WO2017108431A1 - Procédé de production de pâte mécanique à partir d'une biomasse comprenant un matériau lignocellulosique - Google Patents

Procédé de production de pâte mécanique à partir d'une biomasse comprenant un matériau lignocellulosique Download PDF

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WO2017108431A1
WO2017108431A1 PCT/EP2016/080259 EP2016080259W WO2017108431A1 WO 2017108431 A1 WO2017108431 A1 WO 2017108431A1 EP 2016080259 W EP2016080259 W EP 2016080259W WO 2017108431 A1 WO2017108431 A1 WO 2017108431A1
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seq
biomass
cueo
cota
pulp
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PCT/EP2016/080259
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Klara BIRIKH
Antoine Patrice Noël MIALON
Toni Pekka GRÖNROOS
Matti Wilhelm HEIKKILÄ
Veera Kaarina HÄMÄLÄINEN
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Metgen Oy
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0061—Laccase (1.10.3.2)
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C3/00—Pulping cellulose-containing materials
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C9/00—After-treatment of cellulose pulp, e.g. of wood pulp, or cotton linters ; Treatment of dilute or dewatered pulp or process improvement taking place after obtaining the raw cellulosic material and not provided for elsewhere
    • D21C9/001—Modification of pulp properties
    • D21C9/007—Modification of pulp properties by mechanical or physical means

Definitions

  • the present invention is in the field of producing mechanical pulp from a biomass comprising lignocellulosic material. Such a process is useful in paper production. More in particular the invention relates to the process of wood pulping, such as mechanical wood pulping. It provides useful methods, compounds and compositions for reducing the energy requirements of the production of mechanical pulp.
  • Pulp is a composition comprising lignocellulosic fibrous material, which may be prepared by chemically or mechanically separating cellulose fibers from biomass, such as wood, fiber crops or waste paper.
  • biomass such as wood, fiber crops or waste paper.
  • the timber resources used to make wood pulp are referred to as pulpwood.
  • Wood pulp comes from softwood trees such as spruce, pine, fir, larch and hemlock, and hardwoods such as eucalyptus, aspen and birch.
  • a pulp mill is a manufacturing facility that converts wood chips or other plant fiber source into a thick fiberboard which can be shipped to a paper mill for further processing.
  • pulp and paper facilities may be integrated and wet pulp mass can be used directly for paper production.
  • Pulp is characterized by its ability to absorb and retain water, which may be quantified as Canadian Standard Freeness (CSF) measured in milliliters. Defibrated wood material can be considered as pulp if its CSF can be determined.
  • CSF Canadian Standard Freeness
  • Pulp can be manufactured using mechanical, semi-chemical or fully chemical methods (Kraft and sulfite processes).
  • the finished product may be either bleached or non-bleached, depending on the customer's requirements.
  • Wood and other plant materials that may be used to make pulp contain three main components (apart from water): cellulose fibers (desired for papermaking), lignin (a three-dimensional polymer that binds the cellulose fibers together) and hemicelluloses, (shorter branched carbohydrate polymers).
  • a minor component of wood is pectin comprising galacturonic or rhamnogalacturonic acid polymers. Pectin adds in interlacing hemicellulose and lignin layers, thereby enhancing the strength of the material. In grassy plants, pectin is a major component providing rigidity.
  • the aim of the pulping process is to break down the bulk structure of the fiber source, be it chips, stems or other plant parts, into the constituent fibers.
  • Chemical pulping achieves this by chemically degrading the lignin and hemicellulose into small, water-soluble molecules which can be washed away from the cellulose fibers without depolymerizing the cellulose fibers.
  • this process of chemically depolymerizing the hemicellulose weakens the fibers.
  • the various mechanical pulping methods such as groundwood (GW) and refiner mechanical pulping (RMP), physically tear the cellulose fibers one from another. Much of the lignin remains adhered to the fibers. Strength may also be impaired because the fibers may be cut.
  • GW groundwood
  • RMP refiner mechanical pulping
  • hybrid pulping methods that use a combination of chemical and thermal treatment, for instance an abbreviated chemical pulping process, followed immediately by a mechanical treatment to separate the fibers.
  • These hybrid methods include chemi-thermomechanical pulping, also known as CTMP.
  • CTMP chemi-thermomechanical pulping
  • enzymes capable of oxidizing lignin were proposed to be used for pretreatment of wood chips in order to decrease the energy required for grinding. This idea was perceived from natural observation that fungi, especially white-rot fungi are able to decay wood material by secreting lignolytic enzymes such as peroxidases and laccases.
  • WO2014146712 A1 describes a bacterial laccase COTA (Spore Coat Protein) to be advantageous for reducing refining energy requirement in TMP.
  • EP1552052 B1 describes a method comprising pretreatment of cellulose fiber material with pectin-degrading enzymes, so-called pectinases, alone or in combination with a chelating agent, for instance DTPA and/or sulfite, followed by mechanical defibration and refining to produce a mechanical pulp.
  • pectinases so-called pectinases
  • a chelating agent for instance DTPA and/or sulfite
  • a combination of two different enzymes was capable of lowering the energy consumption of a commercial mechanical pulping process in a synergistic manner. More in particular, we found that a composition comprising a combination of a CotA laccase and a blue copper oxidase CueO was capable of synergistically lowering the energy consumption of such a process.
  • the invention relates to a method for producing mechanical pulp from a biomass comprising lignocellulosic material, the method comprising the steps of contacting the biomass with a CotA laccase and a blue copper oxidase CueO and refining the biomass to obtain a mechanical pulp.
