JP5758079B2 - Biomass saccharification accelerator, saccharification treatment agent, and saccharification treatment method using the same - Google Patents

Description

  The present invention relates to a biomass saccharification promoter, a saccharification treatment agent, and a saccharification treatment method using the same.

  Along with the decrease in petroleum resources, in recent years, saccharification treatment of biomass to produce ethanol and supply new energy sources has been actively attempted. However, since the use of corn and other fruits including starch is important for food, non-edible waste such as corn husk and wood are effectively used as cellulosic biomass. It is important.

  Since biomass such as wood contains cellulose, hemicellulose, and lignin, it is usually hydrolyzed using a saccharifying enzyme such as acid, alkali, or cellulase, converted from a polymer to an oligosaccharide, and further to a monosaccharide, The sugar is converted into a raw material for chemical products such as a fuel alcohol such as ethanol or an organic acid by fermentation using microorganisms or the like. In recent years, since saccharification with acid and alkali as described above is accompanied with environmental pollution, saccharification using an enzyme is required worldwide. However, with only a single treatment with the cellulase agent as the main enzyme, a large amount of enzyme is required to increase the saccharification efficiency of cellulose and hemicellulose. For example, when the saccharification efficiency is increased from 90% to 99%, it is generally said that 10 enzyme amounts are required when the saccharification efficiency is 99%, assuming that the enzyme amount required is 1 when the saccharification efficiency is 90%. Cellulosic biomass, in particular, increases in viscosity due to the binding of cellulose molecules, but the viscosity decreases dramatically by improving the saccharification efficiency of only about 10%. As a result, the burden on the fermentation process can be greatly reduced. It is said. Thus, attempts have been made to improve saccharification efficiency and reduce the amount of cellulase by using cellulase in combination with other enzymes, but sufficient efficiency has not yet been obtained (Patent Document 1, Non-Patent Document 1). This raises the cost of saccharification treatment, and is an obstacle to the practical use of the production of chemical raw materials such as fuel alcohol such as ethanol and organic acid from cellulosic biomass.

Kumagai et al., Characteristic of Hydrothermal Decomposition and Saccharification of Various Lignocellulosic Biomass and Enzymatic Saccarification of the Obtained Hydrothermal-Residue. Journal of the Japan Institute of Energy, 86, 712-717 (2007)

Japanese National Patent Publication No. 11-500465

  Accordingly, an object of the present invention is to provide a new saccharification accelerator, a saccharification treatment agent, and a biomass saccharification treatment method using the same, which promote saccharification treatment of biomass by cellulase.

  To achieve the above object, the first saccharification promoting agent of the present invention is a saccharification promoting agent that promotes saccharification treatment of biomass by cellulase, and cultures cells that express at least one of the xynR gene and the fae gene. It is characterized by including a supernatant.

  The first saccharification treatment agent of the present invention is a saccharification treatment agent for use in biomass saccharification treatment, and includes cellulase and the first saccharification promoter of the present invention.

  The first saccharification treatment method of the present invention is a biomass saccharification treatment method, wherein the biomass is treated with cellulase in the presence of the first saccharification promoter of the present invention.

  The second saccharification promoter of the present invention is a saccharification promoter that promotes biomass saccharification treatment with cellulase, and includes a protein comprising an amino acid sequence represented by at least one of SEQ ID NOs: 8 to 14. Features.

  The second saccharification treatment agent of the present invention is a saccharification treatment agent for use in biomass saccharification treatment, and includes cellulase and the second saccharification promoter of the present invention.

  The second saccharification treatment method of the present invention is a biomass saccharification treatment method, characterized in that the biomass is treated with cellulase in the presence of the second saccharification promoter of the present invention.

  According to the present invention, biomass can be saccharified with higher efficiency than saccharification treatment using cellulase alone. For this reason, it can be said that it is extremely useful because it can effectively use wood or its waste as biomass and provide energy resources and chemical products. Further, according to the present invention, for example, since the amount of cellulase used for the saccharification treatment can be reduced, the cost of the saccharification treatment of biomass, which is important in subsequent processes such as fermentation, can be reduced.

FIG. 1 is a schematic diagram showing the structure of an expression vector used in Examples of the present invention.

<First form>
As described above, the first saccharification promoter of the present invention is a saccharification promoter that promotes the saccharification treatment of biomass by cellulase, and the culture supernatant of a cell expressing at least one of the xynR gene and the fae gene is used. It is characterized by including.

The microbial cells preferably belong to the genus Aspergillus , more preferably Aspergillus oryzae .

The xynR gene is a transcriptional regulator gene that positively controls the expression of the xylanase gene, and is also referred to as xlnR. From the xynR gene is not particularly limited, for example, Aspergillus genus are preferable, among them, from Aspergillus oryzae are preferred.

The fae gene is a ferulic acid esterase gene. The fae gene has, for example, a subfamily such as faeA gene, faeB gene, and faeC gene. The fae gene may be, for example, any of the faeA gene, the faeB gene, and the faeC gene, and among them, the faeA gene is preferable. From the fae gene is not particularly limited, for example, Aspergillus genus are preferable, among them, from Aspergillus oryzae are preferred.

  In the present invention, the bacterial cell may express, for example, either the xynR gene or the fae gene, or may express both.

  The cell is preferably a transformant into which an expression vector having at least one of the xynR gene and the fae gene has been introduced. Thus, according to the transformant in which the expression vector is introduced into the host, for example, at least one of the xynR gene and the fae gene can be overexpressed. In the present invention, hereinafter, a vector into which the xynR gene and the fae gene are inserted is referred to as an expression vector, and a vector in which at least one of the xynR gene and the fae gene is inserted into the expression vector is referred to as a recombinant expression vector.

  The gene inserted into the expression vector may be, for example, a cloned natural nucleic acid, a nucleic acid amplified by a gene amplification method, or a synthesized nucleic acid. Examples of the nucleic acid include DNA and RNA. In addition, for example, an artificial nucleic acid such as PNA may be used.

