JP2003533173A - Methods and compositions for preparing lignocellulosic products and products obtained by the methods - Google Patents

Abstract

  (57) [Summary] A method for producing a lignocellulosic product, comprising: (i) a phenolic polymer substituted with a phenolic hydroxy group in a reaction medium; (ii) an enzyme capable of catalyzing the oxidation of the phenolic group and a cellulose bond. A lignocellulose-containing material in which the fusion polypeptide comprising the peptide is immobilized on the cellulose moiety; and (iii) an oxidizing agent. Also disclosed are compositions for use in the method and lignocellulosic products obtained by the method.

Description

Detailed Description of the Invention

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to lignocellulosic products, such as hardboard, from suitable lignocellulosic starting materials (such as wood fiber or vegetable fiber) to which the enzyme is attached via a cellulose-binding peptide. Or medium density fiberboard ("MDF")
Methods and compositions for making fiberboard, particleboard, plywood, paper or paperboard such as cardboard and linerboard, etc. are provided. In this case, such an enzyme would constitute the lignocellulosic starting material in the presence of an oxidizing agent and, if necessary, in the presence of an additional lignocellulosic starting material (eg recycled fiber) without the enzyme. It is possible to catalyze the oxidation of the phenolic groups of a phenolic polymer (eg lignin) which may form an active moiety.

By using the method of the invention, improved mechanical properties are brought to the lignocellulosic products prepared thereby, especially paper products such as linerboard, cardboard and corrugated paperboard.

Lignocellulosic products prepared from lignocellulosic starting materials, among others:
By mechanical or mechanical / chemical methods (the latter is often referred to as "semi-chemical" methods) or by chemical methods without bleaching, or by wood particles (wood "chips", flakes and Products produced starting from plant fibers or wood fibers prepared from (other) are an essential everyday material.

Some of the most familiar types of such products include writing or printing paper,
Includes cardboard, corrugated cardboard, fiberboard (“hardboard”) and particleboard.

Virtually all specifications of paper and cardboard and the like are made from aqueous pulp slurries. Typically, the pulp is suspended in water, mixed with various additives, and then passed through a device such as paper and cardboard to be formed, compressed and dried. From mechanically produced pulp (hereinafter "mechanical pulp"), semi-chemically produced pulp (hereafter "semi-chemical pulp"), unbleached chemical pulp, or recycled fiber Regardless of whether the pulp made (ie pulp prepared from recycled fibers, waste paper and others) is used, in most cases to obtain a final product with sufficient mechanical properties, It is necessary to add various toughening agents to the pulp.

In the case of paper and paperboard used in packaging and the like, the tensile strength and tear strength in a dry state and a wet state are very important. In addition, the compressive strength of the material is also an important factor, especially in the case of certain standard cardboards (for example so-called unbleached paperboards for making boxes of corrugated cardboard used for packaging, shipping and others). Often. There are many environmentally undesirable substances in the tougheners used today that are desired to be replaced by more environmentally acceptable materials. As examples, mention may be made of epichlorohydrin, urea-formaldehyde and melamine-formaldehyde.

“Conventional” lignocellulosic composites used in building structures, flooring, exterior finishes, furniture, packaging and others, such as hardboard (
It is usually produced from wood fibers produced by mechanical or semi-chemical means or by so-called "steam blasting" and particleboard (which is a relatively coarse piece of wood, fragments or "chips"). In the case of composites such as), the fibers / particles may be combined with one or more "extending materials" to obtain aggregates of wood fibers or particles to obtain aggregates with satisfactory strength properties. With a synthetic adhesive (typically a urea-formaldehyde type, a phenol-formaldehyde type or an isocyanate type adhesive) in a mixture with (such as lignosulphonate and / or kraft lignin), and then heat In addition, using a process that is compressed into the desired form (board, sheet, panel, etc.) It can be achieved Te.

However, the use of synthetic adhesives of the above type in the manufacture of wood products generally
Not desirable from an environmental and safety perspective. This is because many such adhesives have direct toxicity and therefore require special handling procedures and / or cause the release of toxic and / or environmentally harmful substances at a later stage. Thus, for example, it has been demonstrated that formaldehyde is released from certain cured formaldehyde-based adhesives (eg, used as binders in particleboards and the like).

Given the shortcomings associated with the use of synthetic adhesives as binders in the production of lignocellulosic products, considerable work has been done in recent years to develop more acceptable binder systems and bonding processes from an environmental and toxic point of view. It is aimed at developing. Patent documents relevant in this respect include:

European Patent EP 0433258A1 discloses a method for producing mechanical pulp from fibrous products using chemical and / or enzymatic treatments. In this case, the "binder" is linked to the lignin in the fibrous product by generating radicals in the lignin part of the fibrous product. This document mentions "hydrocarbonates" and / or proteins such as cationic starch as examples of suitable binders. Examples of suitable enzymes include laccase, lignin peroxidase and manganese peroxidase, and examples of suitable chemical agents include hydrogen peroxide with ferroions, chlorine dioxide, ozone and mixtures thereof.

European Patent EP 0565109A1 discloses a method whereby the binding of mechanically produced wood fragments is achieved by activating lignin in the middle layer of wood cells by incubation with phenol oxidising enzymes. . Therefore, the use of a separate binder is avoided by this method.

US Pat. No. 4,432,921 describes a method of making a binder for wood products from phenolic compounds having phenolic groups. This method in question involves treating the phenolic compound with an enzyme that activates and oxidatively polymerizes the phenolic compound, thereby converting it into a binder. The only phenolic compound specifically referred to in this document or used in the examples shown therein is lignin sulfonate, which is the main compound of the invention described in US Pat. No. 4,432,921. The purpose is produced in large quantities by operating the sulfite process, which is widely used to produce chemical pulp,
Moreover, it is to economically utilize a so-called "sulfite waste liquid" containing lignin sulfonate.

Lignin sulfonate (particularly in the form of sulfite waste liquor) as a phenolic polymer in a system / process for binding wood products (as described in US Pat. No. 4,432,921) Regarding the use, the following comments are appropriate. (I) Further research (H. H. Nimz, Wood A
dhesives, Chemistry and Technology, M
arcel Dekker, New York and Basel, 1983,
Pp. 247-288; Haars et al., Adhesives from Re
newable Resources, ACS Symposium Series 385, A
Merchanical Chemical Society, 1989, p. 126-
(See page 134) is the "conventional" required in the manufacture of wood-based boards.
In order to achieve comparable strength properties by comparison with the amount of synthetic adhesive of
Reveals the need for very high amounts of lignin sulfonate; (i
i) It has been found that the compaction time required when compacting a wood based board prepared using a lignin sulfonate binder is very long (E. Roff.
ael and B. Dix, Holz ass Roh- und Werkst
off, 49 (1991) 199-205); (iii) Lignin sulfonates obtained on a commercial scale are generally very impure and very variable in quality (J. L. Philippou, Journal). of Wood
Chemistry and Technology, 1 (2) (1981) 1
99-227); (iv) The very dark color of the spent sulfite liquor is, for example, a paper product with acceptable color characteristics (non-bleached, such as wrapping paper, linerboard, or cardboard boxes). Not suitable as a source of lignin sulphonate for making paperboard etc.).

US Pat. No. 5,846,788, which provides the above background information and is incorporated by reference as if set forth herein in its entirety, describes substituents containing phenolic hydroxy groups. A lignocellulosic material (vegetable fiber, wood Previously known processes (European Patent EP 0433258A1) in which binding of chips etc. attempted to reduce or avoid the use of toxic and / or otherwise harmful substances. , The same EP05651
09A1 and processes such as those described in US Pat. No. 4,432,921) and at least comparable strength properties, and in many cases significantly better than such strength properties. It teaches that it can be used in the manufacture of lignocellulosic products that exhibit various strength properties. Thus, for example, the amount of binder required to prepare a lignocellulosic product of very satisfactory strength by the process described in US Pat. No. 5,846,788 is generally US Pat. Much lower than the level of binder (based on lignin sulfonate) required to obtain comparable strength properties using the process according to US Pat. No. 4,432,921, typically about 1/3 It is the following. Thus, the process according to U.S. Pat. No. 5,846,788 may not only provide an environmentally noteworthy alternative to the more conventional bonding processes using synthetic adhesives, but perhaps also various such processes. Can compete economically with.

However, the process described in US Pat. No. 5,846,788 is a purified material that is an expensive material when compared to other raw materials and reagents used in the process of making lignocellulosic products. Requires the use of enzymes.

Thus, from a suitable lignocellulosic starting material that does not have the above limitations, lignocellulosic products such as fiberboard (hardboard or medium density fiberboard (such as “MDF”), particleboard, plywood, paper or Paperboard (
It is widely recognized that there is a need for a method for manufacturing cardboard and linerboard, etc., and having such a method is very convenient.

[0017] According to one aspect of the Summary of the Invention The invention, a method for producing a lignocellulosic product is provided. In this method, in a reaction medium, (i) a phenolic polymer substituted with a phenolic hydroxy group; (ii) a fusion polypeptide containing an enzyme capable of catalyzing the oxidation of a phenolic group and a cellulose-binding peptide A lignocellulose-containing material immobilized on the cellulose portion; and (iii) mixing an oxidizing agent.

According to further features in preferred embodiments of the invention described below, the lignocellulosic product is selected from the group consisting of fiberboard, particleboard, flakeboard, plywood and molded composites.

According to still further features in the described preferred embodiments the lignocellulosic 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 genetically engineered or virus infected plants 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 plant fibers and wood fibers derived from genetically engineered or virus infected plants 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 plant fibers and wood fibers previously contacted with an oxidase fused to a cellulose binding peptide.

According to still further features in the described preferred embodiments the phenolic substituent is p-coumaric acid, p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol, ferulic acid, p-hydroxybenzoic acid, and It is selected from the group consisting of any other phenolic group that can be oxidized.

According to still further features in the described preferred embodiments the phenolic polymer forms the structural 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 comprises modified and non-modified starch, modified and non-modified cellulose, and modified and non-modified hemicellulose. Is selected from the group consisting of

According to still further features in the described preferred embodiments the phenolic polysaccharide is selected from the group consisting of ferulylated arabinoxylan and ferrylized pectin.

