WO2015076260A1 - Xylanase résistant à la chaleur - Google Patents

Xylanase résistant à la chaleur Download PDF

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Publication number
WO2015076260A1
WO2015076260A1 PCT/JP2014/080514 JP2014080514W WO2015076260A1 WO 2015076260 A1 WO2015076260 A1 WO 2015076260A1 JP 2014080514 W JP2014080514 W JP 2014080514W WO 2015076260 A1 WO2015076260 A1 WO 2015076260A1
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Prior art keywords
xylanase
amino acid
encoding
protein
positions
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PCT/JP2014/080514
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Japanese (ja)
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高橋　慶
忠弘 小澤
望 柴田
大視 掛下
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花王株式会社
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Publication of WO2015076260A1 publication Critical patent/WO2015076260A1/fr

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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)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases

Definitions

  • the present invention relates to a xylanase having heat resistance.
  • biomass a biomass material containing cellulose
  • biomass a biomass material containing cellulose
  • Biomass is composed of cellulose fibers and hemicellulose and lignin mainly containing xylan surrounding them. In order to increase the saccharification efficiency of cellulose and hemicellulose in biomass, it is necessary to develop an enzyme that hydrolyzes cellulose and hemicellulose. It is also necessary to remove lignin from the biomass.
  • Patent Document 1 Japanese Patent Publication No. 2011-515089
  • Patent Document 2 Japanese Patent Application Laid-Open No. 2012-029678
  • the present invention relates to the following (1) to (6).
  • a xylanase selected from proteins represented by the following (a) to (c): (A) in the amino acid sequence represented by SEQ ID NO: 2, any one or more amino acids of alanine at position 77, serine at position 212, alanine at position 214, glutamine at position 217, serine at position 323, and alanine at position 328 The residue is the amino acid residue: 77th: Proline 212: Alanine 214: Proline 217: Proline 323: Asparagine 328: A protein comprising an amino acid sequence substituted with proline and having xylanase activity.
  • (B) consisting of an amino acid sequence of the protein of (a) and an amino acid sequence having 90% or more identity other than the amino acids at positions 77, 212, 214, 217, 323, and 328, and A protein having xylanase activity.
  • C In the amino acid sequence of the protein of (a), one or several amino acids are deleted or substituted at positions other than the amino acids at positions 77, 212, 214, 217, 323, and 328.
  • a protein comprising an added amino acid sequence and having xylanase activity.
  • the present invention relates to providing a xylanase that can saccharify biomass more efficiently and is excellent in thermal stability.
  • the present inventors have found a novel xylanase derived from bacteria belonging to the genus Cellulomonas that can exhibit high xylanase activity in a wide pH range, and have already filed a patent application (Japanese Patent Application No. 2012-119034). As a result of further investigation, it was found that the thermal stability can be improved by substituting the amino acid residue at a predetermined position of the xylanase with another amino acid residue.
  • the xylanase of the present invention has a wide range of applications because it acts over a relatively wide range of pH conditions and has excellent xylanase activity at 50-65 ° C. Therefore, if this is used, biomass can be efficiently saccharified even under severe conditions.
  • amino acid residue refers to 20 amino acid residues constituting a protein, Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val are meant.
  • identity refers to the number of full-length amino acid residues in the number of positions where identical amino acid residues are present in both sequences when the two amino acid sequences are aligned. The ratio (%) to the number. Specifically, it is calculated by the Lippman-Person method (Lipman-Pearson method; Science, 227, 1435, (1985)) and homology of genetic information processing software Genetyx-Win (Ver. 5.1.1; software development). Using an analysis (Search homology) program, it can be calculated by performing an analysis with Unit size to compare (ktup) of 2.
  • xylanase activity refers to an activity of hydrolyzing a xylose ⁇ -1,4-glycoside bond in xylan.
  • the xylanase activity of a protein can be determined, for example, by reacting with the protein using xylan as a substrate and measuring the amount of xylan degradation product produced. Specific procedures for measuring xylanase activity are described in detail in the examples below.
  • amino acid residue at the corresponding position is identified by comparing the target amino acid sequence with a reference sequence using a known algorithm, and maximizing the conserved amino acid residue present in the amino acid sequence of each xylanase. This can be done by aligning the sequences so as to confer homology. By aligning the amino acid sequence of xylanase in this way, it is possible to determine the position of the homologous amino acid residue in the sequence of each xylanase regardless of insertion or deletion in the amino acid sequence.
  • the alignment can be performed manually based on, for example, the above-described Lippmann-Person method, but the Clustal W multiple alignment program (Thompson, JD et al, (1994) Nucleic Acids Res. 22, p. 4673-4680) with default settings.
  • Clustal W is, for example, the European Bioinformatics Institute (EBI, [www.ebi.ac.uk/index.html]) and the Japan DNA Data Bank (DDBJ, [DDBJ, [ www.ddbj.nig.ac.jp/Welcome-j.html]).
  • an optimal alignment is preferably determined in consideration of amino acid sequence similarity, frequency of inserted gaps, and the like.
  • the similarity of amino acid sequences means the ratio (%) of the number of positions where the same or similar amino acid residues exist in both sequences when two amino acid sequences are aligned to the total number of amino acid residues.
  • a similar amino acid residue means an amino acid residue having properties similar to each other in terms of polarity and charge among 20 kinds of amino acids constituting a protein and causing so-called conservative substitution.
