WO2002092809A1 - Novel hexenuronidase, gene coding for the same, and use of these - Google Patents

Abstract

A novel hexenuronidase characterized in that it has hexenuronidase activity and a molecular weight of about 40,000 to 45,000 and the enzymatic activity is enhanced by 2-mercaptoethanol or dithiothreitol; a process for producing the hexenuronidase; a use of the hexenuronidase; a hexenuroidase gene; and a microorganism which yields the hexenuroidase. This enzymatic protein is useful in bleaching a pulp.

Description

 Description New hexeneduronidase, genes encoding it, and technical fields of their use

 TECHNICAL FIELD The present invention relates to a novel hexeneduronidase, a method for producing the same, and uses thereof, a hexeneduronidase gene, and a microorganism that produces hexeneduronidase.

Background art

 Polysaccharides other than cellulose, so-called hemicelluloses, are present in plant cell walls, both hardwood and coniferous. In particular, these hemicelluloses 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. One of the most abundant and famous hemicellulose is xylane. The xylan backbone is a homopolysaccharide in which xylose is linked to jS 1 → 4, and is called universal hemicellulose, which is contained not only in all terrestrial higher plants but also in the cell walls of seaweeds and other algae in general. Good. Xylan has recently attracted attention as a biomass as a raw material for xylooligosaccharide xylitol.

 About 50% of the earth's biomass can be considered to be lignocellulose derived from plant cells. Furthermore, the content of hemicellulose in this lignocellulose is said to be about 30%, and the proportion of hemicellulose in biomass present on the whole earth is about 15%. This xylan is rarely present as a single chain as a homopolysaccharide, and xylose, which is the main chain in the cell wall, is a) arabinose side chain with a1 → 3 bond from the polysaccharide chain with 31 → 4 bond, α1 → It exists in the form of having 2-linked 4--0-methylglucin chain on the side chain, and it is estimated that these heteropolysaccharides have various physiological activities in living cells.

Currently, the most widely used wood-based biomass is in the paper industry, which uses cellulose as pulp. In order to make pulp, a raw material for paper, it is necessary to process wood chips under high temperature and high alkali conditions. In this step, a part of lignin and hemicellulose other than cellulose is removed and pulping is performed. The pulp bleaching process can be carried advantageously. Chips, when treated under high temperature and high alkali conditions, can remove lignin and dissolve xylan at the same time, but the dissolved xylan re-adsorbs to the cellulose surface as the temperature decreases. When the xylan, which is a polysaccharide, is re-adsorbed, it also re-adsorbs while disturbing coloring substances such as lignin and furan derivatives which are to be bleached in a later step. In the paper industry, pulp is treated with xylanase (EC 3.2.1.8) to remove the re-adsorbed xylan as a pretreatment before bleaching the pulp and to improve the efficiency of the subsequent bleaching process. Some factories have introduced a process to decompose the re-adsorbed xylan and remove colored substances.

 Another important problem that arises when making pulp is that of hexeneduronic acid. As described above, xylan has an arabinose side chain with α 1 → 3 bond and a 4 → 0-methyldalcuronic acid with 1 → 2 bond as side chains in addition to the main chain. In particular, 4-0-methyldalcuronic acid has a methylated hydroxyl group at the 4-position, so under high-temperature and high-pressure conditions, which are the conditions for pulping chips, the 4-methoxy methoxy group undergoes --elimination and is desorbed to methanol. Generate 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 4-0-methyldalcuronic acid is converted into so-called hexeneduronic acid. Hexeneduronic 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.

Hexeneduronic acid reacts with the oxidizing chemicals used in the pulp bleaching process, such as ozone, chlorine and chlorine dioxide, to retain double bonds in its molecule. These bleaching reagents should react with compounds derived from lignin, which is a coloring component, or sugars derivatized with furan.However, hexeneduronic acid, which is not a coloring component, has a double bond in the molecule. The reaction of the bleaching chemicals means that these bleaching chemicals are wasted and reduce the efficiency of the bleaching process. Kappa monovalent is an index that determines the rate of bleaching chemical addition during pulp bleaching. In general, kappa monovalent may be considered as a numerical value of the consumption of permanganate power lime, and more specifically, compounds present in the pulp that may react with oxidizing bleaching chemicals May be considered to indicate the amount of Hula generated from lignin and sugar Colored substances such as butane derivatives have been described as monovalent kappa. Hexeneduronic acid is not a coloring substance, but is counted as a power value because the double bond in the molecule reacts with potassium permanganate. Hexeneduronic acid is also an acidic sugar having a carbonyl group in the molecule. The hexoxyperonic acid group has a negatively charged lipoxyl group, and a heavy metal is chelated to the hexeneduronic acid molecule in the pulp. In the bleaching process using the oxidizing bleaching chemicals, the heavy metal compound (complex) reacts with the bleaching chemicals, so that the chemicals are wasted and the bleaching efficiency deteriorates.

 The L0KP pulp, which has been subjected to oxygen bleaching after the digestion step in the pulp bleaching step, has a cutoff value of about 10 points, of which about 5 points are said to be derived from hexeneduronic acid. If this hexeneduronic acid can be removed in advance before bleaching with an oxidizing bleaching chemical, it can be easily analogized that the bleaching chemicals in the subsequent stage can be reduced.

 Because the harms of hexeneduronic acid in pulp bleaching can be significant, paper companies are eager to remove hexeneduronic acid from the pulp. A common method for removing hexeneduronic acid is to treat the pulp with an acid (WO096 / 12063 pamphlet). When the pH of the pulp slurry is acidified and the temperature is maintained at 80 to 120 ° C for 1 to 2 hours, hexene and oxalic acid are mainly hydrolyzed due to the hydrolysis of glycosidic bonds between them and xylan. It can be removed from the chain and removed.

 However, in these attempts, the pulp itself is hydrolyzed and damaged because the pulp is treated with acid. In particular, if the strength of the pulp decreases due to hydrolysis of cellulose, it is a serious problem, and papermaking with such low strength pulp is considered to be unsuitable for high-speed papermaking with current paper machines. Can be In addition, hydrolysis by acid reduces pulp yield and deteriorates pulp productivity in mills. When hexeneduronic acid is actually removed by dilute acid treatment, a decrease in yield of several percent based on pulp weight is observed. Another problem when hexeneduronic acid is removed by dilute acid treatment is that lignin is condensed with dilute acid and converted into lignin that is not easily bleached in the subsequent bleaching step.

As mentioned above, removal of hexeneduronic acid with dilute acid involves several problems Therefore, the removal of hexeneduronic acid by an enzyme has attracted attention.

Hexeneduronic acid removal method using an enzyme that specifically decomposes acids (International publication

W095 / 33883 pamphlet, W. Hashimoto et al., Arch.Biochem.Biophys. (1999) 368 (2): 367-374) has been proposed. There is a problem with the measurement method, and the nature of the purified enzyme is not clear because the enzyme has not been isolated. Bleaching experiments with pulp purified enzymes have not been conducted because there is no purified enzyme. For this reason, it is impossible to judge from the specification whether the enzyme that degrades hexeneduronic acid is really effective for pulp. On the other hand, hexeneduronidase isolated by W. Hashimoto et al. (Above) is an enzyme derived from Bacillus espii GL1 and has a molecular weight of 42 kDa (SDS-PAGE), optimal pH 6.0- 6.5, Optimum temperature around 45 ° C, pH 7.0, complete inactivation by incubation at 60 ° C for 10 minutes, dithiothreyl or 2-mercaptoethanol activity Has no significant effect on the pulp, but the bleaching effect of pulp is unknown. Disclosure of the invention

 An object of the present invention is to provide a novel hexeneduronidase useful for pulp bleaching, a method for producing the same, and uses thereof.

 The present inventors conducted intensive research based on the above-mentioned problems, searched for hexenduronidase-producing bacteria, and conducted extensive screening, and as a result, produced hexenduronidase from soil in Shinonome, Koto-ku, Tokyo. The present invention has found a novel microorganism and a novel enzyme that degrades hexenduronic acid from a culture of the microorganism, and has succeeded in cloning and highly expressing the gene encoding hexenduronidase. Was completed.

 Therefore, the present invention is summarized as follows.

