U.S. patent application number 12/885611 was filed with the patent office on 2011-01-13 for process and composition for preparing a lignocellulose-based product, and the product obtained by the process.
This patent application is currently assigned to Yissum Research Development Company of the Hebrew University of Jerusalem. Invention is credited to Oded SHOSEYOV.
Application Number | 20110005697 12/885611 |
Document ID | / |
Family ID | 26860299 |
Filed Date | 2011-01-13 |
United States Patent
Application |
20110005697 |
Kind Code |
A1 |
SHOSEYOV; Oded |
January 13, 2011 |
PROCESS AND COMPOSITION FOR PREPARING A LIGNOCELLULOSE-BASED
PRODUCT, AND THE PRODUCT OBTAINED BY THE PROCESS
Abstract
A process for the manufacture of a lignocellulose product, the
process comprising the step of mixing in a reaction medium (i) a
phenolic polymer being substituted with a phenolic hydroxy group;
(ii) a lignocellulose containing material having immobilized to a
cellulosic fraction thereof a fusion polypeptide, the fusion
polypeptide including an enzyme being capable of catalyzing the
oxidation of phenolic groups and a cellulose binding peptide; and
(iii) an oxidizing agent. A composition of matter for use in the
process and a lignocellulose product obtainable by the process are
also disclosed.
Inventors: |
SHOSEYOV; Oded; (Karmei
Yoseef, IL) |
Correspondence
Address: |
MARTIN D. MOYNIHAN d/b/a PRTSI, INC.
P.O. BOX 16446
ARLINGTON
VA
22215
US
|
Assignee: |
Yissum Research Development Company
of the Hebrew University of Jerusalem
Jerusalem
IL
|
Family ID: |
26860299 |
Appl. No.: |
12/885611 |
Filed: |
September 20, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11191964 |
Jul 29, 2005 |
|
|
|
12885611 |
|
|
|
|
10129366 |
May 3, 2002 |
|
|
|
PCT/IL00/00665 |
Oct 19, 2000 |
|
|
|
11191964 |
|
|
|
|
60166389 |
Nov 18, 1999 |
|
|
|
60164140 |
Nov 8, 1999 |
|
|
|
Current U.S.
Class: |
162/174 |
Current CPC
Class: |
D06M 16/003 20130101;
C12Y 302/01004 20130101; D21H 17/005 20130101; C08B 15/00 20130101;
C08B 31/00 20130101; C08B 37/003 20130101; C07K 2319/00 20130101;
C12N 9/2437 20130101; C08B 15/10 20130101; C08B 37/00 20130101;
D21C 9/005 20130101; D06M 15/15 20130101 |
Class at
Publication: |
162/174 |
International
Class: |
D21H 17/22 20060101
D21H017/22 |
Claims
1. A lignocellulose product comprising a mix of: (a) a naive plant
cell wall material; and (b) a modified plant cell wall material
having immobilized to a cellulosic fraction thereof a fusion
polypeptide, said fusion polypeptide including an enzyme being
capable of catalyzing the oxidation of phenolic groups and a
cellulose binding peptide, said modified plant cell wall material
being derived from a genetically modified or virus infected plant
or cultured plant cells expressing said fusion protein.
2. The lignocellulose product of claim 1, wherein said
lignocellulose product is selected from the group consisting of
fiber board, particle board, flakeboard, plywood and molded
composites.
3. The lignocellulose product of claim 1, wherein said
lignocellulose product is selected from the group consisting of
paper and paperboard.
4. The lignocellulose product of claim 1, wherein modified plant
cell wall material is selected from the group consisting of
vegetable fiber and wood fiber derived from a genetically modified
or virus infected plant expressing said fusion polypeptide.
5. The lignocellulose product of claim 1, wherein said enzyme
catalyze formation of oxidized phenolic substituent of lignin
present in said plant cell wall material.
6. The lignocellulose product of claim 5, wherein said phenolic
substituent is selected from the group consisting of p-coumaric
acid, p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol,
ferulic acid and p-hydroxybenzoic acid.
7. The lignocellulose product of claim 1, wherein said enzyme is
selected from the group consisting of oxidases and peroxidases.
8. The lignocellulose product of claim 1, wherein said enzyme is an
oxidase selected from the group consisting of laccases (EC
1.10.3.2), catechol oxidases (EC 1.10.3.1) and bilirubin oxidases
(EC 1.3.3.5).
9. The lignocellulose product of claim 7, wherein said enzyme is a
lacase.
10. The lignocellulose product of claim 9, wherein said lacase is
present in an amount in the range of 0.02-2000 LACU per g of dry
lignocellulose.
11. The lignocellulose product of claim 1, wherein said enzyme is a
laccase encoded by a polynucleotide obtained from a fungus of the
genus Botrytis, Myceliophthora, Trametes or the plant Acer
pseudoplanus.
12. The lignocellulose product of claim 11, wherein the fungus is
Trametes versicolor or Trametes villosa.
13. The lignocellulose product of claim 1, wherein said enzyme is a
peroxidase.
14. The lignocellulose product of claim 13, wherein said peroxidase
is present in an amount in the range of 0.02-2000 PODU per g of dry
lignocellulose.
15. The lignocellulose product of claim 1, wherein an amount of
lignin therein is in the range of 0.1%-10% by weight.
16. The lignocellulose product of claim 15, wherein said naive
plant cell wall material is selected from the group consisting of
vegetable fiber, wood fiber, wood chips, wood flakes, wood veneer
and recycled fibers.
Description
RELATED PATENT APPLICATIONS
[0001] The application is a continuation of U.S. patent application
Ser. No. 11/191,964, filed on Jul. 29, 2005, which is a
continuation of U.S. patent application Ser. No. 10/129,366, filed
on May 3, 2002, which is a National Phase of PCT Application No.
PCT/IL00/00665, filed on Oct. 19, 2000, which claims the benefit
under .sctn.119(e) of U.S. Provisional Patent Application No.
60/164,140, filed on Nov. 8, 1999, and U.S. Provisional Patent
Application No. 60/166,389, filed on Nov. 18, 1999. The contents of
the above Applications are all incorporated herein by
reference.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention provides a process and compositions
for producing a lignocellulose-based product, e.g., fiber board,
such as hardboard or medium-density fiber board ("MDF"), particle
board, plywood, paper or paperboard (such as cardboard and
linerboard), from an appropriate lignocellulosic starting material,
such as wood fiber or vegetable fiber, having an enzyme adhered
thereto via a cellulose binding peptide, which enzyme is capable of
catalyzing the oxidation of phenolic groups of a phenolic polymer
which may form an integral part of the lignocellulosic starting
material, e.g., lignin, in the presence of an oxidizing agent and
optionally in the presence of additional lignocellulosic starting
material devoid of the enzyme, e.g., recycled fibers.
[0003] The use of the process of the invention confers improved
mechanical properties on lignocellulose-based products prepared
thereby, especially paper products such as liner board, cardboard
and corrugated board.
[0004] Lignocellulose-based products prepared from lignocellulosic
starting materials, notably products manufactured starting from
vegetable fiber or wood fiber prepared by mechanical or
mechanical/chemical procedures (the latter often being denoted
"semi-chemical" procedures), or by a chemical procedure without
bleaching, or from wood particles (wood "chips", flakes and the
like), are indispensable everyday materials.
[0005] Some of the most familiar types of such products include
paper for writing or printing, cardboard, corrugated cardboard,
fiber board (e.g. "hardboard"), and particle board.
[0006] Virtually all grades of paper, cardboard and the like are
produced from aqueous pulp slurry. Typically, the pulp is suspended
in water, mixed with various additives and then passed to equipment
in which the paper, cardboard etc. is formed, pressed and dried.
Irrespective of whether mechanically produced pulp (hereafter
denoted "mechanical pulp"), semi-chemically produced pulp
(hereafter denoted "semi-chemical pulp"), unbleached chemical pulp
or pulp made from recycled fibers (i.e., pulp prepared from
recycled fibers, rags and the like) is employed, it is often
necessary to add various strengthening agents to the pulp in order
to obtain an end product having adequate mechanical properties.
[0007] In the case of paper and board for use in packaging and the
like, the tensile strength and tear strength under dry and wet
conditions are of primary importance; moreover, notably in the case
of certain grades of cardboard (e.g., so-called unbleached board
for the manufacture of corrugated cardboard boxes for packaging,
transport and the like), the compression strength of the material
is often also an important factor. Among the strengthening agents
used today there are a number of environmentally undesirable
substances which it would be desirable to replace by more
environmentally acceptable materials. As examples hereof may be
mentioned epichlorohydrin, urea-formaldehyde and
melamine-formaldehyde.
[0008] In the case of "traditional" lignocellulose-based composites
for use in building construction, flooring, cladding, furniture,
packaging and the like, such as hardboard (which is normally made
from wood fibers produced by mechanical or semi-chemical means or
by so-called "steam explosion") and particle board (which is made
from relatively coarse wood particles, fragments or "chips"),
binding of the wood fibers or particles to give a coherent mass
exhibiting satisfactory strength properties can be achieved using a
process in which the fibers/particles are treated--optionally in a
mixture with one or more "extenders", such as lignosulfonates
and/or kraft lignin--with synthetic adhesives (typically adhesives
of the urea-formaldehyde, phenol-formaldehyde or isocyanate type)
and then pressed into the desired form (boards, sheets, panels
etc.) with the application of heat.
[0009] The use of synthetic adhesives of the above-mentioned types
in the production of wood products is, however, generally
undesirable from an environmental and/or safety point of view,
since many such adhesives are directly toxic--and therefore require
special handling precautions--and/or can at a later stage give rise
to release of toxic and/or environmentally harmful substances;
thus, for example, the release of formaldehyde from certain cured
formaldehyde-based adhesives (used as binders in, e.g., particle
board and the like) has been demonstrated.
[0010] In the light of the drawbacks associated with the use of
synthetic adhesives as binders in the manufacture of
lignocellulose-based products, considerable effort has been devoted
in recent years to the development of binder systems and binding
processes which are more acceptable from an environmental and
toxicity point of view, and relevant patent literature in this
respect includes the following:
[0011] EP 0 433 258 A1 discloses a procedure for the production of
mechanical pulp from a fibrous product using a chemical and/or
enzymatic treatment in which a "binding agent" is linked with the
lignin in the fibrous product via the formation of radicals on the
lignin part of the fibrous product. This document mentions
"hydrocarbonates", such as cationic starch, and/or proteins as
examples of suitable binding agents. As examples of suitable
enzymes are mentioned laccase, lignin peroxidase and manganese
peroxidase, and as examples of suitable chemical agents are
mentioned hydrogen peroxide with ferro ions, chlorine dioxide,
ozone, and mixtures thereof.
[0012] EP 0 565 109 A1 discloses a method for achieving binding of
mechanically produced wood fragments via activation of the lignin
in the middle lamella of the wood cells by incubation with
phenol-oxidizing enzymes. The use of a separate binder is thus
avoided by this method.
[0013] U.S. Pat. No. 4,432,921 describes a process for producing a
binder for wood products from a phenolic compound having phenolic
groups, and the process in question involves treating the phenolic
compound with enzymes to activate and oxidatively polymerize the
phenolic compound, thereby converting it into the binder. The only
phenolic compounds which are specifically mentioned in this
document, or employed in the working examples given therein, are
lignin sulfonates, and a main purpose of the invention described in
U.S. Pat. No. 4,432,921 is the economic exploitation of so-called
"sulfite spent liquor", which is a liquid waste product produced in
large quantities through the operation of the widely-used sulfite
process for the production of chemical pulp, and which contains
lignin sulfonates.
[0014] With respect to the use of lignin sulfonates--in particular
in the form of sulfite spent liquor--as phenolic polymers in
systems/processes for binding wood products (as described in U.S.