  • Such groups of similar amino acid residues are well known to those skilled in the art and include, for example, arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; valine, leucine and isoleucine, respectively. However, it is not limited to these.
  • the position of the amino acid residue of the target amino acid sequence aligned with the position corresponding to the arbitrary position of the reference sequence by the above alignment is regarded as the “corresponding position” to the arbitrary position, and the amino acid residue is The amino acid residue at the position ".
  • the xylanase of the present invention includes proteins represented by the following (a) to (c).
  • the residue is the amino acid residue: 77th: Proline 212: Alanine 214: Proline 217: Proline 323: Asparagine 328: A protein comprising an amino acid sequence substituted with proline and having xylanase activity.
  • (B) consisting of an amino acid sequence of the protein of (a) and an amino acid sequence having 90% or more identity other than the amino acids at positions 77, 212, 214, 217, 323, and 328, and A protein having xylanase activity.
  • (C) In the amino acid sequence of the protein of (a), one or several amino acids are deleted or substituted at positions other than the amino acids at positions 77, 212, 214, 217, 323, and 328.
  • a protein comprising an added amino acid sequence and having xylanase activity.
  • amino acid sequence represented by SEQ ID NO: 2 represented by (a) include the xylanase derived from bacteria belonging to the genus Cellulomonas, specifically, the amino acid sequence derived from Cellulomonas fimi ATCC484-derived xylanase (Japanese Patent Application No. 2012-1119034).
  • amino acids at position 77 (77th from the N-terminal), position 214 (214th from the N-terminal) and position 328 (position 328 from the N-terminal) are alanine, and position 212
  • the amino acid at position 212 (position 212 from the N-terminus) and position 323 (position 323 from position N) is serine, and the amino acid at position 217 (position 217 from position N) is glutamine.
  • the xylanase shown in (a) is based on the xylanase consisting of the amino acid sequence shown in SEQ ID NO: 2 as a reference xylanase, and the xylanase has 77th alanine, 212th serine, 214th alanine, 217th glutamine, 323 Any one or more amino acid residues of serine at position and alanine at position 328 are as follows: 77th: Proline, 212: Alanine, 214: Proline, 217: Proline, 323: Asparagine, 328: Proline, Is a mutant (also referred to as mutant xylanase).
  • the amino acid residue substitution may be naturally occurring or artificially introduced.
  • Such a mutant xylanase has improved heat resistance compared to the reference xylanase.
  • a mutant in which any one or more of alanine at positions 77, 214, and 328 is substituted with proline is preferable.
  • the xylanase activity is not impaired.
  • xylanases with mutations eg deletions, substitutions or additions
  • the mutation at the arbitrary position may be naturally occurring or artificially introduced.
  • amino acid sequence having 90% or more identity with the amino acid sequence of the protein of (a) other than the amino acids at positions 77, 212, 214, 217, 323, and 328 Is 90% or more, preferably 95% of the remaining amino acid sequence excluding the amino acids at positions 77, 212, 214, 217, 323, and 328 in the amino acid sequence of the protein (a) More preferably, amino acid sequences having identity of 96% or more, 97% or more, 98% or more, or 99% or more are mentioned.
  • the xylanase represented by (b) has a protein having xylanase activity consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 as a reference xylanase at position 77 of SEQ ID NO: 2.
  • substitution with alanine at the corresponding position as proline substitution with serine at the position corresponding to position 212, substitution with alanine at the position corresponding to position 214, and glutamine at the position corresponding to position 217 with proline
  • An amino acid sequence having at least one amino acid substitution selected from the substitution with serine at the position corresponding to position 323 asparagine and the substitution with alanine at position 328 as proline, and xylanase activity are included.
  • amino acid sequence of the protein of (a) an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the amino acids at positions 77, 214 and 328
  • 1 to 10 amino acids preferably Examples include amino acid sequences in which 1 to 5, more preferably 1 to 3, more preferably 1 to 2, are deleted, substituted, or added.
  • amino acid deletion means deletion or disappearance of an amino acid residue in the sequence
  • amino acid substitution means that an amino acid residue in the sequence is replaced with another amino acid residue.
  • amino acid addition is meant that an amino acid residue has been added.
  • ition includes insertion of another amino acid residue between amino acids in the sequence.
  • xylanase represented by (c) a protein having a xylanase activity consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added to the amino acid sequence represented by SEQ ID NO: 2 is used as a reference xylanase.
  • Substitution of alanine at position 77 corresponding to SEQ ID NO: 2 as proline, substitution at position 212 corresponding to serine as alanine, substitution at position 214 corresponding to alanine as proline, position 217 At least one amino acid substitution selected from a substitution with glutamine at the position corresponding to as a proline, a substitution at position 323 with asparagine at a position corresponding to position 323, and a substitution with alanine at a position corresponding to position 328 as a proline.
  • a protein comprising an amino acid sequence having xylanase activity
  • Arbitrary mutations made in the protein having xylanase activity in (b) and (c) above are, for example, ultraviolet irradiation or site-directed mutagenesis (Site-Directed Mutagenesis) to the gene encoding the target xylanase. It can be produced by introducing a mutation by a known mutagenesis method such as, expressing a gene having the mutation, and selecting a protein having a desired xylanase activity. The procedure for producing such a mutant is well known to those skilled in the art.