 (1) having hexeneduronidase activity, having a molecular weight of about 40,000 to about 45,000,

Hexenduronidase, wherein the enzyme activity is enhanced by 2-mercaptoethanol or dithiothreitol.

(2) The hexene peronidase according to (1), wherein the optimal pH is ρΗ6.0 to 8.0 · 0. (3) The hexene □□ nidase according to (1), wherein the stable pH range in the enzymatic reaction is pH 5.0 to 10.0.

 (4) An optimal temperature of 35 ° (~ 55 ° C, (1), hexeneduronidase.

 (5) The hexene peroni of (1), which retains about 80 or more 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. Daze.

 (6) The hexene peronidase according to any one of (1) to (5), which is derived from a microorganism belonging to the genus Paenibacillus.

 (7) The microorganism is Paenibacillus sp. 7_5, (6) Hexeneperoni

(8) The hexene peronidase according to any one of (1) to (5), which is a recombinant enzyme.

 (9) consisting of the amino acid sequence of SEQ ID NO: 1, or having an amino acid sequence containing deletion, substitution, insertion or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 1; Has xenoperonidase activity, (1)-

(5) Any of the hexene peroxidases.

 (10) The hexeneduronidase of (9), which has 70% or more, preferably 80% or more, more preferably 90% or more homology with the amino acid sequence shown in SEQ ID NO: 1.

 (11) A method for culturing a microorganism belonging to the genus Paenibacillus which produces hexeneduronidase in any one of (1) to (10) in a medium, and collecting hexeneduronidase from the resulting culture. A method for producing hexene peronidase.

(12) Paenibacillus sp. 7-5 capable of producing hexeneduronidase (Accession No. FERM P-18225).

 (13) A hexeneduronidase gene encoding the hexeneduronidase of any one of (1) to (10).

 (14) The hexeneduronidase gene according to (13), comprising the nucleotide sequence represented by SEQ ID NO: 2.

 (15) An expression vector containing the hexeneduronidase gene of (13) or (14).

(16) A host cell transformed by the expression vector of (15). (17) A method for producing a genetically modified hexenduronidase, comprising culturing the host cell of (16) in a medium, and collecting hexenduronidase from the resulting culture.

(18) A bleach containing the hexeneduronidase of any one of (1) to (10) as an active ingredient.

 (19) The bleach according to (18), wherein hexeneduronidase is obtained by the method according to (17).

 (20) A method for bleaching pulp, comprising treating pulp with the bleaching agent according to (18) or (19).

 (21) The method according to (20), wherein treating the pulp with the bleaching agent comprises performing chemical bleaching and Z or alkali extraction either before, after or during the pulp treatment.

 (22) An antibody that recognizes hexeneduronidase according to any one of (1) to (10). This description includes part or all of the contents as disclosed in the description and / or drawings of Japanese Patent Application No. 20 (Π-142019), which is a priority document of the present application.

 FIG. 1 is a diagram showing the optimal ρΗ of hexene peronidase of the present invention.

 FIG. 2 is a diagram showing the stable ρ range of hexene peronidase of the present invention.

 FIG. 3 is a graph showing the optimal temperature of hexeneduronidase of the present invention.

 FIG. 4 is a diagram showing the temperature stability of hexeneduronidase of the present invention. Description of Sequence Listing

 SEQ ID NO: 7 is a primer.

 SEQ ID NO: 8 is a primer. BEST MODE FOR CARRYING OUT THE INVENTION

 Next, the present invention will be described in detail below.

Hexenduronidase activity as used herein refers to hexeneduronate (4-deoxy-i3 -L-threo-hex-4-enopyranosyluronic acid) and 5-glycolyl by hydrolyzing between the glycoside-linked aglycone via the aldehyde group at position 1. Enzyme activity that produces 2-furancarboxylic acid or 2-fruit acid. Specifically, the activity is

Action 1): Hexeneduronic acid bound as a side chain to the xylan main chain; activity of hydrolyzing hexeneduronic acid to form 5-formyl-2-furancarboxylic acid or 2-fluoroic acid Or

Action 2): Hydroxide of hexeneduronic acid, which is produced when mucopolysaccharide, hyaluronic acid, and acidic polysaccharides containing dalc hexonic acid as constituent sugars such as vectin and gellan are degraded by lyase that degrades them. Activity to decompose to form 5-formyl-2-furancarponic acid or 2-froic acid; or

Action 3): Hydrogenation of a synthetic substrate in which hexeneduronic 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-floica. An activity that produces a seed,

Means any of

 Therefore, the enzyme of the present invention is produced by decomposing acidic polysaccharides containing glucuronic acid as constituent sugars such as mucopolysaccharide, hyaluronic acid, and pectin and geran by lyase which degrades them. Sugar chains containing hexuronic acid, xylane containing hexeneduronic acid as a side chain, which is produced when 4-0-methyldarc oxylan in plant cell wall undergoes β-elimination under high temperature and high temperature conditions Acts on substrates such as

 The enzyme of the present invention has, in addition to having the above-mentioned enzyme activity, a molecular weight of about 40,000 to about 45,000, and the presence of a 2-mercaptoethanol or dithiosyl 1 reducing agent. The activity 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 for enzyme reaction is pH 6.0 to 8.0, and the stable pH range for enzyme reaction is pH 5.0 to 10.0.

 (2) Suitable operating temperature range: 35 ° C to 55 ° C.

 (3) Thermal stability: Maintains about 90% or more activity at 30 ° C for 30 minutes, and about 30% residual activity at 60 ° C for 30 minutes.

 (4) Isoelectric point: It is around pH 4.5.

(5) Inhibition: The activity is strongly inhibited by Cu Zn ²⁺ and SDS. EDTA does not inhibit enzyme activity.

 As used herein, the term “stable pH” means a pH capable of maintaining an enzyme reaction activity of 70 to 100% of the enzyme reaction activity at the optimum pH.

 The enzyme of the present invention may be directly isolated from a microorganism, or may be obtained by a gene recombination method. Examples of the microorganism include microorganisms belonging to the genus Paenibacillus which produce the above hexeneduronidase. Specifically, Paenibacillus sp. 7_5 (indication for identification: # 7-5) or a mutant derived from this microorganism is preferred. This microorganism is a new microorganism, Deposited on February 23, 2001 at the National Institute of Advanced Industrial Science and Technology (new name: National Institute of Advanced Industrial Science and Technology (AIST) · Patent Organism Depositary Center 1, Higashi 1-chome 1-Chuo 6th, Tsukuba-shi, Ibaraki Pref. Deposit number FEO P-18225) and transferred to the deposit based on the Budapest Treaty on March 22, 2002 by the original deposit, and given the deposit number FERM BP-7972.

 The microbiological properties of Paenibacillus sp. 7-5 are as follows.

Cell morphology Bacillus (0.5X2.0-3.O ^ m)

Gram stain indefinite

Spores +: edge, swollen, colony forming Round, whole edge smooth, low convex, glossy, translucent

Power tarase +

Saito Sitase +

0 / F test Acid production from carbohydrates

 Glycerol + erythritol

 D-arabinose + L -arabinose + report + D-xylose + L-xylose

 Adnitol

 Mono-methyl-D-xylose + galactose + glucose + fruc I + mannose + sorbose

 Rhamnose

 Sulsi)

 Inositol

 Mannitto lille + sorbitol

 α-methyl-D-mannose α-methyl-D-glucose + Ν-acetyldarcosamine + amygdalin +

+ Esculin + Salicin + Cellobiose + Maltose + Lactose + melibiose + sucrose + trehalose + inulin + melechi] ^ ones + raffinose + starch + glycogen + xylitol gentiobiose +

D—llah North +

D—Lyxose one

D—Evening Gateau

D—Fuco One

L-Four Course I

D-arabitol one

L-arabitol-darconet-

2-ketogluconic acid

5-ketogluconic acid-iQ-galactosidase (ONPG) + arginine dihydrolase-lysine decarboxylase 1 ordinine decarboxylase 1 availability of citrate 1 hydrogen sulfide production 1 protease-tryptophan deaminase 1 Indole production

Acetin production (VP) +

Gelatinase

Nitrate reduction

Viability

 +

 5 0. C one

 1 0% N a C 1

Hydrolysable

 Casein I

 Hippurate

 Strain # 7_5 shows swelling of bacterial cells due to spores and is negative for anaerobic growth positive, ONPG positive, casein hydrolysis, arginine hydrolase, indole, gelatinase, nitrate reduction, etc. It is considered to be a microorganism belonging to the genus Baenibacillus (Paenibaci11us). This strain is presumed to be Paenibacillus lus 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 properties, and is preferably a microorganism belonging to the genus Paenibacillus, more preferably a strain # 7-5 and a strain thereof. Induced strain. Such derived strains can be obtained by using conventional techniques such as mutagenesis by irradiation with ultraviolet rays or mutagenic substances, or by mutagenesis by genetic modification of the microorganism genome. it can.

