WO2013035678A1 - 変異型エンドグルカナーゼ - Google Patents
変異型エンドグルカナーゼ Download PDFInfo
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- WO2013035678A1 WO2013035678A1 PCT/JP2012/072401 JP2012072401W WO2013035678A1 WO 2013035678 A1 WO2013035678 A1 WO 2013035678A1 JP 2012072401 W JP2012072401 W JP 2012072401W WO 2013035678 A1 WO2013035678 A1 WO 2013035678A1
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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)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- 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
- C12P19/02—Monosaccharides
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- 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
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C—CHEMISTRY; METALLURGY
- C13—SUGAR INDUSTRY
- C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
- C13K1/00—Glucose; Glucose-containing syrups
- C13K1/02—Glucose; Glucose-containing syrups obtained by saccharification of cellulosic materials
Definitions
- the present invention relates to a novel mutant endoglucanase.
- Cellulose is abundant in herbaceous plants and woody plants, and these plants are collectively referred to as cellulosic biomass.
- Cell wall of cellulosic biomass is mainly composed of cellulose, hemicellulose, and lignin.
- Cellulose is a linear polysaccharide with ⁇ -1,4-linked glucose molecules
- hemicellulose is a polysaccharide such as xyloglucan, xylan, and mannan
- lignin is an aromatic polymer compound with a complex structure. It is intertwined with hemicellulose to form a three-dimensional network structure.
- saccharification In order to produce ethanol and chemical raw materials from cellulosic biomass, a process called “saccharification” is required in which microorganisms decompose into fermentable monosaccharides.
- Typical saccharification methods include acid treatment methods and enzyme treatment methods, but since acid treatment methods involve a large amount of waste liquid and have a high environmental impact, enzymes that perform reactions under mild conditions using cellulase are now available. Treatment methods have become the mainstream of development.
- Cellulase is a general term for cellulose hydrolase and is classified into three types, cellobiohydrolase, endoglucanase, and ⁇ -glucosidase, based on differences in substrate specificity. It is thought that hydrolysis of cellulose proceeds when these act in concert.
- Non-Patent Documents 1 to 3 the inhibition mechanism is still unclear.
- thermophilic bacterium or a hyperthermophilic bacterium has high stability and can maintain activity for a long period of time even under high temperature conditions. Therefore, application as an industrial enzyme is being studied. So far, cellulase produced by cellulolytic thermophilic bacteria or hyperthermophilic bacteria has been studied, and it has been clarified that most of the cellulase genes possessed by them encode endoglucanase.
- the activity of cellulases such as endoglucanase is inhibited by aromatic compounds derived from lignin.
- the present invention provides a mutant endoglucanase in which the activity inhibition by the lignin-derived aromatic compound is greatly reduced.
- this invention provides the manufacturing method using an enzyme composition with high decomposition
- the present inventors introduced an amino acid mutation at a specific position of a thermophilic bacterium-derived endoglucanase, and succeeded in obtaining a mutant endoglucanase having improved properties.
- the inventors focused on the three-dimensional structure of the wild-type parent endoglucanase and identified amino acids involved in the complex structure formation of the parent endoglucanase and lignin-derived aromatic compound by protein crystal structure analysis.
- the amino acid we succeeded in obtaining endoglucanase in which the activity inhibition by the lignin-derived aromatic compound was significantly reduced.
- the present invention has the following configuration.
- an amino acid sequence of an endoglucanase derived from a thermophilic bacterium an amino acid sequence in which the amino acid residue corresponding to the 273th tryptophan of the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid selected from other than an aromatic amino acid A mutant endoglucanase comprising.
- the amino acid sequence of an endoglucanase derived from a thermophilic bacterium is as follows: (A) an amino acid sequence represented by SEQ ID NO: 1, 7, 13, 19, 25, 31 or 37, which encodes a protein having endoglucanase activity; (B) an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, 7, 13, 19, 25, 31 or 37, and the endoglucanase activity Or (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, 7, 13, 19, 25, 31 or 37, An amino acid sequence encoding a protein having glucanase activity; The mutant endoglucanase according to [1], comprising any amino acid sequence of [1].
- [4] The mutant endoglucanase according to any one of [1] to [3], which comprises the amino acid sequence represented by SEQ ID NO: 2, 8, 14, 20, 26, 32 or 38.
- [7] An expression vector comprising the DNA of [5] or [6].
- [8] A transformed cell produced by transformation using the expression vector of [7].
- a method for producing a mutant endoglucanase comprising the steps of (1) culturing the transformed cell of [8]; and (2) purifying the mutant endoglucanase produced by the transformed cell.
- a composition for degrading biomass comprising a mutant endoglucanase according to any one of [1] to [4] and / or a processed product of transformed cells according to [8].
- a method for producing a sugar liquid from cellulose-derived biomass which comprises adding the composition for biomass decomposition of [10] to a cellulose-containing biomass suspension and hydrolyzing the suspension.
- the activity inhibition by the lignin-derived aromatic compound is greatly reduced. Therefore, when hydrolyzing cellulose, especially cellulosic biomass containing lignin to produce a sugar solution, lignocellulose can be decomposed with high efficiency. Can be manufactured.
- Example 1 it is a figure which shows the alignment of the Pyrococcus holicosi origin endoglucanase (EGPh) of a sequence number 1, and a thermophilic bacterium-derived endoglucanase.
- EGPh Pyrococcus holicosi origin endoglucanase
- the tryptophan at position 273 in SEQ ID NO: 1 is underlined.
- the present invention relates to a mutant endoglucanase in which the activity inhibition by the lignin-derived aromatic compound is greatly reduced as compared with the parent endoglucanase.
- the lignin-derived aromatic compound is obtained by decomposing an aromatic compound generally referred to as monolignol as a lignin precursor, an aromatic compound present in the biosynthetic pathway, and cellulosic biomass.
- an aromatic compound generally referred to as monolignol as a lignin precursor
- an aromatic compound present in the biosynthetic pathway an aromatic compound present in the biosynthetic pathway
- cellulosic biomass As long as it is a thing, it will not specifically limit, One or more types of mixtures may be sufficient.