  • the gene encoding the xylanase of the present invention is a gene encoding the protein shown in the above (a) to (c), and specific examples thereof include the following genes (d) to (f).
  • the invention is not limited to this.
  • the “gene encoding xylanase” means any nucleic acid fragment (including DNA, mRNA, artificial nucleic acid, etc.) encoding the amino acid sequence of xylanase.
  • the “gene” according to the present invention may include other base sequences such as an untranslated region (UTR) in addition to the open reading frame.
  • a base encoding an alanine at positions 229 to 231 of SEQ ID NO: 1 a base encoding a serine at positions 634 to 636, and an alanine at positions 640 to 642
  • At least one of the bases encoding glutamine at positions 649 to 651, the base encoding serine at positions 967 to 969, and the base encoding alanine at positions 982 to 984 are the following amino acids: Encoding base; Positions 229 to 231: Bases encoding proline 634 to 636: Bases encoding alanine 640 to 642: Bases encoding proline 649 to 651: Bases encoding proline 967 to 969: Asparagine-encoding bases 982 to 984: A gene encoding a protein having a base sequence substituted with a proline-encoding base and having xylanase activity.
  • the xylanase of the present invention can be obtained, for example, by the following method. That is, the gene encoding the cloned reference xylanase is subjected to the mutation of the present invention using various mutagenesis techniques known in the art. For example, with respect to the gene encoding xylanase shown in SEQ ID NO: 1, the base sequence encoding the substitution target amino acid residue is mutated to the base sequence encoding the amino acid residue after substitution. Then, by transforming an appropriate host using the obtained mutant gene, culturing the recombinant host, and collecting from the culture, the mutation in which the amino acid residue to be replaced is replaced with the desired amino acid residue Xylanase can be obtained.
  • the gene encoding the reference xylanase can be isolated from bacteria belonging to the genus Cellulomonas, such as Cellulomonas fimi (ATCC 484), using any method used in the art.
  • the gene encoding the xylanase consisting of the amino acid sequence shown in SEQ ID NO: 2 is obtained by extracting the total genomic DNA of the Cellulomonas genus bacteria and then performing PCR using a primer designed based on the base sequence of SEQ ID NO: 1 Can be obtained by selectively amplifying and purifying the amplified gene.
  • the gene can be synthesized genetically or chemically based on the amino acid sequence of the xylanase of the present invention.
  • a means for mutating a gene encoding a reference xylanase basically, PCR amplification using a gene encoding a reference xylanase (for example, a DNA comprising the base sequence represented by SEQ ID NO: 1) as a template DNA or various DNA polymerases
  • site-directed mutagenesis methods well known to those skilled in the art can be used.
  • the site-directed mutagenesis method may be performed by any method such as inverse PCR method or annealing method (edited by Muramatsu et al., “Revised 4th edition Shinshin Genetic Engineering Handbook”, Yodosha, p. 82-88). Can do.
  • various commercially available site-specific mutagenesis kits such as QuickChange II Site-Directed Mutageness ⁇ Kit of Stratagene and QuickChange Multi Site-Directed Mutageness Kit can also be used.
  • the site-specific mutagenesis can be most commonly performed using a mutation primer containing a nucleotide mutation to be introduced.
  • a mutation primer is annealed to a region containing a nucleotide sequence encoding the amino acid residue to be substituted in the reference xylanase gene, and replaced in place of the nucleotide sequence (codon) encoding the amino acid residue to be substituted.
  • the base sequence which has the nucleotide sequence (codon) which codes a subsequent amino acid residue may be included.
  • Those skilled in the art can appropriately recognize and select a nucleotide sequence (codon) encoding a substitution target and a substituted amino acid residue based on a normal textbook.
  • Primers can be prepared by known oligonucleotide synthesis methods such as the phosphoramidite method (Nucleic® Acids® Research, 17, 7059-7071, 1989). Such primer synthesis can also be prepared using, for example, a commercially available oligonucleotide synthesizer (manufactured by ABI, etc.). By using a primer set containing a mutation primer and carrying out site-specific mutagenesis as described above using a reference xylanase gene as a template DNA, a gene encoding a xylanase into which the target mutation has been introduced can be obtained.
  • amino acids 77, 214 and 328 in the amino acid sequence shown in SEQ ID NO: 2 are substituted with proline using the recovered plasmid as a template.
  • a DNA fragment is amplified by PCR using an oligonucleotide containing a base sequence encoding the amino acid sequence as a primer.
  • a heat denaturation reaction for converting a double-stranded DNA into a single strand is performed at 98 ° C. for 10 seconds, and an annealing reaction for hybridizing the primer pair to the single-stranded DNA at 50 ° C.
  • An extension reaction for 15 seconds is carried out at 68 ° C. for 9 minutes, and one cycle of these is carried out for 40 cycles.
  • the DNA amplified by the PCR reaction is treated with a DpnI enzyme that specifically cleaves methylated DNA, E. coli is transformed with the enzyme, and selected on a plate medium containing antibiotics.
  • a plasmid from the transformed Escherichia coli, a gene encoding a xylanase into which the target amino acid mutation has been introduced can be obtained.
  • Production of xylanase using the obtained gene encoding the xylanase of the present invention can be performed by, for example, transforming a host bacterium by ligating the gene with a DNA vector capable of stably amplifying, or maintaining the mutated gene stably. And inoculating the host strain into a medium containing an assimilable carbon source, nitrogen source and other essential nutrients, and culturing according to a conventional method.