  • the invention relates to a mechanical pulp, obtained by a method as described herein.
  • the invention relates to a method, composition or mechanical pulp as described herein, wherein the CotA laccase or the blue copper oxidase CueO are obtained by heterologous expression in Escherichia coli.
  • FIG. 1 Graph showing the specific energy consumption (SEC) expressed in kWh per ton of pulp (dry weight) according to example 3. SEC is plotted against achieved pulp freeness levels expressed as Canadian Standard Freeness levels (CSF) in milliliters. Control samples 1 and 2 were impregnated with impregnation liquid without enzyme and incubated at 65 degrees Celsius. These are shown in dashed lines; the solid line depicts the energy consumption of the same process with a CotA laccase according to SEQ ID NO: 1 . Arrows indicate the difference between the energy requirement at CSF values of 100 and 300 milliliter of the average control and the energy requirement when a CotA laccase is used.
  • SEC specific energy consumption
  • Figure 2 Diagram showing the energy saving in a pulp production method according to example 3, wherein the wood chips are impregnated with CotA(1 ) alone, CueO(1 ) alone and with a composition comprising both CotA(1 ) and CueO(1 ). Energy requirement of processes yielding pulps with SCF 100 ml and 300 ml are shown, interpolated from energy curves as shown on figure 1. Energy saving was calculated as the difference between the refining energy of the runs with and without the enzyme or enzymes at a given CSF (arrows in figure 1 ), divided by the energy of the reference runs without the enzyme(s) (average of 2 runs) times 100%.
  • Figure 3 Diagram showing the energy saving in a pulp production method according to example 3, wherein the wood chips are impregnated with CotA(2) alone, CueO(2) alone and with a composition comprising both CotA(2) and CueO(2). Energy requirement of processes yielding pulps with SCF 100 ml and 300 ml are shown, interpolated from energy curves as shown on figure 1. Energy saving was calculated as the difference between the refining energy of the runs with and without the enzyme or enzymes at a given CSF (arrows in figure 1 ), divided by the energy of the reference runs without the enzyme(s) (average of 2 runs) times 100%.
  • Biomass such as wood and other plant materials used to make pulp contain three main components (apart from water): cellulose fibers (desired for papermaking), lignin (a three-dimensional polymer that binds the cellulose fibers together) and hemicelluloses, (shorter branched carbohydrate polymers).
  • a minor component of wood is pectin (a polymer of galacturonic and/or rhamnogalacturonic acid) which is interlacing hemicellulose and lignin layers, thereby enhancing the strength of the material. In grassy plants, pectin is a major component providing rigidity.
  • Pulping is a process of preparing pulp.
  • Pulp is a material comprising fibers such as cellulose fibers, mostly from wood.
  • the aim of pulping is to break down the bulk structure of the fiber source, be it wood chips, stems or other plant parts, into the constituent fibers.
  • Pulp may be produced in a process called mechanical pulping.
  • wood may be ground, such as for instance against a water lubricated rotating stone. The heat generated by grinding softens the lignin binding the fibers and the mechanical forces separate the fibers to form groundwood. This process is also referred herein as defibration.
  • defibration refers to a process of separating wood fibers from each other.
  • Mechanical pulp comprises a mix of whole fibers and fiber fragments of different sizes. Paper made from mechanical pulp has a yellowish/grey tone with high opacity and a very smooth surface. Mechanical pulping provides a good yield from the pulpwood because it uses the whole of the log except for the bark, but the energy requirement for refining is high and can only be partly compensated by using the bark as fuel.
  • the various mechanical pulping methods such as groundwood (GW) and refiner mechanical (RMP) pulping, physically tear the cellulose fibers one from another. Much of the lignin remains adhered to the fibers. Strength of the fibers may be impaired because the fibers may be cut.
  • thermo-mechanical pulping (TMP)
  • TMP thermo-mechanical pulping
  • Thermo-mechanical pulping is a process in which wood chips are heated and run through a mechanical refiner for defibration (fiber separation), resulting in thermo-mechanical pulp.
  • wood chips are fed to a presteamer and are steamed with process steam (typically 1 to 2 bar or above 100 degrees Celsius, such as 130 to 140 degrees C).
  • Process steam may be obtained from the refiners.
  • the pressurized chips may be fed to the refiner with the feeding screw (plug feeder).
  • the refiner separates the fibers by mechanical force via refiner mechanical means (e.g. between rotating disc plates).
  • the refiner may be fed with fresh steam during startup, to increase the pressure up to 4 or 5 bar and about 150 degrees Celsius.
  • Thermomechanical pulping therefore refers to a process of producing pulp, which includes heating of biomass to a temperature above 100 degrees Celsius and mechanical defibration.
  • CSF Canadian Standard Freeness
  • Pulp is characterized by its ability to absorb and retain water which may be quantified as freeness or CSF, measured in milliliters. Defibrated wood material can be considered as pulp if its CSF can be measured. Often and advantageously, mechanical defibration and refining may be performed at a temperature above 100 degrees Celsius.
  • Pulp is often refined in two stages.
  • the process steam is typically taken to a heat recovery unit to produce clean steam.
  • the refiner discharges the pulp and steam to a cyclone.
  • the cyclone separates the steam from the pulp.
  • thermo-mechanical pulp is pulp produced by processing biomass such as wood chips using heat and a mechanical refining movement.
  • Wood chips are usually produced as follows: the logs are first stripped of their bark and converted into small chips, which have a moisture content of around 25-30%. A mechanical force is applied to the wood chips in a crushing or grinding action which generates heat and water vapour and softens the lignin thus separating the individual fibers.
