Note: Descriptions are shown in the official language in which they were submitted.
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PROCESS AND COMPOSITION FOR PREPARING A
LIGNOCELLULOSE-BASED PRODUCT, AND THE PRODUCT
OBTAINED BY THE PROCESS
FIELD AND BACKGROUND OF THE INVENTION
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
i o 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 fonn 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.
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.
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.
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.
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.
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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
io may be mentioned epichlorohydrin, urea-forinaldehyde and melamine-
formaldehyde.
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.
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
3o 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.
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:
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EP 0 433 258 Al 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
1o mixtures thereof.
EP 0 565 109 Al 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.
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
2o 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.
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.
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Roffael and B. Dix, Holz als Roh- und Werkstoff 49 (1991) 199-205; (iii)
lignin sulfonates available on a commercial scale are generallv 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.
U.S. Pat. No. 5,846,788, from which the above background
lo information is derived,
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
is 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
2o harmful substances, such as the processes described in EP 0 433 258 Al, EP
0 565 109 Al 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
25 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 svnthetic adhesives, but it can probably also compete
30 economically with such processes.
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.
35 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
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and linerboard), from an appropriate lignocellulosic starting material devoid
of the above limitation.
SUMMARY OF THE INVENTION
5 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
io 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.
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.
According to still further features in the described preferred
embodiments the lignocellulose product is selected from the group
consisting of paper and paperboard.
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.
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.
According to still further features in the described preferred
3o 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.
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.
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According to still further features in the described preferred
embodiments the phenolic polymer forms an integral part of the
lignocellulose containing material.
According to still further features in the described preferred
embodiments the phenolic polymer is lignin.
According to still further features in the described preferred
embodiments the phenolic polymer is a phenolic polysaccharide.
According to still further features in the described preferred
embodiments the polysaccharide portion of the phenolic polysaccharide is
io selected from the group consisting of modified and unmodified starches,
modified and unmodified cellulose, and modified and unmodified
hemicelluloses.
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.
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.
According to still further features in the described preferred
2o embodiments the fusion polypeptide is incubated in the presence of the
oxidizing agent for a period of from 1 minute to 10 hours.
According to still further features in the described preferred
embodiments the enzyme is selected from the group consisting of oxidases
and peroxidases.
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.
According to still further features in the described preferred
3o 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.
According to still further features in the described preferred
embodiments the reaction medium is aerated.
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.
According to still further features in the described preferred
embodiments the fungus is Trametes versicolor or Trametes villosa.
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According to still further features in the described preferred
embodiments the enzyme is a laccase from Acer pseudoplanus.
According to still further features in the described preferred
embodiments the enzyme is a peroxidase and the oxidizing agent is
hydrogen peroxide.
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.
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.
According to still further features in the described preferred
embodiments the temperature of the reaction medium is in the range of 10
-120 C.
According to still further features in the described preferred
embodiments the temperature of the reaction medium is in the range of 15
-90 C.
According to still further features in the described preferred
2o embodiments an amount of the phenolic polysaccharide in the range of 0.1
% - 10 % by weight.
According to still further features in the described preferred
embodiments the pH in the reaction medium is in the range of 3-10.
According to still further features in the described preferred
embodiments the pH in the reaction medium is in the range of 4-9.
According to still further features in the described preferred
embodiments the reaction medium further comprising a lignocellulose
containing material devoid of the fusion protein.
According to still further features in the described preferred
3o 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.
Further according to the present invention there is provided a
lignocellulose product obtainable by the process described herein.
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.
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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.
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.
According to still further features in the described preferred
embodiments the fusion protein is compartmentalized within a cellular
to 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.
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.
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.
According to still further features in the described preferred
embodiments the nucleic acid molecule further comprising a sequence
3o 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.
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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.
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
io materials and reagents described in the prior art for use in the process of
manufacturing lignocellulose-based products.
DETAILED DESCRIPTION OF THE INVENTION
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.
The principles and operation of a process according to the present
invention may be better understood with reference to the accompanying
descriptions.
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.
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.
The order of mixing/contacting the three components is unimportant
as long as the process set-up ensures that the activated lignocellulosic
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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.
5 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
t o 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 N2 atmosphere.
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.
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).
Lignocellulose containing material:
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.
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
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serves as a good "gap-filler", which is a big advantage when producing, e.g.,
particle boards from large wood particles.
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 %.