  • the type of the vector is not particularly limited, and vectors usually used for protein production such as plasmid, cosmid, phage, virus, YAC, BAC and the like can be mentioned.
  • a plasmid vector is preferable.
  • a commercially available plasmid vector for protein expression such as shuttle vector pHY300PLK (Takara Bio), pUC19 (Takara Bio), pUC119 (Takara Bio), pBR322 (Takara Bio) and the like can be suitably used.
  • the vector may include a DNA fragment containing a DNA replication initiation region and a DNA region containing a replication origin. Further, in the above vector, upstream of the gene encoding the xylanase of the present invention, a promoter region for initiating transcription of the gene, a secretory signal region for secreting the expressed protein outside the cell, etc.
  • the arrays may be operably linked.
  • a drug resistance gene ampicillin, neomycin, kanamycin, chloramphenicol, etc.
  • control sequences include S237egl promoter and signal sequence (Biosci. Biotechnol.
  • the xylanase gene can be linked to the above regulatory sequences and drug resistance gene by a method such as SOE (splicing by overlap extension) -PCR (Gene, 77, 61, 1989).
  • SOE splicing by overlap extension
  • -PCR Gene, 77, 61, 1989.
  • the procedure for introducing a gene into a plasmid vector is well known in the art.
  • the expression that the gene and the control sequence are “operably linked” means that the gene is arranged so that it can be expressed under the control of the control region.
  • microorganisms for introducing the constructed vector include Staphylococcus genus, Enterococcus genus, Listeria genus, bacteria belonging to the genus Bacillus, etc., among these, Bacillus genus bacteria such as Bacillus subtilis or mutants thereof (for example And a protease 9-deficient strain KA8AX described in JP-A-2006-174707) are preferred.
  • Bacillus genus bacteria such as Bacillus subtilis or mutants thereof (for example And a protease 9-deficient strain KA8AX described in JP-A-2006-174707) are preferred.
  • the introduction method a method usually used in the art such as a protoplast method and electroporation can be used. A strain into which the introduction has been appropriately carried out can obtain the desired transformant by selecting drug resistance or the like as an index.
  • the gene encoded on the vector contained in the transformant is expressed, and the xylanase of the present invention is produced.
  • the medium used for the culture can be appropriately selected by those skilled in the art according to the type of transformant.
  • the protein of the present invention can be obtained by isolating or purifying the produced xylanase from the culture by a conventional method. At this time, when the xylanase gene and the secretory signal sequence are operably linked on the vector, the produced xylanase is secreted outside the cell body, so that recovery becomes easier.
  • the recovered xylanase may be further purified by a known means.
  • biomass saccharification Since the xylanase of the present invention acts in a relatively wide range of pH conditions and has an excellent xylanase activity at 50 to 65 ° C. as shown in the examples described later, a composition containing this is used for biomass saccharification. It can be an enzyme preparation.
  • biomass refers to cellulosic and / or lignocellulosic biomass containing hemicellulose components produced by plants and algae.
  • biomass examples include various types of wood obtained from conifers such as larch and cedar, and broad-leaved trees such as oil palm (stems) and cypress; processed or pulverized products of wood such as wood chips; wood pulp produced from wood Pulp such as cotton linter pulp obtained from fibers around cotton seeds; Paper such as newspaper, cardboard, magazines, fine paper; bagasse (sugar cane residue), palm empty fruit bunch (EFB), rice straw Stalks of plants such as corn stalks or leaves, leaves, fruit bunches, etc .; plant shells such as rice husks, palm husks, coconut husks, algae and the like.
  • bagasse wood, processed or pulverized wood, plant stems, leaves, fruit bunches, etc. are preferred, bagasse, EFB, oil palm (stem) are more preferred, and bagasse is further. preferable. You may use the said biomass individually or in mixture of 2 or more types. Moreover, said biomass may be dried.
  • the enzyme preparation for biomass saccharification preferably further contains cellulase from the viewpoint of improving saccharification efficiency.
  • cellulase refers to an enzyme that hydrolyzes the glycosidic bond of ⁇ -1,4-glucan of cellulose, and is a generic term for enzymes called endoglucanase, exoglucanase or cellobiohydrolase, and ⁇ -glucosidase. It is.
  • commercially available cellulase preparations and cellulases derived from animals, plants and microorganisms may be mentioned. In the present invention, these cellulases may be used alone or in combination of two or more. From the viewpoint of improving saccharification efficiency, the cellulase preferably contains endoglucanase.
  • cellulases include cellulases derived from Trichoderma ; seisei; cellulases derived from Trichoderma viride; Bacillus sp. KSM-N145 (FERM P-19727), Bacillus sp. ) KSM-N252 (FERM P-17474), Bacillus sp. KSM-N115 (FERM P-19726), Bacillus sp. KSM-N440 (FERM P-19728), Bacillus sp.
  • Cellulases derived from various Bacillus strains such as KSM-N659 (FERM P-19730); thermostable cellulases derived from Pyrococcus horikoshii; Humicola insolens Cellulase of years, and the like.
  • KSM-N659 FERM P-19730
  • thermostable cellulases derived from Pyrococcus horikoshii derived from Pyrococcus horikoshii
  • Humicola insolens Cellulase of years and the like.
  • cellulases derived from Trichoderma reesei, Trichoderma vilide, or Humicola insolens are preferable.
