NO178201B - Lignin-degrading enzymatic preparation for the treatment of pulp of wood and method for bleaching of pulp - Google Patents

Description

The present invention relates to a lignin-degrading enzymatic preparation for the treatment of wood pulp.

Furthermore, the present invention relates to a method for bleaching pulp.

These and other features appear in the subsequent patent claims.

In the method according to the invention for bleaching pulp of wood, raw lignin peroxidase can be used in the presence of oxygen instead of hydrogen peroxide as cosubstrate to reduce the lignin content of the pulp of wood. The lignin peroxidase can be used in a modified form.

Wood is a complex material composed of cellulose, hemicellulose and lignin together with other less significant components. The lignin is associated with and even covalently bound to a matrix of cellulose and hemicellulose. In papermaking processes, the lignin should be removed from the pulp of wood as it reduces strength, causes a brownish color and imparts other undesirable properties to the final product. Conventionally, wood chips are first treated with sodium sulphide (Na2S) and sodium hydroxide (NaOH) to significantly break down the lignin. This is called the sulfate process or the Kraft process. Alternatively, other treatments can be used, e.g. the sulphite process. The masses obtained therefrom are called "chemical masses".

Chemical mass such as Kraft pulp usually contains approximately 4-12% by weight of residual lignin, which gives the pulp a characteristic brown colour. At this stage of delignification, the Kappa number, which reflects the lignin content of the pulp, is usually from 10 to 45, most often from 12 to 30. To obtain a pulp with high lightness and lightness stability, the lignin content should be further reduced by means of one or more treatments or step which is usually referred to as bleaching. Many industrial bleaching processes already exist, but most of them are divided into two main parts, namely a complementary delignification followed by "true bleaching" to improve the lightness level. The complementary delignification typically starts with an oxygen stage or a chlorination-extraction (C-E) stage or both. Chlorination and extraction are usually carried out in sequence, where chlorinated lignin compounds are first formed which are then made soluble in the subsequent extraction step. The purpose is exclusively to delignify the pulp as very little lightness increase is achieved at the C-E stage. A complementary process for lightening the lignin can further include the use of components other than chlorine such as e.g. chlorine-containing chemicals such as hypochlorite and chlorine dioxide or oxygen and hydrogen peroxide.

The effluents resulting from the complementary treatment (referred to as E-1 effluents) contain a very large number of chlorinated organic compounds which are harmful to the environment, e.g. dioxins. Also due to their highly corrosive nature, it is quite difficult to recycle the effluents. From an environmental point of view, it is thus clear that new methods of bleaching that can reduce pollution are highly desirable.

In nature, a number of microorganisms occur that delignify wood and break down and modify lignin. The enzymes involved in such a breakdown lead to the classes of oxidases, peroxidases and hemicellulases. An enzymatic treatment can therefore advantageously replace at least one of the bleaching treatments that involve chlorine compounds in pulp bleaching.

Lignin peroxidases (also called ligninases) and MnlI-dependent peroxidase are enzymes of particular interest and which are secreted by many strains of microbes, especially filamentous fungi. Phanerochaete chrysosporium is a fungus that produces mainly both types of peroxidases. These enzymes are able to modify the lignin content of wood so that the lignin is released from the hemicellulose matrix or made releasable after washing or extraction.

However, the optimization of the experimental descriptions by an enzymatic bleaching process has not yet been achieved. This has remained a significant challenge as an enzymatic process must be able to compete with a chemical process on an industrial scale.

Lignin peroxidases have so far been described as enzymes that require the presence of H 2 O 2 to be effective in breaking down lignin with the eventual presence of oxygen. In EP 345715 A1 it is stated that this system works without the use of oxygen, but in the presence of oc-hydroxy acids and detergents. It is also stated that the peroxide must be produced enzymatically in situ. In DE 3636208 Al it is stated that certain oxidizing agents and reducing agents must be present and the redox potential must be maintained at a certain level throughout the course of the reaction. The processes described in these two patents are not commercially feasible due to the high costs of the cosubstrates that are needed. A later publication (Holzforschung 1989, 43(6), 375-384) shows that lignin peroxidases in the presence of hydrogen peroxide alone do not degrade lignin. In yet another publication (Enzyme Microbiol. Technol. 1985, 7(11), 564-566) it is shown that immobilized lignin peroxidases in combination with hydrogen peroxide alone do not delignify lignocellulosic material.