Pat. No. 4,432,921), the following comments are appropriate: (i)
subsequent work (see H. H. Nimz in Wood Adhesives, Chemistry and
Technology, Marcel Dekker, New York and Basel 1983, pp. 247-288),
and A Haars et al. in Adhesives from Renewable Resources, ACS
Symposium Series 385, American Chemical Society 1989, pp. 126-134)
has demonstrated that by comparison with the amounts of
"traditional" synthetic adhesives which are required in the
manufacture of wood-based boards, very large amounts of lignin
sulfonates are required in order to achieve comparable strength
properties; (ii) the pressing time required when pressing
wood-based board products prepared using lignin sulfonate binders
has been found to be very long, see E. Roffael and B. Dix, Holz als
Roh- and Werkstoff 49 (1991) 199-205; (iii) lignin sulfonates
available on a commercial scale are generally very impure and of
very variable quality, see J. L. Philippou, Journal of Wood
Chemistry and Technology 1(2) (1981) 199-227; (iv) the very dark
color of spent sulfite liquor renders it unsuited as a source of
lignin sulfonates for the production of, e.g., paper products (such
as packaging paper, linerboard or unbleached board for cardboard
boxes and the like) having acceptable color properties.
[0015] U.S. Pat. No. 5,846,788, from which the above background
information is derived, and which is incorporated by reference as
if fully set forth herein, teaches that binding of lignocellulosic
materials (vegetable fibers, wood chips, etc.) using a combination
of a polysaccharide having at least substituents containing a
phenolic hydroxy group (in the following often simply denoted a
"phenolic polysaccharide"), an oxidizing agent and an enzyme
capable of catalyzing the oxidation of phenolic groups by the
oxidizing agent can be employed in the manufacture of
lignocellulose-based products exhibiting strength properties at
least comparable to, and often significantly better than, those
achievable using previously known processes which have attempted to
reduce or avoid the use of toxic and/or otherwise harmful
substances, such as the processes described in EP 0 433 258 A1, EP
0 565 109 A1 and U.S. Pat. No. 4,432,921. Thus, for example, the
amount of binder required to prepare lignocellulose-based products
of very satisfactory strength by the process described in U.S. Pat.
No. 5,846,788 is generally much lower typically by a factor of
about three or more--than the level of binder (based on lignin
sulfonate) required to obtain comparable strength properties using
the process according to U.S. Pat. No. 4,432,921. The process
according to U.S. Pat. No. 5,846,788 can thus not only provide an
environmentally attractive alternative to more traditional binding
processes employing synthetic adhesives, but it can probably also
compete economically with such processes.
[0016] However, the process described in U.S. Pat. No. 5,846,788,
requires the use of purified enzymes which are expensive materials
as is compared to other raw materials and reagents used in the
process of manufacturing lignocellulose-based products.
[0017] There is thus a widely recognized need for, and it would be
highly advantageous to have, a process for producing a
lignocellulose-based product, e.g. fiber board, such as hardboard
or medium-density fiber board ("MDF"), particle board, plywood,
paper or paperboard (such as cardboard and linerboard), from an
appropriate lignocellulosic starting material devoid of the above
limitation.
SUMMARY OF THE INVENTION
[0018] According to one aspect of the present invention there is
provided a process for the manufacture of a lignocellulose product,
the process comprising the step of mixing in a reaction medium (i)
a phenolic polymer being substituted with a phenolic hydroxy group;
(ii) a lignocellulose containing material having immobilized to a
cellulosic fraction thereof a fusion polypeptide, the fusion
polypeptide including an enzyme being capable of catalyzing the
oxidation of phenolic groups and a cellulose binding peptide; and
(iii) an oxidizing agent.
[0019] According to further features in preferred embodiments of
the invention described below, the lignocellulose product is
selected from the group consisting of fiber board, particle board,
flakeboard, plywood and molded composites.
[0020] According to still further features in the described
preferred embodiments the lignocellulose product is selected from
the group consisting of paper and paperboard.
[0021] According to still further features in the described
preferred embodiments the lignocellulose containing material is a
cell wall preparation derived from a genetically modified or virus
infected plant or cultured plant cells expressing the fusion
protein.
[0022] According to still further features in the described
preferred embodiments the lignocellulose containing material is
selected from the group consisting of vegetable fiber and wood
fiber derived from a genetically modified or virus infected plant
expressing the fusion polypeptide.
[0023] According to still further features in the described
preferred embodiments the lignocellulose containing material is
selected from the group consisting of vegetable fiber and wood
fiber that has previously made contact with an oxidising enzyme
fused to a cellulose binding peptid.
[0024] According to still further features in the described
preferred embodiments the phenolic substituent is selected from the
group consisting of p-coumaric acid, p-coumaryl alcohol, coniferyl
alcohol, sinapyl alcohol, ferulic acid p-hydroxybenzoic acid and
any other phenolic group that can be oxidized.
[0025] According to still further features in the described
preferred embodiments the phenolic polymer forms an integral part
of the lignocellulose containing material.
[0026] According to still further features in the described
preferred embodiments the phenolic polymer is lignin.
[0027] According to still further features in the described
preferred embodiments the phenolic polymer is a phenolic
polysaccharide.
[0028] According to still further features in the described
preferred embodiments the polysaccharide portion of the phenolic
polysaccharide is selected from the group consisting of modified
and unmodified starches, modified and unmodified cellulose, and
modified and unmodified hemicelluloses.
[0029] According to still further features in the described
preferred embodiments the phenolic polysaccharide is selected from
the group consisting of ferulylated arabinoxylans and ferulylated
pectins.
[0030] According to still further features in the described
preferred embodiments the reaction medium is incubated for a period
of from 1 minute to 10 hours.
[0031] According to still further features in the described
preferred embodiments the fusion polypeptide is incubated in the
presence of the oxidizing agent for a period of from 1 minute to 10
hours.
[0032] According to still further features in the described
preferred embodiments the enzyme is selected from the group
consisting of oxidases and peroxidases.
[0033] According to still further features in the described
preferred embodiments the enzyme is an oxidase selected from the
group consisting of laccases (EC 1.10.3.2), catechol oxidases (EC
1.10.3.1) and bilirubin oxidases (EC 1.3.3.5), and the oxidizing
agent is oxygen.
[0034] According to still further features in the described
preferred embodiments the enzyme is a laccase and is present in an
amount in the range of 0.02-2000 LACU per g of dry
lignocellulose.
[0035] According to still further features in the described
preferred embodiments the reaction medium is aerated.
[0036] According to still further features in the described
preferred embodiments the enzyme is a laccase encoded by a
polynucleotide obtained from a fungus of the genus Botrytis,
Myceliophthora, or Trametes.
[0037] According to still further features in the described
preferred embodiments the fungus is Trametes versicolor or Trametes
villosa.
[0038] According to still further features in the described
preferred embodiments the enzyme is a laccase from Acer
pseudoplanus.
[0039] According to still further features in the described
preferred embodiments the enzyme is a peroxidase and the oxidizing
agent is hydrogen peroxide.
[0040] According to still further features in the described
preferred embodiments the peroxidase is present in an amount in the
range of 0.02-2000 PODU per g of dry lignocellulose, and the
initial concentration of hydrogen peroxide in the reaction medium
is in the range of 0.01-100 mM.
[0041] According to still further features in the described
preferred embodiments the amount of lignocellulose employed
corresponds to 0.1-90% by weight of the reaction medium, calculated
as dry lignocellulose.
[0042] According to still further features in the described
preferred embodiments the temperature of the reaction medium is in
the range of 10.degree.-120.degree. C.
[0043] According to still further features in the described
preferred embodiments the temperature of the reaction medium is in
the range of 15.degree.-90.degree. C.
[0044] According to still further features in the described
preferred embodiments an amount of the phenolic polysaccharide in
the range of 0.1%-10% by weight.
[0045] According to still further features in the described
preferred embodiments the pH in the reaction medium is in the range
of 3-10.
[0046] According to still further features in the described
preferred embodiments the pH in the reaction medium is in the range
of 4-9.
[0047] According to still further features in the described
preferred embodiments the reaction medium further comprising a
lignocellulose containing material devoid of the fusion
protein.
[0048] According to still further features in the described
preferred embodiments the lignocellulose containing material devoid
of the fusion protein is selected from the group consisting of
vegetable fiber, wood fiber, wood chips, wood flakes, wood veneer
and recycled fibers.
[0049] Further according to the present invention there is provided
a lignocellulose product obtainable by the process described
herein.
[0050] According to another aspect of the present invention there
is provided a genetically modified or viral infected plant or
cultured plant cells expressing a fusion protein including an
enzyme being capable of catalyzing the oxidation of phenolic groups
and a cellulose binding peptide.
[0051] According to still further features in the described
preferred embodiments the fusion protein being compartmentalized
within cells of the plant or cultured plant cells, so as to be
sequestered from cell walls of the cells of the plant or cultured
plant cells.
[0052] According to still further features in the described
preferred embodiments expression of the fusion protein is under a
control of a constitutive or tissue specific plant promoter.
[0053] According to still further features in the described
preferred embodiments the fusion protein is compartmentalized
within a cellular compartment selected from the group consisting of
cytoplasm, endoplasmic reticulum, golgi apparatus, oil bodies,
starch bodies, chloroplastids, chloroplasts, chromoplastids,
chromoplasts, vacuole, lysosomes, mitochondria, and nucleus.
[0054] According to still another aspect of the present invention
there is provided a composition of matter comprising a cell wall
preparation derived from a genetically modified or virus infected
plant or cultured plant cells expressing a fusion protein including
an enzyme being capable of catalyzing the oxidation of phenolic
groups and a cellulose binding peptide, the fusion protein being
immobilized to cellulose in the cell wall preparation via the
cellulose binding peptide.
[0055] According to still another aspect of the present invention
there is provided a nucleic acid molecule comprising (a) a promoter
sequence for directing protein expression in plant cells; and (b) a
heterologous nucleic acid sequence including (i) a first sequence
encoding a cellulose binding peptide; and (ii) a second sequence
encoding an enzyme being capable of catalyzing the oxidation of
phenolic groups, wherein the first and second sequences are joined
together in frame.
[0056] According to still further features in the described
preferred embodiments the nucleic acid molecule further comprising
a sequence element selected from the group consisting of an origin
of replication for propagation in bacterial cells, at least one
sequence element for integration into a plant's genome, a
polyadenylation recognition sequence, a transcription termination
signal, a sequence encoding a translation start site, a sequence
encoding a translation stop site, plant RNA virus derived
sequences, plant DNA virus derived sequences, tumor inducing (Ti)
plasmid derived sequences, a transposable element derived sequence
and a plant operative signal peptide for directing a protein to a
cellular compartment of a plant cell.
[0057] According to still further features in the described
preferred embodiments the cellular compartment is selected from the
group consisting of cytoplasm, endoplasmic reticulum, golgi
apparatus, oil bodies, starch bodies, chloroplastids, chloroplasts,
chromoplastids, chromoplasts, vacuole, lysosomes, mitochondria, and
nucleus.
[0058] The present invention successfully addresses the
shortcomings of the presently known configurations by providing a
process and compositions for producing a lignocellulose-based
product which obviates the need for purified enzymes which are
expensive materials as is compared to other raw materials and
reagents described in the prior art for use in the process of
manufacturing lignocellulose-based products.
DETAILED DESCRIPTION OF THE INVENTION
[0059] The present invention is of a process and composition of
matter for the manufacture of a lignocellulose-based product from a
lignocellulosic material, which process obviates the need for using
purified enzymes.
[0060] The principles and operation of a process according to the
present invention may be better understood with reference to the
accompanying descriptions.
[0061] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details of steps and components set forth
in the following description. The invention is capable of other
embodiments or of being practiced or carried out in various ways.
Also, it is to be understood that the phraseology and terminology
employed herein is for the purpose of description and should not be
regarded as limiting.
[0062] The present invention thus provides a process for the
manufacture of a lignocellulose-based product from a
lignocellulosic material. The process according to the present
invention is effected by mixing in a reaction medium (i) a phenolic
polymer substituted with a phenolic hydroxy group (e.g., lignin or
a polysaccharide which is substituted with at least substituents
containing a phenolic hydroxy group); (ii) a lignocellulose
containing material having immobilized to a cellulosic fraction
thereof a fusion polypeptide, the fusion polypeptide including an
enzyme being capable of catalyzing the oxidation of phenolic groups
and a cellulose binding peptide; and (iii) an oxidizing agent.
[0063] The order of mixing/contacting the three components is
unimportant as long as the process set-up ensures that the
activated lignocellulosic material and the activated phenolic
polysaccharide are brought together in a way that enables them to
react in the desired manner. Thus, for example, the oxidizing agent
may be mixed with the lignocellulose containing material before or
after being mixed with the phenolic polymer.