  • cellulase preparations containing the above cellulase include Cellcrust (registered trademark) 1.5 L (manufactured by Novozymes), TP-60 (manufactured by Meiji Seika Co., Ltd.), Cellic (registered trademark) CTec2 (manufactured by Novozymes), Examples include Accelerase TM DUET (manufactured by Genencor) and Ultraflo (registered trademark) L (manufactured by Novozymes).
  • Cellic (registered trademark) CTec2 and AcceleraseTM DUET are preferable, and Cellic (registered trademark) CTec2 is more preferable from the viewpoint of improving saccharification efficiency, reducing manufacturing costs, and obtaining availability.
  • ⁇ -glucosidase which is a kind of cellulase
  • ⁇ -glucosidase derived from Aspergillus niger (for example, Novozymes 188 Novozymes 188 and Megazyme ⁇ -glucosidase), and Trichoderma lyase (Trichoderma ligase). reesei) or ⁇ -glucosidase derived from Penicillium emersonii.
  • Novozyme 188 and ⁇ -glucosidase derived from Trichoderma reease are preferable, and ⁇ -glucosidase derived from Trichoderma lyse is more preferable from the viewpoint of improving saccharification efficiency.
  • endoglucanase which is a kind of cellulase
  • Trichoderma reesei Acremonium celluloriticus, Humicola insolens, Clostridium thermocellum (Bacillus) ), Enzymes derived from Thermobifida, Cellulomonas, and the like.
  • Enzymes derived from Thermobifida Cellulomonas, and the like.
  • endoglucanase derived from Trichoderma reeze, Humicola insolens, Bacillus, Cellulomonas is preferable, and endoglucanase derived from Trichoderma reesei is more preferable.
  • the enzyme preparation of the present invention may contain a hemicellulase other than the xylanase of the present invention.
  • hemicellulase refers to an enzyme that hydrolyzes hemicellulose, and is a general term for enzymes called xylanase, xylosidase, galactanase, and the like.
  • Specific examples of hemicellulases other than the xylanase of the present invention include a hemicellulase derived from Trichoderma reesei; a xylanase derived from Bacillus sp.
  • KSM-N546 (FERM P-19729); an Aspergillus niger ), Trichoderma viride, Humicola insolens, or xylanase from Bacillus alcalophilus; Thermomyces, Aureobasidium, Streptomyces, Streptomyces (Clostridium), Thermomotoga, Thermoascus, Caldocellum, or Thermomonospora xylanase, Bacillus Pumilus (Bacillus pumilus) derived from ⁇ - xylosidase include Serenomonasu Ruminantiumu (Selenomonas ruminantium) from ⁇ - xylosidase.
  • the enzyme preparation of the present invention preferably contains a xylanase derived from Bacillus sp., Aspergillus niger, Trichoderma viride or Streptomyces, or ⁇ -xylosidase derived from Selenomonas luminantium, Alternatively, it is more preferable to contain Trichoderma viride-derived xinalase or Selenomonas luminantium-derived ⁇ -xylosidase.
  • the content of the xylanase of the present invention in the total protein amount of the enzyme preparation of the present invention is preferably 0.5% by mass or more, more preferably 1% by mass or more, further preferably 2% by mass from the viewpoint of improving saccharification efficiency.
  • the content of the cellulase in the total protein amount of the enzyme preparation of the present invention is preferably 10% by mass or more, more preferably 30% by mass or more, and further preferably 50% by mass or more, from the viewpoint of improving saccharification efficiency. And preferably 99% by mass or less, more preferably 95% by mass or less. Further, it is preferably 10 to 99% by mass, more preferably 30 to 95% by mass, and still more preferably 50 to 95% by mass. Further, the content of the endoglucanase in the total protein amount of the enzyme preparation of the present invention is preferably 1% by mass or more, more preferably 5% by mass or more, and further preferably 10% by mass from the viewpoint of improving saccharification efficiency.
  • 70% by mass or less more preferably 50% by mass or less, and still more preferably 40% by mass or less. Further, it is preferably 1 to 70% by mass, more preferably 5 to 50% by mass, and still more preferably 10 to 40% by mass. Further, the content of hemicellulase other than the xylanase of the present invention in the total protein amount of the enzyme preparation of the present invention is preferably 0.01% by mass or more, more preferably 0.8% from the viewpoint of improving saccharification efficiency. 1 mass% or more, More preferably, it is 0.5 or more mass%, Preferably it is 30 mass% or less, More preferably, it is 20 mass% or less. Further, it is preferably 0.01 to 30% by mass, more preferably 0.1 to 20% by mass, and further preferably 0.5 to 20% by mass.
  • the protein amount ratio of the xylanase of the present invention to the cellulase is preferably 0.01 or more, more preferably 0.05 or more, from the viewpoint of improving saccharification efficiency. And preferably 100 or less, more preferably 5 or less, still more preferably 1 or less, and still more preferably 0.5 or less. Further, it is preferably 0.01 to 100, more preferably 0.05 to 5, further preferably 0.05 to 1, and still more preferably 0.05 to 0.5.
  • the protein amount ratio of the xylanase of the present invention to the above endoglucanase is preferably 0.05 or more, more preferably 0.1, from the viewpoint of improving saccharification efficiency. And preferably 10 or less, more preferably 5 or less, still more preferably 2 or less, and still more preferably 1 or less. Further, it is preferably 0.05 to 10, more preferably 0.1 to 5, further preferably 0.1 to 2, and still more preferably 0.2 to 1.
