WO2016056662A1 - Réducteur de viscosité pour substance contenant du mannane et utilisation de ce dernier - Google Patents

Réducteur de viscosité pour substance contenant du mannane et utilisation de ce dernier Download PDF

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WO2016056662A1
WO2016056662A1 PCT/JP2015/078844 JP2015078844W WO2016056662A1 WO 2016056662 A1 WO2016056662 A1 WO 2016056662A1 JP 2015078844 W JP2015078844 W JP 2015078844W WO 2016056662 A1 WO2016056662 A1 WO 2016056662A1
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amino acid
polypeptide
acid sequence
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dna
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元亨 志水
雅士 加藤
哲夫 小林
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学校法人名城大学
国立大学法人名古屋大学
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    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

  • This specification relates to a viscosity reducing agent for mannan-containing materials and use thereof.
  • Mannan is a general term for polysaccharides mainly composed of mannose, and is widely distributed in nature.
  • Mannan includes glucomannan having mannose and glucose contained in the cell walls of conifers and konjac as the main chain, and galactomannan containing mannose and galactose contained in coffee beans and fruits as the main chain. Since these exist in the form of a gel, they are used as food thickeners and stabilizers.
  • mannanase mannan degrading enzyme
  • mannanases Molds and mushrooms secrete mannanase outside the fungus body, lower the molecular weight of mannan, and then take it into the fungus body for use.
  • a wide variety of mannanases has been found so far and they are used in the production of food.
  • Mannan is considered as one of the unused biomass.
  • Cellulase is mainly used for degradation of polysaccharide cellulose mainly composed of glucose, which is currently attracting attention as a biomass, and substances that enhance the degradation have already been reported (Patent Document 1).
  • Patent Document 2 Patent Document 2.
  • Mannan is contained in many plants, but its high viscosity is an obstacle to industrial use. Therefore, the possibility of technology transfer is expected in the manufacture of foods containing mannan and the use of biomass by enabling efficient mannan decomposition.
  • a decomposition enhancing substance such as cellulose has not been found yet.
  • methods other than lowering molecular weight have not yet been discovered for improving mannan utilization.
  • This specification is intended to provide a mannan-containing material viscosity reducing agent capable of improving the availability of mannan, and to provide the utilization thereof.
  • the inventors of the present invention have found a protein derived from A. nidulans that causes a decrease in the viscosity of mannan without hydrolyzing the main chain of mannan in a study on the decrease in the viscosity of mannan. It was also clarified that when this protein acts on mannan, the viscosity of mannan hydrate is reduced, and when it is used together with mannanase, the decomposition efficiency of mannan is increased. Based on such knowledge, the disclosure of the present specification provides the following means.
  • the upper shows the case where glucomannan is added after incubating mannanase and HP for 30 minutes, and the lower shows the case where mannanase is added after incubating glucomannan and HP for 30 minutes. It is the figure which showed the viscosity of the glucomannan at the time of adding rHP. It is the figure which showed the viscosity when adding rHP to glucomannan and adding rHP to glucomannan for 12 hours. It is a figure which shows the weight change of foodstuffs at the time of adding rHP and mannanase to foodstuffs. It is a figure which shows the alignment result with the protein considered to be an ortholog of this polypeptide.
  • the disclosure of this specification relates to the use of a novel mannan-containing material as a viscosity reducing agent and a processing agent for mannan-containing foods.
  • Mannan is a water-soluble dietary fiber that becomes viscous by becoming a hydrate.
  • the degree of viscosity varies depending on the type of mannan contained, the amount of water, etc., but since it often exists in the form of a gel, it is widely used as a thickener and stabilizer because of its high viscosity. .
  • the high viscosity often becomes an obstacle to the use for food and the industrial use.
  • a method for reducing the viscosity of mannan at present, lowering of mannan using mannanase has become the mainstream.
  • the viscosity of the hydrate can be lowered by allowing the polypeptide to act on mannan. Further, by using such an action of the present polypeptide, mannanase can be used together with the present polypeptide to improve the efficiency of mannan degradation.
  • Mannan is a general term for polysaccharides mainly composed of mannose. Mannan is a kind of hemicellulose that is generally present in yeast, mold, plant seeds and fruits, and woody parts of conifers.
  • glucomannan is a ⁇ -1,4 bond between glucose and mannose.
  • Glucomannan is a large amount of glucomannan in which glucose and mannose are combined at a ratio of about 2: 3 is contained in conifers and konjac potatoes.
