Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
  • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
  • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
  • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B15/00—Preparation of other cellulose derivatives or modified cellulose, e.g. complexes
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B15/00—Preparation of other cellulose derivatives or modified cellulose, e.g. complexes
  • C08B15/10—Crosslinking of cellulose
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B31/00—Preparation of derivatives of starch
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• • D—TEXTILES; PAPER
  • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
  • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
  • D06M15/00—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment
  • D06M15/01—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment with natural macromolecular compounds or derivatives thereof
  • D06M15/15—Proteins or derivatives thereof
• • D—TEXTILES; PAPER
  • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
  • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
  • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
  • D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C9/00—After-treatment of cellulose pulp, e.g. of wood pulp, or cotton linters ; Treatment of dilute or dewatered pulp or process improvement taking place after obtaining the raw cellulosic material and not provided for elsewhere
  • D21C9/001—Modification of pulp properties
  • D21C9/002—Modification of pulp properties by chemical means; preparation of dewatered pulp, e.g. in sheet or bulk form, containing special additives
  • D21C9/005—Modification of pulp properties by chemical means; preparation of dewatered pulp, e.g. in sheet or bulk form, containing special additives organic compounds
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
  • D21H17/00—Non-fibrous material added to the pulp, characterised by its constitution; Paper-impregnating material characterised by its constitution
  • D21H17/005—Microorganisms or enzymes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention provides a process and compositions for producing a lignocellulose-based product, e.g., fiber board, such as hardboard or medium-density fiber board ("MDF"), particle board, plywood, paper or paperboard (such as cardboard and linerboard), from an appropriate lignocellulosic starting material, such as wood fiber or vegetable fiber, having an enzyme adhered thereto via a cellulose binding peptide, which enzyme is capable of catalyzing the oxidation of phenolic groups of a phenolic polymer which may fonri an integral part of the lignocellulosic starting material, e.g., lignin, in the presence of an oxidizing agent and optionally in the presence of additional lignocellulosic starting material devoid of the enzyme, e.g., recycled fibers.
• fiber board such as hardboard or medium-density fiber board (“MDF"), particle board, plywood, paper or paperboard (such as cardboard and linerboard
• MDF medium-den
• lignocellulose-based products prepared thereby especially paper products such as liner board, cardboard and corrugated board.
• Lignocellulose-based products prepared from lignocellulosic starting materials notably products manufactured starting from vegetable fiber or wood fiber prepared by mechanical or mechanical/chemical procedures (the latter often being denoted “semi-chemical” procedures), or by a chemical procedure without bleaching, or from wood particles (wood “chips", flakes and the like), are indispensable everyday materials.
• Some of the most familiar types of such products include paper for writing or printing, cardboard, corrugated cardboard, fiber board (e.g. "hardboard”), and particle board.
• binding of the wood fibers or particles to give a coherent mass exhibiting satisfactory strength properties can be achieved using a process in which the fibers/particles are treated - optionally in a mixture with one or more "extenders", such as lignosulfonates and/or kraft lignin - with synthetic adhesives (typically adhesives of the urea- formaldehyde, phenol-formaldehyde or isocyanate type) and then pressed into the desired form (boards, sheets, panels etc.) with the application of heat.
• "extenders" such as lignosulfonates and/or kraft lignin - with synthetic adhesives (typically adhesives of the urea- formaldehyde, phenol-formaldehyde or isocyanate type) and then pressed into the desired form (boards, sheets, panels etc.) with the application of heat.
• EP 0 433 258 Al discloses a procedure for the production of mechanical pulp from a fibrous product using a chemical and/or enzymatic treatment in which a "binding agent" is linked with the lignin in the fibrous product via the formation of radicals on the lignin part of the fibrous product.
• a "binding agent” is linked with the lignin in the fibrous product via the formation of radicals on the lignin part of the fibrous product.
• hydrocarbonates such as cationic starch, and/or proteins as examples of suitable binding agents.
• suitable enzymes are mentioned laccase, lignin peroxidase and manganese peroxidase, and as examples of suitable chemical agents are mentioned hydrogen peroxide with ferro ions, chlorine dioxide, ozone, and mixtures thereof.
• EP 0 565 109 Al discloses a method for achieving binding of mechanically produced wood fragments via activation of the lignin in the middle lamella of the wood cells by incubation with phenol-oxidizing enzymes. The use of a separate binder is thus avoided by this method.
• U.S. Pat. No. 4,432,921 describes a process for producing a binder for wood products from a phenolic compound having phenolic groups, and the process in question involves treating the phenolic compound with enzymes to activate and oxidatively polymerize the phenolic compound, thereby converting it into the binder.
• phenolic compounds which are specifically mentioned in this document, or employed in the working examples given therein, are lignin sulfonates, and a main purpose of the invention described in U.S. Pat. No. 4,432,921 is the economic exploitation of so-called "sulfite spent liquor", which is a liquid waste product produced in large quantities through the operation of the widely-used sulfite process for the production of chemical pulp, and which contains lignin sulfonates.
• the amount of binder required to prepare lignocellulose-based products of very satisfactory strength by the process described in U.S. Pat. No. 5,846,788 is generally much lower typically by a factor of about three or more - than the level of binder (based on lignin sulfonate) required to obtain comparable strength properties using the process according to U.S. Pat. No. 4,432,921.
• the process according to U.S. Pat. No. 5,846,788 can thus not only provide an environmentally attractive alternative to more traditional binding processes employing synthetic adhesives, but it can probably also compete economically with such processes.
• a process for the manufacture of a lignocellulose product comprising the step of mixing in a reaction medium (i) a phenolic polymer being substituted with a phenolic hydroxy group; (ii) a lignocellulose containing material having immobilized to a cellulosic fraction thereof a fusion polypeptide, the fusion polypeptide including an enzyme being capable of catalyzing the oxidation of phenolic groups and a cellulose binding peptide; and (iii) an oxidizing agent.
• the lignocellulose product is selected from the group consisting of fiber board, particle board, flakeboard, plywood and molded composites.
• the lignocellulose product is selected from the group consisting of paper and paperboard.
• the lignocellulose containing material is a cell wall preparation derived from a genetically modified or virus infected plant or cultured plant cells expressing the fusion protein.
• the lignocellulose containing material is selected from the group consisting of vegetable fiber and wood fiber derived from a genetically modified or virus infected plant expressing the fusion polypeptide.
• the lignocellulose containing material is selected from the group consisting of vegetable fiber and wood fiber that has previously made contact with an oxidising enzyme fused to a cellulose binding peptid.
• the phenolic substituent is selected from the group consisting of p-coumaric acid, p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol, ferulic acid p-hydroxybenzoic acid and any other phenolic group that can be oxidized.
