JPH03104993A - Bleaching of wood pulp with enzyme - Google Patents

Abstract

PURPOSE: To effectively bleach wood pulp by treating the pulp with lignin peroxidase in the absence of peroxide and in the presence of oxygen. CONSTITUTION: Bleaching of wood pulp is carried out by treating the wood pulp with at least one lignin peroxidase in the substantial absence of added peroxide and in the presence of oxygen. As the above enzyme, those obtained from crude extracts, filtrates or supernatants of white-rot fungi are preferable, and one obtained from a crude extract. a filtrate or a supernatant of Phanerochaete chrysosporium is more preferable.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application] The present invention relates to a new enzymatic process for bleaching wood pulp, in which a co-substrate (CO- substrat
As e) crude lignin peroxidase is used in the presence of oxygen rather than hydrogen peroxide.

This lignin peroxidase can be used in modified form.

BACKGROUND OF THE INVENTION Wood is a complex material consisting of cellulose, hemicellulose and lignin as well as other trace elements.

Lignin is associated and even copolymerized with cellulose and hemicellulose matrices. In the papermaking process, lignin should be removed from the wood pulp. This is because lignin reduces strength, imparts a brown color to the final product, and imparts other undesirable properties. Traditionally, wood chips are first treated with sodium sulfide (NazS) and sodium hydroxide (NaOH) to substantially degrade the lignin. This is called the sulfation or kraft process. Alternatively, other treatments can be used, such as a sulfite process. The pulp obtained in this way is called ``chemical pulp.'' Chemical pulps, such as kraft pulps, usually contain about 4-12% by weight of residual lignin, which gives the pulp its characteristic brown color. In this delignification stage, the kappa number, which reflects the lignin content of the pulp, is usually 10 to 45.
, more often 12-30. In order to obtain high brightness and brightness stability, the lignin content must be further reduced in one or more treatments or steps, commonly referred to as bleaching. Many industrial bleaching processes already exist, but almost all of them can be divided into two main parts: supplementary delignification, followed by "true bleaching" to improve brightness levels. Supplemental delignification typically begins with an oxygen step or a chlorination-extraction (C-E) step or both. Chlorination and extraction are generally carried out sequentially, first the chlorinated lignin compounds are formed and these compounds are then solubilized in the extraction step. Since little bleaching occurs in the C-E stage, its purpose is solely to delignify the pulp. Supplementary steps for bleaching lignin may further include the use of substances other than chlorine, such as chlorine-containing chemicals such as hypochlorite and chlorine dioxide, or oxygen and hydrogen peroxide. The effluent resulting from the supplementary treatment (referred to as E-1 effluent) is
It contains a large number of chlorinated organic compounds, such as dioxins, which pose a hazard to the environment. Furthermore, due to their highly corrosive nature, it is very difficult to recycle the waste fluids. From an environmental point of view, new techniques that can reduce pollution are therefore highly desirable.

In nature, there are numerous microorganisms that delignify wood and degrade and modify lignin.

The enzymes involved in such digestion belong to the group of oxidases, peroxidases and hemicellulases.

Therefore, enzymatic treatment can be used to replace at least one of the chemical treatments using chlorine compounds in pulp bleaching.

Lignin peroxidase (also called ligninase) and MnlI-dependent peroxidase are secreted by many microbial strains, especially filamentous fungi. An enzyme of particular interest, Phanerochaete chrysosporium (Ph
anerochaete chr sos orius
+) One is a fungus that produces essentially both types of peroxidase. These enzymes can modify the lignin of the wood so that it is released from the hemicellulose matrix or can be released during washing or extraction.

However, optimization of the experimental conditions in the enzymatic bleaching process has not yet been achieved. Will enzymatic processes compete with chemical processes at the industrial level? This is a big challenge because we have to be able to do it.

Up until now, lignin peroxidase has been shown to require 11 to 11 ml with the optimal presence of oxygen to be effective in degrading lignin.
■It was described as an enzyme that requires the presence of Of. E
P 345715 At describes that this system works without the use of oxygen, but in the presence of α-hydroxy acids and detergents. At the same time, it is described whether peroxide needs to be produced enzymatically in situ.

