JPH07501587A - Cellulose pulp processing method and enzyme preparation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Cellulose bulb treatment method and enzyme preparation The present invention provides lignocellulosic materials, The method according to claim 1 relates in particular to the treatment of cellulose bulbs.

The present invention also relates to an enzyme preparation useful for valve treatment according to claim 20.

Traditional chlorine bleaching uses chlorine or chlorine dioxide to remove residual lignin. dissolve. Currently, cellulose valves are also manufactured using oxygen gas, hydrogen peroxide, and ozone. or the bleaching agents mentioned above and combinations containing these substances. be whitened. Enzymatic treatment carried out by hemicellulases or lignin-degrading enzymes is Combined with traditional and new bleaching methods to improve the bleaching properties of textiles came. Only a small amount of enzyme is required to obtain improved bleaching properties, and enzyme treatment is easy. It is incorporated into the valve production process.

According to conventional technology, enzyme treatment is performed directly on the fibers obtained in the pulping process. was. According to the results of the present inventors, the properties of cellulosic fibers are It was found that the effectiveness of the enzymes used was greatly affected. Therefore, for example, Silanase works well when the surface potential (i.e. zeta potential) is very low. Doesn't work. The present inventors discovered that the hemicellulose carboxyl present in the valve At low pH values, where the group is in the acid form and therefore contains no metal counterions, If Kraft Valve is treated with We also observed that it is a poorly suited substrate for microprocessing.

In prior art methods, these factors are completely overlooked and do not alter the fiber condition. No attempt has been made to make it a more suitable substrate for enzymatic treatment.

The carboxyl groups present in cellulosic bulbs are primarily responsible for the surface charge of the bulb. (SjOstr (1m, 1989)). The number of groups essentially depends on the valving method used. Therefore, during mechanical valves In addition, carboxyl groups in kraft are mainly methyl groups in xylan. It consists of a chloric acid group (SjOstrQm, 1989). However, in the sulfurous acid valve, in addition to the methyl glucuronic acid group of xylan, Carboxyl groups are present in lignin produced during the sulfurous acid valve manufacturing process. It consists of sulfonic acid.

The strength and extent of carboxyl group dissociation depend on the counterion of the carboxyl group. (Scallan and Grignon, 1) 979, Mecha52, 1983, Lindström (Lindstr5m) and and Carlsson.

(1982). Usually the counterion is hydrogen or a metal ion. In general, In fact, most of the metals present in the valve are bound to these carboxyl groups. Ru. This applies to both chemical and mechanical valves. cellulose bar Lube metal ions are both useful and harmful in the valve manufacturing process. first time As described in Lube is hardly suitable for enzymatic treatment. On the other hand, the metal ions present in the valve , is undesirable in many respects for the processing of cellulose bulbs. Therefore, many Metal ions, especially iron and manganese, are harmful in the peroxidation process and make things unstable. These substances must be removed for optimal bleaching results. stomach.

Removal of metal ions is also necessary for the manufacture of certain special valves.

According to known methods, the pH of the valve is lowered to a pH value at which metal ions do not dissociate. or treat the valve with known complexing agents such as DTPA or EDTA. (Basta et al., 1991). The genus content can be reduced. However, these methods have considerable deficiencies. There is a point. Therefore, after metal removal under acidic conditions, enzymatic treatment before bleaching is recommended. Produces a highly resistant valve. On the other hand, some non-natural drugs For example, it may cause problems with wastewater purification systems.

Eliminate methodological shortcomings and create a completely new concept in cellulose valve processing is the object of the present invention.

The present invention enzymatically removes carboxyl groups of hemicellulose from cellulose valves. changes both the surface charge and the metal ion content of the bulb. Based on that. Therefore, according to the method of the present invention, The carboxyl groups are removed by treating the cellulose bulb with an enzyme preparation. Ru.

In particular, the method of the invention is characterized by the statement in the characterizing part of claim 1. Ru.

The enzyme preparation of the present invention is characterized by the description in the characterization part of claim 20. .

In this application, "enzyme preparation" refers to a product containing at least one enzyme.

Such enzyme preparations therefore include a culture medium containing one or more enzymes; It may be an isolated enzyme or a mixture of one or more enzymes.

"Essential glucuronidase activity" means that the glucuronidase activity of the enzyme preparation is The enzyme activity is more potent than other enzymes and removes the essential part of the glucuronic acid group from the substrate. means to remove.

The "glucuronic acid group" used in this application refers to the 4-0-methyl-D-curcuronic acid group. It is an abbreviation.

Several enzyme preparations used in the pretreatment step to improve bulb bleaching properties It is known to have some glucuronidase activity that promotes the hydrolysis of lulase. It is being The purpose of these known methods is to obtain (Kantelinen) was used to improve the hydrolysis of en) et al.

(1988; Poutanen et al., 1987). however , the specific enzymatic removal of carboxyl groups has not been studied, but rather Efficient hemicellulose hydrolysis was studied. By the method of the present invention Specific removal of carboxyl groups is difficult depending on the enzyme preparations used so far. This was not possible due to relatively low glucuronidase activity.

Enzymatic hydrolysis of hardwood xylan can be accelerated by glucuronidase treatment Also known in the art: Hydrolysis of 4-0-methylglucuronoxylan Effects of xylanase, β-xylosidase and α-glucuronidase on is described by Ishihara et al. (Ishihara et al., 1999). 1990).

