Classifications

• • 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
  • D21C9/10—Bleaching ; Apparatus therefor
  • D21C9/147—Bleaching ; Apparatus therefor with oxygen or its allotropic modifications
• • 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

Definitions

• This invention relates to a novel enzymatic process for bleaching wood pulp in which a crude lignin peroxidase is used in the presence of oxygen rather than hydrogen peroxide as co-substrate to reduce the lignin content of wood pulp.
• the lignin peroxidase can be used in a modified form.
• Wood is a complex material which is composed of cellulose, hemi­cellulose and lignin along with other minor components.
• the lignin is associated with and even covalently bound to a matrix of cellulose and hemicellulose.
• lignin should be removed from the wood pulp since it reduces the strength, confers a brownish colour and imparts other undesirable characteristics to the finished product.
• wood chips are first treated vith sodium sulphide (Na2S) and sodium hydroxide (NaOH) to degrade the lignin substantially. This is called the sulphate or Kraft process. Alterna­tively other treatments may be of use e.g. the sulphite process.
• the pulps obtained therefrom are called "chemical pulps".
• Chemical pulp e.g. Kraft pulp usually contains about 4-12% by weight of residual lignin which gives the pulp a characteristic brown colour.
• the kappa number which reflects the lignin content of the pulp is usually fro 10 to 45, more frequently from 12 to 30.
• the lignin content should be further reduced in one or more treatments or stages commonly referred to as bleaching.
• bleaching Many industrial bleaching processes already exist but almost all of them are divided into two main parts: A complementary delignification followed by a "true bleaching" for improving the brightness level.
• the complementary delignification typically starts with an oxygen stage or a chlorination-extraction step (C-E) stage or both.
• Chlorination and extraction are usually carried out in sequence, first forming chlori­nated lignin compounds which are then solubilized in the subsequent extraction step.
• the objective is exclusively to delignify the pulp as very little brightening occurs at the C-E stage.
• a complementary pro­cess for brightening the lignin may further include the use of compo­nents other than chlorine such as chlorine containing chemicals e.g. hypochlorite and chlorine dioxide; or oxygen and hydrogen peroxide.
• E-1 effluents contain a very large number of chlorinated organic compounds which are hazardous for the environment e.g. dioxines. Also, due to their highly corrosive nature, it is quite difficult to recycle the effluents. Thus, from the environmental point of view, it is clear that new techniques for bleaching which may reduce pollution are highly desirable.
• an enzymatic treatment may be usefully substituted for at least one of the chemical treatments involving chlorine com­pounds in pulp bleaching.
• Lignin peroxidases also called ligninases
• MnII-dependent peroxidase are enzymes of particular interest which are secreted by many microbial strains, especially filamentous fungi.
• Phanerochaete chrysosporium is a fungus which produces essentially both types of peroxidases. These enzymes are able to modify the lignin content of wood so that lignin is released from the hemicellulose matrix or made releasable upon washing or extraction.
• Lignin peroxidases have been described up to now as enzymes which require the presence of H2O2 to be effective in degrading lignin with the optional presence of oxygen.
• EP 345715 A1 it is claimed that this system works without the use of oxygen but in the presence of ⁇ -hydroxy acids and detergents.
• the peroxide needs to be produced in situ enzymatically.
• DE 3636208 A1 it is claimed that certain oxidation and reducing agents have to be present and the redox potential has to be maintained at a certain level throughout the course of the reaction.
• the processes described in these two patents are commercially not feasible because of the high costs of the co-substrates needed.
• the invention provides a process for bleaching wood pulp which comprises treating the pulp with at least one lignin peroxidase in the substantial absence of added peroxide and in the presence of oxygen.
• the invention provides an enzymatic composition (enzyme preparation) comprising at least one lignin peroxidase derived from a fungal culture which is chemically modified so that it cannot be adsorbed onto pulp.
• bleaching process as used herein is meant a process for delignifying wood pulp or improving the whiteness or brightness of wood pulp or both.
• lignin as used herein is meant not only natural, unmodified forms but also the forms as found in chemically treated pulps which are, in whole or in part, chemically modified by various agents such as those used in the Kraft, organosolv or sulphite pulping process and in the effluent of these processes.
• substantially absence of added peroxide is meant the absence of a substantial amount of added peroxide which would be effective in in­ducing degradation of lignin.
• the scope of the invention intends to encompass a process in which a peroxide is added to the reaction medium in an uneffective amount.
• lignin-degrading enzyme as used herein is meant to en­compass any enzyme which modifies the lignin or hemicellulose compo­nent of wood so that lignin is released from the hemicellulose matrix or made releasable upon washing or extraction.
• Suitable lignin-­degrading enzymes are hemicellulases, oxidases and peroxidases, the latter being particularly preferred.
