JP2013243953A - Xylanase and method for producing sugar therewith - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method which can more efficiently and economically saccharify biomass.SOLUTION: There is provided an isolated or purified protein which has xylanase activity and comprises an amino acid sequence selected from (a) to (c), i.e., (a) an amino acid sequence shown by a specific sequence, (b) an amino acid sequence having the same sequence of ≥85% as the amino acid sequence shown by the specific sequence, and (c) the amino acid sequence represented by the specific sequence, wherein at least one amino acid is deleted, replaced or added.

Description

  The present invention relates to a novel xylanase and a method for producing a sugar using the xylanase.

  A technique for producing sugar from cellulose in a biomass material containing cellulose (hereinafter sometimes referred to as “biomass”) and converting it into ethanol, lactic acid or the like by a fermentation method has been known. Furthermore, in recent years, development of technology that efficiently uses biomass as a resource has attracted attention from the viewpoint of tackling environmental problems.

  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.

  Conventionally, saccharification of biomass by cellulase and enzymatic degradation of biomass by hemicellulase and ligninase have been performed. For example, an enzyme composition in which hemicellulase such as Trichoderma reesei-derived xylanase or arabinofuranosidase is added to cellulase has been developed (Patent Document 1). Further, a protein having xylanase activity derived from a microorganism group present in bagasse compost, and a method for producing sugar by reacting the protein and cellulase with biomass resources are known (Patent Document 2). Furthermore, xylanase preparations (such as Cellic (registered trademark) HTec) manufactured and sold by Novozymes are also known.

Special table 2011-515089 gazette JP 2012-029678 A

However, conventional biomass saccharification methods using hemicellulase or xylanase have not yet been satisfactory in terms of saccharification efficiency, monosaccharide productivity, or enzyme cost. Furthermore, the conventional xylanase has a relatively narrow optimum pH range, which may limit its application.
The present invention relates to a novel xylanase that can saccharify biomass more efficiently and economically, can act in a wide range of pH, and has excellent versatility, and a method for producing sugar using the xylanase.

  The present inventors have found a novel xylanase derived from bacteria belonging to the genus Xylanimonas that can exhibit high xylanase activity in a wide pH range, and have found that biomass saccharification can be efficiently performed by using the xylanase. The present invention has been completed.

That is, the present invention is an isolated or purified protein comprising an amino acid sequence selected from the following (a) to (c) and having xylanase activity (hereinafter sometimes referred to as “the xylanase of the present invention”). I will provide a.
(A) Amino acid sequence represented by SEQ ID NO: 2 (b) Amino acid sequence having 85% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 (c) In the amino acid sequence represented by SEQ ID NO: 2, Amino acid sequence with one amino acid deleted, substituted or added

  The present invention also provides an enzyme preparation for biomass saccharification (hereinafter sometimes referred to as “enzyme preparation of the present invention”) containing the xylanase of the present invention.

  Furthermore, the present invention provides a method for producing sugar (hereinafter sometimes referred to as “the method for producing a sugar of the present invention”) including a step of saccharifying biomass with the enzyme preparation of the present invention.

  The present invention provides a xylanase having high xylanase activity in a wide pH range. By using the xylanase, it is possible to realize more efficient saccharification of biomass than when using a known xylanase such as a commercially available xylanase preparation or a composition containing the same. Therefore, according to the enzyme preparation and the sugar production method of the present invention using the xylanase, sugar can be produced efficiently and inexpensively from biomass.

Amount of sugar produced from biomass by various enzyme preparations.

  In this specification, the sequence identity of a base sequence and an amino acid sequence is calculated by Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing an analysis with Unit size to compare (ktup) as 2, using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win.

  In the present specification, “a sequence in which one to a plurality of amino acids are deleted, substituted or added” is 1 to 50, preferably 1 to 40, more preferably 1 to 30, more preferably. 1 to 20, more preferably 1 to 10, still more preferably 1 to 5 amino acid sequences deleted, substituted or added. In the present specification, “the sequence from which one to a plurality of bases are deleted, substituted or added” is 1-150, preferably 1-120, more preferably 1-90, and even more preferably 1. -60, even more preferably 1-30, still more preferably 1-15, still more preferably 1-10 bases are deleted, substituted or added.

  In this specification, “biomass” refers to cellulosic and / or lignocellulosic biomass containing hemicellulose components produced by plants and algae. Specific examples of biomass 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. Of these, from the viewpoints of availability and raw material costs, 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. The biomass may be dried.

  In the present specification, “xylanase activity” refers to an activity of hydrolyzing a xylose β-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.

(1. Xylanase)
The xylanase of the present invention includes the following (a) to (c):
(A) the amino acid sequence represented by SEQ ID NO: 2;
(B) an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2; and
(C) an amino acid sequence in which one to a plurality of amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2,
And a protein having an xylanase activity.

  The xylanase of the present invention is 85% or more, preferably 90% or more, more preferably 92% or more, still more preferably 95% from the amino acid sequence represented by SEQ ID NO: 2 from the viewpoint of improving pH stability and improving saccharification efficiency. As mentioned above, it consists of an amino acid sequence having a sequence identity of 98% or more. Moreover, as a xylanase of this invention, the xylanase derived from the genus Xylanimonas bacteria (for example, Xylanimonas cellulosilytica DSM15894) which consists of an amino acid sequence shown by sequence number 2 is mentioned from a viewpoint of the improvement of pH stability and saccharification efficiency.

  An example of a protein comprising an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and having xylanase activity includes one to a plurality of amino acids in the amino acid sequence shown in SEQ ID NO: 2. Examples include proteins having a deleted, substituted or added amino acid sequence and having xylanase activity. For example, a natural or artificially produced xylanase derived from the genus Xylanimonas belonging to the amino acid sequence represented by SEQ ID NO: 2 described above is used. Mutants. The mutant is, for example, introduced a mutation by a known mutagenesis method such as ultraviolet irradiation or site-directed mutagenesis to the gene encoding the xylanase derived from the genus Xylanimonas, It can be produced by 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 xylanase of the present invention has an amino acid sequence different from that of a conventionally isolated or purified xylanase. For example, a known xylanase having an amino acid sequence having the highest sequence identity with the sequence shown in SEQ ID NO: 2 is xylanase derived from Xylanimicrobium pachnodae (App. Environ. Microbiol., 1999, 65 (9): 4099-4107). However, the sequence identity with the xylanase of SEQ ID NO: 2 is 81%.