According to the experimental results, in the conventional technology, when glucuronidase is used, The relative hydrolysis rate of (a mixture of xylanase, xylosidase and glucuronidase) (The combined effect is taken as 100%). However, these results do not apply to isolated xylan. It is related to the hydrolysis of. Traditionally, glucuronidase has been used for industrial valves. Regarding applications and varying the metal content and surface charge of such valves. has not been made public at all.

According to the method of the present invention, by enzymatically removing the carboxyl group of the valve, The industrial properties of valve paper can be varied.

According to a preferred embodiment of 1, the glucuronidase treatment is performed by bleaching the cellulose bulb. Do it before the stage. In this way, the consumption of bleach can be reduced.

Glucuronidase treatment reduces the metal ion content of the valve, so hydrogen peroxide can be advantageously used as a bleaching agent.

According to another preferred embodiment, no hemicellulose in the fibers is hydrolyzed. A method in which glucuronidase action on fibrous substances is essentially optimized. The amount of carboxyl groups in the valve is changed using glucuronidase. Made of enzyme The higher the glucuronidase activity in the drug, the easier it is to reach the target. Become. Therefore, the bleaching properties of TCF-bulbs (valves that do not contain any chlorine) are improved. go up

Glucuronidase treatment can be carried out separately (at the same time as another enzyme treatment) or before such processing). Glucuronidase treatment with hemicellulase (e.g. When performed with lanase, mannase) treatment, glucuronidase is added. Enzyme preparations, or genetically modified to produce high glucuronidase activity Using enzyme preparations produced by bacterial strains to create formulations that are inherently high in glucuronidase. It is preferable to obtain According to the present invention, glucuronidase treatment can be performed using other cellulases or and/or ligninase treatment.

Besides chemical valves, the present invention also applies to any lignocellulosic valve (i.e. mechanical valve). or mechanical/chemical valves). In our research, we discovered something unexpected. In addition, the present inventors have demonstrated that the deterioration of bulb whiteness may be reduced by glucuronidase treatment. I found out that.

According to one preferred embodiment of the invention, the enzyme preparation used is a glucuronidase It consists of a culture solution or culture of microorganisms produced by the plant. Preferably, before use, such cultures are Concentrate. According to another preferred embodiment, the enzyme preparation is a Consists of manufactured enzymes.

According to a further preferred embodiment, the glucuronidase is derived from the fungus Trichoderma. ・Glucoro from the culture solution of Trichoderma reisei Nidase enzyme was isolated using ion exchanger and hydrophobic interaction chromatography. Purify using Surprisingly, the enzyme can be easily isolated by these methods. I found out that I can. The new purified glucuronidase was purified by 5DS-PAGE method. It has a molecular weight of about 95 kDa. The glucuronidase was added to Schizophyllum ・Schizophyllum commune or Aspergillus A simple method from the culture of Spergillus niger It can also be isolated. These new enzymes are detailed in Examples 3 and 4. Describe it.

Enzyme preparations useful in the treatment of lignocellulosic materials contain essential glucuronidase enzymes. Although it contains only a small amount of hemicellulase (contains a small amount of hemicellulase) are doing.

However, the invention is not limited to the described enzyme sources, nor is the invention limited to the described enzyme origins. Nor is it limited to law. Enzymes can also be obtained by other methods.

Therefore, Trichoderma or mutated or inherited to produce the desired enzyme. Introducing other microbial strains or genes encoding the enzyme It is possible to produce the enzyme using other production host strains.

The glucuronidase preparation is prepared from Trichoderma (e.g. T. reithei (T. r. eisei)), Aspergillus (e.g. A. niger) ), x I Awamori (＾, awamori), Ei Tellus (＾, ter rus), A. oryzae), Nzophyllum If (e.g. Banis commune (S, commune), Aureobasidium (Aureob) genus Acidium (e.g. A. pullulans) , Phanerochaete genus (e.g. Bee chryso Sporium (P, chrysosporium), Fusarium (Fusari) um) genus (e.g. F, oxysporu+n), Aga The genus Garicus (e.g. A. bisporus) ) Penicillium! (For example, Bee Ja janthinelum (P, janthinelum), B. digitatum (P, d Streptomyces genera (e.g. S, olivochromogenes) ), Nis flavogriseus (S, flavogriseus), and Genus Bacillus (e.g. B, 5ubtil) is), essentially derived from B, circulars. It may be derived from a microorganism selected from the group consisting of:

All these microorganisms were used to develop glucuridase (glucuronin adjacent to xylan). Hydrolyzing acid groups) can also be produced.

According to a preferred embodiment, any of the above-mentioned glucuronides is added to a medium consisting of xylan. The enzyme preparation can also be obtained by culturing enzyme-producing microorganisms.

The method of the invention offers significant advantages. Therefore, as described herein, separate Treating the valve with glucuronidase at the same time as other enzymes (e.g. xylanase) This increases the degree of hydrolysis and allows xylanase treatment to occur at low pH. Ru. However, in this case too, the treatment effect depends exclusively on the removal of charged groups. and not on the total amount of hemicellulose hydrolysis. The present invention The effect of the method is based on the enzymatic removal of glucuronic acid groups, which further reduces the surface charge. to form a form suitable for chemical or enzymatic treatment. The factors listed (such as surface charge) ), e.g. by changing the enzyme's effect on the most advantageous part of the fibrous matrix The effect can be adjusted. These factors, which have been completely neglected in the past, need to be changed. The most effective extraction of residual lignin from fibers can be achieved by therefore , the adjustment of these factors by the method of the present invention also improves the industrial scale rig from fibers. Directly affects the amount of chemicals used for nin extraction, as well as low-chlorine or no-chlorine bleaching methods. Vine used to improve. Therefore, environmental pollution is reduced.