According to still further features in the described preferred embodiments the reaction medium is incubated for 1 minute to 10 hours.

According to still further features in the described preferred embodiments the fusion polypeptide is incubated in the presence of an oxidizing agent for 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 enzymes are laccase (EC 1.10.3.2), catechol oxidase (EC 1.10.
3.1) and bilirubin oxidase (EC 1.3.3.5), and the oxidant is oxygen.

According to still further features in the described preferred embodiments the enzyme is laccase and 0.02 LACU-20 per gram of dry lignocellulose.
Present in amounts ranging from 00 LACU.

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 the genus Botrytis, Myceliophth.
Ora) or laccase encoded by a polynucleotide obtained from a fungus of the genus Trametes.

According to still further features in the described preferred embodiments the fungus is Trametes versicolor or Trametes villasa.

According to still further features in the described preferred embodiments the enzyme is an enzyme obtained from maple (Acer pseudoplanus).

According to still further features in the described preferred embodiments the enzyme is peroxidase and the oxidant is hydrogen peroxide.

According to still further features in the described preferred embodiments the peroxidase is 0.02 PODU-2000 POD per gram of dry lignocellulose.
Present in an amount in the range of U and the initial concentration of hydrogen peroxide in the reaction medium is 0.01 mM
˜100 mM.

According to still further features in the described preferred embodiments the amount of lignocellulose used corresponds to 0.1% to 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 ° C to 120 ° C.

According to still further features in the described preferred embodiments the temperature of the reaction medium is in the range of 15 ° C to 90 ° C.

According to still further features in the described preferred embodiments the amount of phenolic polymer is in the range of 0.1% to 10% by weight.

According to still further features in the described preferred embodiments the pH of the reaction medium is in the range of 3-10.

According to still further features in the described preferred embodiments the pH of the reaction medium is in the range 4-9.

According to still further features in the described preferred embodiments the reaction medium further comprises a lignocellulose-containing material without fusion protein.

According to still further features in the described preferred embodiments the lignocellulose-containing material without fusion protein comprises plant fibers, wood fibers, wood chips,
It is selected from the group consisting of wood flakes, wood veneers and recycled fibers.

Additionally, the present invention provides lignocellulosic products obtainable by the methods described herein.

Another aspect of the present invention provides genetically engineered or virus infected plants or cultured plant cells that express a fusion protein comprising an enzyme capable of catalyzing the oxidation of phenolic groups and a cellulose binding peptide.

According to still further features in the described preferred embodiments the fusion protein is compartmentalized within the cell of the plant or cultured plant cell and is thus isolated from the cell wall of the cell of the plant or cultured plant cell.

According to still further features in the described preferred embodiments the expression of the fusion protein is under the control of a constitutive or tissue-specific plant promoter.

According to still further features in the described preferred embodiments the fusion protein comprises a cytoplasm, an endoplasmic reticulum, a Golgi apparatus, an oil body, a starch body, chloroplastid,
It is compartmentalized within a cell compartment selected from the group consisting of chloroplasts, chromoplastides, chromophores, vacuoles, lysosomes, mitochondria and nuclei.

According to yet another aspect of the invention, a cell wall preparation derived from a genetically engineered plant or virus infected plant or cultured plant cells expressing a fusion protein comprising an enzyme capable of catalyzing the oxidation of phenolic groups and a cellulose binding peptide. A composition comprising a fusion protein, wherein the fusion protein is immobilized on cellulose in a cell wall preparation via a cellulose-binding peptide.

According to yet another aspect of the invention, (a) a promoter sequence that directs expression of the protein in plant cells, (b) (i) a first sequence encoding a cellulose binding peptide and (ii) a phenolic sequence. A nucleic acid molecule comprising a heterologous nucleic acid sequence comprising a second sequence encoding an enzyme capable of catalyzing the oxidation of a group, wherein the first sequence and the second sequence are linked in an open reading frame. A molecule is provided.

According to still further features in the described preferred embodiments the nucleic acid molecule comprises an origin of replication required for propagation in bacterial cells, at least one sequence element required for integration into the plant genome, a polyadenylation recognition sequence, a transcription termination. signal,
Sequence encoding translation start site, sequence encoding translation stop site, sequence derived from plant RNA virus, sequence derived from plant DNA virus, tumor induction (Ti)
It further comprises a sequence element selected from the group consisting of a sequence derived from a plasmid, a sequence derived from a transposable element, and a plant functional signal peptide that directs the protein to the cell compartment of the plant cell.

According to still further features in the described preferred embodiments the cell compartments are cytoplasmic, endoplasmic reticulum, Golgi apparatus, oil bodies, starch bodies, chloroplastids, chloroplasts, chromoplastides, colored bodies, vacuoles, lysosomes. , Mitochondria and nuclei.

The present invention avoids the need for purified enzymes, which are expensive materials when compared to other raw materials and reagents described in the prior art used in the process of manufacturing lignocellulosic products, By providing methods and compositions for producing lignocellulosic products, the shortcomings of the currently known compositions are overcome.

DETAILED DESCRIPTION OF THE INVENTION The present invention is a method and composition for producing a lignocellulosic product from lignocellulosic material that avoids the need to use purified enzymes.

The principles and operation of the method according to the present invention may be better understood with reference to the accompanying description.

Before describing at least one embodiment of the present invention in detail, it is to be understood that the invention, in its application, is not limited to the details of the steps and components set forth in the description below. The invention is capable of other embodiments or of being carried out in various ways. Also,
The phraseology and terminology used herein is for the purpose of description and may include
It must be understood that it should not be considered as a limitation.

Accordingly, the present invention provides a method for producing a lignocellulosic product from a lignocellulosic material. The method according to the invention comprises the steps of (i)
Phenolic polymers substituted with phenolic hydroxy groups (eg lignin or polysaccharides which are at least substituted with substituents containing phenolic hydroxy groups); (ii) enzymes and cellulose capable of catalyzing the oxidation of phenolic groups. It is performed by mixing a lignocellulose-containing material in which a fusion polypeptide containing a binding peptide is immobilized in its cellulose portion; and (iii) an oxidizing agent.

The mixing / contacting sequence of these three components is such that the activated lignocellulosic material and the activated phenolic polysaccharide are combined in such a way that they can react in a desired manner. It does not matter as long as it is ensured by the composition. Thus, for example, the oxidizing agent can be mixed with the lignocellulose-containing material before or after it is mixed with the phenolic polymer.

As described in more detail below, the lignocellulose-containing material is preferably a cell wall preparation derived from genetically engineered or virus infected plants or cultured plant cells expressing the fusion protein. As such, the phenolic polymer may form a structural part of the lignocellulose-containing material. This is because the cell walls of plants contain the phenolic polymer lignin. Thus, cell wall preparations can be made to contain lignin. In this case, and in order to prevent the enzyme from exerting its catalytic activity beforehand, the cell wall preparation can be kept under a non-oxidizing atmosphere such as a N ₂ atmosphere.

It is generally appropriate to incubate the reaction medium containing these three components for at least a few minutes. Incubation times of 1 minute to 10 hours may generally be suitable, but periods of 1 minute to 10 hours are preferred.

As already indicated, the process according to the invention is suitable for all types of lignocellulosic products, for example various types of fiberboard (such as hardboard), particle board, oriented strand board (OSB). Flake board,
Plywood, molded composites (eg shaped bodies based on wood particles, often in combination with other non-lignocellulosic materials (eg certain plastics)), paper and board (cardboard, linerboard and others etc. ) Is well suited for manufacturing.

Lignocellulose-Containing Material: The lignocellulose-containing material used in the method of the invention may be in any suitable form, provided that it is derived from a genetically engineered plant or virus infected plant expressing the fusion polypeptide, eg It can be in the form of vegetable fibers (such as wood fibers).

If appropriate, the lignocellulose-containing material can be used in combination with non-lignocellulosic materials having phenolic hydroxy functional groups. By using the process of the present invention, intermolecular bonds between each of the lignocellulosic and non-lignocellulosic materials (ie, intermolecular bonds when the lignocellulosic material is used alone in this process). Can be formed) in a manner similar to that in which the composite is formed, resulting in a composite product. In addition to acting as a good adhesive / binder, the phenolic polysaccharide also serves as a good "crevice filler". This is a great advantage, for example, when making particleboard from large wood particles.

The lignocellulosic material in question was added to the reaction medium in an amount of 0.1% to 9%.
It is usually appropriate to use an amount corresponding to the weight percentage of dry lignocellulosic material [dry substance (DS)] in the range of 0%.

The temperature of the reaction mixture in the process of the present invention may suitably be in the range of 10 ° C to 120 ° C. However, temperatures in the range of 15 ° C to 90 ° C are generally preferred. As illustrated by the examples described in US Pat. No. 5,846,788, the reactions involved in the process of the present invention can occur very satisfactorily at ambient temperatures near 20 ° C. is expected.

In addition to the lignocellulose-containing material on which the fusion protein is immobilized, the reaction medium according to the invention comprises a lignocellulose-containing material without such fusion protein (plant fiber, wood fiber, wood chips, wood flakes, Wood veneer and recycled fibers can be included, including but not limited to.

Phenolic Polymers: Phenolic polymers used in the process of the present invention are preferably from natural sources or polymers that have been chemically modified by the introduction of substituents bearing phenolic hydroxy groups. It can be a material that can be obtained. Examples of the latter category include phenolic substituents (eg hydroxy-substituted benzoic acids (eg 2-hydroxybenzoic acid, 3-hydroxybenzoic acid or 4
-Acyl-type substituents derived from -hydroxybenzoic acid, etc.).

The phenolic substituents in the phenolic polysaccharides suitable for use in connection with the present invention can be suitably linked to the polymeric species by, for example, ester or ether linkages.

