1,4-xylanase (EX3) and a lower proportion of
endo-b1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized
enzyme mixture containing 16 components (by addition of a-glucuronidase, a GH11 endoxylanase [EX2], Cel5A,
Cel61A, Cip1, Cip2, b-mannanase, amyloglucosidase, a-arabinosidase, and Cel12A to the core set) was determined
for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover
contained more exo-b1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-b1,4-glucanase (EG1 + Cel5A)
than the optimal mixture for AFEX-corn stover. Amyloglucosidase and b-mannanase were the two most important
enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or
AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover
reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover.
Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but
the 16-component set was as effective as the commercial enzyme mixtures at 48 h.
Conclusion: The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails
for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the
Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve
synthetic enzyme mixtures further by the addition of additional accessory enzymes.
* Correspondence: walton@msu.edu
Department of Energy Great Lakes Bioenergy Research Center and
Department of Energy Plant Research Laboratory, Michigan State University,
East Lansing MI 48824, USA
Banerjee et al. Biotechnology for Biofuels 2010, 3:22
http://www.biotechnologyforbiofuels.com/content/3/1/22
© 2010 Banerjee et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

Page 2

Background
The cost of enzymes for the release of fermentable sugars
from plant cell wall polysaccharides remains one of the
major hurdles to the development of an economically
viable cellulosic ethanol industry [1,2]. Currently avail-
able enzyme mixtures are complex and only partially
defined mixtures of more than 80 proteins [3]. A better
understanding of which enzymes, and their proportions,
are important for lignocellulosic degradation could even-
tually lead to the rational design of more efficient, and
hence less expensive, enzyme mixtures. As an approach
to addressing this problem, a high-throughput analysis
platform, called GENPLAT, has been developed. GEN-
PLAT utilizes individual purified enzymes, statistical
experimental design, robotic pipetting of slurries and
enzymes, and automated colorimetric determination of
released sugars [4,5]. With GENPLAT, it is possible to
optimize mixtures of pure enzymes for the release of Glc
and Xyl from different substrates.
Several laboratories have shown that it is possible to
construct multicomponent mixtures of pure endo- and
exocellulases, endoxylanases and debranching enzymes
that equal (for Glc) or surpass (for Xyl) commercial pre-
parations [4-8]. Our earlier studies focused on a single
pretreatment/substrate, namely, ammonia fiber expansion
(AFEX)-treated corn stover. It would be desirable, how-
ever, for ethanol producers to be able to utilize a variety of
lignocellulosic feedstocks, including different grass stovers
(e.g., sorghum, switchgrass or Miscanthus) as well as other
biomass materials such as corn cobs, dried distillers’ grains
(DDG) or mixed native prairie [9,10]. Because different
feedstocks respond differently to different pretreatment
conditions (e.g., steam, hot water, ionic liquids, dilute acid,
AFEX, or alkaline peroxide), there are a large number of
possible pretreatment/substrate combinations. The ligno-
cellulosic industry of the future will likely use many differ-
ent pretreatment/biomass combinations, and it will
therefore be necessary to have enzyme mixtures that can
handle all of these different pretreatment/biomass combi-
nations. Currently available commercial enzyme prepara-
tions are limited in number and composition and have
generally been optimized for acid-pretreated stover from
corn and other grasses. They are therefore unlikely to be
well adapted for the spectrum of pretreatment/biomass
combinations that exist now or that may emerge in the
future.
One solution to this problem is to design enzyme mix-
tures for specific applications. Given a sufficiently abun-
dant source of individual pure enzymes, cocktails could
be designed for specific pretreatment/biomass combina-
tions, plant species, cultivars or even individual batches
of biomass as they are delivered to the processing site.
To do so, there must be a method to rapidly optimize
custom cocktails. We here show that GENPLAT can be
used to construct multicomponent synthetic enzyme
cocktails for release of Glc and Xyl from a variety of dif-
ferent pretreatment/biomass combinations.