[0064] As is further detailed hereinunder, the lignocellulose
containing material is preferably a cell wall preparation derived
from a genetically modified or virus infected plant or cultured
plant cells expressing the fusion protein. As such, the phenolic
polymer may form an integral part of the lignocellulose containing
material because cell walls of plants contain lignin which is a
phenolic polymer and thus the cell wall preparation can be made to
contain lignin. In this case, and in order to prevent from the
enzyme to exert its catalytic activity ahead of time, the cell wall
preparation may be kept under a non-oxidizing atmosphere, such as
an N.sub.2 atmosphere.
[0065] It will generally be appropriate to incubate the reaction
medium containing the three components for a period of at least a
few minutes. An incubation time of from 1 minute to 10 hours will
generally be suitable, although a period of from 1 minute to 10
hours is preferable.
[0066] As already indicated, the process of the invention is well
suited to the production of all types of lignocellulose-based
products, e.g., various types of fiber board (such as hardboard),
particle board, flakeboard, such as oriented-strand board (OSB),
plywood, molded composites (e.g., shaped articles based on wood
particles, often in combination with other, non-lignocellulosic
materials, e.g., certain plastics), paper and paperboard (such as
cardboard, linerboard and the like).
[0067] Lignocellulose Containing Material:
[0068] The lignocellulose containing material employed in the
method of the invention can be in any appropriate form, e.g., in
the form of vegetable fiber (such as wood fiber) with the provision
that it is derived from a genetically modified or virus infected
plant expressing the fusion polypeptide.
[0069] If appropriate, a lignocellulosic material can be used in
combination with a non-lignocellulosic material having phenolic
hydroxy functionalities. Using the process of the invention,
intermolecular linkages between the lignocellulosic material and
the non-lignocellulosic material, respectively, may then be formed
(i.e., in a manner analogous to that in which intermolecular
linkages are formed when lignocellulosic materials alone are
employed in the process), resulting in a composite product. Besides
functioning as a good adhesive/binder, the phenolic polysaccharide
also serves as a good "gap-filler", which is a big advantage when
producing, e.g., particle boards from large wood particles.
[0070] It will normally be appropriate to employ the
lignocellulosic material in question in an amount corresponding to
a weight percentage of dry lignocellulosic material [dry substance
(DS)] in the reaction medium in the range of 0.1-90%.
[0071] The temperature of the reaction mixture in the process of
the invention may suitably be in the range of 10.degree.
C.-120.degree. C., as appropriate; however, a temperature in the
range of 15.degree. C.-90.degree. C. is generally to be preferred.
As illustrated by the working examples described in U.S. Pat. No.
5,846,788, it is anticipated that the reactions involved in a
process of the invention may take place very satisfactorily at
ambient temperatures around 20.degree. C.
[0072] In addition to lignocellulose containing material to which
the fusion protein is immobilized, the reaction medium according to
the present invention may include a lignocellulose containing
material devoid of such fusion protein, such as, but not limited
to, vegetable fiber, wood fiber, wood chips, wood flakes, wood
veneer and recycled fibers.
[0073] Phenolic Polymers:
[0074] The phenolic polymers employed in the process of the
invention may suitably be materials obtainable from natural sources
or polymers which have been chemically modified by the introduction
of substituents having phenolic hydroxy groups. Examples of the
latter category are modified starches containing phenolic
substituents, e.g., acyl-type substituents derived from
hydroxy-substituted benzoic acids (such as, e.g., 2-, 3- or
4-hydroxybenzoic acid).
[0075] The phenolic substituent(s) in phenolic polysaccharides
suited for use in the context of the present invention may suitably
be linked to the polymer species by, e.g., ester linkages or ether
linkages.
[0076] Very suitable phenolic polymers are phenolic polysaccharides
in which the phenolic substituent of the phenolic polysaccharide is
a substituent derived from a phenolic compound which occurs in at
least one of the following plant-biosynthetic pathways: from
p-coumaric acid to p-coumaryl alcohol, from p-coumaric acid to
coniferyl alcohol and from p-coumaric acid to sinapyl alcohol;
p-coumaric acid itself and the three mentioned "end products" of
the latter three biosynthetic pathways are also relevant compounds
in this respect. Examples of relevant "intermediate" compounds
formed in these biosynthetic pathways include caffeic acid, ferulic
acid (i.e., 4-hydroxy-3-methoxycinnamic acid), 5-hydroxy-ferulic
acid and sinapic acid.
[0077] Particularly suitable phenolic polysaccharides are those
which exhibit good solubility in water, and thereby in aqueous
media in the context of the invention. In this and other respects,
a number of types of phenolic polysaccharides which are readily
obtainable in uniform quality from vegetable sources have been
found to be particularly well-suited for use in the process of the
present invention. These include, but are in no way limited to,
phenolic arabino and heteroxylans, and phenolic pectins. Very
suitable examples thereof are ferulylated arabinoxylans
(obtainable, e.g., from wheat bran or maize bran) and ferulylated
pectins (obtainable from, e.g., beet pulp), i.e., arabinoxylans and
pectins containing ferulyl substituents attached via ester linkages
to the polysaccharide molecules.
[0078] The amount of phenolic polysaccharide or other phenolic
polymers, such as lignin, employed in the process of the invention
will generally be in the range of 0.01-10 weight percent, based on
the weight of lignocellulosic material (calculated as dry
lignocellulosic material), and amounts in the range of about 0.02-6
weight percent (calculated in this manner) will often be very
suitable.
[0079] Enzymes and Polynucleotides Encoding Same
[0080] In principle, any type of enzyme capable of catalyzing
oxidation of phenolic groups may be employed in the process of the
invention, with the provision that a polynucleotide encoding same
has been isolated or is readily isolateable using conventional
genetic engineering isolation techniques and which can therefore be
expressed as a part of a fusion polypeptide.
[0081] Preferred enzymes are, however, oxidases, e.g., laccases (EC
1.10.3.2), catechol oxidases (EC 1.10.3.1) and bilirubin oxidases
(EC 1.3.3.5) and peroxidases (EC 1.11.1.7). In some cases it may be
appropriate to employ two or more different enzymes in the process
of the invention.
[0082] Among types of oxidases (in combination with which
oxygen--e.g., atmospheric oxygen--is an excellent oxidizing agent),
laccases have proved to be well suited for use in the method of the
invention.
[0083] Polynucleotides encoding laccases have been or are readily
isolateable from a variety of plant and microbial sources, notably
bacteria and fungi (including filamentous fungi and yeasts), see,
for example, U.S. Pat. Nos. 5,843,745; 5,795,760; 5,770,418; and
5,750,388, which are incorporated herein by reference. Suitable
examples of polynucleotides encoding laccases include those
obtained or obtainable from strains of Aspergillus, Neurospora
(e.g., N. crassa), Podospora, Botrytis, Collybia, Fomes, Lentinus,
Pleurotus, Trametes--some species/strains of which are known by
various names and/or have previously been classified within other
genera; e.g. Trametes villosa=T. pinsitus=Polyporus pinsitis (also
known as P. pinsitus or P. villosus)=Coriolus pinsitus, Polyporus,
Rhizoctonia (e.g., R. solani), Coprinus (e.g., C. plicatilis),
Psatyrella, Myceliophthora (e.g., M. thermophila), Schytalidium,
Phlebia (e.g. P. radita; see WO 92/01046), or Coriolus (e.g., C.
hirsutus; see JP 2-238885,).
[0084] A preferred laccase in the context of the invention is that
obtainable from Trametes villosa or Acer pseudoplanus.
[0085] Polynucleotides encoding peroxidase enzymes (EC 1.11.1)
employed in the method of the invention are preferably those
obtained or obtainable from plants (e.g., horseradish peroxidase or
soy bean peroxidase) or from microorganisms, such as fungi or
bacteria. In this respect, some preferred fungi include strains
belonging to the sub-division Deuteromycotina, class Hyphomycetes,
e.g., Fusarium, Humicola, Tricoderma, Myrothecium, Verticillum,
Arthromyces, Caldariomyces, Ulocladium, Embellisia, Cladosporium or
Dreschlera, in particular Fusarium oxysporum (DSM 2672,), Humicola
insolens, Trichoderma resii, Myrothecium verrucana (IFO 6113,),
Verticillum alboatrum, Verticillum dahlie, Arthromyces ramosus
(FERM P-7754), Caldariomyces fumago, Ulocladium chartarum,
Embellisia alli or Dreschlera halodes.
[0086] Other preferred fungi include strains belonging to the
sub-division Basidiomycotina, class Basidiomycetes, e.g., Coprinus,
Phanerochaete, Coriolus or Trametes, in particular Coprinus
cinereus f. microsporus (IFO 8371), Coprinus macrorhizus,
Phanerochaete chrysosporium (e.g., NA-12) or Trametes versicolor
(e.g. PR4 28-A).
[0087] Further preferred fungi include strains belonging to the
sub-division Zygomycotina, class Mycoraceae, e.g., Rhizopus or
Mucor, in particular Mucor hiemalis.
[0088] Some preferred bacteria include strains of the order
Actinomycetales, e.g., Streptomyces spheroides (ATTC 23965),
Streptomyces thermoviolaceus (IFO 12382) or Streptoverticillum
verticillium ssp. verticillium.
[0089] Other preferred bacteria include Bacillus pumilus (ATCC
12905), Bacillus stearothermophilus, Rhodobacter sphaeroides,
Rhodomonas palustri, Streptococcus lactis, Pseudomonas purrocinia
(ATCC 15958) or Pseudomonas fluorescens (NRRL B-11).
[0090] Further preferred bacteria include strains belonging to
Myxococcus, e.g., M. virescens.
[0091] Other potential sources of useful sources for
polynucleotides encoding peroxidases are listed in B. C. Saunders
et al., Peroxidase, London 1964, pp. 41-43.
[0092] Cellulose Binding Peptides:
[0093] As used herein in the specification and in the claims
section below, the phrase "cellulose binding peptide" includes
peptides e.g., proteins and domains (portions) thereof, which are
capable of, when expressed in plant cells, affinity binding to a
plant derived cellulosic (e.g., lignocellulosic) matter, e.g.,
following homogenization and cell rupture or during plant growth
and development. The phrase thus includes, for example, peptides
which were screened for their cellulose binding activity out of a
library, such as a peptide library or a DNA library (e.g., a cDNA
expression library or a display library) and the genes encoding
such peptides isolated and are expressible in plants. Yet, the
phrase also includes peptides designed and engineered to be capable
of binding to cellulose and/or units thereof.
[0094] Such peptides include amino acid sequences expressible in
plants that are originally derived from a cellulose binding region
of, e.g., a cellulose binding protein (CBP) or a cellulose binding
domain (CBD). The cellulose binding peptide according to the
present invention can include any amino acid sequence expressible
in plants which binds to a cellulose polymer. For example, the
cellulose binding domain or protein can be derived from a
cellulase, a binding domain of a cellulose binding protein or a
protein screened for, and isolated from, a peptide library, or a
protein designed and engineered to be capable of binding to
cellulose or to saccharide units thereof, and which is expressible
in plants. The cellulose binding domain or protein can be naturally
occurring or synthetic, as long as it is expressible in plants.
Suitable polysaccharidases from which a cellulose binding domain or
protein expressible in plants may be obtained include
.beta.-1,4-glucanases. In a preferred embodiment, a cellulose
binding domain or protein from a cellulase or scaffoldin is used.
Typically, the amino acid sequence of the cellulose binding peptide
expressed in plants according to the present invention is
essentially lacking in the hydrolytic activity of a
polysaccharidase (e.g., cellulase, chitinase), but retains the
cellulose binding activity. The amino acid sequence preferably has
less than about 10% of the hydrolytic activity of the native
polysaccharidase; more preferably less than about 5%, and most
preferably less than about 1% of the hydrolytic activity of the
native polysaccharidase, ideally no activity altogether.
[0095] The cellulose binding domain or protein can be obtained from
a variety of sources, including enzymes and other proteins which
bind to cellulose which find use in the subject invention.
