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WO2017013438A1 - Procédés d'extraction de sucres et de produits dérivés de la lignine à partir d'un matériau de biomasse lignocellulosique - Google Patents

Procédés d'extraction de sucres et de produits dérivés de la lignine à partir d'un matériau de biomasse lignocellulosique Download PDF

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WO2017013438A1
WO2017013438A1 PCT/GB2016/052219 GB2016052219W WO2017013438A1 WO 2017013438 A1 WO2017013438 A1 WO 2017013438A1 GB 2016052219 W GB2016052219 W GB 2016052219W WO 2017013438 A1 WO2017013438 A1 WO 2017013438A1
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polypeptide
biomass material
lignocellulosic biomass
amino acid
lignin
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PCT/GB2016/052219
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English (en)
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Guy Barker
Daniel Eastwood
Irnia NURIKA
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University Of Warwick
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Publication of WO2017013438A1 publication Critical patent/WO2017013438A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07G—COMPOUNDS OF UNKNOWN CONSTITUTION
    • C07G1/00—Lignin; Lignin derivatives
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H6/00—Macromolecular compounds derived from lignin, e.g. tannins, humic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H8/00—Macromolecular compounds derived from lignocellulosic materials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
    • C12Y101/99018—Cellobiose oxidase (1.1.99.18)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis

Definitions

  • the present invention relates to polypeptide-based methods for extracting sugars and /or lignin derived products from lignocellulosic biomass materials, uses of a polypeptide for extracting sugars and/or lignin derived products from lignocellulosic biomass materials, and recombinant micro-organisms that express said polypeptide.
  • Lignocellulose is the major structural component of woody plants, and non-woody plants such as grass, and consists of three main components: lignin, cellulose and hemicellulose. These components provide essential resources for the production of components for use in various industries including agriculture, food production, waste management, textiles and paper, as well as renewable energy and high-value chemical products. Much focus has therefore been placed on identifying effective methods for isolating and/ or further processing lignin, cellulose and hemicellulose from waste lignocellulosic biomass material.
  • Biofuels such as biogasoline, biodiesel and bioethanol, offer an alternative renewable energy source to petroleum-based fuels, and have the potential to provide a sustainable source of energy with reduced greenhouse emissions.
  • Lignocellulosic biomass material provides a key source of biological material for production of biofuels.
  • the cellulose and hemicellulose components can be broken down into simple sugars using enzymatic or chemical hydrolysis reactions. Hydrolysis of cellulose results in the release of hexose sugars, such as glucose, mannose and galactose; whilst hemicellulose can be hydrolysed into pentose sugars, such as xylose and arabinose.
  • the extracted sugars can be fermented to alcohol, such as ethanol, and used to produce biogasoline.
  • the extracted sugars may be processed into esters for use in the production of biodiesel.
  • Lignin may also be used to produce biofuels, for example through the generation of syngas (carbon monoxide/ hydrogen), which in turn can be used to generate chemical alcohols and produce biogasoline.
  • High value chemical products can also be produced from the lignin component of lignocellulosic biomass material.
  • lignin can be depolymerised and used to produce a variety of aromatic and polyaromatic compounds, such as phenol, BTX l chemicals (such as benzene, toluene and xylene) and terephthalic acid.
  • Lignin can also be used to produce a wide variety of other products, including carbon fibre materials, plastic materials, polymer modifiers, resins, adhesives, binders, dispersants, emulsifiers, wetting agents, fertilisers, dyestuffs, agglomerants and chelants.
  • Lignocellulosic biomass material is a relatively inexpensive potential source of cellulose, hemicellulose and lignin because it can be obtained from widely available waste materials, such as wood waste, grass and agricultural waste.
  • lignocellulose has a complex structure, in which the cellulose is in the form of fibres that are locked within a rigid structure of hemicellulose and lignin.
  • lignocellulosic biomass material In order to access the cellulose, hemicellulose and lignin components, lignocellulosic biomass material is therefore typically subjected to an initial 'pre-treatment' process that damages or alters the lignocellulose structure to separate and release the individual lignocellulose components.
  • the requirement for this pre-treatment process increase the costs associated with using lignocellulosic biomass material. There is therefore an increasing emphasis on improving the methods of extraction from lignocellulosic biomass materials.
  • pre-treatment methods include thermochemical methods such as steam explosion, in which the lignocellulosic material is heated to 130°C - 230°C by steam injection (optionally including the addition of a catalyst or caustic agent to facilitate degradation); or wet oxidation, which involves exposure of the biomass material to oxygen at 150-185°C.
  • thermochemical methods such as steam explosion, in which the lignocellulosic material is heated to 130°C - 230°C by steam injection (optionally including the addition of a catalyst or caustic agent to facilitate degradation); or wet oxidation, which involves exposure of the biomass material to oxygen at 150-185°C.
  • Another conventional pre-treatment method is acid hydrolysis, in which the lignocellulosic material is subjected to an acid (such as sulphuric acid) that hydrolyses the cellulose and hemicellulose components to their monomeric sugars. Acid hydrolysis is also often performed at high temperatures to promote further hydrolysis of the biomass material.
  • Organosolve pre-treatment methods also exist and involve the use of solvents together with acids or bases, and may be performed at high temperatures. These organosolve methods act by disrupting the interactions between the lignin, hemicellulose and cellulose and permit the lignin and hemicellulose components to be separated from cellulose in conjunction with the solvent. Although these pre-treatment methods allow for efficient breakdown of lignocellulosic biomass material, they require expensive reaction vessels, and are energy intensive.
  • the thermochemical and acid hydrolysis methods occur at high temperatures and extreme pH conditions which are often incompatible with downstream processes, such as the enzymatic hydrolysis reaction used to release sugars for production of biofuels.
  • the pre-treatment processes also result in the production of compounds or by-products that inhibit downstream processes.
  • these pre-treatment processes have been shown to result in the production of furfural from xylose and phenolic fragments from lignin, both of which inhibit the downstream fermentation process used to produce biogasoline.
  • the organosolve pre-treatment methods are also hindered by drawbacks - in particular, because they require large volumes of solvent.
  • the solvents used are not easily recyclable, and it has been shown that any solvent remaining in the extracted sugars can inhibit the downstream fermentation process.
  • these pre- treatment methods are often performed separately to the downstream processes that utilise the cellulose, hemicellulose and lignin components of the lignocellulosic biomass material.
  • biological based approaches have also been proposed, with the aim of providing a more efficient, eco-friendly and cost-effective way of pre-treating lignocellulosic biomass material.
  • biological pre-treatment methods include the use of intact micro-organisms that naturally degrade lignocellulosic biomass material.
  • filamentous fungi such as the white rot fungi basidiomycete P. chrysosporium
  • filamentous fungi have been identified as potentially suitable candidates for use in pre-treatment processes, because they produce enzymes that permit degradation of lignin, hemicellulose and cellulose, via oxidative and hydrolytic mechanisms (Dashtban et al.
  • White rot fungi degrade lignin via an oxidative mechanism, in which the lignin is attacked by reactive oxygen species, such as hydroxyl free radicals.
  • White rot fungi are also known to attack lignin using enzymes such as laccases, which catalyse free radical mediated lignin degradation.
  • White rot fungi also produce hydrolytic enzymes (including those with endocellulase, exocellulase and cellobiohydrolase activities), which degrade glycosidic linkages in cellulose and hemicellulose to release monomeric sugars.
  • hydrolytic enzymes including those with endocellulase, exocellulase and cellobiohydrolase activities
  • endocellulase endocellulase
  • exocellulase and cellobiohydrolase activities hydrolytic enzymes
  • the present invention presents a solution to the above problems by providing a method for extracting sugars and/or lignin-derived products from lignocellulosic biomass material, comprising: contacting the lignocellulosic biomass material with a composition comprising a polypeptide in the presence of a reducible substrate and an oxidising agent, wherein said polypeptide comprises an amino acid sequence that has at least 70% identity to the amino acid sequence of SEQ ID NO: 1 or 2, or a fragment thereof; wherein said polypeptide is capable of extracting sugars and/or lignin-derived products from the lignocellulosic biomass material in the presence of a reducible substrate and an oxidising agent.
  • the present invention also provides a method for extracting sugars from lignocellulosic biomass material, wherein the lignocellulosic biomass material has been subjected to a pre-treatment process that at least partially depolymerises lignin and/ or hemicellulose present in the lignocellulosic structure; the method comprising: contacting the pre- treated lignocellulosic biomass material with a composition comprising a polypeptide, wherein said polypeptide comprises an amino acid sequence that has at least 70% identity to the amino acid sequence of SEQ ID NO: 1 or 2, or a fragment thereof; wherein said polypeptide is capable of extracting sugars from the pre-treated lignocellulosic biomass material.
  • the 'lignocellulosic biomass material' used in the present invention includes plant and/or wood material, such as herbaceous material, softwood, hardwood, wood waste, sawdust, agricultural crop, plant residue, forestry residue, municipal solid waste, pulp or paper mill residue, waste paper, recycling paper, or construction debris.
  • suitable wood material include, but are not limited to, spruce, pine, hemlock, fir, birch, aspen, maple, poplar, alder, salix, cottonwood, rubber tree, marantii, eucalyptus, sugi, and acase.
  • suitable plant material include, but are not limited to aquatic plants such as kelp, algae, lily, and hyacinth; and fibrous plants such as grass (including switch grass, cord grass, rye grass, reed canary grass, mixed prairie grass, miscanthus and Napier grass), straw (including rice straw, barley straw, cereal straw, sugarcane straw, wheat straw, canola straw, and oat straw), sugarcane bagasse, agricultural wastes, rice hulls, corn cobs, oat hulls, corn fiber, stover, soybean stover and corn stover.
  • grass including switch grass, cord grass, rye grass, reed canary grass, mixed prairie grass, miscanthus and Napier grass
  • straw including rice straw, barley straw, cereal straw, sugarcane straw, wheat straw, canola straw, and oat straw
  • sugarcane bagasse agricultural wastes, rice hulls, corn cobs, oat hulls, corn fiber, stover, soybean stover and corn stover
  • the lignocellulosic biomass material obtained from these plant and/ or wood materials comprises lignin, hemicellulose, and cellulose.
  • lignin used throughout the present specification broadly refers to a biopolymer that may be part of secondary cell walls in plants, such as complex highly cross-linked aromatic polymer that covalently links to hemicellulose.
  • hemicellulose used throughout the present specification broadly refers to a branched sugar polymer composed mostly of pentoses, such as with a generally random amorphous structure and up to hundreds of thousands of pentose units.
  • cellulose used throughout the present specification broadly refers to an organic compound with a formula (C 6 H 10 O 5 )z where z includes any suitable integer.
  • Cellulose may include a polysaccharide with a linear chain of several hundred to over ten thousand hexose units and a high degree of crystalline structure.
  • the lignocellulosic biomass material used in the present invention may have been subjected to a pre-treatment step such that lignin and/ or hemicellulose present in the lignocellulosic structure is at least partially depolymerised.
  • Subjecting lignocellulosic biomass material to a pre-treatment step may result in the separation of the cellulose, hemicellulose and lignin components of the lignocellulosic biomass material so as to increase the surface area and/ or accessibility of the material.
