JP2002345472A - New hexene uronidase, gene encoding the same and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new hexene uronidase, to provide a method for producing the same and uses thereof, and to obtain a hexene uronidase gene and a microorganism capable of producing the hexene uronidase. SOLUTION: The hexene uronidase is characterized by having a hexene uronidase activity and about 40,000 to about 45,000 molecular weight and accelerating the enzyme activity with 2-mercaptoethanol or dithiothreitol. The method for producing the enzyme protein and a gene encoding the enzyme protein are provided. The use of the enzyme protein for a bleaching agent is provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel hexene uronidase, a method for producing the same and its use, a hexene uronidase gene, and a microorganism producing hexene uronidase.

[0002]

2. Description of the Related Art Regardless of hardwood or softwood, a polysaccharide other than cellulose, so-called hemicellulose, exists in the plant cell wall. In particular, these hemicellulose are essential components in living cells for binding between lignin and cellulose, which are components of the cell wall, to form a strong cell wall. Xylan is one of the most abundant and famous hemicellulose. The xylan main chain is a homopolysaccharide in which xylose is linked by β1 → 4, and may be referred to as a universal hemicellulose contained not only in all land higher plants but also in the cell walls of algae in general, including seaweed. Xylan has recently attracted attention as a biomass as a raw material for xylooligosaccharides and xylitol.

[0003] About 50% of the earth's biomass can be considered to be lignocellulose derived from plant cells. Further, the hemicellulose content in this lignocellulose is said to be about 30%, and the ratio of hemicellulose in biomass present on the whole earth is as high as about 15%. It is rare that this xylan exists as a single chain as a homopolysaccharide. In the cell wall, the main chain xylose is arabinose side chain having α1 → 3 bond as a side chain from a polysaccharide chain having β1 → 4 bond, α
The heteropolysaccharide exists in a form having a 1 → 2 linked 4-O-methylglucuron chain as a side chain, and it is estimated that these heteropolysaccharides have various physiological activities in living cells.

Currently, the most widely used woody biomass is in the papermaking industry using cellulose as pulp. In order to produce pulp, which is a raw material for paper, a process of treating wood chips under high temperature and high alkali conditions is required. In this step, a part of lignin and hemicellulose other than cellulose is removed and pulping is performed, so that the subsequent pulp bleaching step can be carried advantageously. When the chips are pulped, when treated under high-temperature and high-alkaline conditions, they remove lignin and simultaneously dissolve xylan, but the dissolved xylan re-adsorbs to the surface of cellulose as the temperature decreases. When the xylan, which is a polysaccharide, is re-adsorbed, it also re-adsorbs while disturbing a coloring substance such as lignin or a furan derivative which should be bleached in a subsequent step. In the paper industry, pulp is treated with xylanase (EC 3.2.1.8) to remove the re-adsorbed xylan as a pretreatment for bleaching the pulp in order to increase the efficiency of the subsequent bleaching process, and decompose the xylan. Some factories incorporate a process for removing colored substances.

[0005] Another important problem that occurs when making pulp is the problem of hexenuronic acid. As described above, xylan has an arabinose side chain having α1 → 3 bond and a 4-O-methylglucuronic acid having α1 → 2 bond as side chains in addition to the main chain. In particular, 4-O-methylglucuronic acid has a methylated hydroxyl group at the 4-position, so under high-temperature and high-alkali conditions, which are the conditions for pulping chips, the 4-methoxy will undergo β-elimination and will be eliminated to produce methanol. . At this time, a double bond is introduced between the carbon at the 4-position and the carbon at the 5-position of glucuronic acid, and 4-O-methylglucuronic acid is converted into so-called hexeneuronic acid. Hexenuronic acid is present not only as a side chain in xylan re-adsorbed to pulp, but also in a xylan side chain present in the cell wall of pulp fibers.

Hexenuronic acid reacts with an oxidizing chemical used in the pulp bleaching process, for example, a bleaching reagent such as ozone, chlorine or chlorine dioxide, to retain a double bond in the molecule. These bleaching reagents should react with compounds derived from lignin, which is a coloring component, or sugars derivatized with furan, but the hexeneuronic acid, which is not a coloring component, has a double bond in the molecule and a bleaching agent. The reaction means that these bleaching chemicals are wasted and the efficiency of the bleaching process is deteriorated. The kappa number is an index that determines the rate of addition of bleaching chemicals during pulp bleaching. In general, the kappa number may be considered as a numerical value of the amount of potassium permanganate consumed, and more specifically, the amount of a compound that may react with an oxidizing bleaching chemical among compounds present in pulp. You may think that it shows. Colored substances such as furan derivatives formed from lignin and sugar have been represented as kappa numbers. Hexenuronic acid is not a coloring substance, but is counted as a Kappa number because a double bond in the molecule reacts with potassium permanganate. Hexenuronic acid is also an acidic sugar having a carboxyl group in the molecule. The carboxyl group of hexenuronic acid is negatively charged, and a heavy metal is chelated to a molecule of hexenuronic acid in the pulp. In the bleaching step using the oxidizing bleaching chemical, the heavy metal compound (complex) reacts with the bleaching chemical and wastefully consumes the chemical,
Decreases bleaching efficiency.

In the pulp bleaching process, LOKP pulp subjected to oxygen bleaching after the digestion process has a kappa number of about 10 points, of which about 5 points are said to be derived from hexeneuronic acid. If this hexenuronic acid can be removed in advance before bleaching with an oxidizing bleaching chemical, it can easily be inferred that the bleaching chemical in the subsequent stage can be reduced.

[0008] Because the harmful effects of hexenuronic acid in pulp bleaching are significant, paper companies are keen on methods for removing hexenuronic acid from pulp. A common method of removing hexenuronic acid is to treat the pulp with an acid (WO 96/12063).
No. pamphlet). Acidify the pH of the pulp slurry,
When the temperature is maintained at about 80 ° C. to 120 ° C. and held for 1 to 2 hours, hexenuronic acid is separated from the xylan main chain due to hydrolysis of a glycosidic bond with xylan and can be removed.

However, in these attempts, since the pulp is treated with an acid, the pulp itself is hydrolyzed and damaged. In particular, it is a serious problem that the strength of the pulp is reduced by hydrolysis of cellulose, and it is considered that papermaking with such a low strength pulp is unsuitable for high-speed papermaking by a current paper machine. In addition, hydrolysis by acid reduces pulp yield and deteriorates pulp productivity in a mill. In fact, when hexenuronic acid is removed by dilute acid treatment, a decrease of several percent in yield based on pulp weight is observed. Another problem when hexenuronic acid is removed by a dilute acid treatment is that lignin is condensed with a dilute acid and converted into lignin which is hardly bleached in a subsequent bleaching step.

[0010] As described above, the removal of hexenuronic acid with a dilute acid has several problems. Therefore, the removal of hexenuronic acid by an enzyme has attracted attention. Hexenuronic acid removal method using an enzyme that specifically degrades hexenuronic acid (WO95 / 33883, W. Has
himoto et al., Arch. Biochem. Biophys. (1999) 368 (2): 36
7-374) has been proposed, but the pamphlet of International Publication WO95 / 33883 has a problem in the method for measuring the enzyme activity, and the nature of the purified enzyme is not clear because the enzyme has not been isolated. In addition, bleaching experiments with pulp purified enzymes have not been conducted since no purified enzymes are present. For this reason, it cannot be determined from the specification whether the enzyme that degrades hexenuronic acid is actually exerting an effect on pulp.
On the other hand, hexene uronidase isolated by W. Hashimoto et al. (Above) is an enzyme derived from Bacillus sp. GL1 and has a molecular weight of 42 kDa (SDS-PAGE), an optimal pH of 6.0 to 6.5,
The optimum temperature is around 45 ° C, pH 7.0, and 10 ° C at 60 ° C.
It has properties such as being completely inactivated by incubation for one minute, and that dithiothreitol or 2-mercaptoethanol does not significantly affect the enzyme activity, but the bleaching effect of pulp is unknown.

[0011]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel hexene uronidase useful for pulp bleaching, a method for producing the same, and uses thereof.

[0012]

Means for Solving the Problems The present inventors have conducted intensive studies based on the above-mentioned problems, searched for hexene uronidase-producing bacteria, and conducted extensive and extensive screening.
A novel microorganism that produces hexenuronidase is found in the soil of Shinonome, Koto-ku, Tokyo, and a novel enzyme that degrades hexenuronic acid is found in a culture of the microorganism, and further encodes the hexenuronidase. The gene was successfully cloned and highly expressed, thereby completing the present invention.