It has now surprisingly been found that very good results can be achieved by enzymatic delignification of wood pulp when the pulp is treated with a lignin peroxidase in the absence of a peroxide and when the enzymes are first chemically modified in such a way that they are not adsorbed to the pulp.

The invention thus provides a lignin-degrading enzymatic preparation for treating pulp of wood, which is characterized in that it comprises at least one lignin peroxidase derived from a fungal culture that is chemically modified so that it cannot be adsorbed on the pulp.

The invention further provides a method for bleaching pulp, which is characterized in that it comprises treating the pulp with the lignin-degrading preparation in accordance with the invention.

In one embodiment, the method according to the invention is carried out in the substantial absence of added peroxide and in the presence of added oxygen.

By "bleaching process" as used herein is meant a method for delignifying pulp of wood or improving the whiteness or lightness of the pulp of wood or both.

By "lignin" as used herein is meant not only natural, unmodified forms, but also the forms found in chemically treated masses which have been chemically modified in whole or in part by means of various means such as e.g. those used in the Kraft, organosolv or sulphite mass process and in the effluents from these processes.

By "substantial absence of added peroxide" is meant the absence of a substantial amount of added peroxide which would be effective in inducing the degradation of the lignin. In the method according to the invention, a peroxide can thus be added to the reaction mixture in an ineffective amount.

The term "lignin-degrading enzyme" as used herein is meant to include any enzyme that modifies the lignin or hemicellulose component of wood such that the lignin is released from the hemicellulose matrix or rendered releasable after washing or extraction. Suitable lignin-degrading enzymes are hemicellulases, oxidases and peroxidases, the latter being particularly preferred. Examples of 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 and especially filamentous fungi. The term "lignin peroxidases" as used herein is meant to include the crude enzyme preparation produced by the fungus under ligninolytic conditions as well as the individual lignin peroxidase isoenzymes from natural or recombinant producers.

The lignin peroxidase from a white rot fungus such as e.g. P. chrysosporium either from its native source or in recombinant form. The recombinant form of a lignin peroxidase of P. chrysosporium can be obtained as described in PCT patent application 88/2023.

In an embodiment of the method in accordance with the invention, the enzymatic preparation can be in a substantially purified form. However, it is preferred that the enzymatic preparation is a crude extract, a filtrate or a supernatant of a culture of white rot fungi, e.g. P. chrysosporium.

Strains of P. chrysosporium are generally available and methods for growing them in an N- or C-limited medium are already known. As an example, a suitable culture medium is the nitrogen-limited Blll/glucose medium containing 1.08 x 10"<3> M ammonium tartrate, 1.47 x 10"<2> M KH2PO4, 2.03 x 10"<3> M MgSO4-7H20 , 6.8 x 10"4 M CaCl2- 2H20,

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 nitriloacetic acid, 1.2 x 10"<2> M MgS04-7H20, 1.7 x 10~<2> M NaCl, 3.59 x 10"<4> M FeS04-7H20, 7.75 x 10~<4> M CoCl2, <9.>0 x 10" <4> M CaCl2, 3.48 x 10~<4> M ZnS04, 4 x IO"<5> M CuS04- 5H20, 2.1 x IO"<5> M A1K(S04)2-12H20, 1, 6 x IO"<4> M H3B03, 4.1 x IO"<5> M NaMo04-2H20 and 2.9 x IO"<3> M MnSO4-<H>2<0.>

For use in the method according to the invention, the lignin-degrading enzyme can be chemically modified by means of covalent or non-covalent binding to water-soluble or water-insoluble polymeric compounds which prevent the enzyme from being adsorbed on the pulp during the treatment. Suitable polymeric compounds are e.g. polyethylene glycol (PEG), polypropylene glycol (PPG), polyacrylamides and polymeric sugars with different degrees of polymerization and compositions corresponding to CM cellulose, cellulose, agarose, alginate and chitosan. PEG is a preferred polymeric compound.