  • Galactose is ⁇ -1,6 linked to the side chain of glucomannan, and the one with a high ratio is called galactoglucomannan.
  • galactomannan is ⁇ -1,4-linked mannose and ⁇ -1,6-linked galactose as a side chain, and the ratio of galactose varies depending on the plant containing galactomannan. High in guar gum and coffee beans.
  • the mannan preferably contains an acetyl group, and the distribution of the acetyl group may be regular or irregular. Further, the degree of acetyl group contained in mannan is not limited. For example, glucomannan can contain an acetyl group every 9 to 19 sugar units on average.
  • Xylan refers to a polysaccharide in which D-xylose is linked by ⁇ -1,4. Naturally, it exists as a heterosaccharide in which various side chains are bonded to the main chain of xylose. It is known as a kind of hemicellulose present in plant cell walls.
  • Xylan can be collected from, for example, birch. Xylan can be broken down into xylose, reduced to xylitol, and used as a food additive.
  • xylan include arabinoxylan and glucuronoxylan. In arabinoxylan, ⁇ -1,3 linked L-arabinose and ⁇ -1,2 linked 4-O-methylglucuronic acid are bonded as side chains to the ⁇ -1,4 linked xylose main chain. The ratio is about 10: 1: 2.
  • Arabinoxylan is abundant in corn cobs and grasses such as wheat and oat straw and conifers. Coniferous hemicellulose is the second most common after glucomannan, has a content of about 10%, and does not have an acetyl group.
  • ⁇ -1,2-linked 4-O-methylglucuronic acid is bonded to the main chain of ⁇ -1,4-linked xylose as a side chain, and the ratio is about 5: 1. . It is abundant in broad-leaved trees, and on average, has an acetyl group at the C2-position and C3-position at a ratio of 5-7 to 10 xylose.
  • the polypeptide disclosed in the present specification may be a polypeptide having the amino acid sequence represented by SEQ ID NO: 2.
  • Another aspect of the amino acid sequence that can be possessed by such a polypeptide can have one or more amino acid mutations in the amino acid sequence represented by SEQ ID NO: 2.
  • the number of amino acid mutations is not particularly limited. For example, 1 to 50, preferably 1 to 40, more preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 10, and still more preferably. Means 1 to 5, particularly preferably about 1 to 2.
  • the amino acid mutation may be any of substitution, deletion, and addition, and may include two or more mutation modes at the same time. As an example of amino acid substitution, conservative substitution is preferable, and specific examples include substitution within the following groups.
  • the polypeptide has an amino acid sequence having an identity of 70% or more with respect to the amino acid sequence represented by SEQ ID NO: 2, and exhibits the activity of reducing the stickiness of mannan hydrate.
  • the amino acid sequence represented by SEQ ID NO: 2 is preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, and still more preferably 95% or more. Even more preferably 98% or more, most preferably 99% or more identity is preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, More preferably, it is 95% or more. More preferably, it is 98% or more, and most preferably 99% or more.
  • identity or similarity is a relationship between two or more proteins or two or more polynucleotides determined by comparing sequences, as is known in the art.
  • identity means between protein or polynucleotide sequences as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of such sequences. Means the degree of sequence invariance. Similarity also refers to the degree of correlation between alignments of protein or polynucleotide sequences or, in some cases, between a series of partial sequences. More specifically, it is determined by sequence identity and retention (substitutions that maintain specific amino acids in the sequence or physicochemical properties in the sequence).
  • the similarity is referred to as “Similarity” in the BLAST homology search result described later.
  • the method for determining identity and similarity is preferably a method designed to align the longest between the sequences to be compared. Methods for determining identity and similarity are provided as programs available to the public. For example, BLAST (Basic Alignment Search Tool) program by Altschul et al. (Eg Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990), Altschul SF, Madden TL, Schaffer AA, Zhang Z, Miller W, Lipman DJ., Nucleic Acid Res. 25: p3398-3402 (1997)).
  • the conditions for using software such as BLAST are not particularly limited, but it is preferable to use default values.
  • the polypeptide is stringent with DNA comprising the base sequence encoding the amino acid sequence represented by SEQ ID NO: 2 (or a part thereof) or DNA comprising the base sequence complementary to the DNA.
  • examples thereof include a polypeptide having an amino acid sequence encoded by DNA that hybridizes under conditions and having a viscosity reducing activity of a mannan-containing material.