The host is not particularly limited, and examples thereof include living cells that can be transformed. Examples of the living cells include eukaryotic organisms such as fungi, insects and higher animals, and prokaryotic organisms such as eubacteria and archaea. Examples of the fungi include fungi such as filamentous fungi and yeast. As the filamentous fungus is not particularly limited, for example, Aspergillus spp, Rhizopus spp, Trichoderma sp, Penicillium sp, Mucor spp, Phanerochaetae genus, Acremonium spp, Humicola spp, Fusarium spp, Neurospora spp and the like. Among them, for example, filamentous fungi are preferable, and Aspergillus genus is particularly preferable. The genus Aspergillus, is not particularly limited, for example, Aspergillus oryzae, Aspergillus niger, Aspergillus awamori, Aspergillus kawachii, Aspergillus usamii, Aspergillus sojae, Aspergillus nidulans, Aspergillus glaucus, Aspergillus aculeatus, Aspergillus terreus , and the like, more preferably Aspergillus oryzae . Other examples of the living cells include yeast cells, and specific examples thereof include Saccharomyces cerevisiae , Pichia pastoris , and Schizosaccharomyces pombe (Candida) Methanol assimilating yeast containing yeast genus etc. are mention | raise | lifted. Examples of the eubacteria include Escherichia coli, Bacillus genus, Brevivacillus genus, Lactobacillus genus and the like. Examples of the insect include insect cultured cells such as Sf9 cells, and insect bodies such as silkworms. Examples of the higher animals include cultured cells derived from vertebrates including humans, and individuals such as vertebrates.

  The expression vector is not limited as long as it can insert at least one of the xynR gene and the fae gene. The recombinant expression vector can be produced, for example, by using a conventionally known vector as an expression vector and inserting at least one of the aforementioned xynR gene and fae gene into the vector. The expression vector is not particularly limited, and can be appropriately determined depending on, for example, the type of host into which the recombinant expression vector is introduced, the introduction method, and the like. Examples of the expression vector include non-viral vectors such as plasmid vectors, viral vectors, and the like. Examples of the non-viral vector include pISI (Appl. Microbiol. Biotechnol. 81, 711-719, (2008)), YIp5, YEp24, pBR322, pUC12, pUC18 / 19, pUC118 / 119, pcDNA3.1 (Invitrogen). ), PZeoSV (Invitrogen), pBK-CMV (Stratagene), pCAGGS (Gene 108, 193-200, (1991)), pBluescript plasmid vector (Stratagene), pHSG298 / 299, pHSG396 / 8, etc. Plasmid vectors and the like. In particular, when the host is a filamentous fungus, for example, plasmids per host such as pISI (Appl. Microbiol. Biotechnol. 81, 711-719, (2008).), PUC plasmid vectors (for example, pUC18 / 19, etc.) Those having a high copy number are preferred. Moreover, when the said host is yeast, YIp5, YEp24 etc. are preferable, for example. When the host is Escherichia coli, for example, pBR322, pUC12, pUC18 / 19, pUC118 / 119 and the like are preferable. When the host is a higher animal, for example, pcDNA3.1 (Invitrogen), pZeoSV (Invitrogen), pBK-CMV (Stratagene), pCAGGS (Gene 108, 193-200, (1991).), Etc. Is preferred. Examples of the viral vector include baculovirus vectors, retrovirus vectors, lentivirus vectors such as human immunodeficiency virus (HIV), adenovirus vectors, adeno-associated virus vectors (AAV vectors), herpes viruses, Examples thereof include DNA viruses such as vaccinia virus, pox virus, polio virus, symbis virus, Sendai virus, and simian virus-40 (SV-40), and RNA viruses. In particular, when the host is an insect, for example, an AcNPV (Autographa california nuclear polyhydrovirus) (Vaculovirus) vector is preferable. These vectors can be inserted so that the inserted gene can be expressed, for example, based on a conventionally known method.

  The expression vector may further include a regulatory sequence that regulates the expression of the inserted gene. Examples of the regulatory sequence include a promoter, terminator, enhancer sequence, polyadenylation signal, replication origin sequence (ori) and the like. These regulatory sequences can be appropriately set according to the type of vector, host, target protein, and the like. In addition, the regulatory sequence includes, for example, a sequence that can function as a regulatory sequence in a desired host, and includes an improved regulatory sequence with modifications. The promoter is not particularly limited. For example, when the host is a filamentous fungus, sodM, amyA, amyB, amyC, glaA, agdA, glaB, TEF1, xynF1 tannase, No. 8AN, gpdA, pgkA, enoA, melO, catA, catB, Histone H2A, small v-ATPase, cruciform binding protein, peptidyl primary cis-trans isomerase, etc. When the host is yeast, examples thereof include PHO5, PGK, GAP, ADH1, GAL, SED1, and the like. When the host is a higher animal, examples include cytomegalovirus (CMV) and rous sarcoma virus (RSV). , SV-40, herpes simplex virus (HSV) -derived promoters, constitutive promoters such as muscle β-actin promoter, tissue-specific promoters such as thymidine kinase promoter, growth hormone-regulated promoters, zinc-inducible metallothionein promoters, etc. Examples include promoters and regulatable promoters. The terminator is not particularly limited, and examples thereof include glaB terminator (Gene 207, 127-134, (1998).), AmyA terminator (Gene 84, 319-327, (1989)). The regulatory sequence may be arranged or bonded to a site capable of functionally regulating the expression of the gene, for example, based on a conventionally known method.

  The expression vector may have a sequence (selection marker gene) encoding a selection marker such as an auxotrophic marker, drug resistance marker, fluorescent protein marker, enzyme marker, cell surface receptor marker, and the like. These selectable markers can be appropriately set depending on the type of vector, host and the like. Examples of the selectable marker gene include niaD, argB, sC, leuA, ptrA, pyrG, amdS, aureobasidin resistance gene, benomyl resistance gene, phleomycin resistance gene, cycloheximide resistance gene, ampicillin resistance gene, tetracycline resistance gene, kanamycin Resistance gene, G418 resistance gene, chloramphenicol resistance gene, blasticidin resistance gene, bleomycin resistance gene, puromycin resistance gene and hygromycin B resistance gene, GFP (Green Fluorescent Protein), EGFP (Enhanced GFP), ECFP, Mutant GFP such as EYFP, RFP (Red Fluorescent Protein), luciferase gene, β Galactosidase gene, β- glucuronidase, and the like. When the host is a filamentous fungus, the selection marker gene includes, for example, niaD, argB, sC, leuA, ptrA, pyrG, amdS, an aureobasidin resistance gene, a benomyl resistance gene, a hygromycin B resistance gene, and a carboxin resistance gene. Is more preferable, and niaD is more preferable. When the host is yeast, the selection marker gene is preferably a cycloheximide resistance gene, a G418 resistance gene, a hygromycin B resistance gene, a chloramphenicol resistance gene, a blasticidin resistance gene, a phleomycin resistance gene, or the like. . When the host is Escherichia coli, the selection marker gene is preferably, for example, an ampicillin resistance gene, a tetracycline resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, or the like. When the host is a higher animal, the selection marker gene is preferably a G418 resistance gene, a hygromycin B resistance gene, a blasticidin resistance gene, a puromycin resistance gene, a bleomycin resistance gene, or the like.