Bulb treatment with glucuronidase enzyme hydrolyzes metal-bound glucuronic acid groups. This facilitates the removal of metal ions from the valve. Surprisingly, the treatment of the present invention It has been found that chemical metal ion removal becomes unnecessary. Because of this This further reduces the environmental impact of peroxide or ozone bleaching.

Some types of valves, such as metal-free valves, are produced using enzymatic removal of glucuronic acid groups. can improve productivity.

paper for chemical and mechanical valves by using glucuronidase treatment. Being able to vary the technical properties is typical for the method according to the invention. That's true.

The method of the invention is described below by means of some non-limiting examples.

As mentioned above, the number of carboxyl groups and the metal ions bonded to them and The number affects the charge on the bulb. These factors are based on different chemical and physical parameters. The surface charge of the fiber (bulb) is measured by the zeta potential. (Pullulan, Melzer, 1972). valve By analyzing the metal inside using atomic absorption spectrometry, we can identify the metal in the valve. Content can be measured. The carboxylic acid content of the valve is determined, for example, by Nigerström (SjO Stre)m) method (KCL method) 192: It can be measured by 68). Enzyme action in fibers is controlled by the release of sugar after enzyme treatment and It can be expressed by lignin fragment extraction ability. Another method is to reduce the surface charge Enzyme that alters (i.e. brings the zeta potential and carboxylic acid content to the desired shape) The objective is to measure the increase in whiteness obtained after primary processing.

Example 1. A total of 18 different α-glucuronidase production by different bacterial strains The strains were tested for their α-glucosidase production.

Medium A is (duram per liter), wheat bran 10, solka floc cell Loin (Solka floc cellulose) 10, distiller distiller’s 5 pent grain ) 10, KH2PO45, and (NH<)zso45. this The medium composition is generally suitable for the production of enzymes that degrade lignocellulosic materials. considered to be a thing. Medium B (duram per liter), beach xylan (beech xylan) (Lenzing AG (Austria) nzing AG) 10, glucuronoxylan derived from birch tree (Roto ( Roth) 7500) 5, oligosaccharide obtained by steaming birch wood 5, daisu Contains Taylor's Spent Grain 10 and the same inorganic salts as Medium A. there was. Glucuronic acid substitute xylooligosaccharide and xylan are included in the substrate This is why we considered this medium to be potentially advantageous for α-glucuronidase production. I saw it.

Screening cultures were performed in 250 ml shake flasks containing 50 ml of medium. . Cultures were carried out for 4 days and samples were taken from the cultures on the second day. thermophilic bacterium miseriophthus MyceLi, ophthora thermophila ) was grown at 37°C, while all other strains were grown at 30°C. Culture supernatant 100 μm with 900 μm of 1% glucuronoquinane (Roto 7500) and pH 4,8 (0.05M sodium acetate buffer), incubated at 40°C for 24 hours Assay α-glucuronidase in the culture supernatant by reaction. Kan The organic acids produced using the method of Handke et al. (1989) were Measured as chloric acid. Based on the absorbance of the reaction mixture at 660 nm, Results were expressed as relative production units. Screening for α-glucuronidase production The results of the cleaning test are shown in Table 1.

Table 1. α-glucuronidase production by different strains in shake flasks Medium 2nd day 4th day α Glucuronidaya α Glucuronidaya A660 Ass.

7 SHE/lz KILLS-, tlJ SE VTT-D-85248A 1.90 2.2 4j-I.7 Wamo IJVTT-D-75028A O,901,16 A.N. -VTT-D-85240A 1.97 1.93 A/N -VTT-D-7 9106A 2.36 2.49 E-N-VTT-D-85246A 1. 28 1.41 A Tellus VTT-D-82209A 0.86 0.82 A Ureohasisium pullula:/su VTT-D-89397A 2.50 1.9 3 Curvularia inequali s) VTT-D-79121A O, 380, 55 Penicillium cigitatum V TT-D-87328B 0.08 0.11 Cryptonectria parasiti Power (Cryptonectria parasitica) VTT-D-821 90A 1.39 1.38 Fusarium oxosporum VTT-D-80i3 4 A O, 300, 00 Juanellosiaete Chrysosporium VTT-D-85242A 0.06 0.59 Penicillium chantinerum vTT-D-7809OAO1410,22 Trichoderma-LzEMVTT- D-86271A 1.76 0.32 Myceliophthora thermophilia VTT-D-76003 (37°C) A 2.32 0.36 Medium A Wheat flour Suma 10g1-I Solca flock cellulose 10 Spent grain 10 Medium B: Beech xylan (manufactured by Lenzing) 10 glucuronate derived from birch trees Noxyran (manufactured by Rohto) 5 Heat-treated Kinran (alternative oligosaccharide) 5 spec nt grain 10 IJ: +Dell 7 Ray-MRUT C-30 (VTT-D-86271, AT CC56765) in a bioreactor (Chemap LF2). 0. Effective volume 16　), 30 g 1-' (Austrian Lenz (L enz) manufactured by Lenzing AG) and Corn Steep Nonnos (corn 5teep 5olids) (Jig v (Sigma) company C C-8160)15]” as the main carbon and nitrogen sources (KH 2P 04 5 g l -' and (NH4) 2S 04 D g] -' supplement). Culture conditions: temperature 29°C, pH control 6.0 to 6. ．． 5, aeration: approximately 91 m1n-1, culture time: 3 days. The bacterial cells were centrifuged.

The clarified supernatant was first fractionated by cation exchange chromatography.