A very suitable phenolic polymer is a phenolic polysaccharide in which the phenolic substituent of the phenolic polysaccharide is a substituent derived from a phenolic compound present in at least one of the following plant biosynthetic pathways: : P-coumaric acid to p-coumaryl alcohol biosynthetic pathway, p-coumaric acid to p-coniferyl alcohol biosynthetic pathway, and p-coumaric acid to synapyl biosynthetic pathway; p-coumaric acid The three mentioned “end products” of the self and the latter three biosynthetic pathways are also relevant compounds in this regard. The related "that is generated in these biosynthetic pathways
Examples of "intermediate" compounds include caffeic acid, ferulic acid (i.e. 4-hydroxy-3-methoxycinnamic acid, 5-hydroxyferulic acid and sinapic acid.

Particularly preferred phenolic polysaccharides are phenolic polysaccharides which show good solubility in water and thus in aqueous medium within the scope of the present invention. In this and other respects, many types of phenolic polysaccharides, which are readily obtainable from plant sources in uniform quality, have been found to be particularly well suited for use in the process of the invention. These include, but are in no way limited to, phenolic arabinoxylans and heteroxylans, and phenolic pectins. Very suitable examples thereof are ferrilled arabinoxylan (for example obtainable from wheat bran or soybean bran) and ferrilled pectin (for example obtainable from beet pulp): ie via an ester linkage Arabinoxylan and pectin containing a ferryl substituent attached to a polysaccharide molecule.

The amount of phenolic polysaccharide or other phenolic polymer (such as lignin) used in the process of the present invention is generally 0 based on the weight of lignocellulosic material, which is calculated as dry lignocellulosic material. 0.01 wt% ~
It is within the range of 10% by weight. Amounts in the range of about 0.02% to 6% by weight (calculated in this fashion) are often very suitable.

Enzymes and Polynucleotides Encoding Enzymes: In principle, any type of enzyme capable of catalyzing the oxidation of phenolic groups may be isolated from a polynucleotide encoding such an enzyme, or Use in the process of the invention provided it can be readily isolated using conventional genetic engineering isolation techniques, and thus such enzymes can be expressed as part of a fusion polypeptide. You can

However, preferred enzymes are laccase (EC 1.10.3.2), catechol oxidase (EC 1.10.3.1) and bilirubin oxidase (EC1).
． 3.3.5) and other oxidases, as well as peroxidases (EC 1.11)
． 1.7). In some cases it may be appropriate to use two or more different enzymes in the process of the invention.

Among the oxidases (in combination with which oxygen (eg atmospheric oxygen) is an excellent oxidant), laccase has proved to be well suited for use in the method of the invention. Has been done.

The laccase-encoding polynucleotides are obtained from various plant and microbial sources, especially bacteria and fungi, including filamentous fungi and yeasts, or can be easily isolated. . For example, US Pat. No. 5,843,74
No. 5; No. 5,795,760; No. 5,770,418 and No. 5,7.
50,388, which are incorporated herein by reference.
. Suitable examples of polynucleotides encoding laccase include Aspergillus (
Aspergillus genus, Neurospora genus (for example, N. crassa), Podospora genus, Botrytis genus, Molinokaratake (Collybia) genus, Psyllium genus (Lomatia, Fomaes, Fomaes).
A polynucleotide obtained from, or obtainable from, a strain of the genus urotus, genus Trametes. Some of these species / strains are known by various names and / or have previously been classified into other genera; for example, Trametes villasa = T. pinsit
us = Polyporus pinsitis (P. pinsitis or P.
． (also known as villasus) = Coriolus pinsit
us, Polyporus, Rhizoctonia (eg, R. solan
i), Coprinus (eg, C. plicatilis), Psatyr
ella, Myceliophthora (for example, M. thermophile
a), Schytalidium, Phlebia (eg P. radita
See International Patent Application Publication WO 92/01046) or Coriolu.
s (see, for example, C. hirsutus; JP 2-238885).

A preferred laccase within the scope of the present invention is Trametes virosa (Tram)
ets villasa) or maple (Acer pseudoplane)
lacsase which can be obtained from s).

Peroxidase enzyme (EC1.11.1) used in the method of the present invention
Is preferably obtained or obtainable from a plant (e.g. horseradish peroxidase or soybean peroxidase), or obtained from a microorganism such as a fungus or a bacterium,
Or a polynucleotide that can be obtained. In this regard, some preferred fungi include strains belonging to the filamentous incomplete fungal class of the subphylum Incomplete, such as Fusarium spp., Humicola spp.
The genus Trichoderma, Myrotheci
um), Verticillum, and Arthromyces (Ar)
genus thromyces, Caldariomyces genus, Ulocladium genus, Embellii
strains belonging to the genus sia), the genus Cladosporium or the genus Dreschlera, in particular Fusarium oxysporum (DSM2672),
Humicola insolens, Trichoderma resii, Myrothecium verrucana (IFO6113), Verticillum arboratrum (Verticillum)
Verticillum dahlie, Arthromyces ramosus (FERM P)
-7754), Caldariomyces fumago (Caldariomyces fu)
mago, Ulocladium chart
arum), Embellisia alli, or Dreschlera halodes.

Other preferred fungi include strains belonging to the class Basidiomycetes of the subphylum Basidiomycetes, for example the genus Coprinus, Phaneroch.
aete), Coriolus or Enamel (Tra)
strains belonging to the genus Methes), especially Coprinus cinereus f. Microsporus (Coprinus cinereus f. Microsporu
s) (IFO 8371), Coprinus macrolithus (Coprinus ma)
crorhizus), Fanerocaete chrysosporium (Phaneroc)
haete chrysosporium (eg NA-12) or Trametes versicolor (eg
PR4 28-A) is included.

Further preferred fungi include Mycoracea of the subphylum Zygomycetes.
e) Strains belonging to the class are included, for example, strains belonging to the genus Rhizopus or genus Mucor are included, and in particular, Mucor himalis is included.

Some preferred bacteria include strains of the order Actinomycetes, for example, Streptomyces spheroides.
(ATCC 23965), Streptomyces thermoviolaceus (Strep
tomyces thermoviolaceus (IFO 12382) or Streptoverticillum verticillium ssp. Berticillium (Stre
ptoverticillum verticillium ssp. vert
icillium) is included.

Other preferred bacteria include Bacillus pumil.
us) (ATCC12905), Bacillus stearothermophilus (Baci)
llus stearothermophilus, Rhodobacter sphaeroides, Rhodomonas palustri, Streptococcus acrocus lacunas (Streptococcus lacunas), Streptococcus procus lactis (Streptococcus lacudos),
C15958) or Pseudomonas fluorescens (Pseudomona
s fluorescens) (NRRL B-11).

Further preferred bacteria include strains belonging to the genus Myxococcus, for example M. include viruses.

Another potential source of useful sources of polynucleotides encoding peroxidases is B. C. Saunders and others Peroxidase (Londo
n, 1964, pp. 41-43).

Cellulose-Binding Peptide: As used herein and in the claims below, the expression “cellulose-binding peptide” refers to when expressed in plant cells, eg after homogenization and cell disruption, Or a peptide capable of affinity binding to plant-derived cellulose (eg, lignocellulosic) material during plant growth and development,
For example, proteins and domains (parts) thereof are included. Thus, this expression includes, for example, peptide libraries or DNA libraries (eg, cDNA
Peptides screened for their cellulose binding activity from libraries such as NA expression libraries or display libraries) are included, and genes encoding such peptides that are isolated and expressible in plants are included. In addition, this expression also includes peptides designed and engineered to be able to bind to cellulose and / or its units.

Such peptides can be traced back to eg, cellulose binding proteins (
CBP) or a cellulose-binding domain (CBD) -derived cellulose-binding region. The cellulose-binding peptide according to the present invention can comprise any plant-expressible amino acid sequence that binds to a cellulose polymer. For example, a cellulose-binding domain or cellulose-binding protein bound to a cellulase, a binding domain of a cellulose-binding protein, or a binding domain of a protein screened for and isolated from a peptide library, or to a cellulose or sugar unit thereof. Can be designed and engineered, and can be derived from plant expressible proteins. The cellulose binding domain or cellulose binding protein may be naturally occurring or synthetic, as long as it is expressible in plants. Suitable polysaccharides from which plant-expressable cellulose-binding domains or cellulose-binding proteins can be obtained include β-1,4-glucanase. In a preferred embodiment, cellulase or scaffoldin derived cellulose binding domains or proteins are used. Typically, the amino acid sequence of a cellulose-binding peptide expressed in plants according to the present invention is essentially free of polysaccharidase (eg, cellulase, chitinase) hydrolyzing activity, although Holding The amino acid sequence preferably has less than about 10% of the hydrolytic activity of the native polysaccharide, preferably less than about 5% of the native polysaccharide, and most preferably the native polysaccharide. It has less than about 1% of the hydrolytic activity of the lipase and ideally has no activity at all.

Cellulose-binding domains or cellulose-binding proteins can be obtained from a variety of sources including the cellulose-binding enzymes and other proteins used in the present invention.

In Table 4 below, one or more soluble / one or more soluble / containing all binding domains with affinity for soluble glucans (α, β and / or mixed binders)
Such binding domains that bind insoluble polysaccharides have been listed. C. fim
The N1 cellulose-binding domain from the endoglucanase CenC of i is the only protein known to bind soluble cellosaccharides, and of a small protein population known to bind all soluble polysaccharides. There is one. Tables 1 to 3 show examples of proteins containing a hypothetical β-1,3-glucan-binding domain (Table 1); examples of proteins containing streptococcal glucan-binding repeats (Cpl superfamily). (Table 2); and examples of enzymes with a chitin-binding domain that can also bind cellulose (Table 3). The genes encoding each of the peptides listed in Tables 1 to 4 have been isolated or can be isolated as further detailed below, and such peptides are therefore Can be expressed in. Clostridium
Various scaffoldin proteins, such as the scaffoldin protein produced by Clostridium cellulovorans (Shoseyov et al., International Patent Application PCT / US94 / 04132), or portions thereof, contain a cellulose binding domain, but these are also It can be used as a cellulose-binding peptide expressible in plants according to the invention.
Some fungi, including Trichoderma spp., Also produce polysaccharides capable of isolating plant-expressible polysaccharide binding domains or proteins. Further examples can be found in, for example, Microbiological Hydrolysis.
of Polysaccharides, R.A. A. J. Warren, Ann
u. Rev. Microbiol. 1996, 50: 183-212; "Adv.
ances in Microbiology Physiology ", R.A. K. P
ool, 1995, Academic Press Limited (the two are incorporated by reference as if set forth herein in their entirety).

