JP2007020539A - Arabinofuranosidase b-presenting yeast and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing a xylan hydrolysate, and to provide a material used therefor. <P>SOLUTION: A polynucleotide comprises a secretion signal sequence acting in a yeast cell, a sequence encoding a polypeptide exhibiting an arabinofuranosidase activity, a sequence encoding a part of a protein located in the surface layer of the cell, and a GPI anchor-adhered signal sequence sequentially from the 5'-terminal side. The method for producing the xylan hydrolyzate comprises a process for hydrolyzing xylan in the presence of a yeast holding the polynucleotide and xylosidase and/or xylanase, and a process for recovering the xylan hydrolyzate. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a yeast that retains arabinofuranosidase on the cell surface and a method for producing a xylan degradation product using the yeast.

  In recent years, with increasing interest in the environment and energy, direct use of ethanol as an automobile fuel and partial addition to existing fuels such as gasoline are being studied. As a result, the degree of dependence on fossil fuels is reduced, and the depletion of fossil fuels that are concerned can be prevented. Ethanol is a fuel with very little environmental pollution compared to ordinary gasoline.

  Conventionally, sugars obtained from sugarcane, starches obtained from food crops such as corn, and the like are mainly used as raw materials for producing industrial alcohol by fermentation. However, since these crops are expensive, it is not practical to use as a raw material for producing ethanol for fuel (bioethanol). In addition, it is not practical to use agricultural crops as a raw material for ethanol fermentation because it is predicted that food shortages will occur on a global scale in the future.

  On the other hand, woody biomass is promising as a bioethanol raw material because it can be obtained in large quantities and at a low price and is not used for food. Woody biomass is composed of about 50% cellulose, about 20-30% hemicellulose, and about 20-30% lignin. Cellulose is a polymer of glucose. The main component of hemicellulose is xylan. In xylan, for example, in broad-leaved trees and gramineous plants, L-arabinose, glucuronic acid, 4-O-methylglucuron is bonded to the xylan main chain in which D-xylose residues are linked by β-1,4. It is a polysaccharide in which an acid or the like is bound as a short side chain.

  Polymeric polysaccharides can only be used as a substrate for ethanol fermentation by hydrolysis with a glycolytic enzyme into monosaccharides. Since woody biomass has a higher hemicellulose content ratio than food crops, the efficiency of the hydrolysis reaction of hemicellulose is a major factor in determining the ethanol production efficiency from woody biomass.

  Xylanase and xylosidase are known as enzymes that degrade the β-1,4 bond between xylose in the xylan main chain. However, since these enzymes are unlikely to act on the branched part to which the arabinose side chain is added, these enzymes alone cannot sufficiently hydrolyze xylan, and the xylose recovery rate is low, which in turn produces bioethanol. Efficiency is lowered.

  The enzyme that breaks the bond between the side chain arabinose and the main chain xylose is arabinofuranosidase. It has been reported that when arabinofuranosidase is allowed to act on xylan together with xylosidase or further xylanase, the yield of xylose due to the decomposition of xylan is improved (Non-patent Document 1).

  However, unlike xylanase and xylosidase, arabinofuranosidase has not been established with a technique for producing it in large quantities to be industrially usable. It is also conceivable to produce xylose by decomposing xylan using microorganisms that produce these enzymes. However, since xylan is a polymer, it is difficult to permeate the cell membrane, resulting in poor xylose production efficiency.

Abstracts of the 55th Annual Meeting of the Biotechnology Society of Japan, p.15 p19 Gifu Univ./Nori Takamizawa et al.

  An object of the present invention is to provide an efficient method for producing a xylan decomposition product and a material used therefor.

In order to solve the above-mentioned problems, the present inventor repeated research and obtained the following knowledge.
(1) From upstream, a secretory signal sequence that functions in yeast cells, a sequence that encodes a polypeptide having arabinofuranosidase activity, a sequence that encodes a part of a cell surface localized protein, and a GPI anchor attachment In yeast that holds a polynucleotide containing a signal sequence in an expressible state, a fusion protein of arabinofuranosidase and a cell surface localized protein having a GPI anchor attachment signal at the N-terminus is immobilized at a high density on the cell surface. It will be.

Whether or not a protein can be fixed at a high density on the cell surface as a fusion protein with a cell surface localized protein varies greatly depending on the type of protein and may not be successful. Moreover, even if the surface layer is presented, whether or not the activity can be exhibited in a fixed state varies greatly depending on the type of protein, and the activity may be lost by being immobilized on the cell surface layer. Under such circumstances, the present inventor succeeded in presenting arabinofuranosidase useful for xylose production on the surface of yeast cells as a fusion protein with cell surface localized proteins and expressing high enzyme activity. did.
(2) By incubating xylan in the presence of the above arabinofuranosidase-presenting yeast, xylosidase, or further xylanase, the bond between the main chain xylose and the side chain arabinose and main chain xylose Bonds can be efficiently decomposed, and xylan decomposition products such as xylose and arabinose can be produced in high yield.

  The present invention has been completed based on the above findings, and provides the following polynucleotide, yeast, and method for producing a xylan degradation product.

  Item 1. In order from the 5 ′ end, a secretory signal sequence that functions in yeast cells, a sequence that encodes a polypeptide exhibiting arabinofuranosidase activity, a sequence that encodes part of a cell surface localized protein, and a GPI anchor attachment signal sequence A polynucleotide comprising.