- Examples of the aromatic compound referred to as monolignol and the aromatic compound present in the biosynthetic pathway include coniferyl alcohol, sinapil alcohol, p-coumaryl alcohol, phenylalanine, cinnamic acid, p-coumaric acid, Caffeic acid, 5-hydroxyferulic acid, synaptic acid, p-coumaroylcoenzyme, caffeoylcoenzyme, feruloylcoenzyme, 5-hydroxyferuloylcoenzyme, sinapoylcoenzyme, p-coumarylaldehyde, cafe Illaldehyde, 5-hydroxyconiferyl aldehyde, sinapyraldehyde, caffeyl alcohol, 5-hydroxyconiferyl alcohol, 5-dehydroshikimic acid, shikimic acid, shikimic acid-5-phosphate, 3-enolpyruvylshikimi 5-phosphate, chorismate, Purifen acid, phenylpyru
- Examples of those obtained by decomposing cellulosic biomass include syringaldehyde, p-hydroxybenzaldehyde, 5-formylvanillin, vanillic acid, syringic acid, 5-formylvanillic acid, 5-carboxyvanillin, acetoguaiacon, Guaiacol, vanillyl alcohol, dihydroconiferyl alcohol, syringaldehyde, 5-hydroxylmethylvanillin, 1-guayacyl-1-buten-3-one, p-methoxyazobenzene, benzoic acid, p-hydroxybenzoic acid, o-phthal Acid, terephthalic acid, isophthalic acid, trimethyl gallic acid, vanilloyl formic acid, hemimellitic acid, trimellitic acid, isohemipic acid, trimeditic acid, plenitic acid, pyromellitic acid, merophanic acid, benzenepentacarboxylic acid, Zenhex
- “endoglucanase” is an enzyme that hydrolyzes ⁇ -1,4-glycosyl bonds such as cellulose to produce glucose, cellobiose, cellooligosaccharide, and the like.
- the enzyme group attributed to endoglucanase is described as EC number: EC 3.2.1.4, in the present invention, the protein having the endoglucanase activity is also endotoxin although it is not attributed to endoglucanase in the EC number. It is assumed that it is contained in glucanase.
- xylanase, xyloglucanase, mannanase, chitinase, chitosanase, galactanase, etc. are mentioned.
- parent endoglucanase is an endoglucanase having an amino acid sequence before introducing a mutation, and exhibits the endoglucanase activity.
- parent endoglucanase may be described as “wild type”. In this case, the descriptions “parent endoglucanase” and “wild type” are used interchangeably.
- the “parent endoglucanase” is preferably derived from a thermophilic bacterium.
- thermophilic bacterium is a general term for a group of microorganisms that can grow at 50 ° C. or higher, and the hyperthermophilic bacterium particularly refers to a group of microorganisms that can grow at 80 ° C. or higher.
- thermophilic bacterium include Pyrococcus, Ignisphaera, Staphylothermus, Acidthermus, Spirochaeta, Sulfomoplasma, and Sulfolobus plasma (Thermoplasma), Caldivira (Caldivirga), Thermosphaera (Thermophaera), Picophyllus (Picophilus), Ferbidobacterium (Ferbidobacterium), etc. can be illustrated.
- Thermophilic bacterium-derived endoglucanases are known, for example, registered as AAQ31833 in GenBank and the like, and in the present invention, these can be used as “parent endoglucanases”.
- the parent endoglucanase comprises the amino acid sequence shown in SEQ ID NO: 1, 7, 13, 19, 25, 31 or 37.
- the parent endoglucanase has a deletion, substitution, addition or insertion of one or more or one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1, 7, 13, 19, 25, 31 or 37.
- a protein having endoglucanase activity is not particularly limited, but is, for example, within 10 pieces, more preferably within 5 pieces, particularly preferably within 4 pieces, or 1 piece or 2 pieces.
- the parent endoglucanase includes an amino acid sequence represented by SEQ ID NO: 1, 7, 13, 19, 25, 31 or 37 and BLAST (Basic Local Alignment Search the National Center for Biologics Information) (USA) National Biological Information Center Basic Local Alignment Search Tool)) etc. (eg default or default parameters) amino acids having 90%, 95%, 99% or more identity
- BLAST Basic Local Alignment Search the National Center for Biologics Information
- a protein comprising a sequence, preferably consisting of the amino acid sequence and having endoglucanase activity is also included.
- identity refers to identical amino acids and similarities to all overlapping amino acid residues in an optimal alignment when two amino acid sequences are aligned with or without introducing a gap. It means the percentage of amino acid residues.
- the identity can be determined using methods well known to those skilled in the art, sequence analysis software, and the like (for example, known algorithms such as BLAST and FASTA).
- Endoglucanase activity is as defined above, and the activity is measured by adding an enzyme solution to a substrate solution of phosphate-swelled cellulose dissolved in, for example, 50 mM diacetate-sodium acetate buffer (pH 5.2). After the reaction at 30 to 85 ° C. for 1 hour, the reaction is stopped by changing the pH if necessary, and then the glucose concentration in the reaction solution is quantified using a glucose quantification kit.
- the “mutant endoglucanase” in the present invention is a substitution of an amino acid residue corresponding to the 273th tryptophan of the amino acid sequence of SEQ ID NO: 1 with an amino acid selected from other than an aromatic amino acid in the amino acid sequence of the parent endoglucanase. And a protein having endoglucanase activity.
- the present inventors have analyzed the amino acid sequence of the parent endoglucanase, that is, the amino acid sequence represented by SEQ ID NO: 1 (19 amino acid sequences represented by SEQ ID NO: 1).
- the 273th tryptophan located near the active site is hydrophobic with coniferyl aldehyde, which comprises a total of 73 aromatic amino acid residues, including 20 tryptophans, 20 phenylalanines, 11 histidines, 23 tylosins) It was clarified that an interaction was formed.
- this amino acid forms a hydrophobic interaction with the lignin-derived aromatic compound in the vicinity of the active site, and is found to be strongly involved in inhibiting the hydrolysis reaction of cellulose, which is a substrate for endoglucanase. It was.
- the purpose of introducing mutations into endoglucanase in the present invention is to destroy this hydrophobic interaction involved in activity inhibition, and as a result, uptake of lignin-derived aromatic compounds in the vicinity of the active site is suppressed. is there.
- amino acid corresponding to the 273th tryptophan of the amino acid sequence of SEQ ID NO: 1 is derived from the thermophilic bacterium when the three-dimensional structure of the amino acid sequence of the parent endoglucanase and the amino acid sequence of SEQ ID NO: 1 is compared.
- amino acid sequence of endoglucanase it means an amino acid that is present at the same position as the 273th tryptophan in the amino acid sequence of SEQ ID NO: 1 and is involved in the formation of a hydrophobic interaction with the lignin-derived aromatic compound.
- the amino acid species of “amino acid corresponding to the 273th tryptophan of the amino acid sequence of SEQ ID NO: 1” is preferably tryptophan.
- the method for determining “the amino acid corresponding to the 273th tryptophan of the amino acid sequence of SEQ ID NO: 1” can be carried out by the following procedures 1) to 3).
- the starting methionine is defined as position 1 in the amino acid sequence of Pyrococcus horikoshii-derived endoglucanase (hereinafter referred to as “EGPh”) described in SEQ ID NO: 1.