Equilibrate the sample with sodium acetate to pH 4.5 and add 50mM sodium acetate. Electrotransfer equivalent to buffer solution (pH 4, 5, 0, 14 mol containing 1" NaCl) I made it conductive. The diluted supernatant was added to DEAE Sepha, which had been equilibrated with this buffer. Apply to a column (113 x 200 mm) of Loos FF (manufactured by Farmana). Ta.

Elution was first performed with equilibration buffer to remove unadsorbed proteins, followed by sodium chloride. was added linearly from 150 to 400 mM for elution. NaC] During treatment, fractions containing eluted α-glucuronidase (each 450 m 1) were combined for the next step by hydrophobic chromatography.

By increasing the NaC1a concentration to 1.0M, other adsorbed proteins are eluted and the carbon The ram was washed with 10mM sodium hydroxide.

The enzyme preparation obtained from the first chromatography step was washed with sodium hydroxide. Adjust the pH to 5.5 and increase electrical conductivity by adding (NH4)zso4. The sample was prepared in advance in an amount of 0.8 mol]-' 25mM sodium acetate buffer containing (NH4)2SO4, pH 5.5 Phenyl Sepharose FF' (Pharmacia) column equilibrated with (113 x 110 mm). Elution is first carried out with equilibration buffer, Thereafter, a gradient of ammonium sulfate from 0.8M to 0.6M was performed. 0 ．． 8M ammonium sulfate and α-glue eluted at decreasing concentrations. Fractions containing clonidase were combined. Other adsorbed proteins, (NH4)2s c) Elute by decreasing the 4 concentration to OM and wash the column with 6M urea.

The preparation obtained by hydrophobic chromatography was subjected to ultrafiltration (Amicon Even small batches (150 to 250 m1) can be made using PM-10 membrane manufactured by Con). Concentrate and use Sephacryl S-100HR column (manufactured by Pharmacia, 50X74 5mm). Purified α-glucuronidase protein was dissolved in 50mM sodium acetate. Elution was performed with um buffer, pH 5.0.

α-glucuronide based on the method of Khandke et al. (1989) Dase activity was assayed.

Methylated triuronic acid (2°-0-(4-0-methyl-α-(1,2)-gluc Ronic acid-β-DMF-xylobiose) (mG1uAX2, 2.5mg ml- ’, 5.3mM) in 0.5mM sodium acetate, pH 4.8. Incubate for 10 or 60 minutes with 10 μl of diluted enzyme. The assay was performed by: Milner and Avigado (1967) by adding 200 μm of copper sulfate reagent. The mixture was then heated in boiling water for 10 minutes. After cooling, Nella Nelson (1944) Molybdenum Arsenic Reagent B was added and then The absorbance at 690 nm was measured using glucuronic acid as a standard.

The properties of purified α-glucuronidase were investigated using standard methods of protein chemistry.

These properties are shown in Table 2. In 50mM sodium citrate buffer, pH 5.3 1.0% (W/V) 4-0-gluconoxylan derived from birch tree (7% manufactured by Rohto) Bailey et al. (1992) using 500) as a substrate. Xylanase activity was assayed as follows. β-xylosidase, Poutane As described by Poutanen and Puls (1988). were assayed.

All enzyme units were expressed in Sl units (Qatar).

1.5 liter column of Sephacryl 5100HR (column height 74.5 cm) T. Raysay VTT-D-86 was analyzed by gel filtration chromatography using Samples of 271 culture filtrates were examined for their α-glucuronidase content. 9 In addition to the glucuronidase labeled as 5kDa, there is also 2 with an apparent molecular weight of 50kDa. Two small glucuronidase activities were observed.

Table 2. α-glucuronidase derived from Trichoderma reesei RUT C-30 nature ' in 50mM sodium phosphate-citrate buffer from pH 3.0 to 7.5 50 μg ml-1 enzyme protein; incubation at 40°C for 24 hours 5. Hydrolysis Substrate concentration in solution experiments: 4mM for xylo-oligomers, or xylan 8 mg m1-': 6 mg protein per mmol of xylo oligomer; 3 mg of protein per g of gluconoxylan; 24 hours at 40°C at pH 4,8 incubation Example 3. Purification of α-glucuronidase from Aspergillus niger 7s: , Le Jr. Luz 2ff-VTT-D-77050 (ATCC12846), Wheat bran in a bioreactor (Kemap CF3000. Effective volume 10 l) 10g 1”, Solca Flock Cellulose 2g 1-’, Corn Stape ・Medium containing Nonnos 2g+-1 (K　H2PO22g　1-' and (N H4) Cultured with 2SO42g + arches supplemented). Culture conditions: temperature 30°C, pH Adjust the temperature between 4.0 and 6.0, aeration for about 41 min, and culture time for 3 days.

The bacterial cells were separated by centrifugation.

A portion of the supernatant was adjusted to pH 4.5 with acetic acid and diluted with an equal volume of distilled water. Keiso Celite Standard Super Cell (Celite 5tanda) rd 5upper Ce1).

M'anville) in 5QmM sodium acetate buffer, pH 4,5, and adsorb α-glucuronidase protein from the diluted supernatant onto this carrier. I set it. The diatomaceous earth was washed once with 50mM sodium acetate buffer, then 0.5mM sodium acetate buffer. alpha-glucose in 0.1M sodium phosphate buffer containing up to 5M sodium chloride. Lonidase was eluted. The solution containing the eluted protein was diluted to 10mM using gel filtration. Equilibrate with sodium phosphate buffer, pH 6.5, and D It was applied to a column of EAE Sepharose FF (Pharmacia). First, equilibrium Elution was performed with a buffer solution to remove unadsorbed proteins, followed by 150 mM sodium chloride. It was eluted with a linear gradient from to 0.2M. During the NaCl gradient The fraction containing α-glucuronidase eluted was subjected to cation exchange chromatography. We came together for the next step with graphics. Other adsorbed proteins are NaCl concentrated degree 1. It was eluted by increasing to OM.