• the phenolic polymer forms an integral part of the lignocellulose containing material.
• the phenolic polymer is lignin.
• the phenolic polymer is a phenolic polysaccharide.
• the polysaccharide portion of the phenolic polysaccharide is selected from the group consisting of modified and unmodified starches, modified and unmodified cellulose, and modified and unmodified hemicelluloses.
• the phenolic polysaccharide is selected from the group consisting of ferulylated arabinoxylans and ferulylated pectins.
• reaction medium is incubated for a period of from 1 minute to 10 hours.
• the fusion polypeptide is incubated in the presence of the oxidizing agent for a period of from 1 minute to 10 hours.
• the enzyme is selected from the group consisting of oxidases and peroxidases.
• the enzyme is an oxidase selected from the group consisting of laccases (EC 1.10.3.2), catechol oxidases (EC 1.10.3.1) and bilirubin oxidases (EC 1.3.3.5), and the oxidizing agent is oxygen.
• the enzyme is a laccase and is present in an amount in the range of 0.02-2000 LACU per g of dry lignocellulose.
• reaction medium is aerated.
• the enzyme is a laccase encoded by a polynucleotide obtained from a fungus of the genus Botrytis, Myceliophthora, or Trametes.
• the fungus is Trametes versicolor or Trametes villosa.
• the enzyme is a laccase from Acer pseudoplanus.
• the enzyme is a peroxidase and the oxidizing agent is hydrogen peroxide.
• the peroxidase is present in an amount in the range of 0.02- 2000 PODU per g of dry lignocellulose, and the initial concentration of hydrogen peroxide in the reaction medium is in the range of 0.01-100 mM.
• the amount of lignocellulose employed corresponds to 0.1-90 % by weight of the reaction medium, calculated as dry lignocellulose.
• the temperature of the reaction medium is in the range of 10 ° - 120 ° C.
• the temperature of the reaction medium is in the range of 15 ° - 90 ° C.
• an amount of the phenolic polysaccharide in the range of 0.1 % - 10 % by weight.
• the pH in the reaction medium is in the range of 3-10.
• the pH in the reaction medium is in the range of 4-9.
• reaction medium further comprising a lignocellulose containing material devoid of the fusion protein.
• the lignocellulose containing material devoid of the fusion protein is selected from the group consisting of vegetable fiber, wood fiber, wood chips, wood flakes, wood veneer and recycled fibers.
• a lignocellulose product obtainable by the process described herein.
• a genetically modified or viral infected plant or cultured plant cells expressing a fusion protein including an enzyme being capable of catalyzing the oxidation of phenolic groups and a cellulose binding peptide.
• the fusion protein being compartmentalized within cells of the plant or cultured plant cells, so as to be sequestered from cell walls of the cells of the plant or cultured plant cells.
• expression of the fusion protein is under a control of a constitutive or tissue specific plant promoter.
• the fusion protein is compartmentalized within a cellular compartment selected from the group consisting of cytoplasm, endoplasmic reticulum, golgi apparatus, oil bodies, starch bodies, chloroplastids, chloroplasts, chromoplastids, chromoplasts, vacuole, lysosomes, mitochondria, and nucleus.
• composition of matter comprising a cell wall preparation derived from a genetically modified or virus infected plant or cultured plant cells expressing a fusion protein including an enzyme being capable of catalyzing the oxidation of phenolic groups and a cellulose binding peptide, the fusion protein being immobilized to cellulose in the cell wall preparation via the cellulose binding peptide.
• nucleic acid molecule comprising (a) a promoter sequence for directing protein expression in plant cells; and (b) a heterologous nucleic acid sequence including (i) a first sequence encoding a cellulose binding peptide; and (ii) a second sequence encoding an enzyme being capable of catalyzing the oxidation of phenolic groups, wherein the first and second sequences are joined together in frame.
• the nucleic acid molecule further comprising a sequence element selected from the group consisting of an origin of replication for propagation in bacterial cells, at least one sequence element for integration into a plant's genome, a polyadenylation recognition sequence, a transcription termination signal, a sequence encoding a translation start site, a sequence encoding a translation stop site, plant RNA virus derived sequences, plant DNA virus derived sequences, tumor inducing (Ti) plasmid derived sequences, a transposable element derived sequence and a plant operative signal peptide for directing a protein to a cellular compartment of a plant cell.
• a sequence element selected from the group consisting of an origin of replication for propagation in bacterial cells, at least one sequence element for integration into a plant's genome, a polyadenylation recognition sequence, a transcription termination signal, a sequence encoding a translation start site, a sequence encoding a translation stop site, plant RNA virus derived sequences, plant DNA virus derived sequence
• the cellular compartment is selected from the group consisting of cytoplasm, endoplasmic reticulum, golgi apparatus, oil bodies, starch bodies, chloroplastids, chloroplasts, chromoplastids, chromoplasts, vacuole, lysosomes, mitochondria, and nucleus.
• the present invention successfully addresses the shortcomings of the presently known configurations by providing a process and compositions for producing a lignocellulose-based product which obviates the need for purified enzymes which are expensive materials as is compared to other raw materials and reagents described in the prior art for use in the process of manufacturing lignocellulose-based products.
• the present invention is of a process and composition of matter for the manufacture of a lignocellulose-based product from a lignocellulosic material, which process obviates the need for using purified enzymes.
• the present invention thus provides a process for the manufacture of a lignocellulose-based product from a lignocellulosic material.
• the process according to the present invention is effected by mixing in a reaction medium (i) a phenolic polymer substituted with a phenolic hydroxy group (e.g., lignin or a polysaccharide which is substituted with at least substituents containing a phenolic hydroxy group); (ii) a lignocellulose containing material having immobilized to a cellulosic fraction thereof a fusion polypeptide, the fusion polypeptide including an enzyme being capable of catalyzing the oxidation of phenolic groups and a cellulose binding peptide; and (iii) an oxidizing agent.
• a reaction medium e.g., a phenolic polymer substituted with a phenolic hydroxy group (e.g., lignin or a polysaccharide which is substituted with at least substitu
• the order of mixing/contacting the three components is unimportant as long as the process set-up ensures that the activated lignocellulosic material and the activated phenolic polysaccharide are brought together in a way that enables them to react in the desired manner.
• the oxidizing agent may be mixed with the lignocellulose containing material before or after being mixed with the phenolic polymer.
• the lignocellulose containing material is preferably a cell wall preparation derived from a genetically modified or virus infected plant or cultured plant cells expressing the fusion protein.