Methods
Plant materials and pretreatments
All feedstocks including corn stover were ground before
pretreatment with a Wiley mill to 0.25-mm particle size
instead of 0.5 mm as used earlier [4]. All biomass in the
original plant samples was retained in the final ground
material. For corn (Zea mays) stover, the AFEX condi-
tions have been described previously [4], and this mate-
rial is referred to as “GLBRC stover.” The switchgrass
(Panicum virgatum) variety was Cave-in-Rock, harvested
in October 2005, and the AFEX conditions were 2:1
ammonia to biomass, 50% moisture, and 30-minute resi-
dence time at 140°C [11]. Miscanthus (Miscanthus ×
gigantea), harvested in the spring of 2005, was originally
obtained from Dr. Steven Long, University of Illinois
(Urbana-Champaign, IL, USA). AFEX conditions for Mis-
canthus were 2:1 ammonia to biomass loading, 233%
moisture, and 5-minute residence time at 160°C [12].
Dried distillers’ grains with solubles (DDGS) were a gen-
erous gift of Carbon Green Bioenergy Woodbury LLC
(Lake Odessa, MI, USA). The AFEX conditions for
DDGS were 1:1 ammonia to biomass loading, 60% moist-
ure, and 15-minute residence time at 140°C [13]. Poplar
(Populus nigra), originally obtained from the National
Renewal Energy Lab (Golden, CO, USA), was treated
with AFEX at 2:1 ammonia to biomass loading, 233%
moisture, and 30-minute residence time at 180°C [14].
For mild base pretreatment of all feedstocks, 50 ml of
0.25% (wt/vol) NaOH (62.5 mM) was added to 1 g of
feedstock in a 250-ml Erlenmeyer flask [15]. The flasks
were incubated at 90°C for 4 h without shaking. The
slurry was neutralized by dropwise addition of 12 N
HCl, and the entire contents were dried by lyophiliza-
tion before use in the enzymatic digestions. In every
case, all of the plant material entering the pretreatment
cycle was still present at the end of the cycle. Final glu-
can loadings in the enzyme digestions were adjusted to
compensate for the mass of the salt introduced by the
pretreatment.
For alkaline peroxide (AP) pretreatment of all feed-
stocks, 50 ml of 1% H2O2was adjusted to pH 11.5 with
5 M NaOH and mixed with 1 g of feedstock in a 250-ml
Erlenmeyer flask. Final concentrations were 1% H2O2
(300 mM), 0.8% NaOH (200 mM) and 2% biomass. The
flasks were incubated for 24 h at 24°C with shaking at 90
rpm. The slurries were then neutralized to pH 7 by drop-
wise addition of 12 N HCl. Residual H2O2was inactivated
by addition of 50 μl of catalase (28 mg protein/ml, 21,600
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U/mg protein; catalog no. C30-100MG, Sigma-Aldrich,
St. Louis, MO, USA). Following inactivation of the catalase
by heating at 90°C for 15 minutes, the entire contents of
the flasks were lyophilized before use in the enzymatic
digestions. As noted above, the final glucan loadings were
adjusted to compensate for the mass of the salt introduced
by the pretreatment.
Enzymes
The commercial enzyme mixtures used as benchmarks
were from the same lots described in Banerjee et al. [5].
Multifect-Pectinase (lot no. A216353001) was a gift
from Genencor, Inc. (Palo Alto, CA, USA). Individual
enzymes were produced from native Trichoderma reesei
genes expressed in Pichia pastoris or T. reesei and were
from the same batches used earlier [4,5]. CBH1 (Cel7A)
(from T. longibrachiatum), endo-b1,4-mannanase (from
Aspergillus niger) and amyloglucosidase (g-amylase)
(from A. niger) were purchased from Megazyme, Ltd.
(Brea, Ireland) (catalog nos. E-CBH1, E-BMANN, and
E-AMGDF100, respectively).