Therefore, the present invention is summarized as follows. (1) has hexene uronidase activity and a molecular weight of about 40,0
Hexene uronidase, wherein the enzyme activity is enhanced by 2-mercaptoethanol or dithiothreitol. (2) The hexene uronidase of (1), which acts on xylan. (3) The hexene uronidase according to (1) or (2), wherein the optimum pH is 6.0 to 8.0. (4) The hexene uronidase according to (1) or (2), wherein the stable pH range in the enzyme reaction is pH 5.0 to 10.0. (5) The hexene uronidase of (1) or (2), wherein the optimal temperature is 35 ° C to 55 ° C. (6) retains about 80% or more of activity by heat treatment at 45 ° C for 30 minutes, and shows about 30% residual activity by heat treatment at 60 ° C for 30 minutes;
The hexene uronidase of (1) or (2). (7) The hexene uronidase according to any one of (1) to (6), which is derived from a microorganism belonging to the genus Paenibacillus. (8) The hexene uronidase of (7), wherein the microorganism is Paenibacillus sp. 7-5. (9) The hexene uronidase according to any one of (1) to (6), which is a recombinant enzyme. (10) Consisting of the amino acid sequence of SEQ ID NO: 1, or having the amino acid sequence of SEQ ID NO: 1
Alternatively, the hexene uronidase according to any one of (1) to (6), which has an amino acid sequence containing deletion, substitution, insertion or addition of a plurality of amino acids and has hexene uronidase activity. (11) The hexene uronidase of (10), which has a homology of 70% or more, preferably 80% or more, and more preferably 90% or more with the amino acid sequence shown in SEQ ID NO: 1. (12) culturing a microorganism belonging to the genus Paenibacillus which produces hexene uronidase according to any one of (1) to (11) in a medium, and collecting hexene uronidase from the obtained culture; , A method for producing hexene uronidase. (13) The method according to (12), wherein the microorganism is Paenibacillus sp. 7-5. (14) Paenibacillus sp. 7-5 capable of producing hexenuronidase (accession number FERM P-18225). (15) A hexene uronidase gene encoding the hexene uronidase according to any one of (1) to (11). (16) consisting of the amino acid sequence of SEQ ID NO: 1 or
Or encoding hexene uronidase having an amino acid sequence containing deletion, substitution, insertion or addition of a plurality of amino acids and having hexene uronidase activity, (1)
5) Hexene uronidase gene. (17) a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 2,
5) Hexene uronidase gene. (18) An expression vector comprising the hexene uronidase gene of (15) to (17). (19) A host cell transformed by the expression vector of (18). (20) A method for producing a recombinant hexenuronidase, comprising culturing the host cell of (19) in a medium and collecting hexene uronidase from the resulting culture. (21) A bleach containing the hexene uronidase according to any one of (1) to (11) as an active ingredient. (22) hexene uronidase is a recombinant enzyme,
The bleach of (21). (23) The bleach according to (21), wherein hexene uronidase is obtained by the method according to (20). (24) A method for bleaching pulp, comprising treating pulp with the bleaching agent according to any of (21) to (22). (25) treating the pulp with the bleaching agent, including performing chemical bleaching and / or alkali extraction either before, after or during the pulp treatment;
Method 4). (25) An antibody that recognizes hexene uronidase according to any one of (1) to (11).

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION As used herein, hexeneuronidase activity refers to hexeneuronic acid (4-deoxy-β-L
-threo-hex-4-enopyranosyluronic acid) and 5-formyl-2-furancarboxylic acid or 2-froic acid by hydrolyzing between an aglycone linked via a aldehyde group at position 1 and a glycosidic bond. Refers to the enzymatic activity that produces Specifically, the activity is as follows: Action 1): Hydrolysis of hexeneuronic acid having β1 → 2 bond as a side chain to the xylan main chain to give 5-formyl-2
Activity to form -furancarboxylic acid or 2-froic acid; or action 2): mucopolysaccharide, hyaluronic acid, and pectin;
Hydrolysis of hexeneuronic acid produced when acidic polysaccharides containing glucuronic acid as a constituent sugar such as gellan are decomposed by a lyase that decomposes them to form 5-formyl-2-furancarboxylic acid or 2-floic acid. Action to be produced; or action 3): hydrolyze a synthetic substrate in which hexeneuronic acid and a substance such as a fluorescent substance, a chemiluminescent substance or a sugar are glycosidically bonded, to form 5-formyl-2-furancarboxylic acid or 2-floic acid. Acid-generating activity.

[0015] Accordingly, the enzyme of the present invention is a 4-deoxy-hexu produced when mucopolysaccharide, hyaluronic acid and acidic polysaccharides containing glucuronic acid as constituent sugars such as pectin and gellan are decomposed by a lyase which decomposes them.
The sugar chain containing ronic acid and 4-O-methylglucuronoxylan in the plant cell wall are β-
It acts on substrates such as xylan containing hexeneuronic acid as a side chain, which is generated upon elimination.

The enzyme of the present invention, in addition to having the above-mentioned enzymatic activity, has a molecular weight of about 40,000 to about 45,000, and has an enzymatic activity in the presence of a reducing agent of 2-mercaptoethanol or dithiothreitol. Is enhanced. The degree of enhancement is usually 1.2 times or more, preferably 1.5 times or more, more preferably 1.7 times or more, most preferably 2 times or more, and still more preferably, compared to the activity in the absence of a reducing agent. Is more than 2.5 times.

Further, the enzyme of the present invention can have at least one or all of the following properties. (1) Optimum pH and stable pH range: The optimum pH range in enzyme reaction is pH 6.0-8.0, and stable in enzyme reaction
The pH range is between pH 5.0 and 10.0. (2) Suitable operating temperature range: 35 ° C to 55 ° C. (3) Thermal stability: about 90% or more of the activity is maintained at 45 ° C. for 30 minutes, and about 30% of the remaining activity is left at 60 ° C. for 30 minutes. (4) Isoelectric point: around pH 4.5. (5) Inhibition: The activity is strongly inhibited by Cu ²⁺ , Zn ²⁺ , and SDS. EDTA does not inhibit enzyme activity.

The enzyme of the present invention may be directly isolated from a microorganism, or may be obtained by a genetic recombination method. Examples of the microorganism include a microorganism belonging to the genus Paenibacillus which produces the above hexene uronidase. Specifically, Paenibacillus sp. 7-5 (indication for identification: # 7-5) or a mutant derived from this microorganism is preferable, and this microorganism is novel, Deposited at a research institute (new name: National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, 1-1-1, Higashi 1-1, Tsukuba, Ibaraki Prefecture, Japan) on February 23, 2001, accession number FERM P -18225 was given.

The bacteriological properties of Paenibacillus sp. 7-5 are as follows. Cell morphology Bacillus (0.5 × 2.0-3.0μm) Gram stain Indefinite spores +: Standing, swollen Motility − Colony formation Circular, whole edge, low convex, glossy, translucent catalase + oxidase + O / F test -Acid production from carbohydrates glycerol + erythritol-D-arabinose + L-arabinose + ribose + D-xylose + L-xylose-adonitol-β-methyl-D-xylose + galactose + glucose + fructose + mannose + sorbose-rhamnose- Dulcitol-inositol-mannitol + sorbitol-α-methyl-D-mannose-α-methyl-D-glucose + N-acetylglucosamine + amygdalin + arbutin + esculin + salicin + cellobiose + maltose + lactose + meribio + Sucrose + trehalose + inulin + meletitose + raffinose + starch + glycogen + xylitol-gentiobiose + D-thuranose + D-lyxose-D-tagatose-D-fucose-L-fucitol-L-arabitol-L-arabitol-L-arabitol 2-ketogluconic acid-5-ketogluconic acid-β-galactosidase (ONPG) + arginine dihydrolase-lysine decarboxylase-ornithine decarboxylase-availability of citric acid-hydrogen sulfide production-urease-tryptophan deaminase-indole production-acetoin production ( VP) + gelatinase-nitrate reduction-viable anaerobic + 50 ° C-10% NaCl-hydrolyzable casein-hippurate-

Strain # 7-5 shows swelling of the cells by spores, and is negative for anaerobic growth positive, ONPG positive, casein hydrolysis, arginine hydrolase, indole, gelatinase, nitrate reduction, etc., so that Paenibacillus ( It is considered to be a microorganism belonging to the genus Paenibacillus).
This strain is presumed to be Paenibacillus lautus or its closest relative, since acid production from carbohydrates does not involve gas evolution.

In the present invention, the microorganism that produces the enzyme of the present invention is not limited to the exemplified microorganisms as long as it has an enzyme having the above-mentioned characteristics, and is preferably a microorganism belonging to the genus Paenibacillus, and more preferably # 7-5. Strains and their derivatives. Such a derivative strain can be obtained by using a conventional technique such as mutagenesis by irradiation of ultraviolet rays or the like or a mutagen, or mutagenesis by genetic recombination of a microorganism genome. . When producing the enzyme of the present invention from a microorganism,
The enzyme can be obtained by a method comprising culturing a microorganism producing the enzyme in a medium and recovering hexene uronidase from the resulting culture.

In an embodiment of the present invention, the microorganism is Paenibacillus sp. 7-5 capable of producing hexenuronidase, and the enzyme produced by the microorganism has an amino acid sequence represented by SEQ ID NO: 1. . The invention also encompasses variants of the enzyme, which specifically include deletions, substitutions, insertions or additions of one or more, preferably one or several, amino acids in the amino acid sequence of SEQ ID NO: 1. Hexenuronidase having an amino acid sequence and hexenuronidase activity. here,
Several means usually 2 to 9, preferably 2 to 7, and more preferably 2 to 5. Alternatively, the variant has 70% or more homology with the amino acid sequence shown in SEQ ID NO: 1, preferably 80% or more, more preferably 90% or more, most preferably 95% or more, and most preferably 98% or more. Hexene uronidase.

