WO2002092809A1 - Hexenuronidase, gene codant cette enzyme, et utilisation de ceux-ci - Google Patents

Hexenuronidase, gene codant cette enzyme, et utilisation de ceux-ci Download PDF

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Publication number
WO2002092809A1
WO2002092809A1 PCT/JP2002/004390 JP0204390W WO02092809A1 WO 2002092809 A1 WO2002092809 A1 WO 2002092809A1 JP 0204390 W JP0204390 W JP 0204390W WO 02092809 A1 WO02092809 A1 WO 02092809A1
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Prior art keywords
hexeneduronidase
enzyme
pulp
activity
acid
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PCT/JP2002/004390
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English (en)
Japanese (ja)
Inventor
Yoshiya Izumi
Makoto Sakaino
Akira Tsukamoto
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Oji Paper Co., Ltd.
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Publication of WO2002092809A1 publication Critical patent/WO2002092809A1/fr

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    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C9/00—After-treatment of cellulose pulp, e.g. of wood pulp, or cotton linters ; Treatment of dilute or dewatered pulp or process improvement taking place after obtaining the raw cellulosic material and not provided for elsewhere
    • D21C9/10—Bleaching ; Apparatus therefor
    • D21C9/1063—Bleaching ; Apparatus therefor with compounds not otherwise provided for, e.g. activated gases

Definitions

  • 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.
  • hemicelluloses 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.
  • hemicellulose 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.
  • 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, ⁇ 1 ⁇ 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.
  • the xylan which is a polysaccharide
  • it also re-adsorbs while disturbing coloring substances such as lignin and furan derivatives which are to be bleached in a later step.
  • 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.
  • xylanase EC 3.2.1.8
  • Some factories have introduced a process to decompose the re-adsorbed xylan and remove colored substances.
  • xylan has an arabinose side chain with ⁇ 1 ⁇ 3 bond and a 4 ⁇ 0-methyldalcuronic acid with 1 ⁇ 2 bond as side chains in addition to the main chain.
  • 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.
  • 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.
  • 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.
  • the heavy metal compound 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.
  • a common method for removing hexeneduronic acid is to treat the pulp with an acid (WO096 / 12063 pamphlet).
  • an acid WO096 / 12063 pamphlet
  • 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.
  • 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.
  • Hexenduronidase wherein the enzyme activity is enhanced by 2-mercaptoethanol or dithiothreitol.
  • the microorganism is Paenibacillus sp. 7_5, (6) Hexeneperoni
  • (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.
  • Paenibacillus sp. 7-5 capable of producing hexeneduronidase (Accession No. FERM P-18225).
  • (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.
  • a bleach containing the hexeneduronidase of any one of (1) to (10) as an active ingredient is provided.
  • a method for bleaching pulp comprising treating pulp with the bleaching agent according to (18) or (19).
  • FIG. 1 is a diagram showing the optimal ⁇ of hexene peronidase of the present invention.
  • FIG. 2 is a diagram showing the stable ⁇ 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.
  • Hexenduronidase activity 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.
  • 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,
  • 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.
  • 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.
  • the enzyme of the present invention can have at least one or all of the following properties.
  • Isoelectric point It is around pH 4.5.
  • 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.
  • 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.
  • microbiological properties of Paenibacillus sp. 7-5 are as follows.
  • 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.
  • 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.
  • the enzyme of the present invention 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. .
  • the microorganism is Paenibacillus sp. 7-5 capable of producing hexeneduronidase
  • 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.
  • several means usually 2 to 9, preferably 2 to 7, and more preferably 2 to 5.
  • 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%.
  • any source capable of producing hexeneduronidase can be used.
  • 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.
  • nitrogen compounds such as yeast extract, peptone, various amino acids, soybean, corn steep liquor, and various inorganic nitrogen can be used.
  • 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.
  • 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.
  • 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.
  • the mutant has the above-mentioned meaning.
  • 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.
  • 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.
  • 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.
  • Tm normal melting temperature
  • ionic strength eg, 0.1 to 1 x SSC
  • 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.
  • 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).
  • PCR polymerase chain reaction
  • 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.
  • 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.>
  • examples include cytomegalovirus immediate early gene promoter, SV40 early promoter, retrovirus promoter, heat shock promoter, etc.
  • Hosts include bacteria such as E.
  • 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.
  • 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.
  • 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.
  • 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.
  • a bleach containing hexeneduronidase as an active ingredient is disclosed.
  • the present invention also provides a pulp bleaching method, comprising treating pulp with the bleaching agent.
  • a pulp bleaching method comprising treating pulp with the bleaching agent.
  • chemical bleaching and / or alkali extraction can be performed either before, after or during the pulp treatment.
  • the definition of enzyme activity was defined as the decrease in the absorption per unit time ( ⁇ 232) of the double bond in the hexeneduronic acid molecule due to the degradation of hexeneduronic acid.
  • 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.
  • 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.
  • 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.
  • 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 232 ⁇ 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 ⁇ .
  • 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.
  • 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.
  • Z represents 0 (oxygen) or S (sulfur)
  • 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.
  • fluorophore groups include, but are not limited to, substituted or unsubstituted cumaryl, fluoresynyl, naphthyl (eg, i3-naphthyl) and the like.
  • substitution group examples 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.
  • 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.
  • 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.
  • a chromophore group is p-nitrophenoxy group.
  • p-nitrophenoxy group yellow-yellow to yellow because p-ditrophenol dissociates as a result of the action of hexene ⁇ -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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the protecting group of the protected hexeneduronic acid moiety of the glycosylated compound is deprotected and converted to the hexeneduronic acid moiety.
  • 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.
  • 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.
  • a fluorescent, chemiluminescent or chromogenic substance and the intensity of that signal is measured directly or indirectly by physicochemical methods
  • 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.
  • 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.
  • 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.
  • an alkaline buffer such as glycine-NaOH buffer (pH about 10)
  • 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.
  • 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.
  • 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.
  • 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).
  • a buffer pH 9.0 to 10.5
  • 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).
  • the enzyme was stable in the range of pH 5.0 to 10.0.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • DM Library One is Paenibacill It can be prepared by extracting chromosome ⁇ 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).
  • a gene fragment of an appropriate size is inserted into a cloning vector having a similar cut surface.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the hexeneduronidase of the present invention can also be purified by expressing a cloned gene.
  • an enzyme obtained by expressing a gene is referred to as “recombinant hexeneduronidase”.
  • high production can be achieved by expressing the hexeneduronidase gene obtained by an appropriate method using an appropriate host vector.
  • 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.
  • any source that can assimilate and produce thermostable xylanase can be used.
  • the carbon source agricultural waste such as wheat bran, pulp, bacas, corn fiber, rice straw, or plant fiber can be used.
  • 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.
  • 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.
  • 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.
  • pulp is bleached by chemical bleaching and Z or alkali extraction before, during or after the enzyme treatment.
  • 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).
  • 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).
  • 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).
  • 100 Hi of glycine-NaOH buffer ( ⁇ 5: 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.
  • the hexene peronidase activity in the crude enzyme solution was 0.05 U / ml.
  • steam sterilization was performed at 121 ° C for 15 minutes.
  • 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 20 ⁇ phosphate buffer containing 0.9M ammonium sulfate ( ⁇ 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).
  • 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.
  • 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.
  • 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).
  • the concentrated enzyme sample was desalted and subjected to polyacrylamide gel 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.
  • 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.
  • the target enzyme 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.
  • 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.
  • hexeneduronidase was purified by the method of Example 2.
  • 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.
  • HEX4 SEQ ID NO: 4
  • HEX5 SEQ ID NO: 5
  • HEX6 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.
  • a primer for PCR was designed. Chemical synthesis is performed based on the designed primer,
  • R is G or A
  • I is inosine
  • H is A or C or T
  • ⁇ is ⁇ or C
  • N is A or C or G or T, respectively.
  • 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.
  • DNA-labeled kit (Gene Images TM, 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.
  • washing buffer 40 mM church phosphate buffer containing 1% SDS
  • the washing buffer was discarded, and the same method was repeated four times.
  • detection was performed using a labeled nucleic acid detection kit (Gene Images TM).
  • 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).
  • 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.
  • a positive clone having a hexeneduronidase gene was selected.
  • the PCR fragment used in Southern hybridization was fluorescently labeled and used.
  • two positive clones were obtained in about 800 libraries. These are called B15 and B26.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 was the same as Example 7 except that the hexene peronidase treatment was not performed.
  • 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