The DOE Joint Genome Institute (JGI) identifiers for the
T. reesei proteins used in this paper, and their alternate
names and abbreviations, are CBH1 (Cel7A), Tr_123989;
EG1 (Cel7B), Tr_122081; CBH2 (Cel6A), Tr_72567;
Cel5A, Tr_120312; Cel12A, Tr_123232; b-glucosidase
(BG), Tr_76672; endo-b1,4-xylanase 2 (EX2), Tr_123818;
endo-b1,4-xylanase 3 (EX3), Tr_120229; b-xylosidase
(BX), Tr_121127; Cip1, Tr_73638; Cip2 (glucuronyl ester-
ase), Tr_123940; Cel61A, Tr_73643; a-arabinosidase 2
(Abf2), Tr_76210; and a-glucuronidase (a-Glr), Tr_72526)
http://genome.jgi-psf.org/Trire2/Trire2.home.html.
Hydrolysis
Enzyme hydrolysis was performed in a 96-deep well for-
mat using the GLBRC Enzyme Platform (GENPLAT) as
described earlier [4,5]. Feedstocks were suspended and
dispensed at 0.5% glucan, and final glucan loadings were
0.2%. Unless otherwise specified, enzyme loadings for all
commercial benchmarks and for all mixture experiments
were kept constant at 15 mg/g glucan, and reaction
mixtures were incubated for 48 h at 50°C.
Design-Expert software (Stat-Ease Inc., Minneapolis,
MN, USA) was used for experimental design and analy-
sis. An augmented quadratic design was used through-
out; thus, mixtures containing 6 and 16 components
required 28 and 153 individual reactions, respectively.
The lowest proportion of any enzyme in the core set
(defined as CBH1, CBH2, EG1, BG, EX3, and BX) was
set to 4%, because earlier studies indicated that for most
of the core set, allowing them to go to 0% led to such
poor Glc yields that reliable models could not be pre-
dicted [4]. The lowest proportion of all other enzymes
(“accessory” proteins) was set to 0%. All assays were
replicated once, sampled twice and assayed for Glc and
Xyl twice, for a total of eight replicates of each mixture.
Glc and Xyl were assayed colorimetrically [4]. Model
predictions were tested experimentally as indicated in
each table.
The monosaccharide composition of feedstocks was
determined by the GLBRC Analytical Laboratory at
Michigan State University. Briefly, samples were ground
and washed sequentially with water, 70% ethanol, 1:1
chloroform:methanol, and acetone. The samples were
then treated with amyloglucosidase + a-amylase, and
the released Glc was quantitated as starch. The remain-
ing material was then hydrolyzed with 2 N trifluoroace-
tic acid, and the released sugars were quantitated by GC
of the alditol acetates. The insoluble residue from this
step was treated with Updegraff’s reagent, and the inso-
luble material was hydrolyzed with strong sulfuric acid
and quantitated as cellulose using anthrone [16,17].
The proteins in the commercial preparation Novo-
zyme 188 and in b-mannanase (Megazyme catalog
E-BMANN) were analyzed using standard mass spectro-
metry-based proteomics [3]. Scaffold version 01_07_00
(Proteome Software, Portland, OR, USA) was used to
probabilistically validate protein identifications (DOE
Joint Genome Institute) using the X!Tandem and Pro-
teinProphet computer algorithms.
Results
Commercial enzyme benchmarks
Two commercial T. reesei enzyme preparations, Spe-
zyme CP and Accellerase 1000, were used as bench-
marks. Spezyme CP contains more than 80 proteins of
comparable quantity and identity to those found in the
secretome of T. reesei RUTC30 grown on corn stover [3].
Accellerase 1000 is a T. reesei-based product with
enhanced b-glucosidase activity http://www.genencor.
com.
All combinations of three pretreatments (ammonia
fiber expansion [AFEX], dilute base [0.25% NaOH] or
alkaline peroxide [AP]) and five feedstocks (corn stover,
switchgrass, Miscanthus, dried distillers’ grains + solubles
[DDGS] or poplar) were treated with the two commercial
enzymes at loadings of 15 mg/g glucan (Table 1).