  • the pulp is then screened and cleaned, any material that was not sufficiently refined (did not pass in screening procedure) is separated as "reject" and reprocessed.
  • the TMP process gives a high yield of fiber from the timber (around 95%) and as the lignin has not been removed, the fibers are hard and rigid.
  • delignification process which uses sodium hydroxide and sodium sulfide to chemically remove lignin. After delignification, the color of the pulp is dark brown. If white paper is desired, the pulp is bleached. Delignified, bleached pulp is fed into paper machines after undergoing other chemical processes that produce the desired quality and characteristics for the paper. A chemical pulp or paper is called wood-free, although in practice a small percentage of mechanical fiber is usually accepted.
  • Chemical pulping applies so called cooking chemicals to degrade the lignin and hemicellulose into small, water-soluble molecules which can be washed away from the cellulose fibers without depolymerizing the cellulose fibers. This is advantageous because the de-polymerization of cellulose weakens the fibers.
  • Using chemical pulp to produce paper is more expensive than using mechanical pulp or recovered paper, but it has better strength and brightness properties.
  • CTMP chemical thermo-mechanical pulping
  • the wood chips are impregnated with a chemical such as sodium sulphite or sodium hydroxide before the refining step.
  • the end result is a light-coloured pulp with good strength characteristics.
  • the chemical and thermal treatments reduce the amount of energy subsequently required by the mechanical refining, and also reduce the loss of strength suffered by the fibers.
  • wood chips can be pretreated with sodium carbonate, sodium hydroxide, sodium sulfite and other chemicals prior to refining with equipment similar to a mechanical mill.
  • the conditions of the chemical treatment are less vigorous (lower temperature, shorter time, less extreme pH) than in a chemical pulping process since the goal is to make the fibers easier to refine, not to remove lignin as in a fully chemical process.
  • Wood chips for TMP or CTMP are usually obtained from bark free and fresh tree wood. After manufacturing, the chips are screened to have specified size. For superior quality pulp, and optimal energy consumption, chips usually have thickness of 4-6 mm and length (dimension along the fibers) of 10 - 50 mm, such as 15 - 40 mm or 16-22 mm. Before refining, the chips may be washed and steamed, these chips have a typical moisture content of above 20% such as around 25-30%.
  • the pulp After grinding, the pulp is sorted by screening to suitable grades. It can then be bleached with peroxide for use in higher value-added products.
  • Freeness is a measure of drainability of a pulp suspension. It characterizes how fine the pulp has been refined. It can be determined by Canadian Standard Freeness - (CSF) Method (Thode, E. F., and Ingmanson, W. L, Tappi 42(1 ): 74 (1959) especially p. 82.; Technical Section, Canadian Pulp & Paper Association, Official Standard Testing Method C.1 , "The Determination of Freeness”) and is measured in milliliters. Higher CSF numbers mean faster draining, less refined pulp. Energy requirement for refining depends on the targeted freeness. Reaching lower freeness requires more energy. "Energy saving" in refining refers to a situation when the same freeness is achieved with less energy.
  • Pulp mill is a manufacturing facility that converts biomass such as wood chips or other plant fiber sources into a thick fiber board which can be shipped to a paper mill for further processing. Pulp can be manufactured using mechanical, thermo-mechanical, chemo thermo-mechanical or fully chemical methods. The finished product may be either bleached or non-bleached, depending on the customer requirements.
  • Pulp is intended to mean a composition comprising lignocellulosic fibrous material prepared by chemically and/or mechanically separating cellulose fibers from wood, fiber crops or waste paper. Pulp is characterized by its ability to absorb water, which can be measured in milliliters as Canadian
  • Wood pulp is the most common raw material in
  • lignocellulosic material refers to a material that comprises (1 ) cellulose, hemicellulose, or a combination and (2) lignin.
  • Wood pulp comes from softwood trees such as spruce, pine, fir, larch and hemlock, and hardwoods such as eucalyptus, aspen and birch.
  • Wood chipping is the act and industry of chipping wood for pulp, but also for other processed wood products and mulch. Only the heartwood and sapwood are useful for making pulp. Bark contains relatively few useful fibers and is removed and used as fuel to provide steam for use in the pulp mill.
  • An advantageous effect of applying a mechanical force to the wood chips in a crushing or grinding action is that it generates heat which softens the lignin thus adds in the separation of individual cellulose fibers.
  • Pretreatment is a process when chips are exposed to a certain chemical or enzymatic solution, or a mechanical treatment before refining.
  • the purpose of pretreatment is to reduce refining energy consumption or to improve pulp properties.
  • Physical pretreatment is often called size reduction and is aiming to reduce the physical size of the chips. This is also called low energy mechanical treatment.
  • Chemical pretreatment is to remove chemical barriers so the cellulose fibers are more easily recoverable.
  • low energy mechanical treatment is used herein to indicate a process wherein the biomass containing the lignocellulosic material is subjected to mechanical forces such that the temperature of the biomass does not exceed 95 degrees Celsius.
  • Impregnation is a process when chips are first pressurized and upon slow release of pressure, the pretreatment solution is added to the chips.
  • the pressure can be build up by mechanical force (e.g. impregnation screw) or by a steam-cooker principle.
  • impregnation can sometimes combine chemical and mechanical pretreatment.
  • impregnation is usually done on steamed chips, which facilitates impregnation.
  • Impregnation improves the penetration of the pretreatment solution inside the wood. Pretreatment may be continued in a reaction vessel following the impregnation stage in the process.
  • pretreatment includes low energy mechanical treatment (the energy is low as compared to the refining energy) of wood chips to increase the surface of contact with the pretreatment solution.
