U.S. patent application number 16/677198 was filed with the patent office on 2020-04-23 for degradation of lignocellulosic material.
The applicant listed for this patent is DSM IP ASSETS B.V.. Invention is credited to Wilhelmus Theodorus Antonius Maria DE LAAT, Manoj Kumar, Margot Elisabeth Francois Schooneveld-Bergmans.
Application Number | 20200123577 16/677198 |
Document ID | / |
Family ID | 40377618 |
Filed Date | 2020-04-23 |
United States Patent
Application |
20200123577 |
Kind Code |
A1 |
DE LAAT; Wilhelmus Theodorus
Antonius Maria ; et al. |
April 23, 2020 |
DEGRADATION OF LIGNOCELLULOSIC MATERIAL
Abstract
The present invention describes a method for the treatment of
lignocellulosic material which method comprises contacting said
lignocellulosic material with a composition comprising two or more
enzyme activities, said enzyme activities being cellulase and/or
hemicellulase activities, wherein the pH during the treatment is
about 4.5 or lower, and the treatment is carried out at a dry
matter content of 15% or more.
Inventors: |
DE LAAT; Wilhelmus Theodorus
Antonius Maria; (Echt, NL) ; Kumar; Manoj;
(Echt, NL) ; Schooneveld-Bergmans; Margot Elisabeth
Francois; (Echt, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DSM IP ASSETS B.V. |
Heerlen |
|
NL |
|
|
Family ID: |
40377618 |
Appl. No.: |
16/677198 |
Filed: |
November 7, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13057598 |
Feb 4, 2011 |
10557153 |
|
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PCT/EP2009/060098 |
Aug 4, 2009 |
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16677198 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 7/10 20130101; C12P
19/14 20130101; Y02E 50/16 20130101; Y02E 50/17 20130101; C13K 1/02
20130101; C12P 19/02 20130101; Y02E 50/10 20130101 |
International
Class: |
C12P 7/10 20060101
C12P007/10; C12P 19/14 20060101 C12P019/14; C13K 1/02 20060101
C13K001/02; C12P 19/02 20060101 C12P019/02 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 11, 2008 |
EP |
08162154.2 |
Claims
1. A method for the treatment of lignocellulosic material, which
method comprises contacting said lignocellulosic material with a
composition comprising two or more enzyme activities, said enzyme
activities being cellulase and/or hemicellulase activities, wherein
the pH during the treatment is about 4.5 or lower, and the
treatment is carried out at a dry matter content of 15% or
more.
2. The method according to claim 1, wherein the pH during the
treatment is 4,0 or lower.
3. The method according to claim 1, wherein at least one of the
enzyme activities is derived from Talaromyces emersonii.
4. The method according to claim 1, wherein two or more of the
enzyme activities are derived from Talaromyces emersonii.
5. The method according to claim 1, wherein the enzyme activities
are thermostable.
6. The method according to claim 1, wherein the enzyme activities
are capable of acting at low pH.
7. The method according to claim 1, wherein the composition
comprises one, two or all of endoglucanase, cellobiohydrolase or
.beta.-glucosidase activity.
8. The method according to claim 6, wherein the composition
comprises an endoglucanase, cellobiohydrolase and
.beta.-glucosidase all of which are derived from Talaromyces
emersonii.
9. The method according to claim 1, wherein the composition
comprises endo-1,3(1,4)-.beta. glucanase activity and
endo-.beta.-1,4-glucanase activity, both of which activities are
derived from Talaromyces emersonii.
10. The method according to claim 6, wherein the composition
comprises one or more xylanase activities.
11. The method according to claim 1, wherein the composition
comprises an expansin, an expansin-like protein, a cellulose
induced protein, a cellulose integrating protein, a scaffoldin or a
scaffoldin-like protein, optionally derived from Talaromyces
emersonii.
12. The method according to claim 1, wherein the treatment
comprises the degradation, optionally hydrolysis, and/or
modification of cellulose and/or hemicellulose.
13. A method for producing a sugar or sugars from lignocellulosic
material which method comprises contacting said lignocellulosic
material with the composition as defined in claim 1.
14. The method according to claim 13, wherein the sugars are
monomeric and/or multimeric sugars.
15. The method according to claim 1 herein at least one of the
sugars produced is a fermentable sugar.
16. The method according to claim 15, wherein at least one of the
sugars produced is glucose, cellobiose, xylose, arabinose,
galactose, galacturonic acid, glucuronic acid, mannose, rhamnose,
sucrose or fructose,
17. The method according to claim 1, wherein the lignocellulosic
material is subjected to a pretreatment prior to being contacted
with the composition.
18. The method according to claim 17, wherein the pretreatment
comprises exposing the lignocellulosic material to an acid, a base,
a solvent, heat, a peroxide, ozone, mechanical shredding, grinding,
milling or rapid depressurization, or a combination of any two or
more thereof.
19. The method according to claim 1, wherein the lignocellulosic
material is orchard primings, chaparral, mill waste, urban wood
waste, municipal waste, logging waste, forest thinnings,
short-rotation woody crops, industrial waste, wheat straw, oat
straw, rice straw, barley straw, rye straw, flax straw, soy hulls,
rice hulls, rice straw, corn gluten feed, oat hulls, sugar cane,
corn stover, corn stalks, corn cobs, corn husks, switch grass,
miscanthus, sweet sorghum, canola stems, soybean stems, prairie
grass, gamagrass, foxtail; sugar beet pulp, citrus fruit pulp, seed
hulls, cellulosic animal wastes, lawn clippings, cotton, seaweed,
trees, softwood, hardwood, poplar, pine, shrubs, grasses, wheat,
wheat straw, sugar cane bagasse, corn, corn husks, corn hobs, corn
kernel, fiber from kernels, products and by-products from wet or
dry milling of grains, municipal solid waste, waste paper, yard
waste, herbaceous material, agricultural residues, forestry
residues, municipal solid waste, waste paper, pulp, paper mill
residues, branches, bushes, canes, corn, corn husks, an energy
crop, forest, a fruit, a flower, a grain, a grass, a herbaceous
crop, a leaf, bark, a needle, a log, a root, a sapling, a shrub,
switch grass, a tree, a vegetable, fruit peel, a vine, sugar beet
pulp, wheat midlings, oat hulls, hard or soft wood, organic waste
material generated from an agricultural process, forestry wood
waste, or a combination of any two or more thereof.
20. A method for producing a fermentation product, which method
comprises: producing a fermentable sugar using the method according
to claim 1; and fermenting the resulting fermentable sugar, thereby
to produce a fermentation product.
21. The method according to claim 20, wherein the fermentation
product is ethanol, butanol, lactic acid, a plastic, an organic
acid, a solvent, an animal feed supplement, a pharmaceutical, a
vitamin, an amino acid, an enzyme or a chemical feedstock,
22. The method according to claim 20, wherein no acid or base is
added in the process.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/057,598, filed 4 Feb. 2011, which is a
National Stage entry of International Application No.
PCT/EP2009/060098, filed 4 Aug. 2009, which claims priority to
European Patent Application No. 08162154.2, filed 11 Aug. 2008. The
disclosure of the priority applications are incorporated in their
entirety herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to methods for degrading
lignocellulosic material and to methods for producing a sugar or
sugars from such material. The invention also relates to methods
for producing a fermentation product.
BACKGROUND OF THE INVENTION
[0003] Carbohydrates constitute the most abundant organic compounds
on earth. However, much of this carbohydrate is sequestered in
complex polymers including starch (the principle storage
carbohydrate in seeds and grain), and a collection of carbohydrates
and lignin known as lignocellulose. The main carbohydrate
components of lignocellulose are cellulose, hemicellulose, and
pectins. These complex polymers are often referred to collectively
as lignocellulose.
[0004] Bioconversion of renewable lignocellulosic biomass to a
fermentable sugar that is subsequently fermented to produce alcohol
(e.g., ethanol) as an alternative to liquid fuels has attracted an
intensive attention of researchers since 1970s, when the oil crisis
broke out because of decreasing the output of petroleum by OPEC.
Ethanol has been widely used as a 10% blend to gasoline in the USA
or as a neat fuel for vehicles in Brazil in the last two decades.
More recently, the use of E85, an 85% ethanol blend has been
implemented especially for clean city applications. The importance
of fuel bioethanol will increase in parallel with increases in
prices for oil and the gradual depletion of its sources.
Additionally, fermentable sugars are being used to produce
plastics, polymers and other biobased products and this industry is
expected to grow substantially therefore increasing the demand for
abundant low cost fermentable sugars which can be used as a feed
stock in lieu of petroleum based feedstocks.
[0005] The sequestration of such large amounts of carbohydrates in
plant biomass provides a plentiful source of potential energy in
the form of sugars, both five carbon and six carbon sugars that
could be utilized for numerous industrial and agricultural
processes. However, the enormous energy potential of these
carbohydrates is currently under-utilized because the sugars are
locked in complex polymers, and hence are not readily accessible
for fermentation. Methods that generate sugars from plant biomass
would provide plentiful, economically-competitive feedstocks for
fermentation into chemicals, plastics, and fuels.
[0006] Regardless of the type of cellulosic feedstock, the cost and
hydrolytic efficiency of enzymes are major factors that restrict
the commercialization of the biomass bioconversion processes. The
production costs of microbially produced enzymes are tightly
connected with a productivity of the enzyme-producing strain and
the final activity yield in the fermentation broth.
[0007] In spite of the continued research of the last few decades
to understand enzymatic lignocellulosic biomass degradation and
cellulase production, it remains desirable to discover or to
engineer new highly active cellulases and hemicellulases. It would
also be highly desirable to construct highly efficient enzyme
compositions capable of performing rapid and efficient
biodegradation of lignocellulosic materials.
[0008] In addition, currently available enzymes having cellulase
activity, typically derived from Trichoderma, function at
mesophilic temperatures, such as from 45.degree. C. to 50.degree.
C. and at pH 5.0. This, however, may lead to bacterial infection
reducing product yield, so it is desirable to carry out
saccharification at a temperature of 65.degree. C. or higher. In
addition, the use of mesophilic temperatures increases the
viscosity of the biomass being used such that the dry matter
content used is limited. Also, when acid pretreated biomass is used
as a substrate, the pH must be raised so that the enzyme can
saccharify the sugars in the biomass. In the context of a
commercially viable fuel ethanol industry, this implies a
requirement for, for example, sodium hydroxide or calcium sulphate
and the production of huge quantities of the corresponding salts,
for example gypsum in the case of sodium hydroxide. Accordingly, it
is desirable to carry out saccharification using an enzyme which
can operate at a pH of pH 4.0 or lower.