  • the lignocellulosic biomass material may be exposed to a hydrolysis process, an acidic process (pH 7 and below), a basic or alkali process (pH above 7), an enzymatic process, a solvent-based process, a thermo-mechanical process, a thermo- chemical process, a steam based process, including but not limited to steam explosion and hot water based treatment, and/ or a supercritical process.
  • Acid processes may include concentrated and/ or dilute acid steps, such as with sulphuric acid, sulphurous acid, hydrochloric acid, phosphoric acid, and/ or organic acids.
  • Basic processes may include caustic materials, such as ammonia, calcium hydroxide, calcium oxide, magnesium hydroxide, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, and/or potassium bicarbonate.
  • Pre-treatment processes may also involve subjecting lignocellulosic biomass material to a mechanical de-sizing process which includes any method for reducing the particle size of biomass such as, but not limited to, processes involving grinding, milling or crushing.
  • Other pre- treatment processes known to a skilled person in the art may also be used to prepare pre-treated lignocellulosic material for use in the present invention.
  • the present invention provides polypeptide based methods for extracting sugars and/or lignin derived products from lignocellulosic biomass material.
  • 'sugars' used throughout the present specification means any carbohydrate compound having the general formula C x H 2 xO x , where x includes any suitable integer, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, and/ or the like.
  • Sugars may be building blocks or components of more complex molecules, such as cellulose, which comprises hexose sugars; and hemicellulose, which comprises pentose sugars.
  • lignin derived products used throughout the present specification means any product obtained from lignin that can be used to produce other useful compounds or products, including high value chemicals and/ or biofuels, such as biogasoline, bioethanol or biodiesel. A skilled person will be familiar with the various types of lignin derived products that can be obtained from lignin, and used to produce other useful compounds or products.
  • Non-limiting examples of lignin derived products that can be used to produce other useful compounds or products include guaiacols (such as methylguaiacol, ethylguaiacol, vinylguaiacol and guaiacylacetone, eugenol and isoeugenol); syringols; phenols and phenolic aldehydes (such as vanillin, acetovanillone, veratraldehyde, acetoveratron, trimethoxy benzaldehyde, 4-hydroxy-benzaldehyde, acetaniso!e, acetosyringone and syringaldehyde).
  • guaiacols such as methylguaiacol, ethylguaiacol, vinylguaiacol and guaiacylacetone, eugenol and isoeugenol
  • syringols such as vanillin
  • the polypeptides used in the methods of the present invention are derived from two iron reductase enzymes, IR1 (defined in Genbank accession number EGN95518.1 ) and IR2 (defined in Genbank accession number EGN95519.1 ). These enzymes are encoded by the genome of the brown rot fungus, S. lacrymans, which is a natural decomposer of lignocellulosic material.
  • IR1 and IR2 were previously reported following sequencing and analysis of the S. lacrymans genome (Eastwood et al. The Plant Cell Wall-Decomposing Machinery Underlies the Functional Diversity of Forest Fungi; Science; 201 1 ; 333; 762- 765). These studies revealed that the IR1 enzyme (protein ID 452187) contained sequences corresponding to an iron reductase domain (also known as a 'heme' domain) and a cellulose binding module, whilst the IR2 enzyme (protein ID 417465) contained sequences corresponding to an iron reductase domain, but did not contain sequences corresponding to a cellulose binding module.
  • Figure 1 shows a comparison of the structures of the IR1 and IR2 enzymes.
  • IR1 was proposed to play a role in the process of lignocellulose breakdown by S. lacrymans. It has been suggested that brown rot fungi mediate lignocellulose breakdown using a non-enzymatic reaction in which hydroxyl free radicals are generated via the Fenton reaction (Fe 2+ + H 2 0 2 + H + > Fe 3+ + OH " + H 2 0) and used to depolymerise the lignocellulosic structure. Hydrogen peroxide (H 2 0 2 ) for use in the Fenton reaction has been shown to be metabolically generated by oxidase enzymes produced by brown rot fungi.
  • Brown rot fungi have also been shown to produce metabolites (such as variegatic acid) that reduce Fe 3+ to Fe 2+ for use in the Fenton reaction. It was therefore proposed that S. lacrymans may initiate lignocellulose depolymerisation by producing metabolites such as variegatic acid. Following this initial lignocellulose depolymerisation, it was proposed that IR1 may localise to cellulose in the partially depolymerised lignocellulosic structure via its cellulose binding module, and promote further generation of Fe 2+ using its iron reductase domain, leading to localised hydroxyl free radical production (via the Fenton reaction) and further depolymerisation of the lignocellulosic structure.
  • metabolites such as variegatic acid
  • both IR1 and IR2 can induce depolymerisation of the lignocellulosic structure without requiring any other fungal metabolites or fungal derived compounds (such as variegatic acid and 2,5 dimethoxyhydroquinone) to initiate depolymerisation of the lignocellulosic structure.
  • a simplified method has been developed for targeting depolymerisation of the lignocellulosic structure using IR1 or IR2 polypeptides together with a reducible substrate (such as Fe 3+ ions) and an oxidising agent (such as H 2 0 2 ).
  • sugars and/or lignin derived products can be extracted from lignocellulosic biomass material, without the need to perform any pre-treatment process, and without the need to utilise intact S. lacrymans or any other S. lacrymans derived metabolites or compounds.
  • both IR1 and IR2 have the ability to directly depolymerise polysaccharides present in lignocellulosic biomass material (such as cellulose) to produce sugars.
  • This depolymerisation activity has been shown to occur independently of the iron reductase activity shared by both IR1 and IR2, and does not require the presence of a cellulose binding domain, such as that present in IR1.
  • the enzymatic activity of these enzymes also does not require the presence of any other fungal metabolites or fungal derived compounds.
  • a simplified method has therefore been developed for extracting sugars from lignocellulosic biomass material that avoids the need to add other cellulase enzymes, or utilise intact S. lacrymans.
  • polypeptide based methods for extraction of sugars and/ or lignin derived products from lignocellulosic biomass material have been developed using polypeptides derived from IR1 or IR2.
  • the polypeptide used in the methods of the present invention may comprise (or consist of) an amino acid sequence having at least 70% identity to the sequence of SEQ ID NO: 1 or 2, or a fragment of said sequence.
  • the amino acid sequence defined in SEQ ID NO: 1 corresponds to the polypeptide sequence of IR1 (Genbank accession number EGN95518.1 ) but does not include the IR1 signal peptide sequence.
  • the amino acid sequence defined in SEQ ID NO: 2 corresponds to the polypeptide sequence of IR2 (Genbank accession number EGN95519.1 ) but does not include the IR2 signal peptide sequence.
  • polypeptide used throughout the present specification is synonymous with the terms “oligopeptide”, “peptide” and “protein”. These terms are used interchangeably and do not refer to a specific length of the product. These terms embrace post- translational modifications such as glycosylation, acetylation and phosphorylation.
  • the polypeptide may comprise (or consist of) an amino acid sequence having at least 70% identity (eg. at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to the sequence of SEQ ID NO: 1 , or a fragment of said sequence.
  • the polypeptide may comprise (or consist of) the amino acid of SEQ ID NO: 1 , or a fragment of said sequence.
  • the polypeptide may comprise (or consist of) an amino acid sequence having at least 70% identity (eg. at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to the sequence of SEQ ID NO: 2, or a fragment of said sequence.
  • the polypeptide may comprise (or consist of) the amino acid sequence of SEQ ID NO: 2, or a fragment of said sequence.
  • a fragment of a polypeptide comprises (or consists of) a truncated form of the polypeptide.
  • a fragment of a polypeptide may comprise a series of consecutive amino acid residues from the sequence of the polypeptide.
  • a fragment of a polypeptide may have an N-terminal truncation or a C-terminal truncation (as compared with the polypeptide).
  • the polypeptide may comprise (or consist of) an amino acid sequence having at least 70% identity (eg.
  • the polypeptide may comprise (or consist of) the amino acid sequence of SEQ ID NO: 1 , or a fragment of said sequence having at least 10 consecutive amino acids thereof (eg. least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 or 230 consecutive amino acids thereof).
  • the polypeptide fragment may comprise (or consist of) an amino acid sequence having at least 70% identity to the sequence of the IR1 iron reductase domain (defined as amino acid residues 4-175 of SEQ ID NO: 1 ), or a fragment thereof; and/ or an amino acid sequence having at least 70% identity to the sequence of the IR1 cellulose binding domain (defined as amino acid residues 202-229 of SEQ ID NO: 1), or a fragment thereof.
  • the polypeptide fragment may comprise (or consist of) an amino acid sequence having at least 70% identity (eg.
  • the polypeptide may comprise (or consist of) an amino acid sequence having at least 70% identity (eg. at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to the sequence of SEQ ID NO: 2, or a fragment of said sequence having at least 10 consecutive amino acids thereof (eg. at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180 or 185 consecutive amino acids thereof).
  • at least 70% identity eg. at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
  • SEQ ID NO: 2 or a fragment of said sequence having at least 10 consecutive amino acids thereof (e
  • the polypeptide may comprise (or consist of) the amino acid sequence of SEQ ID NO: 2, or a fragment of said sequence having at least 10 consecutive amino acids thereof (eg. at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180 or 185 consecutive amino acids thereof).
  • the polypeptide fragment may comprise (or consist of) an amino acid sequence having at least 70% identity to the sequence of the IR2 iron reductase domain (defined as amino acid residues 4-175 of SEQ ID NO: 2), or a fragment thereof.
  • the polypeptide fragment may comprise (or consist of) an amino acid sequence having at least 70% identity (eg. at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to the sequence spanning amino acid residues 4 to 175 of SEQ ID NO: 2, or a fragment thereof having at least 50 consecutive amino acids thereof (eg. least 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, or 170 consecutive amino acids thereof).
  • the polypeptides, polypeptide variants and polypeptide fragments for use in the methods of the present invention are capable of extracting sugars from lignocellulosic biomass material that has been previously treated so as to at least partially depolymerise lignin and/ or hemicellulose present in the lignocellulosic structure.
  • the polypeptides, polypeptide variants and polypeptide fragments for use in the methods of the present invention are capable of extracting sugars and/or lignin derived products from lignocellulosic biomass material in the presence of a reducible substrate and an oxidising agent.
  • polypeptides, polypeptide variants and polypeptide fragments for use in the methods of the present invention have reductase activity and/ or polysaccharide cleavage activity.
  • the polypeptides, polypeptide variants and polypeptide fragments for use in the methods of the present invention may have iron reductase activity and/ or cellulase and/ or hemicellulase activity.
  • the identity may exist over a region of the sequences that is at least 10 amino acid residues in length (eg. at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 or 230 amino acid residues in length - eg. up to the entire length of the reference sequence).
  • Polypeptides used in the methods of the present invention may have one or more amino acid substitutions, deletions, or additions. In many embodiments, those changes are of a minor nature, for example, involving only conservative amino acid substitutions. Conservative substitutions are those made by replacing one amino acid with another amino acid within the following groups: Basic: arginine, lysine, histidine; Acidic: glutamic acid, aspartic acid; Polar: glutamine, asparagine; Hydrophobic: leucine, isoleucine, valine; Aromatic: phenylalanine, tryptophan, tyrosine; Small: glycine, alanine, serine, threonine, methionine.