The temperature of the reaction mixture in the process of the
invention may suitably be in the range of 10 C - 120 C., as appropriate;
however, a temperature in the range of 15 C - 90 C is generally to be
lo 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 C.
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.
Phenolic Polymers:
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).
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.
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,
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ferulic acid (i.e., 4-hydroxy-3-methoxycinnamic acid), 5-hydroxy-ferulic
acid and sinapic acid.
Particularly suitable phenolic polysaccllarides are those which
exhibit good solubility in water, and thereby in aqueous media in the context
s 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 bcen 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
io suitable examples thereof are ferulylatcd arabinoxylans (obtainable, e.g.,
from wheat bran or maize bran) and ferulvlaLed pectins (obtainable from,
e.g., beet pulp), i.e., arabinoxylans and pectins containing ferulyl
substituents attached via ester linkages to the polysaccharide molecules.
The amounl of phenolic polysaccharide or other phenolic polymers,
15 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 per cent (calculated in this manner) will often
be very suitable.
20 Elzzymes and polynucleotides encoding same
In principle, any type of enzyme capable of catalyzing oxidation of
phenolic groups tnay 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
25 techniques and which can therefore be expressed as a part of a fusion
polypeptide.
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
30 to employ two or more different enzymes in the process of the invention.
Among types of oxidases (in combination with which oxygen - e.g.,
atmospheric oxygen - is an excellent oxidizing agent), laccases have proved
to bc well suited for use in the method of the invention.
Polynucleotides encoding laccases have been or are readily
35 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.
Suitable examples of polynucleotides
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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,).
A preferred laccase in the context of the invention is that obtainable
from Trametes villosa or Acer pseudoplanus.
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
2o 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.
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).
Further preferred fungi include strains belonging to the sub-division
Zygomycotina, class Mycoraceae, e.g., Rhizopus or Mucor, in particular
Mucor hiemalis.
Some preferred bacteria include strains of the order Actinomycetales,
e.g., Streptomyces spheroides (ATTC 23965), Streptomyces
thermoviolaceus (IFO 12382) or Streptoverticillum verticillium ssp.
verticillium.
Other preferred bacteria include Bacillus pumilus (ATCC 12905),
Bacillus stearothermophilus, Rhodobacter sphaeroides, Rhodomonas
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palustri, Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) or
Pseudomonas fluorescens (NRRL B-11).
Further preferred bacteria include strains belonging to Myxococcus,
e.g., M. virescens.
Other potential sources of useful sources for polynucleotides
encoding peroxidases are listed in B. C. Saunders et al., Peroxidase, London
1964, pp. 41-43.
Cellulose binding peptides:
As used herein in the specification and in the claims section below,
1o 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.
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
3o 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 P-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
WQ 01/34902 CA 02390639 2008-02-07 PCT/IL00/00665
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.
5 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.
In Table 4 below are listed those binding domains which bind to one
or more soluble/insoluble polysaccharides including all binding domains
io with affinity for soluble glucans (a, R, and/or mixed linkages). The NI
cellulose-binding domain from endoglucanase CenC of C. fimi is the only
protein known to bind soluble eellosaccharides 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 P=1,3-glucan-
15 binding domains (Table 1); proteins containing Streptococcal glucan-
binding repeats (Cpl 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
2o 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., WO 94/24158) 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.
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Table 1
Overview of proteins containing putative [~-1,3 glucan-binding
domains
Source (strain) Protein accession No. Reel
Type I
B. circulans (WL-12) GLCA1 P23903/M34503/JQ0420 1
B. circulans (IAM 1165) Bg1H JN0772/D17519/S67033 2
Type II
Actinomadura sp. (FC7) Xynll U08894 3
Arthrobacter sp. (YCWD3) GLCI D23668 9
0. 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) XInA P26514/M64551/JS07986 7