As the carbon source and the nitrogen source for cultivation, any source capable of producing hexene uronidase can be used. For example, as a carbon source,
Xylan or wheat bran, pulp containing xylan,
Agricultural waste such as bagasse, corn fiber, rice straw, or plant fiber can be used. As a nitrogen source,
Nitrogen compounds such as yeast extract, peptone, various amino acids, soybean, corn steep liquor, and various inorganic nitrogens can be used. In addition, various salts, vitamins, minerals, and the like can be appropriately used.

The cultivation temperature and pH may be any as long as the bacterium grows and produces hexene uronidase. The cultivation temperature may be from 20 to 50 ° C and the pH may be from 5 to 9. If the bacterium is a thermostable bacterium, it may exceed 50 ° C. After culturing the microorganism of the present invention, the cells are separated, and the cells are treated enzymatically or physicochemically to obtain hexene uronidase present in the cells.

Hexenuronidase can be concentrated or solidified by dialysis, salting out, ultrafiltration, freeze-drying, or the like. Furthermore, hexene uronidase can be purified by appropriately combining and repeating the culture filtrate with ammonium sulfate fractionation, molecular weight fractionation by gel filtration, various ion exchange resins, hydroxyapatite, isoelectric point fractionation, and the like.

The present invention relates to the amino acid sequence represented by SEQ ID NO: 1, the nucleotide sequence of SEQ ID NO: 2,
coli JM109 / pUC19 (7-5) (Accession number FERM P-18226; Heisei 1
(Deposited with the National Institute of Advanced Industrial Science and Technology and the Patent Organism Depositary on February 23, 3), and a recombinant hexenuronidase containing an amino acid sequence encoded by the target DNA sequence or a mutant thereof. Here, the mutant has the above-mentioned meaning. In the case of producing a recombinant enzyme, an expression vector containing the hexene uronidase gene is prepared, an appropriate host cell is transformed with the vector, the obtained host cell is cultured in a medium, and a system is prepared from the obtained culture. The enzyme can be obtained by a method including collecting a recombinant hexene uronidase. The hexene uronidase gene is a gene (DNA or cDNA) encoding an enzyme having the above-mentioned properties, and specifically, a gene encoding the amino acid sequence of SEQ ID NO: 1, or having a homology of 70% with the amino acid sequence. It is a gene encoding hexene uronidase that is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%. Specifically, it is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 2 or a gene containing the nucleotide sequence shown in SEQ ID NO: 2.

When the hexene uronidase gene of the present invention is cloned from a bacterial source, it can be cloned by a method commonly used in the art (for example, Sambrook et al., Molecular).
Cloning A Laboratory Manual, Second edition, Cold
Spring Harbor Laboratory Press, 1989). Specifically, after preparing a genomic library or a cDNA library from a bacterial source, a DNA capable of encoding an appropriate partial sequence in the amino acid sequence of SEQ ID NO: 1 is chemically synthesized (for example, automatic DNA synthesis). The target gene can be screened by using this as a probe or by hybridization with a DNA comprising the nucleotide sequence of SEQ ID NO: 2 or a fragment thereof. Hybridization is carried out under low, medium or high stringency conditions, followed by washing under relatively high temperature, relatively low ionic strength washing conditions. Generally, hybridization is performed in a suitably diluted SSC at a suitable temperature. The higher the temperature (eg, about 5-10 ° C. below the normal melting temperature (Tm)) and the lower the ionic strength (eg, 0.1-1 × SSC), the higher the stringency. Examples of hybridization conditions are, for example, Ausbel et al., Short Protocols In Molecular
Biology (third edition), John Wiley & Sons, Inc.
And its disclosure can be used. After screening the gene of interest, the gene is amplified by polymerase chain reaction (PCR) using primers (usually 10 to 30 bases) derived from the nucleotide sequence of SEQ ID NO: 2, or the gene is cloned into an appropriate plasmid or The gene of interest can be amplified by incorporating it into a cloning vector such as phage and introducing it into bacteria such as E. coli (Sambrook et al., Supra).

The amplified gene is then treated with a restriction enzyme, if necessary, and inserted into an appropriate expression vector.
After introduction into a suitable host cell, the host cell is cultured in a suitable medium, and the enzyme of the present invention can be extracted from the medium or from the inside of the cultured cell. An expression vector generally contains a promoter, and may contain a replication origin, a terminator, a ribosome binding site, a signal sequence, an enhancer, and the like, if necessary. Examples of the expression vector include commercially available vectors and vectors described in the literature. For example, pQE (Qiagen), pET (Novagen), pBluescript II SK (Stratagene), pUC118 (Takara Shuzo) can be used as a bacterial vector. PHS19, pHS15, pG-1, pXT1 (Stratagene), pBPV, pMSG (Pharmacia) as yeast vectors, and pcDNAI, pcDM8 (Funakoshi), pREP4 as animal cell vectors
(Invitrogen). The promoter is selected depending on the host, and in the case of a bacterial host, a trp promoter, a lac promoter, a P _L promoter, a P _R promoter and the like are exemplified.In the case of a yeast host, a PH05 promoter, a PGK promoter, a GAP promoter, an ADH promoter, a GPD promoter, Heat shock polypeptide promoter and the like, and in the case of animal cell hosts, cytomegalovirus immediate early gene promoter, SV
40 early promoter, retrovirus promoter, heat shock promoter and the like are exemplified. Hosts include bacteria such as Escherichia coli, Bacillus, Brevibacterium, Corynebacterium, Pseudomonas, and yeasts such as Saccharomyces, Pichia, and Candida, as well as fungi (basidiomycetes, filamentous fungi). ), Insect cells, plant cells, animal cells, and the like. For construction and transformation of an expression vector, see Sambrook et al. (Supra).
Can be used.

The expressed enzyme can be purified from the medium or the cells in the same manner as described above. When recovering from the culture medium,
After concentration as necessary, salting out, solvent extraction, precipitation, various chromatography (ion exchange, gel filtration, affinity,
Hydrophobic interaction), HPLC, electrophoresis, chromatofocusing, etc., alone or in an appropriate combination. When the cells are collected from the cells, the cells can be mechanically disrupted using a mill or the like or chemically degraded using a hypotonic solution, and then the enzyme can be purified by the same method as described above.

The present invention also relates to an antibody that recognizes the above hexene uronidase protein. Preferably, the antibody is an antibody that specifically recognizes the protein of the present invention. The term “specifically” means that the enzyme reacts immunologically with the enzyme of the present invention but does not react with other related enzyme proteins. The antibody is a monoclonal or polyclonal antibody, or an antibody fragment thereof (eg, Fab, Fab ′, F (ab ′) ₂ , Fv, etc.). Antibodies can be prepared by conventional techniques. The polyclonal antibody can be obtained, for example, by immunizing an animal (such as a rabbit) with the protein or a fragment thereof and obtaining an antibody from blood collected from the animal by a known method. Monoclonal antibodies are obtained by immunizing mice and rats with the protein and removing spleen cells, fusing the spleen cells with myeloma cells, screening for hybridomas producing the desired antibody, and transplanting the animals intraperitoneally. The desired antibody can be obtained from the ascites. Antibodies can be used, for example, to detect hexene clonidase from various microbial sources.

Furthermore, the present invention relates to a culture obtained by culturing a microorganism belonging to the genus Paenibacillus producing the hexene uronidase or the transformant in a medium, or a hexene uronidase or a group having the above-mentioned properties. Disclosed is a bleaching agent containing a modified hexene uronidase as an active ingredient.

[0032] The present invention also provides a method for bleaching pulp, comprising treating pulp with the bleaching agent. In this method, in treating the pulp with the bleaching agent, chemical bleaching and / or alkali extraction can be performed before, after or during the pulp treatment. Hereinafter, the present invention will be described in more detail with respect to hexene uronidase derived from the genus Paenibacillus, particularly from Paenibacillus sp. 7-5.

[0033] 1. Physicochemical properties of hexene uronidase (method for measuring enzyme activity) The first document showing the possibility of hexene uronidase being present is a report by Peter and Alfred. (Biochem. J. (1977) 165,287-
293). At this time, they discovered an enzyme that hydrolyzes unsaturated sugars present on the non-reducing terminal side of oligosaccharides produced when digested with lyase using chondroitin sulfate, heparin, heparan, and iduronic acid as substrates. They call this glycuronidase, but the enzymatic reaction itself is exactly the same as hexenuronidase, and although this enzyme is not completely purified, it is an enzyme that catalyzes the reaction that specifically hydrolyzes hexeneuronic acid. You can go. They are cultivating the bacterial Flavobacterium heparinum and trying to isolate and purify it from the culture. The definition of enzymatic activity was defined as the decrease per unit time of the absorption (ΔA232) of the double bond in the hexenuronic acid molecule accompanying the degradation of hexenuronic acid.