  Item 2. Item 2. The polynucleotide according to Item 1, wherein the sequence consisting of a sequence encoding a part of the cell surface localized protein and a GPI anchor adhesion signal sequence is included in the sequence encoding yeast α-agglutinin.

  Item 3. Item 2. A sequence comprising a sequence encoding a part of a cell surface localized protein and a GPI anchor attachment signal sequence is contained in a sequence encoding 320 to 600 amino acids from the C-terminal of yeast α-agglutinin. The polynucleotide described.

  Item 4. Item 4. The polynucleotide according to Item 1, 2, or 3, wherein the polypeptide having arabinofuranosidase activity is an enzyme derived from Aspergillus oryzae.

  Item 5. Item 5. The polynucleotide according to any one of Items 1 to 4, which is operably linked to a promoter that can function in yeast cells.

  Item 6. An expression vector comprising the polynucleotide of Item 5.

  Item 7. A yeast that retains the polynucleotide according to Item 5 or the expression vector according to Item 6.

  Item 8. Item 8. A method for producing a xylan degradation product, comprising the step of decomposing xylan in the presence of the yeast and xylosidase of Item 7 or further xylanase, and a step of recovering the xylan degradation product.

  Item 9. Item 9. The method according to Item 8, wherein the yeast is immobilized on a carrier.

  Item 10. A step (a) of decomposing xylan in the presence of the yeast according to item 7, and a step (b) of decomposing the xylan partial degradation product obtained in step (a) in the presence of xylosidase or further xylanase. And a step of recovering the xylan decomposition product.

  Item 11. Item 11. The method according to Item 10, wherein the yeast is immobilized on a carrier.

  According to the present invention, a yeast that retains active arabinofuranosidase in the cell surface at a high density was obtained. By allowing this yeast cell and xylosidase or further xylanase to act on xylan, xylan can be efficiently decomposed to obtain xylan degradation products including xylose and arabinose.

  More specifically, xylanase and xylosidase that break down the β-1,4 bond in the main chain of xylan can be obtained in industrially available quantities, but the technology to produce arabinofuranosidase in an amount commensurate with it is low. Did not exist.

  Since the yeast of the present invention holds active arabinofuranosidase on the cell surface, a large amount of arabinofuranosidase can be obtained at low cost by culturing this yeast.

  In addition, since arabinofuranosidase is immobilized at a high density on the cell surface in the yeast of the present invention, the concentration of arabinofuranosidase can be increased by increasing the yeast density in the reaction solution. Thus, by allowing the yeast of the present invention to be present in the reaction solution together with xylosidase or further xylanase, the side chain of xylan is efficiently decomposed, and the decomposition of xylan main chain by xylosidase or further xylanase is promoted, As a result, the recovery rate and production rate of the xylan decomposition product containing xylose are improved.

  In addition, since arabinofuranosidase is immobilized on the cell surface, the yeast of the present invention can efficiently decompose high-molecular xylan that is difficult to permeate the cell membrane.

  According to the present invention, xylan, which has conventionally been difficult to hydrolyze, can be efficiently hydrolyzed, thus opening the way for utilization of woody biomass by ethanol fermentation.

Hereinafter, the present invention will be described in detail.
(1) Polynucleotide The polynucleotide of the present invention comprises, in order from the 5 ′ end, a secretory signal sequence that functions in yeast cells, a sequence that encodes a polypeptide exhibiting arabinofuranosidase activity, and a cell surface localized protein. A polynucleotide comprising a sequence encoding a region and a GPI anchor attachment recognition signal sequence. This is shown in FIG.
Secretion signal sequence The secretion signal sequence contained in the polynucleotide of the present invention is a polynucleotide encoding a secretion signal peptide.

  In the present invention, the polynucleotide may be DNA or RNA unless otherwise specified. Further, unless specifically mentioned, it may be either double-stranded or single-stranded. Unless otherwise specified, the DNA may be any of cDNA, genomic DNA, and synthetic DNA, and the RNA may be any of total RNA, mRNA, rRNA, and synthetic RNA.

  A secretory signal peptide is a peptide that is normally bound to the N-terminus of a secretory protein that is secreted extracellularly, including the periplasm. This peptide has a similar structure between organisms, consists of about 20 amino acids, has a basic amino acid sequence in the vicinity of the N-terminus, and then contains many hydrophobic amino acids. The secretory signal is usually removed by degradation by a signal peptidase when the secreted protein is secreted from inside the cell through the cell membrane to the outside of the cell.

In the present invention, any polynucleotide sequence encoding a secretory signal peptide capable of secreting arabinofuranosidase outside the yeast can be used, and the origin thereof is not limited. For example, secretory signal peptide sequence of glucoamylase such as Rhizopus oryzae, secretory signal peptide sequence of Aspergillus oryzae glucoamylase, α- or a-agglutinin of yeast (Saccharomyces cerevisiae) Peptide sequences, secretory signal peptide sequences of α-factor derived from yeast (Saccharomyces cerevisiae), and the like can be suitably used. In particular, the secretion signal peptide sequence of Rhizopus oryzae-derived glucoamylase is preferable from the viewpoint of secretion efficiency. In addition, a secretory signal peptide sequence of Aspergillus oryzae-derived glucoamylase is also preferred.
Sequence encoding a polypeptide exhibiting arabinofuranosidase activity In the present invention, arabinofuranosidase activity refers to an activity of decomposing a bond between xylan main chain xylose and side chain arabinose. The sequence encoding the polypeptide exhibiting arabinofuranosidase activity may be a polynucleotide encoding the full length of arabinofuranosidase. It may be an encoding polynucleotide. Moreover, as long as arabinofuranosidase activity is shown, in the sequence which codes natural arabinofuranosidase, the sequence by which 1 or 2 or more nucleotide was deleted, added, or substituted may be sufficient.