SUMMARY OF THE INVENTION
[0009] We have shown that an enzyme preparation derived from
Talaromyces emersonii can extremely effectively hydrolyze
lignocellulolytic material, for example corn stover or wheat straw,
into monomeric sugars which can then be converted into a useful
product, such as ethanol. The enzyme preparation comprises
cellulase and hemicellulase activities.
[0010] Surprisingly, this invention now shows that the said enzyme
preparation can be used to carry out highly effective hydrolysis of
a lignocellulosic substrate (achieving in excess of 90% conversion
of cellulose). The preparation has a higher specific activity than
other products available in the market. This is highly significant
in the context of commercially viable fuel ethanol production from
lignocellulosic biomass since lower amounts of enzyme will be
required (as compared with currently available products).
[0011] Moreover, this hydrolysis may be carried out at a high
temperature which (i) reduces the risk of bacterial infection and
(ii) results in a less viscous biomass pulp. The effect of the
latter is significant since it enables the better blending of
enzymes, resulting in a higher operational dry matter in the plant
and allows a consequent higher ethanol concentration to be
achieved. Thus, less energy need be used improving sustainability
and a smaller fermentation process will be required requiring lower
investment.
[0012] Also, this hydrolysis may be carried out at low pH. This is
desirable since biomass is often pretreated with acid. Biomass
treated in this way does not have to be pH adjusted if the enzymes
subsequently used for saccharification are capable of acting at low
pH. This implies a lower requirement of, for example, sodium
hydroxide or calcium sulphate and a process in which there is no
waste salt. This is significant in a process in which, for example,
fuel ethanol is to be produced since huge quantities of material
are consumed in such processes. This allows a process to be carried
out in which no pH adjustment is required, i.e. there is no
requirement for the addition of acids or bases. The process may
thus be carried out as a zero waste process and/or as a process in
which no inorganic chemical input is required.
[0013] In addition, it has been shown that the enzyme composition
can effectively hydrolyze biomass when high dry matter contents are
used. It is highly desirable that enzymes used in the production
of, for example, fuel ethanol are able to operate on substrates
having high viscosity (i.e. high dry weight composition) since this
allows higher amounts of the final product, for example, fuel
ethanol, to be achieved.
[0014] According to the invention, there is thus provided a method
for the treatment of lignocellulosic material which method
comprises contacting said lignocellulosic material with a
composition comprising two or more enzyme activities, said enzyme
activities being cellulase and/or hemicellulase activities, wherein
the pH during the treatment is about 4.5 or lower, and the
treatment is carried out at a dry matter content of 15% or
more.
[0015] The invention also provides: [0016] method for producing a
sugar or sugars from lignocellulosic material which method
comprises contacting said lignocellulosic material with a
composition as defined above; [0017] a method for producing a
fermentation product, which method comprises: [0018] producing a
fermentable sugar using a method as set out above; and [0019]
fermenting the resulting fermentable sugar, thereby to produce a
fermentation product; [0020] use of a composition as defined above
in the treatment of lignocellulosic material; and [0021] use of a
composition as defined above in the production of a sugar or sugars
from lignocellulosic material.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1a-1c show glucose formation from dilute acid
pretreated corn stover using different enzyme dosages at 50.degree.
C., pH 4.5-5: FIG. 1a=GC 220; FIG. 1b=Filtrase.RTM.NL; and FIG.
1c=Laminex BG.
[0023] FIGS. 2a-2c and 3a-3c show specific activity data for sugar
formation from dilute acid pretreated corn stover: FIGS. 2a and
3a=specific activity at 21 hours; FIGS. 2b and 3b=specific activity
at 93 hours; and FIGS. 2c and 3c=specific activity at 140
hours.
[0024] FIG. 4 shows a schematic for simultaneous saccharification
and fermentation and distillation experiments.
[0025] FIG. 5 shows ethanol production from dilute acid pretreated
corn stover in simultaneous saccharification and fermentation and
distillation experiments using Filtrase.RTM.NL.
[0026] FIG. 6 shows the hydrolysis yield from dilute acid
pretreated corn stover in simultaneous saccharification and
fermentation and distillation experiments using
Filtrase.RTM.NL.
[0027] FIG. 7 shows sugar formation from wheat straw pretreated
with steam, at 60.degree. C., pH 3.8 using Filtrase.RTM.NL.
[0028] FIG. 8 shows glucose production using Talaromyces
cellulases
DETAILED DESCRIPTION OF THE INVENTION
[0029] Throughout the present specification and the accompanying
claims, the words "comprise" and "include" and variations such as
"comprises", "comprising", "includes" and "including" are to be
interpreted inclusively. That is, these words are intended to
convey the possible inclusion of other elements or integers not
specifically recited, where the context allows.
[0030] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to one or at least one) of the grammatical
object of the article. By way of example, "an element" may mean one
element or more than one element.
[0031] The present invention provides relates to a composition
which comprises cellulolytic and/or hemicellulolytic enzyme
activity and which has the ability to modify, for example degrade,
a non-starch carbohydrate material. A non-starch carbohydrate
material is a material which comprises, consists of or
substantially consists of one or more non-starch carbohydrates.
Carbohydrate in this context includes all saccharides, for example
polysaccharides, oligosaccharides, disaccharides or
monosaccharides.
[0032] A composition as described herein typically modifies a
non-starch carbohydrate material by chemically modification of such
material. Chemical modification of the carbohydrate material may
result in the degradation of such material, for example by
hydrolysis, oxidation or other chemical modification such as by the
action of a lyase.
[0033] A non-starch carbohydrate suitable for modification by a
composition as described herein is lignocellulose. The major
polysaccharides comprising different lignocellulosic residues,
which may be considered as a potential renewable feedstock, are
cellulose (glucans), hemicelluloses (xylans, heteroxylans and
xyloglucans). In addition, some hemicellulose may be present as
glucomannans, for example in wood-derived feedstocks. The enzymatic
hydrolysis of these polysaccharides to soluble sugars, including
both monomers and multimers, for example glucose, cellobiose,
xylose, arabinose, galactose, fructose, mannose, rhamnose, ribose,
galacturonic acid, glucoronic acid and other hexoses and pentoses
occurs under the action of different enzymes acting in concert.
[0034] In addition, pectins and other pectic substances such as
arabinans may make up considerably proportion of the dry mass of
typically cell walls from non-woody plant tissues (about a quarter
to half of dry mass may be pectins).
[0035] Cellulose is a linear polysaccharide composed of glucose
residues linked by 3-1,4 bonds. The linear nature of the cellulose
fibers, as well as the stoichiometry of the .beta.-linked glucose
(relative to .alpha.) generates structures more prone to
interstrand hydrogen bonding than the highly branched
.alpha.-linked structures of starch. Thus, cellulose polymers are
generally less soluble, and form more tightly bound fibers than the
fibers found in starch.
[0036] Endoglucanases (EG) and exo-cellobiohydrolases (CBH)
catalyze the hydrolysis of insoluble cellulose to
cellooligosaccharides (cellobiose as a main product), while
.beta.-glucosidases (BG) convert the oligosaccharides, mainly
cellobiose and cellotriose to glucose.
[0037] Hemicellulose is a complex polymer, and its composition
often varies widely from organism to organism, and from one tissue
type to another. In general, a main component of hemicellulose is
.beta.-1,4-linked xylose, a five carbon sugar. However, this xylose
is often branched at 0-3 and/or 0-2 atom of xylose, and can be
substituted with linkages to arabinose, galactose, mannose,
glucuronic acid, galacturonic acid or by esterification to acetic
acid (and esterification of ferulic acid to arabinose).
Hemicellulose can also contain glucan, which is a general term for
.beta.-linked six carbon sugars (such as the .beta.-(1,3)(1,4)
glucans and heteroglucans mentioned previously) and additionally
glucomannans (in which both glucose and mannose are present in the
linear backbone, linked to each other by .beta.-linkages).
[0038] Xylanases together with other accessory enzymes, for example
.alpha.-L-arabinofuranosidases, feruloyl and acetylxylan esterases,
glucuronidases, and .beta.-xylosidases) catalyze the hydrolysis of
hemicelluloses.
[0039] Pectic substances include pectins, arabinans, galactans and
arabinogalactans. Pectins are the most complex polysaccharides in
the plant cell wall. They are built up around a core chain of
.alpha.(1,4)-linked D-galacturonic acid units interspersed to some
degree with L-rhamnose. In any one cell wall there are a number of
structural units that fit this description and it has generally
been considered that in a single pectic molecule, the core chains
of different structural units are continuous with one another.
[0040] The principal types of structural unit are: galacturonan
(homogalacturonan), which may be substituted with methanol on the
carboxyl group and acetate on O-2 and O-3; rhamnogalacturonan I
(RGI), in which galacturonic acid units alternate with rhamnose
units carrying (1,4)-linked galactan and (1,5)-linked arabinan
side-chains. The arabinan side-chains may be attached directly to
rhamnose or indirectly through the galactan chains;
xylogalacturonan, with single xylosyl units on O-3 of galacturonic
acid (closely associated with RGI); and rhamnogalacturonan II
(RGII), a particularly complex minor unit containing unusual
sugars, for example apiose. An RGII unit may contain two apiosyl
residues which, under suitable ionic conditions, can reversibly
form esters with borate.
[0041] A composition for use in a method of the invention will
comprise enzymatic activities typically derived from a saprophyte
fungal microorganism of the class Penicillium and from the genus
Talaromyces, for example Talaromyces emersonii. Talaromyces
emersonii may also be referred to as Geosmithia emersonii or
Penicillium emersonii. Talaromyces emersonii has also been referred
to as Talaromyces duponti and Penicillium duponti.
[0042] A composition for use in a method of the invention comprises
at least two activities, although typically a composition will
comprise more than two activities, for example, three, four, five,
six, seven, eight, nine or more. Typically, a composition of the
invention may comprise at least one cellulase and at least one
hemicellulase. However, a composition of the invention may comprise
cellulases, but no xylanases. In addition, a composition of the
invention may comprise auxiliary enzyme activity, i.e. additional
activity which, either directly or indirectly leads to
lignocellulose degradation. Examples of such auxiliary activities
are mentioned herein.