 When the enzyme of the present invention is produced from a microorganism, the enzyme can be obtained by a method comprising culturing a microorganism producing the enzyme in a medium and recovering hexeneduronidase from the resulting culture. .

In an embodiment of the present invention, the microorganism is Paenibacillus sp. 7-5 capable of producing hexeneduronidase, and the enzyme produced by the microorganism has the amino acid sequence represented by SEQ ID NO: 1. The invention also encompasses variants of the enzyme, specifically one or more, preferably in the amino acid sequence of SEQ ID NO: 1, Is a hexeneduronidase having an amino acid sequence containing deletion, substitution, insertion or addition of one or several amino acids and having hexeneduronidase activity. Here, several means usually 2 to 9, preferably 2 to 7, and more preferably 2 to 5. Alternatively, the variant has a homology of 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more, with the amino acid sequence shown in SEQ ID NO: 1. Even more preferably hexeneduronidase which is at least 98%.

 As a carbon source and a nitrogen source for culturing, any source capable of producing hexeneduronidase can be used. For example, as the carbon source, agricultural waste such as wheat bran, pulp, bagasse, corn fiber, rice straw or plant fiber containing xylan or xylan, 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 nitrogen 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 grow and produce hekinperonidase. The culture temperature may be 20 to 50, and the pH may be 5 to 9. If the bacteria are thermotolerant, the temperature 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-nidase present in the cells.

 Hexeneduronidase can be concentrated or solidified by dialysis, salting out, ultrafiltration, lyophilization or the like. Furthermore, hexeneduronidase can be purified by appropriately combining the culture filtrate with ammonium sulfate fractionation, molecular weight fractionation by gel filtration, various ion exchange resins, hydroxyapatite, isoelectric focusing, etc., and repeating the same. .

The present invention includes the amino acid sequence represented by SEQ ID NO: 1, or the nucleotide sequence of SEQ ID NO: 2 or the amino acid sequence encoded by the target DNA sequence contained in E. coli JM109 / pUC19 (7-5). It also includes recombinant hexenduronidase or a mutant thereof. E. col i JM109 / pUC 19 (7-5) is a registered trademark of the National Institute of Advanced Industrial Science and Technology (AIST) (new name: National Institute of Advanced Industrial Science and Technology) Was deposited on February 23, 2001 at Tsukuba East 1-chome, 1-Chome No. 6), Ibaraki Prefecture (Accession No.FE-18182), and by the original deposit on March 22, 2002 It has been transferred to a deposit under the Pust Treaty and has been assigned the accession number FERM BP-7973. Here, the mutant has the above-mentioned meaning. In the case of producing a recombinant enzyme, an expression vector containing the hexene peronidase 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. Alternatively, the enzyme can be obtained by a method including collecting hexendronidase. The hexeneduronidase 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 homology to the amino acid sequence. Is 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more. . 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.

Cloning of the hexeneduronidase gene of the present invention from a bacterial source can be carried out by a method commonly used in the art (for example, Sambrook et al., Molecular Cloning A Laboratory Manual, Second edition, Coll. Spring Harbor Laboratory Press, 1989). Specifically, after preparing a genomic library or a cDNA library from a bacterial source, chemically synthesizing DNA capable of encoding an appropriate partial sequence in the amino acid sequence of SEQ ID NO: 1 (for example, automated DNA synthesis). However, the target gene can be screened by using this as a probe, or the target gene can be screened by hybridization with DNA having the nucleotide sequence of SEQ ID NO: 2 or a fragment thereof. Hybridization is performed under low, medium or high stringency conditions and then at relatively high temperature and relatively low ionic strength. In general, hybridization is performed in an appropriately diluted SSC at an appropriate temperature. The higher the temperature (eg, about 5 to 10 ° C below the normal melting temperature (Tm)) and the lower the ionic strength (eg, 0.1 to 1 x SSC), the higher the stringency Become. Examples of hybridization conditions are, for example, Ausbel et al., Short Pro tocols In. It is described 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 a primer (usually 10 to 30 bases) derived from the nucleotide sequence of SEQ ID NO: 2, or Into a suitable cloning vector such as plasmid phage and introduced into a bacterium such as Escherichia coli to amplify the gene of interest (Sambrook et al., Supra).

The amplified gene is then treated with a restriction enzyme, if necessary, and then inserted into an appropriate expression vector, introduced into an appropriate host cell, and the host cell is cultured in an appropriate medium. Or the enzyme of the present invention can be extracted from cultured cells. An expression vector generally contains a promoter, and can optionally contain an origin of replication, a terminator, a ribosome binding site, a signal sequence, an enhancer, and the like. Examples of expression vectors include commercially available vectors and those described in the literature. For example, bacterial vectors such as pQE (Qiagen), pET (Novagen), pBluescript II SK (Stratagene), Vectors for yeast such as pUC118 (Takara Shuzo), pHS19, pHS15, pG-1, XTl (Stratagene), BPV, pMSG (Pharmacia), etc. pcDNAI, pcDM8 (Funakoshi) as vector for animal cells , PREP4 (Invitrogen) and the like. Promoter is selected depending on the host, in the case of a bacterial host trp promoter, lac promoter, P _L promoter, is illustrated and P _R promoter and foremost, Myu05 promoter if a yeast host, PGK promoter Isseki one, GAP promoter mono-, Examples include ADH promoter, GPD promoter, heatshock, and oocyte polypeptide promoters.> For animal cell hosts, examples include cytomegalovirus immediate early gene promoter, SV40 early promoter, retrovirus promoter, heat shock promoter, etc. You. Hosts include bacteria such as E. coli, Bacillus, Brevipacterium, Corynebacterium, Pseudomonas, and other yeasts such as Saccharomyces, Pichia, Candida, and fungi (basidiomycetes). Fungi, filamentous fungi, etc.), insect cells, plant cells, animal cells, and the like. For construction and transformation of an expression vector, the methods described in 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. Culture medium If necessary, after concentration, salting out, solvent extraction, precipitation, various chromatographies (ion exchange, gel filtration, affinity, hydrophobic interaction, etc.), HPLC, electrophoresis, chromatofocusing, Etc. can be performed alone or in combination as appropriate. When recovering from cells, the enzyme can be purified by the same method as described above after mechanically destroying the cells using a mill or the like or chemically using a hypotonic solution.

The present invention also relates to an antibody that recognizes the above hexeneduronidase 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. A polyclonal antibody can be obtained, for example, by immunizing an animal (eg, a heron) 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, removing spleen cells, fusing the spleen cells with myeloma cells, screening for hybridomas producing the desired antibody, and implanting them into the animal's peritoneal cavity. Later, antibodies from the eyes ^) can be obtained from the ascites. Antibodies can be used, for example, to detect hexendocynidase from various microbial sources.

 Further, the present invention relates to a culture obtained by culturing a microorganism belonging to the genus Paenibacillus producing the hexeneduronidase or the transformant in a medium, or a hexeneduronidase or a recombinant having the above-mentioned properties. Disclosed is a bleach containing hexeneduronidase as an active ingredient.

 The present invention also provides a pulp bleaching method, 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 either before, after or during the pulp treatment.

 Hereinafter, the present invention will be described in more detail with respect to hexeneduronidase derived from the genus Paenibacillus, in particular, derived from Paenibacillus sp. 7-5.