The origin of arabinofuranosidase is not particularly limited, and may be an enzyme derived from any organism such as a microorganism, a plant, and an animal. Especially, the thing derived from microorganisms is preferable at the point which preparation and handling are easy, and the thing derived from Aspergillus oryzae is more preferable. Since Aspergillus oryzae-derived arabinofuranosidase is excellent in heat resistance, it is a highly practical enzyme in that it can be used in an industrial scale reactor in which the temperature in the reaction vessel is difficult to control.
The present inventors have already obtained a gene abfA encoding arabinofuranosidase from Aspergillus oryzae. In addition, this enzyme has also been successfully presented on the surface of yeast (Japanese Patent Application No. 2004-61379). Furthermore, as an additional gene, attention was paid to the abfB gene derived from Aspergillus oryzae already found. And about abfB, the enzyme activity of the arabinofuranosidase in the yeast cell surface layer was examined.
The DNA sequence encoding Aspergillus oryzae-derived arabinofuranosidase used this time is shown in SEQ ID NO: 8. Further, as long as the encoded polypeptide has arabinofuranosidase activity, SEQ ID NO: 8 or one having two or more polynucleotides added, deleted or substituted in this sequence may be used. For example, substitution can be performed such that the corresponding amino acid after substitution has the same properties as the amino acid before substitution in terms of polarity, charge, solubility, hydrophilicity / hydrophobicity, and the like.
Cell surface localized protein <Cell surface localized protein>
In the method of the present invention, any protein may be used as long as it is a protein that is immobilized on, adhered to, or adhered to the cell surface and is localized there, and any known protein can be used without limitation.

Examples of proteins localized in the cell membrane include transmembrane proteins and cell surface localized proteins. The transmembrane protein is a protein that penetrates the lipid bilayer at the hydrophobic amino acid region, and is often found in receptor proteins. On the other hand, a protein modified with a lipid is known as a cell surface localized protein, and this lipid is immobilized on a cell membrane by covalently binding to a membrane component. In addition, there are cell surface localized proteins whose mechanism of immobilization has not been clarified, and these can be used in the method of the present invention. As cell surface localized proteins, various GPI anchoring proteins described later, BGL2 and the like are known. BGL2 is a yeast β-glucanase and is known to bind strongly to the cell wall, but its mechanism is unknown. BGL2 does not have a motif in common with GPI anchor protein.
<GPI anchoring protein>
As a representative example of cell surface localized protein, GPI (glycosylphosphatidylinositol: glycolipid based on ethanolamine phosphate-6 mannose α1-2 mannose α1-6 mannose α1-4 glucosamine α1-6 inositol phospholipid) anchoring protein Can be mentioned. The GPI anchoring protein has GPI which is a glycolipid at its C-terminal, and this GPI is bound to the surface of the cell membrane by covalently binding to PI (phosphatidylinositol) in the cell membrane.

  GPI binding to the C-terminus of the GPI anchoring protein is performed as follows. That is, the GPI anchoring protein is secreted into the endoplasmic reticulum lumen by the action of a secretion signal existing on the N-terminal side after transcription and translation. A region called a GPI anchor attachment signal that is recognized when the GPI anchor binds to the GPI anchoring protein exists in the region near or at the C-terminus of the GPI anchoring protein. In the endoplasmic reticulum lumen and the Golgi apparatus, this GPI anchor attachment signal region is cleaved, and GPI binds to the newly generated C-terminus.

  The protein to which GPI is bound is transported to the cell membrane by secretory vesicles, and is fixed to the cell membrane by GPI covalently binding to PI of the cell membrane. Further, the GPI anchor is cleaved by phosphatidylinositol-dependent phospholipase C (PI-PLC), and is incorporated into the cell wall so that it is fixed to the cell wall and presented on the cell surface.

The state of processing and intracellular transport of GPI anchoring protein is shown in FIG.
<GPI anchoring protein as one example of cell surface localized protein of the present invention>
Thus, the GPI anchoring protein is one of the cell surface localized proteins referred to in the present invention and has a GPI anchor adhesion signal. In the present invention, a polynucleotide encoding a cell membrane binding region of a GPI anchoring protein containing a GPI anchor attachment signal region is preferably used as a sequence consisting of a part of a cell surface localized protein and a GPI anchor attachment signal sequence. Can be used.

  The cell membrane binding region of the GPI anchoring protein is usually a C-terminal region including a GPI anchor attachment signal region. This cell membrane binding region only needs to contain a GPI anchor attachment signal region, and may contain any part of other GPI anchoring proteins as long as it does not inhibit arabinofuranosidase activity.

  The GPI anchoring protein may be any protein that functions in yeast cells, and any known GPI anchoring protein can be used without limitation. Known GPI anchoring proteins include, for example, α- or a-agglutinin, which is a sex aggregation protein of yeast, Flo1 protein, and outer membrane protein OmpA of E. coli (Georgiou, G. et al. Trends Biotechnol., 11, 6). -10, 1993), Escherichia coli maltose transporter LamB, Escherichia coli flagellar protein flagellin, Bacillus subtilis cell wall lytic enzyme CwlB, and the like.