Preferably, the xylanase of the present invention further has the following enzymological properties.
(I) Optimum reaction pH
In the present specification, “optimum reaction pH in xylanase activity” means a pH at which the activity is maximum when xylan is decomposed at 50 ° C., and specifically, measured by the method described in the examples below. Is done.
In the xylanase of the present invention, the optimum reaction pH for xylanase activity is preferably in the range of pH 5.5 to 8.5, more preferably from the viewpoint of improving pH stability and facilitating cocktail formation with other enzymes. Has a pH of 6.0 to 8.0, more preferably a pH of 6.0 to 7.5, and even more preferably a pH of 6.0 to 7.0.
(ii) Maximum Activity In this specification, the “maximum activity” of xylanase refers to the xylanase activity at the optimum reaction pH at 50 ° C. The “maximum activity” of the xylanase of the present invention is preferably 1000 U / mg protein or more, more preferably 1100 U / mg protein or more, still more preferably 1200 U / mg protein or more, more preferably 1300 U / mg, from the viewpoint of improving saccharification efficiency. Protein or higher, more preferably 1400 U / mg protein or higher, more preferably 1500 U / mg protein or higher.
(iii) pH Dependence The xylanase of the present invention has a maximum activity of 70 at a reaction temperature of 50 ° C. and a pH of 7.0 to 9.0 from the viewpoint of improving pH stability and facilitating cocktail formation with other enzymes. % Or more, more preferably 74% or more. Moreover, it is 75% or more of maximum activity in the range of pH 7.0-8.5, Preferably it is 80% or more, More preferably, it is 85% or more. Here, “maximum activity” refers to xylanase activity at an optimum reaction pH at a reaction temperature of 50 ° C.

(Iv) Optimum reaction temperature In this specification, “optimum reaction temperature in xylanase activity” means a temperature at which xylanase activity is maximized, and is specifically measured by the method described in the examples below. . In the xylanase of the present invention, the optimum reaction temperature for xylanase activity is preferably 40 ° C. or higher and lower than 70 ° C., more preferably 45 to 65 ° C., and further preferably 50 to 65 ° C.

(2. Production method of xylanase)
The xylanase of the present invention can be produced, for example, by a transformant obtained by introducing a vector containing a gene encoding the xylanase of the present invention into a microorganism. Therefore, the present invention may be referred to as a xylanase production method (hereinafter referred to as “the xylanase production method of the present invention”) comprising a step of culturing a transformant containing a vector containing a gene encoding the xylanase of the present invention. )I will provide a. That is, when the transformant is cultured in an appropriate medium, the gene encoded on the vector contained in the transformant is expressed and the xylanase of the present invention is produced. The xylanase of the present invention can be obtained by isolating or purifying the produced xylanase from the culture.

  Examples of the gene encoding the xylanase of the present invention include a gene consisting of the base sequence represented by SEQ ID NO: 1. Alternatively, as a gene encoding the xylanase of the present invention, for example, 85% or more, preferably 90% or more, preferably 95% or more, more preferably 98% or more of the sequence identity with the base sequence represented by SEQ ID NO: 1. And a base sequence in which one or more bases have been deleted, substituted or added in the base sequence represented by SEQ ID NO: 1.

  The gene encoding the xylanase of the present invention can be isolated from bacteria belonging to the genus Xylanimonas, such as Xylanimonas cellulosilytica (DSM 15894), using any method used in the art. For example, after extracting the whole genomic DNA of a genus Xylanimonas bacterium, the gene is selectively amplified by PCR using a primer designed based on the nucleotide sequence of SEQ ID NO: 1, and the amplified gene is purified. Can be obtained. Alternatively, the gene can be synthesized genetically or chemically based on the amino acid sequence of the xylanase of the present invention.

  Alternatively, the gene encoding the xylanase of the present invention may be a known mutagenesis method such as ultraviolet irradiation or site-directed mutagenesis described above with respect to the base sequence of the gene isolated or synthesized by the above procedure. Can be made by introducing mutations. For example, the gene encoding the xylanase of the present invention is mutated to the nucleotide sequence represented by SEQ ID NO: 1 by a known method, and the resulting nucleotide sequence is expressed to examine the xylan-degrading activity to obtain the desired xylan-degrading activity. Can be obtained by selecting a gene encoding a protein having

  The type of vector into which the gene encoding the xylanase of the present invention is to be introduced is not particularly limited, and vectors usually used for protein production such as plasmids, cosmids, phages, viruses, YACs, BACs and the like can be mentioned. A plasmid vector is preferable. For example, commercially available plasmid vectors for protein expression such as shuttle vectors pHY300PLK, pUC19, pUC119, and pBR322 (all manufactured by Takara Bio Inc.) can be preferably used.

  The vector may include a DNA fragment containing a DNA replication initiation region and a DNA region containing a replication origin. In the above vector, upstream of the gene encoding the xylanase of the present invention, a control region such as a promoter region for initiating transcription of the gene and a secretory signal region for secreting the expressed protein outside the cell. May be operably connected. Alternatively, a drug resistance gene (such as ampicillin, neomycin, kanamycin, chloramphenicol) for selecting a microorganism into which the plasmid of the present invention has been appropriately introduced may be further incorporated. Examples of the control sequences include S237egl promoter and signal sequence (Biosci. Biotechnol. Biochem., 2000, 64 (11): 2281-9), and Bacillus sp. KSM-S237 strain (FERM BP-7875) and Bacillus sp. Examples include the transcription initiation regulatory region, translation initiation region and secretory signal peptide region of the cellulase gene derived from the KSM-64 strain (FERM BP-2886). Alternatively, as the control region, the base sequence of base numbers 1 to 662 of the base sequence represented by SEQ ID NO: 3, the base sequence of base numbers 1 to 696 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 4, or these Having a sequence identity of 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more, and a transcription initiation control function, Examples thereof include a base sequence having a translation initiation control function and a function as a secretory signal peptide. The xylanase gene can be linked to the above regulatory sequences and drug resistance genes by a method such as SOE (splicing by overlap extension) -PCR (Gene, 1989, 77: 61-68). The procedure for introducing a gene into a plasmid vector is well known in the art.

  In the present specification, “operably linked” between a gene and a regulatory sequence means that the gene is arranged so that it can be expressed under the control of the regulatory region.

  Next, the constructed vector is introduced into a microorganism to obtain a transformant. Examples of the microorganism for introduction include bacteria belonging to the genus Staphylococcus, Enterococcus, Listeria, and Bacillus. Among these, bacteria belonging to the genus Bacillus, such as Bacillus subtilis or mutants thereof (for example, Japanese Patent Application Laid-Open No. 2006-174707). And the protease 9-deficient strain KA8AX, etc., described in Japanese Patent Publication No. H10. As the introduction method, a method usually used in the art such as a protoplast method and an electroporation method can be used. A target transformant can be obtained by selecting a strain into which introduction is appropriately performed using drug resistance or the like as an index.

  When the transformant thus obtained is cultured in an appropriate medium, the gene encoded on the vector contained in the transformant is expressed, and the xylanase of the present invention is synthesized. 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 cells and can be more easily recovered. The recovered xylanase may be further purified by a known means.

(3. Enzyme preparation for biomass saccharification)
The xylanase of the present invention has a remarkably high activity of saccharifying biomass as compared to conventionally known xylanases and xylanase preparations, as shown in the examples described later. Therefore, the xylanase of the present invention can be suitably used for biomass saccharification.
That is, the present invention also provides a biomass saccharifying agent containing the xylanase of the present invention as an active ingredient and an enzyme preparation for biomass saccharification containing the xylanase of the present invention.