1. Physicochemical properties of hexeneduronidase (Method for measuring enzyme activity)

 The first document showing possible presence of hexeneduronidase is Beata

— And Alfred. (Biochem. J. (1977) 165, 287-293). At this time, they discovered an enzyme that hydrolyzes unsaturated sugars on the non-reducing terminal side of oligosaccharides produced when digested with lyases using chondroitin sulfate, heparin, heparan, and iduronic acid as substrates. I have. They call this dalicuronidase, but the enzymatic reaction itself is exactly the same as hexenduronidase, and although this enzyme is not completely purified, it catalyzes a reaction that specifically hydrolyzes hexenduronic acid. It can be called a smooth enzyme. They are cultivating the bacterial Flavobacterim liepariiiuiii and trying to isolate and purify it from the culture. The definition of enzyme activity was defined as the decrease in the absorption per unit time (ΔΔ232) of the double bond in the hexeneduronic acid molecule due to the degradation of hexeneduronic acid.

 In recent years, Hashimoto et al. Have separated and purified hexeneduronidase from Bacillus sp. GL1 using oligosaccharides containing unsaturated sugars, which are generated when geland-xanthan gum is degraded by lyase, as a substrate. (Archives of Biochemistry and Biophysics Vol. 368, No. 2, August 15, pp. 367-374, 1999). The method of measuring enzyme activity by Hashimoto et al. Is exactly the same as that of Peter et al., Utilizing the fact that the double bond of the unsaturated sugar has an absorption near 232 nm. The decrease is measured by an absorption spectrophotometer and the enzyme activity is defined.

On the other hand, Buhyat et al. Have proposed hexeneduronidase treatment of kraft pulp to enzymatically remove hexeneduronic acid generated during kraft cooking of hardwood chips and to efficiently perform pulp bleaching in the subsequent stage. (W095 / 33883). Buhyat et al. Decompose xylan containing hexene and oxalic acid in the side chain of pulp hemicellulose, which is produced when cooking and pulping wood chips, using xylan-degrading enzymes such as xylanase and xylosidase. The activity of hexene mouth nidase is measured by using a xylo-oligosaccharide containing hexeneduronic acid as a substrate. Their method for measuring the activity of hexene peroxidase utilizes the fact that aglycone, which is glycosidically bound to hexenduronic acid, is liberated by hexenduronidase. Specifically, the xylobiose or xylotriose at the non-reducing end It is stated that the reaction between hexeneduronic acid and hexeneduronidase, which is / 3-linked to the loose, hydrolyzes the bond between hexeneduronic acid and xylo-oligosaccharide, and that xylobiose or xylotriose is generated at this time. ing. 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.

 Hexenduronidase has a background in which the method for measuring enzyme activity is extremely difficult and research has been slow. A method that utilizes the fact that the absorption of 232ηπι of unsaturated sugars is reduced by an enzymatic reaction is also used in the enzyme sample. It is difficult to measure the decrease in absorption around 232 nm because it contains compounds with absorption around 25 Οηπι. The method using an unsaturated saccharide bonded to a xylo-oligosaccharide as a substrate also cannot rule out the possibility that other enzymes such as xylanase entrapped in an enzyme sample will produce xyloligosaccharide if the purification of the substrate is insufficient. Therefore, it is not practical as a true enzyme activity method. Therefore, the present inventors have been required to develop a substrate for enzyme activity with sufficient specificity and detection sensitivity of the enzyme activity.

 (Preparation of synthetic substrate for hexene peronidase measurement)

 Substrates for measuring the activity of glycosidase were studied, and a new xenperonic acid derivative was prepared using 4-0-methylpumbelliferone.

 A hexeneduronic acid derivative is a synthetic substrate for the hexeneduronidase enzyme, and forms a glycosidic bond between the hydroxyl group at position 1 of hexeneduronic acid and a group on a fluorescent substance, a chemiluminescent substance, or a coloring substance. A compound represented by the following formula (I) obtained by When the derivative is subjected to the action of hexeneduronidase, it is cleaved at this glycosidic bond to be decomposed into hexeneduronic acid and a fluorescent, chemiluminescent or chromogenic substance. It is possible to measure the enzyme activity of hexenduronidase by detecting the fluorescence, chemiluminescence, or the signal directly or indirectly emitted by the coloring substance released by the enzymatic action by a physicochemical method. It is.

 [Formula 1]

[In the formula, Z represents 0 (oxygen) or S (sulfur), and X represents a fluorophore group, a chemiluminescence group, or a chromophore group.] The X group in the above formula (I) is a fluorophore group, a chemiluminescence group or a chromophore group. Examples of fluorophore groups include, but are not limited to, substituted or unsubstituted cumaryl, fluoresynyl, naphthyl (eg, i3-naphthyl) and the like. Examples of the substitution group include a group capable of forming a glycosyl bond with the hydroxyl group at position 1 of hexeneduronic acid (preferably a hydroxyl group and a -SH group), which substantially affect the enzymatic action of hexeneduronidase. Any substituent may be used as long as it has no effect on the group, and the like. When X is a cumaryl group, a fluoresynyl group, or a naphthyl group, a glycosidic bond with hexeneduronic acid is formed via a hydroxyl group of these groups.

 An example of a chemiluminophore group is luciferin derived from fireflies or mushrooms.Enzymatically released luciferin can detect the light emitted when luciferase is oxidized to oxyluciferin by the action of luciferase. it can.

 An example of a chromophore group is p-nitrophenoxy group.For example, in the case of p-nitrophenoxy group, yellow-yellow to yellow because p-ditrophenol dissociates as a result of the action of hexene ゥ -nidase The degree of brown coloring can be measured spectroscopically. Hexeneduronic acid derivatives containing a fluorophore group as the X group in the above formula (I) are preferred. The fluorophore group is preferably a substituted coumaryl group, ie, a derivatized phenyl derivative group. Examples of such a group include a 7-hydroxycoumaryl group (also referred to as a deumbelliferyl group) and a 4-methyl-7-hydroxy group. And a coumaryl group (also referred to as a 4-methylumberiphenyl group). Accordingly, an example of a preferred hexeneduronic acid derivative is umbelliferyl-14-dexoxy-1-hex-14-enopyranoside hydroic acid represented by the following formula (II).

The hexeneduronic acid derivative can be produced or synthesized according to the following steps. First, using -galactopyranosideuronic acid as a starting material, the hydroxyl group of which is substituted or unsubstituted alkanol group (eg, acetyl group, acetyl group substituted with 1 to 3 halogen atoms (eg, monochloroacetyl group, Trialkylsilyl group [for example, tri-lower alkyl A protective group such as an aryl group (eg, a trimethylsilyl group, a triethylsilyl group, etc.), an arylalkyl group (eg, a substituted or unsubstituted phenyl lower alkyl group, eg, a substituted or unsubstituted benzyl group), or a similar protecting group. The carboxyl group can be substituted with a substituted or unsubstituted alkyl ester (eg, methyl ester, ethyl ester, etc.), arylalkyl ester (eg, benzyl ester, P-methoxybenzyl ester, etc.), or a similar compound. Protect in ester form. Preferably, the hydroxyl group is protected in the form of an acetyl group and the hydroxyl group in the form of a methyl ester. The lower alkyl used herein refers to Cj-(^ alkyl, preferably methyl and ethyl. The halogen atom used in the present specification is fluorine, chlorine, bromine or It means an iodine atom.

The hydroxyl group at the 1-position of i3-galactobilanosiduronic acid protected as described above is replaced with, for example, a halogen atom (fluorine, chlorine or bromine atom), -S (= 0) R, -0-P (NR ₂ ) _Γ -0-Ρ (=

_{0) (OP) Γ _SR,} -SP (= NPh) (NR 2) 2 ( where, R is a lower alkyl such as methyl, Echiru, propyl, Ph is selected such as by representing.) The Ariru such phenyl Replace. When the halogen atom is a bromine atom, the bromination can be performed, for example, in a bromic acid / acetic acid solution. Next, a glycosylation reaction is performed between the substituted product and a fluorescent substance, a chemiluminescent substance or a coloring substance having a hydroxyl group (for example, Toshinia, K. et al., Chem. Rev. 93: 1503-1531, 1993; Shunichi Hashimoto) And Makoto Nakajima, Fulmasia 32: 1375-1 380, 1996). In the case of a bromine-substituted product, a glycosylation reaction can be carried out in the presence of a silver salt or a mercury salt according to the known Koenigs-Knorr method.