  The method for introducing the recombinant expression vector into the host cell is not particularly limited, and can be appropriately set depending on the type of expression vector, host, and the like. Specific examples of the introduction method include, for example, a protoplast method, a lithium acetate method, a Hanahan method, an electroporation method, an infection introduction method using a viral vector, a calcium phosphate method, a lipofection method, an electroporation method, an ultrasonic nucleic acid, and the like. Examples thereof include an introduction method, a gene gun introduction method, and a DEAE-dextran method. In particular, when the host is a filamentous fungus, examples thereof include a protoplast method, an electroporation method, an infection introduction method using a viral vector, and the like, and the protoplast method is preferable. Further, when the host is yeast, for example, a lithium acetate method, a protoplast method, an electroporation method and the like are preferable. When the host is Escherichia coli, for example, Hanahan method, electroporation method and the like are preferable. When the host is an insect, for example, an infection introduction method using the aforementioned AcNPV vector or the like is preferable. When the host is a higher animal cell, for example, a calcium phosphate method, a lipofection method, an electroporation method, an ultrasonic nucleic acid introduction method, a gene gun introduction method, a DEAE-dextran method, or a virus vector as described above was used. Infection introduction methods are preferred.

  In the first saccharification promoter of the present invention, the method for producing the culture supernatant is not particularly limited, and can be obtained, for example, by culturing a cell that expresses either the xynR gene or the fae gene. The culture supernatant may be, for example, a bacterial cell culture solution itself or a liquid fraction obtained by removing cultured bacterial cells and insoluble matter from the culture solution. The liquid fraction may be, for example, a supernatant obtained by centrifuging the culture solution or a filtrate obtained by filtering the culture solution. The liquid fraction may be, for example, a purified fraction such as the filtrate or supernatant from which the insoluble matter or the like has been removed. The purification method can be carried out by appropriately combining, for example, concentration with an ultrafiltration membrane, salting out such as ammonium sulfate precipitation, dialysis, various chromatography, and the like.

The culture conditions for the cells are not particularly limited, and can be appropriately determined according to the type of the cells. Moreover, when the said microbial cell is the transformant which introduce | transduced the said recombinant expression vector, it can determine suitably according to the kind of host and an expression vector, for example. As a specific example, when the microbial cell belongs to the genus Aspergillus , the medium is preferably, for example, a GPY medium, a DPY medium, a Czapek-Dox medium, a medium containing corn steep liquor (CSL), or a concentrated medium thereof. Examples of components of the medium include carbon sources such as glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, organic acids, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or peptone, yeast. Nitrogen sources such as extract, corn steep liquor, casein hydrolyzate, bran and meat extract, and inorganic salts such as potassium, magnesium, sodium, phosphate, manganese, iron, and zinc salts . The medium may contain vitamins, amino acids and the like, for example, in order to promote the growth of the transformant used. The pH of the medium is, for example, about 3-8, and preferably about 5-7. The culture temperature is, for example, about 10-50 ° C., preferably about 25-40 ° C., and the culture period is, for example, 1-15 days, preferably 3-7 days. The culture is preferably performed, for example, under aerobic conditions. As the culture method, for example, a shaking culture method or an aerobic deep culture method using a jar fermenter can be used.

  In addition, the 1st saccharification promoter of this invention should just contain the said culture supernatant, and another structure is not restrict | limited in particular, For example, you may contain another component further. The other components are not particularly limited. Moreover, the form of the culture supernatant is not limited, and may be liquid or solid, for example. In the case of a solid, for example, a dried product containing the culture supernatant component obtained by drying the culture supernatant can be used. The form of the first saccharification accelerator of the present invention is not particularly limited, and may be, for example, a liquid or a solid such as a powder or a tablet.

  Next, as described above, the first saccharification treatment agent of the present invention is a saccharification treatment agent for use in biomass saccharification treatment, comprising cellulase and the first saccharification promoter of the present invention. It is characterized by including. The 1st saccharification processing agent of this invention should just contain the said cellulase and the said 1st saccharification promoter, and another structure is not restrict | limited at all. The first saccharification treatment agent of the present invention may further contain other components, for example. Examples of the other components include saccharides, sugar alcohols, organic acids, enzyme stabilizers such as surfactants, and the like. Examples of the saccharide include D-glucose, D-galactose, D-mannose, D-sorbose, D-talose, D-fructose, D-ribose, D-xylose and other monosaccharides, lactose, saccharose, maltose, trehalose. Disaccharides such as isomaltose and cellobiose, maltotriose, maltotetraose, maltopentaose, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin and other oligosaccharides, inulin, amylose, amylopectin Water-soluble neutral polysaccharides such as dextrin and pullulan. Examples of the sugar alcohols include glucitol, mannitol, inositol, xylitol and the like. Examples of the organic acid include gluconic acid, citric acid, maleic acid, succinic acid, malic acid, lactic acid, acetic acid, and sodium or potassium salts thereof. Examples of the surfactant include alkyl glycosides such as octyl glycoside and octyl thioglycoside, cholic acids such as cholic acid, deoxycholic acid and lithocholic acid, sodium or potassium salts of cholic acids, polyethylene glycol ethers, polyoxy Examples include ethylene glycol ethers, lauroyl sarcosine, lauroyl sarcosine salts, lauryl sulfate, and lauryl sulfate.

  Next, a first saccharification treatment method of the present invention is a biomass saccharification treatment method, wherein the biomass is treated with cellulase in the presence of the first saccharification promoter of the present invention. The present invention is characterized in that cellulase treatment is performed in the presence of the first saccharification promoter, and other configurations and conditions are not limited at all.