In recent years, Hashimoto et al. Isolated and purified hexene uronidase from Bacillus sp. GL1 using oligosaccharides having unsaturated sugars generated when gellan and xanthan gum were decomposed by lyase, and cloned genes. (Archives of Biochemistry and Biophysics Vo
l.368, No.2, August 15, pp.367-374,1999). The method of measuring enzyme activity by Hashimoto et al. Is exactly the same as the method of Peter et al., Utilizing the fact that the double bond of unsaturated sugars absorbs at around 232 nm, and the absorption around 232 nm is reduced by enzymatic decomposition. This is measured by an absorption spectrophotometer to define the enzyme activity.

On the other hand, Buchert et al. Have proposed hexene uronidase treatment of kraft pulp in order to enzymatically remove hexenuronic acid generated during kraft cooking of hardwood chips and to efficiently perform pulp bleaching in the subsequent stage. (WO
95/33883). Buchert et al. Decompose xylan containing hexenuronic acid in the side chain of pulp hemicellulose produced when cooking and pulping wood chips using xylan-degrading enzymes such as xylanase and xylosidase, and then xylo-oligosaccharides containing hexene uronic acid. Is used as a substrate to measure the activity of hexene uronidase. Their hexene-uronidase activity measurement method utilizes the fact that aglycone, which is glycosidically bound to hexenuronic acid, is liberated by hexene-uronidase. Specifically, the bond between hexenuronic acid and xylo-oligosaccharide is hydrolyzed by reacting hexeneuronic acid β-bonded to xylose on the non-reducing terminal side of xylobiose or xylotriose with hexenuronidase, whereby xylobiose, Or, xylotriose is produced. However, in this patent document, there is no example in which the enzyme activity was measured, and the enzyme was not purified. Therefore, the existence of the enzyme as a protein has not been proved.

Hexenuronidase has a background that the method for measuring the enzyme activity is extremely difficult and research has been difficult to proceed. A method using the fact that the absorption at 232 nm of unsaturated sugars is reduced by an enzyme reaction is also used in enzyme samples.
It is difficult to measure the decrease in absorption around 232 nm because it contains compounds having absorption around 230 nm to 250 nm. In the method using an unsaturated saccharide bound to xylo-oligosaccharide as a substrate, the possibility that xylo-oligosaccharide is generated by other enzymes such as xylanase that is entangled in the enzyme sample cannot be ruled out if the substrate is not sufficiently purified. However, it is not practical as a true enzyme activity method. Therefore, the present inventors have been required to develop a substrate for enzyme activity which has sufficient specificity and detection sensitivity of the enzyme activity.

(Preparation of synthetic substrate for measuring hexene uronidase) A novel substrate for measuring activity using 4-O-methylumbelliferone, which is used as a substrate for measuring glycosidase activity, was prepared (see Example 6). .

(Method for Measuring Enzyme Titer) The concentration of the synthesized substrate was adjusted to 100 μM in the reaction system and reacted with various sample solutions so that the activity of hexene uronidase could be measured. . Specifically, a 200 μM substrate solution was prepared in a phosphate buffer (pH 7.0: 100 mM). 10 μl of the enzyme solution was added to 10 μl of the substrate solution, and for 30 minutes,
Incubated at 37 ° C. Thereafter, 100 μl of glycine-NaOH buffer (pH 10.5: 500 mM) was added to the reaction system to stop the enzyme reaction. The fluorescence intensity of 4-O-methylumbelliferone was excited at 355 nm and measured at 455 nm. The amount of the enzyme that decomposes 1 micromole of hexenuronic acid per minute in the reaction system was defined as 1 unit. This method is an unprecedented method for measuring enzyme activity with high sensitivity.By using this synthetic substrate, hexene uronidase can be directly concentrated from culture solutions of various bacteria and cultured cells of multicellular organisms without concentrating the sample solution. Can be measured.

(Optimal pH and stable pH range) Optimum enzyme reaction
The pH and pH stability were adjusted to acetate buffer (pH 5.0 or less), phosphate buffer (pH 6.0-7.0), Tris-HCl (pH 7.0-8.
5) The enzyme activity was measured using a glycine-NaOH buffer (pH 9.0 to 10.5) (FIG. 1). As can be seen from FIG. 1, the optimum pH for hexene uronidase activity is 6-8, especially 6.
It was found to be between 5 and 7.5. In addition, 50
After being kept in a predetermined mM buffer at 4 ° C. for 24 hours, the enzyme activity was measured in a phosphate buffer at pH 6.5 (FIG. 2). As can be seen from the results in FIG.
It was stable in the range of 0. (Optimum temperature and temperature stability)
5 was measured in a 50 mM phosphate buffer (FIG. 3). All enzyme reaction times were 30 minutes. As can be seen from FIG. 3, the optimum temperature was found to be around 50 ° C. Regarding temperature stability, 50 mM
After holding in phosphate buffer for 30 minutes, pH 6.5, 50 mM
The measurement was performed at 37 ° C. in a phosphate buffer (FIG. 4). As shown in FIG. 4, the stability of the enzyme showed about 80% or more of the residual activity at 45 ° C. for 30 minutes.

<Isoelectric Point of Enzyme> The isoelectric point was measured using Ampholine manufactured by Amersham-Pharmacia. The equipment used was Rotofar (Bio-Rad), and a pH gradient was created from around pH 2.0 to around pH 11.5, and the activity at each pH was measured. The isoelectric point of hexene uronidase was pH 4.5. there were.

<Measurement of Molecular Weight of Enzyme> The molecular weight of the purified hexene uronidase was measured by polyacrylamide gel electrophoresis. As a result, the molecular weight of the enzyme was about 40,000.

(Effects of metal salts, etc. on enzyme activity) Various metal salts, etc. were added to the enzyme solution to a concentration of 1 mM, and the mixture was added at 4 ° C.
After holding for 24 hours, the same type of metal salt or the like was added to the reaction solution to 1 mM, and the activity of the enzyme was measured (Table 1).

[0043]

[Table 1]

This enzyme was strongly inhibited by Cu ²⁺ . This enzyme was also strongly inhibited by Zn ²⁺ and SDS (relative enzyme activity when SDS was contained was 19.9% when the activity without additives was 100%). EDTA did not inhibit enzyme activity. Surprisingly, it has been found that 2-mercaptoethanol (2-ME) enhances the activity about twice or more. Specifically, the relative activity of the hexene uronidase of the present invention in the presence of 2 mM or 10 mM 2-mercaptoethanol was increased 2.0-fold and 2.7-fold, respectively, as compared to the absence of 2-ME (control).

[0045] 2. Microbes producing hexene uronidase
To separate the 7-5 shares than the soil of Koto-ku, Tokyo Shinonome as bacteria to produce an object-hexene Paulo Nida zero. The 7-5 strain was a gram-negative bacillus, and the nucleotide sequence of 16S rRNA showed that there was no 100% match with any of the existing strains. Paenibacillus lautus is the most closely related 16S
It was the most closely related bacterium with rRNA sequence difference of 0.58% (M. Heyndrickx et al., Int. J. Syst.
Bacteriol. (1996) 46: 988-1003). The mycological properties of this bacterium are as described above. Strain # 7-5 is a new species of the genus Paenibacillus,
No. 5 and the National Institute of Advanced Industrial Science and Technology (AIST) (new name: National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, 1-1-1, Higashi 1-chome, Tsukuba, Ibaraki Prefecture) Deposit dated February 23, 2013, accession number FER
M P-18225 was given.

[0046] 3. Hexen uronidase gene claw
To produce a DNA library of Paenibacillus sp. 7-5 in order to clone the training hexene Paulo Nida synthase gene. A DNA library can be prepared by extracting chromosomal DNA from Paenibacillus sp. 7-5, treating it with an appropriate restriction enzyme, ligating it to an appropriate vector, and introducing the resulting DNA into a suitable host. Conventional methods can be used to extract chromosomal DNA (for example, Samb
rook et al., Molecular Cloning; Cold SpringHarbor Labo
ratory Press (1989) Vol.1 Blin and Stafford's method)
.

It is possible to construct a probe using information based on the constituent amino acids of hexene uronidase derived from Paenibacillus sp. 7-5 which has already been purified. A protein with very low expression of an enzyme gene,
If the introduced protein causes toxicity to the host Escherichia coli, it is difficult to perform shotgun cloning from a DNA library in transformed Escherichia coli. For this reason, in order to obtain a gene encoding a target protein directly from a DNA library, a probe for raising the gene was prepared based on information on amino acids constituting the enzyme protein. The probe was prepared using the amino acid sequence of the N-terminal side of the protein and the amino acid sequence of the digested product with lysyl endopeptidase which is a protease.

A gene fragment of an appropriate size is inserted into a cloning vector having a similar cut surface. As a vector containing this gene fragment, any vector such as a pUC system can be used, but a phage vector or a cosmid vector may be used. Vectors containing these gene fragments are transformed into hosts such as Escherichia coli and yeast. The transformed host is cultured on a normal plate plate on which the host can grow, and colony hybridization is performed to select a host containing a fragment of the target gene. As a probe for performing colony hybridization, a probe prepared based on amino acid information of an enzyme protein is used. The nucleotide sequence of the gene fragment thus cloned can be analyzed by a dideoxy method using a radioactive label or a fluorescent label, the Maxam-Gilbert method, or the like.