Throughout this paper, Glc yields are expressed as a per-
centage of total Glc. Because at no stage of grinding, pre-
treatment, or digestion was any of the biomass discarded,
the percentages reported are identical for the original
feedstocks. Glc yields ranged from 17.1% to 68.6%, and
Xyl yields ranged from 3.5% to 54.8% (Table 1). Acceller-
ase 1000 was usually superior to Spezyme CP for Glc
release, probably because of its enhanced b-glucosidase
activity. Spezyme CP consistently released less Glc from
substrates pretreated with 0.25% NaOH compared to the
other pretreatments, a trend not seen with Accellerase
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1000. For Xyl yields, Spezyme CP and Accellerase 1000
were approximately equal for all AFEX-pretreated bio-
mass materials, but Spezyme CP was consistently super-
ior for 0.25% NaOH and AP (Table 1).
Comparing pretreatments, AP released more Glc than
either 0.25% NaOH or AFEX from corn stover, switch-
grass or Miscanthus, but AFEX and AP were approxi-
mately equally effective on DDGS. AP also generally
outperformed the other two pretreatments for Xyl yield.
None of the pretreatments with either Spezyme CP or
Accellerase 1000 released Glc effectively from poplar
(maximum of 18.0%), although 0.25% NaOH and AP
released reasonable levels (37.3%-44.4%) of Xyl from
poplar.
Comparing substrates, corn stover consistently yielded
the most Glc and poplar the least. DDGS consistently
yielded the least Xyl. Overall, Accellerase 1000 digestion
of AP-corn stover gave the highest Glc yield (68.6%),
and Spezyme CP digestion of AP-Miscanthus gave the
highest Xyl yield (54.8%).
Core set optimization
The relative proportions of a “core” set composed of six
pure enzymes (CBH1 [Cel7A], CBH2 [Cel6A], Cel7B
[EG1], a GH10 xylanase [EX3], b-glucosidase [BG], and
b-xylosidase [BX]) [4,5] were independently optimized
for all 15 pretreatment/substrate combinations. Total
enzyme loading was held constant at 15 mg/g glucan.
Table 2 shows the model prediction and experimental
results for Glc, Table 3 shows the results for Xyl, and
Additional file 1, Table S1 shows the optimized model
and results for a 1:1 ratio of Glc and Xyl. Glc yields
ranged from 9.8% (poplar pretreated with 0.25% NaOH)
to 58.2% (corn stover pretreated with AP). For every
substrate except poplar, for which no pretreatment gave
> 13.8% yield, AP was superior to the other two pre-
treatments, as was also seen with the commercial
enzymes (Table 1). Corn stover gave the highest yields
of any substrate, followed by switchgrass.
Comparing Glc yields obtained with the commercial
enzymes to the core set optimized for each pretreat-
ment/substrate combination, the core set mixtures per-
formed about as well as Spezyme CP for most substrate/
pretreatment combinations, although yields with the
core set were higher (25.9% yield vs. 16.4%) for 0.25%
NaOH-switchgrass and lower for both AFEX-Mis-
canthus (22.8% vs. 36.2%) and AFEX-DDGS (22.6% vs.
32.6%). Accellerase 1000 consistently outperformed the
core set for Glc yield but gave about the same yield of
Xyl, consistent with our earlier results [4].
With regard to the optimal proportions of the core
enzymes for Glc release from the different pretreat-
ment/substrate combinations, CBH1 ranged from 16%
(for AP-DDGS) to 49% (for 0.25% NaOH-corn stover).
A noticeable trend was that the optimal proportions of
EX3 were higher for AFEX-pretreated corn stover,
switchgrass, and DDGS than for the other two pretreat-
ments. For all substrates treated with 0.25% NaOH, EX3
was required only at the lowest proportion (4%-5%).