• hemicellulases are mannanases, xylanases, galactomannanases and arabinosidases, while laccases fall under the group of oxidases.
• Preferred peroxidases are MnII-dependent peroxidases, and lignin peroxidases (also called ligninases). Lignin-degrading enzymes are secreted by many microbial strains particularly filamentous fungi.
• lignin peroxidases as used herein is meant to encompass the crude enzyme preparation produced by the fungus under ligninolytic conditions as well as the individual lignin peroxidase isoenzymes from natural or recombinant producers.
• lignin peroxidase of a white-rot fungus e.g. P. chrysosporium either from its native origin or in recombinant form.
• the recombinant form of a lignin peroxidase of P. chrysosporium may be obtained as described in PCT patent application No. 88/2023.
• the enzymatic composition may be in a substantially purified form. It is, however, preferred that the enzymatic composition be a crude extract, a fil­trate or a supernatant of a culture of a white-rot fungus, e.g. P. chrysosporium.
• a suitable culture medium is the nitrogen-limited BIII/glu­cose medium which contains 1.08 x 10 ⁇ 3 M ammonium tartrate, 1.47 x 10 ⁇ 2 M KH2PO4, 2.03 x 10 ⁇ 3 M MgSO4.7H2O, 6.8 x 10 ⁇ 4 M CaCl2 ⁇ 2H2O, 2.96 x 10 ⁇ 6 M thiamine ⁇ HCl and 10 ml ⁇ L ⁇ 1 of a trace element solution.
• the trace element solution contains 7.8 x 10 ⁇ 3 M nitrilo-acetic acid, 1.2 x 10 ⁇ 2 M MgSO4 ⁇ 7HO2, 1.7 x 10 ⁇ 2 M NaCl, 3.59 x 10 ⁇ 4 M FeSO4 ⁇ 7H2O, 7.75 x 10 ⁇ 4 M CoCl2, 9.0 x 10 ⁇ 4 M CaCl2, 3.48 x 10 ⁇ 4 M ZnSO4, 4 x 10 ⁇ 5 M CuSO4 ⁇ 5H2O, 2.1 x 10 ⁇ 5 M AlK(SO4)2 ⁇ 12H2O, 1.6 x 10 ⁇ 4 M H3BO3, 4.1 x 10 ⁇ 5 M NaMoO4 ⁇ 2H2O and 2.9 x 10 ⁇ 3 M MnSO4 ⁇ H2O.
• the lignin-degrading enzyme may be chemically modified by covalent or non-covalent linkage to water-soluble or insoluble polymeric compounds which prevent the enzyme from being adsorbed onto pulp during the treatment.
• Suitable polymeric compounds are for example, polyethylene glycol (PEG), po3y­propylene glycol (PPG), polyacrylamides and polymeric sugars of various degrees of polymerization and composition like CM-cellulose, cellulose, agarose, alginate and chitosan.
• PEG is a preferred poly­meric compound.
• the enzyme may be deglycosylated so that the carbohydrate residues which are usually involved in the mechanism of adsorption are at least partially removed.
• Deglycosylation may be performed by known methods, for example, by treating a sample of lignin-degrading enzyme with an enzyme such as an endoglycosidase capable of degrading carbohydrate residues on a glycoprotein.
• composition of the invention may be produced by chemical modi­fication of a crude extract, filtrate, or supernatant obtained from a fungal culture, preferably after concentration.
• the enzymes may be purified from a fungal material before any chemical treatment. It is particularly advantageous to use lignin peroxidases from a species or strain which does not produce cellulases especially when the enzyme is not purified.
• lignin perox­idase of a white-rot fungus e.g. P. chrysosporium as indicated above.
• the process of the invention may be applied to a wide variety of wood pulps the residual lignin content of which is to be reduced.
• bleached wood pulps which may be treated vith the process of the in­vention are advantageously mechanical pulps, e.g. groundwood pulp, including the thermomechanical pulps such as thermomechanical pulps (TMP), chemimechanical pulps (CMP), chemithermomechanical pulps (CTMP) and chemical pulps (CP) such as sulphite and Kraft pulps, these latter being preferred.
• the enzyme concentration may range from 0.001 to 1000 VAO units/g pulp (a VAO unit is determined by the conversion of veratrylalcohol to veratrylaldehyde at 310 nm 9.3 ⁇ mol.cm ⁇ 1 at 30°C, pH 3.5), preferably from 0.1 to 50 VAO units/g pulp, more preferably from 1 to 20 VAO units/g pulp.
• Optimal enzyme concentration depends upon the commercial origin and type of pulp.
• Wood pulp is advantageously submitted to alkaline extraction be­fore being enzymatically treated.
• the enzymatic treatment is advanta­geously carried out at a pulp consistency of from 0.1 % to 15 %, pre­ferably of from 1 % to 5 %.