• the phenolic polymer may form an integral part of the lignocellulose containing material because cell walls of plants contain lignin which is a phenolic polymer and thus the cell wall preparation can be made to contain lignin.
• the cell wall preparation may be kept under a non-oxidizing atmosphere, such as an N2 atmosphere.
• reaction medium containing the three components It will generally be appropriate to incubate the reaction medium containing the three components for a period of at least a few minutes.
• An incubation time of from 1 minute to 10 hours will generally be suitable, although a period of from 1 minute to 10 hours is preferable.
• the process of the invention is well suited to the production of all types of lignocellulose-based products, e.g., various types of fiber board (such as hardboard), particle board, flakeboard, such as oriented-strand board (OSB), plywood, molded composites (e.g., shaped articles based on wood particles, often in combination with other, non- lignocellulosic materials, e.g., certain plastics), paper and paperboard (such as cardboard, linerboard and the like).
• fiber board such as hardboard
• particle board such as oriented-strand board (OSB)
• OSB oriented-strand board
• plywood molded composites (e.g., shaped articles based on wood particles, often in combination with other, non- lignocellulosic materials, e.g., certain plastics)
• paper and paperboard such as cardboard, linerboard and the like.
• the lignocellulose containing material employed in the method of the invention can be in any appropriate form, e.g., in the form of vegetable fiber (such as wood fiber) with the provision that it is derived from a genetically modified or virus infected plant expressing the fusion polypeptide.
• a lignocellulosic material can be used in combination with a non-lignocellulosic material having phenolic hydroxy functionalities.
• intermolecular linkages between the lignocellulosic material and the non-lignocellulosic material, respectively may then be formed (i.e., in a manner analogous to that in which intermolecular linkages are formed when lignocellulosic materials alone are employed in the process), resulting in a composite product.
• the phenolic polysaccharide also serves as a good "gap-filler", which is a big advantage when producing, e.g., particle boards from large wood particles.
• lignocellulosic material in question in an amount corresponding to a weight percentage of dry lignocellulosic material [dry substance (DS)] in the reaction medium in the range of 0.1 -90 %.
• the temperature of the reaction mixture in the process of the invention may suitably be in the range of 10 °C - 120 °C, as appropriate; however, a temperature in the range of 15 °C - 90 °C is generally to be preferred. As illustrated by the working examples described in U.S. Pat. No. 5,846,788, it is anticipated that the reactions involved in a process of the invention may take place very satisfactorily at ambient temperatures around 20 °C.
• the reaction medium according to the present invention may include a lignocellulose containing material devoid of such fusion protein, such as, but not limited to, vegetable fiber, wood fiber, wood chips, wood flakes, wood veneer and recycled fibers.
• a lignocellulose containing material devoid of such fusion protein such as, but not limited to, vegetable fiber, wood fiber, wood chips, wood flakes, wood veneer and recycled fibers.
• Phenolic Polymers The phenolic polymers employed in the process of the invention may suitably be materials obtainable from natural sources or polymers which have been chemically modified by the introduction of substituents having phenolic hydroxy groups.
• modified starches containing phenolic substituents e.g., acyl-type substituents derived from hydroxy-substituted benzoic acids (such as, e.g., 2-, 3- or 4- hydroxybenzoic acid).
• phenolic substituent(s) in phenolic polysaccharides suited for use in the context of the present invention may suitably be linked to the polymer species by, e.g., ester linkages or ether linkages.
• Very suitable phenolic polymers are phenolic polysaccharides in which the phenolic substituent of the phenolic polysaccharide is a substituent derived from a phenolic compound which occurs in at least one of the following plant-biosynthetic pathways: from p-coumaric acid to p- coumaryl alcohol, from p-coumaric acid to coniferyl alcohol and from p- coumaric acid to sinapyl alcohol; p-coumaric acid itself and the three mentioned "end products" of the latter three biosynthetic pathways are also relevant compounds in this respect.
• relevant "intermediate" compounds formed in these biosynthetic pathways include caffeic acid, ferulic acid (i.e., 4-hydroxy-3-methoxycinna
• Particularly suitable phenolic polysaccharides are those which exhibit good solubility in water, and thereby in aqueous media in the context of the invention.
• phenolic polysaccharides which are readily obtainable in uniform quality from vegetable sources have been found to be particularly well-suited for use in the process of the present invention. These include, but are in no way limited to, phenolic arabino and heteroxylans, and phenolic pectins.
• ferulylated arabinoxylans obtainable, e.g., from wheat bran or maize bran
• ferulylated pectins obtainable from, e.g., beet pulp
• arabinoxylans and pectins containing ferulyl substituents attached via ester linkages to the polysaccharide molecules are particularly suitable examples thereof.
• the amount of phenolic polysaccharide or other phenolic polymers, such as lignin, employed in the process of the invention will generally be in the range of 0.01-10 weight percent, based on the weight of lignocellulosic material (calculated as dry lignocellulosic material), and amounts in the range of about 0.02-6 weight per cent (calculated in this manner) will often be very suitable. Enzymes and poiynucleotides encoding same
• any type of enzyme capable of catalyzing oxidation of phenolic groups may be employed in the process of the invention, with the provision that a polynucleotide encoding same has been isolated or is readily isolateable using conventional genetic engineering isolation techniques and which can therefore be expressed as a part of a fusion polypeptide.
• Preferred enzymes are, however, oxidases, e.g., laccases (EC 1.10.3.2), catechol oxidases (EC 1.10.3.1) and bilirubin oxidases (EC 1.3.3.5) and peroxidases (EC 1.1 1.1.7). In some cases it may be appropriate to employ two or more different enzymes in the process of the invention.
• laccases have proved to be well suited for use in the method of the invention.
• Poiynucleotides encoding laccases have been or are readily isolateable from a variety of plant and microbial sources, notably bacteria and fungi (including filamentous fungi and yeasts), see, for example, U.S. Pat. Nos. 5,843,745; 5,795,760; 5,770,418; and 5,750,388, which are incorporated herein by reference.
• Suitable examples of poiynucleotides encoding laccases include those obtained or obtainable from strains of Aspergillus, Neurospora (e.g., N.
• thermophila Schytalidium, Phlebia (e.g. P. radita; see WO 92/01046), or Coriolus (e.g., C. hirsutus; see JP 2-238885,).
• a preferred laccase in the context of the invention is that obtainable from Trametes villosa or Acer pseudoplanus.
• Poiynucleotides encoding peroxidase enzymes (EC 1.1 1.1) employed in the method of the invention are preferably those obtained or obtainable from plants (e.g., horseradish peroxidase or soy bean peroxidase) or from microorganisms, such as fungi or bacteria.