  The enzyme preparation of the present invention contains the above-described xylanase of the present invention. In addition, from the viewpoint of improving saccharification efficiency, the enzyme preparation of the present invention preferably further contains cellulase. Here, cellulase refers to an enzyme that hydrolyzes the glycosidic bond of β-1,4-glucan of cellulose, and is a generic term for enzymes called endoglucanase, exoglucanase or cellobiohydrolase, β-glucosidase, and the like. It is. As the cellulase used in the present invention, 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 one or more selected from the group consisting of cellobiohydrolase and endoglucanase.

Specific examples of cellulases include cellulases derived from Trichoderma reesei; 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 (Bacillus sp.) .) Cellulases derived from various Bacillus strains such as KSM-N659 (FERM P-19730); thermostable cellulases derived from Pyrococcus horikoshii; cellulases derived from Humicola insolens, etc. It is done. Among these, cellulase derived from Trichoderma reesei, Trichoderma viride, or Humicola insolens is preferable from the viewpoint of improving saccharification efficiency. Specific examples of cellulase preparations containing the 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 ^™ DUET (manufactured by Genencor) and Ultraflo (registered trademark) L (manufactured by Novozymes). Among these, Cellic (registered trademark) CTec2 and Accelerase ^™ DUET (manufactured by Genencor) are preferable, Cellic (registered trademark) CTec2 is more preferable, from the viewpoints of improving saccharification efficiency, manufacturing cost reduction, and availability. .

  Specific examples of β-glucosidase, which is a kind of cellulase, include β-glucosidase derived from Aspergillus niger (for example, Novozymes Novozyme 188 and Megazyme β-glucosidase), and Trichoderma lysase (Trichoderma lysase). reesei) or Penicillium emersonii-derived β-glucosidase. Among these, from the viewpoint of improving saccharification efficiency, Novozyme 188 and β-glucosidase derived from Trichoderma reesei are preferable, and β-glucosidase derived from Trichoderma reesei is more preferable.

  Specific examples of endoglucanase, which is a kind of cellulase, include Trichoderma reesei, Acremonium celluloriticus, Humicola insolens, Clostridium thermocellum, Bacillus. ), Enzymes derived from Thermobifida, Cellulomonas, and the like. Among these, from the viewpoint of improving saccharification efficiency, endoglucanases derived from Trichoderma lysae, Humicola insolens, Bacillus and Cellulomonas are preferable, and endoglucanases derived from Trichoderma lyse are more preferable.

  Moreover, the enzyme preparation of the present invention may contain a hemicellulase other than the xylanase of the present invention. Here, 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, Cross (Clostridium), Thermomotoga, Thermoascus, Caldocellum, or Thermomonospora xylanase, Bacillus pumilus Bacillus pumilus) derived from β- xylosidase include Serenomonasu Ruminantiumu (Selenomonas ruminantium) from β- xylosidase. Among these, from the viewpoint of improving saccharification efficiency, the enzyme preparation of the present invention preferably contains a xylanase derived from Bacillus sp., Aspergillus niger, Trichoderma viride or Streptomyces, or β-xylosidase derived from Selenomonas luminantium, and Bacillus sp. Alternatively, it is more preferable to contain a xylalase derived from Trichoderma viride or β-xylosidase derived from Selenomonas ruminantium.

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 to 70% by mass, more preferably 1 to 50% by mass from the viewpoint of improving saccharification efficiency. It is 2-40 mass%, More preferably, it is 2-30 mass%.
The content of the cellulase in the total protein amount of the enzyme preparation of the present invention is preferably 10 to 99% by mass, more preferably 30 to 95% by mass, and still more preferably 50 to 50% from the viewpoint of improving saccharification efficiency. 95% by mass.
The content of the endoglucanase in the total protein amount of the enzyme preparation of the present invention is preferably 1 to 70% by mass, more preferably 5 to 50% by mass, and still more preferably 10 from the viewpoint of improving saccharification efficiency. -40 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 to 30% by mass, more preferably 0, from the viewpoint of improving saccharification efficiency. 0.1 to 20% by mass, more preferably 0.5 to 20% by mass.

  In the enzyme preparation of the present invention, the protein amount ratio of the xylanase of the present invention to the above cellulase (the xylanase / cellulase of the present invention) is preferably 0.01-100, more preferably 0.05-, from the viewpoint of improving saccharification efficiency. 5, more preferably 0.05 to 1, and still more preferably 0.05 to 0.5.

  In the enzyme preparation of the present invention, the protein amount ratio of the xylanase of the present invention to the endoglucanase (xylanase / endoglucanase of the present invention) is preferably 0.05 to 10, more preferably 0. 0 from the viewpoint of improving saccharification efficiency. It is 1-5, More preferably, it is 0.1-2, More preferably, it is 0.2-1.

  The xylanase and enzyme preparation of the present invention are suitably used in the method for producing the sugar of the present invention described later for biomass saccharification. Biomass is as described above. From the viewpoint of more effectively expressing the saccharification effect of the xylanase and enzyme preparation of the present invention, the biomass is preferably wood, processed or pulverized wood, plant stems, leaves, fruit bunches, etc., bagasse, EFB, oil palm (Stem) is more preferable, and bagasse is more preferable. You may use the said biomass individually or in mixture of 2 or more types. The biomass may be dried.

(4. Method for producing sugar)
The sugar production method of the present invention includes a step of saccharifying biomass with the xylanase or enzyme preparation of the present invention. By the sugar production method of the present invention, the saccharification efficiency of biomass is significantly improved. The reason why the saccharification efficiency is improved is not clear, but by using the xylanase or enzyme preparation of the present invention, hemicellulose in the biomass is efficiently decomposed so that the enzyme easily comes into contact with the cellulose, and the saccharification efficiency as a whole Is presumed to be improved.

  The biomass in the sugar production method of the present invention is as described above. From the viewpoint of availability, raw material cost, and improvement of saccharification efficiency, the biomass is preferably wood, processed or pulverized wood, plant stems, leaves, fruit bunches, etc., and bagasse, EFB, oil palm (stems). More preferably, bagasse is more preferable. You may use the said biomass individually or in mixture of 2 or more types. The biomass may be dried.

The method for producing sugar of the present invention includes the biomass before the step of saccharifying biomass with the xylanase or enzyme preparation of the present invention from the viewpoint of improving grinding efficiency, improving saccharification efficiency, and improving production efficiency (reducing production time). It is preferable to further include a step of pretreating.
Examples of 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.

  In the sugar production method of the present invention, alkali treatment refers to reacting biomass with a basic compound described later. Examples of the alkali treatment method include a method of immersing biomass in an alkali solution containing a basic compound to be described later (hereinafter sometimes referred to as “immersion treatment”), and mixing biomass and a basic compound to be described later. And a method of subjecting to a pulverization treatment (hereinafter sometimes referred to as “alkali mixed pulverization treatment”).