• the pulp consistency is determined by a standard procedure as the dry weight of pulp after drying for 2 to 10 hours at about 105°C.
• the un­bleached wood pulp may be diluted with deionized vater, fresh water or tap water during the bleaching process.
• fresh water or tap water is preferred since it has been found that the characteristics of the water do not influence the final re­sults.
• fresh water is meant water pumped directly e.g. from lakes, ponds or rivers.
• the period of time necessary for treating the pulp may greatly vary with respect to the quality of the substrate and the nature of the enzyme modification from a fern minutes to several hours.
• Optimal temperature and pH conditions should be adapted to the particular enzyme of use.
• temperature is generally in the range from 20 to 50°C, preferably from 40 to 50 °C.
• the pH of the system is usually in the range of from 2 to 5, preferably from 3 to 4.
• the reaction time is usually 30 to 60 minutes.
• extractants include, for example, bases such as alkali metal hydroxides, dimethylformamide, dioxane, acetone and alcohol.
• bases such as alkali metal hydroxides, dimethylformamide, dioxane, acetone and alcohol.
• a dilute aqueous sodium hydroxide ex­traction is generally preferred.
• a typical extraction step may be carried out at a pulp consistency from 1 to 20%, preferably from 1 to 5% at a temperature between 40 and 60°C.
• the final pH is preferably from 10 to 11.
• Reaction time may be from 30 minutes to 3 hrs, preferivelyably from 45 minutes to 2 hrs.
• the extent of delignification of the pulp may be indicated by the Kappa number as measured in a standard method described in TAPPI Test Methods (Tappi, Atlanta, Ga.) Vol. 1, 1988 "Kappa number of pulp - T 236 cm 85".
• the Kappa number is the volume (in millilitres) of 0.1N potassium permanganate solution consumed by one gram of moisture-free pulp under the conditions specified in the above method.
• a lower Kappa number is desirable as it indicates that a smaller amount of lignin is present in the pulp.
• Another similar process of particular interest involves also the treatment of aqueous waste water released from the pulping process of wood or from the bleaching process of wood pulp in order to further degrade the lignin component.
• a typical waste water which may be treated with a lignin peroxidase in the exclusive presence of oxygen as a co-substrate is the E1 effluent of the Kraft process.
• the recombinant apo-ligninase of P. chrysosporium is recovered from the pellet fraction of a culture lysate of E. coli (pBSR3) NRRL-18068 by extraction in 4Murea 50 mM sodium acetate 10mM dithiothreitol (DTT). The supernatant extract is then separated from the pellet by appropriate centrifugation and applied on a DEAE-­Sepharose anion exchange column. A gradient of 0 to 1M NaCl is run in the extraction buffer. Fractions are collected and analysed for their immunoreactivity with an anti-ligninase antibody.
• the most strongly immunoreactive fractions are pooled and applied to a sizing column (S-300 Sephacryl; Pharmacia) in 4M urea 50mM KH2PO4 4mM DTT pH 7. Again, the fractions are checked for their immunoreactivity and the most strongly reactive fractions are pooled and dialysed against Tris-HCl pH 8 1mM DTT 20% (v/v) glycerol.
• Protoheme IX (Sigma) dissolved in 0.1N KOH is added to the dialysed solution. This is then dialysed against 50mM Tris HCl pH 8. 1mM reduced glutathione 100 ⁇ M oxidized glutathione overnight at 4°C. Finally the sample is dialysed against 10mM sodium acetate pH 6.
• 2.5 g of Kraft pulp obtained from hardwood are extracted first with 2.5% sodium hydroxide for one hour at 50°C and then washed with tap water to neutrality. The consistency of the pulp is adjusted to 2.5% (approximately corresponding to 2.5 g pulp diluted in 100 ml tap water) and the pH is lowered to pH 3.5 with hydrochloric acid. The mixture is then flushed with oxygen whilst stirring.
• VAO units/g pulp of the reconstituted ligninase are added to the mixture and the reaction is performed for one hour at 40°C.
• a control sample with heat denatured enzyme is also prepared as well as a sample without enzyme.
• the reaction is stopped by washing with tap water and the Kappa number of the enzymatically treated preparation and the control sample is measured.
• the preparation treated with the recombi­nant ligninase in the exclusive presence of oxygen as co-substrate exhibits a lower Kappa number in comparison with the control sample, revealing that a significant delignification has been achieved.
• a concentrated ligninolytic enzyme mixture essentially containing ligninases and Mn-dependent peroxidases is obtained by ultrafiltration (MW cut off 10,000) of a culture of P. chrysosporium ATCC 24 725 produced by the method of Linko, Enzyme Microb. Technol. 1988, 10 , 410-417. Such a mixture has an enzymatic activity of 125 VAO units/ml. The protein content of the mixture is 5 mg/ml as determined by the method of Bradford et al, Anal. Biochem. (1976) 72 : 248.