• some preferred fungi include strains belonging to the sub-division Deuteromycotina, class Hyphomycetes, e.g., Fusarium, Humicola, Tricoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia, Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672,), Humicola insolens, Trichoderma resii, Myrothecium verrucana (IFO 61 13,), Verticillum alboatrum, Verticillum dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alii or Dreschlera halodes.
• class Hyphomycetes e.g., Fusarium, Humicola, Tricoderma, Myrothecium, Verticillum, Arthro
• fungi include strains belonging to the sub-division Basidiomycotina, class Basidiomycetes, e.g., Coprinus, Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f. microsporus (IFO 8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g., ⁇ N-12) or Trametes versicolor (e.g. PR4 28-A).
• Basidiomycotina class Basidiomycetes
• Coprinus cinereus f. microsporus IFO 8371
• Coprinus macrorhizus Coprinus macrorhizus
• Phanerochaete chrysosporium e.g., ⁇ N-12
• Trametes versicolor e.g. PR4 28-A.
• fungi include strains belonging to the sub-division Zygomycotina, class Mycoraceae, e.g., Rhizopus or Mucor, in particular Mucor hiemalis.
• Some preferred bacteria include strains of the order Nctinomycetales, e.g., Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382) or Streptoverticillum verticillium ssp. verticillium.
• Streptomyces spheroides e.g., Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382) or Streptoverticillum verticillium ssp. verticillium.
• Bacillus pumilus ATCC 12905
• Bacillus stearothermophilus Rhodobacter sphaeroides
• Rhodomonas palustri Streptococcus lactis
• Pseudomonas purrocinia ATCC 15958
• Pseudomonas fluorescens NRRL B-l 1).
• Further preferred bacteria include strains belonging to Myxococcus, e.g., M. virescens.
• Other potential sources of useful sources for poiynucleotides encoding peroxidases are listed in B. C. Saunders et al., Peroxidase, London 1964, pp. 41-43.
• cellulose binding peptide includes peptides e.g., proteins and domains (portions) thereof, which are capable of, when expressed in plant cells, affinity binding to a plant derived cellulosic (e.g., lignocellulosic) matter, e.g., following homogenization and cell rupture or during plant growth and development.
• the phrase thus includes, for example, peptides which were screened for their cellulose binding activity out of a library, such as a peptide library or a DNA library (e.g., a cDNA expression library or a display library) and the genes encoding such peptides isolated and are expressible in plants.
• the phrase also includes peptides designed and engineered to be capable of binding to cellulose and/or units thereof.
• Such peptides include amino acid sequences expressible in plants that are originally derived from a cellulose binding region of, e.g., a cellulose binding protein (CBP) or a cellulose binding domain (CBD).
• CBP cellulose binding protein
• CBD cellulose binding domain
• the cellulose binding peptide according to the present invention can include any amino acid sequence expressible in plants which binds to a cellulose polymer.
• the cellulose binding domain or protein can be derived from a cellulase, a binding domain of a cellulose binding protein or a protein screened for, and isolated from, a peptide library, or a protein designed and engineered to be capable of binding to cellulose or to saccharide units thereof, and which is expressible in plants.
• the cellulose binding domain or protein can be naturally occurring or synthetic, as long as it is expressible in plants. Suitable polysaccharidases from which a cellulose binding domain or protein expressible in plants may be obtained include ⁇ -l,4-glucanases.
• a cellulose binding domain or protein from a cellulase or scaffoldin is used.
• the amino acid sequence of the cellulose binding peptide expressed in plants according to the present invention is essentially lacking in the hydrolytic activity of a polysaccharidase (e.g., cellulase, chitinase), but retains the cellulose binding activity.
• the amino acid sequence preferably has less than about 10 % of the hydrolytic activity of the native polysaccharidase; more preferably less than about 5 %, and most preferably less than about 1 % of the hydrolytic activity of the native polysaccharidase, ideally no activity altogether.
• the cellulose binding domain or protein can be obtained from a variety of sources, including enzymes and other proteins which bind to cellulose which find use in the subject invention.
• binding domains which bind to one or more soluble/insoluble polysaccharides including all binding domains with affinity for soluble glucans ( ⁇ , ⁇ , and/or mixed linkages).
• the Nl cellulose-binding domain from endoglucanase CenC of C. fimi is the only protein known to bind soluble cellosaccharides and one of a small set of proteins which are known to bind any soluble polysaccharides.
• Tables 1 to 3 are examples of proteins containing putative ⁇ -l,3-glucan- binding domains (Table 1); proteins containing Streptococcal glucan- binding repeats (Cpl superfamily) (Table 2); and enzymes with chitin- binding domains, which may also bind cellulose (Table 3).
• the genes encoding each one of the peptides listed in Tables 1 -4 are either isolated or can be isolated as further detailed hereinunder, and therefore, such peptides are expressible in plants.
• Scaffoldin proteins or portions thereof, which include a cellulose binding domain such as that produced by Clostridium cellulovorans (Shoseyov et al, PCT/US94/04132) can also be used as the cellulose binding peptide expressible in plants according to the present invention.
• Clostridium cellulovorans Shoseyov et al, PCT/US94/04132
• Several fungi, including Trichoderma species and others also produce polysaccharidases from which polysaccharide binding domains or proteins expressible in plants can be isolated. Additional examples can be found in, for example, Microbial Hydrolysis of Polysaccharides, R. A. J. Warren, Annu. Rev. Microbiol. 1996, 50: 183-212; and "Advances in Microbial Physiology" R. K. Poole, Ed., 1995, Academic Press Limited, both are incorporated by reference as if fully set forth herein.
• New cellulose binding peptides with interesting binding characteristics and specificities can be identified and screened for and the genes encoding same isolated using well known molecular biology approaches combined with a variety of other procedures including, for example, spectroscopic (titration) methods such as: NMR spectroscopy (Zhu et al Biochemistry (1995) 34: 13196-13202. Gehring et al Biochemistry (1991) 30:5524-5531), UV difference spectroscopy (Belshaw et al Eur. J. Biochem. (1993) 21 1 :717-724), fluorescence (titration) spectroscopy (Miller et al J Biol.
• spectroscopic (titration) methods such as: NMR spectroscopy (Zhu et al Biochemistry (1995) 34: 13196-13202. Gehring et al Biochemistry (1991) 30:5524-5531), UV difference spectroscopy (Belshaw et al Eur. J. Biochem. (1993) 21 1
• the K a for binding of the cellulose binding domains or proteins to cellulose is at least in the range of weak antibody-antigen extractions, i.e., > ⁇ , preferably 10 ⁇ , most preferably 10 ⁇ M" 1 . If the binding of the cellulose binding domain or protein to cellulose is exothermic or endothermic, then binding will increase or decrease, respectively, at lower temperatures, providing a means for temperature modulation of the binding step.