Alternatively, the enzyme can be deglycosylated so that the carbohydrate residues that are usually involved in the adsorption mechanisms are at least partially removed. Deglycosylation can be carried out using known methods, e.g. by treating a sample of lignin-degrading enzyme with an enzyme such as an endoglycosidase capable of breaking down the carbohydrate residues on a glycoprotein.

The preparations according to the invention can be prepared by chemical modification of a crude extract, filtrate or supernatant obtained from a mushroom culture, preferably after concentration. Alternatively, the enzymes can be purified from a fungal material before any chemical treatment. It is particularly advantageous to use lignin peroxidases from a species or strain that does not produce cellulases, especially when the enzyme is not purified. Preferably used is lignin peroxidase from a white rot fungus, e.g. P. chrysosporium as indicated above.

The method according to the invention can be used on a wide range of different masses of wood where the residual lignin content is to be reduced. Unbleached pulps of wood that can be used in the method according to the invention are advantageously mechanical pulps, e.g. grinding mass, including the thermomechanical masses such as thermomechanical masses (TMP), chemical mechanical masses (CMP), chemical thermomechanical masses (CTMP) and chemical masses (CP) such as e.g. sulphite pulps and Kraft pulps, the latter being preferred.

As a general rule, the enzyme concentration can be from

0.001 to 1000 VAO units/g mass (one VAO unit is determined by the conversion of veratryl alcohol to veratryl aldehyde at 310 nm =

9.3 (imol.cm-<1> at 30°C, pH 3.5), preferably from 0.1 to 50 VAO units/g mass, more preferably from 1 to 20 VAO units/g mass. Optimum enzyme concentration depends on the commercial origin and type of pulp.

Wood pulp is preferably subjected to alkaline extraction before it is treated enzymatically. The enzymatic treatment is advantageously carried out with a mass consistency of from 0.1 to 15%, preferably from 1 to 5%. The pulp consistency is determined by a standard procedure as the dry weight of the pulp after drying for 2 to 10 hours at about 105°C. To achieve an optimal pulp consistency, the unbleached pulp of wood can be diluted with deionized water, fresh water or tap water during the bleaching process. For economic reasons, however, fresh water or tap water is preferred as it has been found that the properties of the water do not affect the final results. By "fresh water" is meant water that has been pumped directly, e.g. from water, ponds or rivers.

It is preferred to wash the mass with an alkaline solution before the enzymatic treatment or to carry out the enzymatic treatment after an alkaline step, e.g. oxygen bleach stage or E stage.

The time period required for processing the pulp can vary greatly with respect to the quality of the substrate and the nature of enzyme modification from a few minutes to several hours. Optimal temperature and pH conditions should be adapted to the particular enzyme used. However, the 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 from 2 to 5, preferably from 3 to 4. The reaction time is usually 30 to 60 minutes.

After the enzymatic treatment, the solubilized lignin can be removed from the pulp either by washing, filtration or by extraction, preferably by extraction. Suitable extractants include e.g. bases such as alkali metal hydroxides, dimethylformamide, dioxane, acetone and alcohol. A dilute aqueous sodium hydroxide extraction is generally preferred. A typical extraction step can be carried out at a mass consistency of 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. The reaction time can be from 30 minutes to 3 hours, preferably from 45 minutes to 2 hours.

The degree of delignification of the pulp can be indicated by the Kappa number as measured by the 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 milliliters) of 0.1 N aluminum permanganate solution consumed by 1 gram of moisture-free pulp under the conditions specified in the above-mentioned method. A lower Kappa number is desirable as this indicates that a smaller amount of lignin is present in the pulp.

A further similar process of particular interest also involves the treatment of aqueous waste water released from the wood pulping processes or from the wood pulp bleaching process to further degrade the lignin component. A typical wastewater that can be treated with a lignin peroxidase in the exclusive presence of oxygen as a cosubstrate is the El effluent from the Kraft process.

The invention is further illustrated as follows:

Example 1

Treatment of wood pulp with recombinant lignin peroxidase (ligninase).