The enzyme preparation obtained from the first chromatography step was purified using gel filtration. Buffered with 10mM sodium acetate buffer, pH 4.4 and equilibrated with the same buffer. The mixture was applied to CM Sepharose FF (Pharmacia). First, equilibration Elution was performed with buffer to remove unadsorbed proteins, and the OM was then eluted with 0.2 M sodium chloride. Elution was carried out with the addition of lium. α- eluted at the end of the NaCl gradient The fractions containing glucuronidase were combined and the α-glucuronidase described in Example 2 was obtained. Dase, β-glucuronidase, β-xylanase and β-xylosidase activities Analyzed using standard methods for sex. Other adsorbed proteins, NaCl concentration 1.0 It was eluted by increasing to M.

The preparation obtained by cation-exchange chromatography has α-glucuronidase It had activity but was detectable against gluconoxylan (Rohto 7500) β-xylanase activity or p-nitrophenyl-β-DMF-xylopyra It did not show β-xylosidase activity against nondase. 5DS-PAGE At , the formulation showed a major band corresponding to a molecular weight of approximately 120 kDa.

Example 4. Purification of α-glucuronidase from Schizophyllum commune Lum comune VTT-E-88362 (ATCC38548) was added to bioreactor. Wheat bran 10g in tar (Kemap CF2000. Effective volume 161) +” , Solca Flock Cellulose 10g ]-', Distillers Spent ・Medium containing 5 g l-1 of grains (45 g l-1 of KH2PO and ( NH4)2S04 supplemented with 5 g I-'). Culture conditions: Temperature 3 0°C, adjust pH between 4.0 and 6.0, aerate approximately 4 1 m1n-1, culture Cultivation time: 8 days. The bacterial cells were separated by centrifugation, and ultrafiltration (PM-10 membrane, Amicon The mixture was concentrated 15 times using the following methods:

Solids were removed from the supernatant by centrifugation, followed by anion exchange chromatography. It was fractionated by A sample with a pH of 5.6 was preliminarily diluted with 0.05 mol I” of sodium chloride. Equilibrated with 50mM sodium acetate buffer containing thorium, pH 5,6. It was applied to DEAE Sepharose FF (Pharmacia). First, equilibrate the elution chlorination buffer to remove unadsorbed proteins, and then diluted with 200mM to 450mM chloride buffer. Elution was performed with linear addition of sodium. Elute during NaCl gradient The fraction containing α-glucuronidase was analyzed by hydrophobic chromatography. were combined for the next purification step. Other adsorbed proteins, NaCl concentration 1.0 It was eluted by increasing M.

Sodium chloride concentration of the enzyme preparation obtained in the first chromatography step was adjusted to 1.5M. Front side, 1.5mal l-' NaC] containing 10m Phenyl Sepharose FF equilibrated with M sodium acetate buffer, pH 5.5 ( The sample was applied to a column manufactured by Pharmacia. first with equilibration buffer, then Elution was carried out with a linear gradient from 1.5M sodium chloride to OM. Salt concentration Contains α-glucuronidase eluted during a linear gradient of decreasing degrees of Fractions were combined and concentrated by ultrafiltration. Other adsorbed proteins were added with 10mM vinegar. The column was washed with 6M urea.

The concentrated fraction obtained from hydrophobic chromatography was purified using α as described in Example 2. -glucuronidase, β-glucuronidase, β-xylanase and β-kin Analyzed by standard methods for londase activity. Obtained α-glucuronida These activities in α-glucuronidase 133 nanocatalyst, It was less than 22 nanocatalysts for lanase and 1 nanocatalyst for β-xylosidase.

Example 5. Removal of carboxyl group from kraft valve by glucuronidase Leaving Bulb of Hippo (kappa number 15.5) in 5QmM acetate buffer at pH 5. It was treated with T. Raysay glucuronidase for 4 hours. Enzyme dose is 100 nano It was qatar/g. Control valves were incubated in the buffer for 4 hours. Ta. After treating the valve, wash it with distilled water to remove the carboxyl groups present in the valve. The amount was determined by conductometric titration as described by Katz et al. (1984). It was measured from The results are shown in Table 3.

As the results show, even with small enzyme doses, glucuronidase treatment reduces the It was possible to remove 7% of the carboxylic acid groups from the bulb. Increasing the amount of enzyme , further glucuronic acid groups were removed from the bulb.

Example 6. Kraft valve peroxide bleaching cup pretreated with glucuronidase Cava kraft valve with a pH of 14.8 was used at a pH of 5 with a valve content of 5%. and treated with T. reisei glucuronidase for 4 hours. Adjust the pH of the valve to 5N Combined with H, So. The glucuronidase dose was 500 nanotar/g. It was. After treatment, the bulbs were rinsed with distilled water and bleached with peroxide. Dosage of chemical agents C: Hydrogen peroxide 3%, NaOH 1.5%, Mg5O*0.5%, DTPAo , 2%. The chemicals were used at the above percentages per bulb. Valve content 1 Bleaching was carried out for 1 hour at 0%. The bleaching temperature was 80°C. After bleaching, close the valve Acidic, kappa number (SCANCl: 1977), whiteness (Is○ 4270) and hand strained to measure viscosity (SCAN-C-15+1988).