[0049] 4. Method for producing the enzyme will be explained a manufacturing method of the enzyme of the present invention. (Purification of Natural Hexenuronidase) Hexenuronidase can be produced by culturing Paenibacillus sp. 7-5. Any carbon source and nitrogen source for culture can be used as long as they can be assimilated by 7-5 strains. For example, as a carbon source,
Xylan or wheat bran, pulp containing xylan,
Agricultural waste such as bagasse, corn fiber, rice straw, or plant fiber can be used. As a nitrogen source,
Nitrogen compounds such as yeast extract, peptone, various amino acids, soybean, corn steep liquor, and various inorganic nitrogens can be used. In addition, various salts, vitamins, minerals, and the like can be appropriately used.

The culture temperature and pH may be any range as long as the bacteria grow and produce hexene uronidase. The culture temperature is 20 to 50 ° C., preferably 35 to 45 ° C., and the pH is 5 to 9; Preferably it is 6-8. After culturing the microorganism of the present invention, the cells are separated, and the cells are treated enzymatically or physicochemically to obtain hexene uronidase present in the cells. Such a crude hexene uronidase enzyme solution has a reaction optimum temperature of about 35 to about 55 ° C, preferably about 40 to about 50 ° C.
° C, the optimum pH is about 5.5 to about 8.5, preferably about 6.0 to about 7.5.

Further, hexene uronidase can be concentrated or solidified by dialysis, salting out, ultrafiltration, freeze-drying or the like. Further, hexene uronidase can be purified by appropriately combining and repeating the culture filtrate with ammonium sulfate fractionation, molecular weight fractionation by gel filtration, various ion exchange resins, hydroxyapatite, isoelectric point fractionation, and the like. The specific purification method will be described in Examples.

(Purification of Recombinant Hexenuronidase by Genetic Engineering) The hexenuronidase of the present invention can also be purified by expressing a cloned gene. In the present specification, an enzyme obtained by expressing a gene is referred to as “recombinant hexene uronidase”. According to this technique, hexene uronidase gene obtained by an appropriate method can be highly produced by expressing it using an appropriate host vector. As a vector used for expression, a plasmid vector, a phage vector and the like are mainly used.
Escherichia coli, Bacillus subtilis, yeast and the like are mainly used as hosts.
As the carbon source and the nitrogen source for cultivation, any source that can assimilate and produce thermostable xylanase can be used. For example, as the carbon source, agricultural waste such as wheat bran, pulp, bacas, corn fiber, rice straw, or plant fiber can be used. Nitrogen sources include yeast extract, peptone, various amino acids,
Nitrogen compounds such as soybean, corn steep liquor and various inorganic nitrogens can be used. In addition, various salts, vitamins, minerals, and the like can be appropriately used. The culture temperature and pH may be any as long as the bacteria produce thermostable xylanase. The culture temperature is preferably 37 ° C., and the pH is preferably 7. Enzyme purification methods include ammonium sulfate fractionation, molecular weight fractionation by gel filtration and various ion exchange resins,
Purification can be performed by appropriately combining and repeating hydroxyapatite, isoelectric focusing, and the like. Whether the obtained purified enzyme is the desired xylanase can be determined by determining the molecular weight, optimum pH, optimum temperature, N
It can be determined by comparing the terminal amino acid sequence and the like with hexene uronidase produced by Paenibacillus sp. Strain 7-5. The specific acquisition of the enzyme will be described in Examples.

[0053] 5. Bleaching process of pulp will be described bleaching process of the pulp using an enzyme of the present invention. In the chemical pulp production process, bleaching may be carried out by treating pulp with a culture of a strain Paenibacillus sp. 7-5 belonging to the hexene uronidase (including a recombinant type) Paenibacillus genus of the present invention. it can. Further, pulp can be bleached by performing chemical bleaching and / or alkali extraction before, during or after the enzyme treatment.

Regarding the amount of the culture or the enzyme to be processed into the pulp, about 0.1 to about 5 U, preferably about 0.5 to about 3 U, per gram of the absolute dry weight of the pulp, as hexene uronidase units.
U may be added. The reaction conditions for the culture filtrate (crude enzyme solution) are a reaction temperature of about 40 to about 70 ° C. and a pH of about 5 to about 8,
In the case of a purified enzyme, the reaction temperature is about 40 to about 70 ° C, and the pH is about 5 to about 8. The reaction time is about 0.2 to about 24 hours, preferably about 0.5 to about 8 hours. Reagents used for chemical bleaching include chlorine, chlorine dioxide, nitrogen dioxide, hypochlorite, oxygen, hydrogen peroxide, ozone and the like. Many alkaline compounds known to those skilled in the art can be used for the alkali extraction. In the alkali extraction, an alkali of about 0.5 to about 3% (based on absolute dry pulp) in terms of sodium hydroxide can be used, and the alkali treatment can be performed while adding oxygen, hydrogen peroxide and the like.

[0055]

EXAMPLES The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples. Example 1 Preparation of Crude Enzyme Solution 1.0% glucose, 0.5% peptone, 0.5% yeast extract,
_{K 2 HPO 4 0.1%, MgSO} 4 · 7H 2 O liquid medium 10ml containing 0.02%
(PH 7.0) into a 25 mm inner diameter test tube
Steam sterilized at 121 ° C. for 15 minutes. This is Paenibacillus
One platinum loop of SP7-5 was inoculated and cultured at 37 ° C with reciprocating shaking (amplitude: 25 mm, 300 reciprocations / min). After culturing for 24 hours under the above conditions, the cells were separated by centrifugation (10,000 rpm × 10 minutes), and the cells were suspended in 10 ml of 50 mM phosphate buffer. The cell suspension was placed on ice. Ultrasonic crusher (SONIFIER 45
(Branson) at an output of 10 kHz for 5 minutes to obtain a crude enzyme solution of hexene uronidase.

A 200 μM synthetic substrate solution was prepared in a phosphate buffer (pH 7.0: 100 mM). 10 μl of the enzyme solution was added to 10 μl of the substrate solution, and the mixture was incubated at 37 ° C. for 30 minutes. The substrate is umbelliferyl-4-deoxy-hex-4-enopyransideuronic acid (see Example 6).
Then, add glycine-NaOH buffer (pH10.5: 500mM) to 100
μl was added to the reaction system to stop the enzyme reaction. The calibration curve is a known concentration of 4-O-methylumbelliferone (Sigma)
It was produced using. The hexene uronidase activity was defined as the amount of an enzyme that produces 1 μmol of hexenuronic acid per minute under the above conditions as 1 unit. As a result, the hexene uronidase activity in the crude enzyme solution was 0.05 U / ml.

[Example 2] Purification of hexene uronidase 1.0% glucose, 0.5% peptone, 0.5% yeast extract
%, K ₂ HPO ₄ 0.1%, MgSO ₄ .7H ₂ O 0.02%, pH 7.0, 50 ml of a liquid medium was placed in a 500 ml Sakaguchi flask, stoppered with cotton, and steam sterilized at 121 ° C. for 15 minutes. 1 ml of the culture solution obtained in Example 1 was added thereto, followed by shaking culture at 37 ° C. for 1 day with shaking (amplitude: 10 cm, 100 reciprocations / minute). After completion of the culture, a culture supernatant was obtained by centrifugation (8,000 rpm × 10 minutes). The culture supernatant was fractionated with ammonium sulfate, and the 20-60% fraction was centrifuged (20,000 rp).
(m × 10 minutes), and dialysis was performed using 20 mM phosphate buffer (pH 7.0) containing 0.9 M ammonium sulfate as an external solution. About the obtained crude enzyme solution, butyl Toyopearl 650-M (Tosoh, 2.5 cm in diameter) equilibrated with 20 mM phosphate buffer (pH 7.0) containing 0.9 M ammonium sulfate.
30 cm) for hydrophobic chromatography. After thoroughly washing the column with the buffer used for equilibration, adjust the ammonium sulfate concentration in the phosphate buffer to 0.
The adsorbed fraction was eluted with a concentration gradient of 9M to 0M, and 3.5m
Each l was fractionated. As a result, hexene uronidase activity eluted in one peak.

Next, the active fraction was collected, dialyzed against phosphate buffer (pH 7.0) as an external solution, and DEAE Toyopearl 650-M (Tosoh, 2.5 cm in diameter) equilibrated with the buffer.
× 30 cm) to perform anion exchange chromatography. After washing with the same buffer, adjust the NaCl concentration in the buffer.
The adsorbed fraction was eluted by applying a gradient increasing from 0 M to 0.5 M, and fractionated by 3.0 ml. Hexenuronidase was recovered as one peak.

The collected hexene uronidase fraction was concentrated to a volume of 2.0 ml with Centricon (Amicon). Hexeneuronidase was applied to a column of Sephacryl S-200 (2.5 cm x 95 cm) (Pharmacia) equilibrated with phosphate buffer (pH 7.0), and 50 mM phosphate buffer (pH 7.0) containing 50 mM NaCl Gel filtration was performed using. The flow rate was 30 ml / hr and 5 ml was collected. Hexenuronidase was recovered as a single peak. The recovered hexene uronidase fraction was concentrated to a volume of 1.0 ml with Centricon (Amicon).