  Examples of basic compounds used for alkali treatment include alkali metal hydroxides such as sodium hydroxide, potassium hydroxide and lithium hydroxide, alkaline earth metal hydroxides such as magnesium hydroxide and calcium hydroxide, sodium oxide, Alkaline metal oxides such as potassium oxide, alkaline earth metal oxides such as magnesium oxide and calcium oxide, alkali metal sulfides such as sodium sulfide and potassium sulfide, alkaline earth metal sulfides such as magnesium sulfide and calcium sulfide, etc. Can be mentioned. Of these, from the viewpoint of reducing production costs and improving saccharification efficiency, it is preferable to use an alkali metal hydroxide or an alkaline earth metal hydroxide, more preferably an alkali metal hydroxide, and sodium hydroxide. Alternatively, it is more preferable to use potassium hydroxide. These basic compounds may be used alone or in combination of two or more.

  The concentration of the basic compound in the alkaline solution in the immersion treatment is preferably 0.1 to 60% by mass, more preferably 0.1 to 50% by mass from the viewpoint of increasing saccharification efficiency and increasing the amount of glucose produced. More preferably, it is 0.1 to 40% by mass. The amount of the alkaline solution used in the immersion treatment is preferably 50 to 99% by mass, more preferably 60 to 99% by mass, and still more preferably 80 to 99% by mass with respect to the dry mass of the biomass. The treatment time for the immersion treatment is preferably 0.5 to 50 hours, more preferably 1 to 24 hours, and further preferably 1.5 to 10 hours.

  In the sugar production method of the present invention, the pulverization treatment refers to mechanically pulverizing biomass into small particles. By making the biomass into smaller particles, the saccharification efficiency is further improved. In addition, when the crystal structure of cellulose contained in biomass is destroyed by pulverization, saccharification efficiency is still improved. The pulverization treatment can be performed using a known pulverizer. There is no restriction | limiting in particular in the grinder used, What is necessary is just an apparatus which can make biomass small particles.

  Specific examples of the pulverizer include a roll mill such as a high pressure compression roll mill and a roll rotating mill; a vertical roller mill such as a ring roller mill, a roller race mill or a ball race mill; a rolling ball mill, a vibration ball mill, a vibration rod mill, and a vibration tube. Container driven media mills such as mills, planetary ball mills or centrifugal fluidization mills; medium agitation mills such as tower crushers, stirring tank mills, flow tank mills or annular mills; high speed centrifugal roller mills, ang mills, etc. Consolidation shear mill; Mortar; Stone mill; Mass colloider; Fret mill; Edge runner mill; Knife mill; Pin mill; Among these, from the viewpoint of improving the grinding efficiency of biomass, reducing the crystallinity of cellulose, and productivity, a container-driven medium mill or a medium stirring mill is preferable, a container-driven medium mill is more preferable, a vibration ball mill, a vibration A vibration mill such as a rod mill or a vibration tube mill is more preferable, and a vibration rod mill is even more preferable. The pulverization method may be either a batch type or a continuous type.

  The pulverization time may be appropriately adjusted so that the pulverized biomass is reduced to a desired size. The pulverization time may vary depending on the pulverizer to be used and the amount of energy to be used, but is usually 1 minute to 12 hours. From the viewpoint of reducing the particle size of biomass, the viewpoint of reducing the crystallinity of cellulose, and the viewpoint of energy cost. Therefore, 2 minutes to 6 hours are preferable, 5 minutes to 3 hours are more preferable, and 5 minutes to 2 hours are more preferable.

  The grinding treatment may be combined with the alkali treatment with the basic compound described above. The pulverization treatment may be performed before or after the alkali treatment, or may be performed in parallel with the pulverization treatment. For example, the biomass immersed in the alkali solution described above may be subjected to a pulverization process (wet pulverization), or may be subjected to a pulverization process together with a solid alkali and biomass (dry pulverization), among which dry pulverization is preferable. When a basic compound is used in the pulverization treatment, the amount of holocellulose in the biomass is all cellulose from the viewpoint of improving the saccharification efficiency, reducing the crystallinity of the cellulose, and removing lignin in the biomass. Is assumed to be 0.01 to 10 times mol per mol of anhydroglucose units (hereinafter sometimes referred to as “AGU”) constituting the cellulose, and 0.05 to 8 times mol. More preferably, it is 0.1 to 5 times mol, still more preferably 0.1 to 1.5 times mol.

  The average particle size of the biomass obtained after the pulverization treatment is preferably 1 to 150 μm, more preferably 5 to 100 μm, from the viewpoint of improving saccharification efficiency and reducing the crystallinity of cellulose. Here, the average particle diameter of biomass can be measured by the method described in Examples.

  In the sugar production method of the present invention, hydrothermal treatment refers to heat treatment of biomass in the presence of moisture. Hydrothermal treatment can be performed using a known reaction apparatus, and the reaction apparatus used is not particularly limited. Preferably, in the hydrothermal treatment, the coarsely pulverized biomass is made into a water slurry dispersed in water, and this is heat-treated. From the viewpoint of improving the fluidity of the slurry, the content of biomass in the water slurry is preferably 1 to 500 g / L, more preferably 5 to 400 g / L, and still more preferably 8 to 300 g / L.

  Hydrothermal treatment is preferably performed under acidic conditions from the viewpoint of improving production efficiency and saccharification efficiency. The pH condition is preferably pH 3 to 7, more preferably pH 4 to 6, from the viewpoints of improving production efficiency, improving saccharification efficiency, and suppressing denaturation of lignin. The pH condition can be adjusted as appropriate by using an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, or an organic acid such as acetic acid or citric acid. As conditions for the hydrothermal treatment, a temperature of 100 to 400 ° C. and a pressure of 0.1 to 4 MPa are preferable, and a temperature of 140 to 200 ° C. and a pressure of 0.5 to 1 MPa are more preferable. The treatment time is preferably from 0.1 to 3 hours. The hydrothermal treatment method may be either a batch type or a continuous type. The biomass obtained by hydrothermal treatment may be in a wet state, or may be further subjected to a drying treatment, but from the viewpoint of improving saccharification efficiency, a biomass in a wet state is preferable.

  The sugar production method of the present invention includes a step of saccharifying biomass (preferably the pretreated biomass) with the xylanase or enzyme preparation of the present invention (hereinafter sometimes referred to as “saccharification treatment”). The xylanase or enzyme preparation used for the saccharification treatment is as described above.