The data in Table 4 shows that the whiteness of Kraft bulbs was significantly reduced by glucuronidase treatment. Indicates improvement.

Example 7. xylanase-treated cover of glucuronidase-treated metal-free valve kraft valve (kappa 14.8) with a valve content of 2% and 0. IMHC It was converted to the hydrogen form (metal-free form) in I at room temperature. After cleaning, adjust the pH of the valve to 5N. Adjusted to 5 with NaOH. Enzyme treatment was performed at 45°C for 4 hours with a valve content of 5%. It was. Enzyme dose: T. Raysay's xylanase pI 9200 nanokata Le/g: T. Raysay's glucuronidase 500 nanometer tar/go enzyme After treatment or during enzymatic treatment, the valves are cleaned and then as described in Example 6. Bleached.

Table 5. xylanase treatment of valves pretreated with glucuronidase as described above. , xylanase did not work well on metal-free valves. However, By treating the valve with glucuronidase before silanase treatment, the valve The xylanase treatment effect was improved by removing the carboxyl group.

Example 8. Effect on the industrial properties of paper for mechanical valves and chemical valves Effect of glucuronidase treatment The industrial properties of cellulose bulb paper can be improved by bleaching it with glucuronidase or by unbleaching it. The fiber surface roughness can be changed by treating the fiber surface. The rubonic acid groups are totally or partially removed.

Example 9. Agaricus bisporus against whiteness deterioration of mechanical valves Effect of glucuronidase treatment CTMP　j　a　PGW valve, Agaricus bisporus glucuronider 20 hours at 40°C in 0.2M sodium acetate buffer (pH 5). did. Handmade seaweed made from bulbs) (95cm long, 27℃, relative humidity 47%) Xenon test (Xen test) equipped with a xenon lamp with a total output of 1.3 kW otesO150S device (Heraeus Hanau) ). The reflectance (R+ and Rz) of the sample before and after irradiation was measured at 457 nm. With an Elrepho reflectance photometer equipped with a brightness filter. It was measured. The post-color values are converted to Janso. n) and described by Forsskahl (1989) I calculated it as follows. The results are shown in Table 6 (PGW) and Table 7. Glucuronider Yellowing of both bulbs due to heat treatment (P It is clear that the z-value) has decreased.

Table 6. Effect of glucuronidase on yellowing of PGW Example 10. Trichoderma reisei glucuronin for whiteness deterioration of mechanical valves Effect of dase treatment and xylanase at 40° C. and pH 4.5 for 20 hours. handmade seaweed The post color values were calculated as in Example 9. Glucuronider The yellowing (PC, value) of the bulb was reduced by the enzyme treatment.

Example 11. Removal of metal ions from valves by α-glucuronidase treatment Pine kraft (kappa value 19) was combined with T. reisei α-glucuronidase. (Dose: 100 nanokatals/g) at 45° C. for 24 hours. After treatment, bar The amount of metal ions was measured as described by Buchert et al. (printed). Measured by AAS. The results are shown in Table 9.

Even with small enzyme doses, glucuronidase treatment reduces the metal content of the valve. A significant decrease occurred.

Example 12. Improvement of xylan hydrolysis of bulbs by α-glucuronidase As described in Example 5, Kava Kraft Bulb was treated with T. Raysay's Xylaner. (Tenkanen et al., 1992). xylaner The dosage was 300 nanotar/g. Degree of hydrolysis of xylan in bulb was carried out by measuring reducing sugars. The results are shown in Table 10.

The addition of glucuronidase increased the total amount of xylan hydrolysis in the bulb.

Literature: Bay saw. Bailey, M, J, Biley, P. (Biely, P.) and Poutanen, K. ) (1992) Interlaboratory Testing on Methods Four assays on xylanase activity (Interlab laboratory testing of methods for assay of xylanase acti magician.

Journal on Biotechnology (J, Biotechnol, ) No. Volume 23, pages 257-270 Buchert, J., Tenkanen, M. nkanen, M, ), Piquet Neji. M (Pikanen, M,) and Viikari, L. The Roll on Surf Es Charge And Swelling On The Akunyong On Kisira Nurses on Birch Craft Valve (The role of sur facecharge and swelling on the action n of xylanase on birch craft pu blast j, Tat Tappi Journal (Tappi, J,) Printing page. Kant elinen, A,), Ratto, M, Sund Sundquist, J., Ranu, M. a, M, ), Viikari, Elle (Viikari.

L.) and Linko, M. (1988) Hemicellular. This and There Votential Roll in Bleaching (hemic ellulases and their potential role in 　bleaching)　　　　　　A　　　P　P　I　Pr　oc　c　1988 International Valve Breaching Conference, April ( TAPM Proc1988 int Pu1p Bleaching Con f, April) (1988) pp. 1-9 Melzer, I. 1. ) (1972) Zeterpotentzial Land Seine Bedoy Zeta-Poten tial und 5eine Bedeutung bei der Her Stellung van Papier),_Spa Poutanen, Das Papier, Volume 26 (7): 305-332. Kei (Poutanen, K, ), Ratto, M () Puls, G.I.

J.) and Viikari, L. (1987) Journal on Biotechnology (J, Biot, ) Volume 6 = 49-6 Page 0 Puls, J., Schmi, O. dt, O,) and Grandsif.