T. tridentatus FactorGa D16622 8
B. Bacillus, O. : Oerskovia, R. faecitabidus : Rarobacter faecitabidus, R.
cominunis: Ricinus
conznzunis, S. : Streptomyces, T. : Tachypleus (Horseshoe Crab)
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. Bio1. Chein. 266, 1058-1063
5a) Shimoi et al. (1992) J. Biol. Chenz. 267, 25189-25195
5b) Shimoi et al. (1992) J Biochein l 10, 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 2
Overview of proteins containing Streptococcal glucan-binding repeats
(Cpl superfamily)
Source Protein Accession No. Ref.2
S. downei (sobrinus) (0MZ176) GTF-I D13858 I
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. niutants (GS-5) GTF-B A33128 7
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Table 2 (Continued)
S. mutants (GS-5) GTF-B P08987/M17361/B33135 8
S. mutants GTF-B3'-GRF 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 12
Z 11873/M64111
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
Pha-e 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 21
X51797/S08638
C. difficile (BARTS W 1) ToxA A60991 /X ] 7194 22
C. difficile (VPI 10463) ToxB P18177/X53138/X60984 23,24
S10317
C. difficile (1470) ToxB S44271/Z23277 25,26
C. novyi a-toxin S44272/Z23280 27
C. novyi a-toxin Z48636 28
C. acetobutylicuin (NCIB8052) CspA S49255/Z37723 29
C. acetobattvlicum (NCIB8052) CspB Z50008 30
C. acetobutylicum (NCIB8052) CspC Z50033 30
C. acetobutylicum (NC1B8052) CspD Z50009 30
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. Imnzun. 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
WO 01/34902 CA 02390639 2002-05-08 PCT/ILOO/00665
18
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) Hofinann 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
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.
The Ka for binding of the cellulose binding domains or proteins to
cellulose is at least in the range of weak antibody-antigen extractions, i.e.,
_
103, preferably 104, most preferably 106 M-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
WO 01/34902 CA 02390639 2002-05-08 PCT/IL00/00665
19
temperatures, providing a means for temperature modulation of the binding
step.
Table 3
Overview of enzymes with chitin-binding domains
Source (strain) Enzyme Accession No. Ref.3
Bacterial enzymes
Type I
Aeromonas sp. (NolOS-24) Chi D31818 1
Bacillus circulans (WL-12) ChiAl P20533/M57601/A38368 2
Bacillus circulans (WL-12) ChiD P27050/D10594 3
Janthinobacteriznn lividuni Chi69 U07025 4
Streptomvices griseus Protease C A53669 5
Type 11
Aeromonas cavia (KI) Chi U09139 6
Alteroinonas sp (0-7) Chi85 A40633/P32823/D13762 7
Autographa californica (C6) NPH-128a P41684/L22858 8
Serratia marcescens ChiA A25090/X03657/L01455/P07254 9
Type I11
Rhizopus oligosporus (IF08631) Chi] P29026/A47022/DI0157/S27418 10
Rhizopus oligosporus (IF08631) Chi2 P29027/B47022/D10158/S27419 10
Saccharomvices cerevisiae Chi S5037I /U 17243 11
Saccharomyces cerevisiae Chil P29028/M74069 12
(DBY939)
Saccharonzvices cerevisiae Chi2 P29029/M7407/B41035 12
(DBY918)
Plant enzymes
Hevein superfamily
Allium sativzim Chi M94105 13
Anzaranthus caudatus AMP-lb P27275/A40240 14, 15
Amaranthus caudatus AMP-2b S37381/A40240 14, 15
Arabidopsis thaliana ChiB P19171/M38240/B45511 16
(cv. colombia)
Arabidopsis thaliana PHPc U01880 17
Brassica napus Chi U21848 18
Brassica napus Chi2 Q09023/M95835 19
Hevea brasiliensis Hevld P02877/M36986/A03770/A38288 20, 21
Hordeum vulgare Chi33 L34211 22
Lycopersicon esculentuin Chi9 Q05538/Z15140/S37344 23
Nicotiana tabacum CBP20e S72424 24
Nicotiana tabacum Chi A21091 25
Nicotiana tabacum (cv. Havana) Chi A29074/M15173/S20981/S19855 26
Nicotiana tabacum (FB7-1) Chi JQ0993/S0828 27
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WO O1/34902 PCT/IL00/00665
Table 3 (Continued)
Nicotiana tabaczim (cv. Samsun) Chi A16119 28
Nicotiana tabacum (cv. Havana) Chi P08252/X 16939/S08627 27