  • low energy mechanical pretreatment there is no significant production of heat, in other words, the temperature of the wood chips in this step may not exceed 95 degrees C or less, such as 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, or 40 degrees Celsius.
  • Destructured wood chips are wood chips which were partially destroyed as a result of impregnation or low energy mechanical pretreatment.
  • low energy mechanical pretreatment in this respect is to be interpreted as a process wherein the wood chips are partially destructured but not fiberized.
  • Defibration is herein defined as the process of separating fibers, preferably by a mechanical process.
  • composition comprising a particular combination of two different enzymes was capable of reducing the energy consumption of a commercial mechanical pulping process by at least 20%.
  • the invention therefore relates to a method for producing mechanical pulp from a biomass comprising lignocellulosic material, the method comprising the steps of contacting the biomass with a CotA laccase and a blue copper oxidase CueO and refining the biomass to obtain a mechanical pulp.
  • Laccases (EC 1 .10.3.2) are enzymes having a wide taxonomic distribution and belonging to the group of multicopper oxidases. Laccases are eco- friendly catalysts, which use molecular oxygen from air to oxidize various phenolic and non-phenolic lignin-related compounds as well as highly recalcitrant environmental pollutants, and produce water as the only side-product. These natural "green” catalysts are used for diverse industrial applications including the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, use as bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases are also used as cleaning agents for certain water purification systems. In addition, their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes.
  • Laccases were originally discovered in fungi, they are particularly well studied in White-rot fungi and Brown-rot fungi. Later on, laccases were also found in plants and bacteria. Laccases have broad substrate specificity; though different laccases can have somewhat different substrate preferences. Main characteristic of laccase enzyme is its redox potential, and according to this parameter all laccases can be divided in three groups (see, for example, Morozova, O. V., Shumakovich, G. P., Gorbacheva, M. a., Shleev, S. V., & Yaropolov, a. I. (2007). "Blue" laccases.
  • CotA is a bacterial laccase and is a component of the outer coat layers of bacillus endospore. It is a 65-kDa protein encoded by the cotA gene (Martins, O., Soares, M., Pereira, M. M., Teixeira, M., Costa, T., Jones, G. H., & Henriques, A. O. (2002). Molecular and Biochemical Characterization of a Highly Stable Bacterial Laccase That Occurs as a Structural Component of the Bacillus subtilis Endospore Coat . Biochemistry, 277(21 ), 18849 -18859. doi:10.1074/jbc.M200827200).
  • CotA belongs to a diverse group of multi-copper "blue" oxidases that includes the laccases. This protein demonstrates high thermostability, and resistance to various hazardous elements in accordance with the survival abilities of the endospore. The redox-potential of this protein has been reported to be around 0.5 mV, which places it in the range of medium-redox-potential laccases. CotA laccases are herein also referred to as 'CotA'.
  • the endospore coat protein CotA is a laccase required for the formation of spore pigment and was recently shown to have also bilirubin oxidase (EC 1 .3.3.5) activity.
  • CotA and CueO come from functionally and structurally different families [Hoegger et al., FEBS J., (2006) 273: 2308-2326].
  • the Escherichia coli blue copper oxidase CueO from Escherichia coli (herein also referred to as 'CueO') and the copper resistance protein CopA from Pseudomonas campestris are usually considered pseudo-laccases due to the dependence of the 2,6-dimethoxyphenol oxidation on Cu2+ addition.
  • CueO is involved in periplasmic detoxification of copper by oxidizing
  • Cu+ to Cu2+ and thus preventing its uptake into the cytoplasm The enzyme possesses phenoloxidase and ferroxidase activities and is thought to be involved in the production of polyphenolic compounds and the prevention of oxidative damage in the periplasm. It has a medium redox potential of 0.5 V.
  • CueO is also often referred to as Copper efflux oxidase, and requires a copper cation as co-factor. It binds 4 copper cations per monomer.
  • laccases are Multicopper oxidases (MCOs) able to couple oxidation of substrates with reduction of dioxygen to water.
  • the copper ions are bound in several sites: Type 1 , Type 2, and/or Type 3.
  • the ensemble of types 2 and 3 copper is called a trinuclear cluster.
  • Most multicopper oxidases (including COTA and CueO), consists of three Cupredoxin domains often abbreviated as CuRO domains.
  • the cupredoxin domain 1 contains part of the trinuclear copper binding site, which is located at the interface of domains 1 and 3.
  • the cupredoxin domain 2 has no ability to bind copper.
  • the cupredoxin domain 3 contains the Type 1 (T1 ) copper binding site and part of the trinuclear copper binding site, which is located at the interface of domains 1 and 3.
  • Cupredoxin domains of multicopper oxidases are categorized in families according to their structural features. These categorization is featured, for example, in conserveed Domains database in National Center for Biotechnology Information (NCBI) public internet resource (http://www.ncbi.nlm.nih.gov/cdd/).
  • the CURO_1 domain belongs to a distinct conserved domain family according to NCBI classification
  • cd13844 CuRO_1_BOD_CotA_like.
  • Other proteins sharing this domain are bilirubin oxidase, catalyzing the oxidation of bilirubin to biliverdinphenoxazinone and phenoxazinone synthase (PHS), which catalyzes the oxidative coupling of substituted o-aminophenols to produce phenoxazinones.
  • PHS phenoxazinone synthase
  • Cupredoxin domains 2 and 3 of COTA protein are characteristic for COTA laccases and are forming their own conserved domain subfamilies cd13868: CuRO_2_CotA_like and cd13891 : CuRO_3_CotA_like respectively.