The conditions for the saccharification treatment are not particularly limited as long as the xylanase of the present invention and other enzymes to be mixed at the same time are not inactivated. Appropriate conditions can be determined by those skilled in the art depending on the type of biomass, the procedure of the pretreatment step, and the type of enzyme used.
In the saccharification treatment, the xylanase or enzyme preparation is preferably added to the suspension containing the biomass. The content of biomass in the suspension is preferably 0.5 to 20% by mass, more preferably 3 to 15% by mass, and still more preferably 5 from the viewpoint of improving saccharification efficiency and productivity (reducing production time). -10 mass%.
The amount of xylanase or enzyme preparation used for the suspension is appropriately determined depending on pretreatment conditions, the type and nature of the enzyme to be blended, and is preferably 0.04 to 600% by mass relative to the biomass mass. Preferably, it is 0.1-100 mass% more preferably, More preferably, it is 0.1-50 mass%.
The reaction pH at the time of saccharification treatment is preferably pH 4 to 9, more preferably pH 5 to 8, more preferably pH 5 to 5 from the viewpoints of improving saccharification efficiency, improving productivity (shortening production time), and reducing production costs. 7.
The reaction temperature during the saccharification treatment is preferably 20 to 90 ° C., more preferably 25 to 85 ° C., and further preferably 30 from the viewpoints of improving saccharification efficiency, improving productivity (shortening production time), and reducing production costs. It is -80 degreeC, More preferably, it is 40-75 degreeC, More preferably, it is 45-65 degreeC, More preferably, it is 50-65 degreeC, More preferably, it is 50-60 degreeC. The 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, Preferably it is 1-5 days, More preferably, it is 1-4 days, More preferably, it is 1-3 days.

  As exemplary embodiments of the present invention, the following compositions, manufacturing methods, uses, or methods are further disclosed herein. However, the present invention is not limited to these embodiments.

<1> An isolated or purified protein comprising an amino acid sequence selected from the following (a) to (c) and having xylanase activity:
(A) the amino acid sequence represented by SEQ ID NO: 2;
(B) an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more of the amino acid sequence represented by SEQ ID NO: 2; and
(C) In the amino acid sequence represented by SEQ ID NO: 2, 1 to plural, preferably 1 to 50, more preferably 1 to 40, still more preferably 1 to 30, even more preferably 1 to 20, An amino acid sequence in which preferably 1 to 10, more preferably 1 to 5 amino acids are deleted, substituted or added.

<2> The xylanase activity is 70% or more of the maximum activity in the range of pH 7.0 to 9.0, preferably 74% or more, and 75% or more of the maximum activity in the range of pH 7.0 to 8.5, More preferably, it is 80% or more, More preferably, it is 85% or more, The protein of said <1> description.

<3> The optimum reaction pH is in the range of pH 5.5 to 8.5, more preferably in the range of pH 6.0 to 8.0, still more preferably in the range of pH 6.0 to 7.5, still more preferably pH 6.0. The protein according to <1> or <2> above, which is in a range of ˜7.0.

<4> The protein according to any one of <1> to <3>, encoded by a gene selected from the following (d) to (f):
(D) a gene consisting of the base sequence represented by SEQ ID NO: 1;
(E) a gene comprising a nucleotide sequence having a sequence identity of 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more with the base sequence represented by SEQ ID NO: 1;
(F) 1 to plural, preferably 1 to 150, more preferably 1 to 120, still more preferably 1 to 90, and even more preferably 1 to 60, in the base sequence represented by SEQ ID NO: 1. Preferably a gene comprising a base sequence from which 1 to 30, more preferably 1 to 15 and even more preferably 1 to 10 bases have been deleted, substituted or added.

<5> The protein according to <4> above, wherein the gene is a gene derived from the genus Xylanimonas.

<6> The protein according to <4> or <5>, which is produced by a transformant obtained by introducing a vector containing the gene into a microorganism.

<7> An enzyme preparation for biomass saccharification, comprising the protein according to any one of <1> to <6> above.

<8> The content of the protein according to any one of <1> to <6> in the total protein amount of the enzyme preparation is preferably 0.5 to 70% by mass, more preferably 1 to 50. The enzyme preparation for biomass saccharification according to <7>, wherein the enzyme preparation is mass%, more preferably 2 to 40 mass%, and still more preferably 2 to 30 mass%.

<9> The enzyme preparation for biomass saccharification according to <7> or <8>, further comprising cellulase.

<10> The enzyme preparation for biomass saccharification according to <9>, wherein the cellulase contains endoglucanase.

<11> The content of the endoglucanase in the total protein amount of the enzyme preparation is 1 to 70% by mass, preferably 5 to 50% by mass, and more preferably 10 to 40% by mass. The enzyme preparation for biomass saccharification as described.

<12> The protein amount ratio of the protein according to any one of <1> to <6> and the cellulase (the protein / the cellulase) is 0.01 to 100, preferably 0.05 to 5. The enzyme preparation for biomass saccharification according to any one of the above <9> to <11>, preferably 0.05 to 1, more preferably 0.05 to 0.5.

<13> The protein amount ratio of the protein according to any one of <1> to <6> and the endoglucanase (the protein / the endoglucanase) is 0.05 to 10, preferably 0.1 to 5. The enzyme preparation for biomass saccharification according to any one of the above <10> to <12>, more preferably 0.1 to 2, and still more preferably 0.2 to 1.

<14> 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 any one of the above <7> 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 according to any one of <7> to <14>.

<16> The method further includes a step of pretreating the biomass, wherein the pretreatment is selected from the group consisting of alkali treatment, pulverization treatment and hydrothermal treatment, preferably selected from the group consisting of alkali treatment and pulverization treatment. The method according to <15> above, wherein the alkali treatment and the pulverization treatment are more preferably performed, and the alkali treatment and the pulverization treatment are more preferably performed simultaneously.

<17> The basic compound used for the alkali treatment is selected from the group consisting of alkaline earth metal hydroxides, alkali metal oxides, alkaline earth metal oxides, alkali metal sulfides, and alkaline earth metal sulfides. One or more selected, preferably an alkali metal hydroxide or alkaline earth metal hydroxide, more preferably an alkali metal hydroxide, still more preferably sodium hydroxide or potassium hydroxide, according to the above <16> Method.

<18> The above <16>, wherein the grinding time in the grinding treatment is 1 minute to 12 hours, preferably 2 minutes to 6 hours, more preferably 5 minutes to 3 hours, and further preferably 5 minutes to 2 hours. <17> The method described.

<19> The method according to any one of the above <16> to <18>, wherein an average particle size of the biomass obtained after the pulverization treatment is 1 to 150 μm, preferably 5 to 100 μm.

<20> In the saccharification treatment, the protein according to any one of the above <1> to <6> or the enzyme preparation according to any one of the above <7> to <14> in the suspension containing the biomass. The method according to any one of <15> to <19> above, wherein

<21> The method according to <20> above, wherein the content of the biomass in the suspension is 0.5 to 20% by mass, preferably 3 to 15% by mass, and preferably 5 to 10% by mass.

<22> The amount of the protein according to any one of the above <1> to <6> or the enzyme preparation according to any one of the above <7> to <14> relative to the suspension is the amount of the biomass. Preferably it is 0.04-600 mass% with respect to mass, More preferably, it is 0.1-100 mass%, More preferably, it is 0.1-50 mass%, It is described in said <20> or <21>. Method.

<23> The method according to any one of <15> to <22> above, wherein a reaction pH at the time of the saccharification treatment is pH 4 to 9, preferably pH 5 to 8, more preferably pH 5 to 7.