 Next, the protected glycosylated compound is subjected to deoxylation at the 4-position of the protected << _ galactovilanosidonic acid moiety to convert it to a protected hexeneduronic acid moiety. Finally, the protecting group of the protected hexeneduronic acid moiety of the glycosylated compound is deprotected and converted to the hexeneduronic acid moiety.

 (Method of measuring enzyme titer)

The basic principle of measuring the enzyme activity of hexeneduronidase is that a hexeneduronic acid derivative to which a fluorophore group, a chemiluminescent group or a chromophore group is bound causes hexeneduronidase to act, thereby breaking the glycosidic bond. To produce a fluorescent, chemiluminescent or chromogenic substance, and the intensity of that signal is measured directly or indirectly by physicochemical methods Thereby, the enzyme activity of the hexeneduronidase can be measured. That is, the hexeneduronic acid derivative produced or synthesized as described above can be used as a substrate for measuring the activity of a hexeneduronidase enzyme. When the signal generated by the action of hexeneduronidase on the substrate is fluorescence, the released fluorescent substance is excited at a specific wavelength, and the fluorescence intensity is measured based on the emission wavelength emitted by the excitation. At this time, the amount of the enzyme that releases l / mol of hexeneduronic acid per minute in the reaction system can be defined as 1 unit.

 The reaction may be prepared so that the substrate is prepared at an appropriate concentration (for example, about 100 M) in the reaction system, and reacted with various sample solutions to measure the activity of hexene-nidase. . More specifically, a substrate solution is prepared in a buffer such as a phosphate buffer or Tris-HCl (for example, at a pH of about 6 to about 8), and an enzyme solution is added to the substrate solution at an enzyme to substrate ratio of about 1 : 10 to about 1: 100 and incubate for about 1 to 30 minutes, for example at room temperature to 55. Thereafter, the enzyme reaction is stopped by a method such as adding an alkaline buffer such as glycine-NaOH buffer (pH about 10) to the reaction system.

 The fluorescence, chemiluminescence or chromogenic signal generated at this time can be measured using a fluorimeter or an absorptiometer. The excitation wavelength, emission wavelength, or absorbance are appropriately selected depending on the characteristics of the signal. For example, in the case of fluorescent 4-methyl-7-hydroxycoumarin released as a result of an enzymatic reaction, the excitation wavelength is about 355 nm and the emission wavelength is about 455 nm.

As in the case for example that use e Yuru or Umishii Yuke derived luciferin chemiluminescers group, when enzymatically liberated luciferin luciferase enzyme is allowed to act in the presence of Mg ^2t, is oxidized to O carboxymethyl luciferin The light emitted from the light can be detected spectroscopically.

By using this hexeneduronic acid derivative as a synthetic substrate for hexeneduronidase measurement, the enzyme of hexeneduronidase can be directly concentrated without concentrating the sample solution from various bacterial cultures or cultured cells of multicellular organisms. Activity can be measured. An example of a specific measurement method will be described below (see Example 6). The concentration of the synthesized hexeneduronidase measurement substrate was adjusted to 100 M in the reaction system, and it was set to react with various sample solutions to measure hexeneduronidase activity. Specifically, a 200 M substrate solution was added to a phosphate buffer.

(pH 7.0: lOOniM). An enzyme solution was added to 10/1 of the substrate solution, and the mixture was incubated at 37 ° C for 30 minutes. Thereafter, 100 1 of glycine-NaOH buffer (ρΗΙΟ: 5: 500 mM) was added to the reaction system to stop the enzyme reaction. The fluorescence intensity of 4-0-Methyl Pumbelliferone was excited at 355 nm and measured at 455 nm. The amount of enzyme that decomposes 1 micole mole of hexeneduronic acid per minute in the reaction system was defined as 1 unit. '

(Optimal pH and stable pH range)

 The optimal pH and pH stability of the enzyme were determined using acetate buffer (pH 5.0 or less), phosphate buffer (pH 6.0-7.0), Tris-HC1 (pH 7.0-8.5), glycine-NaOH The enzyme activity was measured using a buffer (pH 9.0 to 10.5) (Fig. 1). As can be seen from FIG. 1, it was found that the optimum pH for hexeneduronidase activity was in the range of 6 to 8, especially 6.5 to 7.5. The enzyme activity was measured in a phosphate buffer at pH 6.5 after each enzyme was kept in a predetermined buffer of 50 mM at 4 ° C. for 24 hours (FIG. 2). As can be seen from the results in FIG. 2, the enzyme was stable in the range of pH 5.0 to 10.0.

 (Optimum temperature and temperature stability)

 The optimal temperature for the enzymatic reaction was measured in a pH 6.5 50 phosphate buffer (Figure 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. In addition, the temperature stability was measured at 37 ° C in a pH 6.5, 50 mM phosphate buffer after maintaining at a predetermined temperature for 30 minutes in a pH 6.5, 50 mM phosphate buffer (Fig. 4). As shown in FIG. 4, the stability of the enzyme was about 80% or more after 45 minutes of treatment at 45 ° C.

 <Isoelectric point of enzyme>

The isoelectric point was measured using Amersham-Pharmacia Ampholine. The equipment used was a Lottopher (Piorad), and a pH gradient was created from around pH 2.0 to around pH 11 and the activity at each pH was measured. As a result, the isoelectric point of hexene peronidase was PH 4.5. there were. <Measurement of molecular weight of enzyme>

 The molecular weight of the purified hexeneduronidase was measured by polyacrylamide gel electrophoresis. As a result, the molecular weight of the enzyme was about 40,000.

 (Effect of metal salts etc. on enzyme activity)

 After adding various metal salts and the like to the enzyme solution so as to be ImM, and keeping at 4 ° C for 24 hours, the same type of metal salts and the like are also added to the reaction solution so as to be ImM to increase the enzyme activity. It was measured (Table 1).

[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 two-fold or more. Specifically, the relative activity of the hexeneduronidase of the present invention in the presence of 2 or lOmM 2 _mercaptoethanol is 2.0 times higher than that in the absence of 21 ME (control). 2.7 times higher.

2. Microorganisms that produce hexeneduronidase

 7-5 strains of hexene peronidase producing bacteria were isolated from the soil of Shinonome, Koto-ku, Tokyo. Seven to five strains were Gram-negative rods, and the analysis of the base sequence of 16 S rRNA revealed that none of the existing strains matched 100%. As the most closely related species, Paenibaci llus l autus was the most closely related bacterium with a difference in nucleotide sequence of 16 S rRNA of 0.58% (M. Heyndrickx et al., Int. J. Syst. Bacterio l. (1996) 46: 988-1003). The microbiological properties of this bacterium are as described above. Strain # 7-5 is a new species of the genus Paenibacillus, named Baenibacillus sp. 7-5, and deposited at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the accession number FERM BP-7972. I have.

3-Cloning of the hexeneduronidase gene

Produce a DNA library of Paenibacillus sp. 7-5 to clone the hexeneduronidase gene. DM Library One is Paenibacill It can be prepared by extracting chromosome 丽 A from SP7-5, treating it with an appropriate restriction enzyme, ligating it into an appropriate vector, and introducing it into a compatible host. Conventional methods can be used to extract chromosomal DNA (eg, Sambrook et al., Molecular Cloning; Cold Spring Harbor Laboratory Press (1989) Vol. 1, Blin and Stafford).

 It is possible to make a probe using information based on the constituent amino acids of hexeneduronidase which is already purified from Paenibacillus sp. 7-5. If the expression of the enzyme gene is very low, or if the introduced protein causes toxicity to the host Escherichia coli, shotgun cloning of the transformed Escherichia coli from the DNA library is difficult. For this reason, in order to obtain the gene encoding the target protein directly from the DNA library, a probe was prepared for lifting the gene based on information on the 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 the proteolytic enzyme lysylendopeptidase.

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

4. Enzyme production method

 Next, a method for producing the enzyme of the present invention will be described.