  • Examples of the base sequence encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 include the base sequence represented by SEQ ID NO: 1.
  • the viscosity-reducing activity of the mannan-containing material is observed for 100 minutes by adding the polypeptide to an aqueous solution of mannan (1% to 2%), as will be apparent from the examples described later. Can be evaluated.
  • stringent conditions refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed.
  • Such stringent conditions are known to those skilled in the art, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) and Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987 ) To set.
  • a nucleic acid having high nucleotide sequence identity that is, 70% or more, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, more preferably, the nucleotide sequence represented by SEQ ID NO: 1. 95% or more.
  • the sodium salt concentration is 15 to 750 mM, preferably 50 to 750 mM, more preferably 300 to 750 mM
  • the temperature is 25 to 70 ° C., preferably 50 to 70 ° C., more preferably 55 to 65 ° C.
  • formamide The condition is that the concentration is 0 to 50%, preferably 20 to 50%, more preferably 35 to 45%.
  • the washing conditions of the filter after hybridization are usually sodium salt concentrations of 15 to 600 mM, preferably 50 to 600 mM, more preferably 300 to 600 mM, and the temperature is 50 to 70 ° C., preferably 55. -70 ° C, more preferably 60-65 ° C.
  • hybridization solution 50% formamide, 10 ⁇ SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 5 ⁇ Denhardt solution, 1% SDS, 10% dextran sulfate, 10 ⁇ g / ml denaturation
  • 5 ⁇ Denhardt solution 1% SDS
  • 10% dextran sulfate 10 ⁇ g / ml denaturation
  • incubation at about 42 ° C to about 50 ° C using salmon sperm DNA, 50 mM phosphate buffer (pH 7.5), followed by washing at about 65 ° C to about 70 ° C using 0.1 x SSC, 0.1% SDS can be mentioned.
  • Further preferable stringent conditions include, for example, 50% formamide, 5 ⁇ SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 1 ⁇ Denhardt solution, 1% SDS, 10% dextran sulfate, 10 ⁇ g / ml as a hybridization solution.
  • such a polypeptide it is 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 93% or more, still more preferably 94, with the nucleotide sequence represented by SEQ ID NO: 1. % Or more, more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, still more preferably 98% or more, and still more preferably 99% or more.
  • examples thereof include polypeptides encoded by DNA and having the viscosity reducing activity of mannan-containing materials.
  • Examples of the base sequence encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 include the base sequence represented by SEQ ID NO: 1.
  • LAIXMLE (X is an arbitrary amino acid, X) at positions 55 to 61 of the amino acid sequence represented by SEQ ID NO: 2 of the polypeptide. Preferably, it is alanine or valine, more preferably alanine. (Hereinafter referred to as the first motif), and WFXGHNG at positions 138 to 145 (X is an arbitrary amino acid, preferably Alanine or serine, more preferably alanine) (hereinafter, secondly referred to as a motif).
  • this polypeptide has a GLGXRK (X is any amino acid, preferably glutamine, alanine, serine, threonine, lysine, more preferably at positions 37 to 42 of the amino acid sequence represented by SEQ ID NO: 2. (Hereinafter referred to as the third motif), and PX 1 TX 2 DI (X 1 and X 2 are each independently any amino acid) at positions 152 to 157 X 1 is preferably asparagine, glycine, leucine or tyrosine, more preferably asparagine, and X 2 is preferably aspartic acid, glutamine or glutamic acid, more preferably aspartic acid. has called.
  • a) fourth motif RX 1 WVX 2 V (X 1 and X 2 in the # 183 ⁇ # 188, respectively Any amino acid in the standing, X 1 is preferably, phenylalanine, tryptophan, and more preferably phenylalanine, X 2 is preferably aspartic acid, asparagine, tyrosine, and more preferably is aspartic acid. (Hereinafter referred to as the fifth motif).
  • amino acid sequence represented by SEQ ID NO: 2 31G, 34T, 42K, 49G, 67T, 83G, 122D, 126R, 134G, 148G, 170Y, 176W, 180G and 191I are conserved.
  • still another embodiment of the present polypeptide includes a polypeptide having a first motif and a second motif when aligned with the amino acid sequence of the polypeptide. Furthermore, in addition to the first and second motifs, a polypeptide having one or more motifs of the third to fifth motifs can be mentioned, preferably two, more preferably all three of these motifs. A polypeptide. Furthermore, the aspect by which 14 types of amino acids mentioned above are maintained is mentioned.