DE 3636208 Al states that certain oxidizing and reducing agents must be present throughout the course of the reaction and that the redox potential must be maintained at a certain level. The processes described in these two patent specifications are not commercially viable because they require expensive auxiliary substrates. Recent announcements (Holz
forschung 19B9. 43(6), 37
5-3843, it has been shown that lignin peroxidase does not degrade lignin in the presence of hydrogen peroxide alone.

Other announcements (t!ngyme Micrbiol.Tec
hnol. 1985+ 7(11), 564-56
63, it has been shown that immobilized lignin peroxidase does not delignify lignocellulosic materials in combination with hydrogen peroxide alone.

[Summary of the invention]

It is now surprising that enzymatic desorption of wood pulp is possible if the pulp is treated with lignin peroxidase in the absence of peroxide and if the enzyme is first chemically modified so that it is not adsorbed to the pulp. It has been found that very good results are achieved with lignin.

The present invention therefore provides a method for bleaching wood pulp comprising treating the wood pulp with at least one lignin peroxidase in the substantial absence of added peroxide and in the presence of oxygen. Furthermore, the present invention
An enzyme composition (enzyme preparation) comprising at least one lignin peroxidase derived from a fungal culture, chemically modified so that it cannot be adsorbed to pulp.

[Specific description]

As used herein, "bleaching process" means a process for delignifying wood pulp, or for improving the whiteness and/or gloss of wood pulp.

As used herein, "lignin" refers not only to its natural, unmodified form, but also to chemical lignin, which may be wholly or partially chemically produced by various agents, such as those used in kraft pulp, organosolv pulp, or sulfite pulp manufacturing processes. Also meant is the form as found in modified chemically processed pulps and in the effluents of these processes.

By "substantial absence of added peroxide" is meant the absence of a substantial amount of added peroxide that may be effective in inducing decomposition of lignin. Accordingly, the scope of the present invention is intended to include such processes in which peroxide is added to the reaction medium in non-effective amounts.

As used herein, the term "lignin-degrading enzyme" refers to any enzyme that modifies the lignin or hemicellulose fraction of wood so that it is released from the lignin or hemicellulose matrix or made available for release during washing or extraction. meant to include enzymes. Suitable lignin degrading enzymes are hemicellulases, oxidases and baroxidases, the latter being particularly preferred. Examples of hemicellulases include mannanase, xylanase, galactomannanase and arabinosidase, while laccase belongs to the group of oxidases. Preferred peroxidases are MnII-dependent peroxidase and lignin peroxidase (also referred to as ligninase). Ligninolytic enzymes are secreted by many microbial strains, especially filamentous fungi.

As used herein, the term "lignin peroxidase" refers to crude enzyme preparations produced by manganese under lignin-degrading conditions, as well as individual lignin peroxidases from natural or recombinant methods. It is meant to include zeisoenzyme.

Preferably used are white rot fungi, native or recombinant, such as P. Chrysosporium ginin peroxidase. P. Recombinant lignin peroxidase of Chrysosporium is published in PCT patent application Nα88/2023
can be obtained as described in .

For use in preferred methods of the invention, enzyme compositions can be in substantially pure form. However, enzyme compositions are not suitable for white rot fungi such as P. It can be a crude extract, filtrate or supernatant of a culture of Chrysosporium.

P. Strains of Chrysosporium are publicly available and methods of culturing them in C1 or N1 restricted media are already known. For example, a suitable medium is 1.08
X 10-'M ammonium tartrate, 1.47x10
~2M KHzP04, 2.03XIO 1st publication MgSOn
・711■0. 6.8x10-'M CaClz
-2 HzO, 2.96 x 10-''M thiamine HCI and a nitrogen-limiting BIII/glucose medium containing 10 d/ffi of a trace element solution. The trace element solution is 7.8 x 10-3 M nitriloacetic acid, 1.2×10-”M MgSO4' 711z0,
1. 7XIO-”M NaCl, 3.59XIO
-'M FeSO4H 7tlzO, 7. 75 X
10-'M CoC E z, 9. OXIO-'
M CaCl2'3.48X10-'M ZnSOa
, 4xio-'MCuSOa ・511zO, 2.
lX10-'M AfK(SO4)112Hzo,
1. 6 XIO-'M HJOi, 4, I XI
O-'MNaM604・2tl20 and 2. 9 X
Contains 10-'M MnSOn'lIzo.