- EGPh Pyrococcus horikoshii-derived endoglucanase
- positions 2, 3, 4. . . The tryptophan corresponding to the 273th is defined as the 273th tryptophan in SEQ ID NO: 1.
- Procedure 2 Next, the amino acid corresponding to the 273th tryptophan of the amino acid sequence represented by SEQ ID NO: 1 in the amino acid sequence of the parent endoglucanase is determined.
- the position of the corresponding amino acid can be determined by aligning the amino acid sequence of the parent endoglucanase, particularly the amino acid sequence near the active site, with the amino acid sequence of SEQ ID NO: 1. Such an operation is called amino acid sequence alignment.
- the alignment tool many well-known software such as ClustalW is used, and default parameters are used.
- a person skilled in the art can determine the position of the amino acid corresponding to the 273th tryptophan of the amino acid sequence represented by SEQ ID NO: 1 in the parent endoglucanase by alignment between amino acid sequences of different lengths.
- the parent endoglucanase is accompanied by mutation such as amino acid deletion, addition, or insertion at a position other than the above-mentioned “amino acid corresponding to the 273th tryptophan of the amino acid sequence represented by SEQ ID NO: 1,” from the N-terminus
- the counted position of “amino acid corresponding to the 273th tryptophan of the amino acid sequence represented by SEQ ID NO: 1” may not be the 273rd position. Even in such a case, the “amino acid corresponding to the 273th tryptophan of the amino acid sequence represented by SEQ ID NO: 1” determined by the above method is substituted with an amino acid other than the aromatic amino acid, and the mutant according to the present invention is used. Endoglucanase.
- amino acid residues such as tryptophan, tylosin, phenylalanine and histidine
- amino acid residues that can be substituted include lysine (Lys), arginine (Arg), histidine (His), glutamic acid (Glu), aspartic acid (Asp), valine (Val), isoleucine (Ile), threonine. (Thr), serine (Ser), cysteine (Cys), methionine (Met), glutamine (Gln), asparagine (Asn), glycine (Gly), leucine (Leu), preferably alanine (Ala).
- amino acid corresponding to the 273th tryptophan of the amino acid sequence represented by SEQ ID NO: 1 as a result, it can be produced as a protein having endoglucanase activity.
- the amino acid corresponding to the 273th tryptophan of the amino acid sequence represented may be artificially deleted.
- the mutant ⁇ -glucosidase of the present invention comprises the amino acid sequence represented by SEQ ID NO: 2, 8, 14, 20, 26, 32, or 38.
- the mutant ⁇ -glucosidase of the present invention can be produced using techniques known to those skilled in the art. For example, by producing a mutant gene encoding a mutant endoglucanase by introducing a mutation into the gene encoding the amino acid sequence of the parent endoglucanase and expressing the mutant gene using an appropriate host. Can do.
- “gene” includes nucleic acids including DNA, RNA, and DNA / RNA hybrids.
- a mutant gene encoding a mutant endoglucanase can be prepared by using a mutagenesis method known to those skilled in the art.
- EGPh a mutant endoglucanase is produced using EGPh as a parent endoglucanase
- the gene encoding EGPh is Pyrococcus horikoshi (Pyrococcus horikoshii, registration number JCM9974, JCM microbial strain catalog 7th edition, published in January 1999) ) Cells.
- the parent endoglucanase gene is a microorganism that produces the endoglucanase protein (for example, Ignisphaela). ⁇ From Aggrephas aggregans, Staphylothermus hellenicus, Pyrococcus abyssi, etc. can do.
- the gene encoding the parent endoglucanase can be obtained by isolating DNA from a microorganism having these endoglucanases according to a known method and amplifying the DNA by a technique such as PCR. For example, after Pyrococcus horikoshi culture, using BLAST search method, a gene that is similar to the endoglucanase sequence of Pyrococcus horikoshi from the gene sequence and shows this enzyme activity (for example, SEQ ID NO: 1) is subjected to PCR. Amplified and extracted by reaction.
- a parental endoglucanase gene obtained from the above endoglucanase-producing bacterium is artificially mutated at a predetermined site to prepare a mutant endoglucanase gene.
- the parent endoglucanase is substituted so that the amino acid corresponding to the 273th tryptophan of the amino acid sequence represented by SEQ ID NO: 1 is substituted. Artificially cause mutations.
- a site-specific mutagenesis method for causing mutation at a target site of a gene it can be carried out by a conventional and commonly used PCR method.
- a gene encoding the mutant endoglucanase prepared as described above is ligated downstream of a promoter in an appropriate expression vector using a restriction enzyme and DNA ligase, thereby producing an expression vector containing the gene.
- expression vectors include bacterial plasmids, yeast plasmids, phage DNA (such as lambda phage), retrovirus, baculovirus, vaccinia virus, adenovirus and other viral DNA, SV40 derivatives, and other Agrobacterium as a vector for plant cells. Any other vector can be used as long as it can replicate and survive in the host cell. For example, when the host is E.
- coli, pUC, pET, pBAD and the like can be exemplified.
- the host is yeast
- pPink-HC, pPink-LC, pPink ⁇ -HC, pPicZ, pPic ⁇ , pPic6, pPic6 ⁇ , pFLD1, pFLD1 ⁇ , pGAPZ, pGAPZ ⁇ , pPic9K, pPic9 and the like can be mentioned.
- the promoter may be any promoter as long as it is appropriate for the host used for gene expression.
- lac promoter Trp promoter, PL promoter, PR promoter and the like can be used.
- Trp promoter Trp promoter
- PL promoter PL promoter
- PR promoter PR promoter
- yeast AOX1 promoter, TEF1 promoter, ADE2 promoter, CYC1 promoter, GAL-L1 promoter and the like can be mentioned.
- the host cells used in the present invention are preferably Escherichia coli, bacterial cells, yeast cells, fungal cells, insect cells, plant cells, animal cells and the like.
- yeast cells include the genus Pichia, the genus Saccharomyces, and the genus Schizosaccharomyces.
- fungal cells include Aspergillus and Trichoderma.
- Insect cells include Sf9, plant cells include dicotyledonous plants, and animal cells include CHO, HeLa, HEK293, and the like.
- Transformation or transfection can be performed by a known method such as a calcium phosphate method or an electroporation method.
- the mutant endoglucanase of the present invention can be obtained by expressing the product in a host cell transformed or transfected as described above under the control of a promoter and recovering the product.
- transformed or transfected host cells are propagated or grown to an appropriate cell density and then chemically induced means such as temperature shift or addition of isopropyl-1-thio- ⁇ -D-galactoside (IPTG)
- IPTG isopropyl-1-thio- ⁇ -D-galactoside
- mutant endoglucanase When the mutant endoglucanase is excreted extracellularly, it is directly from the medium, and when it is present extracellularly, physical means such as ultrasonic disruption or mechanical disruption, or chemicals such as cytolytic agents are used.