The cellulase is not particularly limited, and various commercially available cellulases can be used. Among them, Trichoderma (Trichoderma) genus Aspergillus (Aspergillus) genus Acremonium (Acremonium) genus Bacillus (Bacillus) derived from the genus of cellulase, more preferably a Trichoderma reesei (Trichoderma reesei) derived cellulase.

  The biomass generally means a biological organic resource excluding fossil fuel. As the biomass, for example, lignocellulosic biomass containing lignin, cellulose and hemicellulose can be used. Specific examples include, for example, wood, rice straw, switchgrass, corn, sugarcane, bamboo and pulp, and disposal thereof. Herbaceous and woody biomass raw materials such as corn stover, bagasse and the like.

  The biomass may be pretreated biomass obtained by pretreating the biomass raw material, for example. Examples of the pretreatment method include hot water treatment in which the biomass raw material is treated with pressurized hot water. The conditions for the hydrothermal treatment are not particularly limited, but for example, Kumagai et al. 712-717 (2007)), JP 2009-183806 A, JP 2009-124973 A, JP 2006-136263 A, and the like.

  The pretreated biomass has a lignin content of 5 to 25 w / v% and a holocellulose content of 75 to 95 w / v%, for example. The contents of cellulose and hemicellulose in the holocellulose are, for example, a cellulose content of 60 to 90 w / v% and a hemicellulose content of 10 to 40 w / v%, where holocellulose is 100 w / v%.

  Next, the first saccharification treatment method of the present invention will be described with an example.

  First, a reaction liquid containing the pretreated biomass obtained by treating the biomass material with pressurized hot water, the first saccharification accelerator, and cellulase is prepared. The reaction solution is preferably prepared, for example, by adding the pretreated biomass, the first saccharification promoter, and the cellulase to a solvent. Examples of the solvent include water and a buffer solution, and salts, acids, alkalis, ionic substances, and the like may be added. In the reaction solution, the amount of each component added is not particularly limited. As a specific example, the pretreated biomass (dry weight) is, for example, 1 to 50 w / v%, preferably 10 to 30 w / v%. In the reaction solution, the content of the first saccharification promoter is, for example, 0.01 to 50 v / v%, preferably 0.1 to 10 v / v% in the culture supernatant. In the reaction solution, the content of the cellulase is, for example, 0.01 to 50 FPU / mL, and preferably 0.1 to 10 FPU / mL.

  Then, a catalytic reaction with cellulase is performed in the reaction solution. The reaction conditions are not particularly limited, and can be appropriately determined according to, for example, the type of cellulase. As a specific example, reaction temperature is 15-90 degreeC, for example, Preferably it is 25-60 degreeC, and reaction time is 0.5-120 hours, for example, Preferably it is 8-72 hours.

  By such saccharification treatment, for example, the ratio of non-solubilized holocellulose when treated only with cellulase without using the first saccharification accelerator is further reduced to a value of 30 to 70%, for example. Can be reduced.

<Second form>
As described above, the second saccharification promoter of the present invention is a saccharification promoter that promotes saccharification of biomass by cellulase, and comprises a protein comprising the amino acid sequence represented by at least any one of SEQ ID NOs: 8 to 14. It is characterized by including. The second saccharification promoter of the present invention is the same as the first saccharification promoter of the present invention unless otherwise specified.

  The 2nd saccharification promoter of this invention may contain any one type among the proteins containing the amino acid sequence represented by said sequence number 8-14, and may contain 2 or more types, for example. The proteins containing the amino acid sequences represented by SEQ ID NOs: 8 and 9 are pectinases, respectively, and the proteins containing the amino acid sequences represented by SEQ ID NOs: 10-14 are hemicellulases, and have xylanase activity.

  When two or more kinds of the above proteins are used in combination, the combination is not particularly limited. For example, a protein including the amino acid sequence represented by SEQ ID NO: 8 and a protein including the amino acid sequence represented by SEQ ID NO: 9 are used in combination. It is preferable. In addition, any two or more of the proteins containing the amino acid sequences represented by SEQ ID NOs: 10 to 14 may be used in combination, and preferably all of them are used in combination. You may use together at least 1 type of the protein containing the amino acid sequence represented by the said sequence number 8 and the sequence number 9, and at least 1 type of protein containing the amino acid sequence represented by the said sequence numbers 10-14.

The protein containing the amino acid sequence represented by each SEQ ID NO can be generated, for example, by preparing a DNA consisting of a base sequence in which the amino acid sequence is replaced by a codon, and performing transcription and translation in vivo or in vitro. Moreover, as shown below, it can also isolate from various microbial cells, for example. Specifically, the protein consisting of the amino acid sequence represented by SEQ ID NO: 9 is, for example, from Aspergillus niger strain DMS1957, and the protein consisting of the amino acid sequence represented by SEQ ID NO: 9 is from Aspergillus niger strain SCTCC 4000026, from SEQ ID NO: 10 to The protein consisting of each amino acid sequence represented by 14 can be isolated from Aspergillus niger CBS 513.88.

  Commercially available products can also be used. Examples of the protein containing the amino acid sequence represented by at least one of SEQ ID NO: 8 and SEQ ID NO: 9 include trade name pectinase G Amano (Amano Enzyme), As a protein containing the amino acid sequence represented by at least one of SEQ ID NOs: 10 to 14, for example, trade name hemicellulase “Amano” 90 (Amano Enzyme) can be used.

  Next, as described above, the second saccharification treatment agent of the present invention is a saccharification treatment agent for use in biomass saccharification treatment, comprising cellulase and the second saccharification promoter of the present invention. It is characterized by including. The second saccharification treatment agent of the present invention is the same as the first saccharification treatment agent of the present invention unless otherwise specified.