  • CueO laccases comprise three copredoxin domains that belong to the respective families cd04232: CuRO_1_CueO_FtsP,
  • cd13867 CuRO_2_CueO_FtsP
  • cd13890 CuRO_3_CueO_FtsP. All three domains are shared structurally with another protein family FtsP (also named Sufi). This protein is a component of the cell division apparatus. FtsP belongs to the multicopper oxidase superfamily but lacks metal cofactors and has no catalytic activity, thus can be easily functionally distinguished from CUEO laccase.
  • a CotA laccase is defined as a polypeptide with laccase activity comprising an amino acid sequence that is at least 50% identical with the consensus sequence according to SEQ ID NO: 1 1 .
  • a blue copper oxidase CueO is defined as a polypeptide with laccase activity comprising an amino acid sequence that is at least 50% identical with the consensus sequence according to SEQ ID NO: 12.
  • NCBI also features a conserveed Domain Database (CDD) http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml (Marchler-Bauer et al., Nucl. Acids res. (2015) 43: D222-D226).
  • CDD conserved Domain Database
  • CotA laccases may be very divers with respect to their primary amino acid sequence.
  • CotA(1 ) and CotA(2) The primary amino acid of CotA(1 ) is shown as SEQ ID NO: 1 whereas CotA(2) is shown as SEQ ID NO: 3.
  • SEQ ID NO: 1 has 154 residues in common with the consensus sequence according to SEQ ID NO: 1 1 and is therefore 55% identical
  • SEQ ID NO: 3 has 173 residues in common with the consensus sequence according to SEQ ID NO: 1 1 and is therefore 62% identical.
  • the blue copper oxidase CueO enzymes may also be very divers with respect to their primary amino acid sequence.
  • the primary amino acid of CueO(1 ) is shown as SEQ ID NO: 2
  • CueO(2) is shown as SEQ ID NO: 4
  • CueO(3) is shown as SEQ ID NO: 9.
  • SEQ ID NO: 2 has 142 residues in common with the CueO consensus sequence according to SEQ ID NO: 12 and is therefore 82% identical
  • SEQ ID NO: 4 has 90 residues in common with the CueO consensus sequence according to SEQ ID NO: 12 and is therefore 52% identical
  • SEQ ID NO: 9 has 125 residues in common with the CueO consensus sequence according to SEQ ID NO: 12 and is therefore 72,3% identical (Table 4).
  • CueO(3) behaved in all aspects comparable to CueO(1 ) and is not further shown herein.
  • the degree of identity between a given amino acid sequence and the consensus sequence is determined by aligning the two sequences using alignment software such as BLAST, determining the number of residues that are identical over the entire length of the consensus sequence and divide that number of identical residues by the total length of the consensus sequence.
  • CotA and CueO enzymes were used in a method for producing mechanical pulp from a biomass comprising lignocellulosic material, either alone or in combination.
  • combination refers to a sequential or simultaneous use of the enzymes. This means that the step of contacting the biomass with a CotA laccase and a blue copper oxidase CueO is done either separately with each enzyme or together in one composition, before refining the biomass to obtain a mechanical pulp.
  • Energy saving as shown herein is calculated by determining the difference in energy required for the production of a certain amount of pulp starting from lignocellulosic material. Energy saving was calculated as the difference between the refining energy of the runs with and without the enzyme or enzymes at a given CSF (arrows in figure 1 ), divided by the energy of the reference runs without the enzyme(s)). This is done for pulp of two different levels of freeness as exemplified in figure 1.
  • Table 1 Energy saving by enzymes in a method of producing pulp from biomaterial.
  • Optimal dosage may easily be determined by trial and error methods for a given setting in a traditional pulp mill operation.
  • the skilled person will be well aware of methods for optimizing the conditions for optimizing the use of the enzymes as disclosed and described herein.
  • a skilled person will also be well aware of the amount of enzyme to use in order to reach an optimum between effect and costs.
  • the optimal dose of each enzyme may easily be found empirically, and will usually be in the range of 3 - 1 .000.000 microkatal per ton of dry substrate, such as wood.
  • the lower range of the dose for each enzyme may be at least 5 microkatal per ton of dry substrate, such as 10, 15, 20, 25, 30, 50, 100 or even 300 microkatal per ton of dry substrate.
  • CotA(1 ) and CotA(2) performed equally well and both showed the synergistic effect when used in combination with CueO(1 ), CueO(2) and CueO(3), despite of the fact that the COTA enzymes differed a great deal in their primary sequence, as did the three CueO enzymes.
  • the invention therefore relates to a method as decribed above wherein the CotA laccase comprises an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3 or an amino acid sequence at least 50% identical with the CotA consensus sequence according to SEQ ID NO: 1 1.
  • the invention relates to a method as described herein wherein the CotA laccase is a polypeptide with laccase activity comprising an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3 or an amino acid sequence at least 50% identical with the CotA consensus sequence according to SEQ ID NO: 1 1.
  • the invention relates to a method as described above, wherein the blue copper oxidase CueO comprises an amino acid sequence according to SEQ ID NO: 2, Seq ID NO: 4 or SEQ ID NO: 9 or an amino acid sequence at least 50% identical with the CueO consensus sequence according to SEQ ID NO: 12.
  • the invention relates to a method as described herein wherein the blue copper oxidase CueO is a polypeptide with laccase activity comprising an amino acid sequence according to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 9 or an amino acid sequence at least 50% identical with the CueO consensus sequence according to SEQ ID NO: 12.