  • Such a polypeptide of various aspects is not particularly limited as long as it has the viscosity reducing activity of the mannan-containing material.
  • the polypeptide may be purified by a separation means such as gel electrophoresis, or may be an unpurified or crudely purified product coexisting with other proteins.
  • a separation means such as gel electrophoresis
  • examples of the unpurified or crude purified polypeptide include a culture supernatant of a transformant that secretes and produces the polypeptide described later, or a crude purified product thereof.
  • the method for producing the present polypeptide is not particularly limited. The production method of this polypeptide will be described in detail later.
  • the polynucleotide disclosed herein (hereinafter referred to as the present polynucleotide) encodes the polypeptide.
  • the present polynucleotide include polynucleotides encoding the present polypeptide and having the viscosity reducing activity of a mannan-containing material.
  • This polynucleotide includes a plurality of forms of base sequences generated by degeneracy of the genetic code, corresponding to one amino acid.
  • the polynucleotide may be DNA (single and double stranded), RNA (single stranded), DNA / RNA hybrid (DNA single stranded and RNA single stranded hybrid), DNA and RNA chimera. Further, the present polynucleotide may have only a coding sequence encoding the present polypeptide, such as cDNA, or the like, as long as it is translated into the corresponding present polypeptide in a predetermined host, such as a genome. Two or more introns may be included.
  • This polynucleotide is, for example, DNA extracted from A. nidulans which is a natural source of this polypeptide using primers designed based on the base sequence encoding this polynucleotide, and cDNA libraries of various other organisms.
  • the polynucleotide can be obtained as a fragment by performing PCR amplification using a polynucleotide derived from a genomic DNA library or the like as a template.
  • a polynucleotide fragment can be obtained by performing hybridization using a polynucleotide derived from the above library or the like as a template and a DNA fragment that is a part of the DNA encoding the polypeptide as a probe.
  • the polynucleotide may be synthesized as a DNA fragment or the like by various nucleic acid sequence synthesis methods known in the art such as chemical synthesis methods.
  • the present polypeptide such as DNA encoding a polypeptide of a mode in which a mutation has been introduced into the amino acid sequence represented by SEQ ID NO: 2 is obtained by a known method for introducing a mutation in an amino acid sequence. The mutagenesis method will be described later.
  • those skilled in the art can obtain the present polynucleotides of various aspects based on the nucleotide sequences disclosed for the present polypeptides by referring to the previously described Molecular® Cloning, Current® protocols, in®, Molecular® Biology, etc. it can.
  • the polynucleotide construct disclosed herein comprises the present polynucleotide, and preferably further comprises one or more elements for expressing the polypeptide encoded by the polynucleotide in a host cell. it can. Such elements are appropriately selected based on known techniques, and examples thereof include promoters, terminators, poly A sequences, signal peptide sequences, homologous sequences for genome introduction by homologous recombination with host genomes, and the like.
  • the polynucleotide construct can also include a marker for selecting transformed host cells.
  • Polynucleotide constructs can be circular or linear DNA molecules and can typically take the form of expression vectors.
  • polypeptide of the present embodiment in which a point mutation or the like was introduced into the amino acid sequence represented by SEQ ID NO: 2 used a conventional mutagenesis method, site-directed mutagenesis method, and error-prone PCR. It can be obtained by modifying by a molecular evolution method or the like.
  • a known technique such as the Kunkel method or the Gapped duplex method or a similar method can be mentioned.
  • a mutation introduction kit using site-directed mutagenesis for example, Mutant-K (Takara Bio) Etc.) or Mutant-G (manufactured by TAKARA)
  • the LA PCR in vitro Mutagenesis series kit from TAKARA.
  • the host of the transformant is not particularly limited, and various prokaryotic microorganisms and eukaryotic microorganisms can be used.
  • Prokaryotic microorganisms and eukaryotic microorganisms are not particularly limited in kind, but it is preferable to use microorganisms with established genetic recombination technology, and yeast and E. coli are particularly preferable.
  • the method for producing a degradation product of mannan disclosed in the present specification can comprise a step of degrading mannan using the present polypeptide and mannanase.
  • the degradation efficiency of mannan by mannanase can be improved.
  • mannan degradation products can be obtained efficiently.
  • mannan to be decomposed in this method is not particularly limited. It may be lignocellulosic or other biomass material (non-edible material) that may contain mannan. Moreover, the hemicellulose material isolate
  • separated from these biomass materials may be sufficient. Furthermore, it may be mannan that has been purified to some extent or purified. Moreover, the edible material containing a mannan may be sufficient. Examples of such edible materials include various fruits, coffee beans, strawberries, and various processed foods such as various konjacs and jelly.