  The 2nd saccharification processing agent of this invention should just contain the said cellulase and the said 2nd saccharification promoter, and another structure is not restrict | limited at all. The second saccharification treatment agent of the present invention may further contain other components, and examples thereof include enzyme stabilizers such as saccharides, sugar alcohols, organic acids, and surfactants. Examples of the saccharide include D-glucose, D-galactose, D-mannose, D-sorbose, D-talose, D-fructose, D-ribose, D-xylose and other monosaccharides, lactose, saccharose, maltose, trehalose. Disaccharides such as isomaltose and cellobiose, maltotriose, maltotetraose, maltopentaose, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin and other oligosaccharides, inulin, amylose, amylopectin Water-soluble neutral polysaccharides such as dextrin and pullulan. Examples of the sugar alcohols include glucitol, mannitol, inositol, xylitol and the like. Examples of the organic acid include gluconic acid, citric acid, maleic acid, succinic acid, malic acid, lactic acid, acetic acid, and sodium or potassium salts thereof. Examples of the surfactant include alkyl glycosides such as octyl glycoside and octyl thioglycoside, cholic acids such as cholic acid, deoxycholic acid and lithocholic acid, sodium or potassium salts of cholic acids, polyethylene glycol ethers, polyoxy Examples include ethylene glycol ethers, lauroyl sarcosine, lauroyl sarcosine salts, lauryl sulfate, and lauryl sulfate.

  Next, the second saccharification treatment method of the present invention is a biomass saccharification treatment method, characterized in that the biomass is treated with cellulase in the presence of the second saccharification promoter of the present invention. The second saccharification treatment method of the present invention is the same as the first saccharification treatment method of the present invention unless otherwise specified. The present invention is characterized by cellulase treatment in the presence of the second saccharification promoter, and other configurations and conditions are not limited at all.

  Next, the second saccharification treatment method of the present invention will be described with an example.

  First, a reaction liquid containing the pretreated biomass obtained by treating the biomass material with pressurized hot water, the second saccharification accelerator, and cellulase is prepared. The reaction solution is preferably prepared by adding, for example, the biomass, the second saccharification promoter, and the cellulase to a solvent. Examples of the solvent include water and a buffer solution. Salts, acids, alkalis, ionic substances and the like may be added. In the reaction solution, the amount of each component added is not particularly limited, but the pretreated biomass (dry weight) is, for example, 1 to 50 w / v%, preferably 10 to 30 w / v%. . When the second saccharification accelerator contains hemicellulase, the content of the hemicellulase in the reaction solution is, for example, 1 to 10,000 U / mL as xylanase decomposition activity (Amano method), 10-3,000 U / mL. Further, when the second saccharification accelerator contains pectinase, the content of the pectinase in the reaction solution is, for example, 0.01 to 1000 U / mL as Endo-PG activity (Amano method), preferably 0.1-30 U / mL. Moreover, content of the said cellulase in the said reaction liquid is 0.01-50 FPU / mL, for example, Preferably it is 0.1-20 FPU / mL.

  Then, a catalytic reaction with cellulase is performed in the reaction solution. The reaction conditions are not particularly limited, and can be appropriately determined according to, for example, the type of cellulase, hemicellulase, or pectinase. As a specific example, reaction temperature is 15-90 degreeC, for example, Preferably it is 25-60 degreeC, and reaction time is 0.5-180 hours, for example, Preferably it is 8-72 hours.

  By such saccharification treatment, for example, the non-solubilized holocellulose ratio is, for example, a value that is reduced by 40 to 80% compared to the case where treatment is performed only with cellulase without using the second saccharification promoter. It can be reduced to.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples. Commercially available reagents were used based on their protocol unless otherwise indicated.

Construction of Recombinant Expression Vector Using pUC118 plasmid as a basic skeleton, an expression vector (pLHG) overexpressing the target gene was prepared. An outline of the structure of this expression vector is shown in FIG. As shown in FIG. 1, the expression vector is an Aspergillus oryzae- derived hemolysin promoter (hly promoter, SEQ ID NO: 1) as a promoter, and an Aspergillus oryzae- derived glaB terminator (glaB terminator, SEQ ID NO: 2). A structure having a leuA gene sequence (leuA, SEQ ID NO: 3) was used as a selection marker. And the target gene mentioned later in the said expression vector was inserted in the blunt end by the ligase process between the said promoter and terminator, and the recombinant expression vector was produced.

As a host strain for transformation and culture , Aspergillus oryzae leu-5 (FERM P-20079) lacking the β-isopropylmalate dehydrogenase gene, which is a leucine-requiring mutant, was used.

The recombinant expression vector was introduced into the leucine-requiring mutant strain by a protoplast method, and a transformant was selected using medium A having the following composition containing 1.5 w / v% agar. The spores of the selected transformant were suspended in 0.01% Tween (registered trademark) 80 aqueous solution. Then, the suspension was aseptically inoculated into a Sakaguchi flask (500 ml) containing medium B, medium C or medium D having the following composition so as to be 1.0 × 10 ⁶ sports / mL. The Sakaguchi flask was incubated for 24 to 240 hours under the conditions of 150 times / minute, amplitude of 50 mm, and 30 ° C. to perform culture. The culture broth was centrifuged to recover the supernatant, which was used as a sample for the examples. Moreover, the supernatant was similarly collected using the expression vector into which the target gene was not inserted. This was used as a sample for a comparative example.

(Medium A)
1.5 w / v% purified agar (Nacalai Tesque)
2 w / v% glucose 0.3 w / v% NaNO ₃
0.1 w / v% KH ₂ PO _{4
0.05w / v% MgSO 4 · 7H} 2 O
0.2w / v% KCl
0.001 w / v% FeCl ₃
0.8 mol / L NaCl
(Medium B)
2w / v% glucose 1w / v% polypeptone 0.5w / v% yeast extract (medium C)
2w / v% Birch-derived xylan
(Brand name Xylan from Birchwood, Sigma)
1 w / v% polypeptone (medium D)
2w / v% Oat-derived xylan
(Brand name Xylan from oat spells, Sigma)
1w / v% polypeptone 0.5w / v% yeast extract

For pre-treated biomass biomass material 1 (rice straw produced in Hyogo Prefecture), biomass material 2 (Okinawa bagasse), biomass material 3 (Hokkaido corn stove and soft biomass such as switchgrass from America) A pretreated biomass 1, a pretreated biomass 2 and a pretreated biomass 3 mainly composed of lignocellulose subjected to pressurized hot water treatment were used. The pretreated biomass 1 has a holocellulose content of 15.4 w / v% and a lignin content of 5.1 w / v%. The holocellulose has a cellulose content of 82.9 w / v% and a hemicellulose content of 17 It was 1 w / v%. The pretreated biomass 2 has a holocellulose content of 19.1 w / v%, a lignin content of 8.2 w / v%, and the holocellulose has a cellulose content of 73.1 w / v% and a hemicellulose content of 26 It was 9 w / v%. The pretreated biomass 3 has a holocellulose content of 17.3 w / v% and a lignin content of 6.8 w / v%. The holocellulose has a cellulose content of 78.3 w / v% and a hemicellulose content of 21. 0.7 w / v%.