1) Preparation of recombinant ligninase

Recombinant apo-ligninase of P. chrysosporium is isolated from the pellet fraction of a culture lysate of E. coli (pBSR3) NRRL-18068 by extraction in 4M urea 50 mM sodium acetate 10M dithiothreitol (DTT). The supernatant extract is then separated from the pellet by appropriate centrifugation and applied to a DEAE-Sepharose anion-exchange column. A gradient of 0 to IM NaCl is carried out in the extraction buffer. Fractions are collected and analyzed for their immunoreactivity with an anti-ligninase antibody. The strongest immunoreactive fractions are collected and added to a graduation column (S-300 Sephacryl, Pharmacia) in 4M urea 50 mM KH2P04 4mM DTT pH 7. The fractions are again checked for their immunoreactivity and the strongest reactive fractions are collected and dialyzed against Tris-HCl pH 8 ImM DTT 20%

(volume/volume) glycerol.

Protoheme IX (Sigma) dissolved in 0.1N KOH is added to the dialyzed solution. This is dialyzed against 50mM Tris HC1 pH 8. ImM reduced glutathione 100JJ.M oxidized glutathione overnight at 4°C. Finally, the sample is dialyzed against lOmM sodium acetate pH 6. 2) Bleaching process with the recombinant ligninase 2.5 g Kraft pulp obtained from the leaves is first extracted with 2.5% sodium hydroxide for 1 hour at 50°C and then washed with tap water to neutrality. The consistency of the mass is set to 2.5% (approximately equivalent to 2.5 g of mass diluted in 100 ml of tap water) and the pH is lowered to pH 3.5 with hydrochloric acid. The mixture is then bubbled through with oxygen while stirring. 50 VAO units/g mass of the reconstituted ligninase are added to the mixture and the reaction is carried out for 1 hour at 40°C. A control sample with heat-denatured enzyme is also prepared as a sample without enzyme.

The turnover is stopped by washing with tap water and the Kappa number of the enzymatically treated preparation and the control sample is measured. Surprisingly, the preparation treated with the recombinant ligninase in the exclusive presence of oxygen as cosubstrate shows a lower Kappa number when compared to the control sample, and this shows that a significant delignification had been achieved.

Example 2

Treatment of wood pulp with a filtrate from a culture of

P. chrysosporium.

A concentrated ligninolytic enzyme mixture mainly containing ligninases and Mn-dependent peroxidases is obtained by ultrafiltration (molecular weight limit 10,000) of a culture of P. chrysosporium ATCC 24 725 prepared 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 determined by the method of Bradford et al., Anal. Biochem. (1976) 72: 248. 7.5 g of Kraft pulp obtained from the leaves are first extracted with 2.5% sodium hydroxide for 1 hour at 50°C and then washed with tap water to a neutral reaction. The consistency of the mass is set to 2.5% (approximately equivalent to 2.5 g of mass diluted in 100 ml of tap water) and the pH is lowered to pH 3.5 with hydrochloric acid.

The preparation is divided into three samples. A sample is supplemented with

100 |XM H 2 O 2 , another sample is supplemented with 100 |1M H 2 O 2 and flushed with oxygen, while a further sample is flushed with oxygen only. 50 VAO units/g mass of the enzymatic mixture are added to each sample and the reaction is carried out for 1 hour at 40°C with stirring. The reaction is stopped by washing with tap water and the Kappa number of the samples is measured.

Surprisingly, better results are obtained in the delignification of pulp in the exclusive presence of oxygen as a cosubstrate than in the presence of hydrogen peroxide supplemented or not supplemented with oxygen.

Example 3

Modification of enzyme preparation

Crude lignin peroxidase from Phanerochaete chrysosporium was either prepared according to published procedures (eg, H. Janshekar, A. Fiechter, J. of Biotechnology 1988, 8, 97-112) or purchased from Cultor Ltd., Helsinki, Finland. a) Modification with activated methoxy polyethylene glycol 1 ml of crude lignin peroxidase preparation (activity: 110 VAO units, protein content 5 mg (Bradford et al., Anal. Biochem. 1976, 72, 248)) was diluted in 9 ml of acetate buffer 50 mM. After adjusting the pH to 7.5, 1 g of cyanuric acid chloride-activated methoxy polyethylene glycol (Sigma No. M-3277) was added. This solution was stirred overnight at 4°C. The enzymatic activity after the treatment was 75% of the original mixture. No further purification was carried out. b) Modification with ConA-Sepharose (Concanavalin A-Agarose) 1 ml crude lignin peroxidase preparation (as under a)) was mixed with 1 g ConA-Sepharose (Pharmacia) in 10 ml acetate buffer (100 mM) at pH 7 overnight at 4° C. The solid complex was then washed with 100 ml of the same buffer. The yield with respect to the activity was 50%.

c) Deglycosylation

1 ml of crude lignin peroxidase preparation (as under a)) was diluted in 1 ml of acetate buffer (100 mM, pH 5). Then 10 units of endoglycosidase F (Boehringer) were added to the preparation and the reaction was carried out at 37°C for 2 hours. The yield with regard to activity was 30%.