[Table 1]

[Table 2]

New cellulose-binding peptides with binding properties and binding specificities of interest can be identified and screened, and the genes encoding such cellulose-binding peptides have been identified with well-known molecular biology. The method can be used in combination with various other procedures to isolate. Such methods include
For example, NMR spectroscopy (Zhu et al., Biochemistry (1995) 34.
: 13196-13202; Gehring et al., Biochemistry (1
991) 30: 5524-5531), UV differential light method (Belshaw et al., Eu)
r. J. Biochem. (1993) 211: 717-724), fluorescence (titration) spectroscopy (Miller et al., J. Biol. Chem. (1993) 258: 1).
3665-13672), stop flow analysis by UV or fluorescence (De B
oeck et al., Eur. J. Biochem. (1985) 149: 141-41.
5), affinity method (affinity electrophoresis (Mimura et al., J. c.
chromatography (1992) 597: 345-350) or affinity chromatography with immobilized mono- or oligosaccharides),
Analysis by precipitation or aggregation (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 physicochemical methods (differential scanning calorimetry or isothermal titration calorimetry). Method) (Sigurskjold et al., J. Biol. Chem. (1992) 2
67: 8371-8376; Sigurskjold et al., Eur. J. Biol
． (1994) 225: 133-141), or spectroscopic (titration) such as comparative assays (melting) of protein stability in the absence or presence of oligosaccharides using thermal CD spectroscopy or thermofluorescence spectroscopy. ) Method included.

[0093] K _a of binding to cellulose of the cellulose-binding domain or cellulose binding protein comprises at least a weak antibody - is in the range of antigen extract, i.e.,
It is 10 ³ M ⁻¹ or more, preferably 10 ⁴ M ⁻¹ or more, and most preferably 10 ⁶ M ⁻¹ or more. Where the binding of the cellulose binding domain or cellulose binding protein to cellulose is exothermic or endothermic, the binding may be provided at a lower temperature, respectively, by providing a means of controlling the temperature of the binding step.
Increase or decrease.

[Table 3]

[Table 4]

Accordingly, and as already mentioned, the expression “polysaccharide-binding peptide” comprises an amino acid sequence which comprises at least a functional part of the polysaccharide-binding region (domain) of a polysaccharide-binding protein or a polysaccharide-binding protein. This expression further relates to a polypeptide screened for its cellulose binding activity from a library such as a peptide library or a DNA library (eg, a cDNA library or a display library). By "functional portion" is meant an amino acid sequence that binds to cellulose.

Techniques used in isolating polysaccharidase genes (such as the cellulase gene) and genes for cellulose binding proteins are known in the art,
This includes synthesis, isolation from genomic DNA, preparation from cDNA, or combinations thereof (US Pat. Nos. 5,137,819; 5,202,24).
No. 7; No. 5,340,731; No. 5,496,934; and No. 5,
837,814). The sequences of several binding domains that bind soluble oligopolysaccharides are known (International Patent Application PCT / CA97 / 00033).
(See Figure 1 of WO 97/26358). DNAs encoding various polysaccharidases and polysaccharide binding proteins are also known. Various techniques for manipulating genes are well known and include restriction, digestion, excision, ligation, in vitro mutagenesis, primer repair, using linkers and adapters, etc. (Sambrook et al., Molecular Clon
ing-A Laboratory Manual, Cold Spring
Harbour Laboratory, Cold Spring Harbor
, New York, 1989; which is incorporated herein by reference).

The amino acid sequence of the polysaccharidase is the mRNA derived from the cell of interest as the donor cell for the polysaccharidase gene or the polysaccharide binding protein gene.
Alternatively, it can be used to design a probe to screen a cDNA or genomic library prepared from DNA. By using polysaccharidase cDNA or binding protein cDNA or fragments thereof as hybridization probes, structurally related genes found in other species can be easily cloned, and such genes Provides a cellulose-binding peptide that is expressible in plants according to the present invention. Isolating a gene from an organism expressing polysaccharidase activity using an oligonucleotide probe based on the nucleotide sequence of the gene that can be obtained from the organism where the catalytic and binding domains of the polysaccharidase are distinct , But other polysaccharide-binding proteins can also be used (eg, Shoseyov et al., Proc. Nat'l. Aca).
d. Sci. (USA) (1992) 89: 3483-3487).
.

Of particular interest are probes developed using consensus domains for the binding domains of polysaccharidases or polysaccharide binding proteins. The recently characterized C. Various β-1,4-glucanases from fimi are endoglucanases A, B, C and D (CenA, CenB, CenC and Ce, respectively).
nD), exocellobiohydrolases A and B (CbhA and C, respectively)
bhB), and xylanases A and D (Cex and XylD, respectively).
(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., Bio.
chem. J. (In printing); O'Neill et al. (1986) Gene, 44:
325; Millward-Sadler et al., (1994) Mol. Micro
biol. , 11: 375). These are all modular proteins of varying degrees of complexity, but capable of independent functioning catalytic domains (CD)
And cellulose-binding domain (CBD) have two common features (Mi
llward-Sadler et al., (1994) Mol. Microbiol. ,
11: 375; Gilkes et al., (1988) J. Am. Biol. Chem. , 26
3: 10401; Meinke et al., (1991) J. Am. Bacteriol. 1
73: 7126; Coutinho et al., (1992) Mol. Microbio
l. , 6: 1242). Four of these enzymes (CenB, Ce
nD, CbhA and CbhB), fibronectin type III (F
The n3) repeat separates the N-terminal CD from the C-terminal CBD. The CDs for these enzymes are derived from six families of glycoside hydrolases (H
enrissat, (1991) Biochem. J. 280: 309; He
nrissat, (1993) Biochem. J. 293: 781); all of these enzymes have N- or C-terminal CBD (s) (see Tomme et al., Adv. Microb. Physiol. (In press)). CenC has a tandem CBD from family IV at its N-terminus; CenB and XylD have another internal CBD from family III and II, respectively; Cex and Xy
ID is clearly a xylanase. However, Cex, but not XylD, has a low activity on cellulose. Nevertheless, some other bacterial xylanases (Gilbert et al. (1993) J. Gen. Microbiol.
39: 187), they have multiple CBDs. C. f
imi probably produces other β-1,4-glucanases. A similar system is provided by a family of like bacteria (Wilson, (1992) Crit.
． Rev. Biotechnol. , 12:45; Hazlewood et al., (1
992) J. Appl. Bacteriol. , 72: 244).
Unrelated bacteria also produce glucanase. For example, Clostridium thermocellum produces more than 20 β-1,4-glucanases (Beguin et al., (1992) FEM.
S Microbiol. Lett. , 100: 523). C. f
The CBD from the endo endoglucanase C N1 is the only protein known to bind soluble cellosaccharides, and one of a small population of proteins known to bind all soluble polysaccharides. Is one.

Examples of suitable binding domains are given in International Patent Application PCT / CA97 / 00033 (WO
97/26358). This figure shows an alignment of binding domains from various enzymes that bind to polysaccharides and identifies conserved amino acid residues in most or all of these enzymes. This information can be used to obtain suitable oligonucleotide probes using methods known to those of skill in the art. The probe can be much shorter than the complete sequence, but it is desirable that it be at least 10 nucleotides in length, preferably at least 14 nucleotides. Longer oligonucleotides are also useful, up to the full length of the gene, but preferably 500.
It is not more than nucleotides, and more preferably not more than 250 nucleotides. RN
A probes or DNA probes can be used. In use, the probe is typically labeled in a detectable manner, eg, with ³² P, ³ H, biotin, avidin or other detectable reagent, and is derived from the organism in which the gene is being sought. Incubated with single-stranded DNA or RNA. Hybridization is detected by the label after separating the unhybridized probe from the hybridized probe. The hybridized probe is typically immobilized on a solid matrix such as nitrocellulose paper. Hybridization techniques suitable for use with oligonucleotides are well known to those of skill in the art. The probe is usually used with a detectable label to allow easy identification, but unlabeled probe also serves as a precursor to the labeled probe and also for double-stranded DNA (or DNA /
Both are useful for use in methods that define direct detection of RNA). Thus, the term "oligonucleotide probe" refers to both labeled and unlabeled forms.

Generally, a binding domain identified by probing a nucleic acid from an organism of interest will have at least about 40% identity (conservative) to the binding region (one or more) from which the probe was derived. Appropriate considerations for substitutions, including gaps for better alignment, etc.) and soluble β-1,4
-Binds to glucan with a K _a of 10 ³ M ^-1 or higher. More preferably, the binding domain has at least about 60% identity, and most preferably at least about 70% identity to the binding region used to obtain the probe. The percent identity is greater for such amino acids that are conserved within the binding domain of polysaccharidase. Analysis comparing amino acid sequences
/ Gene (IntelliGenetics, Inc.). PCLUSTAL can be used for multi-sequence alignment and phylogenetic tree generation.

Several genetic methods can be used to isolate a polysaccharide binding protein or domain from a polysaccharide binding enzyme or clustered enzyme. In one method, restriction enzymes are used to remove the part of the gene encoding the protein portion other than its binding portion. The remaining gene fragment is fused with expression control sequences to obtain a mutant gene encoding a truncated protein. Another method uses exonucleases such as Bal31 that systematically delete nucleotides from either the 5'and 3'ends of the DNA from the outside, or from a limited gap within the gene from the inside. Involves doing. These gene deletion methods result in a mutated gene encoding a shortened protein molecule that can be subsequently evaluated for substrate binding, ie, polysaccharide binding.