[Example 1]
Samples of Examples were prepared as described above using an expression vector into which the Aspergillus oryzae xynR gene shown in SEQ ID NO: 4 was inserted as the target gene. Moreover, the sample of the comparative example was prepared as described above using the expression vector into which the xynR gene was not inserted. A reaction solution (about 1000 μL) having the following composition was prepared using each sample. In the following composition, cellulase is a commercially available cellulase (trade name cellulase GC220, Genencor, 160 FPU / mL), xynR (+) (Birch) is an xyR gene obtained using the medium C containing Birch-derived xylan. Example sample xynR (+) (Oat) expressed in the sample sample obtained by using the medium D containing Oat-derived xylan, xynR (-) (Birch) Is a comparative sample obtained by using the medium C containing Birch-derived xylan and not expressing the xyR gene, xynR (-) (Oat) using the medium D containing Oat-derived xylan The obtained comparative example sample that does not express the xynR gene, AcONa is a sodium acetate aqueous solution. A (hereinafter the same). The reaction solution was stirred at 37 ° C. for 72 hours at 500 rpm, and then the amount of holocellulose (total of homocellulose and hemicellulose) was measured by the Wise method for each reaction solution. The residual ratio of holocellulose was calculated for each reaction solution with the amount of holocellulose in Comparative Example 1-1 without addition of cellulase and without addition of sample as 100%. These results are shown in Table 1 below.

  As shown in Table 1, an example sample xynR (+) (Birch) or xynR (overexpressed cellulase and xynR gene compared to cellulase alone Comparative Example 1-2 (residual rate 34.8%) In Examples 1-1 and 1-2 in combination with (+) (Oat), the residual ratio could be reduced to 32.5% and 25.3%, respectively. Thus, since the saccharification was accelerated | stimulated by the sample of the culture supernatant which expressed the xynR gene, it turned out that these samples are useful as a saccharification promoter. Further, in solubilization of biomass, it is important to make the residual rate of holocellulose 30% or less in consideration of ethanol fermentation and distillation steps. Therefore, in particular, the sample xynR (+) (Oat) used in Example 1-2, that is, the saccharification accelerator obtained by excessively reacting the xynR gene in the presence of Oat-derived xylan was combined with cellulase to produce holocellulose. Since the residual ratio can be realized to 30% or less, it can be said that the combined use effect with cellulase is extremely excellent. In addition, Example 1-2 using the saccharification accelerator xynR (+) (Oat) showed a residual rate equivalent to that of Comparative Example 1-3 using 10 times the amount of cellulase. When the final product is bioethanol, cellulase is generally said to account for about 50% of the cost in biomass solubilization. For this reason, if the saccharification accelerator xynR (+) (Oat) is used, the amount of cellulase used can be reduced to 1/10, so that the production cost can be reduced to 55% in calculation. Furthermore, when compared with Comparative Example 1-4 (residual rate 38.1%) and Comparative Example 1-5 (39.5%), which are cellulases alone and xynR is not expressed, Examples 1-1 and 1-2 Each could reduce the residual rate. From this, it was revealed that saccharification was promoted by the culture supernatant in which the xynR gene was overexpressed.

[Example 2]
In this example, the residual rate of holocellulose was calculated in the same manner as in Example 1 except that the pretreated biomass 2 was used as the pretreated biomass. The results are shown in Table 2 below.

  As shown in Table 2, an example sample xynR (+) (Birch) or xynR in which cellulase and the xynR gene were overexpressed as compared to cellulase-only comparative example 2-2 (residual rate 34.2%) In Examples 2-1 and 2-2 in which (+) (Oat) was used in combination, the residual ratio could be reduced to 30% or less, that is, 26.1% and 29.4%, respectively. In addition, the sample xynR (+) (Birch) used in Example 2-2, that is, the saccharification accelerator obtained by excessively reacting the xynR gene in the presence of Birch-derived xylan was combined with cellulase in a 10-fold amount of cellulase. The residual rate equivalent to that of Comparative Example 2-3 using Further, even when compared with Comparative Example 2-4 (residual rate 33.1%) and Comparative Example 2-5 (32.5%), which are cellulases alone and xynR is not expressed, Examples 2-1 and 2-2 Each could reduce the residual rate. From this, it was revealed that saccharification was promoted by the culture supernatant in which the xynR gene was overexpressed.

[Example 3]
In this example, the residual rate of holocellulose was calculated in the same manner as in Example 1 except that the pretreated biomass 2 was used as the pretreated biomass. The results are shown in Table 3 below.

  As shown in Table 3, the sample samples xynR (+) (Birch) or xynR in which cellulase and the xynR gene were overexpressed as compared to cellulase-only comparative example 3-2 (residual rate 32.1%) In Examples 3-1 and 3-2 in combination with (+) (Oat), the residual ratio could be reduced to 30% or less, that is, 29.1% and 25.4%, respectively. In particular, the sample xynR (+) (Oat) used in Example 3-2 showed a residual rate equivalent to that of Comparative Example 3-3 using 10 times the amount of cellulase when used in combination with cellulase. Further, even when compared with Comparative Example 3-4 (residual rate 32.5%) and Comparative Example 3-5 (33.1%), which are cellulase alone and not expressed with xynR, Examples 3-1 and 3-2 were used. Each could reduce the residual rate. From this, it was revealed that saccharification was promoted by the culture supernatant in which the xynR gene was overexpressed.

[Example 4]
In this example, the residual ratio of holocellulose was calculated in the same manner as in Example 1 except that the reaction liquid (about 150 μL) shown in Table 4 below was used and the stirring speed of the reaction liquid was 2500 rpm. The results are shown in Table 4 below.

  As shown in Table 4, an example sample xynR (+) (Birch) or xynR in which cellulase and the xynR gene were overexpressed as compared with the comparative example 4-2 of cellulase alone (residual rate 34.1%) In Examples 4-1 and 4-2 using (+) (Oat) in combination, the residual ratio could be reduced to 30% or less, that is, 29.3% and 28.4%, respectively. In particular, the sample xynR (+) (Oat) of Example 4-2 exhibited a residual rate equivalent to that of Comparative Example 4-3 using 10 times the amount of cellulase when used in combination with cellulase. Furthermore, when compared with Comparative Example 4-4 (residual rate: 34.5%) and Comparative Example 4-5 (35.1%), which are cellulases alone and xynR is not expressed, Examples 4-1 and 4-2 Each could reduce the residual rate. From this, it was revealed that saccharification was promoted by the culture supernatant in which the xynR gene was overexpressed.