Granzov, C., Enzyme and Microvial Technology (Enzyme Microb, Technol, ), 1 987, Volume 9: 83-88 Lindström, T. T.) and Carlsson, G. (1982) The Effect on Chemical Environment on Fiber Swelling (effect of chemical environment) ment on fiberswelling) Svensk Paversdi Döning (Svensk Paperstidn) Volume 85 (3): R14 ~20 Mekaran. Scallan, AM and Grignon, Jay (G Rignon, J.) (1979) The Effect on Cations ・On Valve and Paper Properties (The effect) of cations on pulp and paper property ies).

Svensk Paperstidn , 1979: nro2: pp. 40-47 Scallan (1983) The Effect on Acidi Rock Groups on the Swelling on Pulps (The eff ect of acidic groups on the swelling of pulps) Nireview, Tappi Journal (Tappi, J, ) Volume 66 (11) Near pages 3-75 '/x-Strom, E (SjQstrOm, E,) (1989) The Origin on Charge on Selontsk Fibers (The or igin of charge oncellulosic fibers) , Nordic Valve and Paper Research Journal ic Pu1p and Paper Re5earch Journal) No. Volume 4 (2); pages 90-93 Schuström. E (Sj5strQ+e, E, ), Masan Karpok Massan karboksylipitoisuu s), KCL method, Vol. 192, p. 268 Poutanen, K, Te nkanen, M.), Corte.

Karte, Hl and Puls, J. (1) 991) Accessory Enzymes Impulse in the High Trolley Sith on Xylans (Accessory enzymes 1nvolve d in the hydrolysis of xylans) A CS ■ Podium (AC3Symp,), Ser 460. enzymes in bio Enzymes in Biomass Convert sion), pp. 426-436 Khandke, K. M), Vithayathil.

PJ, ) and Murthy, SK, (19 1989) Arch Biochem Biop hys) Volume 274: pp. 511-517 Basta, J. ), Holtinger, L. and Hawk.

Book, J. (1991) Controlling the Profile Le on Metals in the Valve Before Hydrogen Performance Side treatment (Controlling the profile of metals in the pulp before hydrogen peroxide treatment) International Symposium Wood and Pulping Chemistry -(Proc.

6th Int, Symp, food and Pulping Chem istry), Melbourne, 1X91 Year, pp. 237-244. Jansson, J. and Forsskor, A. Lee (Forsskahl.

1) (1989) Color Changes in Lignin Rich Pulges ・On Irradiation Pie Light (Color changes in 11gn1n rich pulpson 1rradiation by light) Nordic Valve and Paper Research Journal (Nordic Pu1p and Paper Re5earch Jour nal), Volume 3/1989: pp. 197-203 Khandke, KM, Vitayathil, P.G. (vithayathil.

PJ, ) and Murthy, SK, (19 1989) Beauty and Characterization on... An alpha glucuronidase from a thermophilic funga Purification and Characterization of an a-glucuronide ase from a thermophillic fungus, Ther a+oascus aurantia mo floating sword j a Arch Biochem Biophys 2nd Volume 74: pages 511-517 Milner, Y. and Avigado, G.I. igad, G, ^,), Copper Reagent for the Determination Nation on Hexronic Acids and Certan Ketohexo -sis(reagent for thedetermination of hexuronic acids and certain ketohexo ses) Carbohide■ Carbohydr, Res, 1967, No. 4 Volume, pp. 359-361 Nelson, Lang A (Nelson, N, ^,) Fo METRIC ADAPTION ON THE SOMOGI METHOD FOR THE Determination on glucose (photometric adap tation of the Somogyi + aethod for th e　determ Amashi ≠ Ko Amashishi■ of glucose) Journal on Biological Chemistry (J , Biol, Ches, ).

1944, Vol. 153, pp. 375-380 Katz, s. , Beatson, RP and Mecallan. workman Scalln, AM (1984) Svensk Paparsdid Svensk Paperstidn, Volume 87 (6) +R4 8 Poutanen, K. and Prus, J. uls, J.), Journal on Applied Microbiology ・And Biotechnology (J.

Appl, Microbiol, Biotechnol, ), 198 8, Vol. 28: 425-432 Tenkanen, M. , ), Puls, J., and Poutanen, K. outanen, K.), Enzyme and Microvial Tech. Nology (Enzyme Microb, Technol, ), 199 Year 2, Volume 14: Pages 566-574 Continuation of front page (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , 0A (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, SN, TD.

TG), AT, AU, BB, BG, BR, CA, CH.

C3, DE, DK, ES, FI, GB, HU, JP, KP, KR, LK, LU, MG, MN, MW, NL, N.

, PL, PT, RO, RU, SD, SE, US (72) Inventor Rat, Maruya Na No. 9 Shulakinkuya, Vantaa, Nis F-01690, Finland (72) Inventor Vikali, Lisa Finland, Nisf-00200, Bersinki, Rotkikuya 5F (72) Inventor: Bailey, Michael Finland, Nisf-02600, Nispo, Kiryurinkya 3F42 number

Claims (26)