Any cellulose binding protein or cellulose binding domain can be used in the present invention. The term "cellulose binding protein"("CBP")
Refers to any protein or polypeptide that specifically binds to cellulose. The cellulose binding protein may or may not have cellulose activity or cellulose hydrolysis activity. The term "cellulose binding domain"("CBD") refers to any protein or polypeptide that is a region or portion of a larger protein that specifically binds to cellulose. The cellulose binding domain (CBD) is one of the cellulases, xylanases or other polysaccharidases (such as chitinase), sugar-binding proteins (such as maltose binding protein, or scafordin such as CbpA of Clostridium celluvorans). It can be part or part. Many cellulases and hemicellulases (eg, xylanase and mannase) have the ability to bind cellulose. These enzymes typically have a catalytic domain containing an active site for hydrolyzing a substrate, and a carbohydrate or cellulose binding domain for binding to cellulose. CBD may also be derived from a polysaccharide binding protein that has no catalytic activity. Currently, over 100 cellulose binding domains (CBDs) have been classified into at least 13 families called I-XIII (Tomme et al. (1995) "Cellulose Binding Domains: Classification and Properties," ACS Symposium Series 618, Enzymatic Deg
ration and Insoluble Carbohydrates
, 142-161, Saddler and Penner (eds.), Ameri.
can Chemical Chemical Society, Washington, D .; C.
(Tomme I); Tomme et al., Adv. Microb. Physiol.
(1995) 37: 1 (Tomme II); Smant et al., Proc. Nat
l. Acad. Sci. USA, (1998) 95: 4906-4911: all of which are incorporated herein by reference). Tomme I or To
All CBDs described in mme II or any variant thereof, any other currently known CBD, or any new CBD that can be identified can be used in the present invention. As an illustrative, but non-limiting example, the CBP or CBD may be derived from a bacterial, fungal, slime mold or nematode protein or polypeptide. As a more specific and illustrative example, CBD may be a Clostridium cellulovor
ans), Clostridium cellulovorans or Cellulomonas fimi (C
ellulomonas fimi) (eg CenA
, CenB, CenD, Cex). Furthermore, CBDs can be selected from phage display peptide libraries or peptidomimetic libraries (random or otherwise) using, for example, cellulose as a screening agent (Smith, Science, (1985) 228: 1315-1).
317; Lam, Nature, (1991) 354: 82-84). Further, CBD binds to a polysaccharide different from cellulose (or hemicellulose), but a protein or polypeptide that binds cellulose (such as chitinase that specifically binds to chitin), or a portion of a sugar-binding protein such as maltose-binding protein. Can be mutated to thereby allow the moiety to bind to cellulose. In any case,
CBD binds to cellulose or hemicellulose. Shoseyov and Doi (Proc. Natl. Acad. Sci. USA, (1990) 87:
2192-2195) isolated an unprecedented cellulose-binding protein (CbpA) from a cellulose "complex" of the cellulolytic bacterium Clostridium cellulovorans. This major subunit of the cellulose complex was found to bind to cellulose but did not have 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. US
A, (1992) 89: 3483-3487). Using PCR primers flanking the cellulose binding domain of CbpA, the latter was cloned in E. coli at approximately 17k.
It was successfully cloned into an overexpression vector allowing overexpression of Da CBD. This recombinant CBD shows a very strong affinity for cellulose and chitin (US Pat. No. 5,496,934; Goldstein et al.,
J. Bacteriol. (1993) 175: 5762; PCT International Patent Application Publication WO 94/24158: all of which are incorporated by reference as if set forth herein in their entirety).

In recent years, several CBDs have been isolated from various sources. Most of these have been isolated from proteins that have distinct catalytic domains, namely the cellulose domain and the cellulose binding domain, and only two have no apparent hydrolytic activity, but proteins that have cellulose binding activity. (Goldstein et al., J. Bacteriol. (1993) 175:
5762-5768; Morag et al., Appl. Environ. Microb
iol. (1995) 61: 1982-1986).

Cellulose-Binding Peptide-Recombinant Protein Fusions: Fusing two proteins whose genes have been isolated, such as cellulose-binding peptides and oxidases such as laccase, is well known in the art and routine. Have been implemented. Such a fusion involves joining the heterologous nucleic acid sequences in reading frame so that the translation results in the production of a fused protein product or fusion protein. Various methods such as polymerase chain reaction (PCR), restriction, nuclease digestion, ligation, synthetic oligonucleotide synthesis and others are typically used in various combinations in the process of making fusion gene constructs. . One of ordinary skill in the art can readily assemble such constructs for any two or more of the individual proteins. Interestingly,
In most cases where such fusion or chimeric proteins are produced, and in all cases where one of these proteins was a cellulose-binding peptide, both the former and the latter retain their catalytic activity or function. It was In either case, a spacer that matches the reading frame can be included. Its length can vary, for example, from a few amino acids to tens of amino acids. Such spacers may also function to reduce mobilization constraints.

For example, Greenwood et al. (1989, FEBS Lett. 224: 1).
27-131) fused the cellulose-binding domain of Cellulomonas fimi endoglucanase to the enzyme alkaline phosphatase. This recombinant fusion protein retained both its phosphatase activity and cellulose binding capacity.
For a further description of fusion proteins that bind to cellulose, see US Pat.
, 137,819 (issued to Kilburn et al.) And US Pat. No. 5,7.
19, 044 (published to Shoseyov et al.), Both incorporated herein by reference. See also US Pat. No. 5,474,925. All of which are incorporated herein by reference.

Therefore, according to the present invention, a promoter for directing the expression of the protein in plant cells, a first sequence encoding a cellulose-binding peptide and a second sequence encoding an enzyme capable of catalyzing the oxidation of a phenolic group. And a heterologous nucleic acid sequence comprising the sequence of SEQ.

According to a preferred embodiment of the present invention, the nucleic acid molecule is provided with an origin of replication required for propagation in bacterial cells, at least one sequence element required for integration into the plant genome, a polyadenylation recognition sequence, a transcription termination signal, a translation initiation. Sequence coding region, sequence coding translation stop site, sequence derived from plant RNA virus, plant D
Selected from the group consisting of a sequence derived from NA virus, a sequence derived from a tumor-inducing (Ti) plasmid, a sequence derived from a transposable element, and a functional signal peptide of a plant that directs a protein to the cell compartment of a plant cell. Further includes array elements.

In yet a further preferred embodiment, the cellular compartments are from cytoplasm, endoplasmic reticulum, Golgi apparatus, oil bodies, starch bodies, chloroplastids, chloroplasts, chromoplastides, chromophores, vacuoles, lysosomes, mitochondria and nuclei. Is selected from the group consisting of

Genetically Engineered Plant Material: In the present invention, recombinant nucleic acid molecules are used. Such molecules include, for example, a promoter sequence to drive expression of the protein in plant cells; and a heterologous nucleic acid sequence as further detailed below. In this case, the heterologous nucleic acid sequence is downstream of the promoter sequence so that expression of the heterologous nucleic acid sequence can be effected by the promoter sequence. Such nucleic acid molecules need to be effectively introduced into plant cells so that the plant is genetically modified.

There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev. Plant. Physiol.
Mol. Biol. (1991) 42: 205-225; Shimamoto et al., Nature, (1989) 338: 274-276). The principal methods for stably incorporating exogenous DNA into plant genomic DNA include two main methods:

(I) Agrobacterium-mediated gene transfer: Klee et al., (1987) Ann.
u. Rev. Plant Physiol. 38: 467-486; Klee and Rogers, Cell Culture and Somatic Ce.
ll Genetics of Plants, Volume 6: Molecular
Biology of Plant Nuclear Genes, Editor: Sc
hell, J .; And Vasil, L .; K. , Academic Publics
hers, San Diego, Calif. (1989) pp. 2-25; Ga
tenby, Plant Biotechnology, editor: Kung, S. et al.
And Arntzen, C.I. J. , Butterworth Publicize
rs, Boston, Mass. (1989) pages 93-112).

(Ii) Direct DNA uptake: Paszkowski et al., Cell Cu
lture and Somatic Cell Genetics of P
lants, Volume 6, Molecular Biology of Plant
Nuclear Genes, editor: Schell, J. et al. And Vasil,
L. K. , Academic Publishers, San Diego, C
alif. (1989) pages 52-68; this includes DNA in protoplasts.
(Toriyama, K. et al., (1988) Bio)
/ Technology, 6: 1072-1074). DNA uptake induced by short-term electrical shock in plant cells: Zhang et al., Pla
nt Cell Rep. (1988) 7: 379-384. Fromm et al., N
ature (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 / Technol.
Ogy, (1988) 6: 923-926; Sanford, Physiol.
Plant. (1990) 79: 206-209; Method by use of micropipette system: Neuhaus et al., Theor. Appl. Genet. (1
987) 75: 30-36; Neuhaus and Spangenberg, P.
hysiol. Plant. (1990) 79: 213-217; or by direct incubation of DNA with growing pollen, DeWet et al., E.
xperimental Manipulation of Ovule Ti
ssue, editor: Chapman, G .; P. And Mantell, S .; H. And Daniels, W .; , Longman, London, (1985) 19
Pp. 7-209; Ohta, Proc. Natl. Acad. Sci. USA,
(1986) 83: 715-719.

The Agrobacterium system involves the use of plasmid vectors containing defined DNA segments that are integrated within the genomic DNA of the plant. The method of inoculation of plant tissue varies depending on the plant species and Agrobacterium delivery system. A widely used method is the leaf disc method, which can be performed with any tissue explant that provides a good source for initiating complete plant differentiation (Ho.
rsch et al., Plant Molecular Biology Manual
A5, Kluwer Academic Publishers, Dordr
echt (1988), pp. 1-9). The Agrobacterium system is particularly practical in producing transgenic dicotyledonous plants.

There are various methods of direct DNA transfer into plant cells. In electroporation, protoplasts are briefly exposed to a strong electric field. In microinjection, DNA is mechanically injected directly into cells using a very small micropipette. In microparticle bombardment, DNA is adsorbed to microbullets such as magnesium sulfate crystals or tungsten particles, and the microbullets are physically accelerated into cells or plant tissue.

After transformation, plant propagation is carried out. The most common method of plant propagation is by seeds. However, since seeds are produced by plants according to the genetic variations governed by the Mendelian rule, regeneration by seed multiplication has the imperfection that there is no homogeneity in the crop due to heterozygosity. Basically, each offspring is genetically different and each grows with its own specific traits. Therefore, the regenerated plant has the same traits and characteristics as the parental transgenic plant (
Transgenic plants are preferably produced such that they have (for example, reproduction of the fusion protein). Therefore, transgenic plants are preferably regenerated by micropropagation, which provides rapid and consistent reproduction of transgenic plants.