[Example 5]
As the target gene, an expression vector into which an Aspergillus oryzae ferulic acid esterase gene (fae gene) was inserted was prepared. The fae gene was inserted with the full length sequence of the faeA gene shown in SEQ ID NO: 5, the faeB gene shown in SEQ ID NO: 6, and the faeC gene shown in SEQ ID NO: 7. Then, using these expression vectors, using the medium B as a medium, the sample of the example was prepared as described above. In addition, as described above, a sample for a comparative example was prepared using the expression vector into which the fae gene was not inserted. A reaction solution (about 1000 μL) having the following composition was prepared using each sample. In the following composition, the cellulase is a commercially available cellulase (trade name Cellulase GC220, Genencor Corporation, 160 FPU / mL), faeA (+), faeB (+) and faeC (+) are the faeA gene, faeB gene and faeC, respectively. The expressed example sample, fae (−) is a comparative sample in which any fae gene is not expressed, and AcONa is a sodium acetate aqueous solution (hereinafter the same). And about the said reaction liquid, it carried out similarly to the said Example 1, and measured the amount of holocellulose. The residual ratio of holocellulose was calculated for each reaction solution with the amount of holocellulose in Comparative Example 5-1 without addition of cellulase and sample as 100%. The results are shown in Table 5 below.

  As shown in Table 5, the sample samples faeA (+) or faeC (+) in which cellulase and various fae genes were overexpressed as compared with Comparative Example 5-2 (residual rate 34.2%) of cellulase alone. In Examples 5-1 and 5-2, which were used in combination, the residual ratio could be reduced to 29.2%, 34.4%, and 31.6%, respectively. Thus, since the saccharification was accelerated | stimulated by the sample of the culture-medium supernatant which expressed the fae gene, it turned out that these samples are useful as a saccharification promoter. Further, as described above, in solubilization of biomass, it is important to set the holocellulose residual ratio to 30% or less. For this reason, in particular, the sample faeA (+) used in Example 5-1, that is, the saccharification accelerator obtained by overreacting the faeA gene, can achieve a holocellulose residual ratio of 30% or less in combination with cellulase. Therefore, it can be said that the combined effect with cellulase is extremely excellent. Further, Example 5-1 using the saccharification accelerator faeA (+) showed a residual rate equivalent to that of Comparative Example 5-3 using 3 times the amount of cellulase. When the final product is bioethanol, cellulase is said to account for approximately 50% of the cost in biomass solubilization as described above. For this reason, if the saccharification accelerator faeA (+) is used, the amount of cellulase used can be reduced to one third, so that the production cost can be reduced to 66.7% in calculation. Furthermore, even when compared with Comparative Example 5-4 (residual rate 34.5%) which is cellulase alone and does not express fae, Examples 5-1 and 5-2 were able to reduce the residual rate, respectively. This revealed that saccharification was promoted by the culture supernatant in which the fae gene was overexpressed.

[Example 6]
In this example, the residual rate of holocellulose was calculated in the same manner as in Example 5 except that the pretreated biomass 2 was used as the pretreated biomass. The results are shown in Table 6 below.

  As shown in Table 6 above, sample samples faeA (+), faeB (+) in which cellulase and various fae genes were overexpressed, compared to Comparative Example 6-2 (residual rate 34.8%) of cellulase alone. ) Or faeC (+) in Examples 6-1, 6-2 and 6-3, the residual ratio could be reduced to 29.8%, 32.3% and 29.3%, respectively. . Example 6-3 using the saccharification accelerator faeC (+) showed a residual rate equivalent to that of Comparative Example 6-3 using 3 times the amount of cellulase. Furthermore, even when cellulase alone and fae non-expressing Comparative Example 6-4 (residual rate 34.5%) were compared with Examples 6-1, 6-2, and 6-3, respectively, the residual rate was reduced. did it. This revealed that saccharification was promoted by the culture supernatant in which the fae gene was overexpressed.

[Example 7]
In this example, the residual rate of holocellulose was calculated in the same manner as in Example 5 except that the pretreated biomass 3 was used as the pretreated biomass. The results are shown in Table 7 below.

  As shown in Table 7, the sample samples faeA (+) and faeB (+) in which cellulase and various fae genes were overexpressed, compared to Comparative Example 7-2 (residual rate 32.3%) of cellulase alone. ) Or faeC (+) in Examples 7-1, 7-2 and 7-3, the residual ratio could be reduced to 30.8%, 29.3% and 29.8%, respectively. . Example 7-2 using the saccharification accelerator faeB (+) showed a residual rate equivalent to that of Comparative Example 7-3 using 1.5 times the amount of cellulase. Furthermore, even when compared with Comparative Example 7-4 (residual rate 32.9%), which is cellulase alone and does not express fae, Examples 7-1, 7-2, and 7-3 each reduce the residual rate. did it. This revealed that saccharification was promoted by the culture supernatant in which the fae gene was overexpressed.

[Example 8]
In this example, the residual ratio of holocellulose was calculated in the same manner as in Example 5 except that the reaction liquid (about 150 μL) shown in Table 8 below was used and the stirring speed of the reaction liquid was 2500 rpm. The results are shown in Table 8 below.

  As shown in Table 8, the sample samples faeA (+) and faeB (+) in which cellulase and various fae genes were overexpressed, compared to Comparative Example 8-2 (residual rate 33.9%) of cellulase alone. ) Or faeC (+) in Examples 8-1, 8-2 and 8-3, the residual ratio could be reduced to 29.3%, 33.4% and 29.8%, respectively. . Example 8-1 using the saccharification accelerator faeA (+) showed a residual rate equivalent to that of Comparative Example 8-3 using 3 times the amount of cellulase. Furthermore, even in comparison with Comparative Example 8-4 (residual rate: 34.5%), which is cellulase alone and does not express fae, Examples 8-1, 8-2, and 8-3 each reduce the residual rate. did it. This revealed that saccharification was promoted by the culture supernatant in which the fae gene was overexpressed.