  • Basic arginine, lysine, histidine
  • Acidic glutamic acid, aspartic acid
  • Polar glutamine, asparagine
  • Hydrophobic leucine, isoleucine, valine
  • Aromatic phenylalanine, tryptophan, tyros
  • Polypeptides used in the methods of the present invention may also encompass those comprising other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of 1 to about 10 amino acids (such as 1 -5 amino acids); and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
  • the polypeptide may also comprise non-naturally occurring amino acid residues.
  • Essential amino acids in the polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis. Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. The identities of essential amino acids can also be inferred from analysis of homologies with related family members of the polypeptide of interest.
  • nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a polypeptide.
  • DNA molecules can be digested with Bal31 nuclease to obtain a series of nested deletions. These DNA fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for the desired activity.
  • polynucleotide sequence of SEQ ID NO: 3 encodes the IR1 derived polypeptide defined in SEQ ID NO: 1 ; and the polynucleotide sequence of SEQ ID NO: 4 encodes the IR2 derived polypeptide defined in SEQ ID NO: 2.
  • the polypeptide is encoded by a polynucleotide that comprises (or consists of) a sequence having at least 70% identity to the sequence of SEQ ID NO: 3 or 4, or a fragment of said sequence.
  • the polypeptide is encoded by a polynucleotide that comprises (or consists of) a sequence having at least 70% identity (eg. at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to the sequence of SEQ ID NO: 3, or a fragment of said sequence.
  • the polypeptide is encoded by a polynucleotide that comprises (or consists of) the sequence of SEQ ID NO: 3, or a fragment of said sequence.
  • the polypeptide is encoded by a polynucleotide that comprises (or consists of) a sequence having at least 70% identity (eg. at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to the sequence of SEQ ID NO: 4, or a fragment of said sequence.
  • the polypeptide is encoded by a polynucleotide that comprises (or consists of) the sequence of SEQ ID NO: 4, or a fragment of said sequence.
  • a fragment of a polynucleotide comprises (or consists of) a truncated form of the polynucleotide.
  • a "fragment" of a polynucleotide may comprise a series of consecutive nucleotides from the sequence of the polynucleotide.
  • a fragment of a polynucleotide may encode a polypeptide having an N-terminal truncation or a C-terminal truncation (as compared with the reference polypeptide).
  • the polypeptide is encoded by a polynucleotide that comprises (or consists of) a sequence having at least 70% identity (eg.
  • the polypeptide is encoded by a polynucleotide that comprises (or consists of) the sequence of SEQ ID NO: 3, or a fragment of said sequence having at least 30 consecutive nucleotides thereof (eg.
  • the polynucleotide fragment may comprise (or consist of) a nucleotide sequence having at least 70% identity to the nucleotide sequence encoding the IR1 iron reductase domain (corresponding to nucleotides 10-525 of SEQ ID NO: 3), or a fragment thereof; and/ or a nucleotide sequence having at least 70% identity to the sequence encoding the IR1 cellulose binding domain (corresponding to nucleotides 604-687 of SEQ ID NO: 3), or a fragment thereof.
  • the polynucleotide fragment may comprise (or consist of) a nucleotide sequence having at least 70% identity (eg. at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to the sequence spanning nucleotides 10-525 of SEQ ID NO: 3, or a fragment thereof having at least 150 consecutive nucleotides thereof (eg.
  • nucleotide sequence having at least 70% identity eg. at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
  • sequence spanning nucleotides 604-687 of SEQ ID NO: 3 or a fragment thereof having at least 45 consecutive nucleotides thereof (eg. at least 60 or 75 consecutive nucleotides thereof).
  • the polypeptide is encoded by a polynucleotide that comprises (or consists of) a sequence having at least 70% identity (eg. at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to the sequence of SEQ ID NO: 4, or a fragment of said sequence having at least 30 consecutive nucleotides thereof (eg.
  • the polypeptide is encoded by a polynucleotide that comprises (or consists of) the sequence of SEQ ID NO: 4, or a fragment of said sequence having at least 30 consecutive nucleotides thereof (eg. at least 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, 210, 225, 240, 255, 270, 285, 300, 315, 330, 345, 360, 375, 390, 405, 420, 435, 450, 465, 480, 495, 510, 525, 540 or 555 consecutive nucleotides thereof).
  • a polynucleotide that comprises (or consists of) the sequence of SEQ ID NO: 4, or a fragment of said sequence having at least 30 consecutive nucleotides thereof (eg. at least 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, 210, 225, 240, 255, 270, 285, 300,
  • the polynucleotide fragment may comprise (or consist of) a nucleotide sequence having at least 70% identity to the nucleotide sequence encoding the IR2 iron reductase domain (corresponding to nucleotides 10-525 of SEQ ID NO: 4), or a fragment thereof.
  • the polynucleotide fragment may comprise (or consist of) a nucleotide sequence having at least 70% identity (eg. at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to the sequence spanning nucleotides 10-525 of SEQ ID NO: 4, or a fragment thereof having at least 150 consecutive nucleotides thereof (eg.
  • the polynucleotides, polynucleotide variants and polynucleotide fragments for use in the methods of the present invention encode polypeptides that are capable of extracting sugars from lignocellulosic biomass material that has been previously pre-treated so as to at least partially depolymerise lignin and/ or hemicellulose present in the lignocellulosic structure.
  • the polynucleotides, polynucleotide variants and polynucleotide fragments for use in the methods of the present invention encode polypeptides that are capable of extracting sugars and lignin derived products from lignocellulosic biomass material in the presence of a reducible substrate and an oxidising agent.
  • the polynucleotides, polynucleotide variants and polynucleotide fragments for use in the methods of the present invention encode polypeptides that have reductase activity and/ or polysaccharide cleavage activity.
  • the polynucleotides, polynucleotide variants and polynucleotide fragments for use in the methods of the present invention encode polypeptides that have iron reductase activity and/ or cellulase and/ or hemicellulase activity.
  • polynucleotide sequence includes nucleic acid sequences that have been removed from their naturally occurring environment, recombinant or cloned DNA isolates, and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • the polynucleotides may be prepared by any means known in the art. For example, large amounts of the polynucleotides may be produced by replication in a suitable host cell.
  • the natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell.
  • the DNA constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured insect, mammalian, plant or other eukaryotic cell lines.
  • the polynucleotides may also be produced by chemical synthesis, eg. by the phosphoramidite method or the triester method, and may be performed on commercial automated oligonucleotide synthesizers.
  • a double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • the term "recombinant" as used herein intends a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin which, by virtue of its origin or manipulation: (1 ) is not associated with all or a portion of a polynucleotide with which it is associated in nature; or (2) is linked to a polynucleotide other than that to which it is linked in nature; and (3) does not occur in nature.
  • This artificial combination is often accomplished by via conventional chemical synthesis techniques, or by the artificial manipulation of isolated segments of nucleic acids - e.g., by conventional genetic engineering techniques.
  • degenerate codon representative of all possible codons encoding each amino acid.
  • some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of the polypeptide described herein.
  • DNA compounds differing in their nucleotide sequences can be used to encode a given amino acid sequence of the disclosure.
  • the native DNA sequence encoding the polypeptides described are referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides utilised in the methods of the disclosure.
  • the invention contemplates and provides each and every possible variation of nucleic acid sequence encoding the polypeptides could be made by selecting combinations based on possible codon choices.
  • a nucleic acid sequence or fragment thereof is "identical" to a reference sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 70%, 75%, 80%, 85% 90%, 91 %, 92%, 93%, 94%, 95%, 97%, 98%, or 99% of the nucleotide bases. Identity determination is performed as described supra for polypeptides.
  • nucleic acid sequence or fragment thereof is “identical” to a reference sequence if it is capable of hybridizing under stringent (eg. highly stringent) hybridization conditions.
  • Nucleic acid sequence hybridization will be affected by such conditions as salt concentration (e.g. NaCI), temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art.
  • Stringent temperature conditions are preferably employed, and generally include temperatures in excess of 30°C, typically in excess of 37°C and preferably in excess of 45°C.
  • Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM.
  • the pH is typically between 7.0 and 8.3. The combination of parameters is much more important than any single parameter.
  • preferential codon usage refers to codons that are most frequently used in cells of a certain species, thus favouring one or a few representatives of the possible codons encoding each amino acid.
  • the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian host cells ACC is the most commonly used codon; in other species, different Thr codons may be preferential.
  • Preferential codons for a particular host cell species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species.
  • the nucleic acid sequence is codon optimized for expression in a host cell.
  • the polypeptide used in the present invention may be produced using a recombinant micro-organism expressing the polypeptide, or using other recombinant cell-types that express the polypeptide described herein (eg. because they are transformed with recombinant nucleic acids encoding the polypeptide).
  • Expression of the polypeptide from the recombinant micro-organism or cell-type is via a chimeric gene that is operably linked to regulatory sequences and encodes the polypeptide defined herein.
  • Polynucleotides encoding the polypeptide can be prepared using methods that are well known in the art, and described herein.
  • Expression vectors containing the chimeric gene may be used to transform an appropriate host cell. A skilled person will be familiar with methods for recombinant expression of proteins in host cells, and any number of expression vectors are available or can be constructed using routine methods.
  • vectors may include a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC), or a yeast artificial chromosome (YAC).
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • suitable vectors may include derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno- associated virus, retroviruses and many others.
  • Vectors may further comprise regulatory sequences, including, for example, a promoter, operably linked to the protein encoding sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art.
  • the construct may optionally include nucleotide sequences to facilitate integration into a host genome and/or results in amplification of construct copy number in vivo.
  • nucleotide sequences to facilitate integration into a host genome and/or results in amplification of construct copy number in vivo.
  • a promoter sequence may be operably linked to the 5' region of the polypeptide coding sequence. It will be recognized that in making such a construct it is not necessary to define the bounds of a minimal promoter. Instead, the DNA sequence 5' to the polypeptide gene start codon can be replaced with DNA sequence that is 5 ' to the start codon of a given heterologous gene.
  • This 5' "heterologous" sequence thus includes, in addition to the promoter elements per se, a transcription start signal and the sequence of the 5' untranslated portion of the transcribed chimeric mRNA.
  • the promoter-gene construct and resulting mRNA may comprise a sequence encoding the polypeptide described herein and a heterologous 5' sequence upstream to the start codon of the sequence encoding the polypeptide. In some, but not all, cases the heterologous 5' sequence will immediately abut the start codon of the polynucleotide sequence encoding the polypeptide.
  • gene constructs may be employed in which a polynucleotide encoding the polypeptide is present in multiple copies.
  • the polypeptide may be expressed without a signal peptide.
  • the polypeptide may be derived from the sequence defined in SEQ ID NO: 1 , which corresponds to the amino acid sequence of the IR1 polypeptide without its signal peptide; or the polypeptide may be derived from the sequence defined in SEQ ID NO: 2, which corresponds to the amino acid sequence of the IR2 polypeptide without its signal peptide.
  • the polypeptide may be expressed as a pre-protein including the naturally occurring signal peptide of the IR1 or IR2 polypeptides.
  • the polypeptide may be expressed as a pre-protein with the signal peptide of the IR1 polypeptide having the amino acid sequence 'MFSHLLTTIILSIGFRAVTWAQS' (defined in SEQ ID NO: 5).
  • the polypeptide may be expressed as a pre-protein with the signal peptide of the IR2 polypeptide having the amino acid sequence 'MFQKLLVASLLFLGIQFVNAVPN' (defined in SEQ ID NO: 6).