  • the sugar production method of the present invention includes a step of saccharifying biomass with the xylanase or enzyme preparation of the present invention.
  • the conditions for the saccharification treatment are not particularly limited as long as the xylanase of the present invention and other enzymes added at the same time are not inactivated, and those skilled in the art appropriately determine the type of biomass, the procedure of the pretreatment step, and the type of enzyme used.
  • the content of biomass in the suspension is preferably 0.5% by mass or more, more preferably 3% by mass or more, and further preferably 5% by mass, from the viewpoint of improving saccharification efficiency and productivity (reducing production time).
  • 20% by mass or less more preferably 15% by mass or less, and still more preferably 10% by mass or less. Further, it is preferably 0.5 to 20% by mass, more preferably 3 to 15% by mass, and further preferably 5 to 10% by mass.
  • the amount of xylanase or enzyme preparation used for the suspension is appropriately determined depending on the pretreatment conditions, the type and properties of the enzyme to be blended, and is preferably 0.04% by mass or more, more preferably based on the biomass mass. Is 0.1% by mass or more, and preferably 600% by mass or less, more preferably 100% by mass or less, and still more preferably 50% by mass or less.
  • the reaction pH during the saccharification treatment is preferably pH 4 or more, more preferably pH 5 or more, preferably pH 9 or less, from the viewpoints of improving saccharification efficiency, improving productivity (shortening production time), and reducing production costs. More preferably, it is pH 8 or less, More preferably, it is pH 7 or less. Further, the pH is preferably 4 to 9, more preferably 5 to 8, and still more preferably 5 to 7.
  • the reaction temperature during the saccharification treatment is preferably 20 ° C or higher, more preferably 25 ° C or higher, more preferably 30 or higher, from the viewpoints of improving saccharification efficiency, improving productivity (shortening production time), and reducing production costs. Even more preferably 40 ° C or higher, still more preferably 45 ° C or higher, still more preferably 50 ° C or higher, and 90 ° C or lower is preferable, more preferably 85 ° C or lower, still more preferably 80 ° C or lower, still more preferably 75 ° C. or lower, more preferably 65 ° C. or lower, still more preferably 60 ° C. or lower.
  • reaction time of the saccharification treatment can be appropriately set according to the type or amount of biomass, the amount of enzyme, etc., but from the viewpoint of improving saccharification efficiency, improving productivity (shortening production time), and reducing production cost, It is preferably 1 to 5 days, more preferably 1 to 4 days, and further preferably 1 to 3 days.
  • the pretreatment include one or more selected from the group consisting of alkali treatment, pulverization treatment, and hydrothermal treatment. Since the xylanase of the present invention has high enzyme activity even in the alkaline region, the pretreatment is preferably alkali treatment from the viewpoint of improving saccharification efficiency, and alkali treatment and pulverization treatment are performed from the viewpoint of further improving saccharification efficiency. It is preferable to perform the alkali treatment and the pulverization treatment at the same time.
  • a xylanase selected from the proteins represented by the following (a) to (c).
  • the residue is the amino acid residue: 77th: Proline 212: Alanine 214: Proline 217: Proline 323: Asparagine 328: A protein comprising an amino acid sequence substituted with proline and having xylanase activity.
  • (B) consisting of an amino acid sequence of the protein of (a) and an amino acid sequence having 90% or more identity other than the amino acids at positions 77, 212, 214, 217, 323, and 328, and A protein having xylanase activity.
  • C In the amino acid sequence of the protein of (a), one or several amino acids are deleted or substituted at positions other than the amino acids at positions 77, 212, 214, 217, 323, and 328.
  • a protein comprising an added amino acid sequence and having xylanase activity.
  • ⁇ 6> A transformant obtained by introducing the recombinant vector of ⁇ 5> above into a host.
  • ⁇ 8> An enzyme preparation for biomass saccharification containing the xylanase of ⁇ 1> or ⁇ 2> above.
  • the content of the endoglucanase in the total protein amount of the enzyme preparation is preferably 1% by mass or more, more preferably 5% by mass or more, further preferably 10% by mass or more, and preferably 70%. % By mass or less, more preferably 50% by mass or less, further preferably 40% by mass or less, preferably 1 to 70% by mass, more preferably 5 to 50% by mass, and further preferably 10 to 40% by mass.
  • the protein amount ratio between the protein of ⁇ 1> and the cellulase is preferably 0.01 or more, more preferably 0.05 or more, and preferably 100 or less, more preferably Is 5 or less, more preferably 1 or less, even more preferably 0.5 or less, and preferably 0.01 to 100, more preferably 0.05 to 5, and even more preferably 0.05 to 1, more More preferably, the enzyme preparation for biomass saccharification according to ⁇ 9> to ⁇ 11>, which is 0.05 to 0.5.
  • the protein amount ratio of the protein ⁇ 1> or ⁇ 2> to the endoglucanase is preferably 0.05 or more, more preferably 0.1 or more, and preferably Is 10 or less, more preferably 5 or less, still more preferably 2 or less, even more preferably 1 or less, and preferably 0.05 to 10, more preferably 0.1 to 5, and still more preferably 0.1.