<24> The reaction temperature during the saccharification treatment is 20 to 90 ° C., preferably 25 to 85 ° C., more preferably 30 to 80 ° C., still more preferably 40 to 75 ° C., and even more preferably 45 to 65 ° C. The method according to any one of the above <15> to <23>, which is preferably 50 to 65 ° C, more preferably 50 to 60 ° C.

<25> 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 method according to any one of the above <15> to <24>, which is at least one selected from the group, more preferably bagasse.

<26> Use of the protein according to any one of <1> to <6> for the production of a biomass saccharifying agent.

<27> Use of the protein according to any one of the above <1> to <6> for the production of an enzyme preparation for biomass saccharification.

<28> Use of the protein according to any one of the above <1> to <6> for biomass saccharification.

<29> 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 bunches (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 use according to any one of the above <26> to <28>, which is at least one selected from the group, more preferably bagasse.

  In the following examples, “%” means “% by mass” unless otherwise specified.

<Reference Example 1> PCR Reaction In the polymerase chain reaction (PCR) for DNA fragment amplification in the following examples, Applied Biosystems 2720 thermal cycler (Applied Biosystems) is used, and PrimeSTAR Max Premix (Takara Bio) is attached. Amplification of DNA was performed using the above reagents. The PCR reaction solution composition was 1 μL of appropriately diluted template DNA, 20 pmol each of sense primer and antisense primer, and 25 μL PrimeStar Max Premix, so that the total reaction solution volume was 50 μL. PCR reaction conditions were 98 ° C. for 10 seconds, 57 ° C. for 30 seconds and 72 ° C. for 10 to 50 seconds (adjusted according to the target amplification product. The standard is 10 seconds per kb). This was done by repeating.

Reference Example 2 Protein Quantification For protein quantification, protein assay kit (manufactured by BioRad) was used unless otherwise specified, and calculation was performed based on a calibration curve using bovine serum albumin as a standard protein.

Reference Example 3 Measurement of xylanase activity (1) Optimal reaction pH
50 μL of 2.0% (w / v) xylan (Xylan from beechwood, Sigma-Aldrich), 250 mM acetate buffer (pH 4.4, 5.0, or 5.6), 250 mM phosphate buffer (pH 6.2) , 6.6, 7.0, or 7.4), 250 mM Tris-HCl buffer (pH 7.8, 8.2, 8.6, or 9.0), or 250 mM glycine buffer (pH 9.4, 9.8) Or 10.0) 40 μL was mixed to prepare 90 μL of a substrate solution having a different pH. A xylanase solution diluted to an appropriate concentration was added to the substrate solution and reacted at 50 ° C. for 10 minutes.
After the reaction, a DNS solution (sodium hydroxide (Wako Pure Chemical Industries) 1.6% (w / v), dinitrosalicylic acid (Wako Pure Chemical Industries) 0.5% (w / v)), sodium potassium tartrate ( 100 μL of Wako Pure Chemical Industries, Ltd. (30.0% (w / v)) was added and reacted at 100 ° C. for 5 minutes to stop the enzyme reaction. A blank was prepared by adding 100 μL of the DNS solution to 90 μL of the substrate solution, adding 10 μL of the above xylanase solution, and performing the same operation. After cooling the reaction solution, the absorbance at 540 nm was measured with an absorption microplate reader VersaMax (Molecular Device Japan).

(2) Optimal reaction temperature 2.0% (w / v) xylan (xylan from beechwood, Sigma-Aldrich) 50 μL and pH 6.0 250 mM phosphate-citrate buffer 40 μL were mixed to obtain a substrate solution 90 μL. Adjusted. A xylanase solution (10 μL) diluted to an appropriate concentration was added to the substrate solution, and the mixture was reacted at 45 to 70 ° C. (in increments of 5 ° C.) for 10 minutes using a Veriti thermal cycler (Applied Biosystems). Thereafter, 100 μL of the DNS solution was added and reacted at 100 ° C. for 5 minutes to stop the enzyme reaction. After cooling the reaction solution, the absorbance at 540 nm was measured with an absorbance microplate reader. The amount of xylooligosaccharide in the reaction solution was calculated based on a calibration curve prepared with a xylose solution having a known concentration.

<Reference Example 4> Preparation of cellulose-containing raw material (1) Calculation of holocellulose content in cellulose-containing raw material The pulverized cellulose-containing raw material is subjected to Soxhlet extraction with ethanol-dichloroethane mixed solvent (1: 1) for 6 hours, and extracted. The latter sample was vacuum dried at 60 ° C. To 2.5 g of the obtained sample, 150 mL of water, 1.0 g of sodium chlorite and 0.2 mL of acetic acid were added and heated at 70 to 80 ° C. for 1 hour. Subsequently, the operation of adding sodium chlorite and acetic acid and heating was repeated 3 to 4 times until the sample was decolorized white. The white residue was filtered through a glass filter (1G-3), washed with cold water and acetone, and then dried at 105 ° C. until a constant weight was obtained to determine the residue mass. The holocellulose content was calculated by the following formula, and this was defined as the cellulose content.
Cellulose content (mass%) = [residue mass (g) / collection amount of cellulose-containing raw material (g: dry raw material conversion excluding basic compound)] × 100

(2) Calculation of the number of moles of anhydroglucose unit (AGU) The number of moles of AGU was calculated based on the following equation assuming that all the holocellulose in the cellulose-containing raw material was cellulose.
AGU mole number = Holocellulose mass (g) / 162

(3) Measurement of moisture content of cellulose-containing raw material For measurement of the moisture content of cellulose-containing raw material, an infrared moisture meter (product name “FD-610”, manufactured by Kett Scientific Laboratory Co., Ltd.) was used. The measurement was performed at 150 ° C., and the point at which the mass change rate for 30 seconds was 0.1% or less was taken as the end point of the measurement. The value of the measured water content was converted to mass% with respect to the dry mass of the cellulose-containing raw material.

(4) Measurement of average particle size of pulverized product The average particle size of the pulverized product was measured using a laser diffraction / scattering particle size distribution measuring device “LA-950” (manufactured by Horiba, Ltd.). As a measurement sample, 0.1 g of the pulverized product was added to 5 mL of water, and a sample dispersion treated with ultrasonic waves for 1 minute was used. The volume-based median diameter was measured at a temperature of 25 ° C., and this was defined as the average particle diameter.

<Example 1> Preparation of xylanase (extraction of genomic DNA)
Xylanimonas cellulosilytica DSM15894 strain was inoculated into Trypticase Soy Agar medium (3.0% Trypticase Soy, 2.0% Agar) and cultured at 28 ° C. for 1 day. Genomic DNA was obtained from the cells obtained by culturing using UltraClean ^™ Microbial DNA Isolation Kit (manufactured by Mo Bio Laboratories, Inc.).