  In particular, yeast α-agglutinin can be preferably used. A region consisting of 320 or 600 amino acids from the C-terminal side of α-agglutinin (ie, a region consisting of 320 amino acids from the C terminus, a region consisting of 321 amino acids from the C terminus, 599 from the C terminus) It is preferable to use a region consisting of amino acids or a region consisting of 600 amino acids from the C-terminal), and it is more preferable to use a polynucleotide sequence encoding a region consisting of 320 amino acids from the C-terminal side. In a sequence consisting of 320 amino acids from the C-terminal side of α-agglutinin, there are four sugar chain binding sites. After the GPI anchor is cleaved by PI-PLC, the sugar chain and the polysaccharide constituting the cell wall are covalently bound to enhance the fixation of α-agglutinin to the cell wall.

A sequence encoding a cell membrane binding region of a GPI anchoring protein such as α-agglutinin inherently contains both a sequence encoding a part of a cell surface localized protein and a GPI anchor attachment signal sequence. In the present invention, the sequence encoding a part of the cell surface localized protein and the GPI anchor attachment signal sequence may be prepared from different sources.
Production method of polynucleotide Production of DNA having secretory signal sequence, sequence encoding polypeptide exhibiting arabinofuranosidase activity, sequence encoding part of cell surface localized protein, and GPI anchor attachment signal sequence in this order The method is a technique well known to those skilled in the art.

For example, the secretory signal sequence and the arabinofuranosidase structural gene can be bound using a DNA ligase (T4 DNA Ligase TakaRa) or the like. Furthermore, this sequence can be similarly performed when, for example, a sequence encoding a cell membrane binding region of a GPI anchoring protein is bound.
(2) Expression vector The expression vector of the present invention is an expression vector linked so that the polynucleotide (here, DNA) of the present invention can be expressed in yeast cells. That is, in the expression vector of the present invention, the polynucleotide of the present invention is linked downstream of a promoter capable of functioning in yeast cells so that expression can be performed by this promoter.

  The expression vector may be a plasmid vector or an artificial chromosome. A plasmid vector is preferable in that the preparation of the vector is easy and the transformation of yeast cells is easy. Further, it is preferably a shuttle vector between yeast and E. coli so that the polynucleotide of the present invention can be constructed and the polynucleotide of the present invention can be easily incorporated into an expression vector using E. coli. . Any known shuttle vector can be used without limitation.

  Examples of the yeast-E. Coli shuttle vector include those obtained by inserting a yeast 2 μm plasmid or a yeast chromosome origin of replication (Ori) into an E. coli plasmid vector. This shuttle vector has this yeast replication origin and E. coli replication origin (ColE1). Moreover, it is usually sufficient to have a yeast selection marker (for example, drug resistance gene, URA3, TRP1, LEU2, etc.) and an E. coli selection marker (drug resistance gene, etc.). Moreover, in order to express the arabinofuranosidase structural gene, what is necessary is just to contain what is called regulatory sequences, such as an operator, a promoter, a terminator, an enhancer, etc. which normally regulate the expression of this gene. Examples of the yeast-E. Coli shuttle vector into which the polynucleotide of the present invention is inserted include pYE22m and pYGA2270. What is necessary is just to insert the polynucleotide of this invention in the position which can be expressed with this promoter between the GAPDH (glyceraldehyde 3'-phosphate dehydrogenase) promoter and GAPDH terminator of these vectors.

The state of the polynucleotide of the present invention in the expression vector is shown in FIG.
(3) Yeast The yeast of the present invention is a yeast that retains the polynucleotide or expression vector of the present invention. The yeast of the present invention can be obtained by introducing the polynucleotide of the present invention (here, mainly DNA) or an expression vector into yeast as a host. “Introduction of a polynucleotide or expression vector” means introducing a polynucleotide or expression vector into a yeast cell to express the fusion protein.

  The introduction method is not particularly limited, and a known method can be adopted. A typical example is a method of transforming yeast using the expression vector of the present invention described above. Transformation methods are not particularly limited, and known methods such as transfection methods such as calcium phosphate method, electroporation method, lipofection method, DEAE dextran method, lithium acetate method, protoplast method, and microinjection method are used without limitation. it can.

  A linear polynucleotide obtained by cleaving between the promoter of the expression vector of the present invention and the GPI anchor attachment signal using an appropriate restriction enzyme can also be introduced into yeast cells by the transformation method exemplified above.

  In addition, a DNA fragment in which the polynucleotide of the present invention is operably linked to a promoter that can function in yeast cells can also be introduced into yeast cells by the exemplified transformation method.

  The DNA introduced into the yeast of the present invention may be present in any form in the yeast cell as long as it can express a polypeptide having arabinofuranosidase activity. This DNA may be present, for example, in the state of a plasmid, in a state integrated into a host chromosome by homologous recombination, or in a state inserted into a host extranuclear gene.

  Yeast into which the polynucleotide of the present invention has been introduced can be selected according to a conventional method using, for example, a trait by a selection marker such as TRP1 or arabinofuranosidase activity as an index.

It can be confirmed by a conventional method that arabinofuranosidase is immobilized on the surface layer of the obtained yeast. For example, a method in which an anti-arabinofuranosidase antibody and a secondary antibody labeled with a fluorescent dye such as FITC, an enzyme-labeled secondary antibody such as alkaline phosphatase, etc. are allowed to act on a test yeast, anti-arabinofuranosidase antibody And a method of reacting a biotin-labeled secondary antibody with a fluorescent-labeled streptavidin.
<Host>
The yeast used as a host is not particularly limited as long as it belongs to Ascomycetou yeast. Among them, those belonging to Saccharomycesaceae are preferable, and those belonging to Saccharomyces are more preferable.