• 7.5 g of Kraft pulp obtained from hardwood are extracted first with 2.5% sodium hydroxide for one hour at 50°C and then washed with tap water to neutrality. The consistency of the pulp is adjusted to 2.5% (approximately corresponding to 2.5 g pulp diluted in 100 ml tap water) and the pH is lowered to pH 3.5 with hydrochloric acid.
• the preparation is divided into three samples. One is supplemented with 100 ⁇ M H2O2, another one is supplemented with 100 ⁇ M H2O2 and flushed with oxygen, yet another one is only flushed with oxygen.
• Crude lignin peroxidase from Phanerochaete chrysosporium was either produced according to published procedures (e.g. H. Janshekar, A. Fiechter; J. of Biotechnology 1988, 8 , 97-112) or purchased from Cultor Ltd., Helsinki; Finland.
• the enzyme preparation prepared as described in Example 3 is then added to the pulp suspension and the reaction is then performed for one hour at 40°C.
• the reaction is terminated by filtration and a sub­sequent sodium hydroxide extraction as described above.
• the degree of delignification is measured by determination of the Kappa number.
• the lignin is also analytically detectable in the com­bined filtrate/alkaline extract e.g. by gel filtration high perfor­mance liquid chromatography using UV/Vis spectroscopy for detection.
• a PEG modified bovine serum albumine prepared in the same way as the modified enzyme preparation, bovine Heme (Sigma Nr. H-2250), 10 ⁇ g/ml.
• b 5 VAO-units.g pulp.
• c 50 VAO-units.g pulp.
• Table 2 Effect of Hydrogen Peroxide vs.
• Oxygen alone TYPE OF ENZYME PREPARATION DELIGNIFICATION (% Kappa Number Decrease) H2O2 O2 no enzyme 0 0 non-modified 50 units/g 0 6 recombinant 50 units/g 0 18 PEG-modified 5 units/g 0 15 ConA-modified 5 units/g 0 16 Hydrogen peroxide concentration was 100 ⁇ Mol/litre; oxygen was as in Table 1.
• Table 3 Delignification of Different Pulp Types PULP TYPE DELIGNIFICATION (% Kappa Number Decrease) hardwood kraft 15 softwood kraft 8 mixed mechanical 4 softwood sulfite 7 Enzyme concentration was 5 VAO-units/g pulp; A PEG-modified enzyme preparation was used.
• Example 4 is repeated using 50 VAO units/g pulp of the enzymatic mixture as prepared in Example 3 c). When added at the same concentra­tion, the enzymatic mixture treated with endoglycosidase F is more effective in delignifying the pulp than a non-modified mixture.
• reaction products were analysed by gel permeation high perfor­mance liquid chromatography (HPLC) on two serially connected TSK (GMP W&L, 7.8 x 300 mm) columns (Toya Soda, Japan).
• HPLC gel permeation high perfor­mance liquid chromatography
• TSK GMP W&L, 7.8 x 300 mm
• the flow rate was 1 ml/min. and sodium carbonate (10 mM, pH 10.5) with 0.05% polyethylene glycol (PEG 6000) was used as eluent.
• Absorption at 250, 310 and 360 nm was recorded using a diode array UV-detector.
• the enzyme treated lignin was extensively modified. Substantial brightening of the lignin suspension was observed after the enzyme treatment. The absorption spectra at 250, 310 and 360 nm of the individual lignin components after separation by gel permeation chro­matography was extensively altered.

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• Life Sciences & Earth Sciences (AREA)
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• Wood Science & Technology (AREA)
• Chemical & Material Sciences (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• Paper (AREA)
• Chemical And Physical Treatments For Wood And The Like (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Enzymes And Modification Thereof (AREA)

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• 1990
  • 1990-09-10 AU AU62323/90A patent/AU646403B2/en not_active Ceased
  • 1990-09-10 ES ES90810681T patent/ES2067719T3/es not_active Expired - Lifetime
  • 1990-09-10 DE DE1990615294 patent/DE69015294T2/de not_active Expired - Fee Related
  • 1990-09-10 FI FI904456A patent/FI904456A0/fi not_active IP Right Cessation
  • 1990-09-10 EP EP90810681A patent/EP0418201B1/de not_active Expired - Lifetime
  • 1990-09-10 NO NO903924A patent/NO178201C/no unknown
  • 1990-09-11 BR BR909004525A patent/BR9004525A/pt unknown
  • 1990-09-11 PT PT9527390A patent/PT95273A/pt not_active Application Discontinuation
  • 1990-09-11 CA CA 2025079 patent/CA2025079A1/en not_active Abandoned
  • 1990-09-12 JP JP24017190A patent/JPH03104993A/ja active Pending

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