For use in the method of the invention, the lignin-degrading enzyme is chemically modified, either covalently or non-covalently, with a water-soluble or insoluble polymeric compound that prevents the narcotic from being adsorbed to the pulp during processing. You can. Suitable polymeric compounds include, for example, polyethylene glycol (PEG).
, polypropylene glycol (PPG), polyacrylamide and various degrees of polymerized sugars and CM
- M1 substances such as cellulose, cellulose, agarose, alginate and chitosan. PEG is a preferred polymeric compound. Alternatively, the enzyme may be deglycosylated to at least partially remove the carbonide residues normally involved in the mechanism of adsorption. Deglycosylation can be carried out in known manner, for example by treating lignin-degrading enzymes with enzymes such as endoglycosidases, which are capable of degrading carbohydrate residues on glycoproteins.

&IIIi. of the present invention. The product can be produced by chemically modifying the crude extract, filtrate or supernatant obtained from the fungal culture, preferably after filtration.

Alternatively, enzymes can be purified from fungal material prior to chemical treatment. It is particularly advantageous to use lignin peroxidase from species or strains that do not produce cellulase, especially if the enzyme is not purified. As mentioned above, white rot fungi such as P. Preferably, Chrysosporium gluenin peroxidase is used.

The method of the invention can be applied to a wide variety of wood pulps whose residual lignin content is to be reduced. The pulp that can be treated by the method of the invention is advantageously a mechanical pulp, such as a thermomechanical pulp.
chan ica 1) Groundwood pulp including pulp such as thermo-mechanical pulp (TMP), chemical-mechanical pulp (ch
eIIIin+echan ica 1) Pulp (CM
P), chemical thermomechanical
chan ica I) pulp (CTMP), and chemical pulps such as sulphite pulp and kraft pulp, the latter being preferred.

As a general rule, the enzyme concentration should be between 0.001 and 1000 VA.
O unit/g pulp (VAO unit is 310mm
=9.3 μmol. cm-', 30°C. ．． pH3.
(determined by the conversion of veratryl alcohol to veratryl aldehyde at step 5), will range from 0.1 to 50 vAO units/g half, more preferably from 1 to 20 VAO units/g pulp. The optimum enzyme concentration depends on the commercial origin and type of pulp.

The wood pulp is advantageously subjected to alkaline extraction before being enzymatically treated. Enzyme treatment advantageously ranges from 0.1% to 15%
, preferably at a pulp consistency of I% to 5%. Pulp consistency is approximately 105℃
It is determined by standard methods as the dry weight of the pulp after drying for 2 to 10 hours. To achieve optimal pulp consistency, unbleached wood pulp can be diluted with deionized water, fresh water, or tap water during the bleaching process. However, fresh water or tap water is preferred for economic reasons, as it has been found that the properties of the water do not affect the final result. "Freshwater" means water drawn directly from, for example, a lake, pond or river. Preferably, the pulp is washed with an alkaline solution before the enzyme treatment or the enzyme treatment is carried out after an alkaline stage, such as an oxygen bleaching stage or an E-1 stage. The time required to process the pulp can vary widely from several minutes to several hours in view of the nature of the substrate and the nature of the enzyme modification. Optimal temperature and p11 conditions should depend on the particular enzyme used. However, the temperature will generally range from 20°C to 50°C, preferably 40°C.
~50°C. The p value of the system is usually in the range of 2 to 5, preferably in the range of 3 to 4. Reaction time is usually 30
~60 minutes.

After the enzymatic treatment, removal of solubilized gunins from the pulp can be carried out by washing, filtration or extraction, preferably by extraction. Suitable extractants include, for example, bases such as alkali metal hydroxides, dimethylformamide,
Contains acetone and alcohol. Dilute aqueous sodium hydroxide extraction is generally preferred. A typical extraction step is
It can be carried out at a pulp consistency of 1 to 20%, preferably 1 to 5%, and a temperature between 40 and 60C. The final pH is preferably between 10 and 1 l. The reaction time is 30 minutes to 3 hours, preferably 45 minutes to 2 hours.