  • mannanase is an endo-type enzyme that hydrolyzes the ⁇ -1,4 bond of mannan and is also called ⁇ -mannanase or ⁇ -mannosidase.
  • EC enzyme classification can be classified into EC 3.2.1.25, and CAZy can be classified into GH1, GH2, and GH5.
  • GH represents Glycoside Hydrolase Family.
  • Mannanase may be naturally derived or artificially modified.
  • mannanases derived from Aspergillus genus including Aspergillus niger and filamentous fungi such as Trichoderma seisei, Bacillus genus bacteria and the like are known.
  • mannanase can be used alone or in combination of two or more.
  • mannanases having different substrate specificities can be used in combination as long as they act as mannanases.
  • the form of contact between the present polypeptide, mannanase and mannan-containing material is not particularly limited.
  • the present polypeptide and mannan may be contacted in advance, and then the mannanase and mannan may be contacted, or the mannanase and mannan may be contacted and then the present polypeptide and mannan may be contacted. .
  • a method for producing a biomass raw material for use in a method for producing various organic compounds derived from biomass can be provided by carrying out the decomposition step of this method. it can.
  • the decomposition efficiency of hemicellulose by various enzymes can be improved.
  • Cellulase decomposition process using biomass raw materials with loose hemicellulose or biomass raw materials containing cellulose separated from hemicellulose improves the accessibility of cellulase to cellulose and efficiently obtains constituent monosaccharides such as glucose It is also possible to provide a method for producing a degradation product of biomass material.
  • the method for producing a mannan viscosity-decreasing product disclosed in the present specification can include a step of reducing the viscosity of mannan hydrate using the polypeptide. By using this polypeptide, the viscosity of mannan hydrate can be effectively reduced without decomposing mannan.
  • mannanase may be used together with the polypeptide.
  • mannanase decomposition efficiency of mannanase can be enhanced, and the use of mannanase can be reduced.
  • the various embodiments of the polypeptide and mannanase disclosed in the present specification can be applied, as in the production method of mannan degradation products.
  • the various embodiments described above can be applied to the manner of contact with mannan.
  • mannan of various aspects can be targeted for viscosity reduction.
  • a pretreatment method for producing various organic compounds derived from biomass by performing the viscosity reduction step of this method can be provided.
  • the decomposition efficiency of hemicellulose by various enzymes can be improved.
  • hemicellulose can be separated and removed from cellulose.
  • the efficiency reduction can be achieved by performing the viscosity reduction process of the present method.
  • An effective extraction (processing) method can be provided.
  • it since it is not accompanied by decomposition of mannan, it can also be used as mannan.
  • the viscosity and elasticity of this type of food can be reduced by performing the viscosity reduction step of this method. It is possible to provide a method of producing a food that can be moderately reduced, improves the swallowability of the food, and has a new texture. In this case, foods having various characteristics such as texture and elasticity can be produced by appropriately selecting the action site of the polypeptide on the target food as the inside of the food or near the surface.
  • the production method of mannan degradation products and the production method of mannan viscosity-reduced product can also be carried out as a food processing method or a food production method including the steps included in these methods.
  • the food in these methods is not particularly limited, and any food that is effective in degrading mannan or reducing the viscosity of mannan using the polypeptide can be used.
  • Each protein was identified by performing a MASCOT search using the obtained data (peptide fingerprint and MS / MS spectrum). As in the case of endo-1,4- ⁇ -mannanase and mannosidase, in the table of FIG. No. As shown in FIG. 5, a protein with unknown function (HP) secreted in large quantities outside the cell was identified. The amino acid sequence of this protein was shown by SEQ ID NO: 2.
  • the number of spores was combined using a counting chamber in 100 ml of 1.0% glucomannan liquid minimal medium in a 500 ml Erlenmeyer flask (2,000 spores per ⁇ l). Spores of WT and ⁇ HP strains 500 ⁇ l of each suspension was added and cultured with shaking (100 rpm). Thereafter, the bacterial cells and the culture solution were separated using a Buchner funnel, the obtained bacterial cells were placed on a filter paper and dried at 50 ° C. for 2 days, and the weight of the bacterial cells was measured (FIG. 3). As a result, since the growth of the ⁇ HP strain was suppressed as compared with the WT strain, HP was considered to be involved in the degradation of mannan.