  Among these, practical yeasts are preferable because they are resistant to various culture stresses and exhibit stable cell growth even in industrial production in which strict control is difficult. Practical yeasts include yeasts that are deeply involved in fermented foods, such as sake yeast, shochu yeast, wine yeast, beer yeast, and baker's yeast. Among practical yeasts, sake yeast having high fermentability and high ethanol tolerance and genetically stable is preferable.

In general, ethanol is toxic to microorganisms. Yeast that undergoes ethanol fermentation is no exception, and when the ethanol concentration exceeds 8% (v / v), it is gradually killed by the ethanol it produces. Here, sake yeast is a strain that has been selected and bred for many years with sake moromi with an ethanol concentration as high as 20%, and has extremely high ethanol resistance compared to general yeast.
In the present invention, xylose and arabinose are produced by decomposition of xylan using yeast, and these pentoses are used as an ethanol fermentation raw material. In ethanol fermentation, since it is important to increase the ethanol concentration in the culture solution to the ultimate, the yeast used therein is required to have high ethanol resistance.
Examples of sake yeast include “Kyokai Yeast” distributed by Japan Brewing Association and mutant strains using these as parent strains. In addition, any yeast that is used in sake brewing is useful. When an auxotrophic gene is used as a transformation marker, a strain obtained by imparting auxotrophy to these sake yeasts using a technique such as mutation may be used. Such auxotrophy is uracil auxotrophy, tryptophan. Requirement, leucine requirement, histidine requirement, adenine requirement and the like. Saccharomyces cerevisiae GRI-117-U obtained by a mutation method using “Kyokai No. 9 yeast” as a host is given as an example of imparting uracil requirement to sake yeast.
<Arabinofuranosidase activity>
The arabinofuranosidase activity of the yeast of the present invention obtained as described above is usually about 3 to 10 × 10 ⁻⁵ U per 1 mg of cells. The arabinofuranosidase activity of yeast is a value measured by the method described in Examples.
(4) Production method of first xylan degradation product The first production method of xylan degradation product of the present invention comprises a step of decomposing xylan in the presence of the yeast of the present invention and xylosidase, or further xylanase, and xylan degradation. Recovering the product.
Xylan wood such as cedar, beech and bamboo; weed; seed husks such as rice husk and bran; rice straw; From plant biomass, hemicellulose containing xylan and arabinoxylan as the main component can be extracted by methods such as explosion, solvent extraction, steam distillation, supercritical carbon dioxide extraction, and pressurized hot water extraction.

In the method of the present invention, xylan can be subjected to decomposition in the form of hemicellulose as it is, or xylan separated from hemicellulose can also be subjected to decomposition.
Yeast density in the yeast reaction solution varies depending on the number and activity of arabinofuranosidase immobilized per yeast cell, but the arabinofuranosidase activity in the reaction solution is about 0.02 to 5 U / l. The density is preferable, and the density is more preferably about 0.05 to 2 U / l.

  Yeast may be included in the reaction solution as it is, or may be immobilized, but is immobilized in that it can be used continuously by continuous reaction, batch reaction or semi-batch reaction. Is preferred. “Immobilization” means a state in which the yeast is not in a free state, for example, a state in which the yeast is bound to, attached to, or incorporated into the carrier. The immobilization method is not particularly limited, and a known microorganism immobilization method can be employed. Examples of such known immobilization methods include a method of holding yeast on a carrier having pores, a method of enclosing yeast with a lattice or microcapsule, and the like.

  As the carrier, a carrier made of a substance insoluble in water or a solvent in the reaction solution is used. Specifically, for example, a porous body made of a resin such as polyvinyl alcohol, polyacrylamide, polyvinyl formal, or cellulose can be suitably used. In addition, foams such as polyurethane foam, polystyrene foam, and silicon foam can be used as the porous body. A porous carrier is desirable because yeast can be successfully grown in the carrier, and further, yeast with reduced activity or dead yeast can be retained for a long time without detachment.

  Although the diameter of the pores (openings) of the porous body varies depending on the kind of yeast, it is usually preferably about 50 to 1000 μm, more preferably about 50 to 100 μm. If it is the range of the said opening part diameter, invasion and multiplication of yeast will be easy, and it can hold | maintain yeast with high density.

  The outer shape of the carrier is not particularly limited, and may be any shape such as a spherical shape, a cubic shape or an indefinite shape; a mesh shape or a sheet shape. It is preferable that it is granular in terms of easy preparation. The size of the particles may be, for example, about 2 to 50 mm in diameter in the case of a sphere, and about 2 to 50 mm in the case of a cube.

As a method of immobilizing yeast by inclusion, a method of including yeast in a lattice form using polyacrylamide, calcium alginate, κ-carrageenan, synthetic prepolymer (photocrosslinkable resin prepolymer, urethane prepolymer, etc.); The method of covering this with the microcapsule which consists of a polymer semipermeable membrane by methods, such as a separation method, an interfacial polymerization method, and an underwater drying method, is mentioned. Moreover, the method of bridge | crosslinking yeast mutually is mention | raise | lifted.
In order to decompose the β-1,4 bond between xylose of the main chain of xylanase / xylosidase xylan, xylosidase or further xylanase is added to the reaction solution. Both xylanase and xylosidase have the activity of cleaving the β-1,4 bond of the xylan main chain. Among them, xylanase is an endo-type enzyme and has an activity of cleaving regardless of the site of the xylan main chain. On the other hand, xylosidase is an exo-type enzyme that cleaves xylose one by one from the end of the xylan main chain. Therefore, xylanase alone cannot produce xylose from xylan. In addition, xylosidase is slow in reaction rate due to the property of decomposing from the xylan end. In the method of the present invention, it is preferable to use a combination of xylanase and xylosidase.