  • the biomass is selected from the group consisting of processed or pulverized wood, pulp, paper, bagasse, rice straw, corn stalk or leaf, palm empty fruit bunch (EFB), plant shells, and algae At least one selected from the group consisting of wood, processed or pulverized wood, plant stems, leaves, and fruit bunches, more preferably bagasse, EFB, oil palm (stem)
  • the enzyme preparation for biomass saccharification according to the above ⁇ 8> to ⁇ 13> which is at least one selected from the group, more preferably bagasse.
  • ⁇ 15> A method for producing sugar, comprising a step of saccharifying biomass with the enzyme preparation of ⁇ 8> to ⁇ 14> above.
  • ⁇ 16> The method according to ⁇ 15>, wherein, in the saccharification treatment, the protein according to ⁇ 1> or the enzyme preparation according to ⁇ 8> to ⁇ 14> is added to the suspension containing the biomass.
  • the content of the biomass in the suspension is 0.5 to 20% by mass, preferably 3 to 15% by mass, preferably 5 to 10% by mass.
  • the amount of the protein ⁇ 1> or the enzyme preparation ⁇ 8> to ⁇ 14> used in the suspension is preferably 0.04% by mass or more, more preferably, based on the mass of the biomass. Is 0.1% by mass or more, and preferably 600% by mass or less, more preferably 100% by mass or less, still more preferably 50% by mass or less, and preferably 0.04 to 600% by mass, more preferably Is 0.1 to 100% by mass, more preferably 0.1 to 50% by mass, according to the above ⁇ 16> or ⁇ 17>.
  • the reaction pH during the saccharification treatment is preferably pH 4 or more, more preferably pH 5 or more, preferably pH 9 or less, more preferably pH 8 or less, further preferably pH 7 or less, and preferably pH 4 to 9.
  • the reaction temperature during the saccharification treatment is preferably 20 ° C or higher, more preferably 25 ° C or higher, still more preferably 30 or higher, still more preferably 40 ° C or higher, still more preferably 45 ° C or higher, still more preferably.
  • a ′ a protein having an amino acid sequence in which at least one of alanine at positions 77, 214, and 328 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with proline, and having xylanase activity.
  • B ′ a protein comprising an amino acid sequence having 90% or more identity other than the amino acids at positions 77, 214 and 328 with the amino acid sequence of the protein of (a ′) and having xylanase activity.
  • PCR polymerase chain reaction
  • Applied Biosystems Applied Biosystems 2720 thermal cycler
  • PrimeSTAR Max Premix GXL Takara Bio
  • DNA amplification was performed.
  • the PCR reaction solution composition was 1 ⁇ L of appropriately diluted template DNA, 10 pmol of each of the sense primer and antisense primer, and 1 ⁇ L of PrimeSTAR Max Premix GXL, so that the total amount of the reaction solution was 50 ⁇ L.
  • PCR reaction conditions were carried out by repeating 40 steps of three steps of temperature changes of 98 ° C. for 10 seconds, 50 ° C. for 15 seconds and 68 ° C. for 9 minutes.
  • Example 1 Cloning of Cellulomonas-derived cellulase gene 1-1 Extraction of genomic DNA Cellulomonas fimi ATCC 484 strain was inoculated into Growth medium (1.0% Polypeptone, 0.2% Yeast extract, 0.1% MgSO4 ⁇ 7H2O, pH 7.0) at 30 ° C. For 1 day. Genomic DNA was obtained from the cells obtained by the culture using UltraClean TM Microbial DNA Isolation Kit (manufactured by Mo Bio Laboratories, Inc.).
  • a strain carrying the plasmid into which the target gene was inserted was selected by colony PCR.
  • the selected transformant was cultured using the same LB agar medium (37 ° C., 1 day), and the plasmid was recovered and purified from the obtained bacterial cell using High® Pure® Plasmid Isolation® kit (Roche).
  • the plasmid was introduced into the host bacteria according to the protoplast transformation method (Mol. Gen. Genet., 168, 111 (1979)). At this time, Bacillus subtilis 168 protease 9-deficient strain (KA8AX) (JP 2006-174707) was used as the host bacterium.
  • Transformant regeneration medium includes tetracycline-containing DM3 regeneration agar medium (xylan from beechwood (Sigma-Aldrich) 1.0% (w / v), bactocasamino acid (Difco) 0.5% (w / v), yeast extract (Difco) 0.5% (w / v), L-tryptophan (Wako Pure Chemical Industries) 0.01% (w / v), disodium succinate hexahydrate (Wako Pure Chemical Industries) 8.1% (w / v), monophosphate Dipotassium hydrogen (Wako Pure Chemical Industries) 0.35% (w / v), Monopotassium dihydrogen phosphate (Wako Pure Chemical Industries) 0.15% (w / v), Glucose (Wako Pure Chemical Industries) 0.5% (w / v) ), Magnesium chloride (Wako Pure Chemical Industries) 20 mM, bovine serum albumin (Wako Pure Chemical Industries) 0.01% (w /
  • Example 2 Xylanase production Transformant grown on DM3 regenerated agar medium After seed culture using 5 mL of LB medium containing 15 ppm tetracycline (30 ° C., 250 rpm, 16 hours), 0.6 mL of seed culture solution was added to 20 mL of 2 ⁇ L Mal medium.