• Aeromonas cavia Chi U09139 Alteromonas sp (0-7) Ch ⁇ 85 A40633/P32823/D13762 Autographa californica (C6) NPH-128 3 P41684/L22858 Serratia marcescens ChiA A25090/X03657/L01455/P07254
• Rhizopus ohgosporus (IF08631 ) Chil P29026/A47022/D 10157/S27418 10 Rhizopus ohgosporus (IF08631 ) Ch ⁇ 2 P29027/B47022/D 10158/S27419 10
• Nicotiana tabacum (FB7-1) Chi JQ0993/S0828 27 Table 3 (Continued)
• Nicotiana tabacum (cv BY4) Chi P24091/X51599/X64519//S13322 26,27,29
• Phaseolus vulgaris (cv Saxa) Chi P06215/M 13968/M 19052/A25898 43,44,45
• ⁇ -glucanases avicelases, CMCases, Domains ' cellodext ⁇ nases
• exoglucanses or cellobiohydrolases cellulose binding proteins xylanases mixed xylanases/glucanases esterases chitinases ⁇ -l,3-glucanases ⁇ - 1 ,3-( ⁇ - 1 ,4)-glucanases
• Dextran Binding Domains (Streptococcal) glycosyl transferases ⁇ dextran sucrases (unconfirmed) Clostridial toxins 13, 14 glucoamylases" dextran binding proteins ⁇ -Glucan Binding Domains ⁇ -l ,3-glucanases ' ⁇ ' ° ⁇ -l,3-( ⁇ -l ,4)-glucanases (unconfirmed) ⁇ -l ,3-glucan binding protein
• polysaccharide binding peptide includes an amino acid sequence which comprises at least a functional portion of a polysaccharide binding region (domain) of a polysaccharidase or a polysaccharide binding protein.
• the phrase further relates to a polypeptide screened for its cellulose binding activity out of a library, such as a peptide library or a DNA library (e.g., a cDNA library or a display library).
• a library such as a peptide library or a DNA library (e.g., a cDNA library or a display library).
• polysaccharidase genes such as cellulase genes, and genes for cellulose binding proteins are known in the art, including synthesis, isolation from genomic DNA, preparation from cDNA, or combinations thereof.
• polysaccharidase genes such as cellulase genes, and genes for cellulose binding proteins.
• the sequences for several binding domains, which bind to soluble oligosaccharides are known (See, Figure 1 of PCT/CA97/00033, WO 97/26358).
• the DNAs coding for a variety of polysaccharidases and polysaccharide binding proteins are also known.
• the amino acid sequence of a polysaccharidase can be used to design a probe to screen a cDNA or a genomic library prepared from mRNA or DNA from cells of interest as donor cells for a polysaccharidase gene or a polysaccharide binding protein gene.
• a polysaccharidase cDNA or binding protein cDNA or a fragment thereof as a hybridization probe, structurally related genes found in other species can be easily cloned and provide a cellulose binding peptide which is expressible in plants according to the present invention.
• genes from organisms that express polysaccharidase activity using oligonucleotide probes based on the nucleotide sequences of genes obtainable from an organism wherein the catalytic and binding domains of the polysaccharidase are discrete, although other polysaccharide binding proteins also can be used (see, for example, Shoseyov, et al, Proc. Nat'l. Acad. Sci. (USA)
• Probes developed using consensus sequences for the binding domain of a polysaccharidase or polysaccharide-binding protein are of particular interest.
• the ⁇ -l,4-glycanases from C. fimi characterized to date are endoglucanases A, B, C and D (CenA, CenB, CenC and CenD, respectively), exocellobiohydrolases A and B (CbhA and CbhB, respectively), and xylanases A and D (Cex and XylD, respectively) (see Wong et al. (1986) Gene, 44:315; Meinke et al. (1991) J. Bacteriol., 173 :308; Coutinho et al, (1991) Mol. Microbiol. 5: 1221 ; Meinke et al,
• C. fimi probably produces other ⁇ - 1,4-glycanases. Similar systems are produced by related bacteria (see Wilson (1992) Crit. Rev. Biotechnol., 12:45; and Hazlewood et al, (1992) J. Appl. Bacteriol., 72:244). Unrelated bacteria also produce glycanases; Clostridium thermocellum, for example, produces twenty or more ⁇ -1,4- glycanases (see Beguin et al, (1992) FEMS Microbiol. Lett., 100:523).
• the CBD derived from C. fimi endoglucanase C Nl is the only protein known to bind soluble cellosaccharides and one of a small set of proteins that are known to bind any soluble polysaccharides.
• Suitable binding domains are shown in Figure 1 of PCT/CA97/00033 (WO 97/26358), which presents an alignment of binding domains from various enzymes that bind to polysaccharides and identifies amino acid residues that are conserved among most or all of the enzymes.
• This information can be used to derive a suitable oligonucleotide probe using methods known to those of skill in the art.
• the probes can be considerably shorter than the entire sequence but should at least be 10, preferably at least 14, nucleotides in length. Longer oligonucleotides are useful, up to the full length of the gene, preferably no more than 500, more preferably no more than 250, nucleotides in length.
• RNA or DNA probes can be used.
• the probes are typically labeled in a detectable manner, for example, with 32p ? 3H, biotin, avidin or other detectable reagents, and are incubated with single-stranded DNA or RNA from the organism in which a gene is being sought. Hybridization is detected by means of the label after the unhybridized probe has been separated from the hybridized probe.
• the hybridized probe is typically immobilized on a solid matrix such as nitrocellulose paper. Hybridization techniques suitable for use with oligonucleotides are well known to those skilled in the art.
• oligonucleotide probe refers to both labeled and unlabeled forms.
• the binding domains identified by probing nucleic acids from an organism of interest will show at least about 40 % identity (including as appropriate allowances for conservative substitutions, gaps for better alignment and the like) to the binding region or regions from which the probe was derived and will bind to a soluble ⁇ -1,4 glucan with a K a of > 10 ⁇ M"l . More preferably, the binding domains will be at least about 60 % identical, and most preferably at least about 70 % identical to the binding region used to derive the probe. The percentage of identity will be greater among those amino acids that are conserved among polysaccharidase binding domains. Analyses of amino acid sequence comparisons can be performed using programs in PC/Gene (IntelliGenetics, Inc.).
• PCLUSTAL can be used for multiple sequence alignment and generation of phylogenetic trees.
• several genetic approaches can be used.
• One method uses restriction enzymes to remove a portion of the gene that codes for portions of the protein other than the binding portion thereof. The remaining gene fragments are fused with expression control sequences to obtain a mutated gene that encodes a truncated protein.