[0043] Thus, a composition for use in the invention may comprise
endoglucanase activity and/or cellobiohydrolase activity and/or
.beta.-glucosidase activity. A composition for use in the invention
may comprise more than one enzyme activity in one or more of those
classes. For example, a composition for use in the invention may
comprise two endoglucanase activities, for example,
endo-1,3(1,4)-.beta. glucanase activity and
endo-.beta.-1,4-glucanase activity. Such a composition may also
comprise one or more xylanase activities. Such a composition may
comprise an auxiliary enzyme activity.
[0044] A composition for use in the invention may be derived from
Talaromyces emersonii. In the invention, it is anticipated that a
core set of (lignocellulose degrading) enzyme activities may be
derived from Talaromyces emersonii. Talaromyces emersonii can
provide a highly effective set of activities as demonstrated herein
for the hydrolysis of lignocellulosic biomass. That activity can
then be supplemented with additional enzyme activities from other
sources. Such additional activities may be derived from classical
sources and/or produced by a genetically modified organism.
[0045] The activities in a composition for use in the invention may
be thermostable.
[0046] Herein, this means that the activity has a temperature
optimum of 40.degree. C. or higher, for example about 50.degree. C.
or higher, such as about 60.degree. C. or higher, for example about
70.degree. C. or higher, such as about 75.degree. C. or higher, for
example about 80.degree. C. or higher such as 85.degree. C. or
higher. Activities in a composition for use in the invention will
typically not have the same temperature optima, but preferably
will, nevertheless, be thermostable.
[0047] In addition, enzyme activities in a composition for use in
the invention may be able to work at low pH. For the purposes of
this invention, low pH indicates a pH of about 5.5 or lower, about
5 or lower, about 4.9 or lower, about 4.8 or lower, about 4.7 or
lower, about 4,6 or lower, about 4.5 or lower, about 4.4 or lower,
about 4.3 or lower, about 4.2 or lower, about 4,1 or lower, about
4.0 or lower about 3.9 or lower, or about 3.8 or lower, about 3.7
or lower, about 3.6 or lower, or about 3.5 or lower.
[0048] Activities in a composition for use in the invention may be
defined by a combination of any of the above temperature optima and
pH values.
[0049] The composition used in a method of the invention may
comprise, in addition to the activities derived from Talaromyces, a
cellulase (for example one derived from a source other than
Talaromyces) and/or a hemicellulase (for example one derived from a
source other than Talaromyces) and/or a pectinase.
[0050] A composition for use in the invention may comprise one, two
or three classes of cellulase, for example one, two or all of an
endoglucanase (EG), an exo-cellobiohydrolase (CBH) and a
.beta.-glucosidase (BG). A composition for use in the invention may
comprise two or more of any of these classes of cellulase.
[0051] The .beta.-glucosidase enzyme native to Talaromyces is known
to be very active, Vmax value for the Talaromyces
.beta.-glucosidase Cel3a is 512 IU/mg which is considerably higher
than the values reported for the .beta.-glucosidases from the other
fungal sources (P. Murray et al./Protein Expression and PuriWcation
38 (2004) 248-257) Despite the high activity of the
.beta.-glucosidase in the compositions according to the invention,
and the high glucose levels achieved, no glucose inhibition occurs.
This is advantageous since high activities and high glucose levels
may be combined using the compositions according to the
invention.
[0052] A composition of the invention may comprise an activity
which has a different type of cellulase activity and/or
hemicellulase activity and/or pectinase activity than that provided
by the composition for use in a method of the invention. For
example, a composition of the invention may comprise one type of
cellulase and/or hemicellulase activity and/or pectinase activity
provided by a composition as described herein and a second type of
cellulase and/or hemicellulase activity and/or pectinase activity
provided by an additional cellulose/hemicellulase/pectinase.
[0053] Herein, a cellulase is any polypeptide which is capable of
degrading or modifying cellulose. A polypeptide which is capable of
degrading cellulose is one which is capable of catalysing the
process of breaking down cellulose into smaller units, either
partially, for example into cellodextrins, or completely into
glucose monomers. A cellulase according to the invention may give
rise to a mixed population of cellodextrins and glucose monomers
when contacted with the cellulase. Such degradation will typically
take place by way of a hydrolysis reaction.
[0054] Herein, a hemicellulase is any polypeptide which is capable
of degrading or modifying hemicellulose. That is to say, a
hemicellulase may be capable of degrading or modifying one or more
of xylan, glucuronoxylan, arabinoxylan, glucomannan and xyloglucan.
A polypeptide which is capable of degrading a hemicellulose is one
which is capable of catalysing the process of breaking down the
hemicellulose into smaller polysaccharides, either partially, for
example into oligosaccharides, or completely into sugar monomers,
for example hexose or pentose sugar monomers. A hemicellulase
according to the invention may give rise to a mixed population of
oligosaccharides and sugar monomers when contacted with the
hemicellulase. Such degradation will typically take place by way of
a hydrolysis reaction.
[0055] Herein, a pectinase is any polypeptide which is capable of
degrading or modifying pectin. A polypeptide which is capable of
degrading pectin is one which is capable of catalysing the process
of breaking down pectin into smaller units, either partially, for
example into oligosaccharides, or completely into sugar monomers. A
pectinase according to the invention may give rise to a mixed
population of oligosacchardies and sugar monomers when contacted
with the pectinase. Such degradation will typically take place by
way of a hydrolysis reaction.
[0056] Accordingly, a composition of the invention may comprise any
cellulase, for example, a cellobiohydrolase, an
endo-.beta.-1,4-glucanase, a .beta.-glucosidase or a
.beta.-(1,3)(1,4)-glucanase.
[0057] Herein, a cellobiohydrolase (EC 3.2.1.91) is any polypeptide
which is capable of catalysing the hydrolysis of
1,4-.beta.-D-glucosidic linkages in cellulose or cellotetraose,
releasing cellobiose from the ends of the chains. This enzyme may
also be referred to as cellulase 1,4-.beta.-cellobiosidase,
1,4-.beta.-cellobiohydrolase, 1,4-.beta.-D-glucan
cellobiohydrolase, avicelase, exo-1,4-.beta.-D-glucanase,
exocellobiohydrolase or exoglucanase.
[0058] Herein, an endo-.beta.-1,4-glucanase (EC 3.2.1.4) is any
polypeptide which is capable of catalysing the endohydrolysis of
1,4-.beta.-D-glucosidic linkages in cellulose, lichenin or cereal
.beta.-D-glucans. Such a polypeptide may also be capable of
hydrolyzing 1,4-linkages in .beta.-D-glucans also containing
1,3-linkages. This enzyme may also be referred to as cellulase,
avicelase, .beta.-1,4-endoglucan hydrolase, .beta.-1,4-glucanase,
carboxymethyl cellulase, celludextrinase,
endo-1,4-.beta.-D-glucanase, endo-1,4-.beta.-D-glucanohydrolase,
endo-1,4-.beta.-glucanase or endoglucanase.
[0059] Herein, a .beta.-glucosidase (EC 3.2.1.21) is any
polypeptide which is capable of catalysing the hydrolysis of
terminal, non-reducing .beta.-D-glucose residues with release of
.beta.-D-glucose. Such a polypeptide may have a wide specificity
for .beta.-D-glucosides and may also hydrolyze one or more of the
following: a .beta.-D-galactoside, an .alpha.-L-arabinoside, a
.beta.-D-xyloside or a .beta.-D-fucoside. This enzyme may also be
referred to as amygdalase, .beta.-D-glucoside glucohydrolase,
cellobiase or gentobiase.
[0060] Herein a .beta.-(1,3)(1,4)-glucanase (EC 3.2.1.73) is any
polypeptide which is capable of catalyzing the hydrolysis of
1,4-.beta.-D-glucosidic linkages in .beta.-D-glucans containing
1,3- and 1,4-bonds. Such a polypeptide may act on lichenin and
cereal .beta.-D-glucans, but not on .beta.-D-glucans containing
only 1,3- or 1,4-bonds. This enzyme may also be referred to as
licheninase, 1,3-1,4-.beta.-D-glucan 4-glucanohydrolase,
.beta.-glucanase, endo-.beta.-1,3-1,4 glucanase, lichenase or mixed
linkage .beta.-glucanase. An alternative for this type of enzyme is
EC 3.2.1.6, which is described as endo-1,3(4)-beta-glucanase. This
type of enzyme hydrolyses 1,3- or 1,4-linkages in beta-D-glucanse
when the glucose residue whose reducing group is involved in the
linkage to be hydrolysed is itself substituted at C-3. Alternative
names include endo-1,3-beta-glucanase, laminarinase,
1,3-(1,3;1,4)-beta-D-glucan 3 (4) glucanohydrolase; substrates
include laminarin, lichenin and cereal beta-D-glucans.
[0061] A composition of the invention may comprise any
hemicellulase, for example, an endoxylanase, a .beta.-xylosidase, a
.alpha.-L-arabionofuranosidase, an .alpha.-D-glucuronidase, an
acetyl xylan esterase, a feruloyl esterase, a coumaroyl esterase,
an .alpha.-galactosidase, a .beta.-galactosidase, a
.beta.-mannanase or a .beta.-mannosidase.
[0062] Herein, an endoxylanase (EC 3.2.1.8) is any polypeptide
which is capable of catalyzing the endohydrolysis of
1,4-.beta.-D-xylosidic linkages in xylans. This enzyme may also be
referred to as endo-1,4-.beta.-xylanase or 1,4-.beta.-D-xylan
xylanohydrolase. An alternative is EC 3.2.1.136, a
glucuronoarabinoxylan endoxylanase, an enzyme that is able to
hydrolyse 1,4 xylosidic linkages in glucuronoarabinoxylans.
[0063] Herein, a .beta.-xylosidase (EC 3.2.1.37) is any polypeptide
which is capable of catalyzing the hydrolysis of
1,4-.beta.-D-xylans, to remove successive D-xylose residues from
the non-reducing termini. Such enzymes may also hydrolyze
xylobiose. This enzyme may also be referred to as xylan
1,4-.beta.-xylosidase, 1,4-.beta.-D-xylan xylohydrolase,
exo-1,4-.beta.-xylosidase or xylobiase.
[0064] Herein, an .alpha.-L-arabinofuranosidase (EC 3.2.1.55) is
any polypeptide which is capable of acting on
.alpha.-L-arabinofuranosides, .alpha.-L-arabinans containing (1,2)
and/or (1,3)- and/or (1,5)-linkages, arabinoxylans and
arabinogalactans. This enzyme may also be referred to as
.alpha.-N-arabinofuranosidase, arabinofuranosidase or
arabinosidase.