The degree of pulp delignification is determined by TAPPI TestM
methods (Tappi Atlanta, G.
a) Vol. 1. 1988 'Kappanum
It can be expressed by the kappa number measured by the standard method described in "Ber of Pulp-T236c+++85". The kappa number is the O. 1N potassium permanganate? '8 is the volume (d) of the liquid. A low kappa number is preferred since a low kappa number indicates that less lignin is present in the pulp.

Other similar methods of particular interest include the treatment of wastewater released from wood pulping processes or from wood pulp bleaching processes to further degrade lignin content. A typical wastewater that can be treated by lignin peroxidase in the presence of only oxygen as a co-substrate is the Kraft process El
It is waste water.

Next, the invention will be further explained.

P. Chrysosporium &ll recombinant averligninase was purified by Escherichia coli (pBSR3) NRRL-180
From the pellet fraction of 68 cultured cell lysates, 5M urea,
501. Recover by extraction in sodium acetate and 10mM dithiothreitol (DTT). The extraction supernatant is then separated from the pellet by appropriate centrifugation and DE
Apply to AR-Sepharose anion exchange power ram. 0 to I M NaCj in extraction buffer! Perform a gradient of Both aliquots are collected and analyzed for immunoreactivity using anti-ligninase antibodies. The most strongly immunoreactive fractions were pooled and treated with 4M urea, 50mM KMzPO.
n, 4 nM DTT (pH 7) medium sizing column (S-300 Sephacryl; Pharmacia)
Applicable to. Again, both fractions were checked for their immunoreactivity, and the most strongly reactive fractions were pooled and Tris-11cj! (pH 8), 1sM
Dialyze against DTT, 20% (V/V) glycerol. Protoheme (proto) is added to this dialyzed solution.
Add heme) IX (Sigma). Next, add this to 50
a+M Tris-HCi! (pll8), 1 mM
Dialyze overnight at 4°C against reduced glutathione and roomM oxidized glutathione. Finally, add this sample to 1
Dialyze against 0mM sodium acetate (pH 6).

2) Containing ligninase First, 2.5 g of kraft pulp obtained from engineered hardwood was subjected to 2.
Extract with 5% sodium hydroxide for 1 hour at 50°C and then wash with tap water until neutral.

Pulp consistency 2.5% (approximately equivalent to 2.5 g pulp diluted in 100 d tap water)
and pl1 was lowered to pH 3.5 with hydrochloric acid. The mixture is then flushed with oxygen while stirring.

50 VAO units/g pulp of redissolved ligninase is added to the mixture and the reaction is run for 1 hour at 40''C. A control sample with heat-transformed enzyme is also prepared along with a sample without enzyme.

The reaction is stopped by washing with tap water and the kappa numbers of the enzyme-treated preparations and control samples are determined. Surprisingly, preparations treated with recombinant gninase in the presence of only oxygen as a cosubstrate showed lower kappa numbers compared to control samples, indicating that significant delignification was achieved. .

Linko, Enzyme Microb. Tec
hnol. 198B+ Lot 410-41
P. produced by the method of 7. Chrysosporium ATC
Ultracentrifugation (MW power off 10,000) of a culture of C 24.725 removes ligninase and Mn?
A concentrated lignin-degrading enzyme mixture is obtained which essentially contains oxidase. Such a mixture is 125
It has an enzymatic activity of VAO units/unit.

The protein content of this mixture was determined by Bradford et al., An.
al. Biochem (1976) 72:248.

First, 7.5g of kraft pulp obtained from hardwood was mixed into 2.
Extract with 5% sodium hydroxide for 1 hour at 50°C and then wash neutrally with tap water.

Pulp consistency 2.5% (approximately equivalent to 2.5 g pulp diluted in 100 aj! of tap water)
and the pH is lowered to pl 13.5 with hydrochloric acid.