• Another method involves the use of exonucleases such as BaB l to systematically delete nucleotides either externally from the 5' and the 3' ends of the DNA or internally from a restricted gap within the gene.. These gene deletion methods result in a mutated gene encoding a shortened protein molecule which can then be evaluated for substrate or polysaccharide binding ability.
• cellulose binding protein refers to any protein or polypeptide which specifically binds to cellulose.
• the cellulose binding protein may or may not have cellulose or cellulolytic activity.
• cellulose binding domain refers to any protein or polypeptide which is a region or portion of a larger protein, said region or portion binds specifically to cellulose.
• the cellulose binding domain may be a part or portion of a cellulase, xylanase or other polysaccharidase, e.g., a chitinase, etc., a sugar binding protein such as maltose binding protein, or scaffoldin such as CbpA of Clostridium celluvorans, etc.
• Many cellulases and hemicellulases e.g., xylanases and mannases
• These enzymes typically have a catalytic domain containing the active site for substrate hydrolysis and a carbohydrate-binding domain or cellulose-binding domain for binding cellulose.
• the CBD may also be from a non-catalytic polysaccharide binding protein.
• CBDs cellulose-binding domains
• I-XIII Tomme et al (1995) "CelluloseBinding Domains: Classification and Properties", in ACS Symposium Series 618 Enzymatic Degradation and Insoluble Carbohydrates, pp. 142-161, Saddler and Penner eds., American Chemical Society, Washington, D.C. (Tomme I) ; Tomme et al. Adv. Microb. Physiol. (1995) 37:1 (Tomme II); and Smant et al, Proc. Natl. Acad. Sci U.S.A.
• the CBDs described in Tomme I or II or any variants thereof, any other presently known CBDs or any new CBDs which may be identified can be used in the present invention.
• the CBP or CBD can be from a bacterial, fungal, slime mold, or nematode protein or polypeptide.
• the CBD is obtainable from Clostridium cellulovorans, Clostridium cellulovorans, or Cellulomonas fimi (e.g., CenA, CenB, CenD, Cex).
• the CBD may be selected from a phage display peptide or peptidomimetic library, random or otherwise, using e.g., cellulose as a screening agent.
• a phage display peptide or peptidomimetic library random or otherwise, using e.g., cellulose as a screening agent.
• the CBD may be derived by mutation of a portion of a protein or polypeptide which binds to a polysaccharide other than cellulose (or hemicellulose) but also binds cellulose, such as a chitinase, which specifically binds chitin, or a sugar binding protein such as maltose binding protein, rendering said portion capable of binding to cellulose.
• the CBD binds cellulose or hemicellulose.
• CBDs have been isolated from different sources. Most of these have been isolated from proteins that have separate catalytic, i.e., cellulose and cellulose binding domains, and only two have been isolated from proteins that have no apparent hydrolytic activity but possess cellulose-binding activity (Goldstein et al J. Bacteriol. (1993) 175:5762-5768; Morag et al Appl. (1995) Environ. Microbiol. 61 : 1980- 1986). Cellulose binding peptide-recombinant protein fusions:
• fusion of two proteins for which genes has been isolated is well known and regularly practiced in the art.
• Such fusion involves the joining together of heterologous nucleic acid sequences, in frame, such that translation thereof results in the generation of a fused protein product or a fusion proteins.
• Methods such as the polymerase chain reaction (PCR), restriction, nuclease digestion, ligation, synthetic oligonucleotides synthesis and the like are typically employed in various combinations in the process of generating fusion gene constructs.
• PCR polymerase chain reaction
• restriction nuclease digestion, ligation, synthetic oligonucleotides synthesis and the like are typically employed in various combinations in the process of generating fusion gene constructs.
• nuclease digestion ligation
• synthetic oligonucleotides synthesis synthetic oligonucleotides synthesis and the like are typically employed in various combinations in the process of generating fusion gene constructs.
• One ordinarily skilled in the art can readily form such constructs
• an in frame spacer can be included.
• the length thereof may range, for example, from several to several dozens of amino acids.
• Such a spacer may also function to reduce mobilization constraints.
• nucleic acid molecule comprising a promoter sequence for directing protein expression in plant cells and a heterologous nucleic acid sequence including a first sequence encoding a cellulose binding peptide; and a second sequence encoding an enzyme being capable of catalyzing the oxidation of phenolic groups, wherein the first and second sequences are joined together in frame.
• the nucleic acid molecule further comprising a sequence element selected from the group consisting of an origin of replication for propagation in bacterial cells, at least one sequence element for integration into a plant's genome, a polyadenylation recognition sequence, a transcription termination signal, a sequence encoding a translation start site, a sequence encoding a translation stop site, plant RNA virus derived sequences, plant DNA virus derived sequences, tumor inducing (Ti) plasmid derived sequences, a transposable element derived sequence and a plant operative signal peptide for directing a protein to a cellular compartment of a plant cell.
• a sequence element selected from the group consisting of an origin of replication for propagation in bacterial cells, at least one sequence element for integration into a plant's genome, a polyadenylation recognition sequence, a transcription termination signal, a sequence encoding a translation start site, a sequence encoding a translation stop site, plant RNA virus derived sequences, plant DNA virus derived sequence
• the cellular compartment is selected from the group consisting of cytoplasm, endoplasmic reticulum, golgi apparatus, oil bodies, starch bodies, chloroplastids, chloroplasts, chromoplastids, chromoplasts, vacuole, lysosomes, mitochondria, and nucleus.
• the present invention employs recombinant nucleic acid molecules.
• a molecule includes, for example, a promoter sequence for directing protein expression in plant cells; and a heterologous nucleic acid sequence as further detailed herein, wherein, the heterologous nucleic acid sequence is down stream the promoter sequence, such that expression of the heterologous nucleic acid sequence is effectable by the promoter sequence.
• Such a nucleic acid molecule needs to be effectively introduced into plant cells, so as to genetically modify the plant.
• There are various methods of introducing foreign genes into both monocotyledonous and dicotyledenous plants Panets, I., Annu. Rev. Plant. Physiol, Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al, Nature (1989) 338:274-276).
• the principle methods of causing stable integration of exogenous DNA into plant genomic DNA include two main approaches:
• the Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. Horsch et al in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. The Agrobacterium system is especially viable in the creation of transgenic dicotyledenous plants.
• DNA transfer into plant cells There are various methods of direct DNA transfer into plant cells.
• electroporation the protoplasts are briefly exposed to a strong electric field.
• microinjection the DNA is mechanically injected directly into the cells using very small micropipettes.
• microparticle bombardment the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.