  • the polypeptide may be expressed as a pre-protein with a heterologous signal peptide.
  • the polypeptide may be produced using recombinant microorganisms using techniques, such as using yeast cells and filamentous fungal cells; algal cells; and prokaryotic cells, including gram positive, gram negative and gram- variable bacterial cells.
  • the recombinant micro-organism may be a fungal host cell including, but are not limited to, Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, Fungi imperfecti.
  • fungal host cells may be yeast cells and filamentous fungal cells. Filamentous fungal host cells may include all filamentous forms of the subdivision Eumycotina and Oomycota.
  • the filamentous fungal host cell may be a cell of a species of, but not limited to Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothia, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium, Volvariella, or teleomorph
  • the recombinant micro-organism may be a yeast host cell, including a cell of a species of, but not limited to Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, and Yarrowia.
  • the yeast cell may be Hansenula polymorpha, Saccharomyces cerevisiae, Saccaromyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, and Yarrowia lipolytica.
  • the recombinant micro-organism may be an algal host cell such as, Chlamydomonas (e.g., C. reinhardtii) and Phormidium (P. sp. ATCC29409).
  • algal host cell such as, Chlamydomonas (e.g., C. reinhardtii) and Phormidium (P. sp. ATCC29409).
  • the recombinant micro-organism may be a prokaryotic cell.
  • Suitable prokaryotic cells include gram positive, gram negative and gram-variable bacterial cells.
  • the host cell may be a species of, but not limited to, Agro bacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, llyobacter, Micrococcus, Microbacterium, Mes
  • the bacterial host strain may be non-pathogenic to humans.
  • the bacterial host strain may be an industrial strain. Numerous bacterial industrial strains are known and suitable in the present invention. Strains that may be used in the practice of the invention including both prokaryotic and eukaryotic strains, are readily accessible to the public from a number of culture collections such as American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
  • ATCC American Type Culture Collection
  • DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • compositions that comprises the polypeptide, as defined herein.
  • composition used throughout the present specification is intended to designate a reagent for use in the methods of the present invention which comprises the polypeptide isolated from its natural wild type S. lacrymans host (ie. when the polypeptide is not present in its natural wild type S. lacrymans host).
  • the present invention encompasses only compositions that comprise the polypeptide when it is isolated from its natural wild type host (ie. when the polypeptide is not present in its natural wild type S. lacrymans host).
  • the composition used in the methods of the present invention may comprise (or consist of) the polypeptide in the form of an isolated polypeptide.
  • the composition used in the methods of the present invention may comprise a crude, semi-purified, or purified preparation of polypeptide produced using a recombinant micro-organism or other recombinant cell-type, as described herein.
  • polypeptide produced using a recombinant micro-organism may be provided in a crude cell mass fermentation broth, which may be treated to prevent further microbial growth (for example, by heating or addition of antimicrobial agents).
  • the polypeptide produced using other recombinant cell-types may be provided in cell culture media or in a cell lysate obtained from the recombinant cells expressing the polypeptide.
  • the polypeptide need not be isolated from the culture medium (i.e., if the polypeptide is secreted into the culture medium) or cell lysate (i.e., if the polypeptide is not secreted into the culture medium) or used in a purified form to be useful.
  • Any composition, cell culture medium, or cell lysate containing the polypeptide may be suitable for use in the methods of the present invention.
  • the expressed polypeptide may be purified by, for instance, a combination of hydrophobic interaction chromatography, ion exchange chromatography and ceramic hydroxyl apatite chromatography. Other chromatographic techniques well known to the art of protein purification, such size exclusion chromatography, may be used. Polypeptide purity or homogeneity may be indicated by, for example, polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel, or using HPLC.
  • the isolated polypeptide is substantially free from other proteins with which it is co-produced as well as from other contaminants.
  • an isolated polypeptide is substantially free of material or other proteins from the cell, bacterial, or tissue source from which it was derived.
  • a “purified” molecule is substantially free of its original environment and is sufficiently pure for use in pharmaceutical compositions.
  • a substantially pure polypeptide refers to a polypeptide that is at least about 50% (w/w) pure; or at least about 60%, 70%, 80%, 85%, 90% or 95% (w/w) pure; or at least about 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.
  • the composition used in the methods of the present invention may comprise (or consist of) a recombinant micro-organism that expresses the polypeptide.
  • the polypeptide may be recombinantly expressed using one or more of the recombinant micro-organisms described herein, including one or more of the specific yeast cells and filamentous fungal cells; algal cells; and prokaryotic cells, including gram positive, gram negative and gram-variable bacterial cells described herein.
  • composition used in the methods of the present invention may comprise (or consist of) a recombinant micro-organism that expresses the polypeptide in combination with one or more additional polypeptides that facilitate extraction of sugars and lignin derived products from the lignocellulosic biomass material.
  • the recombinant micro-organism may be modified so as to express one or more enzymes that degrade the cellulose, lignin and hemicellulose components of the lignocellulosic structure.
  • the one or more enzymes may be polysaccharases including cellulases, beta-glucanases, xylanases, pectinases, alpha glucuronidases, alpha-L-arabinefuranosidases, alpha amylases, beta-amylases, glucoamylases, pullulanases, beta-glucanases, hemicellulases, arabinosidases, mannanases, pectin hydrolyases, polygalacturonases, exopolygalaturonases and/ or pectate lyases.
  • the one or more enzymes may facilitate depolymerisation of lignin, including oxidases, peroxidases, and laccases.
  • the composition used in the methods of the present invention may further comprise one or more chelating agents.
  • chelating agents available in the art.
  • the composition may further comprise 2,3 dihydroxybenzoic acid (2,3-DHBA).
  • the composition used in the methods of the present invention may further comprise 2,3 dihydroxybenzoic acid (2,3-DHBA) at a concentration from about 10 ⁇ to about 100 ⁇ .
  • the composition used in the methods of the present invention may further comprise 2,3 dihydroxybenzoic acid (2,3-DHBA) at a concentration of at least 10 ⁇ , at least 20 ⁇ , at least 30 ⁇ , at least 40 ⁇ , at least 50 ⁇ , at least 60 ⁇ , at least 70 ⁇ , at least 80 ⁇ , at least 90 ⁇ or at least 100 ⁇ .
  • composition used in the methods of the present invention may further comprise 2,3 dihydroxybenzoic acid (2,3-DHBA) at a concentration of up to 10 ⁇ , up to 20 ⁇ , up to 30 ⁇ , up to 40 ⁇ , up to 50 ⁇ , up to 60 ⁇ , up to 70 ⁇ , up to 80 ⁇ , up to 90 ⁇ or up to 100 ⁇ .
  • composition used in the methods of the present invention may further comprise 2,3 dihydroxybenzoic acid (2,3-DHBA) at a concentration of about 50 ⁇ .
  • compositions used in the methods of the present invention further improves the efficiency of the sugar and/ or lignin derived product extraction methods.
  • the present invention further provides a recombinant micro-organism that expresses a polypeptide comprising an amino acid sequence that has at least 70% identity (eg. up to
  • polypeptide is capable of extracting sugars and/or lignin derived products from lignocellulosic biomass material in the presence of a reducible substrate and an oxidising agent; and wherein said polypeptide is capable of extracting sugars from lignocellulosic biomass material that has been subjected to a pre-treatment process that at least partially depolymerises lignin and/ or hemicellulose present in the lignocellulosic structure.
  • the recombinant micro-organism expresses a polypeptide that is encoded by a polynucleotide comprising a nucleic acid sequence having at least 70% identity (eg. up to 100% identity) to the sequence of SEQ ID NO: 3 or 4, or a fragment thereof, wherein said polypeptide is capable of extracting sugars and/or lignin derived products from lignocellulosic biomass material in the presence of a reducible substrate and an oxidising agent; and wherein said polypeptide is capable of extracting sugars from lignocellulosic biomass material that has been subjected to a pre-treatment process that at least partially depolymerises lignin and/ or hemicellulose present in the lignocellulosic structure.
  • polypeptides, polypeptide variants and polypeptide fragments, and polynucleotides, polynucleotide variants and polynucleotide fragments, as described herein with respect to the methods of the present invention apply equally to said recombinant micro-organism of the present invention.
  • All embodiments of the recombinant micro-organisms as described herein with respect to the methods of the present invention apply equally to said recombinant microorganism of the present invention.
  • the present invention provides a method for extracting lignin derived products from lignocellulosic biomass material, comprising contacting the lignocellulosic biomass material with a composition comprising a polypeptide, as defined herein, in the presence of a reducible substrate and an oxidising agent, wherein said polypeptide is capable of extracting lignin derived products from lignocellulosic biomass material in the presence of a reducible substrate and an oxidising agent.
  • extracting lignin derived products refers to the process by which the polypeptide induces the depolymerisation of lignocellulosic biomass material to produce lignin derived products.
  • reducible substrate used throughout the present specification means any substrate that can be reduced by the iron reductase domain of the IR1 or IR2 polypeptide. By 'reduction', we mean the process whereby electrons are donated to the reducible substrate.
  • the reducible substrate may comprise Fe 3+ ions, such as those present in FeCI 3 .
  • Fe 3+ ions may be reduced using the iron reductase domain of IR1 or IR2 to generate Fe 2+ ions.
  • oxidising agent used throughout the present specification means any compound that is capable of accepting electrons.
  • the oxidising agent may comprise hydrogen peroxide.
  • the polypeptide has reductase activity and permits electrons to be donated to a reducible substrate, which in turn promotes the production of free radicals in the presence of an oxidising agent.
  • the polypeptide is able to induce depolymerisation of the lignocellulosic structure to produce lignin derived products.
  • the polypeptide may have iron reductase activity and may act by reducing Fe 3+ ions to generate Fe 2+ ions.
  • Fe 2+ ions promote the generation of hydroxyl free radicals (OH " ) via the Fenton Reaction: Fe 2+ + H 2 O 2 + H + ⁇ Fe 3+ + OH " + H 2 0.
  • the lignocellulosic biomass material for use in these methods may or may not have been subjected to a pre-treatment process so as to at least partially depolymerise lignin and/ or hemicellulose present in the lignocellulosic structure.
  • the polypeptide used in the method has reductase activity and is able to promote the production of free radicals that induce depolymerisation of the lignocellulosic biomass material. There is therefore no need to subject the lignocellulosic biomass material to a pre-treatment process (such as the conventional thermo-chemical methods described herein) prior to performing the method of the present invention.
  • the lignocellulosic biomass material for use in these methods may therefore comprise pre- treated or untreated lignocellulosic biomass material.
  • the present method for extracting lignin derived products from lignocellulosic biomass material is performed using a polypeptide that is capable of extracting lignin derived products from lignocellulosic biomass material in the presence of a reducible substrate and an oxidising agent.
  • the method does not require the presence of any other S. lacrymans metabolites or S. lacrymans derived compounds.
  • the method does not require the presence of intact S. lacrymans.
  • the present invention also provides methods for extracting sugars from lignocellulosic biomass material.
  • extracting sugars refers to the process by which the polypeptide induces the depolymerisation of lignocellulosic biomass material to produce sugars.
  • the present invention provides a method for extracting sugars from lignocellulosic biomass material, wherein the lignocellulosic biomass material has been subjected to a pre-treatment process that at least partially depolymerises lignin and/ or hemicellulose present in the lignocellulosic structure; the method comprising: contacting the pre-treated lignocellulosic biomass material with a composition comprising a polypeptide, as defined herein, that is capable of extracting sugars from the pre-treated lignocellulosic biomass material.