This trend did not follow for Miscanthus, perhaps
because Miscanthus has the lowest Xyl content of the
three grass feedstocks (Additional file 1, Table S2). The
elevated EX3 requirement was generally compensated by
a lower loading of CBH1, i.e., CBH1 was present at a
Table 1 Commercial enzyme benchmarks for release of Glc and Xyl from 15 pretreatment/feedstock combinations*
FeedstockPretreatmentGlc release (%)
No enzymeSpezyme CPAccellerase 1000
Xyl release (%)
Spezyme CPNo enzyme Accellerase 1000
Corn stover AFEX1.0 ± 0.0
1.1 ± 0.0
0.4 ± 0.0
2.1 ± 0.0
0.6 ± 0.1
1.4 ± 0.0
1.2 ± 0.3
0.0 ± 0.0
0.4 ± 0.1
0.8 ± 0.1
1.2 ± 0.1
2.8 ± 0.1
2.1 ± 0.3
0.1 ± 0.1
0.8 ± 0.0
44.0 ± 0.0
40.7 ± 0.1
58.4 ± 0.1
22.9 ± 0.1
16.4 ± 0.1
41.6 ± 0.4
36.2 ± 0.3
13.0 ± 0.0
37.7 ± 0.6
32.6 ± 0.4
26.4 ± 0.2
31.5 ± 0.2
18.0 ± 0.1
10.3 ± 0.3
17.8 ± 0.2
50.6 ± 0.3
54.1 ± 0.3
68.6 ± 0.3
28.1 ± 0.0
31.1 ± 0.1
50.6 ± 0.1
35.1 ± 0.0
23.1 ± 0.3
43.8 ± 0.3
42.6 ± 0.4
32.0 ± 0.2
38.1 ± 0.3
17.6 ± 0.0
17.1 ± 0.4
18.0 ± 0.0
1.2 ± 0.0
1.2 ± 1.2
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.7 ± 0.3
0.8 ± 0.3
1.1 ± 0.7
0.8 ± 0.1
2.1 ± 0.5
0.0 ± 0.0
0.3 ± 0.0
0.1 ± 0.1
34.4 ± 0.5
35.4 ± 0.2
49.6 ± 0.6
22.6 ± 0.7
30.5 ± 0.2
45.1 ± 0.2
36.9 ± 1.0
27.8 ± 0.2
54.8 ± 2.2
5.6 ± 0.2
3.8 ± 0.8
7.3 ± 0.6
21.3 ± 0.8
44.4 ± 0.1
43.9 ± 1.4
30.0 ± 0.6
22.9 ± 0.3
38.0 ± 0.6
23.4 ± 0.1
23.2 ± 0.1
34.6 ± 0.3
36.8 ± 0.5
22.2 ± 0.3
42.3 ± 0.2
4.9 ± 1.2
3.5 ± 0.6
6.4 ± 0.8
21.5 ± 0.3
32.3 ± 0.5
37.3 ± 0.7
0.25% NaOH
Alk. peroxide
AFEX
0.25% NaOH
Alk. peroxide
AFEX
0.25% NaOH
Alk. peroxide
AFEX
0.25% NaOH
Alk. peroxide
AFEX
0.25% NaOH
Alk. peroxide
Switchgrass
Miscanthus
DDGS
Poplar
*Glc and Xyl release are expressed as a percentage of total Glc and Xyl in each substrate (Additional file 1, Table S2). Data are expressed as the means ± 1 SD
(n = 8). All enzyme loadings were 15 mg/g glucan.
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lower proportion for AFEX-pretreated corn stover,
switchgrass and DDGS compared to other pretreat-
ments. Owing to the limited importance of EX3 for
releasing Glc from Miscanthus, CBH1 and EG1 consti-
tuted > 80% of the enzyme cocktail for this substrate,
with the other enzymes needed only at their lowest
limit. (Because the minimum proportion of each core
enzyme was set at 4%, it cannot be determined whether
a particular enzyme whose “optimum” is listed in
Table 2 as 4% is needed at this level or at a lower level.)