  • the expression "at least 50%” includes at least 51 %, 52%, 53%, 54% or 55%, such as 56%, 57%, 58%, 59% or 60%, such as 61 %, 62%, 63%, 64%, 65%, 70% or at least 75%, 80% or 85%, such as at least 90%, 91 %, 92%, 93%, 94% or 95%, 96%, such as 97%, 98% or even at least 99%.
  • the invention relates to a method as described herein wherein the blue copper oxidase CueO consists of a polypeptide with laccase activity, comprising an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94% or 95% identical to the sequence of SEQ ID NO:2, SEQ ID NO: 4 or SEQ ID NO: 9, preferably 96%, such as 97%, 98% or even at least 99%.
  • the invention relates to a method as described herein wherein the CotA laccase consists of a polypeptide with laccase activity, comprising an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94% or 95% identical to the sequence of SEQ ID NO:1 or SEQ ID NO: 3, preferably 96%, such as 97%, 98% or even at least 99%.
  • the alignment of two laccase sequences is to be performed over the full length of the polypeptides.
  • homologous enzymes for use in the present invention.
  • the prior art teaches random and directed mutagenesis methods suitable for that purpose.
  • Homologous CotA laccases and blue copper oxidase CueOs may be prepared by conventional recombinant DNA
  • a particularly preferred substrate in the method according to the invention is wood, a wood chip, a destructured wood chip, defibrated wood, hardwood, softwood, non-wood fiber material, bamboo, bagasse, reed or straw.
  • the invention relates to a method as described above wherein the lignocellulosic material comprises a material selected from the group consisting of wood, a wood chip, a destructured wood chip, defibrated wood, hardwood, softwood, non-wood fiber material, bamboo, bagasse, reed, straw and combinations thereof.
  • the invention relates to a method as described above, comprising an additional step of compressing the biomass comprising the lignocellulosic material before contacting it with the CotA laccase and the blue copper oxidase CueO.
  • the method as described above not only decreased the energy consumption of the mechanical pulping or thermo-mechanical pulping process, it was also found to be advantageous in a particular embodiment of the TMP process, namely a chemo thermo-mechanical pulping (CTMP) process.
  • CTMP chemo thermo-mechanical pulping
  • the invention also relates to a method as described above, wherein the TMP is a chemo thermo- mechanical pulping (CTMP) process.
  • a chemo-thermo-mechanical pulping process differs from a TMP process in that at least one additional step is added and wherein the biomass containing the lignocellulosic material is impregnated with a composition such as a chemical composition in order to at least partially degrade lignin.
  • the invention relates to a method as decribed above comprising an additional step of treating the biomass comprising the lignocellulosic materials with a chemical composition before the biomass is subjected to defibration.
  • the chemical composition is able to degrade lignin.
  • the treatment with the CotA laccase and the blue copper oxidase CueO may be employed at different stages in the process.
  • lignocellulosic material may be contacted with the enzymes after it has been provided in the appropriate dimensions, optionally after cleaning and steaming.
  • the invention relates to a method as described herein comprising an additional step of treating the biomass comprising the lignocellulosic material with heat, preferably by steaming, before contacting it with the CotA laccase and the blue copper oxidase CueO.
  • the biomass comprising the lignocellulosic material is heated to a temperature below the inactivation temperature of the CotA laccase and the blue copper oxidase CueO.
  • the wood is treated after it is debarked and chopped in pieces and selected for size. These pieces are usually referred to as wood chips.
  • wood chips typically have a largest dimension of typically in the order of up to 5 cm, such as 2, 3, or 4 cm.
  • the biomass comprising the lignocellulosic material may preferably be contacted with the enzymes after washing and or steaming. This makes the material more accessible for the enzyme and increases the moisture content of the material.
  • the invention relates to a method as described above, wherein the wood has a moisture content of at least 20% and is preheated to a temperature below 100 degrees Celsius before treating the wood with the the CotA laccase and the blue copper oxidase CueO.
  • the invention therefore also relates to a composition, such as a fluidic composition, a suspension or a solution comprising a biomass comprising a lignocellulosic material and at least a CotA and a CueO enzyme.
  • the composition comprises a CotA polypeptide comprising an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3 or an amino acid sequence at least 50% identical with the CotA consensus sequence according to SEQ ID NO: 1 1 , as well as a blue copper oxidase CueO comprising an amino acid sequence according to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 9 or an amino acid sequence at least 50% identical with the CueO consensus sequence according to SEQ ID NO: 12.
  • the contacting of the biomass with the enzymes can be performed for a period of time up to one day. While longer enzymatic digestions are possible, a shorter period of time such as 15 minutes, 60 minutes, 1 hour, 2 hours, 3 hours or any time less than these values or any time between any of two of these values may be used for practical or economic reasons. In another preferred embodiment, the enzymatic digestions can take 50, 100, 150 or 200 hours or any time less than these values or any time between any of two of these values. In a further preferred embodiment, the invention relates to a method as described herein wherein the the CotA laccase and the blue copper oxidase CueO are contacted with the biomass comprising the lignocellulosic material for a duration of between 10 and 120 minutes. Another preferred period of enzyme contact is about 3 days or less.
  • the enzyme pretreatment process may be performed at a specific temperature such as, for example at a temperature from 30 degrees C to about 80 degrees Celsius; 40 degrees C to 70 degrees C; or 45 degrees C to 65 degrees or 45 degrees C to 60 degrees C, such as at least 50 degrees C or 60 degrees Celsius or 65 degrees Celsius or at room temperature or lower.
  • the invention relates to a method according to any one of claims 1 - 10 wherein the treatment with the CotA laccase and the blue copper oxidase CueO is performed after a low energy mechanical treatment step before refining the biomass.