[Claims] 1. Pulp is prepared by contacting the pulp with an oxygen preparation that has essential glucuronidase activity. lignocell, characterized in that the glucuronic acid groups of Process for processing loin materials, especially cellulosic pulp. 2. Glucuronides that contain very little, if any, hemicellulase 2. The method according to claim 1, wherein the Dase preparation and the pulp are reacted. 3. The glucuronidase preparation is essentially a culture of a glucuronidase-producing microbial strain. 3. The method according to claim 1 or 2, comprising: 4. The glucuronidase preparation is essentially a culture of a glucuronidase-producing microbial strain. 3. The method according to claim 1 or 2, comprising an enzyme isolated from. 5. The genus Trichoderma (e.g. T. ．． reisei), Aspergillus spp. (e.g. A.niger, A.awamori, A.niger ・TerrUS (A. terrUS), A. oryzae), Schizov Schizophyllum (e.g. S. comme) une)), Aureobasidium (e.g. A. pullulans), Phane rochaete) (e.g. P. chrysosporium orium), Fusarium spp. (e.g. F. oxyspo. F. oxysporum), Agaricus genus (e.g. A. bisporus), Penicillium llium) (e.g. P. janthinellum ), P. digitatum), Streptomyces (St reptomyces (e.g. S. olivochromogenes) chromogenes), S. flavogris eus)), and the Bacillus genus (e.g. P. subtilis) (B. subtilis), P. circulans glucuronidae derived from a microbial strain selected from the group consisting essentially of microorganisms in 5. The method according to claim 1, wherein the pulp is brought into contact with the enzyme preparation. 6. Thermoascus aurant icus), Curvularia ineq ualiss), Tyromyces palustris tris), Cryptonectria parasitica parasitica), Myceliophthora thermophila thora thermophila), and Thermobacter auranti Selected from the group consisting of Thermobacter auranticus Claim 1: The pulp is brought into contact with a glucuronidase preparation derived from a symptomatic strain. 4. The method according to any one of 4. 7. Glucuronidase preparations contain the gene encoding α-glucuronidase. 6. The method according to claim 4 or 5, which is derived from a genetically modified strain containing. 8. The pulp obtained from the pulping process has the essential glucuronidase activity. 2. The method of claim 1, wherein the pulp is treated with an enzyme preparation to reduce the amount of metal in the pulp. 9. The pulp is treated with an enzyme that has the requisite glucuronidase and xylanase activities. 2. The method according to claim 1, wherein the treatment is performed with an elementary preparation. 10. Pulp is treated with the enzyme preparation and processed using oxygen, peroxide or ozone. 10. A method according to any one of claims 1, 8 and 9, wherein the method promotes chlorine-free bleaching of lube. 11. Claim 1: Pulp is treated with said enzyme preparation to reduce color reversion of the pulp. Method described. 12. A claim in which pulp is treated with said enzyme preparation to improve the paper industrial properties of the pulp. The method described in 1. 13. The pulp is first treated with an enzyme preparation having the requisite glucuronidase activity; and has hemicellulase, cellulase and/or ligninase activity. A method according to any of the preceding claims, comprising contacting with an enzyme preparation. 14. The pulp is treated with an enzyme preparation having glucuronidase activity, hemicellulase, Simultaneous treatment with enzyme preparations having cellulase and/or ligninase activity A method according to any of the preceding claims. 15. Pulp is treated with the enzyme preparation to produce hemicellulases, cellulases or rigs. Claim 1, 14 or 1 which promotes the action of other enzymes such as ninase on parv. 5. The method described in 5. 16. Specifically, using an enzyme that removes the gluconic acid groups of the polymeric xylan chains. A method according to any of the claims. 17. An enzyme preparation produced by the microbial strain Trichoderma reisei, which The enzyme isolates the culture by anion exchange and hydrophobic interaction chromatography. 6. The method according to claim 5, wherein a separated enzyme preparation is used. 18. Cellulose pulp has a molecular weight of approximately 95 kDa by SDS-PAGE method. 18. The method of claim 17, further comprising treating with an alpha-glucuronidase enzyme. 19. An enzyme preparation produced by the microbial strain Aspergillus niger, comprising: α-glucuronidase enzyme with a molecular weight of approximately 120 kDa by DS-PAGE method 6. The method according to claim 5, wherein an enzyme preparation containing 20. Contains minimal, if any, essential glucuronidase enzyme activity a lignocellulosic substance characterized by containing hemicellulase of Enzyme preparations particularly useful in the treatment of cellulose pulp. 21. Glucuronidase enzymes are present in Trichoderma reesei, Schizophyllum com Ne, Aspergillus niger, Thermoasuchus auranthus, Agaricus ・Bisporus, Aspergillus awamori, Curvularia inequalis, Chi Romyces palustris, Streptomyces flavogriseus, S. olivo chromogenes, and other Streptomyces strains, Cryptonectria parasitae ica, Myceliophthora thermophila, Penicillium jantinerum, Penisi Rium digitatum, Aspergillus tellus, Thermobacter aulanti Aureobasidium pullulans Aspergillus oryzae, Fanerosi Chaete chrysosporium, Bacillus circulans and B. subtilis derived from a glucuronidase-producing microbial strain selected from the group consisting essentially of The enzyme preparation according to claim 20. 22. The glucuronidase preparation uses the gene encoding α-glucuronidase. 22. The enzyme preparation according to claim 21, which is derived from a genetically modified strain containing. 23. The glucuronidase preparation is essentially a culture of glucuronidase-producing microbial strains. Claim 20 or 21, wherein the culture is optionally concentrated. Enzyme preparations as described. 24. The glucuronidase preparation is essentially a culture of glucuronidase-producing microbial strains. 22. The oxygen preparation according to claim 20 or 21, comprising an enzyme isolated from a substance. 25. Glucuronidase enzyme is derived from Trichoderma reesei and its molecular weight is about 95 kDa according to SDS-PAGE method, according to claim 20 or 21. Oxygen preparations. 26. Glucuronidase enzyme is derived from Aspergillus niger and its molecular weight is about 120 kDa according to SDS-PAGE method. Enzyme preparations.