Micropropagation is a method of growing new generation plants from a single piece of tissue excised from a selected parent plant or cultivar. This method allows for large-scale reproduction of plants with favorable tissues expressing the fusion protein.
The new generation plant produced is genetically identical to the original plant and has all the characteristics of the original plant. Micropropagation allows for large-scale production of constant quality plant material in a short time and results in rapid growth of the selected cultivar in preserving the characteristics of the original transgenic or transformed plant.
The advantage of cloning plants is the rate of plant reproduction and the quality and homogeneity of the plants produced.

Micropropagation is a multi-step method that requires changes in culture medium or growth conditions between steps. Thus, the micropropagation method involves four basic stages: Stage 1, early tissue culture; Stage 2, tissue culture reproduction; Stage 3, differentiation and plant formation; Stage 4, greenhouse cultivation. And rugged. At the first stage of initial tissue culture, tissue culture is established and confirmed to be free of contamination. During the second stage, the initial tissue culture is grown until a sufficient number of tissue samples are obtained to meet the production objectives. During the third stage, the tissue sample grown in the second stage is split and grown into individual plantlets. In the fourth stage, the transgenic plantlets are transferred to a greenhouse for ruggedness, where the plants' tolerance to light is gradually increased so that they can grow in their natural environment.

The basic bacterial / plant vector construct is preferably a broad host range prokaryotic origin of replication; a prokaryotic selectable marker; and in the case of transformation with Agrobacterium, Agrobacterium into the plant chromosome. It contains the T-DNA sequences required for intermediary transfer. If the heterologous sequence is not easily detected, the construct also preferably
It has a suitable selectable marker gene for determining whether a plant cell has been transformed. For a general review of suitable markers for members of the grass family, see Wi.
lmink and Dons, Plant Mol. Biol. Reptr. (1
993) 11: 165-185.

Suitable sequences are also recommended, which allow integration of the heterologous sequences into the plant genome. Such sequences may include Ti sequences that allow random insertion of heterologous expression cassettes into the plant genome, as well as transposon sequences for performing homologous recombination and the like.

Suitable prokaryotic selectable markers include resistance to antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions can also be present in the vector, as is known in the art.

The construct of the present invention comprises an expression cassette for expressing the fusion protein of interest. Usually, there will be only one expression cassette, but more than one is possible.
The recombinant expression cassette comprises, in addition to the heterologous sequence, one or more of the following sequence elements: promoter sequence, plant 5'untranslated sequence, whether the structural gene is accompanied by a start codon. Initiation codons that depend on and termination sequences for transcription and translation. The various unique restriction enzyme sites at the 5'and 3'ends of the cassette allow for easy insertion into already existing vectors.

Virus-Infected Plant Material: Viruses are a unique class of infectious agents whose unique characteristics are their simple organization and their mechanism of replication. In fact, the complete virus particle (ie,
Virions) are largely capable of autonomous replication and are viewed as a mass of genetic material (either DNA or RNA) surrounded by a protein coat and sometimes by an additional membrane envelope such as in the case of alphaviruses. be able to. The coat protects the virus from the environment and serves as a vehicle for transmission from one host cell to another.

Viruses shown to be useful in the transformation of plant hosts include CaV,
Includes TMV and BV. Transformation of plants using plant viruses is described in US Pat. No. 4,855,237 (BGV), European Patent EP-A67,553 (TM).
V), JP-A-63-14693 (TMV), European Patent EPA 194,809 (B)
V), European Patent EPA 278,667 (BV); Gluzman, Y .; Other, Co
mmunications in Molecular Biology: Vi
Ral Vectors, Cold Spring Harbor Labor
Atory, New York, pages 172-189 (1988). Virus-like particles used in expressing foreign DNA in many hosts, including plants, are described in WO 87/06261.

Plant RNA for introducing and expressing non-viral foreign genes in plants
Viral construction is described in the references above, as well as Dawson, W .; O. Other, Vi
Rology, (1989) 172: 285-292; Takamatsu et al.,
EMBO J.M. , (1987) 6: 307-311; French et al., Scie.
No., (1986) 231: 1294-1297; Takamatsu et al., F.
EBS Letters, (1990) 269: 73-76.

If the virus is a DNA virus, the construct can be made against the virus itself. Alternatively, the virus can be first cloned into a bacterial plasmid to facilitate the construction of the desired viral vector with foreign DNA. The virus can then be excised from the plasmid.
If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacterium. This D
Transcription and translation of NA produces a coat protein that wraps viral DNA to form a capsid. 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 constructs. The RNA virus then transcribes the viral sequences of the plasmid and translates the viral genes,
It is produced by producing a coat protein that wraps the viral RNA to form a capsid.

Plant RNA for introducing and expressing non-viral foreign genes in plants
Viral construction is described by the above references and in US Pat. No. 5,316,9.
No. 31 revealed.

In one embodiment, a plant viral nucleic acid is provided in which the viral native coat protein coding sequence is deleted from the viral nucleic acid, allowing expression in a plant host, packaging the recombinant plant viral nucleic acid, and Non-native plant virus coat protein coding sequences and non-viral promoters that ensure systemic infection of the host with recombinant plant viral nucleic acid (preferably subgenomics of non-viral coat protein coding sequences).
(nomic) promoter) has been inserted. Alternatively, the coat protein gene can be inactivated by inserting into it a non-viral nucleic acid sequence so that a fusion protein is produced. The recombinant plant viral nucleic acid can include one or more additional nonviral native subgenomic promoters. Each non-viral subgenomic promoter is capable of transcribing or expressing flanking genes or nucleic acid sequences in a plant host, but is incapable of recombination with each other and recombination with the viral native subgenomic promoter. When two or more nucleic acid sequences are included, the non-viral (foreign) nucleic acid sequence is adjacent to the viral native plant viral subgenomic promoter, or the viral native plant viral subgenomic promoter and the non-viral native plant viral sub It can be inserted adjacent to the genomic promoter. The non-native nucleic acid sequence is transcribed or expressed in the host plant under the control of a subgenomic promoter to produce the desired product.

In a second embodiment, a recombinant plant viral nucleic acid is provided as in the first embodiment, but in this embodiment, the viral native coat protein coding sequence is a non-viral native coat. Instead of the protein coding sequence,
It is placed adjacent to one of the non-native viral coat protein subgenomic promoters.

In a third embodiment, the recombination wherein the viral native coat protein gene is flanked by its subgenomic promoters and one or more non-viral subgenomic promoters are inserted within the viral nucleic acid. Plant viral nucleic acids are provided. The inserted non-viral subgenomic promoter is capable of transcribing or expressing flanking genes in the plant host, but is incapable of recombining with each other and with the viral original subgenomic promoter. The non-viral nucleic acid sequence can be inserted adjacent to the non-viral subgenomic plant viral promoter such that the sequence is transcribed or expressed in the host plant under the control of the subgenomic promoter to produce the desired product. Will be produced.

In a fourth embodiment, a recombinant plant viral nucleic acid is provided as in the third embodiment, wherein in this embodiment the viral native coat protein coding sequence is a non-viral native coat protein. It has been replaced by a coding sequence.

The viral vector is encapsulated by a coat protein encoded by the recombinant plant viral nucleic acid to form a capsid and produce a recombinant plant virus.
The recombinant plant viral nucleic acid or recombinant plant virus is used to infect suitable host plants. The recombinant plant viral nucleic acid allows replication in the host, systemic spread in the host, and transcription or expression of the foreign gene in the host to produce the desired fusion protein.

Compartmentalization of Fusion Proteins-Signal Peptide: As already mentioned above, compartmentalization of fusion proteins is an important feature of the invention, as it does not disturb the growth of plants. Thus, according to one aspect of the present invention, the fusion protein becomes compartmentalized within the cells of the plant or cultured plant cells and thus becomes sequestered from the cell wall of the cells of the plant or cultured plant cells.

Fusion proteins include, for example, cytoplasm, endoplasmic reticulum, Golgi apparatus, oil bodies, starch bodies, chloroplastids, chloroplasts, chromoplastids, colored bodies, vacuoles, lysosomes,
It can be compartmentalized within a cellular compartment such as the mitochondria or nucleus.

Thus, the heterologous sequence used when performing the method according to this aspect of the invention includes the following sequences: (i) a first sequence encoding a cellulose binding peptide; (ii) A second sequence encoding the recombinant protein, in which case the first and second sequences are ligated in reading frame; and (iii) a signal peptide for directing the protein to the cell compartment. A third coding sequence, which is located upstream of and in reading frame with the first and second sequences.

In the following, signal peptides that may be used to direct the fusion proteins according to the invention to specific cell compartments are described.

It is well known that signal peptides perform the translocation function of the produced protein across the endoplasmic reticulum membrane. Similarly, the transmembrane segment arrests translocation and results in anchoring the protein to the plasma membrane (Johnson et al., The Plant Cell, (1990) 2: 525-532; Saue).
r et al., EMBO J. (1990) 9: 3045-3050; Mueckler.
, Science, (1985) 229: 941-945). Mitochondrial, nuclear, chloroplast or vacuole signals direct the expressed protein to the corresponding organelle via the secretory pathway (Von Heijne).
, Eur. J. Biochem. (1983) 133: 17-21; Yon H.
eijne, J. Mol. Biol. (1986) 189: 239-242; I
Turriaga et al., The Plant Cell, (1989) 1: 381.
~ 390; McKnight et al., Nucl. Acid Res. (1990) 1
8: 4939-4949; Matsuoka and Nakamuma, Proc.
． Natl. Acad. Sci. USA, (1991) 88: 834-838). Recent books by Cunningham and Porter (R
ecobinant proteins from plants, editor: C
． Cunningham and A.M. J. R. Porter, 1998, Hum
ana Press Totowa, N.M. J. ) Describes methods for producing recombinant proteins in plants and targeting proteins to various compartments in plant cells. In particular, two of those chapters (Chapter 14 and 15)
Various methods have been described for introducing targeting sequences that cause the accumulation of recombinant proteins in compartments such as R, vacuole, plastid, nucleus and cytoplasm. C
This book by unningham and Porter is incorporated herein by reference. Currently, the preferred accumulation site for the fusion protein according to the invention is
An ER using a signal peptide such as the signal peptide of Cel1 or rice amylase at the termini and an ER retention peptide (HDEL or KDEL) at the C-terminus.