Next, the concentrated enzyme sample was desalted and subjected to polyacrylamide gel electrophoresis. In the electrophoresis, electrophoresis was carried out at 50 mA on a 4.5% concentrated gel and 10% electrophoretic gel, and the electrophoresis was performed until the tip of the electrophoresis reached a position 5 mm from the end of the gel. After the electrophoresis, the polyacrylamide gel was immersed in a phosphate buffer (pH 7.0) containing 100 μM of a synthetic substrate, kept at 40 ° C. for 15 minutes, and allowed to react the enzyme in the gel with the synthetic substrate. When the band was illuminated with a UV lamp (CHROMATO-VUE CABINET MODEL CC-60, UVP, INC.), A pale fluorescent protein band showing enzymatic activity was confirmed slightly below the center of the gel. The gel containing the protein emitting fluorescence was cut out, kept at 4 ° C. for 24 hours, and the enzyme protein was extracted with 3 ml of 50 mM phosphate buffer (pH 7.0). After desalting the extract containing the enzyme, SDS polyacrylamide gel electrophoresis was performed to test the purity of the protein. For electrophoresis, the concentrated gel was 4.5
%, And electrophoresis gel was performed at a concentration of 10%, and it was confirmed that the enzyme protein was uniformly purified. On the electrophoresis, the target enzyme moved to a position where the molecular weight was about 40,000. The yield of purified hexene uronidase based on the crude enzyme solution was 0.28%, and the specific activity was 3.59 U / mg.

Example 3 Paenibacillus sp. 7
Preparation of -5 chromosomal DNA Glucose 1.0%, Peptone 0.5%, Yeast extract 0.5
%, K_TwoHPO_Four 0.1%, MgSO _Four・ 7H_TwoO 0.02%, pH 7.0
50 ml of liquid medium was placed in a 500 ml Sakaguchi flask and a cotton plug was placed.
Thereafter, steam sterilization was performed at 121 ° C. for 15 minutes. This is Paenibacill
Inoculate one loopful of S.P.S. strain 7-5 and go overnight at 37 ° C.
The cells were cultured with shaking (amplitude 10 cm, 100 reciprocations / minute). End of culture
Thereafter, cells were obtained by centrifugation (10,000 rpm × 10 minutes).

The cells were suspended in 5 ml of a glucose-lysozyme solution (50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl buffer (pH 8.0), 4 mg / ml lysozyme) and allowed to stand at room temperature.
Left for 15 minutes. 5 ml of alkaline solution (0.2N NaOH, 1%
SDS) was added, mixed gently, and cooled in ice for 15 minutes. Thereafter, phenol extraction and chloroform extraction were performed, and the extracted aqueous layer was dialyzed at 4 ° C. against a 10 mM TE solution containing 5 mM EDTA to obtain genomic DNA.

[Example 4] Determination of internal amino acid sequence of hexene uronidase First, hexene uronidase was purified by the method of Example 2. Next, the amino acid sequence on the N-terminal side of the purified protein was analyzed by Edman degradation method using a gas phase sequencer (G1000A).
, Hewlett Packrd).
(SEQ ID NO: 3). Purified hexene uronidase is converted to lysyl endopeptidase (Achromobacter Protease I)
And the digested peptide fragments were separated and purified by polyacrylamide gel electrophoresis. The separated and purified peptide fragments were analyzed by a gas-phase sequencer, and the internal amino acid sequence HEX4 (SEQ ID NO: 4), HEX5
(SEQ ID NO: 5) and HEX6 (SEQ ID NO: 6) were determined. HEX3, HEX4, HEX5 and HEX6 have the following sequences. HEX3: amino acid sequence from N-terminus of hexene uronidase Met-Trp-Glu-Gln-Ala-Ile-Met-Asp-Ala-Val-Glu-Lys-Th
r-Lys-Arg-Asn-Val-Gly-Leu-Phe-Pro-Met-Lys-Phe-Pro-
His-Ile-Thr-Ala (SEQ ID NO: 3) HEX4: internal amino acid sequence of hexene uronidase Gly-Leu-Leu-Thr-Asp-Ala-Val-Glu-Lys (SEQ ID NO: 4) HEX5: hexene uronidase Internal amino acid sequence of Asp-Thr-Ala-Ile-Ala-Gln-Leu-Glu-Asp-Tyr-Lys (SEQ ID NO: 5) HEX6: Internal amino acid sequence of hexene uronidase Lys-Met-Ile-Ser-Leu- Val-Asn-Arg-Tyr-Ser (SEQ ID NO: 6)

[Example 5] Cloning of DNA fragment containing hexene uronidase gene An equivalent of 100 µg of genomic DNA prepared in Example 3 was used for BamHI, Kp
The digestion was carried out at 37 ° C. for 18 hours with 100 units of each restriction enzyme of nI, SalI, and PstI, and further 100 units of restriction enzyme was added and reacted for further 6 hours. An amount corresponding to 10 μg of the amount of DNA fragmented from this reaction solution was subjected to 0.8% agarose gel electrophoresis. After electrophoresis, Cyber Green I (BMA)
After the DNA fragment electrophoresed was stained to confirm electrophoresis, the DNA was transferred to a nylon membrane (Hybond N +: manufactured by Amersham) by Southern blotting.

(Preparation of Southern Hybridization Probe) N-terminal amino acid sequence HEX3 obtained in Example 4
(SEQ ID NO: 3) and internal amino acid sequence HEX4 (SEQ ID NO: 4)
Based on the above, a primer for PCR was designed. Perform chemical synthesis based on the designed primers. Primer 1: 5'-ATGTGGGARCARGCIATHATGGAYGCNGTNG-
3 ′ (SEQ ID NO: 7) (where R is G or A, I is inosine,
H is A or C or T, Y is T or C, N is A or C or
Indicates G or T, respectively. Primer 2: 5'-TTRTARTCYTCIAGYTGIGCDATNG-3 '(SEQ ID NO: 8) (where R is G or A, I is inosine, H is A or C or T, Y is T or C, N is A or C or G or T and D indicate A or G or T, respectively).
Using these two primers, PCR was performed using a thermal cycler (manufactured by PerkinElmer) to obtain a 221 bp full length.
The PCR fragment was obtained. The procedure of the PCR method is not a problem in a general manner, but the only condition to be specified is to specify 55.5 ° C. as the annealing condition.

The double-stranded DNA PCR fragment was cloned using a TA cloning kit (manufactured by INVITROGEN). When the base sequence of the cloned PCR fragment was decoded using a DNA sequencer (Model 310, manufactured by ABI), a DNA sequence encoding the amino acid of SEQ ID NO: 3 was found 32 bp or less from the 5 'end. From this, P
The CR fragment is 2 from the N-terminal side of hexene uronidase.
It is a DNA fragment complementary to a 21 bp gene fragment. This DNA fragment was labeled with a fluorescein label kit (Gene Images
^TM , Amersham-Pharmacia)
Fluorescein-labeled nucleic acid was randomly introduced into the strand to label the DNA strand itself.

A nylon membrane for gene cloning prepared in advance is placed in a hybridization bag, and the mixture is incubated at 60 ° C. with a church phosphate buffer (Ch.
urch and Gilbert, Proc. Natl. Acad. Sci. USA 81: 1.
991-1995 (1984)). Thereafter, 10 ng of a randomly labeled PCR DNA fragment was added thereto and hybridized at 60 ° C. for 24 hours. Next, the plate was washed with a washing buffer (40 mM church phosphate buffer containing 1% SDS) at 60 ° C. for 5 minutes, the washing buffer was discarded, and the same method was repeated four times. After removing excess liquid, detection was performed using a labeled nucleic acid detection kit (Gene Images ^™ ).

In the signal of the probe, an appropriate size
About 4000 bp DN in BamHI digest as DNA fragment
The A fragment was selected. 20μ of genomic DNA from # 7-5
0.8% after complete digestion using BamHI (Takara Shuzo)
Perform agarose gel electrophoresis on Cyber Green I
Lambda HindII electrophoresed on the same gel after staining with
Using a marker I (manufactured by Toyobo) as a mark, a DNA of about 4000 bp was sliced and extracted from the gel to obtain a DNA fragment containing the hexene uronidase gene. At this time, DNA fragments were extracted from the gel using an easy trap (Takara Shuzo).