 (Purification of natural hexeneduronidase)

Hexeneduronidase can be produced by culturing Paenibacillus sp. 7-5. 7-5 strains can be used as carbon and nitrogen sources for cultivation. Any of these can be used. For example, as the carbon source, xylan or xylan-containing wheat bran, pulp, bagasse, corn fiber, agricultural waste such as rice straw, plant fiber, or the like can be used. As a nitrogen source, nitrogen compounds such as yeast extract, peptone, various amino acids, soybean, corn steep liquor, and various inorganic nitrogen can be used. Various salts, minerals, minerals, and the like can be used as appropriate.

 The cultivation temperature and pH may be any as long as the bacteria grow and produce hexene-nidase, and the cultivation temperature is 20 to 50 ° C, preferably 35 to 45 ° C, and the pH is 5 to 50 ° C. 9, preferably 6-8. After culturing the microorganism of the present invention, the cells are separated, and the cells are treated enzymatically or physicochemically to obtain hexenduronidase present in the cells. Such a crude hexeneduronidase enzyme solution has an optimal reaction temperature of about 35 to about 55 ° C, preferably about 40 to about 50 ° C, and an optimum pH of about 5.5 to about 8.5, preferably about 6 ° C. 0 to about 1.5.

 Hexeneduronidase can be concentrated or solidified by dialysis, salting out, ultrafiltration, lyophilization or the like. Furthermore, hexeneduronidase can be purified by appropriately combining the culture filtrate with ammonium sulfate fractionation, molecular weight fractionation by gel filtration, various ion exchange resins, hydroxyapatite, isoelectric point fractionation, and the like, and repeating the same. The specific purification method is described in Examples.

(Purification of Recombinant Hexeneduronidase by Genetic Engineering) The hexeneduronidase 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 hexeneduronidase”. According to this technique, high production can be achieved by expressing the hexeneduronidase gene obtained by an appropriate method 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. As the nitrogen source, nitrogen compounds such as yeast extract, peptone, various amino acids, soybean, corn steep liquor, various inorganic nitrogen, and the like 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 heat-resistant xylanase. The culture temperature is preferably 37 ° C, and the pH is preferably 7. The enzyme can be purified by appropriately combining and repeating ammonium sulfate fractionation, molecular weight fractionation by gel filtration, various ion exchange resins, hydroxyapatite, isoelectric point fractionation, and the like. To confirm whether the obtained purified enzyme is the xylanase required, the molecular weight, optimum pH, optimum temperature, N-terminal amino acid sequence, etc. of the obtained purified enzyme were determined by the production of Paenibacillus sp. It can be determined by comparing with hexeneduronidase. Specific examples of obtaining enzymes will be described in Examples.

5-how to bleach pulp

 Next, a method for bleaching pulp using the enzyme of the present invention will be described. In the chemical pulp manufacturing process, bleaching is performed by treating the pulp with a culture of a strain of Paenibacillus sp 7-5 belonging to the hexene peronidase (including a recombinant type) Paenibacillus genus of the invention. Can do it. In addition, pulp can be bleached by chemical bleaching and Z or alkali extraction before, during or after the enzyme treatment.

 Regarding the amount of culture or enzyme to be processed into the pulp, about 0.1 to about 5 U, preferably about 0.5 to about 3 U, is added as a hexeneduronidase unit per gram of the absolute dry weight of the pulp. do it. The reaction conditions are a reaction temperature of about 40 to about 70 ° C. and a pH of about 5 to about 8 for the culture filtrate (crude enzyme solution), and a reaction temperature of about 40 to about 7 (TC, pH The reaction time is about 0.2 to about 24 hours, preferably about 0.5 to about 8 hours.The reagents used for chemical bleaching include chlorine, chlorine dioxide, and nitrogen dioxide. , Hypochlorite, oxygen, hydrogen peroxide, ozone, etc. Many alkaline compounds known to those skilled in the art can be used for alkali extraction. Alkali treatment can be performed while adding oxygen, hydrogen peroxide, etc. using an alcohol of about 0.5 to about 3% (vs. dry pulp).

【Example】 Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these embodiments.

 Example 1 Preparation of Crude Enzyme Solution

Glucose 1.0%, peptone 0.5%, yeast extract _{0 · 5%, K 2 HP0} 4 0. 1%, MgS0 4 - 7¾0 0. 02% liquid containing medium 10ml of (p H7 0.), It was taken in a test tube with an inner diameter of 25%, sealed with a paper stopper, and then steam-sterilized at 121 ° C for 15 minutes. One loopful of Paenibacillus sp. 7-5 was inoculated with this, and cultured with reciprocal shaking at 37 C (amplitude: 25, 300 reciprocations: Z minutes). After culturing for 24 hours under the above conditions, the cells were separated by centrifugation (10, OOOrpm X 10 minutes), and the cells were suspended in 10 ml of 50-pass phosphate buffer. The solution was treated on ice using an ultrasonic crusher (S0NIFIER 450, manufactured by Branson) at an output of 10 kHz for 5 minutes to obtain a crude enzyme solution of hexeneduronidase.

 A 200 M synthetic substrate solution was prepared with phosphate buffer (pH 7.0: 100 mM). The enzyme solution was added to the substrate solution 10 ^ 1, and the mixture was incubated at 37 ° C for 30 minutes. The substrate is umberyphryl-4-dexoxy-hex-4_enopyrazidou humic acid (see Example 6). Thereafter, 100 Hi of glycine-NaOH buffer (ρΗΙΟ5: 500 mM) was added to the reaction system to stop the enzyme reaction. The calibration curve was prepared using a known concentration of 4-0-methylbenbelliferone (manufactured by Sigma).

 The hexeneduronidase activity was defined as 1 unit (Unit) of the enzyme producing 1 mol of hexeneduronic acid per minute under the above conditions. As a result, the hexene peronidase activity in the crude enzyme solution was 0.05 U / ml.

 [Example 2] Purification of hexeneduronidase

Glucose 1.0%, peptone 0.5%, yeast extract _{0. 5%, K 2 HP0 4} 0. 1%, MgS0 4 · 7H 2 0 0. 02%, a p H7. 0 of the liquid medium 50 ml 500 After being placed in a ml volumetric flask and covered with a cotton plug, steam sterilization was performed at 121 ° C for 15 minutes. To this, 1 ml of the culture solution obtained in Example 1 was added, and the mixture was reciprocally shake-cultured (amplitude 10 cm, 100 reciprocations // minute) at 37 for 1 day. After completion of the culture, the culture supernatant was obtained by centrifugation (8, OOOrpmX10 minutes). The culture supernatant was subjected to ammonium sulfate fractionation, and a 20-60% fraction was collected by centrifugation (20, OOOrpm X 10 minutes). Then, a 20ηιΜ phosphate buffer containing 0.9M ammonium sulfate (ρ 7.0 ) Was used as an external solution for dialysis. 20 mM phosphate buffer containing ammonium sulfate Hydrophobic chromatography was performed using Petiltoyopearl 650-M (Tosoichi, diameter 2.5 cm × 30 cm) equilibrated with — (pH 7.0). After thoroughly washing the buffer using the buffer used for equilibration, the adsorbed fraction is eluted with a concentration gradient that reduces the concentration of ammonium sulfate in the phosphate buffer from 0.9M to 0M. Fractionated. As a result, hexeneduronidase activity eluted in one peak.

 Next, the active fraction was collected, dialyzed against phosphate buffer (pH 7.0) as an external solution, and equilibrated with the same buffer. DEAE Toyopearl 650-M (Tosoichi, diameter 2.5cm x 30cm) ) To perform anion exchange chromatography. After washing with the same buffer, the adsorbed fraction was eluted by applying a gradient to raise the NaC1 concentration in the buffer between 0 M and 0.5 M, and fractionated in 3.0 ml portions. Hexeneduronidase was recovered as a single peak.

 The collected hexene peronidase fraction was concentrated to a volume of 2. Oml with Centricon (Amicon). Hexane peronidase 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. Gel filtration was performed using (0). The flow rate was 30 ml / hr, and 5 ml was collected. Hexeneduronidase was recovered as a single peak.

 The recovered hexene peronidase fraction was concentrated to 1. Oil using Centricon (Amicon).