[0065] Herein, an .alpha.-D-glucuronidase (EC 3.2.1.139) is any
polypeptide which is capable of catalyzing a reaction of the
following form: alpha-D-glucuronoside +H(2)O=an
alcohol+D-glucuronate. This enzyme may also be referred to as
alpha-glucuronidase or alpha-glucosiduronase. These enzymes may
also hydrolyse 4-O-methylated glucoronic acid, which can also be
present as a substituent in xylans.
[0066] Alternative is EC 3.2.1.131: xylan
alpha-1,2-glucuronosidase, which catalyses the hydrolysis of
alpha-1,2-(4-O-methyl)glucuronosyl links.
[0067] Herein, an acetyl xylan esterase (EC 3.1.1.72) is any
polypeptide which is capable of catalyzing the deacetylation of
xylans and xylo-oligosaccharides. Such a polypeptide may catalyze
the hydrolysis of acetyl groups from polymeric xylan, acetylated
xylose, acetylated glucose, alpha-napthyl acetate or p-nitrophenyl
acetate but, typically, not from triacetylglycerol. Such a
polypeptide typically does not act on acetylated mannan or
pectin.
[0068] Herein, a feruloyl esterase (EC 3.1.1.73) is any polypeptide
which is capable of catalyzing a reaction of the form:
feruloyl-saccharide+H(2)O=ferulate+saccharide. The saccharide may
be, for example, an oligosaccharide or a polysaccharide. It may
typically catalyze the hydrolysis of the
4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified
sugar, which is usually arabinose in `natural` substrates.
p-nitrophenol acetate and methyl ferulate are typically poorer
substrates. This enzyme may also be referred to as cinnamoyl ester
hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. It
may also be referred to as a hemicellulase accessory enzyme, since
it may help xylanases and pectinases to break down plant cell wall
hemicellulose and pectin.
[0069] Herein, a coumaroyl esterase (EC 3.1.1.73) is any
polypeptide which is capable of catalyzing a reaction of the form:
coumaroyl-saccharide+H(2)O=coumarate+saccharide. The saccharide may
be, for example, an oligosaccharide or a polysaccharide. This
enzyme may also be referred to as trans-4-coumaroyl esterase,
trans-p-coumaroyl esterase, p-coumaroyl esterase or p-coumaric acid
esterase. This enzyme also falls within EC 3.1.1.73 so may also be
referred to as a feruloyl esterase.
[0070] Herein, an .alpha.-galactosidase (EC 3.2.1.22) is any
polypeptide which is capable of catalyzing the hydrolysis of of
terminal, non-reducing .alpha.-D-galactose residues in
.alpha.-D-galactosides, including galactose oligosaccharides,
galactomannans, galactans and arabinogalactans. Such a polypeptide
may also be capable of hydrolyzing .alpha.-D-fucosides. This enzyme
may also be referred to as melibiase.
[0071] Herein, a .beta.-galactosidase (EC 3.2.1.23) is any
polypeptide which is capable of catalyzing the hydrolysis of
terminal non-reducing .beta.-D-galactose residues in
.beta.-D-galactosides. Such a polypeptide may also be capable of
hydrolyzing .alpha.-L-arabinosides. This enzyme may also be
referred to as exo-(1->4)-.beta.-D-galactanase or lactase.
[0072] Herein, a .beta.-mannanase (EC 3.2.1.78) is any polypeptide
which is capable of catalyzing the random hydrolysis of
1,4-.beta.-D-mannosidic linkages in mannans, galactomannans and
glucomannans. This enzyme may also be referred to as mannan
endo-1,4-.beta.-mannosidase or endo-1,4-mannanase.
[0073] Herein, a .beta.-mannosidase (EC 3.2.1.25) is any
polypeptide which is capable of catalyzing the hydrolysis of
terminal, non-reducing .beta.-D-mannose residues in
.beta.-D-mannosides. This enzyme may also be referred to as
mannanase or mannase.
[0074] A composition of the invention may comprise any pectinase,
for example an endo polygalacturonase, a pectin methyl esterase, an
endo-galactanase, a beta galactosidase, a pectin acetyl esterase,
an endo-pectin lyase, pectate lyase, alpha rhamnosidase, an
exo-galacturonase, an expolygalacturonate lyase, a
rhamnogalacturonan hydrolase, a rhamnogalacturonan lyase, a
rhamnogalacturonan acetyl esterase, a rhamnogalacturonan
galacturonohydrolase, a xylogalacturonase.
[0075] Herein, an endo-polygalacturonase (EC 3.2.1.15) is any
polypeptide which is capable of catalyzing the random hydrolysis of
1,4-.alpha.-D-galactosiduronic linkages in pectate and other
galacturonans. This enzyme may also be referred to as
polygalacturonase pectin depolymerase, pectinase,
endopolygalacturonase, pectolase, pectin hydrolase, pectin
polygalacturonase, poly-.alpha.-1,4-galacturonide glycanohydrolase,
endogalacturonase; endo-D-galacturonase or
poly(1,4-.alpha.-D-galacturonide) glycanohydrolase.
[0076] Herein, a pectin methyl esterase (EC 3.1.1.11) is any enzyme
which is capable of catalyzing the reaction: pectin+n H.sub.2O=n
methanol+pectate. The enzyme may also been known as pectinesterase,
pectin demethoxylase, pectin methoxylase, pectin methylesterase,
pectase, pectinoesterase or pectin pectylhydrolase.
[0077] Herein, an endo-galactanase (EC 3.2.1.89) is any enzyme
capable of catalyzing the endohydrolysis of
1,4-.beta.-D-galactosidic linkages in arabinogalactans. The enzyme
may also be known as arabinogalactan endo-1,4-.beta.-galactosidase,
endo-1,4-.beta.-galactanase, galactanase, arabinogalactanase or
arabinogalactan 4-.beta.-D-galactanohydrolase.
[0078] Herein, a pectin acetyl esterase is defined herein as any
enzyme which has an acetyl esterase activity which catalyzes the
deacetylation of the acetyl groups at the hydroxyl groups of GaIUA
residues of pectin
[0079] Herein, an endo-pectin lyase (EC 4.2.2.10) is any enzyme
capable of catalyzing the eliminative cleavage of
(1.fwdarw.4)-.alpha.-D-galacturonan methyl ester to give
oligosaccharides with
4-deoxy-6-O-methyl-.alpha.-D-galact-4-enuronosyl groups at their
non-reducing ends. The enzyme may also be known as pectin lyase,
pectin trans-eliminase; endo-pectin lyase, polymethylgalacturonic
transeliminase, pectin methyltranseliminase, pectolyase, PL, PNL or
PMGL or (1.fwdarw.4)-6-O-methyl-.alpha.-D-galacturonan lyase.
[0080] Herein, a pectate lyase (EC 4.2.2.2) is any enzyme capable
of catalyzing the eliminative cleavage of
(1.fwdarw.4)-.alpha.-D-galacturonan to give oligosaccharides with
4-deoxy-.alpha.-D-galact-4-enuronosyl groups at their non-reducing
ends. The enzyme may also be known polygalacturonic transeliminase,
pectic acid transeliminase, polygalacturonate lyase, endopectin
methyltranseliminase, pectate transeliminase, endogalacturonate
transeliminase, pectic acid lyase, pectic lyase,
.alpha.-1,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N,
endo-.alpha.-1,4-polygalacturonic acid lyase, polygalacturonic acid
lyase, pectin trans-eliminase, polygalacturonic acid
trans-eliminase or (1-4)-.alpha.-D-galacturonan lyase.
[0081] Herein, an alpha rhamnosidase (EC 3.2.1.40) is any
polypeptide which is capable of catalyzing the hydrolysis of
terminal non-reducing .alpha.-L-rhamnose residues in
.alpha.-L-rhamnosides or alternatively in rhamnogalacturonan. This
enzyme may also be known as .alpha.-L-rhamnosidase T,
.alpha.-L-rhamnosidase N or .alpha.-L-rhamnoside
rhamnohydrolase.
[0082] Herein, exo-galacturonase (EC 3.2.1.82) is any polypeptide
capable of hydrolysis of pectic acid from the non-reducing end,
releasing digalacturonate. The enzyme may also be known as
exo-poly-.alpha.-galacturonosidase, exopolygalacturonosidase or
exopolygalacturanosidase.
[0083] Herein, exo-galacturonase (EC 3.2.1.67) is any polypeptide
capable of catalyzing:
(1,4-.alpha.-D-galacturonide).sub.n+H.sub.2O=(1,4-.alpha.-D-galacturonide-
).sub.n-1+D-galacturonate. The enzyme may also be known as
galacturan 1,4-.alpha.-galacturonidase, exopolygalacturonase,
poly(galacturonate) hydrolase, exo-D-galacturonase,
exo-D-galacturonanase, exopoly-D-galacturonase or
poly(1,4-.alpha.-D-galacturonide) galacturonohydrolase.
[0084] Herein, exopolygalacturonate lyase (EC 4.2.2.9) is any
polypeptide capable of catalyzing eliminative cleavage of
4-(4-deoxy-.alpha.-D-galact-4-enuronosyl)-D-galacturonate from the
reducing end of pectate, i.e. de-esterified pectin. This enzyme may
be known as pectate disaccharide-lyase, pectate exo-lyase,
exopectic acid transeliminase, exopectate lyase,
exopolygalacturonic acid-trans-eliminase, PATE, exo-PATE, exo-PGL
or (1.fwdarw.4)-.alpha.-D-galacturonan
reducing-end-disaccharide-lyase.
[0085] Herein, rhamnogalacturonan hydrolase is any polypeptide
which is capable of hydrolyzing the linkage between
galactosyluronic acid acid and rhamnopyranosyl in an endo-fashion
in strictly alternating rhamnogalacturonan structures, consisting
of the disaccharide
[(1,2-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].
[0086] Herein, rhamnogalacturonan lyase is any polypeptide which is
any polypeptide which is capable of cleaving
.alpha.-L-Rhap-(1.fwdarw.4)-.alpha.-D-GalpA linkages in an
endo-fashion in rhamnogalacturonan by beta-elimination.
[0087] Herein, rhamnogalacturonan acetyl esterase is any
polypeptide which catalyzes the deacetylation of the backbone of
alternating rhamnose and galacturonic acid residues in
rhamnogalacturonan.
[0088] Herein, rhamnogalacturonan galacturonohydrolase is any
polypeptide which is capable of hydrolyzing galacturonic acid from
the non-reducing end of strictly alternating rhamnogalacturonan
structures in an exo-fashion.