- the mutant endoglucanase is purified after disrupting the cells.
- Mutant endoglucanase can be obtained from recombinant cell culture medium by ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, reverse phase high performance liquid chromatography, affinity chromatography, gel filtration chromatography, electrophoresis, etc. The techniques can be combined and partially or fully purified.
- the mutant endoglucanase of the present invention can significantly reduce the inhibition of activity by the lignin-derived aromatic compound as compared with the parent endoglucanase. Therefore, the mutant endoglucanase of the present invention is approximately 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold compared to the parent endoglucanase in the presence of the lignin-derived aromatic compound. Has an endoglucanase activity that is fold, 9, 10, 11, 12, 13, 14, 15, or more times.
- the mutant endoglucanase of the present invention may be either purified or roughly purified.
- the mutant endoglucanase of the present invention may be immobilized on a solid phase.
- the solid phase include, but are not limited to, polyacrylamide gel, polystyrene resin, porous glass, and metal oxide.
- a processed product of a cell transformed with a gene encoding the mutant endoglucanase can also be used as a roughly purified mutant endoglucanase.
- the “processed product of transformed cells” includes transformed cells immobilized on a solid phase, killed and disrupted transformed cells, and those obtained by immobilizing them on a solid phase.
- the mutant endoglucanase of the present invention can be used for hydrolysis of cellulose-containing biomass as an enzyme composition for biomass degradation by mixing with cellulase.
- Cellulase as used herein is not particularly limited as long as it has an activity of decomposing cellulose, and may be one or more kinds of mixtures.
- examples of such enzymes include cellulase, hemicellulase, cellobiohydrase, endoglucanase, exoglucanase, ⁇ -glucosidase, xylanase, mannanase, xyloglucanase, chitinase, chitosanase, galactanase and the like.
- the cellulase is a filamentous fungus-derived cellulase.
- Microorganisms that produce filamentous fungal cellulases include Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces, Humikola, and Humikola. (Acremonium), Irpex genus (Irpex), Mucor genus (Mucor), Talaromyces genus (Talaromyces), and the like. In order to produce cellulase in the culture solution, these microorganisms may be used as they are as an unpurified filamentous fungus cellulase, or the culture solution is purified and formulated into a filamentous fungus cellulase mixture.
- a cellulase preparation containing a substance other than an enzyme such as a protease inhibitor, a dispersant, a dissolution accelerator, and a stabilizer, is added. May be used as
- the filamentous fungus-derived cellulase used in the present invention is preferably a Trichoderma-derived cellulase.
- the Trichoderma-derived cellulase is not particularly limited as long as it has an activity of degrading cellulose, and may be a mixture of one or more kinds.
- Examples of such enzymes include cellulase, hemicellulase, cellobiohydrase, endoglucanase, exoglucanase, ⁇ -glucosidase, xylanase, mannanase, xyloglucanase, chitinase, chitosanase, galactanase and the like.
- Trichoderma reesei ATCC66589 Trichoderma reesei ATCC68589)
- Trichoderma reesei QM9414 Trichoderma reesei QM9414
- Trichoderma reesei QM9123 Trichoderma reesei QM9123 (Trichoderma reesei QM9414)
- Trichoderma reesei QM9123 Trichoderma reesei QM9123
- Trichoderma reesei QM9123 Trichoderma reesei QM9414
- Trichoderma reesei PC3-7 Trichoderma reesei PC3-7
- Trichoderma reesei CL-847 Trichoderma reesei CL-847
- Trichoderma reesei MCG77 Trichoderma re sei MCG77
- Trichoderma reesei MCG80 Trichoderma reesei M
- the mutant endoglucanase obtained as described above can be used alone or in combination with cellulase for the treatment of foods, feeds, detergents, cellulose-containing fabrics, and the production of sugar liquid from cellulosic biomass.
- the food and feed contain at least the mutant endoglucanase of the present invention and, if necessary, further contain other components.
- the content of the mutant endoglucanase of the present invention in the food and feed is not particularly limited and can be appropriately selected depending on the purpose.
- the said foodstuff and feed contain the variant endoglucanase of this invention, the cellulose etc. which are contained in foodstuff and feed can be decomposed
- disassembled and digestion can be made efficient, for example.
- the content of mutant endoglucanase in the detergent is not particularly limited and can be appropriately selected depending on the purpose. Moreover, there is no restriction
- the method for treating a cellulose-containing fabric includes treating the cellulose-containing fabric with the mutant endoglucanase of the present invention (treatment step), and further includes other steps as necessary.
- treatment step There is no restriction
- variant endoglucanase of this invention in the said process process have no restriction
- by treating the jeans with the method for treating a cellulose-containing fabric of the present invention for example, stone washing can be performed.
- the cellulose-containing biomass in the present invention is not limited as long as it contains at least cellulose. Specifically, bagasse, corn stover, corn cob, switch glass, rice straw, wheat straw, tree, wood, waste building materials, newspaper, waste paper, pulp, and the like. These cellulose-containing biomass contains impurities such as polymer aromatic compounds lignin and hemicellulose, but as a pretreatment, lignin and hemicellulose were partially decomposed using acid, alkali, pressurized hot water, etc. Cellulose-containing biomass may be used as cellulose.
- the cellulose-containing biomass suspension in the present invention contains the above-described cellulose-containing biomass at a solid content concentration of 0.1 to 30%.
- a solvent used for suspension According to the objective, it can select suitably.
- “addition” means adding a mutant endoglucanase, a processed product of transformed cells, cellulase, or the like to the cellulose-containing biomass suspension.
- the addition amount is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it is preferably 0.001 mg to 100 mg, more preferably 0.01 to 10 mg, and more preferably 0 to 1 g of the cellulose-containing biomass. .1 to 1 mg is particularly preferred.
- the temperature in the enzyme treatment of the cellulose-containing biomass suspension in the production of sugar liquid is not particularly limited, and the reaction temperature is preferably 30 to 100 ° C, more preferably 40 to 90 ° C, and particularly preferably 50 to 80 ° C.
- the treatment pH is not particularly limited and is preferably pH 2 to 8, more preferably pH 3 to 7, and particularly preferably pH 4 to 6.
- the cellulose-containing biomass solid content concentration is preferably 0.1 to 30%.
- This enzyme treatment may be performed batchwise or continuously. Since the hydrolyzate obtained by such an enzyme treatment contains monosaccharide components such as glucose and xylose, it can be used as a raw sugar such as ethanol and lactic acid.
- thermophilic bacterium-derived endoglucanase Determination of the 273rd amino acid residue in thermophilic bacterium-derived endoglucanase
- a BLAST search was performed.