The approximately 4000 bp fragment thus extracted has both ends.
Since it is a BamHI site, the cloning vector of pUC19 (Takara Shuzo) was digested with BamHI in advance, the terminal was dephosphorylated with alkaline phosphatase, and ligated to this vector. Ligation was performed using Ligation Kit Ver.II (Takara Shuzo).
(Manufactured by Takara Shuzo), and a positive clone having the hexene uronidase gene was selected. For selection, the PCR fragment used for Southern hybridization was fluorescently labeled and used. As a result, two positive clones were obtained in about 800 libraries. These are called B15 and B26. After culturing B15, the plasmid was recovered, and the hexene uronidase gene present on the insert in pUC19 carried by B15 was sequenced. Since the hexene uronidase gene did not have a BamHI site in its sequence, the full length of the structural gene could be obtained and the sequence could be read. The hexene uronidase gene contained in its sequence a peptide having an internal sequence obtained when purified hexene uronidase was treated with lysyl endopeptidase. Further, the molecular weight of the protein estimated from the nucleotide sequence of 1125 bp of the gene was 43410, which almost coincided with about 40,000 obtained from SDS-PAGE of the purified enzyme. The determined amino acid sequence of hexene uronidase and the nucleotide sequence of its gene are shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The plasmid pUC19 (7-5) containing the DNA encoding the hexene uronidase of the present invention was prepared from Escherichia coli JM1
Introduced into strain 09, and under the name E. coli JM109 / pUC19 (7-5), National Institute of Advanced Industrial Science and Technology (AIST) (new name:
National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Center, 1-1-1, Higashi 1-chome, Tsukuba City, Ibaraki Pref.
Deposited on February 23, 2016 and given accession number FERM P-18226. [Example 6] Synthesis of fluorescent hexenuronic acid derivative as substrate for hexene uronidase Synthesis of (tri-O-acetyl-α-galactopyranosyl bromide) uronic acid methyl ester (I)

[0071]

Embedded image 3.76 g (10 mmol) of (tetra-O-acetyl-β-galactopyranoside) uronic acid methyl ester were added at 0 ° C. under nitrogen for 30 minutes.
The solution was dissolved in a 25% bromic acid / acetic acid solution (25 mg), stirred for 1 hour, and further stirred at room temperature for 1 hour. After completion of the reaction, dichloromethane (100 ml) was added, and the mixture was added with ice water and cold aqueous sodium bicarbonate solution for 5 times each.
Extracted times. The organic layer was separated, dried over anhydrous sodium sulfate, the solvent was distilled off, and 4.0 g of the title compound (I) (100% yield) was obtained.
%). 2. Synthesis of (umbelliferyl tri-O-acetyl-galactopyranoside) uronic acid methyl ester (II)

[0072]

Embedded image (Tri-O-acetyl-galactopyranosyl bromide)
3.0 g (7.5 mmol) of uronic acid methyl ester, 1.32 g (7.5 mmol) of umbelliferone, 10 g of 4A molecular sieves, and 20 ml of anhydrous acetonitrile were stirred under nitrogen at room temperature for 1 hour. Next, 9.0 g (3.2 mmol) of silver carbonate was added, and the mixture was stirred overnight. After completion of the reaction, the reaction solution was filtered through Celite and eluted with dichloromethane. The dichloromethane eluate was extracted five times with a saturated aqueous sodium bicarbonate solution, the organic layer was separated, and dried over anhydrous sodium sulfate. After evaporating the solvent, the reaction product was subjected to silica gel chromatography to obtain 2.92 g (yield 79%) of the title compound (II) from a fraction of dichloromethane-methanol (97: 3). 3. Synthesis of (umbelliferyl-di-O-acetyl-4-deoxy-hex-4-enopyranoside) uronic acid methyl ester (III)

[0073]

Embedded image A solution of (umbelliferyl tri-O-acetyl-galactopyranoside) uronic acid methyl ester (200 mg, 0.4 mmol) in anhydrous THF (5 ml) at room temperature under nitrogen at room temperature was treated with DBU (1,8-diaz).
abicyclo [5.4.0] undec-7-ene) was added, and the mixture was stirred for 30 minutes. After completion of the reaction, the reaction solution was diluted with ethyl acetate, washed with distilled water, and then dried over anhydrous sodium sulfate. After evaporating the solvent, 153 mg of the crude product was purified by silica gel chromatography, and 132 mg (76% yield) of the title compound (III) was obtained from the dichloromethane fraction. 4. Synthesis of umbelliferyl-4-deoxy-hex-4-enopyranoside uronic acid (IV)

[0074]

Embedded image (Umbelliferyl-di-O-acetyl-4-deoxy-hex-4-enopyranoside) A solution of 86 mg (0.2 mmol) of uronic acid methyl ester in 9 ml of water-methanol (1: 2) under nitrogen at 0 ° C. under 2N. 1 ml of NaOH was added and stirred for 1 hour. After completion of the reaction, the reaction solution was filtered through a cation exchange resin,
Eluted with methanol. After concentration of the eluate, elution with 30% methanol was carried out using ODS reverse phase chromatography to obtain 43 mg (yield: 64%) of the title compound (IV).

Since the title compound is very unstable and decomposes at a high rate, the title compound is used without purification in measuring the activity of hexene uronidase. When storing the substrate, the hydroxyl group present in the hexenuronic acid portion is stored while being acetylated. At the time of activity measurement, the substrate is previously deacetylated by performing a reaction in an ice bath using 1N NaOH in the reaction system, and then diluted with a buffer to a predetermined concentration before use.

Example 7 Hexenuronidase of the Present Invention
Bleaching of pulp using a (1) pre-treatment (alkali-oxygen bleaching) brightness 50.0%, kappa value 11.2, pulp viscosity 19.7mPa
After s beech-based hardwood oxygen delignification, kraft pulp (hereinafter referred to as LOKP) was diluted to 60% by absolute dry weight with ion-exchanged water to adjust the pulp concentration to 10.0%. (2) Pulp treatment with hexene uronidase A phosphate buffer was added to the pulp slurry in the reaction system.
mM, the pH was adjusted to 7.0, and the hexene uronidase obtained in Example 2 was added at a ratio of 0.5 U to 1 g of LOKP absolute dry weight. Hold for hours. (3) Bleaching with chlorine dioxide The pulp slurry after the hexene uronidase treatment described above was added to 0.8% chlorine dioxide in pulp adjusted to a pulp concentration of 10% and a pH of 8.1, and kept at a pulp concentration of 70 ° C for 2 hours. .
The pH at the end of the chlorine dioxide treatment was 5.4. After the bleached pulp is washed with distilled water, NaOH is adjusted to pH 8.0.
After the extraction operation was completed, the obtained pulp was washed with ion-exchanged water to obtain a finished bleached pulp.

(Comparative Example 1) Comparative Example 1 is the same as Example 7 except that the hexene uronidase treatment was not performed. Comparative Example 2 Comparative Example 2 is the same as Example 7 except that the hexene uronidase treatment was not performed, and the chlorine dioxide bleaching amount was 1.2% in chlorine dioxide bleaching. Table 2 shows the whiteness of the pulp measured in the above Examples and Comparative Examples.

[0078]

[Table 2]

As can be seen from the table, pulp whiteness can be improved by using the hexene uronidase enzyme of the present invention without increasing the amount of chlorine-based chemicals.

[0080]