[0089] Herein, xylogalacturonase is any polypeptide which acts on
xylogalacturonan by cleaving the .beta.-xylose substituted
galacturonic acid backbone in an endo-manner. This enzyme may also
be known as xylogalacturonan hydrolase.
[0090] Herein, an .alpha.-L-arabinofuranosidase (EC 3.2.1.55) is
any polypeptide which is capable of acting on
.alpha.-L-arabinofuranosides, .alpha.-L-arabinans containing (1,2)
and/or (1,3)- and/or (1,5)-linkages, arabinoxylans and
arabinogalactans. This enzyme may also be referred to as
.alpha.-N-arabinofuranosidase, arabinofuranosidase or
arabinosidase.
[0091] Herein, endo-arabinanase (EC 3.2.1.99) is any polypeptide
which is capable of catalyzing endohydrolysis of
1,5-.alpha.-arabinofuranosidic linkages in 1,5-arabinans. The
enzyme may also be know as endo-arabinase, arabinan
endo-1,5-.alpha.-L-arabinosidase, endo-1,5-.alpha.-L-arabinanase,
endo-.alpha.-1,5-arabanase; endo-arabanase or
1,5-.alpha.-L-arabinan 1,5-.alpha.-L-arabinanohydrolase.
[0092] A composition of the invention will typically comprise at
least one cellulase and/or at least one hemicellulase and/or at
least one pectinase (one of which is a polypeptide according to the
invention). A composition of the invention may comprise a
cellobiohydrolase, an endoglucanase and/or a .beta.-glucosidase.
Such a composition may also comprise one or more hemicellulases
and/or one or more pectinases.
[0093] In addition, one or more (for example two, three, four or
all) of an amylase, a protease, a lipase, a ligninase, a
hexosyltransf erase, a glucuronidase or an expansin or a cellulose
induced protein or a cellulose integrating protein or like protein
may be present in a composition of the invention (these are
referred to as auxiliary activities above).
[0094] "Protease" includes enzymes that hydrolyze peptide bonds
(peptidases), as well as enzymes that hydrolyze bonds between
peptides and other moieties, such as sugars (glycopeptidases). Many
proteases are characterized under EC 3.4, and are suitable for use
in the invention incorporated herein by reference. Some specific
types of proteases include, cysteine proteases including pepsin,
papain and serine proteases including chymotrypsins,
carboxypeptidases and metalloendopeptidases.
[0095] "Lipase" includes enzymes that hydrolyze lipids, fatty
acids, and acylglycerides, including phospoglycerides,
lipoproteins, diacylglycerols, and the like. In plants, lipids are
used as structural components to limit water loss and pathogen
infection. These lipids include waxes derived from fatty acids, as
well as cutin and suberin.
[0096] "Ligninase" includes enzymes that can hydrolyze or break
down the structure of lignin polymers. Enzymes that can break down
lignin include lignin peroxidases, manganese peroxidases, laccases
and feruloyl esterases, and other enzymes described in the art
known to depolymerize or otherwise break lignin polymers. Also
included are enzymes capable of hydrolyzing bonds formed between
hemicellulosic sugars (notably arabinose) and lignin. Ligninases
include but are not limited to the following group of enzymes:
lignin peroxidases (EC 1.11.1.14), manganese peroxidases (EC
1.11.1.13), laccases (EC 1.10.3.2) and feruloyl esterases (EC
3.1.1.73).
[0097] "Hexosyltransf erase" (2.4.1-) includes enzymes which are
capable of catalyzing a transferase reaction, but which can also
catalyze a hydrolysis reaction, for example of cellulose and/or
cellulose degradation products. An example of a hexosyltransferase
which may be used in the invention is a
.beta.-glucanosyltransferase. Such an enzyme may be able to
catalyze degradation of (1,3)(1,4)glucan and/or cellulose and/or a
cellulose degradation product.
[0098] "Glucuronidase" includes enzymes that catalyze the
hydrolysis of a glucoronoside, for example .beta.-glucuronoside to
yield an alcohol. Many glucuronidases have been characterized and
may be suitable for use in the invention, for example
.beta.-glucuronidase (EC 3.2.1.31), hyalurono-glucuronidase (EC
3.2.1.36), glucuronosyl-disulfoglucosamine glucuronidase
(3.2.1.56), glycyrrhizinate .beta.-glucuronidase (3.2.1.128) or
.alpha.-D-glucuronidase (EC 3.2.1.139).
[0099] A composition for use in the invention may comprise an
expansin or expansin-like protein, such as a swollenin (see
Salheimo et al., Eur. J. Biohem. 269, 4202-4211, 2002) or a
swollenin-like protein.
[0100] Expansins are implicated in loosening of the cell wall
structure during plant cell growth. Expansins have been proposed to
disrupt hydrogen bonding between cellulose and other cell wall
polysaccharides without having hydrolytic activity. In this way,
they are thought to allow the sliding of cellulose fibers and
enlargement of the cell wall. Swollenin, an expansin-like protein
contains an N-terminal Carbohydrate Binding Module Family 1 domain
(CBD) and a C-terminal expansin-like domain. For the purposes of
this invention, an expansin-like protein or swollenin-like protein
may comprise one or both of such domains and/or may disrupt the
structure of cell walls (such as disrupting cellulose structure),
optionally without producing detectable amounts of reducing
sugars.
[0101] A composition for use in the invention may a cellulose
induced protein, for example the polypeptide product of the cip1 or
cip2 gene or similar genes (see Foreman et al., J. Biol. Chem.
278(34), 31988-31997, 2003), a cellulose/cellulosome integrating
protein, for example the polypeptide product of the cipA or cipC
gene, or a scaffoldin or a scaffoldin-like protein. Scaffoldins and
cellulose integrating proteins are multi-functional integrating
subunits which may organize cellulolytic subunits into a
multi-enzyme complex. This is accomplished by the interaction of
two complementary classes of domain, i.e. a cohesion domain on
scaffoldin and a dockerin domain on each enzymatic unit. The
scaffoldin subunit also bears a cellulose-binding module (CBM) that
mediates attachment of the cellulosome to its substrate. A
scaffoldin or cellulose integrating protein for the purposes of
this invention may comprise one or both of such domains.
[0102] A composition for use in a method of the invention may be
composed of a member of each of the classes of enzymes mentioned
above, several members of one enzyme class, or any combination of
these enzymes classes or helper proteins (i.e. those proteins
mentioned herein which do not have enzymatic activity per se, but
do nevertheless assist in lignocellulosic degradation).
[0103] A composition for use in a method of the invention may be
composed of enzymes from (1) commercial suppliers; (2) cloned genes
expressing enzymes; (3) complex broth (such as that resulting from
growth of a microbial strain in media, wherein the strains secrete
proteins and enzymes into the media; (4) cell lysates of strains
grown as in (3); and/or (5) plant material expressing enzymes.
Different enzymes in a composition of the invention may be obtained
from different sources.
[0104] The enzymes can be produced either exogenously in
microorganisms, yeasts, fungi, bacteria or plants, then isolated
and added, for example, to lignocellulosic feedstock.
Alternatively, the enzymes are produced, but not isolated, and
crude cell mass fermentation broth, or plant material (such as corn
stover or wheat straw), and the like may be added to, for example,
the feedstock. Alternatively, the crude cell mass or enzyme
production medium or plant material may be treated to prevent
further microbial growth (for example, by heating or addition of
antimicrobial agents), then added to, for example, a feedstock.
These crude enzyme mixtures may include the organism producing the
enzyme. Alternatively, the enzyme may be produced in a fermentation
that uses feedstock (such as corn stover or wheat straw) to provide
nutrition to an organism that produces an enzyme(s). In this
manner, plants that produce the enzymes may themselves serve as a
lignocellulosic feedstock and be added into lignocellulosic
feedstock.
[0105] In the uses and methods described herein, the components of
the compositions described above may be provided concomitantly
(i.e. as a single composition per se) or separately or
sequentially.
[0106] The invention thus relates to methods in which the
composition described above are used and to uses of the composition
in industrial processes.
[0107] In principle, a composition of the invention may be used in
any process which requires the treatment of a material which
comprises non-starch polysaccharide. Thus, a polypeptide or
composition of the invention may be used in the treatment of
non-starch polysaccharide material. Herein, non-starch
polysaccharide material is a material which comprises or consists
essential of one or, more typically, more than one non-starch
polysaccharide.
[0108] Typically, plants and fungi and material derived therefrom
comprise significant quantities of non-starch polysaccharide
material. Accordingly, a polypeptide of the invention may be used
in the treatment of a plant or fungal material or a material
derived therefrom.
[0109] An important component of plant non-starch polysaccharide
material is lignocellulose (also referred to herein as
lignocellulolytic biomass). Lignocellulose is plant material that
is composed of cellulose and hemicellulose and lignin. The
carbohydrate polymers (cellulose and hemicelluloses) are tightly
bound to the lignin by hydrogen and covalent bonds. Accordingly, a
polypeptide of the invention may be used in the treatment of
lignocellulolytic material. Herein, lignocellulolytic material is a
material which comprises or consists essential of lignocellulose.
Thus, in a method of the invention for the treatment of a
non-starch polysaccharide, the non-starch polysaccharide may be a
lignocellulosic material/biomass.
[0110] Accordingly, the invention provides a method of treating a
non-starch polysaccharide in which the treatment comprises the
degradation and/or modification of cellulose and/or
hemicellulose.
[0111] Degradation in this context indicates that the treatment
results in the generation of hydrolysis products of cellulose
and/or hemicellulose and/or a pectic substance, i.e. saccharides of
shorter length are present as result of the treatment than are
present in a similar untreated non-starch polysaccharide. Thus,
degradation in this context may result in the liberation of
oligosaccharides and/or sugar monomers.
[0112] All plants and fungi contain non-starch polysaccharide as do
virtually all plant- and fungal-derived polysaccharide materials.
Accordingly, in a method of the invention for the treatment of a
non-starch polysaccharide, said non-starch polysaccharide may be
provided in the form of a plant or a plant derived material or a
material comprising a plant or plant derived material, for example
a plant pulp, a plant extract, a foodstuff or ingredient therefore,
a fabric, a textile or an item of clothing.
[0113] The invention provides a method for producing a sugar from a
lignocellosic material which method comprises contacting a
composition as described herein with the lignocellulosic
material.
[0114] Such a method allows free sugars (monomers) and/or
oligosaccharides to be generated from lignocellulosic biomass.
These methods involve converting lignocellulosic biomass to free
sugars and small oligosaccharides with a polypeptide or composition
of the invention.