[0096] In Table 4 below are listed those binding domains which bind
to one or more soluble/insoluble polysaccharides including all
binding domains with affinity for soluble glucans (.alpha., .beta.,
and/or mixed linkages). The N1 cellulose-binding domain from
endoglucanase CenC of C. fimi is the only protein known to bind
soluble cellosaccharides and one of a small set of proteins which
are known to bind any soluble polysaccharides. Also, listed in
Tables 1 to 3 are examples of proteins containing putative
.beta.-1,3-glucan-binding domains (Table 1); proteins containing
Streptococcal glucan-binding repeats (Cp1 superfamily) (Table 2);
and enzymes with chitin-binding domains, which may also bind
cellulose (Table 3). The genes encoding each one of the peptides
listed in Tables 1-4 are either isolated or can be isolated as
further detailed hereinunder, and therefore, such peptides are
expressible in plants. Scaffoldin proteins or portions thereof,
which include a cellulose binding domain, such as that produced by
Clostridium cellulovorans (Shoseyov et al., PCT/US94/04132) can
also be used as the cellulose binding peptide expressible in plants
according to the present invention. Several fungi, including
Trichoderma species and others, also produce polysaccharidases from
which polysaccharide binding domains or proteins expressible in
plants can be isolated. Additional examples can be found in, for
example, Microbial Hydrolysis of Polysaccharides, R. A. J. Warren,
Annu. Rev. Microbiol. 1996, 50:183-212; and "Advances in Microbial
Physiology" R. K. Poole, Ed., 1995, Academic Press Limited, both
are incorporated by reference as if fully set forth herein.
TABLE-US-00001 TABLE 1 Overview of proteins containing putative
.beta.-1,3 glucan-binding domains Source (strain) Protein accession
No. Ref.sup.11 Type I B. circulans (WL-12) GLCA1
P23903/M34503/JQ0420 1 B. circulans (IAM 1165) BglH
JN0772/D17519/S67033 2 Type II Actinomadura sp. (FC7) XynII U08894
3 Arthrobacter sp. (YCWD3) GLCI D23668 9 O. xanthineolytica GLC
P22222/M60826/A39094 4 R. faecitabidus (YLM-50) RP I
Q05308/A45053/D10753 5a, b R. communis Ricin A12892 6 S. lividans
(1326) XlnA P26514/M64551/JS07986 7 T. tridentatus FactorGa D16622
8 B.:Bacillus, O.:Oerskovia, R. faecitabidus: Rarobacter
faecitabidus, R. communis: Ricinus communis, S.:Streptomyces,
T.:Tachypleus (Horseshoe Crab) .sup.1 References: 1 Yahata et al.
(1990) Gene 86, 113-117 2 Yamamoto et al. (1993) Biosci.
Biotechnol. Biochem. 57, 1518-1525 3 Harpin et al. (1994) EMBL Data
Library 4 Shen et al. (1991) J. Biol. Chem. 266, 1058-1063 5a
Shimoi et al. (1992) J. Biol. Chem. 267, 25189-25195 5b Shimoi et
al. (1992) J. Biochem 110, 608-613 6 Horn et al. (1989) Patent
A12892 7 Shareck et al. (1991) Gene 107, 75-82 8 Seki et al. (1994)
J. Biol. Chem. 269, 1370-1374 9 Watanabe et al. (1993) EMBL Data
Library
TABLE-US-00002 TABLE 2 Overview of proteins containing
Streptococcal glucan-binding repeats (Cpl superfamily) Source
Protein Accession No. Ref..sup.2 S. downei (sobrinus) (0MZ176)
GTF-I D13858 1 S. downei (sobrinus) (MFe28) GTF-I P11001/M17391 2
S. downei (sobrinus) (MFe28) GTF-S P29336/M30943/A41483 3 S. downei
(sobrinus) (6715) GTF-I P27470/D90216/A38175 4 S. downei (sobrinus)
DEI L34406 5 S. mutants (Ingbritt) GBP M30945/A37184 6 S. mutants
(GS-5) GTF-B A33128 7 S. mutants (GS-5) GTF-B P08987/M17361/B33135
8 S. mutants GTF-B.sup.3'-ORF P05427/C33135 8 S. mutants (GS-5)
GTF-C P13470/M17361/M22054 9 S. mutants (GS-5) GTF-C not available
10 S. mutants (GS-5) GTF-D M29296/A45866 11 S. salivarius GTF-J
A44811/S22726/S28809 Z11873/M64111 12 S. salivarius GTF-K
S22737/S22727/Z11872 13 S. salivarius (ATCC25975) GTF-L L35495 14
S. salivarius (ATCC25975) GTF-M L35928 14 S. pneumoniae R6 LytA
P06653/A25634/M13812 15 S. pneumoniae PspA A41971/M74122 16 Phage
HB-3 HBL P32762/M34652 17 Phage Cp-1 CPL-1 P15057/J03586/A31086 18
Phage Cp-9 CPL-9 P19386/M34780/JQ0438 19 Phage EJ-1 EJL A42936 20
C. difficile (VPI 10463) ToxA P16154/A37052/M30307 X51797/S08638 21
C. difficile (BARTS W1) ToxA A60991/X17194 22 C. difficile (VPI
10463) ToxB P18177/X53138/X6098 S10317 23, 24 C. difficile (1470)
ToxB S44271/Z23277 25, 26 C. novyi .alpha.-toxin S44272/Z23280 27
C. novyi .alpha.-toxin Z48636 28 C. acetobutylicum (NCIB8052) CspA
549255/Z37723 29 C. acetobutylicum (NCIB8052) CspB Z50008 30 C.
acetobutylicum (NCIB8052) CspC Z50033 30 C. acetobutylicum
(NCIB8052) CspD Z50009 30 .sup.2References: 1 Sato et al. (1993)
DNA sequence 4, 19-27 2 Ferreti et al. (1987) J. Bacteriol. 169,
4271-4278 3 Gilmore et al. (1990) J. Infect. Immun. 58, 2452-2458 4
Abo et al. (1991) J. Bacteriol. 173, 989-996 5 Sun et al. (1994) J.
Bacteriol. 176, 7213-7222 6 Banas et al. (1990) J. Infect. Immun.
58, 667-673 7 Shiroza et al. (1990) Protein Sequence Database 8
Shiroza et al. (1987) J. Bacteriol. 169, 4263-4270 9 Ueda et al.
(1988) Gene 69, 101-109 10 Russel (1990) Arch. Oral. Biol. 35,
53-58 11 Honda et al. (1990) J. Gen. Microbiol. 136, 2099-2105 12
Giffard et al. (1991) J. Gen. Microbiol. 137, 2577-2593 13 Jacques
(1992) EMBL Data Library 14 Simpson et al. (1995) J. Infect. Immun.
63, 609-621 15 Gargia et al. (1986) Gene 43, 265-272 16 Yother et
al. (1992) J. Bacteriol. 174, 601-609 17 Romero et al. (1990) J.
Bacteriol. 172, 5064-5070 18 Garcia et al. (1988) Proc. Natl. Acad.
Sci, USA 85, 914-918 19 Garcia et al. (1990) Gene 86, 81-88 20 Diaz
et al. (1992) J. Bacteriol. 174, 5516-5525 21 Dove et al. (1990) J.
Infect. Immun. 58, 480-488 22 Wren et al. (1990) FEMS Microbiol.
Lett. 70, 1-6 23 Barroso et a. (1990) Nucleic Acids Res. 18,
4004-4004 24 von Eichel-Streiber et al. (1992) Mol. Gen. Genet.
233, 260-268 25 Sartinger et al. (1993) EMBL Data Library 26 von
Eichel-Streiber et al. (1995) Mol. Microbiol. In Press 27 Hofmann
et al. (1993) EMBL Data Library 28 Hofmann et al. (1995) Mol. Gen.
Genet. In Press 29 Sanchez et al. (1994) EMBL Data Library 30
Sanchez et al. (1995) EMBL Data Library
[0097] New cellulose binding peptides with interesting binding
characteristics and specificities can be identified and screened
for and the genes encoding same isolated using well known molecular
biology approaches combined with a variety of other procedures
including, for example, spectroscopic (titration) methods such as:
NMR spectroscopy (Zhu et al. Biochemistry (1995) 34:13196-13202,
Gehring et al. Biochemistry (1991) 30:5524-5531), UV difference
spectroscopy (Belshaw et al. Eur. J. Biochem. (1993) 211:717-724),
fluorescence (titration) spectroscopy (Miller et al. J. Biol. Chem.
(1983) 258:13665-13672), UV or fluorescence stopped flow analysis
(De Boeck et al. Eur. J. Biochem. (1985) 149:141-415), affinity
methods such as affinity electrophoresis (Mimura et al. J.
chromatography (1992) 597:345-350) or affinity chromatography on
immobilized mono or oligosaccharides, precipitation or
agglutination analysis including turbidimetric or nephelometric
analysis (Knibbs et al. J. Biol. Chem. (1993) 14940-14947),
competitive inhibition assays (with or without quantitative IC50
determination) and various physical or physico-chemical methods
including differential scanning or isothermal titration calorimetry
(Sigurskjold et al. J. Biol. Chem. (1992) 267:8371-8376;
Sigurskjold et al. Eur. J. Biol. (1994) 225:133-141) or comparative
protein stability assays (melts) in the absence or presence of
oligo saccharides using thermal CD or fluorescence
spectroscopy.
[0098] The K.sub.a for binding of the cellulose binding domains or
proteins to cellulose is at least in the range of weak
antibody-antigen extractions, i.e., 10.sup.3, preferably 10.sup.4,
most preferably 10.sup.6 M.sup.-1. If the binding of the cellulose
binding domain or protein to cellulose is exothermic or
endothermic, then binding will increase or decrease, respectively,
at lower temperatures, providing a means for temperature modulation
of the binding step.
TABLE-US-00003 TABLE 3 Overview of enzymes with chitin-binding
domains Source (strain) Enzyme Accession No. Ref..sup.3 Bacterial
enzymes Type I Aeromonas sp. (No10S-24) Chi D31818 1 Bacillus
circulans (WL-12) ChiA1 P20533/M57601/A38368 2 Bacillus circulans
(WL-12) ChiD P27050/D10594 3 Janthinobacterium lividum Chi69 U07025
4 Streptomyces griseus Protease C A53669 5 Type II Aeromonas cavia
(K1) Chi U09139 6 Alteromonas sp (0-7) Chi85 A40633/P32823/D13762 7
Autographa californica (C6) NPH-128.sup.a P41684/L22858 8 Serratia
marcescens ChiA A25090/X03657/L01455/P07254 9 Type III Rhizopus
oligosporus (IFO8631) Chi1 P29026/A47022/D10157/S27418 10 Rhizopus
oligosporus (IFO8631) Chi2 P29027/B47022/D10158/S27419 10
Saccharomyces cerevisiae Chi S50371/U17243 11 Saccharomyces
cerevisiae Chi1 P29028/M74069 12 (DBY939) Saccharomyces cerevisiae
Chi2 P29029/M47407/B41035 12 (DBY918) Plant enzymes Hevein
superfamily Allium sativum Chi M94105 13 Amaranthus caudatus
AMP-1.sup.b P27275/A40240 14, 15 Amaranthus caudatus AMP-2.sup.b
S37381/A40240 14, 15 Arabidopsis thaliana ChiB P19171/M38240/B45511
16 (cv. colombia) Arabidopsis thaliana PHP.sup.c U01880 17 Brassica
napus Chi U21848 18 Brassica napus Chi2 Q09023/M95835 19 Hevea
brasiliensis Hev1.sup.d P02877/M36986/A03770/A38288 20, 21 Hordeum
vulgare Chi33 L34211 22 Lycopersicon esculentum Chi9
Q05538/Z15140/S37344 23 Nicotiana tabacum CBP20.sup.e 572424 24
Nicotiana tabacum Chi A21091 25 Nicotiana tabacum (cv. Havana) Chi
A29074/M15173/S20981/S19855 26 Nicotiana tabacum (FB7-1) Chi
JQ0993/S0828 27 Nicotiana tabacum (cv. Samsun) Chi A16119 28
Nicotiana tabacum (cv. Havana) Chi P08252/X16939/S08627 27
Nicotiana tabacum (cv. BY4) Chi P24091/X51599/X64519//S13322 26,
27, 29 Nicotiana tabacum (cv. Havana) Chi P29059/X64518/S20982 26
Oryza sativum (IR36) ChiA L37289 30 Oryza sativum ChiB
JC2253/S42829/Z29962 31 Oryza sativum Chi 539979/S40414/X56787 32
Oryza sativum (cv. Japonicum) Chi X56063 33 Oryza sativum (cv.