[Sequence List] SEQUENCE LISTING <110> OJI PAPER CO., LTD. <120> NEW HEXENEURONIDASE, GENE ENCODING THE SAME, AND USE OF THE SAME <130> P00-0913 <140> <141> <160> 8 <170 > PatentIn Ver. 2.0 <210> 1 <211> 375 <212> PRT <213> Paenibacillus <400> 1 Met Trp Glu Gln Ala Ile Met Asp Ala Val Glu Lys Thr Lys Arg Asn 1 5 10 15 Val Gly Leu Phe Pro Met Lys Phe Pro His Ile Thr Ala Glu Lys Arg 20 25 30 Tyr Glu Trp Gly Glu Ser Asn Asp Trp Ile Glu Gly Phe Tyr Thr Gly 35 40 45 Met Ile Trp Leu Cys Tyr Glu Tyr Thr Lys Asp Pro Leu Phe Lys Asp 50 55 60 Thr Ala Ile Ala Gln Leu Glu Asp Tyr Lys Ala Arg Leu Glu Glu Lys 65 70 75 80 Arg His Leu Asp His His Asp Ile Gly Phe Leu Tyr Leu Pro Ser Ala 85 90 95 Leu Ala Ala Trp Ile Val Asp Glu His Glu Glu Gly Lys Gln Leu Ala 100 105 110 Leu Lys Ala Ala Asp His Leu Met Leu Arg Trp Arg Glu Glu Gly Gln 115 120 125 Tyr Ile Gln Ala Trp Gly Arg Lys Gly Asp Glu Lys Asn Gly Gly Arg 130 135 140 Ile Ile Ile Asp Cys Met Met Asn Ile Pro Leu Leu Tyr Trp Ala Tyr 145 150 155 160 Asp Gln Thr Gly Asp Glu Arg Tyr Lys Arg Val Ala Val Ala His Ala 165 170 175 Glu Lys Ser Arg Arg Tyr Leu Met Arg Gly Asp Asp Ser Ser Tyr His 180 185 190 Thr Phe Tyr Phe Gln Pro Ser Asp Gly Lys Pro Ile Gly Gly Gly Thr 195 200 205 His Gln Gly Tyr His Asp Gly Ser Thr Trp Thr Arg Gly Gln Ala Trp 210 215 220 Ala Ile Tyr Gly Phe Ala Leu Ser Tyr Arg Tyr Thr Arg Asp Pro Lys 225 230 235 240 Tyr Leu Asp Thr Ala Val Arg Ala Ala Arg Tyr Phe Ile Glu His Leu 245 250 255 Pro Glu Asp His Val Ala Tyr Trp Asp Phe Asp Val Pro Ile Glu Xaa 260 265 270 270 Xaa Thr Pro Arg Asp Ser Ser Ala Ser Ala Ile Ala Ala Cys Gly Met 275 280 285 Leu Glu Ile Leu Glu His Leu Glu Glu Glu Asp Gly Asn Lys Gly Leu 290 295 300 Leu Thr Asp Ala Val Glu Lys Ser Met Ile Ser Leu Val Asn Arg Tyr 305 310 315 320 Ser Thr Ile Gly Glu Ala Ala Glu Glu Gly Leu Ile Lys His Gly Ser 325 330 335 Tyr Tyr Val Arg Gly Gly Met Gly Pro Asp Asp Tyr Met Ile Trp Gly 340 345 350 Asp Tyr Tyr Tyr Leu Glu Ala Leu Leu Arg Leu Thr Lys Gly Ile Pro 355 360 365 Gly TyrTrp Tyr Glu Arg Gly 370 375 <210> 2 <211> 1125 <212> DNA <213> Paenibacillus <400> 2 atgtgggagc aggcgattat ggacgcggtt gaaaagacca agaggaacgt cggtttattt 60 ccgatgaaat tccctcacat cacggcggag aagcgatacg agtggggaga aagcaatgac 120 tggattgaag gattctacac cggaatgatc tggctgtgtt atgaatatac gaaggacccg 180 ctgtttaagg atacggcaat cgctcaattg gaggattaca aggcgcggct ggaggagaag 240 cggcatctgg atcatcatga tatcggattt ctatacctgc cctccgcatt ggccgcgtgg 300 atcgtggatg aacatgaaga gggcaaacag ctggccctaa aagcagccga ccatctgatg 360 ctgcgctggc gggaagaagg ccagtatatt caggcctggg gccggaaggg cgacgagaag 420 aacggcggcc gcattatcat cgactgcatg atgaacatac cgctgctgta ttgggcgtat 480 gaccaaacag gcgacgaacg ctacaaacgc gtggccgtcg cgcacgcgga gaagagccgc 540 cgttatttaa tgcgagggga tgattcttcg taccatacct tctacttcca gccgtcggac 600 gggaagccca tcggcggggg gacgcaccaa ggatatcacg acggctccac ctggacgcgc 660 gggcaagcat gggccatcta cggatttgcg ctttcctacc gttatacccg tgatccgaag 720 tatttggata ccgccgtgcg agctgcccgc tattttatcg agcatttg cc gagaccat 780 tatt gggattttga cgttccgatc gaagcgggta cgcctcgcga cagttccgct 840 tccgccatcg ccgcatgcgg gatgctggag atcctggagc acctggaaga ggaagatggc 900 aataaagggc tattgacaga cgccgtcgag aagtccatga tctcccttgt aaaccgttat 960 tccactatcg gtgaagctgc cgaggaaggc ttgatcaagc atggttccta ttatgtcaga 1020 ggcgggatgg gaccggacga ttacatgatc tggggggatt attattatct cgaggccttg 1080 ctgcgactga ccaaagggat acctggctat tggtacgaga gagga 1125 <210> 3 <211> 29 < 212> PRT <213> Paenibacillus <400> 3 Met Trp Glu Gln Ala Ile Met Asp Ala Val Glu Lys Thr Lys Arg Asn 1 5 10 15 Val Gly Leu Phe Pro Met Lys Phe Pro His Ile Thr Ala 20 25 <210> 4 <211> 9 <212> PRT <213> Paenibacillus <400> 4 Gly Leu Leu Thr Asp Ala Val Glu Lys 1 5 <210> 5 <211> 11 <212> PRT <213> Paenibacillus <400> 5 Asp Thr Ala Ile Ala Gln Leu Glu Asp Tyr Lys 1 5 10 <210> 6 <211> 10 <212> PRT <213> Paenibacillus <400> 6 Lys Met Ile Ser Leu Val Asn Arg Tyr Ser 1 5 10 <210> 7 <211 > 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <2 20> <223> i: inosine <400> 7 atgtgggarc argciathat ggaygcngtn g 31 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <220> < 223> i: inosine <400> 8 ttrtartcyt ciagytgigc datng 25

[0081]

[Sequence listing free text] SEQ ID NO: 7-Description of artificial sequence: primer SEQ ID NO: 8-Description of artificial sequence: primer

[Brief description of the drawings]

FIG. 1 shows the optimal pH of hexene uronidase of the present invention.

FIG. 2 shows the stable pH range of hexene uronidase of the present invention.

FIG. 3 shows the optimal temperature of hexene uronidase of the present invention.

FIG. 4 shows the temperature stability of hexene uronidase of the present invention.

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[Procedure amendment]

[Document name] Notification of change of accession number

[Submission date] May 7, 2002

[Name of the former depositary institution] National Institute of Advanced Industrial Science and Technology

[Old Accession Number] FERM P-18225

[Name of new depositary institution] Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology

[New accession number] FERM BP-7972

[Name of the former depositary institution] National Institute of Advanced Industrial Science and Technology

[Old Accession Number] FERM P-18226

[Name of new depositary institution] Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology

[New accession number] FERM BP-7973

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C12N 1/21 C12N 9/42 5/10 D21C 9/10 Z 9/42 C12N 15/00 ZNAA D21C 9 / 10 5/00 A (72) Inventor Akira Tsukamoto 1-10-6 Shinonome, Koto-ku, Tokyo Oji Paper Co., Ltd. Shinonome Research Center F term (reference) 4B024 AA03 BA12 CA02 HA01 4B050 CC01 CC03 DD02 LL10 4B065 AA01 AB01 BA02 CA31 CA60 4H045 AA11 CA11 DA75 4L055 AD20 BB25 FA12

Claims (25)

[Claims] 1. A hexene uronidase having hexene uronidase activity, having a molecular weight of about 40,000 to about 45,000, wherein the enzyme activity is enhanced by 2-mercaptoethanol or dithiothreitol. 2. The method according to claim 1, wherein the optimum pH is pH 6.0 to 8.0.
The hexene uronidase described. 3. The stable pH range in the enzymatic reaction is pH 5.0-1.
The hexene uronidase according to claim 1, which is 0.0. 4. The method according to claim 1, wherein the optimum temperature is 35 ° C. to 55 ° C.
The hexene uronidase described. 5. The hexene uronider according to claim 1, which retains about 80% or more of the activity by heat treatment at 45 ° C. for 30 minutes and shows about 30% residual activity by heat treatment at 60 ° C. for 30 minutes. Ze. 6. The hexene uronidase according to claim 1, which is derived from a microorganism belonging to the genus Paenibacillus. 7. The microorganism according to claim 7, wherein the microorganism is Paenibacillus sp.
The hexene uronidase according to claim 6, which is 5. 8. The method according to claim 1, which is a recombinant enzyme.
The hexene uronidase according to any one of the above. 9. An amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO: 1 or having an amino acid sequence containing a deletion, substitution, insertion or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 1; The hexene uronidase according to any one of claims 1 to 5, which has uronidase activity. 10. The hexene uronidase according to claim 9, wherein the homology with the amino acid sequence shown in SEQ ID NO: 1 is 70% or more, preferably 80% or more, and more preferably 90% or more. 11. A microorganism belonging to the genus Paenibacillus which produces the hexene uronidase according to any one of claims 1 to 10, which is cultured in a medium.
A method for producing hexenuronidase, comprising collecting hexenuronidase from the obtained culture. 12. The method according to claim 12, wherein the microorganism is Paenibacillus sp.
12. The method of claim 11, wherein the value is -5. 13. A Paenibacillus sp. 7-5 capable of producing hexene uronidase (accession number FERM P-182).
twenty five). A hexene uronidase gene encoding the hexene uronidase according to any one of claims 1 to 10. 15. An amino acid sequence consisting of the amino acid sequence represented by SEQ ID NO: 1, or having an amino acid sequence containing a deletion, substitution, insertion or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 1; Encoding hexene uronidase having uronidase activity,
The hexene uronidase gene according to claim 14. 16. The hexene uronidase gene according to claim 14, which comprises the nucleotide sequence represented by SEQ ID NO: 2. An expression vector comprising the hexene uronidase gene according to any one of claims 14 to 16. 18. A host cell transformed with the expression vector according to claim 17. 19. A method for producing a genetically modified hexenuronidase, comprising culturing the host cell according to claim 18 in a medium and recovering hexene uronidase from the resulting culture. 20. A bleach comprising the hexene uronidase according to claim 1 as an active ingredient. 21. The bleach according to claim 20, wherein the hexene uronidase is a recombinant enzyme. 22. The hexene uronidase according to claim 20.
The bleach according to claim 20, which is obtained by the method according to the above. 23. A method for bleaching pulp, comprising treating pulp with the bleaching agent according to claim 20. 24. The method of claim 23, wherein treating the pulp with the bleaching agent comprises performing chemical bleaching and / or alkali extraction either before, after or during the pulp treatment. An antibody that recognizes hexene uronidase according to any one of claims 1 to 10.