Next, the concentrated enzyme sample was desalted and subjected to polyacrylamide gel electrophoresis. For electrophoresis, electrophoresis was performed at 4.5 mA on a concentration gel 4.5% and electrophoresis gel 10% at 50 mA, and electrophoresis was performed until the tip of the electrophoresis was 5 mm from the end of the gel. After the electrophoresis, the polyacrylamide gel was immersed in phosphate buffer (PH 7.0) containing 100 zM synthetic substrate, kept at 40 for 15 minutes, and the enzyme in the gel was reacted with the synthetic substrate. When the band was illuminated with a UV lamp (CHR0MAT0-VUE CABINET MODEL CC-60, UVP, INC.), A pale fluorescent protein band showing enzyme activity was confirmed slightly below the center of the gel. The gel containing the fluorescent protein 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 was subjected to SDS polyacrylamide gel electrophoresis to test the purity of the protein. Electrophoresis Was performed at a concentration of 4.5% for the concentrated gel and 10% for the electrophoresis gel, and it was confirmed that the enzyme protein was uniformly purified. On the electrophoresis, the target enzyme was moved to a position having a molecular weight of about 40,000. The yield of purified hexenduronidase relative to the crude enzyme solution was 0.28%, and the specific activity was 3.59 U / mg.

 [Example 3] Preparation of Paenibacillus sp. 7_5 chromosomal DNA

Glucose 1.0%., Peptone 0.5%, yeast extract _{0. 5%, K 2 HP0 4} 0. 1%, MgS0 4 · 7H 2 0 0. 02%, a p H7. 0 of the liquid medium 50ml After taking a cotton plug in a 500 ml capacity flask, steam sterilization was performed at 12 C for 15 minutes. One loopful of Paenibacillus sp. 7-5 strain was inoculated into this, and cultured at 37 ° C overnight with reciprocal shaking (amplitude 10 cm, 100 reciprocal Z minutes). After completion of the culture, 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 for 15 minutes. 5 ml of an alkaline solution (0.2 N Na0H, 1% SDS) was added, mixed gently, and cooled on ice for 15 minutes. Thereafter, phenol extraction and black-mouthed form extraction were performed, and the extracted aqueous layer was dialyzed at 4 ° C against an iOmM TE solution containing 5mMEMi to obtain genomic DNA.

 [Example 4] Determination of internal amino acid sequence of hexeneduronidase

 First, hexeneduronidase was purified by the method of Example 2. Next, the amino acid sequence at the N-terminal side of the purified protein was determined by Edman degradation using a gas-phase sequencer 1000A, Hewlett Packard), and this was designated as HEX3 (SEQ ID NO: 3). Purified hexopenidase was digested with lysylendopeptidase (Acryromobacter Protease I), and the digested peptide fragments were separated and purified by polyacrylamide gel electrophoresis. The separated and purified peptide fragments were analyzed using a gas phase sequencer, and the internal amino acid sequences 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 hexeneduronidase

Met-Trp-Glu-Gln-Ala-Ile-Met-Asp-Ala-Val-Glu-Lys-Tr-Lys-Arg-Asii-Yal-Gly- Leu-Phe-Pro-Me t-Lys-P e-Pro-His-11 e-Thr-Al a (SEQ ID NO: 3)

HEX4:

Internal amino acid sequence of hexene peronidase

Gly-Leu-Leu-Thr-Asp-Ala-Val-Glu-Lys (SEQ ID NO: 4)

HEX5:

Internal amino acid sequence of hexene peronidase.

Asp-Tr-Ala-Ile-Ala-Gln-Leu-Glu-Asp-Tyr-Lys (SEQ ID NO: 5)

HEX6:

Internal amino acid sequence of hexene peronidase

Lys-Met-Ile-Ser-Leu-Val-Asn-Arg-Tyr-Ser (SEQ ID NO: 6)

 [Example 5] Cloning of DNA fragment containing hexeneduronidase gene Equivalent to genomic DNA xOO xg prepared in Example 3 was treated with 100 units of each restriction enzyme of BamHI and KpnL Sal L Pstl at 37 units for 18 hours each. After digestion, the reaction was continued for an additional 6 hours with an additional 100 units of restriction enzyme. An amount equivalent to 10 zg of the amount of DNA fragmented from this reaction solution was subjected to 0.8 agarose gel electrophoresis. After electrophoresis, the DNA fragments electrophoresed with Cyber Green I (manufactured by BMA) were stained, and after confirming electrophoresis, the DNA was transferred to nylon membrane (High Pond N +: manufactured by Amersham) by Southern blotting. .

 (Preparation of probe for Southern hybridization)

 Based on the N-terminal amino acid sequence HEX3 (SEQ ID NO: 3) obtained in Example 4 and the internal amino acid sequence HEX4 (SEQ ID NO: 4), a primer for PCR was designed. Chemical synthesis is performed based on the designed primer,

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, Υ is Τ or C, and N is A or C or G or T, respectively.)

Primer 2:

 5'-TTRTARTCYTC I AGYTGI GCDATNG-3 '(SEQ ID NO: 8)

(Where R is G or A, I is inosine, H is A or C or T, Υ is Τ or C and N represent A or C or G or T, and D represents A or G or T, respectively. ) Got. Using these two primers, PCR was carried out using a thermal cycler (manufactured by Pakinkin Elma) to obtain a PCR fragment with a total length of 221 bp. The PCR procedure is performed in a general manner, but the only condition that should be specified is 55.5 ° C as the annealing condition.

 The double-stranded DM PCR fragment was cloned using a TA cloning kit (manufactured by INVITROGEN). The base sequence of the cloned PCR fragment was analyzed using a DNA sequencer (Model 310, manufactured by ABI). As a result, a DNA sequence encoding the amino acid of SEQ ID NO: 3 was found 32 bp or less from the 5 'end. did. From this, the PCR fragment is a DM fragment that is complementary to the 221 bp gene fragment from the N-terminal side of hexeneduronidase. Using a DNA-labeled kit (Gene Images ™, manufactured by Amersham-Pharmacia), the DNA fragment was randomly introduced with a nucleic acid labeled with fluorescein into the DNA strand, and the DM strand itself was labeled. A nylon membrane for gene cloning prepared in advance is placed in a hybridization bag, and a church phosphate buffer (Clmrch and Gilbert, Pro Natl. Acad. Sc. USA 81: 1991-) is used at 60 ° C. 1995 (1984)). Thereafter, 10 ng of a randomly labeled PCR DNA fragment was added thereto and hybridized at 60 ° C. for 24 hours. Next, using a washing buffer (40 mM church phosphate buffer containing 1% SDS) at 6 Ot: 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 ™).

An approximately 4000 bp DM fragment in the BamHI digest was selected as an appropriately sized DNA fragment in the probe signal. Complete digestion of genomic DNA from # 7-5 with BamHI (Takara Shuzo) was performed, followed by 0.8% agarose gel electrophoresis, staining with Cyber Green I, and electrophoresis on the same gel. The DNA of about 4000 bp was extracted by slicing the gel using a marker of Lambda HindIII (manufactured by Toyobo) as a mark to obtain a DNA fragment containing the gene for hexendronidase. At this time, DNA fragments were extracted from the gel using an easy trap (Takara Shuzo). Since the extracted fragment of about 4000 bp has BamHI sites at both ends, the pUC19 cloning vector (Takara Shuzo) was digested with BamHI in advance, and the ends were dephosphorylated with alkaline phosphatase, and ligated to this vector. The ligation was performed using the ligation kit Ver. II (Takara Shuzo) and the host was JM109.

(Manufactured by Takara Shuzo), and a positive clone having a hexeneduronidase gene was selected. For selection, the PCR fragment used in 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 hexeneduronidase gene present on the insert in PUC19 carried by B15 was sequenced. Since the hexeneduronidase 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 hexenduronidase gene contained in its sequence an internal sequence peptide obtained when purified hexenduronidase was treated with lysyl endopeptidase. 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, which was obtained from SDS-PAGE of the purified enzyme. The determined amino acid sequence of hexeneduronidase and the nucleotide sequence of the gene are shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

 The plasmid pUC19 (7-5) containing the DNA encoding the hexeneduronidase of the present invention was introduced into the E. coli JM109 strain, and was named E. coli JM109 / pUC19 (7-5). Deposited with the National Institute of Advanced Industrial Science and Technology · Patent Organism Depositary under the accession number FERM BP-7973.