Japonicum) Chi1 P24626/X54367/S14948 34 Oryza sativum Chi2
P25765/S15997 35 Oryza sativum (cv. Japonicum) Chi3 D16223 Oryza
sativum ChiA JC2252/S42828 30 Oryza sativum Chi1 D16221 32 Oryza
sativum (IR58) Chi U02286 36 Oryza sativum Chi X87109 37 Pisum
sativum (cv. Birte) Chi P36907/X63899 38 Pisum sativum (cv. Alcan)
Chi2 L37876 39 Populus trichocarpa Chi S18750/S18751/X59995/P29032
40 Populus trichocarpa (H11-11) Chi U01660 41 Phaseolus vulgaris
(cv. Saxa) Chi A24215/S43926/Jq0965/P36361 42 Phaseolus vulgaris
(cv. Saxa) Chi P06215/M13968/M19052/A25898 43, 44, 45 Sambucus
nigra PR-3.sup.f Z46948 46 Secale cereale Chi JC2071 47 Solanum
tuberosum ChiB1 U02605 48 Solanum tuberosum ChiB2 U02606 48 Solanum
tuberosum ChiB3 U02607/S43317 48 Solanum tuberosum ChiB4 U02608 48
Solanum tuberosum WIN-1.sup.g P09761/X13497/S04926 49 (cv. Maris
Piper) Solanum tuberosum WIN-2.sup.g P09762/X13497/S04927 49 (cv.
Maris Piper) Triticum aestivum Chi S38670/X76041 50 Triticum
aestivum WGA-1.sub.h.sup.h P10968/M25536/S09623/S07289 51, 52
Triticum aestivum WGA-2.sup.h P02876/M25537/S09624 51, 53 Triticum
aestivum WGA-3 P10969/J02961/S10045/A28401 54 Ulmus americana
(NPS3-487) Chi L22032 55 Urtica dioica AGL.sup.i M87302 56 Vigna
unguiculata Chi1 X88800 57 (cv. Red caloona) .sup.aNHP: nuclear
polyhedrosis virus endochitinase like sequence; Chi: chitinase,
.sup.banti-microbial peptide, .sup.cpre-hevein like protein,
.sup.dhevein, .sup.echitin-binding protein, .sup.fpathogenesis
related protein, .sup.gwound-induced protein, .sup.hwheat germ
agglutinin, .sup.iagglutinin (lectin). .sup.3References: 1 Udea et
al. (1994) J. Ferment. Bioeng. 78, 205-211 2 Watanabe et al. (1990)
J. Biol. Chem. 265, 15659-16565 3 Watanabe et al. (1992) J.
Bacteriol. 174, 408-414 4 Gleave et al. (1994) EMBL Data Library 5
Sidhu et al. (1994) J. Biol. Chem. 269, 20167-20171 6 Jones et al.
(1986) EMBO J. 5, 467-473 7 Sitrit et al. (1994) EMBL Data Library
8 Genbank entry only 9 Tsujibo et al. (1993) J. Bacteriol. 175,
176-181 10 Yanai et al. (1992) J. Bacteriol. 174, 7398-7406 11
Pauley (1994) EMBL Data Library 12 Kuranda et al. (1991) J. Biol.
Chem. 266, 19758-19767 13 van Damme et al. (1992) EMBL Data Library
14 Broekaert et al. (1992) Biochemistry 31, 4308-4314 15 de Bolle
et al. (1993) Plant Mol. Physiol. 22, 1187-1190 16 Samac et al.
(1990) Plant Physiol. 93, 907-914 17 Potter et al. (1993) Mol.
Plant Microbe Interact. 6, 680-685 18 Buchanan-Wollaston (1995)
EMBL Data Library 19 Hamel et al. (1993) Plant Physiol. 101,
1403-1403 20 Broekaert et al. (1990) Proc. Natl. Acad. Sci. USA 87,
7633-7637 21 Lee et al. (1991) J. Biol. Chem. 266, 15944-15948 22
Leah et al. (1994) Plant Physiol. 6, 579-589 23 Danhash et al.
(1993) Plant Mol. Biol. 22 1017-1029 24 Ponstein et al. (1994)
Plant Physiol. 104, 109-118 25 Meins et al. (1991) Patent
EP0418695-A1 26 van Buuren et al. (1992) Mol. Gen. Genet. 232,
460-469 27 Shinshi et al. (1990) Plant Mol. Biol. 14, 357-368 28
Cornellisen et al. (1991) Patent EP0440304-A2 29 Fukuda et al.
(1991) Plant Mol. Biol. 16, 1-10 30 Yun et al. (1994) EMBL Data
Library 31 Kim et al. (1994) Biosci. Biotechnol. Biochem. 58,
1164-1166 32 Nishizawa et al. (1993) Mol. Gen. Genet. 241, 1-10 33
Nishizawa et al. (1991) Plant Sci 76, 211-218 34 Huang et al.
(1991) Plant Mol. Biol. 16, 479-480 35 Zhu et al. (1991) Mol. Gen.
Genet. 226, 289-296 36 Muthukrishhnan et al. (1993) EMBL Data
Library 37 Xu (1995) EMBL Data Library 38 Vad et al. (1993) Plant
Sci 92, 69-79 39 Chang et al. (1994) EMBL Data Library 40 Davis et
al. (1991) Plant Mol. Biol. 17, 631-639 41 Clarke et al. (1994)
Plant Mol. Biol. 25, 799-815 42 Broglie et al. (1989) Plant Cell 1,
599-607 43 Broglie et al. (1986) Proc. Natl. acad. Sci. USA 83,
6820-6824 44 Lucas et al. (1985) FEBS Lett. 193, 208-210 45 Hedrick
et al. (1988) Plant Physiol. 86, 182-186 46 Roberts et al. (1994)
EMBL Data Library 47 Vamagami et al. (1994) Biosci. Biotechnol.
Biochem. 58, 322-329 48 Beerhues et al. (1994) Plant Mol. Biol. 24,
353-367 49 Stanford et al. (1989) Mol. Gen. Genet. 215, 200-208 50
Liao et al. (1993) EMBL Data Library 51 Smith et al. (1989) Plant
Mol. Biol. 13, 601-603 52 Wright et al. (1989) J. Mol. Evol. 28,
327-336 53 Wright et al. (1984) Biochemistry 23, 280-287 54 Raikhel
et al. (1987) Proc. Natl. acad. Sci. USA 84, 6745-6749 55 Hajela et
al. (1993) EMBL Data LibraryI 56 Lerner et al. (1992) J. Biol.
Chem. 267, 11085-11091 57 Vo et al. (1995) EMBL Data Library
TABLE-US-00004 TABLE 4 Sources of polysaccharide binding domains
Proteins Where Binding Binding Domain Domain is Found Cellulose
Binding .beta.-glucanases (avicelases, CMCases, Domains.sup.1
cellodextrinases) exoglucanses or cellobiohydrolases cellulose
binding proteins xylanases mixed xylanases/glucanases esterases
chitinases .beta.-1,3-glucanases .beta.-1,3-(.beta.-1,4)-glucanases
(.beta.-)mannanases .beta.-glucosidases/galactosidases cellulose
synthases (unconfirmed) Starch/Maltodextrin -amylases.sup.2,3
Binding Domains .beta.-amylases.sup.4,5 pullulanases
glucoamylases.sup.6,7 cyclodextrin glucotransferases.sup.8-10
(cyclomaltodextrin glucanotransferases) maltodextrin binding
proteins.sup.11 Dextran Binding Domains (Streptococcal) glycosyl
transferases.sup.12 dextran sucrases (unconfirmed) Clostridial
toxins.sup.13,14 glucoamylases.sup.6 dextran binding proteins
.beta.-Glucan Binding Domains .beta.-1,3-glucanases.sup.15,16
.beta.-1,3-(.beta.-1,4)-glucanases (unconfirmed) .beta.-1,3-glucan
binding protein.sup.17 Chitin Binding Domains chitinases
chitobiases chitin binding proteins (see also cellulose binding
domains) Heivein .sup.1Gilkes et al., Adv. Microbiol Reviews,
(1991) 303-315. .sup.2S?gaard et al., J. Biol. Chem. (1993)
268:22480. .sup.3Weselake et al., Cereal Chem. (1983) 60:98.
.sup.4Svensson et al., J. (1989) 264:309. .sup.5Jespersen et al.,
J. (1991) 280:51. .sup.6Belshaw et al., Eur. J. Biochem. (1993)
211:717. .sup.7Sigurskjold et al., Eur. J. Biochem. (1994) 225:133.
.sup.8Villette et al., Biotechnol. Appl. Biochem. (1992) 16:57.
.sup.9Fukada et al., Biosci. Biotechnol. Biochem. (1992) 56:556.
.sup.10Lawson et al., J. Mol. Biol. (1994) 236:590. .sup.14von
Eichel-Streiber et al., Mol. Gen. Genet. (1992) 233:260.
.sup.15Klebl et al., J. Bacteriol. (1989) 171:6259. .sup.16Watanabe
et al., J. Bacteriol. (1992) 174:186. .sup.17Duvic et al., J. Biol.
Chem. (1990): 9327.
[0099] Thus, and as already stated, the phrase "polysaccharide
binding peptide" includes an amino acid sequence which comprises at
least a functional portion of a polysaccharide binding region
(domain) of a polysaccharidase or a polysaccharide binding protein.
The phrase further relates to a polypeptide screened for its
cellulose binding activity out of a library, such as a peptide
library or a DNA library (e.g., a cDNA library or a display
library). By "functional portion" is intended an amino acid
sequence which binds to cellulose.
[0100] The techniques used in isolating polysaccharidase genes,
such as cellulase genes, and genes for cellulose binding proteins
are known in the art, including synthesis, isolation from genomic
DNA, preparation from cDNA, or combinations thereof. (See, U.S.
Pat. Nos. 5,137,819; 5,202,247; 5,340,731; 5,496,934; and
5,837,814). The sequences for several binding domains, which bind
to soluble oligosaccharides are known (See, FIG. 1 of
PCT/CA97/00033, WO 97/26358). The DNAs coding for a variety of
polysaccharidases and polysaccharide binding proteins are also
known. Various techniques for manipulation of genes are well known,
and include restriction, digestion, resection, ligation, in vitro
mutagenesis, primer repair, employing linkers and adapters, and the
like (see Sambrook et al., Molecular Cloning--A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989,
which is incorporated herein by reference).
[0101] The amino acid sequence of a polysaccharidase can be used to
design a probe to screen a cDNA or a genomic library prepared from
mRNA or DNA from cells of interest as donor cells for a
polysaccharidase gene or a polysaccharide binding protein gene. By
using the polysaccharidase cDNA or binding protein cDNA or a
fragment thereof as a hybridization probe, structurally related
genes found in other species can be easily cloned and provide a
cellulose binding peptide which is expressible in plants according
to the present invention. Particularly contemplated is the
isolation of genes from organisms that express polysaccharidase
activity using oligonucleotide probes based on the nucleotide
sequences of genes obtainable from an organism wherein the
catalytic and binding domains of the polysaccharidase are discrete,
although other polysaccharide binding proteins also can be used
(see, for example, Shoseyov, et al., Proc. Nat'l. Acad. Sci. (USA)
(1992) 89:3483-3487).
[0102] Probes developed using consensus sequences for the binding
domain of a polysaccharidase or polysaccharide-binding protein are
of particular interest. The .beta.-1,4-glycanases from C. fimi
characterized to date are endoglucanases A, B, C and D (CenA, CenB,
CenC and CenD, respectively), exocellobiohydrolases A and B (CbhA
and CbhB, respectively), and xylanases A and D (Cex and XylD,
respectively) (see Wong et al. (1986) Gene, 44:315; Meinke et al.
(1991) J. Bacteriol., 173:308; Coutinho et al., (1991) Mol.
Microbiol. 5:1221; Meinke et al., (1993) Bacteriol., 175:1910;
Meinke et al., (1994) Mol. Microbiol., 12:413; Shen et al.,
Biochem. J., in press; O'Neill et al., (1986) Gene, 44:325; and
Millward-Sadler et al., (1994) Mol. Microbiol., 11:375). All are
modular proteins of varying degrees of complexity, but with two
features in common: a catalytic domain (CD) and a cellulose-binding
domain (CBD) which can function independently (see Millward-Sadler
et al., (1994) Mol. Microbiol., 11:375; Gilkes et al., (1988) J.