- Protein BLAST was used with SEQ ID NO: 1 as a query.
- SEQ ID NO: 1 As a result, as an endoglucanase derived from a thermophilic bacterium having 75% or more identity with EGPh, Ignisphaera aggregans endoglucanase 1 (EGIa1) described in SEQ ID NO: 7 and Ignisphafa described in SEQ ID NO: 13 Endoglucanase 2 (EGIa2) derived from Ignisphaera aggregans, Staphylothermus helenicus described in SEQ ID NO: 19, Endoglucanase (EGSh) derived from Staphyloceramus pycoccy P.
- EGIa1 Ignisphaera aggregans endoglucanase 1
- Ignisphafa described in SEQ ID NO: 13
- Endoglucanase 2 derived from Ignisphaera aggregans
- Endoglucanase (EGPa), Acidthermus cellulolytics described in SEQ ID NO: 31 (Acidth) RMUs cellulolyticus) derived endoglucanase (EGAc), spirochete thermophila SEQ ID NO: 37, wherein (Spirochaeta thermophila) derived endoglucanase (EGST) was confirmed to be applicable.
- the prepared vectors pET-EGPh, EGIa1, EGIa2, EGSh, EGPa, EGAc, and EGSt were isolated using a miniprep kit (Qiagen) and subjected to nucleotide sequence analysis.
- pET-EGPh, EGIa1, EGIa2, EGSh, EGPa, EGAc, and EGSt were transformed into the E. coli BL21 (DE3) pLysS strain for expression to produce a BL21-PfuBGL strain.
- BL21-PfuBGL strain was inoculated into 10 mL of ampicillin-containing LB medium, and cultured with shaking (preculture) at 37 ° C. overnight.
- the obtained cell-free extract was incubated at 85 ° C. for 15 minutes to aggregate and precipitate proteins derived from E. coli other than the endoglucanase.
- the precipitate was removed by centrifugation, and the supernatant was dialyzed against 50 mM acetate buffer (pH 5.0) using a regenerated cellulose dialysis membrane (Spectrum Laboratories) having a fractional molecular weight of 10,000.
- the obtained protein solution was used as wild type EGPh, EGIa1, EGIa2, EGSh, EGPa, EGAc, EGSt.
- Example 2 Preparation of mutant endoglucanase
- the mutant endoglucanase of the present invention was prepared by the following method using the primer pairs shown in Table 1.
- a mutant EGPh (SEQ ID NO: 2) is prepared by site mutation using the oligonucleotides shown in the nucleotide sequences of SEQ ID NOs: 5 and 6 did.
- Example 3 Phosphate-swelling cellulose-decomposing activity of mutants
- the phosphate-swelling cellulose-degrading activities of the mutant obtained in Example 2 and the parent endoglucanase prepared in Reference Example 1 were compared in the following experiment.
- the enzymes prepared in Reference Example 1 and Example 2 were added at a final concentration of 0.5 ⁇ M, respectively, and the enzyme reaction was performed at 50 ° C. for 1 hour. I did it.
- the glucose concentration (g / L) produced by the parent endoglucanase under the above reaction conditions is defined as 100%, and the phosphate-swelling cellulose degradation activity in each mutant is shown in Table 2 as relative values.
- Example 4 Inhibition experiment 1 with lignin-derived aromatic compound 1
- the degradation activity of phosphate-swollen cellulose of wild type and mutant endoglucanase in the presence of coniferyl aldehyde was measured.
- coniferyl aldehyde Sigma Aldrich
- Reference Example 1 After the enzyme prepared in Example 2 was added at a final concentration of 0.5 ⁇ M, an enzyme reaction was performed at 50 ° C. for 1 hour.
- the glucose concentration (g / L) produced by the parent endoglucanase at an added concentration of 0 mM is defined as 100%, and the phosphate-swelling cellulolytic activity in each mutant is shown in Table 3 as relative values.
- Example 5 Inhibition experiment 2 with aromatic compound derived from lignin
- vanillin the degradation activity of phosphate-swollen cellulose of wild type and mutant endoglucanase was measured.
- vanillin Sigma Aldrich
- Reference Example 1 and Example respectively After the enzyme prepared in 2 was added at a final concentration of 0.5 ⁇ M, the enzyme reaction was performed at 50 ° C. for 1 hour.
- the glucose concentration (g / L) produced by the parent endoglucanase at an added concentration of 0 mM is defined as 100%, and the phosphate-swelling cellulolytic activity in each mutant is shown in Table 4 as relative values.
- lignocellulose 1 (ammonia treatment) Rice straw was used as the cellulose. The cellulose was charged into a small reactor (TVS-N2 30 ml, pressure-resistant glass industry) and cooled with liquid nitrogen. Ammonia gas was flowed into the reactor, and the sample was completely immersed in liquid ammonia. The reactor lid was closed and left at room temperature for about 15 minutes. Subsequently, it processed in the 150 degreeC oil bath for 1 hour. After the treatment, the reactor was taken out from the oil bath, and immediately after ammonia gas leaked in the fume hood, the reactor was further evacuated to 10 Pa and dried. This was used as lignocellulose 1 in the following examples.
- lignocellulose 2 (dilute sulfuric acid treatment) Rice straw was used as the cellulose. Cellulose was immersed in a 1% sulfuric acid aqueous solution and autoclaved (manufactured by Nitto Koatsu) at 150 ° C. for 30 minutes. After the treatment, solid-liquid separation was performed to separate into a sulfuric acid aqueous solution (hereinafter, dilute sulfuric acid treatment liquid) and sulfuric acid-treated cellulose. Next, the mixture was stirred and mixed with sulfuric acid-treated cellulose and a dilute sulfuric acid treatment solution so that the solid content concentration was 10% by weight, and then the pH was adjusted to around 5 with sodium hydroxide. This was used as lignocellulose 2 in the following examples.
- lignocellulose 3 (hydrothermal treatment) Rice straw was used as the cellulose. The cellulose was immersed in water and autoclaved (manufactured by Nitto Koatsu Co., Ltd.) at 180 ° C. for 20 minutes while stirring. The pressure at that time was 10 MPa. After the treatment, the solution component (hereinafter, hydrothermal treatment liquid) and the treated biomass component were subjected to solid-liquid separation using centrifugation (3000 G). This treated biomass component was used as lignocellulose 3 in the following examples.
- Example 6 Saccharification of lignocellulose using an enzyme composition comprising a cellulase mixture derived from a filamentous fungus and a mutant endoglucanase 1 The change in the amount of glucose produced when the enzyme composition was allowed to act on a lignocellulose substrate was compared.
- Trichoderma reesei-derived cellulase (cell crust, Sigma) was used as the filamentous fungus-derived cellulase mixture.