  • the polypeptide used in the method of the present invention may have 'cellulase' activity that cleaves the glycosidic bonds present in cellulose to produce sugars, such as hexose sugars.
  • Hexose sugars include six carbon member sugars or saccharides (monomers), corresponding dissacharides (dimers), corresponding trisaccharides (trimmers), corresponding tetrasaccharides (tetramers) and/or the like.
  • Hexose includes glucose, galactose, sucrose, fructose, allose, altrose, gulose, idose, mannose, sorbose, talose, tagatose, any other isomer of six carbon sugars, and/ or the like.
  • the polypeptide used in the method of the present invention may have 'hemicellulase' activity that cleaves the glycosidic bonds present in hemicellulose to produce sugars, such as pentose sugars.
  • Pentose sugars include five carbon member sugars or saccharides (monomers), corresponding dissacharides (dimers), corresponding trisaccharides (trimers), corresponding tetrasaccharides (tetramers) and/or the like.
  • Pentose includes xylose, ribose, arabinose, ribulose, xylulose, lyxose, any other isomer of five carbon sugars, and/ or the like.
  • the polypeptide may be capable of extracting sugars from the cellulose and/or hemicellulose components from the pre-treated lignocellulosic biomass material without requiring the presence of a reducible substrate and/or an oxidising agent.
  • the method of the present invention for extracting sugars from pre-treated lignocellulosic biomass may be performed (and operates) substantially in the absence of a reducible substrate and/ or an oxidising agent.
  • the lignocellulosic biomass material for use in this embodiment must have been subjected to a pre-treatment process so as to at least partially depolymerise lignin and/ or hemicellulose present in the lignocellulosic structure.
  • the phrase "operates substantially in the absence of a reducible substrate and/ or an oxidising agent” means that the reaction is performed in the presence of less than about 1 % of a reducible substrate and/ or an oxidising agent (eg. less than 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1 % or 0.01 % of a reducible substrate and/ or an oxidising agent).
  • the reaction may be performed in the presence of up to about 1 % of a reducible substrate and/ or an oxidising agent (eg.
  • the method of the present invention for extracting sugars from pre-treated lignocellulosic biomass may also be performed (and operates) entirely in the absence of a reducible substrate and/ or an oxidising agent.
  • the lignocellulosic biomass material for use in this embodiment must have been subjected to a pre-treatment process so as to at least partially depolymerise lignin and/ or hemicellulose present in the lignocellulosic structure.
  • the present method for extracting sugars from pre- treated lignocellulosic biomass material is performed using a polypeptide that is capable of extracting sugars from pre-treated lignocellulosic biomass material.
  • the method does not require the presence of any other S. lacrymans metabolites or S. lacrymans derived compounds.
  • the method does not require the presence of intact S. lacrymans.
  • the present invention provides a method for extracting sugars from lignocellulosic biomass material, comprising contacting the lignocellulosic biomass material with a composition comprising a polypeptide, as defined herein, in the presence of a reducible substrate and an oxidising agent, wherein said polypeptide is capable of extracting sugars from the lignocellulosic biomass material in the presence of a reducible substrate and an oxidising agent.
  • the polypeptide used in the methods of the present invention acts as a reductase to add electrons to a reducible substrate, which in turn promotes the production of free radicals in the presence of an oxidising agent.
  • the polypeptide By promoting the production of free radicals, the polypeptide induces depolymerisation of the lignocellulosic biomass material to produce sugars.
  • the polypeptide may have iron reductase activity and may act by reducing Fe 3+ ions to generate Fe 2+ ions.
  • Fe 2+ ions promote the generation of hydroxyl free radicals (OH " ) via the Fenton Reaction: Fe 2+ + H 2 O 2 + H + ⁇ Fe 3+ + OH " + H 2 O.
  • the polypeptide not only has reductase activity, but may additionally have 'cellulase' and/ or 'hemicellulase' activity to produce sugars from the cellulose and/ or hemicellulose components of lignocellulosic biomass material.
  • the polypeptide may additionally have 'cellulase' activity that cleaves the glycosidic bonds present in cellulose to produce sugars, such as hexose sugars.
  • the polypeptide may additionally have 'hemicellulase' activity that cleaves the glycosidic bonds present in hemicellulose to produce sugars, such as pentose sugars.
  • the lignocellulosic biomass material for use in this embodiment may or may not have been subjected to a pre-treatment process so as to at least partially depolymerise lignin and/ or hemicellulose present in the lignocellulosic structure. This is because the polypeptide used in the method has reductase activity and is able to promote the production of free radicals that induce depolymerisation of the lignocellulosic biomass material. There is therefore no need to subject the lignocellulosic biomass material to a pre-treatment process (such as the conventional thermo-chemical methods described herein) prior to performing the method of the present invention.
  • the lignocellulosic biomass material for use in these methods may therefore comprise pre-treated or untreated lignocellulosic biomass material.
  • the present method for extracting sugars from lignocellulosic biomass material is performed using a polypeptide that is capable of extracting sugars from lignocellulosic biomass material in the presence of a reducible substrate and an oxidising agent.
  • the method does not require the presence of any other S. lacrymans metabolites or S. lacrymans derived compounds.
  • the method does not require the presence of intact S. lacrymans.
  • An advantage of the methods of the present invention which relate to the extraction of sugars and/ or lignin derived products in the presence of a reducible substrate and an oxidising agent is that it is not necessary to "pre-treat" the lignocellulosic biomass material so as to at least partially depolymerise lignin and/ or hemicellulose present in the lignocellulose structure.
  • the methods of the present invention overcome the disadvantages associated with performing conventional pre-treatment processes.
  • the methods of the present invention permit lignocellulosic biomass material to be depolymerised without accumulating these 'inhibitory' by-products or breakdown products.
  • the method of the present invention may operate as a single step method.
  • the term "single step method" means that the entire method for extracting sugars and/ or lignin derived products from lignocellulosic biomass material takes place without requiring any intermediate purification or "clean-up" steps to be performed.
  • the methods of the present invention may operate in the presence of a reducible substrate that can be reduced by the iron reductase domain of IR1 or IR2.
  • the reducible substrate may comprise Fe 3+ ions.
  • the reducible substrate may comprise FeCI 3 .
  • Alternative reducible substrates will also work in the methods of the present invention and will be familiar to a person of skill in the art. These can be identified using routine methods known in the art, such as those described herein.
  • the methods of the present invention may take place in the presence of FeCI 3 at a concentration of at least 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.1 mM, 0.2mM, 0.3mM, 0.4mM or 0.5mM FeCI 3 .
  • the methods of the present invention may take place in the presence of FeCI 3 at a concentration of up to 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.1 mM, 0.2mM, 0.3mM, 0.4mM or 0.5mM FeCI 3 .
  • the methods of the present invention may take place in the presence of FeCI 3 at a concentration of from about 0.05mM to about 0.5mM.
  • the methods of the present invention may take place in the presence of 0.1 mM FeCI 3 .
  • the methods of the present invention may operate in the presence of an oxidising agent.
  • the oxidising agent used in the methods of the present invention is hydrogen peroxide.
  • Alternative oxidising agents may include any other inorganic peroxide.
  • a person of skill in the art will also be familiar with other alternative oxidising agents that will work in the methods of the present invention. These can be identified using routine methods known in the art, such as those described herein.
  • the oxidising agent will be used at a concentration sufficient to induce free radical production in the presence of a reducible substrate and the polypeptide described herein.
  • the methods of the present invention may take place in the presence of hydrogen peroxide at a concentration of at least 0.5mM, 1 mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 7.5mM, 8mM, 8.5mM, 9mM, 9.5 M or 10mM H 2 0 2 .
  • the methods of the present invention may take place in the presence of hydrogen peroxide at a concentration of up to 0.5mM,1 mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 7.5mM, 8mM, 8.5mM, 9mM, 9.5 M or 10mM H 2 0 2 .
  • the methods of the present invention may take place in the presence of hydrogen peroxide at a concentration of from about 0.5mM to about 10mM H 2 0 2 (eg.
  • the methods of the present invention may take place in the presence of hydrogen peroxide at a concentration of at least 4mM H 2 0 2 .
  • All methods of the present invention comprise the step of 'contacting' lignocellulosic biomass material with compositions that comprise a polypeptide, as defined herein.
  • the term 'contacting' used throughout the present specification refers to the placing of the polypeptide in sufficiently close proximity to the components of the lignocellulosic biomass material to enable extraction of sugars and/or lignin derived products, in accordance with the methods of the present invention.
  • 'contacting' may comprise a step of mixing or combining the lignocellulosic biomass material with the composition comprising the polypeptide.
  • the 'contacting' step takes place in a solution.
  • the 'contacting' step is not limited only to a mixing or combining step in which the polypeptide directly contacts or interacts with components of the lignocellulosic biomass material, but also includes a mixing or combining step in which the polypeptide is in solution with but spatially separated from components of the lignocellulosic biomass material (ie. the polypeptide does not necessarily need to come into direct contact or interact directly with components of the lignocellulosic biomass material in order to effect the 'contacting' step).
  • the 'contacting' step of the method involves mixing or combining lignocellulosic biomass material with a culture of the recombinant micro-organism expressing the polypeptide.
  • the 'contacting' step of the method involves mixing or combining lignocellulosic biomass material with the isolated polypeptide.
  • the methods of the present invention may be performed using conditions suitable for maintaining the enzymatic activities of the polypeptide.
  • the reaction conditions used in the methods of the present invention may be suitable for maintaining the polysaccharide cleavage activity of the polypeptide.
  • reaction conditions used in the methods of the present invention may be suitable for maintaining the 'cellulase' and/ or 'hemicellulase' activity of the polypeptide.
  • reaction conditions used in the methods of the present invention may be suitable for maintaining the reductase activity of the polypeptide, such as the iron reductase activity described herein.
  • the methods may take place under mild conditions that do not include extreme heat or acid treatment. In one embodiment, the methods may take place at a temperature of from about 20°C to about 70°C, and at a pH range from about pH 4.5 to about pH 9.
  • the methods of the present invention may take place at a temperature of at least 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C or 70°C.
  • the methods of the present invention may take place at a temperature of up to 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C or 70°C.
  • the methods of the present invention may take place at a temperature of from about 20°C to about 70°C (eg.
  • the methods of the present invention take place at a temperature of at least 50°C.
  • the methods of the present invention may take place at a temperature of up to 50°C.
  • the methods of the present invention may take place at a pH of at least pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0, pH 8.5 or pH 9.0.
  • the methods of the present invention may take place at a pH of up to pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0, pH 8.5 or pH 9.0.
  • the methods of the present invention may take place at a pH of from about pH 4.5 to about pH 9.0 (eg.
  • the methods of the present invention may be performed at a least pH 7.5.
  • the methods of the present invention may be performed at up to pH 7.5.
  • the methods of the present invention may take place from several minutes to several hours. In one embodiment, the methods may take place from about 6 hours to about 120 hours, such as from about 6 hours to about 48 hours, from about 6 to about 24 hours, or for about 6 hours. In one embodiment, the methods may take place for at least 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours or 120 hours. In one embodiment, the methods of the present invention may take place for at least 24 hours.