 [Example 6] Synthesis of fluorescent hexeneduronic acid derivative as a substrate for hexeneduronidase

 1. Synthesis of (tree 0-acetyl-α-galactopyranosyl bromide) peronate methyl ester (I)

 [Formula 3]

(Tetra-acetyl-galactopyranoside) 3.76 g (10 ol) of peronic acid methyl ester are dissolved in a 30% bromic acid / acetic acid solution (25 mg) at 0 ° C under nitrogen and stirred for 1 hour. After stirring, the mixture was further stirred at room temperature for 1 hour. After completion of the reaction, dichloromethane (100 ml) was added, and the mixture was extracted five times with ice water and cold sodium bicarbonate water. The organic layer was separated, dried over anhydrous sodium sulfate, and the solvent was distilled off to obtain 4. Og (100% yield) of the title compound (I).

2. Synthesis of (Umbelliferyl Tree O-Acetyl-galactopyranoside) peronic acid methyl ester (II)

 [Formula 4]

(Tree-acetyl-galactopyranosyl bromide) peronic acid methyl ester

3.0 g (7.5 mmol), umbelliferone 1.32 g (7.5 bandol ol), 4A molecular sheep 10 g, and anhydrous acetonitrile 20 ml 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 solution of sodium bicarbonate, and 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 the fraction of dichloromethane-methanol (97: 3).

3. Synthesis of (Umbelliferi L-O-Acetyl-4-Doxy-1-hexyl-4-enopyranoside) Peronic Acid Methyl Ester (I I I)

 [Formula 5]

(Umbelliferyl tri-acetyl-galactovyranoside) A solution of methyluronic acid ester 200 mg (0.4 ol) in 5 ml of anhydrous THF under nitrogen at room temperature under DBU (1, 8-diazabicyclo [ 5.4.0] undec-7-ene) was added to 200 1 (4 equivalents) and stirred for 30 minutes. After completion of the reaction, the reaction solution was diluted with ethyl acetate, washed with distilled water, and dried over anhydrous sodium sulfate. After evaporating the solvent, 153 mg of the crude product was purified by silica gel chromatography to obtain 132 mg (76% yield) of the title compound (III) from the dichloromethane fraction.

 4. Emberbelliferyl 4-deoxy-1-hexyl 4-Synthesis of enoviranoside uronic acid (IV)

(6) (Umbelliferi Rouge O-Acetyl- 4-Doxy-Hex-41-enoviranoside) ゥ Methyl ester 86 mg (0.2 imnol) of water-methanol (1: 2) 9 ml of a solution of 2NNaOHlml In addition, the mixture was stirred for 1 hour. After completion of the reaction, the reaction solution was filtered with a cation exchange resin and eluted with methanol. After the eluate was concentrated, it was eluted with 30% methanol using 0DS reverse phase chromatography to obtain 43 mg (yield: 64%) of the title compound (IV).

Since the title compound is very unstable and decomposes rapidly, the title compound is used without purification in measuring the activity of hexeneduronidase. Hydroxyl groups present in the portion of the Kisenuron acid to case to store the substrate by performing an ice bath the reaction with IN of NaOH in advance substrate reaction system during _c activity measurement to store while Asechiru of de Asechiru Then, it is diluted with a buffer to a predetermined concentration and used. Example 7 Bleaching of pulp using hexeneduronidase of the present invention

 (1) Pretreatment (alkali oxygen bleaching)

 Whiteness 50.0%, Kappa monovalent U.2, Pulp viscosity 19.7mPa-s Beech-based broadleaf Trees After delignification of oxygen, Kraft pulp (hereinafter referred to as L0KP) is 60 g in absolute dry weight, ion exchange The pulp concentration was adjusted to 10.0% by dilution with water.

 (2) Pulp treatment with hexeneduronidase

 A phosphate buffer was added to the pulp slurry to a concentration of 50 mM in the reaction system, the pH was adjusted to 7.0, and 0.5 g of the hexeneduronidase obtained in Example 2 was added to 1 g of L0KP absolute dry weight. The mixture was added at a ratio of U and kept at 45 ° C for 2 hours.

 (3) Bleaching chlorine dioxide

 The pulp slurry after the above-mentioned hexeneduronidase treatment was added with 0.8% of chlorine dioxide to pulp adjusted to a pulp concentration of 10% and PH8.1, and kept at a pulp concentration of 70 ° C for 2 hours. H was 5.4 at the end of the chlorine dioxide treatment. After the bleached pulp was washed with distilled water, NaOH was added to a pH of 8.0, and after the extraction operation, the obtained pulp was washed with ion-exchanged water to obtain a finished bleached pulp.

 (Comparative Example 1)

Comparative Example 1 was the same as Example 7 except that the hexene peronidase treatment was not performed. And

 (Comparative Example 2)

 Comparative Example 2 is the same as Example 7 except that the hexeneduronidase 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. [Table 2] As can be seen from the table, the use of the hexeneduronidase enzyme of the present invention can improve pulp whiteness without increasing the amount of chlorine-based chemicals. Industrial applicability

 By using the hexeneduronidase enzyme according to the present invention in the pulp bleaching process, it is possible to produce pulp with improved whiteness without increasing the amount of oxidizing chemicals such as chlorine dioxide used conventionally. It is very useful in pulp bleaching. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

The scope of the claims

1. A hexene peroxidase having hexeneduronidase activity, a molecular weight of about 40,000 to about 45,000, and an enzyme activity of which is enhanced by 2-mercaptoethanol or dithiothreitol. Ronidase.

2. The hexeneduronidase according to claim 1, wherein the optimal pH is pH 6.0 to 8.0.

3. The hexenduronidase according to claim 1, wherein the stable pH range in the enzymatic reaction is pH 5.0 to 0.

4. The hexeneduronidase according to claim 1, wherein the optimum temperature is 35 ° C to 55 ° C.

5. The hexeneduronidase according to claim 1, which retains about 80% or more of the activity by a heat treatment at 45 ° C for 30 minutes and shows about 30% of a residual activity by a heat treatment at 60 ° C for 30 minutes. .

6. The hexeneduronidase according to any one of claims 1 to 5, which is derived from a microorganism belonging to the genus Paenibacillus.

7. The hexene-tinidase according to claim 6, wherein the microorganism is Paenibacillus sp. 7-5.

8. The hexeneduronidase according to any one of claims 1 to 5, which is a genetically modified enzyme.

9. It consists of the amino acid sequence of SEQ ID NO: 1, or has 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, and The hexeneduronidase according to any one of claims 1 to 5, which has lonidase activity.

10. The hexeneduronidase according to claim 9, which comprises an amino acid sequence having a homology of 70% or more, preferably 80% or more, more preferably 90% or more with the amino acid sequence shown in SEQ ID NO: 1.

11. Culturing a microorganism belonging to the genus Paenibacillus which produces the hexeneduronidase according to any one of claims 1 to 10 in a medium, and collecting hexeneduronidase from the resulting culture. A method for producing hexeneduronidase, comprising:

12. Paenibacillus sp. 7-5 capable of producing hexene peronidase (Accession No. FERM P-18225).

13. A hexeneduronidase gene encoding hexeneduronidase according to any one of claims 1 to 10.

14. The hexeneduronidase gene according to claim 13, which comprises the nucleotide sequence represented by SEQ ID NO: 2.

15. An expression vector comprising the hexeneduronidase gene according to claim 13 or 14.

16. A host cell transformed by the expression vector of claim 15.

17. A method for producing a genetically modified hexoperonidase, comprising culturing the host cell according to claim 16 in a medium and recovering hexendronidase from the obtained culture.

18. The hexene peronidase according to any one of claims 1 to 10 as an active ingredient Including bleach.

19. The bleach according to claim 18, wherein the hexeneduronidase is obtained by the method according to claim 17.

20. A method for bleaching pulp, comprising treating pulp with the bleaching agent according to claim 18 or 19.

21. The method of claim 20, wherein treating the pulp with a bleaching agent comprises performing chemical bleaching and / or alkali extraction either before, after, or during the pulp treatment.

22. An antibody that recognizes hexeneduronidase according to any one of claims 1 to 10.