Nicotiana tabacum (cv. BY4) Chi P24091/X51599/X64519//S13322 26,27,29
5 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 S39979/S40414/X56787 32
Oryza sativum (cv. Japonicum) Chi X56063 33
10 Oryza sativum (cv. Japonicum) Chil P24626/X54367/S14948 34
Oryza sativum Chi2 P25765/S15997 35
Oryza sativum (cv. Japonicum) Chi3 D16223
Oryza sativum ChiA JC2252/S42828 30
Oryzasativutn Chil D16221 32
15 Oryza sativum (IR58) Chi U02286 36
Oryza sativum Chi X87109 37
Pisum sativuzn (cv. Birte) Chi P36907/X63899 38
Pisum sativum (cv. Alcan) Chi2 L37876 39
Populus trichocarpa Chi S18750/S18751/X59995/P29032 40
20 Populustrichocazpa(HII-l1) Chi U01660 41
Phaseolus vulgaris (cv. Saxa) Chi A24215/S43926/Jq0965/P36361 42
Phaseolus vulgaris (cv. Saxa) Chi P06215/M 13968/M 19052/A25898 43,44,45
Sambucus nigra PR-3f Z46948 46
Secale cereale Chi JC2071 47
Solanum tuberosum ChiB 1 U02605 48
Solanum tuberosum ChiB2 U02606 48
Solanunz tuberosum ChiB3 U02607/S43317 48
Solanunz tztberosunz ChiB4 U02608 48
Solanum tuberosum WIN-lg P09761/X13497/SO4926 49
(cv. Maris Piper)
Solanum tuberosum WIN-2g P09762/X13497/SO4927 49
(cv. Maris Piper)
Triticum aestivuni Chi S38670/X76041 50
Triticunz aestivum WGA-1h P10968/M25536/S09623/S07289 51,52
Triticum aestivum WGA-2h P02876/M25537/S09624 51,53
Triticum aestivuni WGA-3 P I 0969/J02961 /S 10045/A28401 54
Ulnzus americana (NPS3-487) Chi L22032 55
Urtica dioica AGLi M87302 56
Vigna unguiculata Chil X88800 57
(cv. Red caloona)
aNHP : nuclear polyhedrosis virus endochitinase like sequence; Chi :
chitinase, banti-microbial
peptide, cpre-hevein like protein, dhevein, echitin-binding protein,
fpathogenesis related protein,
gwound-induced protein, hwheat germ agglutinin, i agglutinin (lectin).
3References:
1) Udea et al. (1994) J. Fernzent. 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 Librarv
5) Sidhu et al. (1994) J. Biol. Chein. 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
CA 02390639 2002-05-08
WO 01/34902 PCT/IL00/00665
21
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) Broekaertet 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) Broekaertet 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 Phvsiol. 6, 579-589
23) Danhash et al. (1993) Plant tLlol. 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 Libraryl
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 Library
56) Lerner et al. (1992) J. Biol. Chem. 267, 11085-11091
57) Vo et al. (1995) EMBL Data Library
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Table 4
Sources ofpolysaccharide binding domains
Binding Domain Proteins Where Binding
Domain is Found
Cellulose Binding (3-glucanases (avicelases, CMCases,
Domains 1 cellodextrinases)
exoglucanses or cellobiohydrolases
cellulose binding proteins
xylanases
mixed xylanases/glucanases
esterases
chitinases
(3-1,3-glucanases
(3-1,3-((3-1,4)-glucanases
((3-)mannanases
(3-glucosidases/galactosidases
cellulose synthases (unconfirmed)
Starch/Maltodextrin a-amylases2,3
Binding Domains (3-amylases4,5
pullulanases
glucoamylases6,7
cyclodextrin glucotransferases8-10
(cyclomaltodextrin glucanotransferases)
maltodextrin binding proteinsl l
Dextran Binding Domains (Streptococcal) glycosyl transferasesl2
dextran sucrases (unconfirmed)
Clostridial toxins 13,14
glucoamylases6
dextran binding proteins
R-Glucan Binding Domains (3-1,3-glucanases15,16
(3-1,3-((3-1,4)-glucanases (unj~nfirmed)
(3-1,3-glucan binding protein
Chitin Binding Domains chitinases
chitobiases
chitin binding proteins
(see also cellulose binding domains)
Heivein
1Gilkes et al., Adv. Microbiol Reviews, (1991) 303-315.
2S?gaard et al., J. Biol. Chem. (1993) 268:22480.
3Weselake et al., Cereal Chem. (1983) 60:98.
4Svensson et al., J. (1989) 264:309.
5Jespersen et al., J. (1991) 280:51.
6Belshaw et al., Eur. J. Biocheni. (1993) 211:717.
7Sigurskjold et al., Eur. J. Biochein. (1994) 225:133.
8Villette et al., Biotechnol. Appl. Biocheni. (1992) 16:57.