Biol. Chem., 263:10401; Meinke et al., (1991) J. Bacteriol.,
173:7126; and Coutinho et al., (1992) Mol. Microbiol., 6:1242). In
four of the enzymes, CenB, CenD, CbhA and CbhB, fibronectin type
III (Fn3) repeats separate the N-terminal CD from the C-terminal
CBD. The CDs of the enzymes come from six of the families of
glycoside hydrolases (see Henrissat (1991) Biochem. J., 280:309;
and Henrissat et al., (1993) Biochem. J., 293:781); all of the
enzymes have an N- or C-terminal CBD or CBDs (see Tomme et al.,
Adv. Microb. Physiol., in press); CenC has tandem CBDs from family
IV at its N-terminus; CenB and XylD each have a second, internal
CBD from families III and II, respectively. Cex and XylD are
clearly xylanases; however, Cex, but not XylD, has low activity on
cellulose. Nonetheless, like several other bacterial xylanases (see
Gilbert et al., (1993) J. Gen. Microbiol., 139:187), they have
CBDs. C. fimi probably produces other .beta.-1,4-glycanases.
Similar systems are produced by related bacteria (see Wilson (1992)
Crit. Rev. Biotechnol., 12:45; and Hazlewood et al., (1992) J.
Appl. Bacteriol., 72:244). Unrelated bacteria also produce
glycanases; Clostridium thermocellum, for example, produces twenty
or more .beta.-1,4-glycanases (see Beguin et al., (1992) FEMS
Microbiol. Lett., 100:523). The CBD derived from C. fimi
endoglucanase C N1, is the only protein known to bind soluble
cellosaccharides and one of a small set of proteins that are known
to bind any soluble polysaccharides.
[0103] Examples of suitable binding domains are shown in FIG. 1 of
PCT/CA97/00033 (WO 97/26358), which presents an alignment of
binding domains from various enzymes that bind to polysaccharides
and identifies amino acid residues that are conserved among most or
all of the enzymes. This information can be used to derive a
suitable oligonucleotide probe using methods known to those of
skill in the art. The probes can be considerably shorter than the
entire sequence but should at least be 10, preferably at least 14,
nucleotides in length. Longer oligonucleotides are useful, up to
the full length of the gene, preferably no more than 500, more
preferably no more than 250, nucleotides in length. RNA or DNA
probes can be used. In use, the probes are typically labeled in a
detectable manner, for example, with .sup.32P, .sup.3H, biotin,
avidin or other detectable reagents, and are incubated with
single-stranded DNA or RNA from the organism in which a gene is
being sought. Hybridization is detected by means of the label after
the unhybridized probe has been separated from the hybridized
probe. The hybridized probe is typically immobilized on a solid
matrix such as nitrocellulose paper. Hybridization techniques
suitable for use with oligonucleotides are well known to those
skilled in the art. Although probes are normally used with a
detectable label that allows easy identification, unlabeled
oligonucleotides are also useful, both as precursors of labeled
probes and for use in methods that provide for direct detection of
double-stranded DNA (or DNA/RNA). Accordingly, the term
"oligonucleotide probe" refers to both labeled and unlabeled
forms.
[0104] Generally, the binding domains identified by probing nucleic
acids from an organism of interest will show at least about 40%
identity (including as appropriate allowances for conservative
substitutions, gaps for better alignment and the like) to the
binding region or regions from which the probe was derived and will
bind to a soluble .beta.-1,4 glucan with a K.sub.a of
.gtoreq.10.sup.3 M.sup.-1. More preferably, the binding domains
will be at least about 60% identical, and most preferably at least
about 70% identical to the binding region used to derive the probe.
The percentage of identity will be greater among those amino acids
that are conserved among polysaccharidase binding domains. Analyses
of amino acid sequence comparisons can be performed using programs
in PC/Gene (IntelliGenetics, Inc.). PCLUSTAL can be used for
multiple sequence alignment and generation of phylogenetic
trees.
[0105] In order to isolate the polysaccharide binding protein or a
polysaccharide binding domain from an enzyme or a cluster of
enzymes that binds to a polysaccharide, several genetic approaches
can be used. One method uses restriction enzymes to remove a
portion of the gene that codes for portions of the protein other
than the binding portion thereof. The remaining gene fragments are
fused with expression control sequences to obtain a mutated gene
that encodes a truncated protein. Another method involves the use
of exonucleases such as Bal31 to systematically delete nucleotides
either externally from the 5' and the 3' ends of the DNA or
internally from a restricted gap within the gene. These gene
deletion methods result in a mutated gene encoding a shortened
protein molecule which can then be evaluated for substrate or
polysaccharide binding ability.
[0106] Any cellulose binding protein or cellulose binding domain
may be used in the present invention. The term "cellulose binding
protein" ("CBP") refers to any protein or polypeptide which
specifically binds to cellulose. The cellulose binding protein may
or may not have cellulose or cellulolytic activity. The term
"cellulose binding domain" ("CBD") refers to any protein or
polypeptide which is a region or portion of a larger protein, said
region or portion binds specifically to cellulose. The cellulose
binding domain (CBD) may be a part or portion of a cellulase,
xylanase or other polysaccharidase, e.g., a chitinase, etc., a
sugar binding protein such as maltose binding protein, or
scaffoldin such as CbpA of Clostridium celluvorans, etc. Many
cellulases and hemicellulases (e.g., xylanases and mannases) have
the ability to associate with cellulose. These enzymes typically
have a catalytic domain containing the active site for substrate
hydrolysis and a carbohydrate-binding domain or cellulose-binding
domain for binding cellulose. The CBD may also be from a
non-catalytic polysaccharide binding protein. To date, more than
one hundred cellulose-binding domains (CBDs) have been classified
into at least thirteen families designated I-XIII (Tomme et al.
(1995) "CelluloseBinding Domains: Classification and Properties",
in ACS Symposium Series 618 Enzymatic Degradation and Insoluble
Carbohydrates, pp. 142-161, Saddler and Penner eds., American
Chemical Society, Washington, D.C. (Tomme I); Tomme et al. Adv.
Microb. Physiol. (1995) 37:1 (Tomme II); and Smant et al., Proc.
Natl. Acad. Sci U.S.A. (1998) 95:4906,-4911, all of which are
incorporated herein by reference). Any of the CBDs described in
Tomme I or II or any variants thereof, any other presently known
CBDs or any new CBDs which may be identified can be used in the
present invention. As an illustrative, but in no way limiting
example, the CBP or CBD can be from a bacterial, fungal, slime
mold, or nematode protein or polypeptide. For a more particular
illustrative example, the CBD is obtainable from Clostridium
cellulovorans, Clostridium cellulovorans, or Cellulomonas fimi
(e.g., CenA, CenB, CenD, Cex). In addition, the CBD may be selected
from a phage display peptide or peptidomimetic library, random or
otherwise, using e.g., cellulose as a screening agent. (See Smith
Science (1985) 228:1315-1317 and Lam, Nature (1991) 354:82-84).
[0107] Furthermore, the CBD may be derived by mutation of a portion
of a protein or polypeptide which binds to a polysaccharide other
than cellulose (or hemicellulose) but also binds cellulose, such as
a chitinase, which specifically binds chitin, or a sugar binding
protein such as maltose binding protein, rendering said portion
capable of binding to cellulose. In any event, the CBD binds
cellulose or hemicellulose. Shoseyov and Doi (Proc. Natl. Acad.
Sci. USA (1990) 87:2192-2195) isolated a unique cellulose-binding
protein (CbpA) from the cellulose "complex" of the cellulolytic
bacterium Clostridium cellulovorans. This major subunit of the
cellulose complex was found to bind to cellulose, but had no
hydrolytic activity, and was essential for the degradation of
crystalline cellulose. The CbpA gene has been cloned and sequenced
(Shoseyov et al. Proc. Natl. Acad. Sci. USA (1992) 89:3483-3487).
Using PCR primers flanking the cellulose-binding domain of CbpA,
the latter was successfully cloned into an overexpression vector
that enabled overproduction of the approximately 17 kDa CBD in
Escherichia coli. The recombinant CBD exhibits very strong affinity
to cellulose and chitin (U.S. Pat. No. 5,496,934; Goldstein et al.,
J. Bacteriol. (1993) 175:5762; PCT International Publication WO
94/24158, all are incorporated by reference as if fully set forth
herein).
[0108] In recent years, several CBDs have been isolated from
different sources. Most of these have been isolated from proteins
that have separate catalytic, i.e., cellulose and cellulose binding
domains, and only two have been isolated from proteins that have no
apparent hydrolytic activity but possess cellulose-binding activity
(Goldstein et al. J. Bacteriol. (1993) 175:5762-5768; Morag et al.
Appl. (1995) Environ. Microbiol. 61:1980-1986).
[0109] Cellulose Binding Peptide-Recombinant Protein Fusions:
[0110] The fusion of two proteins for which genes has been
isolated, such as a cellulose binding peptide and an oxidase, such
as a laccase, is well known and regularly practiced in the art.
Such fusion involves the joining together of heterologous nucleic
acid sequences, in frame, such that translation thereof results in
the generation of a fused protein product or a fusion proteins.
Methods, such as the polymerase chain reaction (PCR), restriction,
nuclease digestion, ligation, synthetic oligonucleotides synthesis
and the like are typically employed in various combinations in the
process of generating fusion gene constructs. One ordinarily
skilled in the art can readily form such constructs for any pair or
more of individual proteins. Interestingly, in most cases where
such fusion or chimera proteins are produced, and in all cases
where one of the proteins was a cellulose binding peptide, both the
former and the latter retained their catalytic activity or
function. In any case, an in frame spacer can be included. The
length thereof may range, for example, from several to several
dozens of amino acids. Such a spacer may also function to reduce
mobilization constraints.
[0111] For example, Greenwood et al. (1989, FEBS Lett. 224:127-131)
fused the cellulose binding region of Cellulomonas fimi
endoglucanase to the enzyme alkaline phosphatase. The recombinant
fusion protein retained both its phosphatase activity and the
ability to bind to cellulose. For more descriptions of cellulose
binding fusion proteins, see U.S. Pat. No. 5,137,819 issued to
Kilburn et al., and U.S. Pat. No. 5,719,044 issued to Shoseyov et
al. both incorporated by reference herein. See also U.S. Pat. No.
5,474,925. All of which are incorporated herein by reference.
[0112] Thus, according to the present invention there is provided a
nucleic acid molecule comprising a promoter sequence for directing
protein expression in plant cells and a heterologous nucleic acid
sequence including a first sequence encoding a cellulose binding
peptide; and a second sequence encoding an enzyme being capable of
catalyzing the oxidation of phenolic groups, wherein the first and
second sequences are joined together in frame.
[0113] According to a preferred embodiment of the invention the
nucleic acid molecule further comprising a sequence element
selected from the group consisting of an origin of replication for
propagation in bacterial cells, at least one sequence element for
integration into a plant's genome, a polyadenylation recognition
sequence, a transcription termination signal, a sequence encoding a
translation start site, a sequence encoding a translation stop
site, plant RNA virus derived sequences, plant DNA virus derived
sequences, tumor inducing (Ti) plasmid derived sequences, a
transposable element derived sequence and a plant operative signal
peptide for directing a protein to a cellular compartment of a
plant cell.
[0114] According to still a preferred embodiment, the cellular
compartment is selected from the group consisting of cytoplasm,
endoplasmic reticulum, golgi apparatus, oil bodies, starch bodies,
chloroplastids, chloroplasts, chromoplastids, chromoplasts,
vacuole, lysosomes, mitochondria, and nucleus.
[0115] Genetically Modified Plant Material:
[0116] The present invention employs recombinant nucleic acid
molecules. Such a molecule includes, for example, a promoter
sequence for directing protein expression in plant cells; and a
heterologous nucleic acid sequence as further detailed herein,
wherein, the heterologous nucleic acid sequence is down stream the
promoter sequence, such that expression of the heterologous nucleic
acid sequence is effectable by the promoter sequence. Such a
nucleic acid molecule needs to be effectively introduced into plant
cells, so as to genetically modify the plant.