Example 4

Treatment of wood pulp.

2.5 g of the mass in question is first extracted with sodium hydroxide (2.5% of g of dry mass, 10% consistency) for 1 hour at 50°C and then washed with tap water to a neutral reaction. After adding 100 ml of tap water, the pH is lowered to 3.5 with hydrochloric acid. The mixture is then bubbled through with oxygen while stirring.

The enzyme preparation prepared as described in example 3 is then added to the mass suspension and the reaction is then carried out for 1 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 determining 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.

Better results when delignifying the pulp are achieved with a modified enzyme preparation than with a non-modified enzyme preparation, even if the enzymatic activity of the modified preparation per g mass was 10 times lower than for the unmodified preparation (table 1).

Better results are obtained when oxygen alone is used than when hydrogen peroxide alone is used (table 2).

Delignification of løwed kraft pulp, softwood kraft pulp mixed mechanical pulp and softwood kraft pulp can be achieved (Table 3).

Example 5

Treatment of wood pulp.

Example 4 is repeated using 50 VAO units/g mass of the enzymatic mixture as prepared in example 3 c). When this is added at the same concentration, the enzymatic mixture treated with endoglycosidase F is more effective at delignifying the pulp than a non-modified mixture.

Example 6

Modification of lignin.

200 µg of Organosolv lignin (87/64003; Organocell, Munich, Germany) from a 2% stock solution in dioxane in 1 ml sodium tartrate buffer (100 mM) pH 3.5 was incubated with 1 VAO unit of a ConA-Sepharose modified enzyme preparation at 40°C for 1 hour while flushing with oxygen. After this, the pH was adjusted

to 10.5 with sodium hydroxide and the sample was filtered through a 0.45 µm filter to remove the enzyme/ConA complex. A sample treated in the same way with ConA-Sepharose, but without enzyme, was prepared at the same time.

The reaction products were analyzed by gel permeation high-performance liquid chromatography (HPLC) on two series-connected TSK (GMP W&L, 7.8 x 300 mm) columns (Toya Soda, Japan). 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. Absorbance at 250, 310 and 360 nm was determined using a diode array UV detector.

The enzyme-treated lignin was highly modified. Substantial lightening of the lignin suspension was observed after the enzyme treatment. The UV absorption spectra at 250, 310 and 360 nm of the individual lignin components after separation by gel permeation chromatography were strongly altered.

All reaction mixtures were flushed with oxygen before and during the course of the reaction (1 hour).

a: PEG-modified bovine serum albumin prepared in the same manner as the modified enzyme preparation, bovine Heme (Sigma No. H-2250), 10flg/ml.

b: 5 VAO units/g mass,

c: 50 VAO units/g mass.

The hydrogen peroxide concentration was 100 jiMol/liter; the oxygen content was as in table 1.

Claims (6)

1. Lignin-degrading enzymatic preparation for treating wood pulp, characterized in that it comprises at least one lignin peroxidase derived from a fungal culture which is chemically modified so that it cannot be adsorbed onto the pulp. 2. Preparation as stated in claim 1, characterized in that the lignin peroxidase is associated with a water-soluble or -insoluble polymeric compound by covalent or non-covalent bonding. 3. Preparation as stated in claim 1, characterized in that it comprises a lignin peroxidase which is at least partially deglycosylated. 4. Preparation as specified in one or more of claims 1-3, characterized in that it is derived from a crude extract, a filtrate or a supernatant of Phanerochaete chrysosporium. 5. Procedure for bleaching pulp, characterized in that it includes treating the pulp with a preparation as specified in one or more of claims 1 to 4. 6. Method as stated in claim 5, characterized in that it is carried out in the substantial absence of added peroxide and in the presence of added oxygen.