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Abstract

L'invention concerne une hexenuronidase caractérisée en ce qu'elle possède une activité hexenuronidase et un poids moléculaire compris entre 40 000 et 45 000, l'activité enzymatique étant améliorée par 2- mercaptoéthanol ou dithiothreitol. L'invention concerne également un procédé de production de l'hexenuronidase, l'utilisation de cette enzyme, un gène hexenuroidase, et un micro-organisme qui produit l'hexenuroidase. Cette protéine enzymatique est utile dans le blanchiment de pâte à papier.
PCT/JP2002/004390 2001-05-11 2002-05-02 Hexenuronidase, gene codant cette enzyme, et utilisation de ceux-ci WO2002092809A1 (fr)

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JP2006219767A (ja) * 2005-02-08 2006-08-24 Univ Of Tsukuba 製紙用化学パルプ中の不飽和ウロン酸の除去方法
JP4750068B2 (ja) * 2007-04-16 2011-08-17 王子製紙株式会社 漂白パルプ中のヘキセンウロン酸含量の測定方法、漂白パルプの製造方法、および紙の製造方法
JP5733711B2 (ja) * 2008-09-17 2015-06-10 国立大学法人 筑波大学 ヘキセンウロン酸特異的遊離酵素およびこの酵素を産生する微生物
CN104926898B (zh) * 2015-05-14 2018-09-28 广东省微生物研究所 一种基于4-甲基伞形酮的合成多种糖苷的方法
CN106946959B (zh) * 2017-03-29 2020-05-05 昆药集团股份有限公司 合成5,6,4＇-三羟基黄酮-7-0-d-葡萄糖醛酸有关物质及其制备方法和应用

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CN109810157B (zh) * 2018-12-03 2021-05-25 广东省微生物研究所（广东省微生物分析检测中心） 一种β-葡萄糖醛酸苷酶沉淀型荧光底物合成方法

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