  • the methods of the present invention may be performed using conditions suitable for maintaining a culture of the recombinant micro-organism expressing the polypeptide, such as under suitable temperature, pH, and/ or culture media formulations. Reaction conditions will vary depending on the micro-organism in question, and will be familiar to a person skilled in the art.
  • the methods of the present invention take place under sterile reaction conditions, such as those routinely used in the manufacture of biofuels.
  • the methods of the present invention are performed under reaction conditions that result in release of substantial amounts of sugar and/ or lignin derived products from the lignocellulosic biomass material.
  • substantial amount is intended at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of available sugars and/ or lignin derived products.
  • the present invention further provides the use of a polypeptide for extracting sugars and/or lignin derived products from lignocellulosic biomass material in the presence of a reducible substrate and an oxidising agent, wherein the polypeptide comprises an amino acid sequence that has at least 70% identity (eg. up to 100% identity) to the amino acid sequence of SEQ ID NO: 1 or 2, or a fragment thereof; and wherein said polypeptide is capable of extracting sugars and/or lignin derived products from the lignocellulosic biomass material in the presence of a reducible substrate and an oxidising agent.
  • the polypeptide that is capable of extracting sugars and lignin derived products from the lignocellulosic biomass material in the presence of a reducible substrate and an oxidising agent is encoded by a polynucleotide that comprises (or consists of) a sequence having at least 70% identity (eg. up to 100% identity) to the sequence of SEQ ID NO: 3 or 4, or a fragment of said sequence.
  • the present invention further provides the use of a polypeptide for extracting sugars from lignocellulosic biomass material that has been subjected to a pre-treatment process that at least partially depolymerises lignin and/ or hemicellulose present in the lignocellulosic structure; wherein the polypeptide comprises an amino acid sequence that has at least 70% identity (eg. up to 100% identity) to the amino acid sequence of SEQ ID NO: 1 or 2, or a fragment thereof; and wherein said polypeptide is capable of extracting sugars from the pre-treated lignocellulosic biomass material.
  • the polypeptide that is capable of releasing sugars from pre-treated lignocellulosic biomass material is encoded by a polynucleotide that comprises (or consists of) a sequence having at least 70% identity (eg. up to 100% identity) to the sequence of SEQ ID NO: 3 or 4, or a fragment of said sequence.
  • the invention further provides a method for the production of high-value chemicals comprising carrying out any one of the methods of the present invention for extracting sugars and/ or lignin derived products from lignocellulosic biomass material, and subsequently using the extracted sugars and/ or lignin derived products to produce high- value chemicals.
  • the term 'high-value chemicals' refers to compounds or products that have a high value relative to the value of the starting materials used to produce the compounds or products.
  • a skilled person will be familiar with the types of high-value chemicals that can be obtained from sugars and lignin derived products that have been extracted from lignocellulosic biomass material.
  • Non- limiting examples include high value chemicals such as eugenol, syringols, coniferols, guaiacols, wood preservatives and nutraceuticals/ drugs.
  • the invention further provides methods for the production of biofuels, such as biogasoline, bioethanol and biodiesel from lignocellulosic biomass material.
  • the invention provides a method for the production of biogasoline comprising carrying out any one of the methods of the present invention for extracting sugars and/ or lignin derived products from lignocellulosic biomass material, and subsequently producing biogasoline using the extracted sugars and/ or lignin derived products.
  • biogasoline used throughout the present specification refers to a gasoline product containing non-oxygenated hydrocarbons that is produced from biomass material.
  • the invention may provide a method for producing biogasoline that comprises carrying out any one of the methods of the present invention for extracting sugars from lignocellulosic biomass material, subsequently fermenting the extracted sugars to produce fermentation end products, and using the fermentation end products to produce biogasoline.
  • fermentation or “fermenting” used throughout the present specification refer to an enzyme controlled aerobic or anaerobic breakdown of an energy-rich compound, such as a carbohydrate to carbon dioxide and an alcohol or an organic acid. Fermentation may also include an enzyme controlled transformation of an organic compound.
  • Fermentation processes may be performed using yeast, bacteria, cyanobacteria, algae, and/ or enzymes to produce fermentation end products, such as alcohol or oxygen containing compounds.
  • fermentation may be performed using naturally occurring hexose or pentose consumers and/ or genetically modified hexose or pentose consumers.
  • Naturally occurring organisms may produce alcohols or other oxygen containing compounds, such as may be used directly or may be converted to an ether.
  • the fermentation end products are then further processed using conventional techniques to produce biogasoline.
  • the fermentation end products may be converted into non-oxygenated hydrocarbons using conventional chemical processing techniques.
  • the biogasoline may be optionally blended with a gasoline supply.
  • the invention may provide a method for producing biogasoline that comprises carrying out any one of the methods of the present invention for extracting lignin derived products from lignocellulosic biomass material, and subsequently producing biogasoline from the lignin derived products.
  • Conventional methods may be used to produce biogasoline using lignin derived products.
  • gasification may be used to convert lignin into syngas (carbon monoxide/ hydrogen), which in turn is used to prepare methanol and/ or dimethyl ether, from which biogasoline can be prepared.
  • Pyrolysis and hydroliquefaction processes may also be used to produce biogasoline from lignin derived products.
  • the invention further provides a method for the production of bioethanol comprising carrying out any one of the methods of the present invention for extracting sugars from lignocellulosic biomass material, subsequently fermenting the extracted sugars to produce ethanol, and processing the ethanol into bioethanol.
  • bioethanol used throughout the present specification refers to the biofuel product containing ethanol that is produced from biomass material.
  • ethanol obtained following fermentation may be subjected to fractional distillation and/ or dehydration processes to remove excess water.
  • the ethanol may be optionally blended with a gasoline supply.
  • a skilled person will be aware of the conventional techniques used to process ethanol into bioethanol.
  • the invention further provides a method for the production of biodiesel comprising carrying out any one of the methods of the present invention for extracting sugars and/ or lignin derived products from lignocellulosic biomass material, and subsequently producing biodiesel using the extracted sugars and/ or lignin derived products.
  • biomass used throughout the present specification refers to the diesel product containing long-chain alkyl esters that is produced from biomass material.
  • conventional processes may be used to convert the extracted sugars and/ or lignin derived products into biodiesel.
  • the extracted sugars may be converted into one or more suitable biodiesel materials, such as triglycerides, fatty acids, alkanes, alkenes, and/or pure hydrocarbons, from which long-chain alkyl esters can be produced.
  • fermentation may be used to convert the extracted sugars into one or more suitable biodiesel materials. All embodiments of the fermentation process described above with respect to the production of biogasoline apply equally to this embodiment of the invention.
  • pentose sugars obtained from hydrolysis of hemicellulose may be processed into fatty acids using naturally occurring pentose consumers or genetically modified pentose consumers.
  • Naturally occurring organisms may produce fatty acids, which may be esterified with an alcohol and/ or hydrogenated with hydrogen to produce long-chain alkyl esters suitable for forming a biodiesel product.
  • Lignin derived products may be converted into biodiesel using processes such as pyrolysis.
  • a skilled person will be aware of alternative conventional processes for converting extracted sugars and/ or lignin derived products into biodiesel.
  • the biodiesel may be optionally blended with a diesel supply.
  • SEQ ID NO: 1 Amino acid sequence of IR1 derived polypeptide (without signal peptide)
  • SEQ ID NO: 2 Amino acid sequence of IR2 derived polypeptide (without signal peptide)
  • SEQ ID NO: 3 Nucleotide sequence encoding IR1 derived polypeptide (without signal peptide)
  • SEQ ID NO: 4 Nucleotide sequence encoding IR2 derived polypeptide (without signal peptide)
  • SEQ ID NO: 5 signal peptide of IR1
  • SEQ ID NO: 6 signal peptide of IR2
  • SEQ ID NO: 7 AttB1-IR1plusSP-F primer
  • SEQ IS NO: 8 AttB2-IR1minSTP-R
  • SEQ ID NO: 9 AttB1-IR2minSP-F
  • SEQ ID NO: 10 AttB2-IR2plusSTP-R
  • SEQ ID NO: 12 AttB2-adapter-R
  • SEQ ID NO: 13 M13 primer
  • SEQ ID NO: 14 M13 primer SEQ ID NO: 1 (IR1 Polypeptide without signal peptide)
  • Heme domain (underlined) - corresponds to residues 4-175
  • Cellulose binding domain (bold and italics) - corresponds to residues 202-229
  • Heme domain (underlined) - corresponds to residues 4-175
  • SEQ ID NO: 3 Polynucleotide encoding the IR1 polypeptide without signal seguence
  • Heme domain (underlined) - corresponds to nucleotides 10-525
  • Cellulose binding domain (bold and italics) - corresponds to nucleotides 604-687
  • SEQ ID NO: 4 Polynucleotide encoding the IR2 polypeptide without signal seguence
  • Heme domain (underlined) - corresponds to nucleotides 10-525
  • Figure 1 shows the domain structure of the (a) IR1 and (b) IR2 enzymes encoded by Serpula lacrymans.
  • the IR1 and IR2 proteins both contain a cellulose binding domain family 9-like (IPR008980) / (iron reductase domain (IPR015920); IR1 also has a cellulose binding module-1 (CBM1 - PDOC00486) domain.
  • Figure 2 shows the domain structure of the (a) IR1 and (b) IR2 enzymes encoded by Serpula lacrymans.
  • the IR1 and IR2 proteins both contain a cellulose binding domain family 9-like (IPR008980) / (iron reductase domain (IPR015920); IR1 also has a cellulose binding module-1 (CBM1 - PDOC00486) domain.
  • Figure 2 shows the domain structure of the (a) IR1 and (b) IR2 enzymes encoded by Serpula lacrymans.
  • Figure 2 shows the results of a western blot analysis performed on purified recombinant protein GST-IR1 and GST-IR2 by SDS-PAGE gels, as described in Example 2.
  • Figure 3 shows the results of a western blot analysis performed on purified recombinant protein GST-IR1 and GST-IR2 by SDS-PAGE gels, as described in Example 2.
  • Figure 3 shows the results obtained from the Ferrozine spot assay performed using extracts of IR1 and IR2 protein, as described in Example 3.
  • Figure 4 shows absorbance at 540nm observed for the dichlorophenol indophenol (DCPIP) assay performed using extracts of IR1 and IR2 protein over a 15 minute period, as described in Example 4.
  • Figure 5 shows absorbance at 540nm observed for the dichlorophenol indophenol (DCPIP) assay performed using extracts of IR1 and IR2 protein over a 15 minute period, as described in Example 4.
  • Figure 5 shows absorbance at 540nm observed for the dichlorophenol indophenol (DCPIP) assay performed using extracts of IR1 and IR2 protein over a 15 minute period, as described in Example 4.
  • DCPIP dichlorophenol indophenol
  • Figure 5 shows the mean change of absorbance at 540nm observed for the dichlorophenol indophenol (DCPIP) assay performed using extracts of IR1 and IR2 protein over a 15 minute period, as described in Example 4.
  • DCPIP dichlorophenol indophenol
  • Figure 6 shows absorbance at 430nm observed for the nitrated lignin assay performed using extracts of IR1 and IR2 protein over a 20 minute period, as described in Example 5.
  • Figure 7 shows absorbance at 430nm observed for the nitrated lignin assay performed using extracts of IR1 and IR2 protein over a 20 minute period, as described in Example 5.