Control of Promoters and Expression: Any promoter capable of expressing the fusion protein according to the invention may be utilized for carrying out the methods of the invention (both constitutive and tissue-specific promoters). Can be used). According to a presently preferred embodiment, the promoter selected is constitutive. This is because such promoters can drive higher levels of fusion protein expression. In this regard, the present invention offers significant advantages over the teaching of US Pat. No. 5,474,925. In that patent, only tissue-specific weak promoters can be used because of the deleterious effects of the described fusion proteins on cell wall development. The reason why the present invention can utilize strong constitutive promoters lies in the method of compartmentalization and sequestration that prevents contact between the expressed fusion protein and the plant cell wall in which such a wall is developing.

CaMV35S promoter (Ode, a constitutive tissue-specific promoter)
11 et al., Nature, (1985) 313: 810-812) and the ubiquitin promoter (Christensen and Quail, Transgen).
ic research, (1996) 5: 213-218) is the most widely used constitutive promoter in plant transformation and is the preferred promoter of choice when practicing the present invention.

In maize, a protein under the control of the ubiquitin promoter accumulates preferentially in the germ within the grain (Kusnadi et al., Biotechn.
ol. Bioeng. (1998) 60: 44-52). The amylose extender (Ae) gene encoding starch branching enzyme IIb (SBEIIb) in maize is predominantly expressed in endosperm and embryo during grain development (
Kim et al., Plant. Mol. Biol. (1998) 38: 945-956.
). Starch branching enzyme (SBE) showed promoter activity after being introduced into endosperm suspension cells of corn by particle bombardment (Kim et al., Gene, (19).
98) 216: 233-243). It has been shown in transgenic wheat that the native HMW-GS gene promoter of wheat can be used to obtain high levels of expression of seed storage proteins and potentially other proteins in endosperm (Blechl and Anderson, Nat.Biotechno
l. (1996) 14: 875-9). The polygalacturonase (PG) promoter has been shown to confer high levels of maturity-specific gene expression in tomato (Nicholass et al., Plant. Mol. Biol. (19).
95) 28: 423-435). ACC oxidase promoter (Blume
And Grierson, Plant. J. (1997) 12: 731-746.
) Is a promoter derived from the ethylene pathway and shows increased expression during fruit ripening and senescence in tomato. The promoter of the tomato 3-hydroxy-3-methylglutaryl coenzyme A reductase gene accumulates at high levels 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., Bio.
chem. J. (1996) 320: 487-492). Proteins linked to chloroplast transit peptides altered protein content in seeds of transgenic soybeans and transgenic canola when expressed from seed-specific promoters (Falco et al., Biotechnolog.
y (NY), (1995) 13: 577-82). Seed-specific bean phaseolin and soybean β-conglycinin promoters are also suitable for the latter case (Keeler et al., Plant. Mol. Biol. (1997).
) 34: 15-29). Promoters expressed in plastids are also suitable in combination with plastid transformation.

Each of these promoters can be used to carry out the method according to the invention.

Therefore, the plant promoter used may be a constitutive promoter, a tissue-specific promoter, an inducible promoter or a chimeric promoter.

Examples of constitutive plant promoters include the CaMV35S and CaMV19S promoters, the FMV34S promoter, the sugarcane rod-shaped budnavirus promoter, the CsVMV promoter, the Arabidopsis (Arabidopsis).
s) ACT2 / ACT8 actin promoter, Arabidopsis ubiquitin UB
Examples include, but are not limited to, the Q1 promoter, the barley leaf thionine BTH6 promoter, and the rice actin promoter.

Examples of tissue-specific promoters include the bean phaseolin storage protein promoter, the DLEC promoter, the PHSβ promoter, the zein storage protein promoter, the soybean conglutin γ promoter, the AT2S1 gene promoter, the Arabidopsis ACT11 actin promoter, and the rape (Bra).
ssica napus) and the tomato patatin gene promoter, but are not limited thereto.

Inducible promoters are promoters that are induced, for example, by specific stimuli such as light, temperature, chemicals, sunlight, high salinity, osmotic shock, stress conditions including oxidative conditions, or in the case of pathogenicity. Which includes a light-inducible promoter derived from the pea rbcS gene, a promoter derived from the alfalfa rbcS gene, DREs, MYCs and MYBs active in sunlight.
, INT and INP active in high salinity and osmotic stress
S, prxEa, Ha hsp17.7G4 and RD21 promoters,
And hsr303J and str246C promoters active in pathogenic stress, but are not limited thereto.

Expression Tracking: Expression of fusion proteins can be monitored by a variety of methods. Qualitatively and / or quantitatively monitoring the expression of the fusion protein in plants using, for example, ELISA analysis or Western blot analysis using antibodies that specifically recognize the recombinant protein or its cellulose-binding peptide counterpart. You can Alternatively, the fusion protein can be SDS-PAGE using various staining techniques such as, but not limited to, Coomassie blue staining or silver staining.
It can be monitored by analysis. Other methods can be used to monitor the expression levels of RNA encoding the fusion protein. Such methods include RNA hybridization methods such as Northern blots and RNs.
A dot blot included.

Accordingly, the present invention provides genetically engineered or virus infected plants or cultured plant cells that express a fusion protein comprising an enzyme capable of catalyzing the oxidation of phenolic groups and a cellulose binding peptide.

According to a preferred embodiment of the present invention, the fusion peptide is compartmentalized within the cells of said plant or cultured plant cell, so that it is sequestered from the cells of said plant or cultured plant cell, and so on. If it is desired to do so, it does not interfere with development and does not result in too much expression. According to a preferred embodiment, the fusion protein is selected from the group consisting of cytoplasm, endoplasmic reticulum, Golgi apparatus, oil bodies, starch bodies, chloroplastides, chloroplasts, chromoplastides, chromophores, vacuoles, lysosomes, mitochondria and nuclei. Compartmentalized within the cell compartment.

Determination of oxidase activity and peroxidase activity: When a laccase-encoding polynucleotide is used in the method of the invention, an amount of laccase in the range of 0.02-2000 laccase units (LACU) per gram of dry lignocellulosic material. Are generally preferred. 0.02 to 200 per gram of dry lignocellulosic material when peroxidase is used
Its amount in the range of 0 peroxidase units (PODU) is generally preferred.

The measurement of oxidase (eg laccase) activity is based on the oxidation of syringaldazine to tetramethoxyazobis-methylenequinone under aerobic conditions. 1 LACU is an enzyme amount that converts 1 μM syringaldazine per minute under the following conditions: 19 μM syringaldazine, 23.2 mM acetate buffer, 30 ° C., pH 5.5, reaction time: 1 Min, shake; monitor reaction spectrophotometrically at 530 nm.

For peroxidase activity, 1 PODU is the amount of enzyme that catalyzes the conversion of 1 μmol hydrogen peroxide per minute under the following conditions: 0.88 mM hydrogen peroxide, 1.67 mM 2,2 ′. -Azinobis (3-ethylbenzothioazoline-
6-sulfonate), 0.1 M phosphate buffer, pH 7.0, incubation at 30 ° C .; reaction is monitored photometrically at 418 nm.

Binding of fusion protein to plant-derived cellulosic material: When sufficient expression is detected, binding of fusion protein to plant-derived cellulosic material is performed. Such binding can be achieved, for example, as follows.
Whole plants, plant-derived tissues or cultured plant cells are homogenized by mechanical methods in the presence or absence of buffers such as, but not limited to, PBS. Thus, the fusion protein is offered the opportunity to bind to plant-derived cellulosics. Non-specific proteins can be washed from the cellulosic material using buffers that can contain salts and / or detergents in optimal concentrations.

Thus, further according to the invention there is provided a cell wall preparation derived from a genetically engineered plant or virus infected plant or cultured plant cells expressing a fusion protein comprising an enzyme capable of catalyzing the oxidation of phenolic groups and a cellulose binding peptide. Compositions are provided that include. In this case, the fusion protein is immobilized on the cellulose in the cell wall preparation via the cellulose-binding peptide.

Oxidizing agent: The enzyme (s) and oxidizing agent (s) used in the method of the invention must obviously be combined with each other, and the oxidizing agent in question is the binding process. It is clearly preferred to participate only in the oxidative reactions involved in and not otherwise have any detrimental effect on the substrates / materials involved in the process.

Oxidases, such as laccases, are well suited within the scope of the invention as they catalyze the oxidation by molecular oxygen, among other reasons. Thus, reactions carried out in a vessel open to the atmosphere and containing oxidase as an enzyme can utilize atmospheric oxygen as an oxidant. However, it may be desirable to force aeration of the reaction medium during the reaction to ensure a sufficient oxygen supply.

In the case of peroxidase, hydrogen peroxide is the preferred oxidizing agent within the scope of the present invention, preferably used at a concentration (in the reaction medium) in the range 0.01 mM to 100 mM.

PH in Reaction Medium: Above all, depending on the characteristics of the enzyme used, the pH in the aqueous medium (reaction medium) in which the reaction of the present invention is carried out is in the range of 3 to 10, preferably 4 to 9. It is a range.