9Fukada et al., Biosci. Biotechnol. Biochem. (1992) 56:556.
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lOLawson et al., J. Mol. Biol. (1994) 236:590.
14von Eichel-Streiber el al., Mol. Gen, Genet. (1992) 233:260.
15K1eb1 et al., J. Bacteriol. (1989) 171:6259.
16Watanabe et aL, J. Bacteriol. (1992) 174:186.
17Duvic ei al., J. Biol. Chern. (1990) :9327.
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
lo 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.
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 sequenccs for
several binding domains, which bind to soluble oligosaccharides are known
(See, Figure 1 of 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 inutagenesis,
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, New York, 1989,
The amino acid sequence of a polysaccharidase can be used to design
a probe to screen a eDNA 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
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24
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).
Probes developed using consensus sequences for the binding domain
of a polysaccharidase or polysaccharide-binding protein are of particular
interest. The R-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 Xy1D, respectively) (see
io 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 vaiying 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. Chein., 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 Xy1D each have a second, internal CBD from
families III and II, respectively. Cex and Xy1D are clearly xylanases;
however, Cex, but not Xy1D, 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 R-
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 (3-1,4-
glycanases (see Beguin et al., (1992) FEMS Microbiol. Lett., 100:523).
The CBD derived from C. fami endoglucanase C N1, is the only protein
WO 01/34402 CA 02390639 2008-02-07 YC:I'/1LUU/UU665
known to bind soluble cellosaccharides and one of a small set of proteins
that are known to bind any soluble polysaccharides.
Examples of suitable binding domains are shown in Figure 1 of
WO 97/26358 , which presents an aligninent of binding
s 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,
io 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 32P, 3H, biotin, avidin or other detectable reagents, and
t s 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
20 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
25 probe" refers to both labeled and unlabeled forms.
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 alignrnent and the like) to the binding region or regions from which
the probe was derived and will bind to a soluble p-1,4 glucan with a Ka of _
103 M-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.
WO 01/34902 CA 02390639 2008-02-07 PCT/IL00/00665
26
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 truncatcd protein. Another method
involves the use of exonucleases such as Ba131 to systematically delete
nucleotides either externally from the 5' and the 3' ends of the DNA or
io 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.
Any cellulose binding protein or cellulose binding domain may be
used in the present invention. The tenn "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. Nati.
3s Acad. Sci U.S.A. (1998) 95:4906,-4911.,
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,
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27
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 exainple, the CBD is obtainable from Clostridium
cellulovorans, Clostridium cellulovorans, or Cellulomonasfimi (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). Furthermore, the CBD may be
derived by mutation of a portion of a protein or polypeptide which binds to
io 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.
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).
Cellulose binding peptide-recombinant protein fusions:
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
WO 01/34902 CA 02390639 2008-02-07 Yl:IALUU/UU665
28
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
to 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.
For example, Greenwood et al. (1989, FEBS Lett. 224:127-131)
fused the cellulose binding region of Cellulomonas f mi 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. Patent No.
5,137,819 issued to Kilburn et al., and U.S. Patent No. 5,719,044 issued to
Shoseyov et al. See also U.S. Pat.
No. 5,474,925.
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.
According to a preferred embodiment of the invention the nucleic
3o 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.
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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.
Genetically modified plaht material:
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
io 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.
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; Shiinamoto et al.,
Nature (1989) 338:274-276). The principle methods of causing stable
integration of exogenous DNA into plant genomic DNA include two main
2o approaches:
(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.
(ii) direct DNA uptake: Paszkowski et al., in Cell Culture and
Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant
3o 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.
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WO 01/34902 PCT/IL00/00665
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,
5 (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-
719.
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
io 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
15 the creation of transgenic dicotyledenous plants.
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
2o adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten
particles, and the microprojectiles are physically accelerated into cells or
plant tissues.
Following transformation plant propagation is exercised. The most
common method of plant propagation is by seed. Regeneration by seed
25 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
30 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.
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
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31
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.
Micropropagation is a multi-stage procedure that requires alteration
of culture medium or growth conditions between stages. Thus, the
1o 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.
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.
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.
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.
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32
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
io a pre-existing vector.
Viral infected plant material:
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
1s 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.
20 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.,
25 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.
Construction of plant RNA viruses for the introduction and
3o 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 Takainatsu et al. FEBS Letters (1990) 269:73-
76.