- the mutant endoglucanase prepared in Example 2 and the wild type endoglucanase prepared in Reference Example 1 were used. Enzyme addition amounts are cellulase 1.0 mg / mL, endoglucanase 0.1 mg / mL (1/10 amount of cellulase).
- Tables 6, 7 and 8 compare the glucose concentration (g / L) produced from lignocellulose 1, 2, 3 24 hours after the reaction, respectively.
- Trichoderma-derived cellulase was prepared by the following method.
- Trichoderma reesei ATCC 66589 spores were inoculated into this preculture medium so as to be 1 ⁇ 10 7 cells / ml medium, and cultured with shaking at 28 ° C. for 72 hours at 180 rpm. (Shaking device: BIO-SHAKER BR-40LF manufactured by TAITEC).
- Trichoderma reesei ATCC 66589 Tricoderderma reesei pre-cultured in a liquid medium by the aforementioned method in advance. 250 ml of ATCC 66589) was inoculated. Thereafter, the cells were cultured at 28 ° C. for 96 hours at 300 rpm and an aeration rate of 1 vvm.
- Example 7 Saccharification of lignocellulose using an enzyme composition comprising a cellulase mixture derived from a filamentous fungus and a mutant endoglucanase 2 Using the lignocellulose 1 to 3 prepared in Reference Example 3 as a substrate, the Trichoderma reesei culture solution prepared in Reference Example 4 was used as a cellulase-derived cellulase mixture, the enzyme addition amount was 1.0 mg / mL cellulase, ⁇ -glucosidase Lignocellulose 1-3 in the same manner as in Example 6 except that 0.1 mg / mL (1/10 amount of cellulase) and ⁇ -glucosidase (Novozyme 188) 0.01 mg / mL (1/100 amount of cellulase) were used. The hydrolysis of was carried out.
- Tables 9, 10 and 11 compare the glucose concentrations (g / L) produced from lignocellulose 1, 2 and 3 after 24 hours of reaction, respectively.
- Comparative Example 1 Production of Mutant Endoglucanase As a comparative example, a mutant in which the 273th tryptophan was substituted with another aromatic amino acid was produced using the primers shown in Table 12.
- EGPh (W273Y) (273th tryptophan was converted to tyrosine using site mutation method using oligonucleotides shown in the nucleotide sequences of SEQ ID NOs: 43 and 44) To: SEQ ID NO: 49).
- EGPh (W273F) (substituting tryptophan at position 73 with phenylalanine: SEQ ID NO: 50) and the oligonucleotides shown in SEQ ID NOs: 47 and 48 were used.
- EGPh (W273H) 73th tryptophan was replaced with histidine: SEQ ID NO: 51) was prepared.
- Comparative Example 2 Phosphate-swelling cellulose-degrading activity of mutant The activity of the mutant obtained in Comparative Example 1 was compared in the same manner as in Example 3. The glucose concentration (g / L) produced by the parent endoglucanase under the above reaction conditions is taken as 100%, and the phosphate-swelling cellulose degradation activity in each mutant is shown in Table 13 as relative values.
- the mutant endoglucanase in the present invention can be used for the production of a sugar solution by dropping lignocellulose. Since the enzyme cost can be significantly reduced by the effect of improving the lignocellulose decomposition efficiency, it is very useful industrially.
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Abstract
Description
(a)配列番号1、7、13、19、25、31または37で示されるアミノ酸配列であって、エンドグルカナーゼ活性を有するタンパク質をコードするアミノ酸配列;
(b)配列番号1、7、13、19、25、31または37で示されるアミノ酸配列において、1から数個のアミノ酸が欠失、置換、または付加されたアミノ酸配列であって、エンドグルカナーゼ活性を有するタンパク質をコードするアミノ酸配列;あるいは
(c)配列番号1、7、13、19、25、31または37で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列であって、エンドグルカナーゼ活性を有するタンパク質をコードするアミノ酸配列、
のいずれかのアミノ酸配列を含む、[1]の変異型エンドグルカナーゼ。
(2)該形質転換細胞により生産された変異型エンドグルカナーゼを精製する工程を含む、変異型エンドグルカナーゼの製造方法。