[0117] There are various methods of introducing foreign genes into
both monocotyledonous and dicotyledenous plants (Potrykus, I.,
Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225;
Shimamoto et al., Nature (1989) 338:274-276). The principle methods
of causing stable integration of exogenous DNA into plant genomic
DNA include two main approaches:
[0118] (i) Agrobacterium-mediated gene transfer: Klee et al. (1987)
Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell
Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular
Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K.,
Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in
Plant Biotechnology, eds. Kung, S, and Arntzen, C. J., Butterworth
Publishers, Boston, Mass. (1989) p. 93-112.
[0119] (ii) direct DNA uptake: Paszkowski et al., in Cell Culture
and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of
Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic
Publishers, San Diego, Calif. (1989) p. 52-68; including methods
for direct uptake of DNA into protoplasts, Toriyama, K. et al.
(1988) Bio/Technology 6:1072-1074. DNA uptake induced by brief
electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988)
7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection
into plant cells or tissues by particle bombardment, Klein et al.
Bio/Technology (1988) 6:559-563; McCabe et al. Bio/Technology
(1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209; by
the use of micropipette systems: Neuhaus et al., Theor. Appl.
Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant.
(1990) 79:213-217; or by the direct incubation of DNA with
germinating pollen, DeWet et al. in Experimental Manipulation of
Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels,
W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad.
Sci. USA (1986) 83:715-719.
[0120] The Agrobacterium system includes the use of plasmid vectors
that contain defined DNA segments that integrate into the plant
genomic DNA. Methods of inoculation of the plant tissue vary
depending upon the plant species and the Agrobacterium delivery
system. A widely used approach is the leaf disc procedure which can
be performed with any tissue explant that provides a good source
for initiation of whole plant differentiation. Horsch et al. in
Plant Molecular Biology Manual A5, Kluwer Academic Publishers,
Dordrecht (1988) p. 1-9. The Agrobacterium system is especially
viable in the creation of transgenic dicotyledenous plants.
[0121] There are various methods of direct DNA transfer into plant
cells. In electroporation, the protoplasts are briefly exposed to a
strong electric field. In microinjection, the DNA is mechanically
injected directly into the cells using very small micropipettes. In
microparticle bombardment, the DNA is adsorbed on microprojectiles
such as magnesium sulfate crystals or tungsten particles, and the
microprojectiles are physically accelerated into cells or plant
tissues.
[0122] Following transformation plant propagation is exercised. The
most common method of plant propagation is by seed. Regeneration by
seed propagation, however, has the deficiency that due to
heterozygosity there is a lack of uniformity in the crop, since
seeds are produced by plants according to the genetic variances
governed by Mendelian rules. Basically, each seed is genetically
different and each will grow with its own specific traits.
Therefore, it is preferred that the transgenic plant be produced
such that the regenerated plant has the identical traits and
characteristics of the parent transgenic plant, e.g., a
reproduction of the fusion protein. Therefore, it is preferred that
the transgenic plant be regenerated by micropropagation which
provides a rapid, consistent reproduction of the transgenic
plants.
[0123] Micropropagation is a process of growing new generation
plants from a single piece of tissue that has been excised from a
selected parent plant or cultivar. This process permits the mass
reproduction of plants having the preferred tissue expressing the
fusion protein. The new generation plants which are produced are
genetically identical to, and have all of the characteristics of,
the original plant. Micropropagation allows mass production of
quality plant material in a short period of time and offers a rapid
multiplication of selected cultivars in the preservation of the
characteristics of the original transgenic or transformed plant.
The advantages of cloning plants are the speed of plant
multiplication and the quality and uniformity of plants
produced.
[0124] Micropropagation is a multi-stage procedure that requires
alteration of culture medium or growth conditions between stages.
Thus, the micropropagation process involves four basic stages:
Stage one, initial tissue culturing; stage two, tissue culture
multiplication; stage three, differentiation and plant formation;
and stage four, greenhouse culturing and hardening. During stage
one, initial tissue culturing, the tissue culture is established
and certified contaminant-free. During stage two, the initial
tissue culture is multiplied until a sufficient number of tissue
samples are produced to meet production goals. During stage three,
the tissue samples grown in stage two are divided and grown into
individual plantlets. At stage four, the transgenic plantlets are
transferred to a greenhouse for hardening where the plants'
tolerance to light is gradually increased so that it can be grown
in the natural environment.
[0125] The basic bacterial/plant vector construct will preferably
provide a broad host range prokaryote replication origin; a
prokaryote selectable marker; and, for Agrobacterium
transformations, T DNA sequences for Agrobacterium-mediated
transfer to plant chromosomes. Where the heterologous sequence is
not readily amenable to detection, the construct will preferably
also have a selectable marker gene suitable for determining if a
plant cell has been transformed. A general review of suitable
markers for the members of the grass family is found in Wilmink and
Dons, Plant Mol. Biol. Reptr. (1993) 11:165-185.
[0126] Sequences suitable for permitting integration of the
heterologous sequence into the plant genome are also recommended.
These might include transposon sequences and the like for
homologous recombination as well as Ti sequences which permit
random insertion of a heterologous expression cassette into a plant
genome.
[0127] Suitable prokaryote selectable markers include resistance
toward antibiotics such as ampicillin or tetracycline. Other DNA
sequences encoding additional functions may also be present in the
vector, as is known in the art.
[0128] The constructs of the subject invention will include an
expression cassette for expression of the fusion protein of
interest. Usually, there will be only one expression cassette,
although two or more are feasible. The recombinant expression
cassette will contain in addition to the heterologous sequence one
or more of the following sequence elements, a promoter region,
plant 5' untranslated sequences, initiation codon depending upon
whether or not the structural gene comes equipped with one, and a
transcription and translation termination sequence. Unique
restriction enzyme sites at the 5' and 3' ends of the cassette
allow for easy insertion into a pre-existing vector.
[0129] Viral Infected Plant Material:
[0130] Viruses are a unique class of infectious agents whose
distinctive features are their simple organization and their
mechanism of replication. In fact, a complete viral particle, or
virion, may be regarded mainly as a block of genetic material
(either DNA or RNA) capable of autonomous replication, surrounded
by a protein coat and sometimes by an additional membranous
envelope such as in the case of alpha viruses. The coat protects
the virus from the environment and serves as a vehicle for
transmission from one host cell to another.
[0131] Viruses that have been shown to be useful for the
transformation of plant hosts include CaV, TMV and BV.
Transformation of plants using plant viruses is described in U.S.
Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Published
Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV);
and Gluzman, Y. et al., Communications in Molecular Biology: Viral
Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189
(1988). Pseudovirus particles for use in expressing foreign DNA in
many hosts, including plants, is described in WO 87/06261.
[0132] Construction of plant RNA viruses for the introduction and
expression of non-viral foreign genes in plants is demonstrated by
the above references as well as by Dawson, W. O. et al., Virology
(1989) 172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311;
French et al. Science (1986) 231:1294-1297; and Takamatsu et al.
FEBS Letters (1990) 269:73-76.
[0133] When the virus is a DNA virus, the constructions can be made
to the virus itself. Alternatively, the virus can first be cloned
into a bacterial plasmid for ease of constructing the desired viral
vector with the foreign DNA. The virus can then be excised from the
plasmid. If the virus is a DNA virus, a bacterial origin of
replication can be attached to the viral DNA, which is then
replicated by the bacteria. Transcription and translation of this
DNA will produce the coat protein which will encapsidate the viral
DNA. If the virus is an RNA virus, the virus is generally cloned as
a cDNA and inserted into a plasmid. The plasmid is then used to
make all of the constructions. The RNA virus is then produced by
transcribing the viral sequence of the plasmid and translation of
the viral genes to produce the coat protein(s) which encapsidate
the viral RNA.
[0134] Construction of plant RNA viruses for the introduction and
expression of non-viral foreign genes in plants is demonstrated by
the above references as well as in U.S. Pat. No. 5,316,931
[0135] In one embodiment, a plant viral nucleic acid is provided in
which the native coat protein coding sequence has been deleted from
a viral nucleic acid, a non-native plant viral coat protein coding
sequence and a non-native promoter, preferably the subgenomic
promoter of the non-native coat protein coding sequence, capable of
expression in the plant host, packaging of the recombinant plant
viral nucleic acid, and ensuring a systemic infection of the host
by the recombinant plant viral nucleic acid, has been inserted.
Alternatively, the coat protein gene may be inactivated by
insertion of the non-native nucleic acid sequence within it, such
that a fusion protein is produced. The recombinant plant viral
nucleic acid may contain one or more additional non-native
subgenomic promoters. Each non-native subgenomic promoter is
capable of transcribing or expressing adjacent genes or nucleic
acid sequences in the plant host and incapable of recombination
with each other and with native subgenomic promoters. Non-native
(foreign) nucleic acid sequences may be inserted adjacent the
native plant viral subgenomic promoter or the native and a
non-native plant viral subgenomic promoters if more than one
nucleic acid sequence is included. The non-native nucleic acid
sequences are transcribed or expressed in the host plant under
control of the subgenomic promoter to produce the desired
products.
[0136] In a second embodiment, a recombinant plant viral nucleic
acid is provided as in the first embodiment except that the native
coat protein coding sequence is placed adjacent one of the
non-native coat protein subgenomic promoters instead of a
non-native coat protein coding sequence.
[0137] In a third embodiment, a recombinant plant viral nucleic
acid is provided in which the native coat protein gene is adjacent
its subgenomic promoter and one or more non-native subgenomic
promoters have been inserted into the viral nucleic acid. The
inserted non-native subgenomic promoters are capable of
transcribing or expressing adjacent genes in a plant host and are
incapable of recombination with each other and with native
subgenomic promoters. Non-native nucleic acid sequences may be
inserted adjacent the non-native subgenomic plant viral promoters
such that said sequences are transcribed or expressed in the host
plant under control of the subgenomic promoters to produce the
desired product.
[0138] In a fourth embodiment, a recombinant plant viral nucleic
acid is provided as in the third embodiment except that the native
coat protein coding sequence is replaced by a non-native coat
protein coding sequence.
[0139] The viral vectors are encapsidated by the coat proteins
encoded by the recombinant plant viral nucleic acid to produce a
recombinant plant virus. The recombinant plant viral nucleic acid
or recombinant plant virus is used to infect appropriate host
plants. The recombinant plant viral nucleic acid is capable of
replication in the host, systemic spread in the host, and
transcription or expression of foreign gene(s) in the host to
produce the desired fusion protein.
[0140] Fusion Protein Compartmentalization--Signal Peptides:
[0141] As already mentioned hereinabove, compartmentalization of
the fusion protein is an important feature of the present invention
because it allows undisturbed plant growth. Thus, according to one
aspect of the present invention, the fusion protein is
compartmentalized within cells of the plant or cultured plant
cells, so as to be sequestered from cell walls of the cells of the
plant or cultured plant cells.
[0142] The fusion protein can be compartmentalized within a
cellular compartment, such as, for example, the cytoplasm,
endoplasmic reticulum, golgi apparatus, oil bodies, starch bodies,
chloroplastids, chloroplasts, chromoplastids, chromoplasts,
vacuole, lysosomes, mitochondria or the nucleus.
[0143] Accordingly, the heterologous sequence used while
implementing the process according to this aspect of the present
invention includes (i) a first sequence encoding a cellulose
binding peptide; (ii) a second sequence encoding a recombinant
protein, wherein the first and second sequences are joined together
in frame; and (iii) a third sequence encoding a signal peptide for
directing a protein to a cellular compartment, the third sequence
being upstream and in frame with the first and second
sequences.
[0144] The following provides description of signal peptides which
can be used to direct the fusion protein according to the present
invention to specific cell compartments.
[0145] It is well-known that signal peptides serve the function of
translocation of produced protein across the endoplasmic reticulum
membrane. Similarly, transmembrane segments halt translocation and
provide anchoring of the protein to the plasma membrane, see,
Johnson et al. The Plant Cell (1990) 2:525-532; Sauer et al. EMBO
J. (1990) 9:3045-3050; Mueckler et al. Science (1985) 229:941-945.
Mitochondrial, nuclear, chloroplast, or vacuolar signals target
expressed protein correctly into the corresponding organelle
through the secretory pathway, see, Von Heijne, Eur. J. Biochem.