  • RNA was extracted from the WT strain using RNeasy Plant Mini Kit (QIAGEN), and cDNA was reverse transcribed using PrimeScript 1st cDNA Synthesis Kit (TaKaRa) to obtain cDNA.
  • PCR is performed using primer A (CCCAAGCTTcg GCCCCCACGACGGACATGACCA) (SEQ ID NO: 3) and primer B (CCG CTC GA G GAT AGC CTG GAC ATC AAC CCA AAA GCG) (SEQ ID NO: 4). The gene was amplified.
  • the sample was centrifuged and passed through a filter (0.22 ⁇ l) to remove insolubles. Thereafter, the sample was passed through a Ni-affinity column, and recombinant HP (rHP) was adsorbed onto the column, washed three times with buffer A, and eluted with buffer A containing 300 mM imidazole to purify rHP (FIG. 4). When used for measuring enzyme activity, it was dialyzed and desalted.
  • rHP recombinant HP
  • the culture filtrate and a final concentration of 0.5% glucomannan were reacted at 37 ° C. for 30 minutes, respectively, and ⁇ -mannanase activity (final concentration)
  • the amount of reducing sugar produced when 0.5% glucomannan was used as a substrate was measured by the DNS method.
  • RHP was added to the culture solution of the ⁇ HP strain so as to have the concentration shown in FIG. As a result, as shown in FIG. 5, rHP itself has no mannanase activity, but it was found that the mannanase activity increases as the concentration of rHP added to the ⁇ HP strain increases. This indicates that the ⁇ HP strain endogenous mannanase activity is promoted.
  • Viscosity reduction of glucomannan by HP Using a viscometer (SV-10), rHP was added to a 1% glucomannan solution under a constant temperature condition (37 ° C.), and the viscosity of the solution was measured every 10 seconds for 100 minutes. As shown in FIG. 8, it was found that when HP was added to the glucomannan solution, the viscosity of the glucomannan solution decreased with time.
  • HP was added to a 2% glucomannan solution and incubated at 37 ° C. for 12 hours.
  • a control to which no rHP was added was used.
  • the glucomannan solution before the addition of rHP has a high viscosity, and the glucomannan used in this example is in a state closer to a solid than a liquid and has a high viscosity that requires time to drop when lifted. It was.
  • the viscosity of the lycomannan solution 12 hours after addition of rHP decreased (FIG. 9) and was almost liquid, and dropped without taking time even when lifted.

Abstract

　L'invention concerne un réducteur de viscosité pour une substance contenant du mannane, moyennant quoi l'utilité du mannane peut être accrue. Le réducteur de viscosité pour une substance contenant du mannane comprend un polypeptide pourvu d'une séquence supplémentaire dans laquelle au moins deux histidines peuvent apparaître à la suite à l'extrémité C d'une séquence d'acides aminés choisie dans le groupe comprenant les polypeptides (a) à (f). (a) : Un polypeptide ayant une séquence d'acides aminés représentée par SEQ ID N° : 2. (b) : Un polypeptide ayant une séquence d'acides aminés identique à hauteur d'au moins 70 % à la séquence d'acides aminés représentée par SEQ ID N° : 2. (c) : Un polypeptide ayant une séquence d'acides aminés dans laquelle un ou plusieurs acides aminés sont remplacés, supprimés et/ou insérés dans la séquence d'acides aminés représentée par SEQ ID N° : 2. (d) : Un polypeptide codé par de l'ADN qui s'hybride dans des conditions de stringence avec de l'ADN comprenant la séquence de bases codant pour la séquence d'acides aminés représentée par SEQ ID N° : 2 ou la séquence de bases complémentaire de cette dernière. (e) : Un polypeptide codé par une séquence de bases identique à hauteur d'au moins 70 % à la séquence de bases représentée par SEQ ID N° : 1. (f) : Un polypeptide codé par de l'ADN qui s'hybride dans des conditions de stringence avec de l'ADN comprenant la séquence de bases codant pour la séquence d'acides aminés représentée par SEQ ID N° : 2 ou la séquence de bases complémentaire de cette dernière.
PCT/JP2015/078844 2014-10-10 2015-10-09 Réducteur de viscosité pour substance contenant du mannane et utilisation de ce dernier WO2016056662A1 (fr)

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US10647973B2 (en) * 2015-02-27 2020-05-12 Meijo University Educational Foundation Mannanase and use thereof

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