EGPhのアミノ酸配列と同一性の高い好熱性菌由来エンドグルカナーゼを探索するため、BLAST検索をおこなった。
EGPh、EGIa1、EGIa2、EGSh、EGPa、EGAc、EGSt遺伝子は、それぞれ配列番号1、7、13、19、25、31、37記載の遺伝子を全合成し、pET11dのNcoIおよびBamHIにDNA Ligation Kit<Mighty Mix>(タカラバイオ)を使用して連結し、JM109(タカラバイオ)に形質転換した。スクリーニングはアンピシリンを抗生物質として含むLB寒天培地を用いて行った。形質転換されたJM109株より、作製したベクターpET-EGPh、EGIa1、EGIa2、EGSh、EGPa、EGAc、EGStをミニプレップキット(キアゲン)により単離し、塩基配列解析を行った。pET-EGPh、EGIa1、EGIa2、EGSh、EGPa、EGAc、EGStは、発現用大腸菌BL21(DE3)pLysS株に形質転換し、BL21-PfuBGL株を作製した。BL21-PfuBGL株を、アンピシリン含有LB培地10mLに植菌し、37℃で一晩振とう培養(前培養)を行った。本培養として、アンピシリン含有LB培地1Lに、前培養で得られた菌体を植菌し、波長600 nmでの吸光度OD600が0.6となるまで振とう培養を行った。その後、最終濃度が0.5mMになるようにイソプロピル-1-チオ-β-D-ガラクトシド(IPTG)を加え、さらに25℃で一晩培養した。培養後、菌体を遠心分離により回収し、 50mM リン酸カリウム緩衝液(pH7.0)に再懸濁した。この溶液を氷冷しながら、超音波破砕を行い、その上清を無細胞抽出液として遠心分離により回収した。得られた無細胞抽出液を、85℃で15分保温し、該エンドグルカナーゼ以外の大腸菌に由来するタンパク質を凝集沈殿した。遠心分離により沈殿物を除去し、上清を分画分子量10000の再生セルロース製透析膜(スペクトラム・ラボラトリーズ製)を使用して、50mM 酢酸緩衝液(pH5.0)に透析した。得られたタンパク質溶液を、野生型のEGPh、EGIa1、EGIa2、EGSh、EGPa、EGAc、EGStとして使用した。
エンドグルカナーゼの加水分解活性を測定する際に基質として使用するリン酸膨潤セルロースは、Walseth (1971) Tappi 35:228(1971)及びWood Biochem J.121:353(1971)に記載の方法に従って、アビセル(Avicel)から調製した。この物質を緩衝液及び水を用いて希釈して2重量%混合物を得て、酢酸ナトリウムの最終濃度が50mM、pH5.2 になるようにした。これをリン酸膨潤セルロースとして、以下の実施例に使用した。
実施例2で得られた変異体と、参考例1で調製した親エンドグルカナーゼのリン酸膨潤セルロース分解活性を以下実験にて比較した。基質に1%リン酸膨潤セルロース/50mM酢酸緩衝液(pH5.2)を用い、それぞれ参考例1、実施例2で調製した酵素を終濃度0.5μMで添加し、50℃で1時間酵素反応をおこなった。上記反応条件下における親エンドグルカナーゼにより生成されたグルコース濃度(g/L)を100%として、各変異体におけるリン酸膨潤セルロース分解活性を相対値で表2に示す。
コニフェリルアルデヒド存在下における、野生型及び変異型エンドグルカナーゼのリン酸膨潤セルロースの分解活性を測定した。基質に1%リン酸膨潤セルロース/50mM酢酸緩衝液(pH5.2)を用い、コニフェリルアルデヒド(シグマアルドリッチ)を終濃度0、5、10、15mMになるように添加し、それぞれ参考例1、実施例2で調製した酵素を終濃度0.5μMで添加後、50℃で1時間酵素反応をおこなった。添加濃度0mMにおいて親エンドグルカナーゼにより生成されたグルコース濃度(g/L)を100%として、それぞれの変異体におけるリン酸膨潤セルロース分解活性を相対値で表3に示す。
バニリン存在下における、野生型及び変異型エンドグルカナーゼのリン酸膨潤セルロースの分解活性を測定した。基質に1%リン酸膨潤セルロース/50mM酢酸緩衝液(pH5.2)を用い、バニリン(シグマアルドリッチ)を終濃度0、5、10、15mMになるように添加し、それぞれ参考例1、実施例2で調製した酵素を終濃度0.5μMで添加後、50℃で1時間酵素反応をおこなった。添加濃度0mMにおいて親エンドグルカナーゼにより生成されたグルコース濃度(g/L)を100%として、それぞれの変異体におけるリン酸膨潤セルロース分解活性を相対値で表4に示す。
フェルラ酸存在下における、野生型及び変異型エンドグルカナーゼのリン酸膨潤セルロースの分解活性を測定した。基質に1%リン酸膨潤セルロース/50mM酢酸緩衝液(pH5.2)を用い、フェルラ酸(シグマアルドリッチ)を終濃度0、5、10、15mMになるように添加し、それぞれ参考例1、実施例2で調製した酵素を終濃度0.5μMで添加後、50℃で1時間酵素反応をおこなった。添加濃度0mMにおいて親エンドグルカナーゼにより生成されたグルコース濃度(g/L)を100%として、それぞれの変異体におけるリン酸膨潤セルロース分解活性を相対値で表5に示す。
エンドグルカナーゼの加水分解活性を測定する際に基質として使用するリン酸膨潤セルロース1~3を以下のように調製した。
セルロースとして、稲藁を使用した。前記セルロースを小型反応器(耐圧硝子工業製、TVS-N2 30ml)に投入し、液体窒素で冷却した。この反応器にアンモニアガスを流入し、試料を完全に液体アンモニアに浸漬させた。リアクターの蓋を閉め、室温で15分ほど放置した。次いで、150℃のオイルバス中にて1時間処理した。処理後、反応器をオイルバスから取り出し、ドラフト中で直ちにアンモニアガスをリーク後、さらに真空ポンプで反応器内を10Paまで真空引きし乾燥させた。これをリグノセルロース1として以下の実施例に使用した。
セルロースとして、稲藁を使用した。セルロースを硫酸1%水溶液に浸し、150℃で30分オートクレーブ処理(日東高圧製)した。処理後、固液分離を行い、硫酸水溶液(以下、希硫酸処理液)と硫酸処理セルロースに分離した。次に硫酸処理セルロースと固形分濃度が10重量%となるように希硫酸処理液と攪拌混合した後、水酸化ナトリウムによって、pHを5付近に調製した。これをリグノセルロース2として以下の実施例に使用した。
セルロースとして、稲藁を使用した。前記セルロースを水に浸し、撹拌しながら180℃で20分間オートクレーブ処理(日東高圧株式会社製)した。その際の圧力は10MPaであった。処理後は溶液成分(以下、水熱処理液)と処理バイオマス成分に遠心分離(3000G)を用いて固液分離した。この処理バイオマス成分をリグノセルロース3として以下の実施例に使用した。
リグノセルロース基質に該酵素組成物を作用させた場合のグルコース生成量の変化を比較した。50mM酢酸緩衝液(pH5.2)に5重量%のリグノセルロース1~3(参考例3で調製)を懸濁したものを基質とした。反応は50℃において24時間まで行い、適宜サンプリングして生成したグルコース濃度の測定を行った。糸状菌由来セルラーゼ混合物としては、市販のトリコデルマ・リーセイ由来セルラーゼ(セルクラスト、シグマ)を用いた。エンドグルカナーゼとしては、実施例2で調製した変異型エンドグルカナーゼと参考例1で調製した野生型エンドグルカナーゼをそれぞれ用いた。酵素添加量は、セルラーゼ1.0mg/mL、エンドグルカナーゼ0.1mg/mL(セルラーゼの1/10量)である。表6、7、8にそれぞれリグノセルロース1、2、3から反応24時間後に生成したグルコース濃度(g/L)を比較した。
トリコデルマ属由来セルラーゼを以下の方法で調製した。
コーンスティップリカー2.5%(w/vol)、グルコース2%(w/vol)、酒石酸アンモニウム0.37%(w/vol)、硫酸アンモニウム0.14%(w/vol)、リン酸二水素カリウム0.2%(w/vol)、塩化カルシウム二水和物0.03%(w/vol)、硫酸マグネシウム七水和物0.03%(w/vol)、塩化亜鉛0.02%(w/vol)、塩化鉄(III)六水和物0.01%(w/vol)、硫酸銅(II)五水和物0.004%(w/vol)、塩化マンガン四水和物 0.0008%(w/vol)、ホウ酸0.0006%(w/vol)、七モリブデン酸六アンモニウム四水和物0.0026%(w/vol)となるよう蒸留水に添加し、100mLを500mLバッフル付き三角フラスコに張り込み、121℃で15分間オートクレーブ滅菌した。放冷後、これとは別にそれぞれ121℃で15分間オートクレーブ滅菌したPE-MとTween80をそれぞれ0.1%添加した。この前培養培地にトリコデルマ・リーセイATCC66589(Tricoderma reesei ATCC66589)の胞子を、1×107個/ml培地になるように植菌し、28℃、72時間、180rpmで振とう培養し、前培養とした(振とう装置:TAITEC社製 BIO-SHAKER BR-40LF)。