Divide this preparation into three samples. One ha100
supplemented with μM I1■0■, and the other one with 100μM
One is supplemented with HtOt and flushed with oxygen, the other is flushed with oxygen only.

50 VAO units/g pulp of the enzyme 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 sample is determined.

Surprisingly, better pulp delignification results are obtained in the presence of oxygen alone as co-substrate than in the presence of supplemented hydrogen peroxide and without oxygen.

Okunushi Hong Meng Dish Alliance Hikio Shukei Crude lignin peroxidase from Phanerochaete chrysosporium was prepared using published methods (for example, H.J.
anshekar, A. Fiechfer;J+of
Biotechnology 1988. Dan, 97-
112) or from CulLor, Helsinki, Finland.

Crude lignin peroxidase preparation of ld [Activity: 1
10 VAO units; protein content 5 mg (B
radford3, eightal, Biochem. 1
976. 7.2, 24B)) was diluted in 9-50mM acetate buffer. After adjusting pl1 to 7.5, 1g
Methoxypolyethylene glycol (Sigma NaM-3277) activated with cyanuric chloride was added. This solution was stirred at 4°C overnight. The enzyme activity after treatment was 75% of the original mixture.

No further molding was done.

1 ml of crude lignin peroxidase preparation (same as a)] was mixed with 1 g of Con A-Sepharose (Fanoremacia) in 10 ml of 100 mM acetate buffer (pH 7) at 4°C overnight. Next, the solid composite was
d with the same buffer. The yield of activity was 50%.

C. The crude lignin peroxidase preparation [same as a)] of L1 Dannojofu LD was added to 1 mfl of 100 mM acetate buffer (pH 5).
diluted to Next, 10 units of endoglycosidase F (Boehringer) were added to the preparation and the reaction was
Run for 2 hours at 7"C. Yield of activity was 30%.

2.5 g of wood pulp treated with Zhuhu and Earthworks was first diluted with sodium hydroxide (2.5% of dry pulp, 10% consistency) to 50%
Extract for 1 hour at °C and then wash to neutrality with tap water. After addition of 100% tap water, the pl+ is lowered to 3.5 with hydrochloric acid. The mixture is then flushed with oxygen while stirring.

Next, the enzyme preparation prepared as described in Example 3 was added to the pulp suspension and the reaction was then carried out for 1 hr at 40'C.
Do time. The reaction is stopped by filtration and subsequent sodium hydroxide extraction is performed as described above.

The degree of delignification is measured by determining the kappa number. Lignin can also be detected analytically in the filtrate/alkaline extract combination, for example by gel filtration velocity liquid chromatography using a UV/Vis spectrometer for detection. The enzyme activity of the modified preparation per gram of pulp is 10 times lower than that of the unmodified preparation (Table 1), but it is important to note that the enzyme activity of the modified enzyme preparation per gram of pulp is 10 times lower than that of the unmodified preparation (Table 1). Better results are obtained.

Better results are obtained when using only oxygen than when using only hydrogen peroxide (Table 2). Can achieve delignification of hardwood kraft pulp, softwood kraft pulp, mixed groundwood pulp and softwood sulfite pulp (
Table 3).

G-modified bovine serum albumin, bovine II e m e (
Sigma No. If-2250), 10 μg/1.

(b): 5 νAO units/g pulp.

(c): 50 VAO Uni. , To/g HaJL/7”.

Type of enzyme preparation Delignification (lowering of kappa number) No enzyme PEG only Con A - Sepharose only PEG one modified BSA, lleme PEG one modified, heat denatured PEG one modified Con A one modified recombinant All reaction mixtures before and after reaction Flushed with oxygen during (1 hour).

(a): Type of PE enzyme preparation prepared in the same manner as the modified enzyme preparation (decrease) H20■ (h 00 06 0 18 0 15 The hydrogen peroxide concentration was 100 μMol/j2, and oxygen was provided as described in Table 1.

Recruitment SL Shiba Delignification of Different Types of Pulp Types of Pulp Delignification (reduction in copper number) Hardwood kraft pulp 15 Softwood kraft pulp 8 Mixed groundwood pulp 4 Softwood sulfite pulp 7 Enzyme concentration is 5 VAO units/g, PEG-1 A modified enzyme preparation was used.