(Production of vector)
Using the obtained genomic DNA as a template, the forward primer 1 (SEQ ID NO: 5) and reverse primer 1 (SEQ ID NO: 6) shown in Table 1 were used, and the xylanase gene region (SEQ ID NO: 1) on the genome was about 1.0 kbp. Fragment (A) was amplified.
The S237 alkaline cellulase gene derived from the Bacillus genus KSM-S237 strain (FERM BP-7875) (see Japanese Patent Laid-Open No. 2000-210081) (SEQ ID NO: 3) at the BamHI restriction enzyme cleavage point of the shuttle vector pHY300PLK (Takara Bio) The recombinant plasmid pHY-S237 into which the DNA fragment to be encoded was inserted was used as a template, and the S237 alkaline cellulase structural gene was removed using the forward primer 2 (SEQ ID NO: 7) and reverse primer 2 (SEQ ID NO: 8) shown in Table 1. An approximately 5.6 kbp fragment (B) was amplified. The amplified gene fragment was purified with High Pure PCR Product Purification kit (Roche).

  1 μL of the purified DNA fragment (A) and 1 μL of the DNA fragment (B), 5 × In-Fusion HD Enzyme Premi × 2 μL contained in In-Fusion HD Cloning Kit (Takara Bio), 6 μL of DNase-free water, The reaction was carried out at 50 ° C. for 15 minutes, and the xylanase gene fragment was cloned into a vector. Thereafter, 20 μL of competent cell E. coli HB101 (Takara Bio) was transformed with 2 μL of the reaction solution. An LB agar medium containing 50 ppm ampicillin sodium (Wako Pure Chemical Industries) and 2.0% agar (Wako Pure Chemical Industries) was used as the regeneration medium for the transformant. From the transformants obtained as ampicillin resistant strains, 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 then the plasmid was recovered and purified from the obtained bacterial cell using High Pure Plasmid Isolation kit (Roche).

(Production of transformants)
The plasmid was introduced into the host bacterium according to the protoplast transformation method (Mol. Gen. Genet., 1979, 168: 111-115). At this time, a Bacillus subtilis 168 protease 9-deficient strain (KA8AX) (Japanese Patent Laid-Open No. 2006-174707) was used as the host bacterium. The regeneration medium of the transformant 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), dipotassium monohydrogen phosphate (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 / v), trypan blue 0 .01% (w / v) (A ROS ORGANICS), tetracycline hydrochloride (Wako Pure Chemical Industries) 0.005% (w / v), were used 1.0% agar (w / v)).

(Xylanase production)
After seed culture using 5 mL of a 15 ppm tetracycline-containing LB medium grown on DM3 regenerated agar medium (30 ° C., 250 rpm, 16 hours), 0.6 mL of the seed culture solution was added to 20 mL of 2 × L Mal medium (Bactotryptone). 2% (w / v), yeast extract 1.0% (w / v), sodium chloride 1.0% (w / v), manganese sulfate pentahydrate (Wako Pure Chemical Industries) 75 ppm, maltose monohydrate Product (Wako Pure Chemical Industries, Ltd.) 7.5% (w / v), tetracycline hydrochloride 15 ppm) and main culture was performed (30 ° C., 230 rpm, 3 days). After culture, the culture supernatant was obtained by centrifugation (11,000 rpm, 15 minutes, 4 ° C.). A recombinant xylanase crude enzyme solution was prepared by exchanging 3 mL of the culture supernatant with 20 mM Tris-HCl pH 7.5 using a desalting column Econo-Pac 10DG (BioRad).

<Test Example 1>
(Xylanase activity and optimum reaction pH)
The xylanase solution (10 μL) prepared in Example 1 or the control xylanase Cellic (registered trademark) HTec (Novozymes A / S) diluted to an appropriate concentration was added to the substrate solution, and the conditions of Reference Example 3 (1) described above were added. The xylanase activity was measured after reaction.
Calculate the amount of xylooligosaccharides in the reaction solution based on a calibration curve created with a xylose solution of known concentration, and subtract the background value (blank) derived from the substrate solution from that value. The amount of sugar produced. Thereafter, the xylanase activity (unit) of each diluted enzyme solution was calculated from the amount of produced sugar. 1 unit (U) was defined as the activity of an enzyme that liberates a reducing sugar equivalent to 1 μmol of D-xylose per minute. The results are shown in Table 2. In the table, the relative activity (%) relative to the maximum activity indicates the relative activity when the activity at the pH (pH 6.6) at which the maximum activity was obtained is defined as 100%, and the relative activity against Cellic (registered trademark) HTec ( %) Indicates relative activity when the specific activity of Cellic (registered trademark) HTec at each pH is defined as 100%.

  The xylanase of the present invention prepared in Example 1 showed the maximum activity at pH 6.6, and the specific activity at that time was about 1717 U / mg. The xylanase showed higher specific activity than commercial xylanase at pH 5.0 or higher. Furthermore, the xylanase had a specific activity of 1200 U / mg or more between pH 7.0 and 9.0, and maintained an activity of 70% or more of the maximum activity. From these results, it was shown that the xylanase has high xylanase activity in a wide range of pH, and particularly has high activity even in the alkaline region.

<Test Example 2>
(Optimum reaction temperature)
The xylanase solution (10 μL) prepared in Example 1 diluted to an appropriate concentration was added to the substrate solution, and the xylanase activity was measured under the measurement conditions described in Reference Example 3 (2) above. The amount of xylooligosaccharide in the reaction solution was calculated based on a calibration curve prepared with a xylose solution having a known concentration. The blank value was subtracted from the calculated value to calculate the amount of xylooligosaccharide produced by the enzyme reaction. The amount of sugar produced at each temperature when the sample showing the maximum amount of sugar produced was taken as 100% was determined as the relative activity. The results are shown in Table 3. The xylanase of the present invention had the highest xylan decomposition activity around 60 ° C., and showed an activity of 60% or more of the activity at 60 ° C. in the range of 45 to 65 ° C.

Example 2 Saccharification of biomass with xylanase (Preparation of biomass)
The bagasse ground material used as a substrate was prepared as follows.
Bagasse (sugarcane pomace, holocellulose content 71.3 mass%, crystallinity 29%, moisture content 7.0 mass%) was placed in a vacuum dryer VO-320 (Advantech Toyo) under nitrogen circulation. It was dried under reduced pressure for 2 hours under the conditions to obtain a dry bagasse having a holocellulose content of 71.3 wt%, a crystallinity of 29%, and a moisture content of 2.0 wt%.

(Mixing and grinding process)
Batch of 100 g of the obtained dry bagasse and 8.8 g of granular sodium hydroxide “Tosoh Pearl” (manufactured by Tosoh Corporation) having a particle size of 0.7 mm (equivalent to 0.5 mol per 1 mol of AGU constituting holocellulose) Type vibration mill MB-1 (Chuo-Kakoki: total volume 3.5L, φ30mm as medium, length 218mm, 13 SUS304 rods with a circular cross section are used, rod filling rate 57%) The bagasse pulverized product (average particle size of about 16.6 μm) was obtained by pulverizing for a time.