  These enzymes may be derived from any organism. For example, the thing of origin, such as bacteria and mold, can be used. Among them, it is preferable to use those originating from mold (genus Aspergillus) in terms of easy preparation of a large amount of enzyme.

The amount of xylosidase and xylanase used may be such that the activity in the reaction solution is usually about 0.1 to 200 U / l, preferably about 1 to 20 U / l. These enzyme activities are values measured by the method described in Examples.
Reaction The decomposition reaction of xylan with arabinofuranosidase and xylosidase or further xylanase can be carried out in a state where free yeast or immobilized yeast is suspended in the reaction solution in the reaction vessel. Moreover, it can also carry out using the bioreactor which filled the yeast free or fix | immobilized in reaction containers like a column. The reaction may be carried out by any of continuous, batch (batch) and semi-batch methods.

  In the reaction solution, only the substrate may be contained, or in addition to this, a buffer for pH adjustment and a known substance necessary for the survival of the yeast may be contained.

  The concentration of the substrate (xylan) in the reaction solution is usually preferably about 0.02 to 20% by weight, more preferably about 0.05 to 5% by weight. If the substrate concentration is too low, practical xylose production efficiency cannot be obtained. On the other hand, if the substrate concentration is too high, the viscosity of the reaction solution may increase, making stirring and temperature control difficult, and the osmotic pressure of the reaction solution may increase, resulting in a state where a great deal of stress is applied to the yeast. . Extreme stress triggers deterioration of cell proliferation and cell death (destruction), and as a result, ideal reaction efficiency cannot be obtained. However, such a problem does not occur within the above-mentioned range.

  The pH of the reaction solution is preferably about 4 to 8 including the optimum pH of arabinofuranosidase and xylosidase, or xylanase. Since the pH of the reaction solution decreases with the progress of the reaction, it is desirable to adjust the pH to be within the above range.

  The reaction temperature should just be the temperature which the arabinofuranosidase and xylosidase which yeast hold | maintain, and also xylanase show activity, Usually, about 20-60 degreeC is preferable. When a thermostable enzyme is used, it can be performed at a higher temperature. In this case, even if the yeast cannot grow, since the arabinofuranosidase on the surface layer is immobilized with activity, there is no problem in the degradation reaction.

  When cellulose or the like is mixed in addition to xylan in the reaction solution, various sugar hydrolases such as cellulase may be used in combination.

What is necessary is just to determine the additional amount of a substrate, reaction time, the addition amount of a pH adjuster, etc. by monitoring each component density | concentration in a reaction liquid by measuring with time, for example with a gas chromatograph or HPLC.
Recovery of xylan degradation product Depending on the species of xylan origin, xylose, arabinose, etc. are mainly produced in the reaction solution by the above reaction. Monosaccharides may be separated from the reaction solution after completion of the reaction by centrifugation or the like. Thereby, the xylan decomposition product can be recovered. These monosaccharides can be used as they are as substrates for ethanol fermentation.
(5) Manufacturing method of 2nd xylan decomposition product The manufacturing method of the 2nd xylan decomposition product of this invention is obtained by the process (a) which decomposes | disassembles xylan in presence of the yeast of this invention, and a process (a). The method comprises a step (b) of decomposing the obtained xylan partial decomposition product in the presence of xylosidase or further xylanase, and a step of recovering the xylan decomposition product.
Decomposition step with arabinofuranosidase (step (a))
The substrate, yeast and reaction method are the same as in the first method. The arabinofuranosidase activity in the reaction solution is the same as in the first method. The reaction solution after completion of the reaction is subjected to the next step as it is or after recovering and removing arabinose by centrifugation or the like.
Decomposition step with xylosidase or further xylanase (step (b))
After decomposing arabinose on the side chain of xylan using the yeast of the present invention, the main chain xylose is decomposed with xylosidase or further xylanase. The reaction method and the recovery method of the xylan decomposition product are the same as in the first method. The enzyme activity in the reaction solution is the same as in the first method.
Examples Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

In the present invention, the enzyme activity is a value measured by the following method.
<Detection of arabinofuranosidase activity>
The presence or absence of arabinofuranosidase activity was determined by adding bacterial cells or culture supernatant to 50 mM acetate buffer (pH 5.0) containing 1 mM 4-Nitrophenyl α-L-arabinofuranoside and allowing to stand at 30 ° C. for 3 hours. The released 4-nitrophenol was visually confirmed. Since free 4-Nitrophenol exhibits a yellow color, this coloration is confirmed.
<Quantification of arabinofuranosidase activity in bacterial cells>
The arabinofuranosidase activity was determined by adding the bacterial cells or culture supernatant to 50 mM acetate buffer (pH 5.0) containing 1 mM 4-Nitrophenyl α-L-arabinofuranoside, leaving it at 30 ° C. for 3 hours, and releasing it. The amount was determined from the amount of 4-nitrophenol. The arabinofuranosidase activity was defined as 1 U = the amount of enzyme required to produce 1 μmol of 4-Nitrophenol from 1-Nitrophenyl α-L-arabinofuranoside per minute.
<Quantification of xylanase activity>
For the measurement of xylanase activity, the cells, culture supernatant or purified enzyme solution was added to 50 mM acetate buffer (pH 5.0) containing 1% xylan from birchwood (Sigma) and allowed to stand at 30 ° C. for 2 hours. Thereafter, the reducing power of the released sugar was quantified by the Somogy Nelson method. Xylanase activity was defined as 1U = the amount of enzyme that produced a reducing power corresponding to 1 μmol of xylose per minute from xylan from birchwood.
<Quantification of xylosidase activity>
The xylosidase activity was determined by adding the bacterial cell or culture supernatant purified enzyme solution to 50 mM acetate buffer (pH 5.0) containing 1 mM 2-Nitrophenyl β-D-xylide, and allowing to stand at 30 ° C. for 5 minutes. It was determined from the amount of 2-nitrophenol. Xylosidase activity 1 U = 2-Nitrophenyl β-D-xylide was defined as the amount of enzyme required to produce 1 μmol of 2-Nitrophenol per minute.