[0115] The process of converting a complex carbohydrate such as
lignocellulose into sugars preferably allows conversion into
fermentable sugars. Such a process may be referred to as
"saccharification." Accordingly, a method of the invention may
result in the liberation of one or more hexose and/or pentose
sugars, such as one or more of glucose, cellobiose, xylose,
arabinose, galactose, galacturonic acid, glucuronic acid, mannose,
rhamnose, sucrose and fructose.
[0116] Lignocellulolytic biomass suitable for use in the invention
includes Biomass can include virgin biomass and/or non-virgin
biomass such as agricultural biomass, commercial organics,
construction and demolition debris, municipal solid waste, waste
paper and yard waste. Common forms of biomass include trees, shrubs
and grasses, wheat, wheat straw, sugar cane bagasse, switch grass,
miscanthus, corn, corn stover, corn husks, corn cobs, canola stems,
soybean stems, sweet sorghum, corn kernel including fiber from
kernels, products and by-products from milling of grains such as
corn, wheat and barley (including wet milling and dry milling)
often called "bran or fibre" as well as municipal solid waste,
waste paper and yard waste. The biomass can also be, but is not
limited to, herbaceous material, agricultural residues, forestry
residues, municipal solid wastes, waste paper, and pulp and paper
mill residues. "Agricultural biomass" includes branches, bushes,
canes, corn and corn husks, energy crops, forests, fruits, flowers,
grains, grasses, herbaceous crops, leaves, bark, needles, logs,
roots, saplings, short rotation woody crops, shrubs, switch
grasses, trees, vegetables, fruit peels, vines, sugar beet pulp,
wheat midlings, oat hulls, and hard and soft woods (not including
woods with deleterious materials). In addition, agricultural
biomass includes organic waste materials generated from
agricultural processes including farming and forestry activities,
specifically including forestry wood waste. Agricultural biomass
may be any of the aforestated singularly or in any combination or
mixture thereof.
[0117] Apart from virgin biomass or feedstocks already processed in
food and feed or paper and pulping industries, the
biomass/feedstock may additionally be pretreated with heat,
mechanical and/or chemical modification or any combination of such
methods in order to enhance enzymatic degradation.
[0118] The fermentable sugars can be converted to useful
value-added fermentation products, non-limiting examples of which
include amino acids, vitamins, pharmaceuticals, animal feed
supplements, specialty chemicals, chemical feedstocks, plastics,
solvents, fuels, or other organic polymers, lactic acid, and
ethanol, including fuel ethanol.
[0119] Specific value-added products that may be produced by the
methods of the invention include, but not limited to, biofuels
(including ethanol and butanol and a biogas); lactic acid; a
plastic; a specialty chemical; an organic acid, including citric
acid, succinic acid, fumaric acid, itaconic acid and maleic acid;
3-hydoxy-propionic acid, acrylic acid; acetic acid;
1,3-propane-diol; ethylene, glycerol; a solvent; an animal feed
supplement; a pharmaceutical, such as a .beta.-lactam antibiotic or
a cephalosporin; vitamins; an amino acid, such as lysine,
methionine, tryptophan, threonine, and aspartic acid; an industrial
enzyme, such as a protease, a cellulase, an amylase, a glucanase, a
lactase, a lipase, a lyase, an oxidoreductases, a transferase or a
xylanase; and a chemical feedstock.
[0120] The composition, nature of substitution, and degree of
branching of hemicellulose is very different in dicotyledonous
plants (dicots, i.e., plant whose seeds have two cotyledons or seed
leaves such as lima beans, peanuts, almonds, peas, kidney beans) as
compared to monocotyledonous plants (monocots; i.e., plants having
a single cotyledon or seed leaf such as corn, wheat, rice, grasses,
barley). In dicots, hemicellulose is comprised mainly of
xyloglucans that are 1,4-.beta.-linked glucose chains with
1,6-.beta.-linked xylosyl side chains. In monocots, including most
grain crops, the principal components of hemicellulose are
heteroxylans. These are primarily comprised of 1,4-.beta.-linked
xylose backbone polymers with 1,3-.alpha. linkages to arabinose,
galactose, mannose and glucuronic acid or 4-O-methyl-glucuronic
acid as well as xylose modified by ester-linked acetic acids. Also
present are .beta. glucans comprised of 1,3- and 1,4-.beta.-linked
glucosyl chains. In monocots, cellulose, heteroxylans and
.beta.-glucans may be present in roughly equal amounts, each
comprising about 15-25% of the dry matter of cell walls. Also,
different plants may comprise different amounts of, and different
compositions of, pectic substances. For example, sugar beet
contains about 19% pectin and about 21% arabinan on a dry weight
basis.
[0121] Accordingly, a composition of the invention may be tailored
in view of the particular feedstock which is to be used. That is to
say, the spectrum of activities in a composition of the invention
may vary depending on the feedstock in question.
[0122] Enzyme combinations or physical treatments can be
administered concomitantly or sequentially. The enzymes can be
produced either exogenously in microorganisms, yeasts, fungi,
bacteria or plants, then isolated and added to the lignocellulosic
feedstock. Alternatively, the enzymes are produced, but not
isolated, and crude cell mass fermentation broth, or plant material
(such as corn stover), and the like are added to the feedstock.
Alternatively, the crude cell mass or enzyme production medium or
plant material may be treated to prevent further microbial growth
(for example, by heating or addition of antimicrobial agents), then
added to the feedstock. These crude enzyme mixtures may include the
organism producing the enzyme. Alternatively, the enzyme may be
produced in a fermentation that uses feedstock (such as corn
stover) to provide nutrition to an organism that produces an
enzyme(s). In this manner, plants that produce the enzymes may
serve as the lignocellulosic feedstock and be added into
lignocellulosic feedstock.
[0123] In the method of the invention, a enzyme or combination of
enzymes acts on a lignocellulosic substrate or plant biomass,
serving as the feedstock, so as to convert this complex substrate
to simple sugars and oligosaccharides for the production of ethanol
or other useful fermentation products.
[0124] Accordingly, another aspect of the invention includes
methods that utilize the composition described above together with
further enzymes or physical treatments such as temperature and pH
to convert the lignocellulosic plant biomass to sugars and
oligosaccharides.
[0125] While the composition has been discussed as a single mixture
it is recognized that the enzymes may be added sequentially where
the temperature, pH, and other conditions may be altered to
increase the activity of each individual enzyme.
[0126] Alternatively, an optimum pH and temperature can be
determined for the enzyme mixture.
[0127] The composition is reacted with substrate under any
appropriate conditions. For example, enzymes can be incubated at
about 25.degree. C., about 30.degree. C., about 35.degree. C.,
about 37.degree. C., about 40.degree. C., about 45.degree. C.,
about 50.degree. C., about 55.degree. C., about 60.degree. C.,
about 65.degree. C., about 70.degree. C., about 75.degree. C.,
about 80.degree. C., about 85.degree. C., about 90.degree. C. or
higher. That is, they can be incubated at a temperature of from
about 20.degree. C. to about 95.degree. C., for example in buffers
of low to medium ionic strength and/or from low to neutral pH. By
"medium ionic strength" is intended that the buffer has an ion
concentration of about 200 millimolar (mM) or less for any single
ion component. The pH may range from about pH 2.5, about pH 3.0,
about pH 3.5, about pH 4.0, about pH 4.5, about pH 5, about pH 5.5,
about pH 6, about pH 6.5, about pH 7, about pH 7.5, about pH 8.0,
to about pH 8.5. Generally, the pH range will be from about pH 3.0
to about pH 9.
[0128] Typically, the reaction may be carried out under low pH
conditions as defined above. Thus, a method of the invention may be
carried out such that no pH adjustment (i.e. to a more neutral pH
is required). That is to say, an acid pretreated feedstock may be
used as is with no requirement to addition of, for example, sodium
hydroxide, prior to addition of a composition of the invention.
[0129] The feedstock may be washed prior to
liquefaction/hydrolysis. Such washing may be with, for example,
water.
[0130] Incubation of a composition under these conditions results
in release or liberation of substantial amounts of the sugar from
the lignocellulosic material. By substantial amount is intended at
least about 20%, at least about 30%, at least about 40%, at least
about 50%, at least about 60%, at least about 70%, at least about
80%, at least about 90%, at least about 95% or more of available
sugar.
[0131] A liquefaction/hydrolysis or presaccharification step
involving incubation with an enzyme or enzyme mixture can be
utilized. This step can be performed at many different temperatures
but it is preferred that the pretreatment occur at the temperature
best suited to the enzyme mix being tested, or the predicted enzyme
optimum of the enzymes to be tested. The temperature of the
pretreatment may range from about 10.degree. C. to about 80.degree.
C., about 20.degree. C. to about 80.degree. C., about 30.degree. C.
to about 70.degree. C., about 40.degree. C. to about 60.degree. C.,
about 37.degree. C. to about 50.degree. C., preferably about
37.degree. C. to about 80.degree. C., more preferably about
50.degree. C. In the absence of data on the temperature optimum, it
is preferable to perform the pretreatment reactions at 37.degree.
C. first, then at a higher temperature such as 50.degree. C. The pH
of the pretreatment mixture may range from about 2.0 to about 10.0,
but is preferably about 3.0 to about 5.0. Again, it may not be
necessary to adjust the pH prior to saccharification since a
composition for use in the invention is typically suitable for use
at low pH as defined herein.
[0132] The liquefaction/hydrolysis or presaccharification step
reaction may occur from several minutes to several hours, such as
from about 1 hour to about 120 hours, preferably from about 2 hours
to about 48 hours, more preferably from about 2 to about 24 hours,
most preferably for from about 2 to about 6 hours. The cellulase
treatment may occur from several minutes to several hours, such as
from about 6 hours to about 168 hours, preferably about 12 hours to
about 96 hours, more preferably about 24 hours to about 72 hours,
even more preferably from about 24 hours to about 48 hours. These
conditions are particularly suitable in case the
liquefaction/hydrolysis or presaccharification step is conducted in
a Separate Hydrolyis and Fermentation (SHF) mode.
SSF Mode
[0133] For Simultaneous Saccharification and Fermentation (SSF)
mode, the reaction time for liquefaction/hydrolysis or
presaccharification step is dependent on the time to realize a
desired yield, i.e. cellulose to glucose conversion yield. Such
yield is preferably as high as possible, preferably 60% or more,
65% or more, 70% or more, 75% or more 80% or more, 85% or more,90%
or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or
more, even 99.5% or more or 99.9% or more.