コーンスティップリカー2.5%(w/vol)、グルコース2%(w/vol)、セルロース(アビセル)10%(w/vol)、酒石酸アンモニウム0.37%(w/vol)、硫酸アンモニウム0.14%(w/vol)、リン酸二水素カリウム0.2%(w/vol)、塩化カルシウム二水和物0.03%(w/vol)、硫酸マグネシウム七水和物0.03%(w/vol)、塩化亜鉛0.02%(w/vol)、塩化鉄(III)六水和物0.01%(w/vol)、硫酸銅(II)五水和物0.004%(w/vol)、塩化マンガン四水和物0.0008%(w/vol)、ホウ酸0.0006%(w/vol)、七モリブデン酸六アンモニウム四水和物0.0026%(w/vol)となるよう蒸留水に添加し、2.5Lを5L容撹拌ジャー(ABLE社製 DPC-2A)容器に張り込み、121℃で15分間オートクレーブ滅菌した。放冷後、これとは別にそれぞれ121℃で15分間オートクレーブ滅菌したPE-MとTween80をそれぞれ0.1%添加し、あらかじめ前記の方法にて液体培地で前培養したトリコデルマ・リーセイATCC66589(Tricoderma reesei ATCC66589)を250mL接種した。その後、28℃、96時間、300rpm、通気量1vvmにて培養を行い、遠心分離後、上清を膜濾過(ミリポア社製 ステリカップ-GV 材質:PVDF)した。
参考例3で調製したリグノセルロース1~3を基質とし、参考例4で調製したトリコデルマ・リーセイ培養液を糸状菌由来セルラーゼ混合物として使用し、酵素添加量をセルラーゼ1.0mg/mL、β―グルコシダーゼ0.1mg/mL(セルラーゼの1/10量)、β―グルコシダーゼ(Novozyme188)0.01mg/mL(セルラーゼの1/100量)とした以外は、実施例6と同様にしてリグノセルロース1~3の加水分解を実施した。
比較例1で得られた変異体を実施例3と同様の手法で活性を比較した。上記反応条件下における親エンドグルカナーゼにより生成されたグルコース濃度(g/L)を100%として、各変異体におけるリン酸膨潤セルロース分解活性を相対値で表13に示す。
コニフェリルアルデヒド存在下における、野生型及び比較例1の変異型エンドグルカナーゼのリン酸膨潤セルロースの分解活性を測定した。実験は実施例4と同じ手順で行い、それぞれの変異体におけるリン酸膨潤セルロース分解活性を相対値で表14に示す。
Claims (12)
- 好熱性菌由来のエンドグルカナーゼのアミノ酸配列において、配列番号1のアミノ酸配列の273番目のトリプトファンに相当するアミノ酸残基が、芳香族アミノ酸以外から選ばれるアミノ酸に置換されているアミノ酸配列を含む、変異型エンドグルカナーゼ。
- 好熱性菌由来のエンドグルカナーゼのアミノ酸配列が、以下:
(a)配列番号1、7、13、19,25、31または37で示されるアミノ酸配列であって、エンドグルカナーゼ活性を有するタンパク質をコードするアミノ酸配列;
(b)配列番号1、7、13、19,25、31または37で示されるアミノ酸配列において、1から数個のアミノ酸が欠失、置換、または付加されたアミノ酸配列であって、エンドグルカナーゼ活性を有するタンパク質をコードするアミノ酸配列;あるいは
(c)配列番号1、7、13、19,25、31または37で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列であって、エンドグルカナーゼ活性を有するタンパク質をコードするアミノ酸配列、
のいずれかのアミノ酸配列を含む、請求項1に記載の変異型エンドグルカナーゼ。 - 配列番号1のアミノ酸配列の273番目のトリプトファンに相当するアミノ酸残基がアラニンに置換されている、請求項1または2に記載の変異型エンドグルカナーゼ。
- 配列番号2、8、14、20、26、32または38で示されるアミノ酸配列を含む、請求項1~3のいずれか1項に記載の変異型エンドグルカナーゼ。
- 請求項1~4のいずれか1項に記載の変異型エンドグルカナーゼをコードするDNA。
- 配列番号4、10、16、22、28、34または40で示される塩基配列を含む、請求項5に記載のDNA。
- 請求項5または6に記載のDNAを含む、発現ベクター。
- 請求項7に記載の発現ベクターを用いた形質転換により作製された、形質転換細胞。
- (1)請求項8に記載の形質転換細胞を培養する工程;および
(2)該形質転換細胞により生産された変異型エンドグルカナーゼを精製する工程を含む、変異型エンドグルカナーゼの製造方法。 - 請求項1~4のいずれか1項に記載の変異型エンドグルカナーゼおよび/または請求項8に記載の形質転換細胞の処理物を含む、バイオマス分解用組成物。
- セルロース含有バイオマス懸濁液に、請求項10に記載のバイオマス分解用組成物を添加して加水分解することを含む、セルロース由来バイオマスより糖液を製造する方法。
- さらに、糸状菌由来セルラーゼを添加することを含む、請求項11に記載の方法。
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CN201280042920.9A CN103797116A (zh) | 2011-09-05 | 2012-09-04 | 突变型内切葡聚糖酶 |
AU2012305442A AU2012305442A1 (en) | 2011-09-05 | 2012-09-04 | Mutant endoglucanase |
JP2013532590A JP5971811B2 (ja) | 2011-09-05 | 2012-09-04 | 変異型エンドグルカナーゼ |
IN2455CHN2014 IN2014CN02455A (ja) | 2011-09-05 | 2012-09-04 | |
EP12830699.0A EP2754712B1 (en) | 2011-09-05 | 2012-09-04 | Mutant endoglucanase |
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US9193963B2 (en) | 2011-09-05 | 2015-11-24 | Toray Industries, Inc. | Mutant endoglucanase |
CN112746064A (zh) * | 2020-12-28 | 2021-05-04 | 苏州科宁多元醇有限公司 | 一种来源于梭菌属的壳聚糖酶基因及其重组菌和在生产壳寡糖上的应用 |
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CN116042554A (zh) * | 2023-01-31 | 2023-05-02 | 中国科学院青岛生物能源与过程研究所 | 具有高酶活性与高热稳定性的葡聚糖单加氧酶及其制备方法与应用 |
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US9193963B2 (en) | 2011-09-05 | 2015-11-24 | Toray Industries, Inc. | Mutant endoglucanase |
CN112746064A (zh) * | 2020-12-28 | 2021-05-04 | 苏州科宁多元醇有限公司 | 一种来源于梭菌属的壳聚糖酶基因及其重组菌和在生产壳寡糖上的应用 |
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EP2754712A1 (en) | 2014-07-16 |
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