  • Figure 7 shows the mean change of absorbance at 430nm observed at 1 and 20 minutes for the nitrated lignin assay performed using extracts of IR1 and IR2 protein, as described in Example 5.
  • Figure 8 shows the amount of total reducing sugars extracted from Avicel and wheat straw following 24 hours incubation with recombinant IR1 and IR2.
  • IR1 and IR2 sequences were amplified from RNA collected from Serpula lacrymans. The RNA was collected after 28 days of culture showing that these polypeptides are expressed in the later stage of Serpula lacrymans growth. Amplified products were cloned and the nucleotide sequences and predicted translated amino acid sequence of IR1 and IR2 were aligned with the sequences of other fungal genes. BLASTN alignment with the NCBI databases found no significant alignments with previously described nucleotide sequences. However, comparison of the iron reductase amino acid sequence by BLASTP showed that IR1 shares high identity (74.2%) with the amino acid sequence of carbohydrate binding module (CBM1 ) of different fungi e.g.
  • CBM1 carbohydrate binding module
  • Coniophora souna (accession number EIW84939), 65.9% identity with that from carbohydrate binding cytochrome (CBcyt b562) from Stereum hirsutum (accession number EIM89944.1 ), 61 .4% identity with cellulose binding cytochrome b562 from Phanerochaete chrysosporium (accession number BAD95668), and 59.8% identity with IR2 (protein ID: 417465).
  • a phylogenetic tree was constructed based on the amino acid sequences for IR1 , IR2 and 14 other fungal genes encoding either cellobiose dehydrogenases (CDH) or cellulose/ carbohydrate binding modules (CBM).
  • CDH cellobiose dehydrogenases
  • CBM cellulose/ carbohydrate binding modules
  • IR1 and IR2 genes were found to contain a cellulose binding (CBD) 9 family cytochrome domain (also referred to as a heme domain).
  • CBD cellulose binding
  • the IR1 gene was also found to contain a C-terminal cellulose binding module (CBM1 ) (see Figure 1 ).
  • IR1 and IR2 contain certain conserved amino acids. These included: a methionine at amino acid residue '65' of the IR1 sequence (as defined in SEQ ID NO: 1 ) and amino acid residue '65' of the IR2 sequence (as defined in SEQ ID NO: 2) and a histidine at amino acid residue '165' of the IR1 sequence (as defined in SEQ ID NO: 1 ) and amino acid residue '165' of the IR1 sequence (as defined in SEQ ID NO: 2).
  • Cysteine residues are also present at amino acid residues '6', '13', '120' and '123' of the IR1 sequence (as defined in SEQ ID NO: 1 ) and at amino acid residues '6', '13', '120' and '123' of the IR2 sequence (as defined in SEQ ID NO: 2).
  • cellulose binding domains normally only contain one aromatic residue, such as tyrosine (Tomme et al., 1998).
  • the recombinant protein IR1 contains three aromatic residues including a tryptophan at amino acid residue '210' of the IR1 sequence (as defined in SEQ ID NO: 1 ), and a tyrosine at amino acid residues '229' and '230' of the IR1 sequence (as defined in SEQ ID NO: 1 ).
  • the brown rot basidiomycete Serpula lacrymans S7.3 was obtained from the culture collection of Warwick HRI (School of Life Sciences) and grown in the dark on malt extract agar (MEA) plate at 20°C for 3-4 weeks.
  • PCR Polymerase Chain Reaction
  • IR1 and IR2 were amplified using primers, designed from the sequence from the Serpula lacrymans genome (Table 1 ). All primers were ordered from INVITROGEN. 5 g RNA and 2 ⁇ Oligo dT18 (Invitrogen) were denatured at 65°C for 5min and cooled on ice for 2 minutes.
  • the 5x cDNA synthesis mix (0.1 M DTT (Invitrogen), IxSuperScript Buffer (Invitrogen), 10mM dNTPs (Invitrogen), x1 RNaseOUT (Invitrogen), x1 Superscript RT (Invitrogen) and DEPC- H 2 O) were added and the following cycles was completed: 1 cycle of 96°C (5min); 30cycles of 95°C (20sec), 59°C(20sec), 73°C(40sec) and 1 cycle of 73°C(10min). The cDNA was then stored at -20°C.
  • the constructs then were transformed into E. coli (DH5a cells) and the transformants were cultured overnight at 37°C in LB agar plate containing 5C ⁇ g/ml kanamycin (KAN) selective media. Plasmid DNA from positive colonies was isolated using the alkaline lysis miniprep method (QIAGEN Plasmid Mini Purification protocol). The presence of a fragment of the correct size was confirmed by PCR using gene specific primers (see Table 1 ) or plasmid specific M13 Forward and M13 Reverse primers (see Table 2). The yield of DNA was determined using a UV spectrophotometer (Nano-drop) and by quantitative analysis on a 1.2% agarose gel.
  • Sequencing reactions were performed using the ABI BigDye terminator V.1.1/3.1 seq Kit. Each reaction contained a vector specific primer (3.2pmol), 2 ⁇ ready reaction mix (Big dye V3.1 ), 1 ⁇ big dye sequencing buffer and 1 ⁇ of plasmid cDNA (100ng) samples. Each reaction was made up to 10 ⁇ with pure distilled water. PCR cycles (25) were as followed: 96°C for 10 sec, 50°C for 5 sec and 60°C for 4 min. The product were then analysed using a ABI3130xl sequencer at the School of Life Sciences- Wellesbourne campus.
  • Example 2 Expression and purification of the recombinant protein in E. coli (BL21 )
  • Figure 2 shows the western blotting results obtained for purified GST-IR1 Figure 2(a) and purified GST-IR2 Figure 2(b).
  • the arrow indicates a band corresponding to recombinant IR1 having a molecular weight of 55kDa.
  • Lanes 1 and 2 correspond to the flow through fraction; lane 3 and 4 correspond to the wash fractions; lanes 5-7 correspond to the elute fractions; lane 8 is empty; and lane 9 corresponds to further elute fractions.
  • the arrow indicates a band corresponding to recombinant IR2 having a molecular weight of 49kDa IR2.
  • Lanes 1 and 2 correspond to the flow through fraction; lanes 3-7 correspond to the wash fractions; and lanes 8-9 correspond to the elute fractions.
  • the Invitrogen Gateway Cloning system (www.invitrogen.com) was used to clone the coding regions of IR1 (SEQ ID NO: 3) and IR2 (SEQ ID NO: 4) genes.
  • This system uses site-specific recombination attB x attP ⁇ attL x attR of a phage, which is schematically presented below: affB1 -gene-affB2 x affP1 -ccc/B-affP2 ⁇ affL1 -gene-affl_2 x affR1 -ccc/B-affR2 (Expression clone) (pDONR) (Entry clone) (Destination vector)
  • the major steps of the Gateway cloning system are the BP and LR reactions.
  • the attB x attP reaction is mediated by Gateway BP clonase II enzyme mix, while the attV. x attR reaction is mediated by Gateway LR clonase II enzyme mix.
  • the BP reactions utilize the recombination between attB of the DNA segment of interest and attP of the donor to create entry clones.
  • IR1 and IR2 fragments with a plus stop codon and without the signal peptide were PCR amplified from plasmids containing the IR1 and IR2 genes. Primers used for PCR amplification are as defined in Table 1 .
  • the first and second steps of the gateway system were carried out as follows. 2 ⁇ (10mM) primers, 2 ⁇ plasmid cDNA (1 OOng), 25 ⁇ taq DNA polymerase were mixed with 21 ⁇ pure water. The PCR reaction was performed using 1 cycle of 94°C (3 min), followed by 5 cycles of denaturation (30sec at 94°C), annealing (30sec at 55°C) and extension (1 .5min at 72°C) and then 25 cycles: 94°C for 30sec, 65°C for 30sec and 72°C for 1 .5min, and a final extension at 72°C for 7 min.
  • BP reactions The product of the recombination reactions (BP reactions) was used to transform competent DH5a E. coli using heat shock. Positive transformants were cultured overnight at 37°C in 5ml LB containing antibiotic selection 30 pg ml "1 Zeocin. The plasmid was extracted using the Qiagen plasmid mini-prep kit. Performing the LR Recombination Reaction
  • pDEST15 is N-terminal fusion vectors which contain an ATG initiation codon upstream of GST tag.
  • the product of recombination of LR reaction was transformed into the DH5a E. coli strain. Positive transformants were cultured overnight at 37°C in 5ml LB containing antibiotic selection 30pg/ml Zeocin. The plasmid was then purified using the Qiagen plasmid mini-prep kit.
  • each sequencing reaction contains 3.2pmol primers, 2 ⁇ ready raction mix (Big dye V3.1 ), 1 ⁇ big dye sequencing buffer and 1 ⁇ of BP product.
  • Each reaction was made up to 10 ⁇ with pure distilled water, and the sequencing condition were as followed: 1 cycles 96°C for 2min; 35cycles: 96°C for 10 sec, 50°C for 10sec and 60°C for 3 min. These were sequenced using an ABI3130XL.
  • the transformant colonies were inoculated into 10ml of LB medium containing the selective antibiotics 50pg/ml carbenicellin and 34pg/ml chloramphenicol and grown overnight at 37°C with shaking 220rpm. 2.5ml of overnight culture was inoculated into 50ml of prewarmed LB media (with antibiotics) on the shaking incubator (220 rpm for approximately 1 .5 hours), until the OD 6 oo is 0.5— 0.7.
  • the transformants were induced using 0.4mM of isopropyl-p-D-thiogalactopiranoside (IPTG) and the culture incubated at 30°C for an additional 5-6 hours. 1 ml induced samples were collected in different time points; 0, 3, 5, 12 and 20 hours.
  • the cell pellet was resuspended in 1 ml of lysis buffer containing 50mM Tris-HCI pH 8; 1 mM EDTA pH 8,0; 1 mM tris2 carboxyethyl-phosphine (TCEP); 1 mM phenyl methylsulfonyl- fluoride (PMSF); 200mM NaCI, and deionized water (dH 2 0).
  • the cell pellet was frozen using liquid nitrogen and thawed in cold water.
  • the cells were then sonicated for 6 x 10sec with 10 sec pauses at 200-300W and the lysate was centrifuged at 5000 x g at 4°C for 20m in. The supernatant was obtained (ie. 'the crude extract') and used for protein analysis.
  • Western blotting was carried out using standard protocols.
  • the protein was transferred onto nitrocellulose membrane for 1 .5 hour and treated for 2-3 hours at room temperature using 5% skimmed milk as the blocking agent.
  • the membrane was then incubated overnight at 4°C with primary antibody (monoclonal anti-GST antibody (SIGMA G-1 160)) at a dilution of 1 :2000.
  • the membrane was washed three times using PBST (Phosphate Buffer Saline with Tween 20) for 5-10min.
  • the membrane was then incubated with secondary antibody (anti-GST antibody- peroxidase conjugate produced in mouse (SIGMA-A4416)) at a dilution of 1 : 10,000). Secondary antibody was incubated for 2 hours at room temperature.
  • the blot was washed three to five times for 15 minutes using buffer PBST. The blot was then incubated with ECL (Enhanced chemiluminesence) Western blotting detection reagents (according to manufacture instructions from Amersham) for 5 minutes at room temperature. Analysis of the blot was performed using a hyperprocessor machine.