(Preparation of DNA having a secretory signal sequence, a structural gene sequence of arabinofuranosidase, a partial sequence of α-agglutinin and a GPI anchor attachment signal sequence in this order)
The procedure for preparing the subject DNA will be described with reference to FIG.
(A) A secretory signal sequence of Aspergillus oryzae glucoamylase was obtained according to a conventional method. Briefly, 5′-GAATTCATCGCGGAACAACCTCTCTTTTTT-3 ′ (SEQ ID NO: 1) and 5′-GTCGGACGTTGAGATCCCACTACTCCTCTT-3 ′ (SEQ ID NO: 2) were synthesized and used as primers for plasmid pNGB1 (Gene 207 127-134 ( 1998)) was used as a template for PCR. As PCR conditions, a cycle of 94 ° C./1 minute-52 ° C./1 minute-72 ° C./30 seconds was repeated 30 times. Here, the restriction enzyme sites EcoRI and SalI were added to the design of both primers in order to later fuse the secretory signal sequence with arabinofuranosidase.

The obtained PCR product was purified with a spin column (Qiagen, PCR Purification Kit) and then cleaved with restriction enzymes EcoRI and SalI to obtain a glucoamylase secretion signal sequence of about 100 bp. The sequence is shown in SEQ ID NO: 7 in the Sequence Listing (B). Aspergillus oryzae arabinofuranosidase B cDNA was obtained according to a conventional method. Briefly, first, total RNA was extracted from Aspergillus oryzae, and then Poly (A) + RNA was obtained using oligo dT cellulose.

  Next, using Poly (A) + RNA as a template, reverse transcription reaction was performed using RT-PCR KIT manufactured by GIBCO BRL to obtain a cDNA mixture. That is, 5'-gtcgacATGTTCCTCAGGATTAAGCCT-3 '(SEQ ID NO: 3) and 5'-gttaacCAGCAAAGCCAGTGCTGACA-3' (SEQ ID NO: 4) were synthesized, and PCR was carried out using this cDNA mixture as a template. PCR was performed under the condition of repeating a cycle of 94 ° C / 1 minute-52 ° C / 1 minute-72 ° C / 2 minutes 30 times after 50 ° C / 30 minutes-94 ° C / 2 minutes. Here, in designing both primers, a restriction enzyme site SalI was added in order to later fuse one end of arabinofuranosidase with a secretory signal sequence, and the other end was part of the C-terminus of α-agglutinin. For fusion, the restriction enzyme site HpaI was added and the translation stop codon was removed.

The obtained PCR product was purified with a spin column (Qiagen, PCR Purification Kit), and then cleaved with restriction enzymes SalI and HpaI to obtain an about 1.5 kbp arabinofuranosidase cDNA fragment. The sequence is shown in SEQ ID NO: 8 in the sequence listing.
(C) Chromosomal DNA is obtained from yeast Saccharomyces cerevisiae MT8-1 by a conventional method in order to obtain a sequence having a gene (320 amino acids) having a sequence encoding a part of the C-terminus of α-agglutinin and a GPI anchor attachment signal sequence. Was isolated and PCR was performed using two primers: 5′-cccggggctcgAGCGCCCAAAAGCTCTTTTATC-3 ′ (SEQ ID NO: 5) and 5′-ggtaccTTTGATTTAGTTTCTTTCTAT-3 ′ (SEQ ID NO: 6). As PCR conditions, a cycle of 94 ° C./1 minute-52 ° C./1 minute-72 ° C./2 minutes was repeated 30 times.

  The obtained PCR product was purified with a spin column (Qiagen, PCR Purification Kit) and then digested with SmaI and KpnI to obtain a SmaI-KpnI fragment. This SmaI-KpnI fragment contains a sequence encoding 320 amino acids from the C-terminal of α-agglutinin and 446 bp of the 3 ′ non-coding region, and this sequence contains a GPI anchor attachment signal sequence. . The sequence is shown in SEQ ID NO: 9 in the sequence listing.

  The target DNA, which is one example of the polynucleotide of the present invention, comprises the secretory signal sequence obtained in (A), the arabinofuranosidase gene obtained in (B), and the SmaI-KpnI fragment obtained in (C). Can be obtained by using a DNA ligation enzyme (T4 Ligase) or the like according to a conventional method.

  The obtained target DNA was inserted into the EcoRI and KpnI cleavage sites of the plasmid pRS406 Psed1 to obtain the target plasmid pK113-abfB which is one example of the present invention.