  • Example 3 Mutation Introduction 77-Pro induced Cfi Fw (SEQ ID NO: 9) and 77-Pro shown in Table 2 below (actually shown) using as a template a gene encoding a wild-type xylanase consisting of the base sequence shown in SEQ ID NO: 1 Induced Cfi Rv (SEQ ID NO: 10) primer pair, 214-Pro induced Cfi Fw (SEQ ID NO: 11) and 214-Pro induced Cfi Rv (SEQ ID NO: 12) primer pair, 328-Pro induced Cfi Fw (SEQ ID NO: 13) And 328-Pro induced Cfi Rv (SEQ ID NO: 14) primer pair, 212-Ala induced Cfi Fw (SEQ ID NO: 15) and 212-Ala induced Cfi Rv (SEQ ID NO: 16) primer pair, 217-Gly induced Cfi Fw ( PCR using a primer pair of SEQ ID NO: 17) and 217
  • the obtained PCR reaction solution was treated with DpnI to digest the template DNA, and then transformed into Escherichia coli to construct a plasmid expressing the mutant xylanase.
  • the obtained plasmid was confirmed to have a gene encoding a mutant xylanase inserted by a DNA sequencing method.
  • Example 4 Analysis of Enzymatic Properties of Mutant Xylanase 4-1 Heat Resistance Heat resistance was measured. First, the crude enzyme solution was appropriately diluted with 50 mM acetate buffer, and incubated at 62-67 ° C. (in 1 ° C. increments) for 20 minutes using a Veriti thermal cycler (Applied Biosystems). Simultaneously, 20 ⁇ L of the synthetic substrate 1 mM p-nitrophenylxylobioside solution, 20 ⁇ L of 250 mM acetate buffer and 50 ⁇ L of water were mixed to prepare 90 ⁇ L of the substrate solution. 10 ⁇ L of the crude enzyme solution after incubation was added to the substrate solution, and reacted at 50 ° C. for 10 minutes.
  • a Veriti thermal cycler Applied Biosystems
  • the xylanase of the present invention showed the maximum activity at pH 6.0 and maintained 70% or more of the maximum activity at pH 5.0 to 7.0.
  • a substrate solution 90 ⁇ L was prepared by mixing 2.0 ⁇ l (w / v) xylan from beechwood 50 ⁇ L, 250 mM acetate buffer (pH 5.0) 20 ⁇ L and water 20 ⁇ L. 10 ⁇ L of the crude enzyme solution of the mutant diluted to an appropriate concentration was added and reacted at 50 ° C. for 10 minutes. After the reaction, 100 ⁇ L of DNS solution was added to stop the reaction, and the reaction was carried out at 100 ° C. for 5 minutes. After cooling, the absorbance at 540 nm was measured with an absorbance microplate reader.
  • a blank was prepared by adding 100 ⁇ L of the DNS solution to 90 ⁇ L of the substrate solution, adding 10 ⁇ L of the enzyme solution, and performing the same operation.
  • the amount of xylooligosaccharides produced by preparing a calibration curve prepared with a xylose solution was calculated, and the value obtained by subtracting the background sugar derived from the substrate solution was taken as the amount of produced sugars.
  • the xylanase activity of the diluted enzyme solution was calculated from the amount of sugar produced, and the relative activity was measured with the wild-type activity as 100%.
  • Table 5 shows the results of specific activities related to the degradation of xylan by mutant xylanase and wild-type xylanase.
  • the xylanase of the present invention had the highest xylan decomposition activity in the vicinity of 50-60 ° C., and showed an activity of 60% or more of the maximum activity in the range of 45-65 ° C.

Abstract

L'invention concerne une xylanase qui peut sacchariser une biomasse de manière plus efficace que la manière classique et qui présente une stabilité supérieure à la chaleur. Cette xylanase est choisie parmi les protéines (a) à (c) suivantes : (a) une protéine présentant une activité de xylanase et comprenant une séquence d'acides aminés dans laquelle au moins l'une parmi une alanine en 77ème position, une sérine en 212ème position, une alanine en 214ème position, une glutamine en 217ème position, une sérine en 323ème position et une alanine en 328ème position dans une séquence d'acides aminés représentée par la séquence SEQ ID NO:2 est substituée par les résidus d'acide aminé suivants : 77ème position : proline, 212ème position : alanine, 214ème position : proline, 217ème position : proline, 323ème position : asparagine et 328ème position : proline ; (b) une protéine xylanase comprenant une séquence d'acides aminés présentant une similarité supérieure ou égale à 90 %, autre que les sites de substitution, par rapport à la protéine (a) ; et (c) une protéine xylanase comprenant une séquence d'acides aminés dans laquelle un ou plusieurs acides aminés en des positions autres que les sites de substitution de la protéine (a) sont soit supprimés, soit substitués soit ajoutés.
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WO2011070101A1 (fr) * 2009-12-09 2011-06-16 Novozymes A/S Méthodes permettant de produire des variantes de xylanase gh8
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WO2007115407A1 (fr) * 2006-04-12 2007-10-18 National Research Council Of Canada Modification de xylanases permettant d'améliorer la thermophilie, la thermostabilité et l'alcalophilie
JP2012513206A (ja) * 2008-12-23 2012-06-14 ダニスコ・アクティーゼルスカブ キシラナーゼ活性を有するポリペプチド
WO2011070101A1 (fr) * 2009-12-09 2011-06-16 Novozymes A/S Méthodes permettant de produire des variantes de xylanase gh8
WO2013176205A1 (fr) * 2012-05-24 2013-11-28 花王株式会社 Xylanase et procédé de production de sucre à l'aide de celle-ci

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