The optimum proportions of core enzymes for DDGS
were different from those for corn stover, switchgrass,
or Miscanthus, which is consistent with its very different
sugar composition (Additional file 1, Table S1). For
DDGS, a higher proportion of b-glucosidase and a lower
proportion of CBH1 were optimal across all pretreat-
ments (Table 2). This might be due to a lower degree of
polymerization (DP) of the glucan of DDGS or to a
lower degree of crystallinity.
EG1, BX, and EX3 were the most important enzymes
for optimal Xyl yield, except in the case of AP-treated
switchgrass, for which EG1 was needed only at the
Table 2 Optimized proportions of six core enzymes for release of Glc from 15 pretreatment/substrate combinations*
Optimized enzyme proportions (%)
FeedstockPre-treatmentCBH1 BGEG1
Glc yield (%)
BX EX3CBH2 MPExptl.
Corn stover AFEX35
49
43
31
46
47
42
36
48
28
27
16
44
47
47
12
5
8
4
12
4
4
4
4
20
27
43
4
4
4
26
34
30
23
30
19
42
46
32
23
34
29
40
36
37
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
19
4
11
35
4
22
4
5
7
20
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
42.9
42.0
58.9
23.0
27.0
38.0
23.0
17.0
30.0
21.3
23.1
31.0
14.5
10.0
10.0
43.9 ± 0.5
40.7 ± 1.0
58.2 ± 0.2
24.4 ± 0.6
25.9 ± 0.8
39.1 ± 0.5
22.8 ± 1.3
17.5 ± 0.7
32.1 ± 1.2
22.6 ± 0.8
23.8 ± 0.5
29.8 ± 0.5
13.8 ± 0.1
9.8 ± 0.1
10.5 ± 0.2
0.25% NaOH
Alk. peroxide
AFEX
0.25% NaOH
Alk. peroxide
AFEX
0.25% NaOH
Alk. peroxide
AFEX
0.25% NaOH
Alk. peroxide
AFEX
0.25% NaOH
Alk. peroxide
Switchgrass
Miscanthus
DDGS
Poplar
*MP, model prediction. Exptl., experimental results. Data are expressed as means ± 1 SD (n = 8). All enzyme loadings were 15 mg/g glucan.
Table 3 Optimized proportions of six core enzymes for release of Xyl from 15 pretreatment/substrate combinations*
Optimized enzyme proportions (%)Xyl yield (%)
Feedstock PretreatmentCBH1 BGEG1BX EX3CBH2MPExptl.
Corn stoverAFEX7
23
4
4
17
4
14
20
8
-
-
-
4
7
4
4
4
4
4
4
4
4
4
4
-
-
-
4
4
4
20
23
30
18
23
4
20
28
24
-
-
-
11
25
23
26
23
35
23
31
37
30
25
29
-
-
-
28
16
19
39
23
23
47
21
47
28
19
31
-
-
-
47
43
46
4
4
4
4
4
4
4
4
4
-
-
-
5
4
4
28.2
29.0
40.4
26.2
27.0
40.5
37.2
22.6
42.3
-
-
-
19.6
23.0
25.2
27.8 ± 0.7
30.1 ± 0.5
40.0 ± 2.0
24.8 ± 0.5
26.3 ± 0.6
39.5 ± 0.3
35.5 ± 1.0
23.0 ± 0.0
40.6 ± 0.9
< 7.0
< 7.0
< 7.0
21.0 ± 0.0
23.8 ± 1.1
26.0 ± 0.5
0.25% NaOH
Alk. peroxide
AFEX
0.25% NaOH
Alk. peroxide
AFEX
0.25% NaOH
Alk. peroxide
AFEX
0.25% NaOH
Alk. peroxide
AFEX
0.25% NaOH
Alk. peroxide
Switchgrass
Miscanthus
DDGS
Poplar
*MP, model prediction. Exptl., experimental results. Data are expressed as means ± 1 SD (n = 8). All enzyme loadings were 15 mg/g glucan. Xyl release from all
DDG/pretreatment combinations was too low to construct statistically significant models.
Banerjee et al. Biotechnology for Biofuels 2010, 3:22
http://www.biotechnologyforbiofuels.com/content/3/1/22
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