35 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
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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.
Construction of plant RNA viruses for the introduction and
1 o 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
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
2o 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.
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.
In a third embodiment, a recombinant plant viral nucleic acid is
provided in which the native coat protein gene is adjacent its subgenomic
WO 01/34902 CA 02390639 2002-05-08 PCT/ILOO/00665
34
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.
In a fourth embodiment, a recombinant plant viral nucleic acid is
lo provided as in the third embodiment except that the native coat protein
coding sequence is replaced by a non-native coat protein coding sequence.
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.
Fusion protein compartmentalization - sigrcal peptides:
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.
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
3o nucleus.
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.
WO 01/34902 CA 02390639 2008-02-07 Y( !/1LUU/UU665
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.
It is well-known that signal peptides serve the function of
5 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,
io 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) 1:3 81-390; McKnight et
al., Nucl. Acid Res. (1990) 18:4939-4943; Matsuoka and Nakamura, Proc.
15 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 Huinaiia Press Totowa, N.J.) describe methods for the
production of rccombinant proteins in plants and methods for targeting the
proteins to different compartments in the plant cell. In particular, two
20 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.
Presently, the preferred site of accumulation of the fusion protein according
25 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.
Promoters and control of expression:
Any proinoter which can direct the expression of the fusion protein
3o 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
35 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
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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.
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.
In corn, within the kernel, proteins under the ubiquitin promoters, are
io preferentially accumulated in the germ (Kusnadi et al., Biotechnol. Bioeng.
(1998) 60:44-52). The amylose-extender (Ae) gene encoding starch-
branching enzyme Ilb (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.
2o 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.
Each of these promoters can be used to implement the process
according to the present invention.
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Thus, the plant promoter employed can a constitutive promoter, a
tissue specific promoter, an inducible promoter or a chimeric promoter.
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.
Examples of tissue specific promoters include, without being limited
to, bean phaseolin storage protein promoter, DLEC promoter, PHS(3
io 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.
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 hspl7.7G4 and RD21 active in high
salinity and osmotic stress, and the promoters hsr303J and str246C active in
pathogenic stress.
Expression follow up:
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.
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.
According to a preferred embodiment of the present invention the
fusion protein is compartmentalized within cells of said plant or cultured
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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, chioroplastids, chloroplasts, chromoplastids,
chromoplasts, vacuole, lysosomes, mitochondria, and nucleus.
Deternzination of Oxidase and Peroxidase Activity:
When employing a polynucleotide encoding a laccase in the process
io 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.
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
M of syringaldazin per minute under the following conditions: 19 M
syringaldazin, 23.2 mM acetate buffer, 30 C., pH 5.5, reaction time 1
minute, shaking; the reaction is monitored spectrophotometrically at 530
nm.
With respect to peroxidase activity, I PODU is the amount of
enzyme which catalyses the conversion of 1 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 C.; the reaction is monitored
photometrically at 418 nm.
Biiiding of the fusion protein to the plant derived cellulosic nzatter:
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.
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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.
Oxidizing agents:
The enzyme(s) and oxidizing agent(s) used in the process of the
io 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.
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.
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.
pH in the Reaction Medium:
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.
General Procedures:
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, Maryland (1989); Perbal, "A Practical Guide to
Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al.,
WO 01/34902 CA 02390639 2008-02-07 Y(rT/1LUUlU0605
"Recombinant DNA", Scientific American Books, New York; Birren et al.
(eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold
Spring I Iarbor 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 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes 1-111 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, CT (1994); Mishell and Shiigi (eds),
"Selected Methods in Cellular Immunology", W. H. Freeman and Co., New
io York (1980); available immunoassays are extensively described in the
patent and scientific literature, see, for exainple, 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.
15 J., ed. (1984); "Nucieic 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.
20 1-317, Academic Press; "PCR Protocols: A Guide To Methods And
Applications", Academic Press, San Diego, CA (1990); Marshak et al.,
"Strategies for Protein Purification and Characterization - A Laboratory
Course Manual" CSHL Press (1996).
Other general references are provided
25 throughout this document. The procedures therein are believed to be well
known in the art and are provided for the convenience of the reader.
Product by Process:
The present invention also relates to a lignocellulose-based product
30 obtainable by a process according to the invention as disclosed herein.
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.
35 Accordingly, it is intended to embrace all such alternatives, modifications
and variations that fall within the spirit and broad scope of the appended
claims.