• transgenic plant propagation is exercised.
• the most common method of plant propagation is by seed.
• Regeneration by seed propagation has the deficiency that due to heterozygosity there is a lack of uniformity in the crop, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. Basically, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferred that the transgenic plant be produced such that the regenerated plant has the identical traits and characteristics of the parent transgenic plant, e.g., a reproduction of the fusion protein. Therefore, it is preferred that the transgenic plant be regenerated by micropropagation which provides a rapid, consistent reproduction of the transgenic plants.
• Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein.
• the new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant.
• Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant.
• the advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.
• Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages.
• the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening.
• stage one initial tissue culturing
• stage two tissue culture multiplication
• stage three differentiation and plant formation
• stage four greenhouse culturing and hardening.
• stage one initial tissue culturing
• the tissue culture is established and certified contaminant- free.
• stage two the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals.
• stage three the tissue samples grown in stage two are divided and grown into individual plantlets.
• the transgenic plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that it can be grown in the natural environment.
• the basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes.
• the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed.
• suitable markers for the members of the grass family is found in Wilmink and Dons, Plant Mol. Biol. Reptr. (1993) 1 1 :165-185. Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended.
• transposon sequences and the like for homologous recombination might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a heterologous expression cassette into a plant genome.
• Suitable prokaryote selectable markers include resistance toward antibiotics such as ampiciliin or tetracycline.
• Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.
• the constructs of the subject invention will include an expression cassette for expression of the fusion protein of interest. Usually, there will be only one expression cassette, although two or more are feasible.
• the recombinant expression cassette will contain in addition to the heterologous sequence one or more of the following sequence elements, a promoter region, plant 5' untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence.
• Unique restriction enzyme sites at the 5' and 3' ends of the cassette allow for easy insertion into a pre-existing vector.
• Viruses are a unique class of infectious agents whose distinctive features are their simple organization and their mechanism of replication.
• a complete viral particle, or virion may be regarded mainly as a block of genetic material (either DNA or RNA) capable of autonomous replication, surrounded by a protein coat and sometimes by an additional membranous envelope such as in the case of alpha viruses.
• the coat protects the virus from the environment and serves as a vehicle for transmission from one host cell to another.
• Viruses that have been shown to be useful for the transformation of plant hosts include CaV, TMV and BV. Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (BGV).
• EP-A 67,553 (TMV) Japanese Published Application No.
• the constructions can be made to the virus itself.
• the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid.
• the virus is a DNA virus
• a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsidate the viral DNA.
• the virus is an RNA virus
• the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions.
• the RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA.
• a plant viral nucleic acid in which the native coat protein coding sequence has been deleted from a viral nucleic acid, a non-native plant viral coat protein coding sequence and a non-native promoter, preferably the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral nucleic acid, and ensuring a systemic infection of the host by the recombinant plant viral nucleic acid, has been inserted.
• the coat protein gene may be inactivated by insertion of the non-native nucleic acid sequence within it, such that a fusion protein is produced.
• the recombinant plant viral nucleic acid may contain one or more additional non-native subgenomic promoters.
• Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or nucleic acid sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters.
• Non-native (foreign) nucleic acid sequences may be inserted adjacent the native plant viral subgenomic promoter or the native and a non-native plant viral subgenomic promoters if more than one nucleic acid sequence is included.
• the non-native nucleic acid sequences are transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.
• a recombinant plant viral nucleic acid is provided as in the first embodiment except that the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a non-native coat protein coding sequence.
• a recombinant plant viral nucleic acid in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral nucleic acid.
• the inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters.
• Non-native nucleic acid sequences may be inserted adjacent the non-native subgenomic plant viral promoters such that said sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.
• a recombinant plant viral nucleic acid is provided as in the third embodiment except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.
• the viral vectors are encapsidated by the coat proteins encoded by the recombinant plant viral nucleic acid to produce a recombinant plant virus.
• the recombinant plant viral nucleic acid or recombinant plant virus is used to infect appropriate host plants.
• the recombinant plant viral nucleic acid is capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene(s) in the host to produce the desired fusion protein.
• Fusion protein compartmentalization - signal peptides As already mentioned hereinabove, compartmentalization of the fusion protein is an important feature of the present invention because it allows undisturbed plant growth. Thus, according to one aspect of the present invention, the fusion protein is compartmentalized within cells of the plant or cultured plant cells, so as to be sequestered from cell walls of the cells of the plant or cultured plant cells.
• the fusion protein can be compartmentalized within a cellular compartment, such as, for example, the cytoplasm, endoplasmic reticulum, golgi apparatus, oil bodies, starch bodies, chloroplastids, chloroplasts, chromoplastids, chromoplasts, vacuole, lysosomes, mitochondria or the nucleus.
• a cellular compartment such as, for example, the cytoplasm, endoplasmic reticulum, golgi apparatus, oil bodies, starch bodies, chloroplastids, chloroplasts, chromoplastids, chromoplasts, vacuole, lysosomes, mitochondria or the nucleus.
• the heterologous sequence used while implementing the process according to this aspect of the present invention includes (i) a first sequence encoding a cellulose binding peptide; (ii) a second sequence encoding a recombinant protein, wherein the first and second sequences are joined together in frame; and (iii) a third sequence encoding a signal peptide for directing a protein to a cellular compartment, the third sequence being upstream and in frame with the first and second sequences.
• signal peptides which can be used to direct the fusion protein according to the present invention to specific cell compartments.
• the preferred site of accumulation of the fusion protein according to the present invention is the ER using signal peptide such as Cel 1 or the rice amylase signal peptide at the N-terminus and an ER retaining peptide (HDEL or KDEL) at the C-terminus.
• signal peptide such as Cel 1 or the rice amylase signal peptide at the N-terminus
• HDEL or KDEL ER retaining peptide
• any promoter which can direct the expression of the fusion protein according to the present invention can be utilized to implement the process of the instant invention, both constitutive and tissue specific promoters.
• the promoter selected is constitutive, because such a promoter can direct the expression of higher levels of the fusion protein.
• the present invention offers a major advantage over the teachings of U.S. Pat. No. 5,474,925 in which only tissue specific and weak promoters can be employed because of the deleterious effect of the fusion protein described therein on cell wall development.
• the reason for which the present invention can utilize strong and constitutive promoters relies in the compartmentalization and sequestering approach which prohibits contact between the expressed fusion protein and the plant cell walls which such walls are developing.
• transgenic wheat it has been shown that a native HMW-GS gene promoter can be used to obtain high levels of expression of seed storage and, potentially, other proteins in the endosperm (Blechl and Anderson, Nat. Biotechnol. (1996) 14:875-9).