(Saccharification treatment)
50 mg of the prepared bagasse pulverized product and 0.4 mL of water were added to a 2 mL-volume microtube, and sodium hydroxide used for pulverization was neutralized with 1N HCl. In the neutralized solution, 0.4 mL of 250 mM sodium acetate buffer (pH 4.0 or 5.0), 250 mM phosphate buffer (pH 6.0 or 7.0), or 250 mM Tris-HCl buffer (pH 8.0 or 9. 0) was added to prepare a substrate solution. As the substrate solution, the xylanase solution prepared in Example 1 or the control xylanase solution was added so as to be 50 μg-protein per sample, and then water was added to make the total volume 1 mL. Thereafter, a saccharification reaction was performed for 1 day while reciprocally shaking at 50 ° C. and 150 rpm. As a control xylanase, Cellic (registered trademark) HTec (Novozymes A / S) or Thermomyces lanuginosus-derived xylanase (Sigma-Aldrich) was used. Thermomyces lanuginosus-derived xylanase was added only to the pH 5.0 substrate solution.

  After completion of the reaction, the supernatant was collected by centrifugation (15000 rpm, 5 min, 4 ° C.), and the concentrations of xylose, xylobiose and xylotriose in the supernatant were measured with a DX500 chromatography system (manufactured by Nippon Dionex). . The sum of these three free sugar amounts was defined as the amount of sugar produced from bagasse. The results are shown in Table 4. The relative amount of sugar produced in the table is a relative value when the amount of sugar produced by Cellic (registered trademark) HTec at each pH is 100%.

  As shown in Table 4, the xylanase of the present invention exhibits higher saccharification activity than Cellic (registered trademark) HTec in the entire measured pH range, and is stable and high saccharification particularly in the alkaline region (pH 7 to 9). The activity was maintained. In addition, commercially available Thermomyces lanuginosus-derived xylanase exhibited a saccharification activity (relatively produced sugar amount 101%) equivalent to Cellic (registered trademark) HTec at pH 5. From these results, it was considered that the xylanase of the present invention can be used for a wide range of pH.

<Example 3> Saccharification of biomass by enzyme preparation (preparation of biomass)
The bagasse ground material (average particle diameter of about 16.6 μm) prepared in Example 2 was used.
(Preparation of cellulase)
Trichoderma reesei QM6a strain-derived endoglucanase I (SEQ ID NO: 10, hereinafter abbreviated as TrEGI) was prepared as follows.
A DNA sequence (SEQ ID NO: 9) encoding TrEGI was amplified by PCR and introduced into pPTR I DNA (Takara Bio), an expression vector for Aspergillus oryzae, after ligation with a promoter. Thereafter, the prepared vector was introduced into A. oryzae RIB40 strain to obtain a transformant. By culturing the obtained transformant, TrEGI was secreted and produced into the culture solution. The protein amount of the expressed endoglucanase I (hereinafter referred to as TrEGI) derived from Trichoderma reesei strain QM6a is determined from the CMC degradation activity of purified TrEGI and the protein amount obtained by DC protein assay kit (manufactured by BioRad) (211.5 U / mg-protein). And the amount of protein in the supernatant was calculated from the CMC degradation activity in the culture supernatant.

(Saccharification treatment)
50 mg of the prepared bagasse pulverized product and 0.4 mL of water were added to a 2 mL-volume microtube, and sodium hydroxide used for pulverization was neutralized with 1N HCl. A substrate solution was prepared by adding 0.4 mL of 250 mM sodium acetate buffer (pH 5.5) to the neutralized solution. To the substrate solution, an enzyme preparation (test product) containing the xylanase of the present invention was added so as to be 100 μg-protein per sample, and then water was added to make the total volume 1 mL. As an object, 50 μg, 100 μg, or 150 μg-protein of Cellic (registered trademark) CTec2 was added per sample, and then water was similarly added to make the total volume 1 mL. Thereafter, a saccharification reaction was carried out for 3 days while reciprocally shaking at 50 ° C. and 150 rpm. The enzyme preparation containing the xylanase of the present invention includes the xylanase of the present invention: TrEGI: exo-1,4-β-D-Xylosidase (Megazyme): Cellic (registered trademark) CTec2 = 10: 20: 1: 31 (protein (Amount ratio) containing 100 μg of Cellic (registered trademark) CTec2 in 100 μg of protein). In addition, the protein amount measured with the DC protein assay kit was used for calculation of each protein amount and ratio in the enzyme preparation.

  After completion of the reaction, the supernatant was collected by centrifugation (15000 rpm, 5 min, 4 ° C.), and glucose, cellobiose, xylose, xylobiose and xylotriose in the supernatant were collected with a DX500 chromatography system (manufactured by Nippon Dionex). Concentration was measured. The sum of these five free sugars was defined as the amount of sugar produced from bagasse. The results are shown in FIG. The relative saccharification rate in the figure is a relative value when Cellic (registered trademark) CTec2 is added with 50 μg-protein per sample and the amount of sugar produced is 100%.

  As shown in FIG. 1, by using an enzyme preparation containing the xylanase of the present invention, the glycanase of the present invention exhibits higher saccharification activity than the same protein amount or 1.5 times the amount of Cellic (registered trademark) CTec2. It has been shown that the enzyme preparation containing it is suitable for biomass saccharification.

Claims (13)

An isolated or purified protein comprising an amino acid sequence selected from the following (a) to (c) and having xylanase activity:
(A) the amino acid sequence represented by SEQ ID NO: 2;
(B) an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2; and
(C) An amino acid sequence in which one to a plurality of amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2.   The protein according to claim 1, wherein the xylanase activity in the range of pH 7.0 to 9.0 is 70% or more of the maximum activity.   The protein according to claim 1 or 2, wherein the optimum reaction pH is 6.0 to 7.0. The protein according to any one of claims 1 to 3, which is encoded by a gene selected from the following (d) to (f):
(D) a gene consisting of the base sequence represented by SEQ ID NO: 1;
(E) a gene consisting of a base sequence having a sequence identity of 85% or more with the base sequence represented by SEQ ID NO: 1, and
(F) A gene comprising a base sequence in which one to a plurality of bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 1.   The protein according to claim 4, wherein the gene is a gene derived from the genus Xylanimonas.   The protein according to claim 4 or 5, which is produced by a transformant obtained by introducing a vector containing the gene into a microorganism.   An enzyme preparation for biomass saccharification comprising the protein according to any one of claims 1 to 6.   The enzyme preparation for biomass saccharification according to claim 7, further comprising cellulase.   The enzyme preparation for biomass saccharification according to claim 8, wherein the cellulase contains endoglucanase.   The biomass is one 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 The enzyme preparation for biomass saccharification according to any one of claims 7 to 9, which is as described above.   The manufacturing method of saccharide | sugar including the process of saccharifying biomass with the enzyme formulation of any one of Claims 7-10.   The method according to claim 11, further comprising a step of pretreating the biomass, wherein the pretreatment is at least one selected from the group consisting of an alkali treatment, a pulverization treatment, and a hydrothermal treatment.   The biomass is one 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 The method according to claim 11 or 12, which is as described above.