(Production of yeast having arabinofuranosidase on the cell surface)
Plasmid pK113-abfB obtained in Example 1 was isolated, and DNA that had been linearized by cutting one site with restriction enzyme StuI was introduced into yeast Saccharomyces cerevisiae GRI-117-U by the lithium acetate method. 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101), cultured on an SD agar medium containing 2% glucose, and grown yeasts were selected. In this medium, plasmid pK113-abfB is introduced into the chromosome, and only strains with complemented uracil requirement can grow. This yeast was named Saccharomyces cerevisiae GRI-117-U-abfB (hereinafter simply referred to as GRI-117-U-abfB).

  The obtained yeast GRI-117-U-abfB was added to an SD liquid medium containing 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101) and 2% glucose. After culturing and centrifuging, the medium and the cells were separated, and the arabinofuranosidase activity of each was measured. As a control, Saccharomyces cerevisiae GRI-117-U was used. As a result, the control showed no arabinofuranosidase activity in the medium and cells. Almost no arabinofuranosidase activity was found in the medium of transformed yeast GRI-117-U-abfB, but yeast GRI-117-U-abfB cells had arabinofuranosidase activity. It was.

(Degradation of xylan by yeast having arabinofuranosidase B on the cell surface)
The yeast GRI-117-U-abfB obtained in Example 2 was grown on a YNB medium, then collected and washed, and the cells were treated with a reaction solution (0.5% Xylan from oat spells (manufactured by SIGMA) ( pH 5.0)) was added so that the arabinofuranosidase activity was 5 mU, and 20 mU each of xylanase and xylosidase was added and reacted at 30 ° C. After 3 hours of reaction, the mixture was centrifuged, the xylose in the supernatant was quantified, and compared with the case where yeast GRI-117-U-abfB was not added.
As a result, the addition of yeast GRI-117-U-abfB increased xylose production by 18%. Since the arabinose content of the used Xylan from oat salts (manufactured by SIGMA) was 10%, it was found from this result that when abfB was used, the arabinose side chain could be almost completely removed from xylan.

(Immobilization of yeast having arabinofuranosidase on the cell surface)
0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101), 6 mm square polyurethane in a bubble column type culture tank containing 3 L of SD medium containing 2% glucose Form BSPs were added at 1,000 cells / L, and cultured with yeast GRI-117-U-abfB at an aeration rate of 6 to 20 L / min. Thereby, yeast GRI-117-U-abfB was fixed to the polyurethane foam.

  The arabinofuranosidase activity of this yeast was 0.01 U per polyurethane foam.

(Degradation of xylan by yeast having arabinofuranosidase B on the immobilized cell surface)
To 3,000 yeast GRI-117-U-abfB fixed to the polyurethane foam obtained in Example 4, reaction solution: crude xylan 100 g, xylanase (total activity 150 U amount), xylosidase (total activity 150 U amount), And 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101), 3 L of SD medium containing 2% glucose, and aeration of 3-20 L / min It was made to contact for 24 hours at 30 degreeC in a bubble column type culture vessel. During the reaction, the pH was controlled at 5.0 to 6.0 using KOH.

After collecting the reaction solution of the first cycle, a new reaction solution was added to perform a reaction of 2 cycles, and this was performed up to 5 cycles. As a result of analyzing the composition of the reaction solution after completion of the first cycle, it was found that 11.5 g of xylose was produced in the culture tank. In addition, when yeast GRI-117-U-abfB (arabinofuranosidase) fixed to the polyurethane foam was not added, 9.5 g of xylose was produced in the culture tank. That is, about 20% increase in xylose production was observed by adding arabinofuranosidase.
The immobilized yeast GRI-117-U-abfB expressed high arabinofuranosidase activity without any decrease in activity after the second cycle.

It is a figure which shows the structure of the polynucleotide of this invention. It is a figure explaining the mode of processing and intracellular transport of GPI anchoring protein. It is a schematic diagram of surface layer presentation vector preparation in an Example.

Claims (11)

In order from the 5 ′ end, a secretory signal sequence that functions in yeast cells, a sequence that encodes a polypeptide exhibiting arabinofuranosidase activity, a sequence that encodes part of a cell surface localized protein, and a GPI anchor attachment signal sequence A polynucleotide comprising. The polynucleotide according to claim 1, wherein the sequence encoding a part of the cell surface localized protein and the sequence consisting of a GPI anchor adhesion signal sequence are included in the sequence encoding yeast α-agglutinin. 3. A sequence comprising a sequence encoding a part of a cell surface localized protein and a GPI anchor attachment signal sequence is contained in a sequence encoding 320 to 600 amino acids from the C-terminal of yeast α-agglutinin. The polynucleotide according to 1. The polynucleotide according to claim 1, 2, or 3, wherein the polypeptide having arabinofuranosidase activity is an enzyme derived from Aspergillus oryzae. The polynucleotide according to any one of claims 1 to 4, wherein the polynucleotide is operably linked to a promoter that can function in yeast cells. An expression vector comprising the polynucleotide of claim 5. A yeast carrying the polynucleotide according to claim 5 or the expression vector according to claim 6. A method for producing a xylan degradation product, comprising the steps of decomposing xylan in the presence of the yeast and xylosidase of claim 7, or further xylanase, and a step of recovering the xylan degradation product. The method according to claim 8, wherein the yeast is immobilized on a carrier. Step (a) for decomposing xylan in the presence of yeast according to claim 7, and step (b) for decomposing the xylan partial degradation product obtained in step (a) in the presence of xylosidase or further xylanase And a process for recovering the xylan decomposition product. The method according to claim 10, wherein the yeast is immobilized on a carrier.

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