  • the biomass such as wood chips are impregnated for better contact between the enzyme and the substrate. This is known to the skilled person.
  • the wood chips are destructured such as defibrated before or after the enzyme treatment.
  • the invention therefore also relates to a method as described herein, further comprising an impregnation step wherein the biomass is impregnated with a CotA laccase and a blue copper oxidase CueO.
  • lignocellulosic material to be treated may be in excess of the optimum temperature or even above the enzyme inactivation temperature of the enzymes according to SEQ ID NO:s 1 - 4 or their homologues. Since a high temperature may inactivate enzymes by irreversibly denaturing its amino acid chain, the enzyme may advantageously be added to the biomass at a point below the enzyme inactivation temperature. The enzymes may be added within the functional temperature range(s) or at the optimal temperature(s) of the enzyme. In case of biomasses with a high temperature, the enzymes may be added after the biomass has cooled below the inactivation
  • a dilution liquid such as water at a certain temperature, may be used to cool the biomass.
  • thermostable enzymes may be used so that the process may be conducted at higher temperatures.
  • the invention also relates to mechanical pulp, obtained by a method as described herein.
  • heterologous expression system or equivalent means a system for expressing a DNA sequence from one host organism in a recipient organism from a different species or genus than the host organism.
  • the most prevalent recipients known as heterologous expression systems, are chosen usually because they are easy to transfer DNA into or because they allow for a simpler assessment of the protein's function.
  • Heterologous expression systems are also preferably used because they allow the upscaling of the production of a protein encoded by the DNA sequence in an industrial process.
  • Preferred recipient organisms for use as heterologous expression systems include bacterial, fungal and yeast organisms, such as for example
  • Escherichia coli Bacillus, Corynebacterium, Pseudomonas, Pichia pastoris,
  • Saccharomyces cerevisiae Saccharomyces cerevisiae, Yarrowia lipolytica, filamentous fungi and many more systems well known in the art.
  • the invention relates to a method or a mechanical pulp as described herein wherein the CotA laccase or the blue copper oxidase CueO are obtained by expression in Escherichia coli.
  • sequences by attaching or inserting, including , but not limited to, affinity tags, facilitating protein purification (S-tag, maltose binding domain, chitin binding domain), domains or sequences assisting folding (such as thioredoxin domain, SUMO protein), sequences affecting protein localization (periplasmic localization signals etc), proteins bearing additional function, such as green fluorescent protein (GFP), or sequences representing another enzymatic activity.
  • affinity tags facilitating protein purification
  • S-tag maltose binding domain, chitin binding domain
  • domains or sequences assisting folding such as thioredoxin domain, SUMO protein
  • sequences affecting protein localization periplasmic localization signals etc
  • proteins bearing additional function such as green fluorescent protein (GFP)
  • GFP green fluorescent protein
  • Other suitable fusion partners for the presently disclosed polypeptides are known to those skilled in the art.
  • CotA and CueO enzymes may be obtained by standard recombinant methods known in the art. Briefly, such a method may comprise the steps of: culturing a recombinant host cell as described above under conditions suitable for the production of the polypeptide, and recovering the polypeptide obtained. The polypeptide may then optionally be further purified.
  • Possible vectors include, but are not limited to, plasmids or modified viruses which are maintained in the host cell as autonomous DNA molecule or integrated in genomic DNA.
  • the vector system must be compatible with the host cell used as is well known in the art.
  • suitable host cells include bacteria (e.g. E.coli, bacilli), yeast (e.g. Pichia Pastoris, Saccharomyces Cerevisiae), fungi (e.g. filamentous fungi) insect cells (e.g. Sf9).
  • Example 1 Preparation of polypeptides according to SEQ ID NO:s 1 - 4 and 9.
  • DNA sequences according to SEQ ID NO:s: 5-8 and 10, encoding the polypeptides according to SEQ ID NO:s 1 -4 and 9 were commercially synthesized and cloned into a standard plasmid vector pET28a+ under the control of T7-RNA- polymerase promoter for expression in Escherichia coli BL21 (DE3).
  • Protein production was carried out in E.coli BL21 (DE3) strain according to the plasmid manufacturer protocol available at
  • Example 2 Laccase activity measurement.
  • laccase activity is used herein to mean the capability to act as a laccase enzyme, which may be expressed as the maximal initial rate of the specific oxidation reaction.
  • Relative activity was measured by oxidation of ABTS (2,2'-Azino- bis(3-ethylbenzothiazoline-6-sulfonic acid). Reaction course was monitored by change in absorbance at 405 nm (extinction coefficient of ABTS at 405 nm is 36 800 M-1 cm-1 ). The appropriate reaction time was determined to provide initial rates of oxidation when color development is linear in time.
  • ABTS concentration in the reaction mixture was 5 mM to provide maximum initial rates (substrate saturation conditions).
  • reaction were carried out in 1 ml volume of 50 mM Succinic acid pH 5, the substrate was preheated to the desired temperature (60 degrees Celsius) and reaction was initiated by the addition of the enzyme. After the reaction time has lapsed, absorbance at 405 nm of the reaction mixtures was determined by a spectrophotometer, and the absorbance of the control sample (containing no enzyme) was subtracted.
  • One unit of laccase activity is defined as the enzyme amount oxidizing 1 micro mole of substrate per minute, one microkatal is the amount of enzyme oxidizing 1 micromol of substrate per second, hence 10 millikatal equals 600,000 units.