The method of Example 4 is repeated using the enzyme mixture prepared in Example 3c) 50 VAO units l-/g pulp. When added at the same concentration, the enzyme mixture treated with endoglycosidase F is more effective in delignifying pulp than the unmodified mixture. Organosol lignin from a 2% stock solution in dioxane (87/64003; organocell,
20 μg (Munich, West Germany) of I in sodium tartrate buffer (100 mM) (pH 3.5).
The VAO unit was incubated with the ConA-Sepharose-modified enzyme preparation at 40"C for 1 hour with an oxygen flush. After this, pl+ was adjusted to 10.5 with sodium hydroxide and the sample was incubated at 0.4
Enzyme/ConA complexes were removed by filtration through a 5 μm filter. Similarly, ConA-
Sepharose-treated samples were prepared at the same time.

The reaction product was connected to two TSKs (GMPW) in series.
&L, 7. The samples were analyzed by gel permeation high performance liquid chromatography (IIPLC) on an 8×300 column (Tosoh). The flow rate was ld/min. Sodium carbonate (lQmM, pll 10.5) containing 0.05% polyethylene glycol (PEG 6000) was used as eluent. 250. 310 and 3601+1
[Absorption at +1 was recorded using a diode array U■ detector.

Enzyme-treated lignin was highly denatured.

After enzyme treatment, lignin suspension 'Im? Substantial whitening of & was observed. 250% of the individual lignin components after separation by gel permeation chromatography. 310 and 36
The UV absorption spectra at 0 ms changed significantly.

Procedural amendment (voluntary) November 2016 (ζ day)

Claims (1)

[Claims] 1. A method for bleaching wood pulp, comprising treating the pulp with at least one lignin peroxidase in the substantial absence of added peroxide and in the presence of added oxygen. A method comprising: 2. The method according to claim 1, wherein the enzyme composition is selected from a crude extract, filtrate or supernatant of white rot fungi. 3. The enzyme composition contains Phanerochaete chrysosponium
3. The method according to claim 2, wherein the extract is selected from crude extract, filtrate or supernatant of P. sporium. 4. The method according to claim 1, wherein the lignin peroxidase is obtained in recombinant form. 5. The method according to any one of claims 1 to 4, wherein kraft pulp, organosolv or sulfite pulp is bleached. 6. A method for treating wastewater released from the pulping process of wood or from the bleaching process of wood pulp, comprising treating said wastewater with at least one lignin peroxidase in the absence of peroxides and in the presence of oxygen. A method comprising processing. 7. Phanerochaete chrysosporium
7. The method according to claim 6, wherein the wastewater is treated with an enzyme composition selected from the crude extract, filtrate or supernatant of P. porium. 8. The method according to claim 6 or 7, wherein the wastewater is E1 wastewater. 9. An enzyme composition for treating wood pulp, comprising at least one lignin peroxidase derived from a fungal culture, chemically modified so that it cannot be adsorbed to the pulp. 10. The composition according to claim 9, which is derived from a crude extract, filtrate or supernatant of white rot fungi. 11. The enzyme is P. Chrysosporium (¥P.¥¥c
11. The composition according to claim 10, which is a peroxidase from P. hrysosporium. 12. The composition according to any one of claims 9 to 11, wherein the lignin peroxidase is covalently or non-covalently associated with a water-soluble or insoluble polymeric compound. 13. The composition of claim 12, wherein the water-soluble polymer compound is selected from polyethylene glycol, polypropylene glycol, polymerized sugar, and polyacrylamide. 14. A composition according to any one of claims 9 to 11, comprising at least partially deglycosylated lignin peroxidase. 15. A method for bleaching wood pulp, comprising treating the pulp with a composition according to any one of claims 9 to 14. 16. The method of claim 15 in the substantial absence of added peroxide and in the presence of added oxygen. 17. A method for treating wastewater released from a wood pulping process or a wood pulp bleaching process according to any one of claims 9 to 14, in the absence of peroxide and in the presence of oxygen.
A method comprising treating wastewater with a composition according to paragraph 1.