[0134] According to the invention very high sugar concentrations in
SHF mode and very high product concentrations (e.g. ethanol) in SSF
mode are realized. In SHF operation the glucose concentration is 25
g/L or more, 30 g/L or more, 35 g/L or more, 40 g/L or more, 45 g/L
or more, 50 g/L or more, 55 g/L or more, 60 g/L or more, 65 g/L or
more, 70 g/L or more, 75 g/L or more, 80 g/L or more, 85 g/L or
more, 90 g/L or more, 95 g/L or more, 100 g/L or more, 110 g/L or
more, 120 g/L or more or may e.g. be 25 g/L-250 g/L, 30 gl/L-200
g/L, 40 g/L-200 g/L, 50 g/L-200 g/L, 60 g/L-200 g/L, 70 g/L-200
g/L, 80 g/L-200 g/L, 90 g/L, 80 g/L-200 g/L.
Product Concentration in SSF Mode
[0135] In SSF operation, the product concentration (g/L) is
dependent on the amount of glucose produced, but this is not
visible since sugars are converted to product in the SSF, and
product concentrations can be related to underlying glucose
concentration by multiplication with the theoretical maximum yield
(Yes max in gr product per gram glucose)
[0136] The theoretical maximum yield (Yps max in gr product per
gram glucose) of a fermentation product can be derived from
textbook biochemistry. For ethanol, 1 mole of glucose (180 gr)
yields according to normal glycolysis fermentation pathway in yeast
2 moles of ethanol (=2.times.46=92 gr ethanol. The theoretical
maximum yield of ethanol on glucose is therefore 92/180=0.511 gr
ethanol/gr glucose.
[0137] For Butanol (MW 74 gr/mole) or iso butanol, the theoretical
maximum yield is 1 mole of butanol per mole of glucose. So Yps max
for (iso-)butanol=74/180=0.411 gr (iso-)butanol/gr glucose.
[0138] For lactic acid the fermentation yield for homolactic
fermentation is 2 moles of lactic acid (MW=90 gr/mole) per mole of
glucose. According to this stoichiometry, the Yps max=1 gr lactic
acid/gr glucose.
[0139] For other fermentation products a similar calculation may be
made.
SSF Mode
[0140] In SSF operation the product concentration is 25 g*Yps g/L/L
or more, 30*Yps g/L or more, 35 g*Yps/L or more, 40*Yps g/L or
more, 45*Yps g/L or more, 50*Yps g/L or more, 55*Yps g/L or more,
60*Yps g/L or more, 65*Yps g/L or more, 70*Yps g/L or more, 75*Yps
g/L or more, 80*Yps g/L or more, 85*Yps g/L or more, 90*Yps g/L or
more, 95*Yps g/L or more, 100*Yps g/L or more, 110*Yps g/L or more,
120 g/L*Yps or more or may e.g. be 25*Yps g/L-250*Yps g/L, 30*Yps
gl/L-200*Yps g/L, 40*Yps g/L-200*Yps g/L, 50*Yps g/L-200*Yps g/L,
60*Yps g/L-200*Yps g/L, 70*Yps g/L-200*Yps g/L, 80*Yps g/L-200*Yps
g/L, 90*Yps g/L, 80*Yps g/L-200*Yps g/L
[0141] Sugars released from biomass can be converted to useful
fermentation products such a one of those including, but not
limited to, amino acids, vitamins, pharmaceuticals, animal feed
supplements, specialty chemicals, chemical feedstocks, plastics,
and ethanol, including fuel ethanol.
[0142] Significantly, a method of the invention may be carried out
using high levels of dry matter (of the lignocellulosic material)
in the hydrolysis reaction. Thus, the invention may be carried out
with a dry matter content of about 5% or higher, about 8% or
higher, about 10% or higher, about 11% or higher, about 12% or
higher, about 13% or higher, about 14% or higher, about 15% or
higher, about 20% or higher, about 25% or higher, about 30% or
higher, about 35% or higher or about 40% or higher.
[0143] Accordingly, the invention provides a method for the
preparation of a fermentation product, which method comprises:
[0144] a. degrading lignocellulose using a method as described
herein; and [0145] b. fermenting the resulting material, thereby to
prepare a fermentation product.
[0146] Such a process may be carried out without any requirement to
adjust the pH during the process. That is to say, the process is
one which may be carried out without the addition of any acid(s) or
base(s). However, this excludes a pretreatment step, where acid may
be added. The point is that the composition of the invention is
capable of acting at low pH and, therefore, there is no need to
adjust the pH of acid of an acid pretreated feedstock in order that
saccharification may take place. Accordingly, a method of the
invention may be a zero waste method using only organic products
with no requirement for inorganic chemical input.
[0147] Fermentation products which may be produced according to the
invention include amino acids, vitamins, pharmaceuticals, animal
feed supplements, specialty chemicals, chemical feedstocks,
plastics, solvents, fuels, or other organic polymers, lactic acid,
and ethanol, including fuel ethanol (the term "ethanol" being
understood to include ethyl alcohol or mixtures of ethyl alcohol
and water).
[0148] Specific value-added products that may be produced by the
methods of the invention include, but not limited to, biofuels
(including ethanol and butanol); lactic acid; 3-hydroxy-propionic
acid; acrylic acid; acetic acid; 1,3-propane-diol; ethylene;
glycerol; a plastic; a specialty chemical; an organic acid,
including citric acid, succinic acid and maleic acid; a solvent; an
animal feed supplement; a pharmaceutical such as a .beta.-lactam
antibiotic or a cephalosporin; a vitamin; an amino acid, such as
lysine, methionine, tryptophan, threonine, and aspartic acid; an
enzyme, such as a protease, a cellulase, an amylase, a glucanase, a
lactase, a lipase, a lyase, an oxidoreductase, a transferase or a
xylanase; a chemical feedstock; or an animal feed supplement.
[0149] A method for the preparation of a fermentation product may
optionally comprise recovery of the fermentation product.
[0150] Such a process may be carried out under aerobic or anaerobic
conditions. Preferably, the process is carried out under
micro-aerophilic or oxygen limited conditions.
[0151] An anaerobic fermentation process is herein defined as a
fermentation process run in the absence of oxygen or in which
substantially no oxygen is consumed, preferably about 5 or less,
about 2.5 or less or about 1 mmol/L/h or less, and wherein organic
molecules serve as both electron donor and electron acceptors.
[0152] An oxygen-limited fermentation process is a process in which
the oxygen consumption is limited by the oxygen transfer from the
gas to the liquid. The degree of oxygen limitation is determined by
the amount and composition of the ingoing gasflow as well as the
actual mixing/mass transfer properties of the fermentation
equipment used. Preferably, in a process under oxygen-limited
conditions, the rate of oxygen consumption is at least about 5.5,
more preferably at least about 6 and even more preferably at least
about 7 mmol/L/h.
[0153] The following Examples illustrate the invention:
Example 1
Saccharification of Corn Stover Hydrolysate using Various
Cellulases
Materials and Methods
[0154] The ability of three different cellulase preparations to
saccharify a corn stover hydrolysate was evaluated. A Talaromyces
emersonii enzyme product called Filtrase.RTM.NL (DSM Food
Specialties, Delft, Netherlands) was compared with Laminex.RTM. BG
and GC 220 (Genencor-Danisco, Rochester, USA). Laminex.RTM. BG and
GC220 are considered to be the benchmark enzymes as presently
available in the market.
[0155] Dilute acid pretreated corn stover prepared by NREL was used
as the substrate for saccharification carried out with the
cellulase preparations. The pretreated corn stover was stored at
4.degree. C. The slurry was about 34% total solids with about 17%
insoluble solids. The composition of the corn stover used for the
pretreatment is set out in Table 1.
TABLE-US-00001 TABLE 1 The composition of the raw stover is as
follows: % (w/w, dry component basis) cellulose 33.9 .+-. 0.7 xylan
24.1 .+-. 1.1 lignin 11.4 .+-. 0.8 extractives 9.7 .+-. 0.3 sucrose
6.2 .+-. 1.0 uronic acid.sup.b 4.0 .+-. 0.2 acetate 3.9 .+-. 0.8
arabinan 3.1 .+-. 0.2 non-structural inorganics 2.0 .+-. 0.4
protein 1.6 .+-. 0.5 galactan 1.5 .+-. 0.1 ash 1.4 .+-. 0.5 .sup.a
Mean .+-. standard deviation of 4 samples. .sup.bCalculated
value.
[0156] The dry matter concentration was verified and turned out to
be 32.8% dry matter pulp (105.degree. C.,48 hrs drying).
[0157] 60 gr of the fiber was mixed with 120 gr water and pH (1.9)
was adjusted to 5.0 using 4N NaOH and after that added up to 200 gr
with water to obtain a 9.45% dry matter sludge. 10 gr portions of
the sludge were divided into a 50 ml Schott-flask and each enzyme
preparation was added at three different dosages (20, 61 and 204
.mu.L respectively).
[0158] Subsequently the flasks were closed and incubated at
50.degree. C. at 280 RPM for 140 hrs and sampled (3 ml) at 0, 21,
93 and 140 hrs, centrifuged using eppendorf centrifuge and the
supernatant was decanted to a vial and analyzed for glucose,
arabinose, xylose and galactose using NMR.
Results and Discussion
[0159] At the start of the incubation it was clear that the dilute
acid pretreatment had done the work on the hemicellulose fraction
since 24 g/L of xylose was present already from theoretical 30 g/L.
The sugar composition at start of the saccharification is set out
in Table 2; the free glucose concentration at time zero was around
4 g/L.
TABLE-US-00002 TABLE 2 Sugar composition at the start of the
saccharification Raw Time Glu Gal Xyl Ara Acetic Lactic Total
material (hrs) g/L g/L g/L g/L g/L g/L sugars Sample 1 0 3.9 0.5
24.4 1.6 2.3 0.1 30.4 Sample 2 0 3.9 0.5 23.9 1.5 2.3 0.1 29.8
Average 0 3.9 0.5 24.2 1.6 2.3 0.1 30.1
[0160] The pH was set at 5.0 at t=0 and was measured at 93 hrs and
140 hrs and was pH 4.5 in both cases, although the organic acid
concentrations did not increase significantly. The drop in pH might
have impacted the enzyme performance although the pH 4.5 is more
ideal for application than pH 5.0 (due to lower bacterial
contamination risk at 50.degree. C.).