• Polygalacturonase (PG) promoter was shown to confer high levels of ripening-specific gene expression in tomato (Nicholass et al. Plant. Mol. Biol. (1995) 28:423-435).
• the ACC oxidase promoter (Blume and Grierson, Plant. J. (1997) 12:731-746) represents a promoter from the ethylene pathway and shows increased expression during fruit ripening and senescence in tomato.
• the promoter for tomato 3- hydroxy-3-methylglutaryl coenzyme A reductase gene accumulates to high level during fruit ripening (Daraselia et al Plant. Physiol. (1996) 112:727- 733).
• Specific protein expression in potato tubers can be mediated by the patatin promoter (Sweetlove et al. Biochem. J. (1996) 320:487-492).
• Protein linked to a chloroplast transit peptide changed the protein content in transgenic soybean and canola seeds when expressed from a seed-specific promoter (Falco et al Biotechnology (NY) (1995) 13:577-82).
• the seed specific bean phaseolin and soybean beta-conglycinin promoters are also suitable for the latter example (Keeler et al. Plant. Mol. Biol. (1997) 34: 15-29). Promoters that are expressed in plastids are also suitable in conjunction with plastid transformation.
• each of these promoters can be used to implement the process according to the present invention.
• the plant promoter employed can a constitutive promoter, a tissue specific promoter, an inducible promoter or a chimeric promoter.
• constitutive plant promoters include, without being limited to, CaMV35S and CaMV19S promoters, FMV34S promoter, sugarcane bacilliform badnavirus promoter, CsVMV promoter, Arabidopsis
• ACT2/ACT8 actin promoter Arabidopsis ubiquitin UBQ1 promoter, barley leaf thionin BTH6 promoter, and rice actin promoter.
• tissue specific promoters include, without being limited to, bean phaseolin storage protein promoter, DLEC promoter, PHS ⁇ promoter, zein storage protein promoter, conglutin gamma promoter from soybean, AT2S1 gene promoter, ACT 1 1 actin promoter from Arabidopsis, napA promoter from Brassica napus and potato patatin gene promoter.
• the inducible promoter is a promoter induced by a specific stimuli such as stress conditions comprising, for example, light, temperature, chemicals, drought, high salinity, osmotic shock, oxidant conditions or in case of pathogenicity and include, without being limited to, the light- inducible promoter derived from the pea rbcS gene, the promoter from the alfalfa rbcS gene, the promoters DRE, MYC and MYB active in drought; the promoters INT, INPS, prxEa, Ha hspl 7.7G4 and RD21 active in high salinity and osmotic stress, and the promoters hsr303J and str246C active in pathogenic stress.
• stress conditions comprising, for example, light, temperature, chemicals, drought, high salinity, osmotic shock, oxidant conditions or in case of pathogenicity and include, without being limited to, the light- inducible promoter derived from the pea r
• Fusion protein can be monitored by a variety of methods. For example, ELISA or western blot analysis using antibodies specifically recognizing the recombinant protein or its cellulose binding peptide counterpart can be employed to qualitatively and/or quantitatively monitor the expression of the fusion protein in the plant. Alternatively, the fusion protein can be monitored by SDS-PAGE analysis using different staining techniques, such as, but not limited to, coomasie blue or silver staining. Other methods can be used to monitor the expression level of the RNA encoding for the fusion protein. Such methods include RNA hybridization methods, e.g., Northern blots and RNA dot blots.
• a genetically modified or viral infected plant or cultured plant cells expressing a fusion protein including an enzyme being capable of catalyzing the oxidation of phenolic groups and a cellulose binding peptide.
• the fusion protein is compartmentalized within cells of said plant or cultured plant cells, so as to be sequestered from cell walls of said cells of said plant or cultured plant cells, so as not to hamper development and to allow higher expression, if so required.
• the fusion protein is compartmentalized within a cellular compartment selected from the group consisting of cytoplasm, endoplasmic reticulum, golgi apparatus, oil bodies, starch bodies, chloroplastids, chloroplasts, chromoplastids, chromoplasts, vacuole, lysosomes, mitochondria, and nucleus.
• oxidase activity is based on the oxidation of syringaldazin to tetramethoxy azo bis-methylene quinone under aerobic conditions, and 1 LACU is the amount of enzyme which converts 1 ⁇ M of syringaldazin per minute under the following conditions: 19 ⁇ M syringaldazin, 23.2 mM acetate buffer, 30 °C, pH 5.5, reaction time 1 minute, shaking; the reaction is monitored spectrophotometrically at 530 nm.
• 1 PODU is the amount of enzyme which catalyses the conversion of 1 ⁇ mol of hydrogen peroxide per minute under the following conditions: 0.88 mM hydrogen peroxide. 1.67 mM 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate), 0.1 M phosphate buffer, pH 7.0, incubation at 30 °C; the reaction is monitored photometrically at 418 nm.
• Binding of the fusion protein to the plant derived cellulosic matter When sufficient expression has been detected, binding of the fusion protein to the plant derived cellulosic matter is effected. Such binding can be achieved, for example, as follows. Whole plants, plant derived tissue or cultured plant cells are homogenized by mechanical method in the presence or absence of a buffer, such as, but not limited to, PBS. The fusion protein is therefore given the opportunity to bind to the plant derived cellulosic matter. Buffers that may include salts and/or detergents at optimal concentrations may be used to wash non specific proteins from the cellulosic matter.
• composition of matter comprising a cell wall preparation derived from a genetically modified or virus infected plant or cultured plant cells expressing a fusion protein including an enzyme being capable of catalyzing the oxidation of phenolic groups and a cellulose binding peptide, said fusion protein being immobilized to cellulose in said cell wall preparation via said cellulose binding peptide.
• Oxidizing agents The enzyme(s) and oxidizing agent(s) used in the process of the invention should clearly be matched to one another, and it is clearly preferable that the oxidizing agent(s) in question participate(s) only in the oxidative reaction involved in the binding process, and does/do not otherwise exert any deleterious effect on the substances/materials involved in the process.
• Oxidases e.g. laccases, are, among other reasons, well suited in the context of the invention since they catalyze oxidation by molecular oxygen.
• hydrogen peroxide is a preferred peroxide in the context of the invention and is suitably employed in a concentration (in the reaction medium) in the range of 0.01-100 mM.
• pH in the Reaction Medium Depending, inter alia, on the characteristics of the enzyme(s) employed, the pH in the aqueous medium (reaction medium) in which the process of the invention takes place will be in the range of 3-10, preferably in the range 4-9.
• the present invention also relates to a lignocellulose-based product obtainable by a process according to the invention as disclosed herein.

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