  • Wood chips were pre-steamed at 90- 100°C during 10 minutes at atmospheric pressure in a bin, in which the chips were introduced and the steam diffused at the bottom with a steam feeding system.
  • MSD modular screw device
  • R retention
  • T temperature
  • the compression zone of the MSD RT-Pressafiner is a screw press with an increasing shaft diameter where the chips are compressed as they are fed forward through the narrowing gap between the shaft and the outer casing. During this process, water and extractives are pressed out of the chips through holes in the outer casing.
  • the chips were directly fed into an impregnation tank containing an impregnation fluid.
  • the impregnation fluid was heated by passing into a hot water heat exchanger and re-circulated continuously during the reaction time.
  • spruce chips For each trial, a quantity of spruce chips was used, corresponding to 7 kilograms of dry weight.
  • the impregnation tank was filled with 65 liter of impregnation fluid, in which the chips were introduced in totality. The impregnation was followed by incubation for 60 minutes at 60 degrees Celsius at atmospheric pressure.
  • control 1 Two reference runs using impregnation fluid without any enzyme were performed, one at the beginning and one at the end of the series. These are referred to herein as control 1 and control 2.
  • Impregnation fluid (65L) contained 150 units of enzyme (either CotA or
  • the impregnation fluid was drained from the impregnation tank and the chips were collected to be defibrated using Sprout Waldron D2A507 Ni Hard refiner plates.
  • the fibers were refined into mechanical pulp using 12SA001 refiner discs. Pulps were refined to four different freeness levels. The freeness level (Canadian Standard Freeness, CSF) was determined for each sample according to the international standard.
  • CSF Canadian Standard Freeness
  • Power in kW was measured on the motors by the pilot plant automation every 15 seconds and the average power was determined that was consumed for the period during which the pulp flow was measured.
  • the pulp flow was evaluated by sampling pulps during an exact period of time, by weighting this sample and by determining the dry matter of this sample.
  • the energy consumption in kWh/t was calculated by dividing the average power in kW by the pulp flow in dry weight of tons per hour.
  • Amino acid sequence and nucleotide sequences are provided herewith in the WIPO ST_25 standard. For convenience the sequences are also provided in table 3.
  • SEQ ID NO: 1 is a mutated COTA laccase from Bacillus subtilis
  • SEQ ID NO: 2 is CUEO laccase from Escherichia coli
  • SEQ ID NO: 3 is COTA laccase from Bacillus coagulans
  • SEQ ID NO: 4 is CUEO laccase from Lysinibacillus boronitolerans
  • SEQ ID NO:s 5 - 8 are the DNA sequences encoding the polypeptides according to SEQ ID NO:s 1 - 4.
  • SEQ ID NO: 9 is CUEO laccase from Proteus mirabilis.
  • SEQ ID NO: 10 is the DNA sequence encoding the polypeptide of SEQ ID NO: 9.
  • SEQ ID NO: 1 1 is a CotA consensus sequence
  • SEQ ID NO: 12 is a CueO consensus sequence

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Abstract

La présente invention concerne le domaine de production de pâte mécanique à partir d'une biomasse comprenant un matériau lignocellulosique. Un tel processus est utile dans la production du papier. Plus particulièrement, l'invention concerne le processus de préparation de pâte de bois, telle que la pâte de bois mécanique. Elle fournit des procédés, des composés et des compositions utiles pour réduire les exigences énergétiques de la production de pâte mécanique. Plus particulièrement, elle fournit un procédé comprenant les étapes de mise en contact de la biomasse avec une CotA laccase et une cuivre bleu oxydase CueO et l'affinage de la biomasse pour obtenir une pâte mécanique.
PCT/EP2016/080259 2015-12-22 2016-12-08 Procédé de production de pâte mécanique à partir d'une biomasse comprenant un matériau lignocellulosique WO2017108431A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112160176A (zh) * 2020-10-20 2021-01-01 深圳市瑞成科讯实业有限公司 用于造纸的木纤维的制备方法
CN114837007A (zh) * 2022-06-01 2022-08-02 齐鲁工业大学 一种利用复合菌剂进行小麦秸秆制浆的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0429422A1 (fr) * 1989-11-17 1991-05-29 Enso-Gutzeit Oy Procédé de fabrication de pâte
US6267841B1 (en) * 1992-09-14 2001-07-31 Steven W. Burton Low energy thermomechanical pulping process using an enzyme treatment between refining zones
WO2013045782A1 (fr) * 2011-09-30 2013-04-04 Arkema France Pretraitement enzymatique de bois dans un procede de fabrication de pate a papier mecanique
WO2014146712A1 (fr) * 2013-03-20 2014-09-25 Metgen Oy Procédé permettant d'économiser de l'énergie en production de papier

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0429422A1 (fr) * 1989-11-17 1991-05-29 Enso-Gutzeit Oy Procédé de fabrication de pâte
US6267841B1 (en) * 1992-09-14 2001-07-31 Steven W. Burton Low energy thermomechanical pulping process using an enzyme treatment between refining zones
WO2013045782A1 (fr) * 2011-09-30 2013-04-04 Arkema France Pretraitement enzymatique de bois dans un procede de fabrication de pate a papier mecanique
WO2014146712A1 (fr) * 2013-03-20 2014-09-25 Metgen Oy Procédé permettant d'économiser de l'énergie en production de papier

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112160176A (zh) * 2020-10-20 2021-01-01 深圳市瑞成科讯实业有限公司 用于造纸的木纤维的制备方法
CN114837007A (zh) * 2022-06-01 2022-08-02 齐鲁工业大学 一种利用复合菌剂进行小麦秸秆制浆的方法

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