  • ECL Enhanced chemiluminesence
  • 500ml LB medium was prepared for the purification of recombinant protein (IR1 and IR2) as described above. All of the protein purification was undertaken at 4°C. The supernatant was centrifuged at 5000 x g for 20 minutes, 4°C using a SORVALL RC 5B and resuspended in lysis buffer (containing 50mM Tris-HCI pH 8; 1 mM EDTA pH 8,0; 1 mM tris2 carboxyethyl-phosphine (TCEP); 1 mM phenyl methylsulfonyl- fluoride (PMSF); 200mM NaCI, and deionized water (dH 2 0)).
  • lysis buffer containing 50mM Tris-HCI pH 8; 1 mM EDTA pH 8,0; 1 mM tris2 carboxyethyl-phosphine (TCEP); 1 mM phenyl methylsulfonyl- fluoride (PMSF);
  • the cells were lysed using a combination of freeze thaw and sonication. Lysed cells were then centrifuged at 13,000 x g for 10 minutes at 4°C, and the supernatant was collected for purification.
  • the soluble fractions of recombinant protein (IR1 and IR2) were purified using the Glutathione Sepharose 4B beads (GE Healthcare, UK) according to the manufacturer's instructions.
  • the crude cell extract was passed through a column pre-equilibrated with binding buffer PBS pH 7.5 (140mM NaCI, 2.7mM KCI, 10mM Na 2 HP0 , and 1 .8mM KH 2 P0 ).
  • the columns were prepared according to the manual (Glutathione Separose 4B, 52-2303-00 AK). After extensive washing using binding buffer, the GST fusion proteins were eluted with elution buffer (50mM Tris- HCI, 20mM reduced glutathione, pH 8.0).
  • the IR proteins were both predicted to have iron reducing activity due to the presence of the CBD9 (iron reductase) domain.
  • the Ferrozine spot assay was used to detect the release of Fe 2+ following reduction of Fe 3+ .
  • 50 ⁇ of 2,3 dihydroxybenzoic acid (2,3- DHBA) was used as a positive control for the assay, and shown to significantly increase absorbance.
  • Figure 3 illustrates the results of the Ferrozine spot assay. Changes in absorbance at 550nm were observed following the addition of recombinant protein IR1 or IR2 or in the presence of the positive control 2,3 dihydroxybenzoic acid (2,3-DHBA) after 30 minutes of incubation. No significant change in absorbance was observed for the negative control buffer without 2,3-DHBA. The error bars represent the least significant different (LSD 5%).
  • the experiment was conducted in 96-well micro titer plates. 50 ⁇ of crude extract/supernatant from soluble fusion protein of IR1 and IR2 cultures were combined with 0.1 mM FeCI 3, 1 M acetate buffer pH4.6, in the presence and absence of 50 ⁇ 2,3 dihydroxybenzoic-acid (DHBA). After 10 minutes incubation 10 ⁇ Ferrozine reagent was added to the reaction. The absorbance was measured at 550nm using a spectrophotometer TECAN-Genious plate reader for 30 minutes kinetically.
  • DHBA 2,3 dihydroxybenzoic-acid
  • Example 4 The ability of iron reductases to act as an electron acceptor as measured by the effect of 2,3-DHBA in the presence of H 2 0 2
  • DCPIP dichlorophenol indophenol
  • Figure 4 shows the results obtained from the DCPIP assay, measuring absorbance at 540nm over a period of 15 minutes.
  • the DCPIP assay was performed in the presence of IR1 (with and without 2,3 DHBA) and IR2 (with and without 2,3 DHBA).
  • Negative controls used in the assay include E.Coli (with and without 2,3 DHBA) and buffer (with and without 2,3 DHBA).
  • Figure 5 shows the mean change in absorbance observed at 540nm for the DCPIP assay following 15 minutes of incubation.
  • the DCPIP assay was performed in the presence of IR1 (with and without 2,3 DHBA) and IR2 (with and without 2,3 DHBA).
  • Negative controls used in the assay include E.Coli (with and without 2,3 DHBA) and buffer (with and without 2,3 DHBA). The error bars represent the least significant different (LSD 5%).
  • DCPIP assay performed was a modified version of the methods described in Baminger et al., (1999) and Nakagame et ai, (2006) (Baminger et al. A simple assay for measuring cellobiose dehydrogenase activity in the presence of laccase; Journal of Microbiological Methods; 1999; 35; 253-259; and Nakagame, S. et al; Purification and characterization of cellobiose dehydrogenase from white-rot basidiomycete Trametes hirsata; Bioscience Biotechnology and Biochemistry; 2006; 70; 1629-1635).
  • Recombinant enzyme activity was determined at room temperature using 0.1 M 2,6- dichlorophenol-indophenol (DCPIP; Sigma-Aldrich) as an electron acceptor in two different buffers 50mM sodium acetate buffer (pH 5) and 50mM tris-HCL (pH 7.5) with cellobiose as the substrate.
  • the reaction mixture was prepared in a total volume 200 ⁇ and contained 10 ⁇ of recombinant IR1 or IR2 protein, 40 ⁇ of 0.6mM cellobiose, 10 ⁇ of Fe 3+ (Ferric chloride), 10 ⁇ 2,3 dihydroxyl-benzoic acid (2,3 DHBA), 10 ⁇ of 4mM H 2 0 2 and 10 ⁇ 0.5mM DPCIP.
  • Reducing activity was measured by following decrease in absorbance of the electron acceptor DCPIP.
  • the decrease in absorbance of DPCIP was monitored using kinetic spectrophotometry at 540nm every minute from the first 60s until 30 minutes. Absorbance was measured using a spectrophotometer TECAN GENious plate reader. The assay was performed also in the absence of cellobiose, 2,3-DHBA and recombinant proteins. All readings were taken in quadruplicate.
  • Example 5 The ability of iron reductases to degrade nitrated lignin In order to demonstrate the role of IR1 and IR2 in the depolymerisation of lignin, the nitrated lignin assay was performed, and the release of phenolic compounds was measured.
  • IR1 and IR2 were tested by incubating IR1 or IR2 (with and without 2,3-DBHA) in the presence of iron (Fe 3+ ) and hydrogen peroxide (H 2 0 2 ). An increase in the absorbance at 430nm was observed in all cases after 20 minutes of incubation. However, IR1 showed greater activity than IR2 in the presence and absence of 2,3-DHBA, which indicates IR1 under these conditions has a greater potential to degrade nitrated lignin compared to IR2. After 20 minutes of incubation, IR2 only showed a significant difference in absorbance at 430nm when it was added in the presence of both Fe 3+ and 2,3-DHBA. The presence of 2,3-DHBA appeared to be additive increasing the potency of both IR1 and IR2.
  • the nitrated lignin assay was performed in the presence of IR1 (with and without 2,3 DHBA) and IR2 (with and without 2,3 DHBA).
  • Figure 7 shows the mean change in absorbance observed at 430nm for the nitrated lignin assay following 1 and 20 minutes of incubation.
  • the nitrated lignin assay was performed in the presence of IR1 (with and without 2,3 DHBA) and IR2 (with and without 2,3 DHBA).
  • the error bars represent the least significant different (LSD 5%).
  • a stock solution of nitrated organosolve lignin was prepared from the mixture of 25mg organosolve lignin with 5ml of glacial acetic acid (80mM of organosolv lignin in glacial acteic acid). The solution was filtered to remove insoluble material. The solution was then added to 750 ⁇ of concentrated nitric acid (HN0 3 ) and stirred on ice for 1 hour. The reaction was neutralized with 1 M NaOH at pH 7.0 and added to 10ml of H 2 0. The nitrated lignin stock solution was stored at 4-5°C and diluted 25- fold in deionized H 2 0.
  • the nitrated lignin assay was performed using a method modified from Ahmad et al., (2010), using Fe 3+ as a substrate (Ahmad, M. et al.; Development of novel assays for lignin degradation: comparative analysis of bacterial and fungal lignin degraders; 2010; Molecular Biosystems; 6; 815-821 ).
  • the DNS (Dinitrosalicylic Acid) assay was used to demonstrate that IR1 and IR2 proteins have the capacity to degrade cellulose into its component sugars.
  • the assay was performed in the presence of either IR1 or IR2 and using cellulose in the form of purified cellulose (Avicel) or in the form of lignocellulosic cellulose (wheat straw powder). Both IR1 and IR2 enzymes were observed to degrade both forms of cellulose into their component sugars (see increase in absorbance in Figure 8).
  • Figure 8 shows the results observed for the DNS (Dinitrosalicylic Acid) assay.
  • DNS Dinitrosalicylic Acid
  • the total amount of reducing sugars (pg/ml) released following 1 hour and 24 hours incubation of Avicel or straw with IR1 or IR2 is shown.
  • a negative control containing buffer was included in the assay.

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Abstract

La présente invention concerne un procédé pour l'extraction de sucres et/ou de produits dérivés de la lignine à partir d'un matériau de biomasse lignocellulosique consistant à mettre en contact le matériau de biomasse lignocellulosique avec une composition comprenant un polypeptide, en présence d'un substrat réductible et d'un agent oxydant, ledit polypeptide comprenant une séquence aminoacide présentant une identité d'au moins 70 % avec la séquence aminoacide SEQ ID Nº : 1 ou 2 ou un fragment de celle-ci. Ledit polypeptide est capable d'extraire des sucres et/ou des produits dérivés de la lignine à partir du matériau de biomasse lignocellulosique en présence d'un substrat réductible et d'un agent oxydant.
PCT/GB2016/052219 2015-07-21 2016-07-21 Procédés d'extraction de sucres et de produits dérivés de la lignine à partir d'un matériau de biomasse lignocellulosique WO2017013438A1 (fr)

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* Cited by examiner, † Cited by third party
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CN108680657A (zh) * 2018-03-23 2018-10-19 中国农业科学院北京畜牧兽医研究所 蒸汽爆破技术降低秸秆中玉米赤霉烯酮含量的方法及应用
WO2022221209A1 (fr) * 2021-04-11 2022-10-20 National Technology and Engineering Solutions of Sandia, LLC Procédés de fenton à médiation par un chélateur (cmf) pour modifier la lignine
CN116676800A (zh) * 2023-07-14 2023-09-01 北京联新望达科技有限公司 一种利用分子筛从木质纤维素原料中去除木质素的方法及其反应装置
US11932557B2 (en) 2020-06-30 2024-03-19 University Of Kentucky Research Foundation Detection and extraction of plastic contaminants within water using hydrophobic deep eutectic solvents

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108680657A (zh) * 2018-03-23 2018-10-19 中国农业科学院北京畜牧兽医研究所 蒸汽爆破技术降低秸秆中玉米赤霉烯酮含量的方法及应用
US11932557B2 (en) 2020-06-30 2024-03-19 University Of Kentucky Research Foundation Detection and extraction of plastic contaminants within water using hydrophobic deep eutectic solvents
WO2022221209A1 (fr) * 2021-04-11 2022-10-20 National Technology and Engineering Solutions of Sandia, LLC Procédés de fenton à médiation par un chélateur (cmf) pour modifier la lignine
CN116676800A (zh) * 2023-07-14 2023-09-01 北京联新望达科技有限公司 一种利用分子筛从木质纤维素原料中去除木质素的方法及其反应装置

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