[Example 9]
The amount of holocellulose was measured in the same manner as in Example 1 except that the reaction solution (about 1000 μL) having the composition shown in Table 9 and Table 10 below was used, and holocellulose of Comparative Example 9-1 without addition of cellulase. The residual rate of holocellulose was calculated for each reaction solution with the amount as 100%. The results are shown in Table 10 below.

In Table 9 and Table 10 below, the pectinase is 10 w / v% pectinase (10 mg / L pectinase derived from Aspergillus (trade name: Pectinase G Amano, Amano Enzyme) diluted with 0.1 mol / L sodium acetate aqueous solution (pH 5). 1-fold dilution), 1 w / v% pectinase (100-fold dilution), 0.1 w / v% pectinase (1000-fold dilution) were used. The pectinase G Amano had an Endo-PGase value by the Amano method of 1200 U / g or more. In Table 9 and Table 10 below, hemicellulase was prepared by diluting Aspergillus- derived hemicellulase (trade name: hemicellulase “Amano” 90, Amano Enzyme) with 0.1 mol / L sodium acetate aqueous solution (pH 5). 10 w / v% hemicellulase (diluted 10 times), 1 w / v% hemicellulase (diluted 100 times), 0.1 w / v% hemicellulase (diluted 1000 times) were used. The hemicellulase G Amano had an xylase power of 90000 U / g or more according to the Amano method. In Table 9 below, the cellulase is Cellulase GC220 (trade name, Genencor, 160 FPU / mL). The concentrations of pectinase and hemicellulase in Table 10 are final concentrations in the reaction solution.

  As shown in Table 10, in Examples 9-1, 9-2, and 9-3, by using pectinase and cellulase together, the residual rate of cellulase alone in Comparative Example 9-2 was 34.1%. Furthermore, it could be reduced to 24.8%, 26.5% and 33.5%. Moreover, Example 9-1 was equivalent to the residual rate of 25.2% of Comparative Example 9-3 using 10 times the amount of cellulase. Moreover, as shown in the said Table 10, in Example 9-4, 9-5, and 9-6, the residual rate 34.1 in the comparative example 9-2 of cellulase single by using hemicellulase and cellulase together. % Could be further reduced to 23.9%, 25.9% and 31.9%. Moreover, compared with 25.2% of the residual rate of Comparative Example 9-3 using 10 times the amount of cellulase, Example 9-4 has a low residual rate, and Example 9-5 has an equivalent residual rate. showed that.

The Aspergillus- derived hemicellulase Amano 90 and the Aspergillus- derived pectinase G Amano and the enzyme protein contained therein were subjected to LC-MSMS analysis, and the resulting ion peak pattern was subjected to MASCOT analysis and NCBI database analysis. And identification.

As a result, it was found that the hemicellulase Amano 90 includes a protein consisting of the amino acid sequence represented by SEQ ID NO: 8 and a protein consisting of the amino acid sequence represented by SEQ ID NO: 9 as two main types of proteins. The amino acid sequence represented by SEQ ID NO: 8 is GenBank Accession No. It was homologous to the amino acid sequence of Aspergillus niger strain DMS1957 endo-1,4-beta-xylanase A registered in EU — 848304. The amino acid sequence represented by SEQ ID NO: 9 is GenBank Accession No. It was homologous with the amino acid sequence of Aspergillus niger strain SCTCC 40000264 xylanase B registered in FJ772090.

The pectinase G amano is represented by the protein consisting of the amino acid sequence represented by SEQ ID NO: 10, the protein comprising the amino acid sequence represented by SEQ ID NO: 11, the protein comprising the amino acid sequence represented by SEQ ID NO: 12, and the sequence SEQ ID NO: 13. It was found to include a protein consisting of an amino acid sequence and a protein consisting of an amino acid sequence represented by SEQ ID NO: 14. The amino acid sequence represented by SEQ ID NO: 10 is GenBank Accession No. It is homologous to the amino acid sequence of Aspergillus niger CBS 513.88 detect lyase plyA registered in XM_001402441. The amino acid sequence shown in SEQ ID NO: 11 is GenBank accession no. It is homologous to the amino acid sequence of Aspergillus niger CBS 513.88 pectin lyase pelB registered in XM — 001389889, and the amino acid sequence shown in SEQ ID NO: 12 is GenBank Accession No. The amino acid sequence shown in SEQ ID NO: 13 is homologous to the amino acid sequence of Aspergillus niger CBS 513.88 endo-polygalacturonase pgaA registered in XM — 001397963. The amino acid sequence shown in SEQ ID NO: 14 is homologous to the amino acid sequence of Aspergillus niger CBS 513.88 endopolygalacturoneses pgaB registered in XM_001399591. It was homologous to the amino acid sequence of Aspergillus niger CBS 513.88 peptidesterase pmeA registered in XM — 001390469.

  As described above, according to the present invention, biomass can be saccharified with higher efficiency than saccharification treatment using cellulase alone. For this reason, it can be said that it is extremely useful because it can effectively use wood or its waste as biomass and provide energy resources and chemical products. In addition, according to the present invention, for example, the amount of cellulase used in the saccharification treatment can be reduced, so that the cost of biomass saccharification treatment, which is important in subsequent steps such as fermentation, can be reduced.

Claims (7)

A saccharification accelerator that promotes saccharification of biomass by cellulase ,
Glycation promoting agent characterized in that the faeA gene transformants expression vector has been introduced having a fae A gene from Aspergillus oryzae comprises overexpressed culture supernatant. The saccharification promoting agent according to claim 1, wherein the transformant belongs to the genus Aspergillus . The saccharification promoter according to claim 1 or 2 , wherein the transformant is Aspergillus oryzae . A saccharification treatment agent for use in biomass saccharification treatment,
A saccharification treatment agent comprising cellulase and the saccharification promoter according to any one of claims 1 to 3 . A method for saccharification of biomass,
A saccharification treatment method, wherein the biomass is treated with cellulase in the presence of the saccharification accelerator according to any one of claims 1 to 3 . The saccharification processing method of Claim 5 whose said biomass is lignocellulosic biomass. The saccharification processing method according to claim 5 or 6 , wherein the biomass is pretreated biomass obtained by pretreating a biomass raw material with pressurized hot water.

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