General Procedures: In general, the terminology used herein and the laboratory procedures utilized in carrying out the present invention include molecular, biochemical, microbiological and Includes recombinant DNA technology. Such techniques are explained fully in the literature. For example,
See below: "Molecular Cloning: A laboratory.
"Tory Manual", Sambrook et al. (1989); "Curren
t Protocols in Molecular Biology ", No. I
Volume-Volume III, Ausubel, R .; M. Ed. (1994); Ausubel et al., “Current Protocols in Molecular Bio”.
LOGY ", John Wiley and Sons, Baltimore,
Maryland (1989); Perbal, "A Practical G.
"ide to Molecular Cloning", John Wile
y & Sons, New York (1988); Watson et al., "Recom
"binant DNA", Scientific American Book
s, New York; Birren et al. (ed.), “Genome Analysis
is: A Laboratory Manual Series ", Volumes 1 to 4, Cold Spring Harbor Laboratory Pre.
ss, New York (1998); U.S. Pat. Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272. , 057; "Cell Biolog.
y: A Laboratory Handbook ", Volumes I-III, C
ellis, J .; E. Hen (1994); "Current Protocols
in Immunology ", Volumes I-III, Celigan J. et al.
E. Hen (1994); Statuses et al. (Hen), "Basic and Clin"
"Inal Immunology" (8th Edition), Appleton & Lange
, Norwalk, CT (1994); Michel and Shiigi (eds.), "Selected Methods in Cellular Immu."
noology ", W.N. H. Freeman and Co. , New York
(1980); available immunoassays are described in detail in the patent and scientific literature, eg, US Pat. Nos. 3,791,932 and 3,839,153.
No. 3, No. 3,850,752, No. 3,850,578, No. 3,853.
No. 987, No. 3,867,517, No. 3,879,262, No. 3,9.
No. 01,654, No. 3,935,074, No. 3,984,533, No. 3,996,345, No. 4,034,074, No. 4,098,876, No. 4,879,219, No. 5,011,771 and No. 5,281.
, 521; "Oligonucleotide Synthes.
is ", Gait, M .; J. Hen (1984), "Nucleic Acid H
ybridization ", Hames, B .; D. And Higgins S
． J. Hen (1985); "Transscription and Transl
", Hames, B .; D. And Higgins S. et al. J. Hen (19
84); "Animal Cell Culture", Freshney, R.
． I. Hen (1986); "Immobilized Cells and En
zymes, "IRL Press (1986);" A Practical
Guide to Molecular Cloning ", Perbal, B
． (1984) and "Methods in Enzymology", No. 1.
-317, Academic Press; "PCR Protocols:
A Guide to Methods And Applications "
, Academic Press, San Diego, CA (1990); M.
Arshak et al., "Strategies for Protein Puri"
fication and Characterization-A Labo
rally Course Manual ", CSHL Press (199
6). All of these are incorporated by reference as if set forth herein in their entirety. Other general procedures are provided herein. The procedures herein are considered well known in the art and are presented for the convenience of the reader. All the information contained therein is incorporated herein by reference.

Product by Method: The present invention also relates to a lignocellulosic product obtainable by the method according to the invention disclosed herein.

Although the present invention has been described with particular embodiments thereof, it will be apparent that many alternatives, modifications and variations will be apparent to those of ordinary skill in the art. Accordingly, the present invention is intended to embrace all such alterations, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications cited herein are incorporated by reference in their entirety.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW F term (reference) 2B030 AA02 AB03 AD08 CA17 CA19                       CB03                 4B024 AA03 BA08 BA80 CA07 DA01                       EA04 FA02 FA04 FA18 FA20                       GA11                 4B065 AA57Y AA71Y AA88X AA88Y                       AA95X AB01 AC14 BA02                       CA24 CA28 CA60                 4L055 AB12 AB15 AB19 AB20 AC01                       AC05 BA39 BA40 EA03 EA20                       EA31 EA32 FA13 GA05 GA06

Claims (38)

[Claims] 1. A method for producing a lignocellulosic product comprising: (i) a phenolic polymer substituted with phenolic hydroxy groups in a reaction medium; (ii) catalyzing the oxidation of phenolic groups. A method comprising the step of mixing a lignocellulose-containing material in which a fusion polypeptide comprising the enzyme to be obtained and a cellulose-binding peptide is immobilized in its cellulose portion; and (iii) an oxidizing agent. 2. The method of claim 1, wherein the lignocellulosic product is selected from the group consisting of fiberboard, particleboard, flakeboard, plywood and molded composites. 3. The method of claim 1, wherein the lignocellulosic product is selected from the group consisting of paper and paperboard. 4. The method of claim 1, wherein the lignocellulose-containing material is a cell wall preparation derived from genetically engineered plants or virus infected plants or cultured plant cells expressing the fusion protein. 5. The method of claim 1, wherein the lignocellulose-containing material is selected from the group consisting of plant fibers and wood fibers derived from genetically engineered plants or virus infected plants expressing the fusion polypeptide. 6. The phenolic substituent is p-coumaric acid, p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol, ferulic acid and p.
A method according to claim 1 selected from the group consisting of: -hydroxybenzoic acid. 7. The method of claim 1, wherein the phenolic polymer forms a constituent part of the lignocellulose-containing material. 8. The method of claim 7, wherein the phenolic polymer is lignin. 9. The method of claim 1, wherein the phenolic polymer is a phenolic polysaccharide. 10. The polysaccharide portion of the phenolic polysaccharide is selected from the group consisting of modified and non-modified starch, modified and non-modified cellulose, and modified and non-modified hemicellulose. Item 9. The method according to Item 9. 11. The method of claim 9, wherein the phenolic polysaccharide is selected from the group consisting of ferryl arabinoxylan and ferryl pectin. 12. The method of claim 1, wherein the reaction medium is incubated for 1 minute to 10 hours. 13. The method of claim 12, wherein the fusion polypeptide is incubated in the presence of the oxidizing agent for 1 minute to 10 hours. 14. The method of claim 1, wherein the enzyme is selected from the group consisting of oxidases and peroxidases. 15. The enzyme is laccase (EC1.10.3.2), catechol oxidase (EC1.10.3.1) and bilirubin oxidase (EC).
The method of claim 1, wherein the oxidizing agent is oxygen and is selected from the group consisting of EC 1.3.3.5). 16. The method of claim 15, wherein the enzyme is laccase and is present in an amount in the range of 0.02 LACU to 2000 LACU per gram of dry lignocellulose. 17. The method of claim 15, wherein the reaction medium is vented. 18. The enzyme is the genus Botrytis, the genus Myceliophthora, or the tranche, Tram.
etes) fungi or plant maple (Acer pseudoplanus)
The method according to claim 15, which is a laccase encoded by the polynucleotide obtained from 19. The fungus is Trametes Trametes.
Versicolor or Trametes virosa
19. The method of claim 18, which is llosa). 20. The method of claim 1, wherein the enzyme is peroxidase and the oxidizing agent is hydrogen peroxide. 21. The peroxidase is present in an amount in the range of 0.02 PODU to 2000 PODU per gram of dry lignocellulose, and the initial concentration of hydrogen peroxide in the reaction medium is in the range of 0.01 mM to 100 mM. Item 21. The method according to Item 20. 22. The method according to claim 1, wherein the amount of lignocellulose used corresponds to 0.1% to 90% by weight of the reaction medium, calculated as dry lignocellulose. 23. The method of claim 1, wherein the temperature of the reaction medium is in the range of 10 ° C to 120 ° C. 24. The method according to claim 23, wherein the temperature of the reaction medium is in the range of 15 ° C to 90 ° C. 25. The method according to claim 1, wherein the amount of the phenolic polymer is in the range of 0.1% to 10% by weight. 26. The method of claim 1, wherein the pH of the reaction medium is in the range of 3-10. 27. The method according to claim 26, wherein the pH of the reaction medium is in the range of 4-9. 28. The method of claim 1, wherein the reaction medium further comprises a lignocellulosic inclusion without the fusion protein. 29. The lignocellulose-containing material without the fusion protein is selected from the group consisting of plant fibers, wood fibers, wood chips, wood flakes, wood veneers and recycled fibers. the method of. 30. A lignocellulosic product obtainable by the method of claim 1. 31. A genetically engineered plant or virus infected plant or cultured plant cell expressing a fusion protein comprising an enzyme capable of catalyzing the oxidation of a phenolic group and a cellulose-binding peptide. 32. The gene according to claim 31, wherein said fusion protein is compartmentalized within the cell of said plant or cultured plant cell and is thus isolated from the cell wall of said cell of said plant or cultured plant cell. Manipulated plants or virus infected plants or cultured plant cells. 33. A genetically engineered plant or virus infected plant or cultured plant cell according to claim 31, wherein expression of said fusion protein is under the control of a constitutive or tissue-specific plant promoter. 34. The fusion protein is selected from the group consisting of cytoplasm, endoplasmic reticulum, Golgi apparatus, oil bodies, starch bodies, chloroplastides, chloroplasts, chromoplastides, chromophores, vacuoles, lysosomes, mitochondria and nuclei. 33. The genetically engineered plant or virus infected plant or cultured plant cell of claim 32, which is compartmentalized within a cell compartment that is 35. A composition comprising a cell wall preparation derived from a genetically engineered plant or virus infected plant or cultured plant cells expressing a fusion protein comprising an enzyme capable of catalyzing the oxidation of phenolic groups and a cellulose binding peptide. And a composition in which the fusion protein is immobilized on cellulose in the cell wall preparation via the cellulose-binding peptide. 36. (a) A promoter sequence that drives protein expression in plant cells; and (b) (i) a first sequence encoding a cellulose-binding peptide; (ii) capable of catalyzing the oxidation of a phenolic group. A nucleic acid molecule comprising a heterologous nucleic acid sequence comprising a second sequence encoding an enzyme, wherein the first sequence and the second sequence are linked in an open reading frame. 37. An origin of replication required for propagation in bacterial cells, at least one sequence element required for integration into the plant genome, a polyadenylation recognition sequence,
A transcription termination signal, a sequence encoding a translation start site, a sequence encoding a translation stop site, a sequence derived from a plant RNA virus, a sequence derived from a plant DNA virus, a sequence derived from a tumor-inducing (Ti) plasmid, a transfer element 37. The nucleic acid molecule of claim 36, further comprising a sequence of origin and a sequence element selected from the group consisting of a functional plant signal peptide that directs the protein to the cell compartment of a plant cell. 38. The cell compartment is selected from the group consisting of cytoplasm, endoplasmic reticulum, Golgi apparatus, oil bodies, starch bodies, chloroplastides, chloroplasts, chromoplastides, chromophores, vacuoles, lysosomes, mitochondria and nuclei. 37. The nucleic acid molecule according to claim 36, which is