TABLE-US-00003 TABLE 3 Enzyme dosage of example 1 in mg enzyme
protein (Bradford) per gram corn stover dry matter (mg EP/g CS dm),
for low, medium and high enzyme dosage Enzyme dosage mg EP/g CS dm
Enzyme Low Medium High GC220 1.0 3.1 10.4 Filtrase .RTM. NL 0.15
0.47 1.5 Laminex .RTM. BG 1.1 3.5 11.5
[0161] FIGS. 1a to c set out the saccharification results for the
three enzyme preparations used in these experiments. From the data
presented in FIGS. 1a to 1c it is clear that Laminex.RTM. BG very
much resembles GC 220. However, the Talaromyces Filtrase.RTM.NL
preparation seems to be very active since it liberates more sugars
per amount of enzyme protein than GC 220 or Laminex.RTM. BG.
Protein content of the enzyme mixtures was determined by Bradford
protein assay and it was determined that the protein amount used in
the experiments was less for the Talaromyces Filtrase.RTM.NL
preparation than for GC 220 or Laminex.RTM. BG. See table 3. This
demonstrates that the specific activity of the Talaromyces
Filtrase.RTM.NL preparation is higher than the other cellulase
preparations used in the comparison.
Specific Activity
[0162] The specific activity of the protein was calculated
according to the following equation:
Productivity={[Glucose+arabinose+galactose+xylose] (gram monomeric
sugars) at time X-[Glucose+arabinose+galactose+xylose] (gram
monomeric sugars) at time 0]}/[Overall incubation time
(hrs)]/[protein amount in incubation (gr protein)] [0163] in gr
fermentable sugars/gr enzyme protein/hr.
[0164] Specific activity data were only calculated for experiments
that produced more than 34 g/L of total sugar compared to the
initially available 30 g/L of sugars at time 0 because small
measurements errors have a high impact on the specific activities
at low net production levels. The specific activity was checked for
the 3 enzyme preparations at different time points and the results
obtained are set out in FIGS. 2a to c and FIGS. 3a to 3c.
[0165] From the results obtained in these experiments, it is
demonstrated that the Filtrase.RTM.NL enzyme preparation
outperforms the other benchmark commercial enzyme preparations on
sugar or glucose production per protein amount. GC 220 was
comparable to Laminex.RTM. BG.
Example 2
SSF Experiments with Filtrase.RTM.NL and Saccharomyces cerevisiae
237NG Using Pretreated Corn Stover
[0166] As follow up of the saccharification experiment described in
Example 1, Simultaneous Saccharification and Fermentation and
Distillation experiments for cellulose ethanol were carried out on
100 ml scale with dilute acid pretreated corn stover using
Filtrase.RTM.NL and Yeast (Saccharomyces cerevisiae 237 NG). Using
this technology, inhibitory glucose is removed and then higher
hydrolysis yields are assumed to be obtained as compared to
hydrolysis as such. A scheme for these experiments is set out in
FIG. 4.
Materials and Methods
[0167] Ethanol yield per biomass dry weight input and hydrolysis
yields were calculated as follows:
[0168] 1. Dry matter input: dry weight determination 48 hrs, 105
C
[0169] 2. Ethanol output: ml ethanol@100% measured on DMA
[0170] 3. Glucan content: NREL sample we used data provided by
NREL
[0171] 4. Sugars in wash liquid were determined by NMR (sum of
glucose, galactose, xylose and arabinose) and total sugars present
in hydrolysate were calculated from the mass balance of the wash
procedure (concentration of sugars multiplied by measured weight of
of wash liquid)
[0172] 5. Theoretical yield of ethanol from glucan was calculated
by: [0173] a) Amount of fiber dry weight in 250 ml flask; exact
(100,0+/-0.1 gr) weight of fiber slurry times dry matter content of
starting material (gr fiber dm)*100/330 (we prepared 3 flasks of
100 gr mash each from a 330 gr fiber-slurry preparation after
adjusting pH) value Around 10 gr fiber in 100 gr slurry=10% dm)
[0174] b) Amount of glucan was calculated by multiplying glucan
content as obtained from feedstock supplyers with dry matter
content as we determined ourselves (3-4 gr glucan in unwashed
case/100 gr fiberslurry and 5-6 gr glucan in washed fiber
preparations) [0175] c) Amount of potential glucose was calculated
by multiplying glucan with 180/162 (chemical gain factor due to
hydrolysis of glucan (glucan=cellulose=polymer of glucose) [0176]
d) Amount of potential ethanol (assuming 0.79 gr ethanol/ml
ethanol@100%) was calculated by multiplying amount of potential
glucose with the theoretical maximum yield of ethanol on glucose
being 0.511 gr ethanol/gr glucose and assuming a fermentation yield
of 91.5% of the theoretical maximum (industrial average is assumed
to be 91.5% +/-1.5% (this means between 90% and 93%). [0177] e)
(Cellulose or glucan) Hydrolysis %=100*amount of ethanol produced
(gr)/theoretical maximum ethanol (gr)
Results & Discussion
[0178] When using different enzyme dosages, low medium and high
enzyme (1, 2 and 3 respectively) dosage increasing amounts of
ethanol were obtained.
[0179] The theoretical maximum amount of ethanol that could have
been produced at 10.2% dry matter and 34% glucan content
(NREL-analysis) would have been 102*0.34*180/162*0.91*0.511=17.9 g
ethanol/L medium. As shown in FIGS. 5 and 6 it can be seen that
this amount of ethanol was reached at the highest enzyme dosage
demonstrating that full saccharification is possible with
Filtrase.RTM.NL. Surprisingly, this thermophilic enzyme is also
very effective at a mesophilic temperature of 33.degree. C.
Example 3
Saccharification of Wheat Straw Using Filtrase.RTM.NL
[0180] Wheat straw was pretreated with steam at 195.degree. C. for
12 minutes as described by Jan Larsen et al. Chem. Eng. Technol.
2008, 31, No.5, 1-9. The fiber was hydrolysed using Filtrase.RTM.NL
at 8% dry matter without any addition of acid or base at pH 3.8 at
60.degree. C. while shaking at 175 RPM in a shaker incubator.
[0181] At a 50% glucan content in the fiber, one would expect
maximum 40 g/L of glucose to be produced in this experiment the
hydrolysis yield is >85% and the total ethanol potential of this
hydrolysate would be 19 g/L at 92% fermentation yield on total
sugars of 40 g/L. FIG. 7 sets out the results of this experiment,
demonstrating that this level of glucose was achieved. Thus, the
acidic properties of the enzyme preparation thus enables ethanol
production without any addition of acid or base as pH 3.8 is also
optimal for the Yeast Saccharomyces cerevisiae.
Example 4
Saccharification of Wheat Straw Using Filtrase.RTM.NL
[0182] In 10 L scale pretreated wheat straw feedstock at 33-34% dry
matter as used in example 3 were mixed with water an enzyme
solution obtained from Filtrase.RTM.NL by dialyzing away glycerol
(which is present in the commercial product as a formulation agent)
with water over a 10 kD dialfiltration UF-unit from the commercial
preparation and concentrating the enzyme 10 fold. The total dry
matter concentration in the preparation after 6 hrs is 28% wheat
straw dry matter. See Table 4.
TABLE-US-00004 TABLE 4 Saccharification of feedstock pretreated
wheatstraw in fed-batch operation, overview of dosages at t = 1 h,
t = 3 h and t = 6 h. at high enzyme dosage (High) and medium enzyme
dosage (Medium). Age (h) Compound High Medium 0.00 Feedstock 1000 g
1000 g Cellulase enzyme 116 g 47 g Water 154 g 227 g 3.00 Feedstock
1600 g 1600 g Cellulase enzyme 36 g 14 g Water 545 g 567 g 6.00
Feedstock 3436 g 3436 g
[0183] pH was controlled at 5.0 +/-0.2 using 8N KOH and 4N
H.sub.2SO.sub.4 [0184] Temperature was controlled between 55 and
60.degree. C. [0185] Stirring was done at 700 RPM using 1 standard
Rushton turbine
[0186] After one day already very high glucose concentrations could
be measured of >110 g/L and after two days of incubation, a
glucose concentration of 128 g/L was measured using NMR sugar
measurement in the supernatant after removing of the remaining
lignin solids by means of centrifugation showing that the enzyme is
less severe inhibited by glucose than expected from literature. See
FIG. 8.
[0187] This glucose concentration is the highest glucose
concentration ever observed with a Talaromyces cellulose
preparation which enables also commercial SHF processes using this
enzyme while achieving a theoretical maximum of 65 g/L of ethanol
when all glucose would be converted to ethanol (=0.511*128
g/L).
Theoretical Maximum Yield
[0188] The theoretical maximum yield (Yps max in gr product per
gram glucose) of a fermentation were calculated as follows. For
ethanol, 1 mole of glucose (180 gr) yields according to normal
glycolysis fermentation pathway in yeast 2 moles of ethanol
(=2.times.46=92 gr ethanol. The theoretical maximum yield of
ethanol on glucose is therefore 92/180=0.511 gr ethanol/gr
glucose.
[0189] For Butanol (MW 74 gr/mole) or iso butanol, the theoretical
maximum yield is 1 mole of butanol per mole of glucose. So Yps max
for (iso-)butanol=74/180=0.411 gr (iso-)butanol/gr glucose.
[0190] For lactic acid the fermentation yield for homolactic
fermentation is 2 moles of lactic acid (MW=90 gr/mole) per mole of
glucose. According to this stoichiometry, the Yps max=1 gr lactic
acid/gr glucose.
TABLE-US-00005 TABLE 5 Achievable glucose concentrations in SHF and
achievable product concentrations in SSF for products with
different Yps max (g/g). SSF SHF Yps max (g/g) Glucose 0.511 0.411
1 Achievable Ethanol butanol lactic acid concentration g/L g/L g/L
g/L Minimum 25 12.8 10.3 25.0 Maximum 250 127.8 102.8 250.0
No Glucose Inhibition:
[0191] Table 6 shows a kinetic comparison of beta glucosidases. It
is clear from that table that the Ki (glucose) of Talaromyces
betaglucosidase is very low (0.045), which shows that the
composition according to the invention is not glucose
repressed.
TABLE-US-00006 TABLE 6 Kinetic comparison of beta glucosidases from
different sources: Talaromyces Trichoderma A. niger A. oryzae Unit
Vmax 1080 470 400 10131 gr/gr (cellobiose) protein/hr Km 0.02 0.68
0.05 2.39 g/L (cellobiose) Ki 0.045 0.108 3 245 g/L (glucose) S 2.5
2.5 2.5 2.5 g/L (Cellobiose)
* * * * *