(1983) 133:17-21; Yon Heijne, J. Mol. Biol. (1986) 189:239-242;
Iturriaga et al. The Plant Cell (1989) 1:381-390; McKnight et al.,
Nucl. Acid Res. (1990) 18:4939-4943; Matsuoka and Nakamura, Proc.
Natl. Acad. Sci. USA (1991) 88:834-838. A recent book by Cunningham
and Porter (Recombinant proteins from plants, Eds. C. Cunningham
and A. J. R. Porter, 1998 Humana Press Totowa, N.J.) describe
methods for the production of recombinant proteins in plants and
methods for targeting the proteins to different compartments in the
plant cell. In particular, two chapters therein (14 and 15)
describe different methods to introduce targeting sequences that
results in accumulation of recombinant proteins in compartments
such as ER, vacuole, plastid, nucleus and cytoplasm. The book by
Cunningham and Porter is incorporated herein by reference.
Presently, the preferred site of accumulation of the fusion protein
according to the present invention is the ER using signal peptide
such as Cel 1 or the rice amylase signal peptide at the N-terminus
and an ER retaining peptide (HDEL or KDEL) at the C-terminus.
[0146] Promoters and Control of Expression:
[0147] Any promoter which can direct the expression of the fusion
protein according to the present invention can be utilized to
implement the process of the instant invention, both constitutive
and tissue specific promoters. According to presently preferred
embodiment the promoter selected is constitutive, because such a
promoter can direct the expression of higher levels of the fusion
protein. In this respect the present invention offers a major
advantage over the teachings of U.S. Pat. No. 5,474,925 in which
only tissue specific and weak promoters can be employed because of
the deleterious effect of the fusion protein described therein on
cell wall development. The reason for which the present invention
can utilize strong and constitutive promoters relies in the
compartmentalization and sequestering approach which prohibits
contact between the expressed fusion protein and the plant cell
walls which such walls are developing.
[0148] Constitutive and tissue specific promoters, CaMV35S promoter
(Odell et al. Nature (1985) 313:810-812) and ubiquitin promoter
(Christensen and Quail, Transgenic research (1996) 5:213-218) are
the most commonly used constitutive promoters in plant
transformations and are the preferred promoters of choice while
implementing the present invention.
[0149] In corn, within the kernel, proteins under the ubiquitin
promoters, are preferentially accumulated in the germ (Kusnadi et
al., Biotechnol. Bioeng. (1998) 60:44-52). The amylose-extender
(Ae) gene encoding starch-branching enzyme IIb (SBEIIb) in maize is
predominantly expressed in endosperm and embryos during kernel
development (Kim et al. Plant. Mol. Biol. (1998) 38:945-956). A
starch branching enzyme (SBE) showed promoter activity after it was
introduced into maize endosperm suspension cells by particle
bombardment (Kim et al. Gene (1998) 216:233-243). In transgenic
wheat it has been shown that a native HMW-GS gene promoter can be
used to obtain high levels of expression of seed storage and,
potentially, other proteins in the endosperm (Blechl and Anderson,
Nat. Biotechnol. (1996) 14:875-9). Polygalacturonase (PG) promoter
was shown to confer high levels of ripening-specific gene
expression in tomato (Nicholass et al. Plant. Mol. Biol. (1995)
28:423-435). The ACC oxidase promoter (Blume and Grierson, Plant.
J. (1997) 12:731-746) represents a promoter from the ethylene
pathway and shows increased expression during fruit ripening and
senescence in tomato. The promoter for tomato
3-hydroxy-3-methylglutaryl coenzyme A reductase gene accumulates to
high level during fruit ripening (Daraselia et al. Plant. Physiol.
(1996) 112:727-733). Specific protein expression in potato tubers
can be mediated by the patatin promoter (Sweetlove et al. Biochem.
J. (1996) 320:487-492). Protein linked to a chloroplast transit
peptide changed the protein content in transgenic soybean and
canola seeds when expressed from a seed-specific promoter (Falco et
al. Biotechnology (NY) (1995) 13:577-82). The seed specific bean
phaseolin and soybean beta-conglycinin promoters are also suitable
for the latter example (Keeler et al. Plant. Mol. Biol. (1997)
34:15-29). Promoters that are expressed in plastids are also
suitable in conjunction with plastid transformation.
[0150] Each of these promoters can be used to implement the process
according to the present invention.
[0151] Thus, the plant promoter employed can a constitutive
promoter, a tissue specific promoter, an inducible promoter or a
chimeric promoter.
[0152] Examples of constitutive plant promoters include, without
being limited to, CaMV35S and CaMV19S promoters, FMV34S promoter,
sugarcane bacilliform badnavirus promoter, CsVMV promoter,
Arabidopsis ACT2/ACT8 actin promoter, Arabidopsis ubiquitin UBQ1
promoter, barley leaf thionin BTH6 promoter, and rice actin
promoter.
[0153] Examples of tissue specific promoters include, without being
limited to, bean phaseolin storage protein promoter, DLEC promoter,
PHS.beta. promoter, zein storage protein promoter, conglutin gamma
promoter from soybean, AT2S1 gene promoter, ACT11 actin promoter
from Arabidopsis, napA promoter from Brassica napus and potato
patatin gene promoter.
[0154] The inducible promoter is a promoter induced by a specific
stimuli such as stress conditions comprising, for example, light,
temperature, chemicals, drought, high salinity, osmotic shock,
oxidant conditions or in case of pathogenicity and include, without
being limited to, the light-inducible promoter derived from the pea
rbcS gene, the promoter from the alfalfa rbcS gene, the promoters
DRE, MYC and MYB active in drought; the promoters INT, INPS, prxEa,
Ha hsp17.7G4 and RD21 active in high salinity and osmotic stress,
and the promoters hsr303J and str246C active in pathogenic
stress.
[0155] Expression Follow Up:
[0156] Expression of the fusion protein can be monitored by a
variety of methods. For example, ELISA or western blot analysis
using antibodies specifically recognizing the recombinant protein
or its cellulose binding peptide counterpart can be employed to
qualitatively and/or quantitatively monitor the expression of the
fusion protein in the plant. Alternatively, the fusion protein can
be monitored by SDS-PAGE analysis using different staining
techniques, such as, but not limited to, coomasie blue or silver
staining. Other methods can be used to monitor the expression level
of the RNA encoding for the fusion protein. Such methods include
RNA hybridization methods, e.g., Northern blots and RNA dot
blots.
[0157] Thus, according to the present invention there is provided a
genetically modified or viral infected plant or cultured plant
cells expressing a fusion protein including an enzyme being capable
of catalyzing the oxidation of phenolic groups and a cellulose
binding peptide.
[0158] According to a preferred embodiment of the present invention
the fusion protein is compartmentalized within cells of said plant
or cultured plant cells, so as to be sequestered from cell walls of
said cells of said plant or cultured plant cells, so as not to
hamper development and to allow higher expression, if so required.
According to a preferred embodiment the fusion protein is
compartmentalized within a cellular compartment selected from the
group consisting of cytoplasm, endoplasmic reticulum, golgi
apparatus, oil bodies, starch bodies, chloroplastids, chloroplasts,
chromoplastids, chromoplasts, vacuole, lysosomes, mitochondria, and
nucleus.
[0159] Determination of Oxidase and Peroxidase Activity:
[0160] When employing a polynucleotide encoding a laccase in the
process of the invention, an amount of laccase in the range of
0.02-2000 laccase units (LACU) per gram of dry lignocellulosic
material will generally be suitable; when employing peroxidases, an
amount thereof in the range of 0.02-2000 peroxidase units (PODU)
per gram of dry lignocellulosic material will generally be
suitable.
[0161] The determination of oxidase (e.g., laccase) activity is
based on the oxidation of syringaldazin to tetramethoxy azo
bis-methylene quinone under aerobic conditions, and 1 LACU is the
amount of enzyme which converts 1 .mu.M of syringaldazin per minute
under the following conditions: 19 .mu.M syringaldazin, 23.2 mM
acetate buffer, 30.degree. C., pH 5.5, reaction time 1 minute,
shaking; the reaction is monitored spectrophotometrically at 530
nm.
[0162] With respect to peroxidase activity, 1 PODU is the amount of
enzyme which catalyses the conversion of 1 .mu.mol of hydrogen
peroxide per minute under the following conditions: 0.88 mM
hydrogen peroxide, 1.67 mM
2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate), 0.1 M phosphate
buffer, pH 7.0, incubation at 30.degree. C.; the reaction is
monitored photometrically at 418 nm.
[0163] Binding of the Fusion Protein to the Plant Derived
Cellulosic Matter:
[0164] When sufficient expression has been detected, binding of the
fusion protein to the plant derived cellulosic matter is effected.
Such binding can be achieved, for example, as follows. Whole
plants, plant derived tissue or cultured plant cells are
homogenized by mechanical method in the presence or absence of a
buffer, such as, but not limited to, PBS. The fusion protein is
therefore given the opportunity to bind to the plant derived
cellulosic matter. Buffers that may include salts and/or detergents
at optimal concentrations may be used to wash non specific proteins
from the cellulosic matter.
[0165] Thus, further according to the present invention there is
provided a composition of matter comprising a cell wall preparation
derived from a genetically modified or virus infected plant or
cultured plant cells expressing a fusion protein including an
enzyme being capable of catalyzing the oxidation of phenolic groups
and a cellulose binding peptide, said fusion protein being
immobilized to cellulose in said cell wall preparation via said
cellulose binding peptide.
[0166] Oxidizing Agents:
[0167] The enzyme(s) and oxidizing agent(s) used in the process of
the invention should clearly be matched to one another, and it is
clearly preferable that the oxidizing agent(s) in question
participate(s) only in the oxidative reaction involved in the
binding process, and does/do not otherwise exert any deleterious
effect on the substances/materials involved in the process.
[0168] Oxidases, e.g. laccases, are, among other reasons, well
suited in the context of the invention since they catalyze
oxidation by molecular oxygen. Thus, reactions taking place in
vessels open to the atmosphere and involving an oxidase as enzyme
will be able to utilize atmospheric oxygen as oxidant; it may,
however, be desirable to forcibly aerate the reaction medium during
the reaction to ensure an adequate supply of oxygen.
[0169] In the case of peroxidases, hydrogen peroxide is a preferred
peroxide in the context of the invention and is suitably employed
in a concentration (in the reaction medium) in the range of
0.01-100 mM.
[0170] pH in the Reaction Medium:
[0171] Depending, inter alia, on the characteristics of the
enzyme(s) employed, the pH in the aqueous medium (reaction medium)
in which the process of the invention takes place will be in the
range of 3-10, preferably in the range 4-9.
[0172] General Procedures:
[0173] Generally, the nomenclature used herein and the laboratory
procedures utilized when practicing the present invention include
molecular, biochemical, microbiological and recombinant DNA
techniques. Such techniques are thoroughly explained in the
literature. See, for example, "Molecular Cloning: A laboratory
Manual" Sambrook et al., (1989); "Current Protocols in Molecular
Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al.,
"Current Protocols in Molecular Biology", John Wiley and Sons,
Baltimore, Md. (1989); Perbal, "A Practical Guide to Molecular
Cloning", John Wiley & Sons, New York (1988); Watson et al.,
"Recombinant DNA", Scientific American Books, New York; Birren et
al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4,
Cold Spring Harbor Laboratory Press, New York (1998); methodologies
as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531;
5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook",
Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in
Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al.
(eds), "Basic and Clinical Immunology" (8th Edition), Appleton
& Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds),
"Selected Methods in Cellular Immunology", W. H. Freeman and Co.,
New York (1980); available immunoassays are extensively described
in the patent and scientific literature, see, for example, U.S.
Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987;
3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;
4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;
"Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid
Hybridization" Hames, B. D., and Higgins S. J., eds. (1985);
"Transcription and Translation" Hames, B. D., and Higgins S. J.,
eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986);
"Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical
Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in
Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To
Methods And Applications", Academic Press, San Diego, Calif.
(1990); Marshak et al., "Strategies for Protein Purification and
Characterization--A Laboratory Course Manual" CSHL Press (1996);
all of which are incorporated by reference as if fully set forth
herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
[0174] Product by Process:
[0175] The present invention also relates to a lignocellulose-based
product obtainable by a process according to the invention as
disclosed herein.
[0176] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications cited herein are incorporated by reference in their
entirety.
* * * * *