JP2007049914A - Method for producing xylooligosaccharide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily producing a xylooligosaccharide having a specific structure. <P>SOLUTION: The method for producing a xylooligosaccharide contains a step to decompose a xylan derivative having a side chain bonded to a main chain composed of β-1,4-xylan with an exo-type xylanase. The xylanase is especially preferably xylanase X (XynX) derived from Aeromonas punctata ME-1 strain. The Fig. 3(a) and (b) show a TLC (thin-layer chromatography) plate obtained by developing a hydrolysis product produced by the decomposition of birchwood xylan with xylanase X, and a TLC plate obtained by developing the hydrolysis product after separating with an anion exchange resin. The spots of the same developing degree to X2 and X4 are xylobiose and 4-O-methylglucuronic acid-α-1,2-xylotriose, respectively. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing a xylooligosaccharide having a specific structure and capable of exhibiting an intestinal regulating action and an immunostimulatory action. For example, the present invention relates to a method for producing a xylooligosaccharide having a specific structure such as 4-O-methylglucuronic acid-α1,2-xylotriose by decomposing a polysaccharide having xylan as a main chain with xylanase.

  In recent years, as environmental problems are attracting attention, great expectations are placed on the effective use of renewable biomass resources that can be produced semi-permanently. At present, woody biomass resources are mainly used for food, fuel, fiber, building materials, etc., but the amount is only a small amount of the annual biomass production on the earth. When using wood as a biomass resource, cell walls are often used. The cell walls of wood contain about 45% cellulose, about 30% hemicellulose, and about 25% lignin. Among these, hemicellulose, which is the main component after cellulose, has hardly been industrially used except that xylitol and some xylooligosaccharides are used as food materials.

  The main component of hemicellulose contained in wood includes polysaccharides composed of xylan and its derivatives. Xylan is a linear polymer in which D-xylose residues are linearly bonded by β1,4 bonds. In addition to the above xylan, natural hemicellulose includes a linear main chain composed of xylan, an L-arabinofuranose residue, a D-glucuronic acid residue, a 4-O-methyl-D-glucuronic acid residue. There are xylan derivatives such as arabinoxylan, glucuronoxylan, arabinoglucuronoxylan, and methylglucuronoxylan (4-O-methyl-D-glucuronoxylan) to which side chains such as groups are bonded.

  Xylooligosaccharides are oligosaccharides having a structure in which about 2 to 7 xyloses are linked by β1,4 → glycoside bonds. Xylooligosaccharides are not hydrolyzed by human digestive enzymes, but are known to have a high intestinal regulating action because they can serve as nutrient sources for useful enteric bacteria such as bifidobacteria. In particular, xylo-oligosaccharides have been reported to be effective at a concentration that is an order of magnitude lower than other oligosaccharides, and since they are excellent in heat resistance and stable in a low pH environment, they are used as food additives, etc. Very useful. Xylooligosaccharide is a natural product obtained from the bark of plants such as corn, corn cob, bagasse and cottonseed. Xylooligosaccharides can also be obtained from xylan as a wood component. At present, 50 million tons of wood waste materials are discharged annually in Japan, so there are great expectations for the industrial effective use of such waste materials to solve environmental problems and waste problems. .

  In general, an enzyme that decomposes a xylan main chain into a xylo-oligosaccharide is called a xylanase. Xylanase is widely recognized in the microbial community. Many xylanases act on hardwood xylan to produce mainly oligoxylose from xylobiose, aldohexauronic acid from aldobiouronic acid, and the like. Xylanases can be classified into an endo type that randomly decomposes the main chain of xylan and an exo type that cleaves the main chain of xylan from the end. Since the exo-type xylanase cleaves the main chain of xylan with an oligosaccharide unit having a specific degree of polymerization, only xylo-oligosaccharide having a specific structure is specifically generated as a degradation product.

Glycosyl hydrolases, including xylanases, are classified into the 97 family based on conformational and hydrophobic cluster analysis of amino acid sequences. Most of the xylanases are classified across 10 families, most of which are classified into families 10 and 11. In general, family 10 is characterized by high molecular weight (> 30 kDa) and low isoelectric point (pI) acidic proteins. Family 10 has a basic structure consisting of a (β / α) ₈ barrel structure, and about 40% of the secondary structure is occupied by an α helix.

For example, Non-Patent Documents 1 to 3 report xylanase X (XynX), which is a xylanase derived from Aeromonas punctata (conventional name: Aeromonas cabie) ME-1. Xylanase X is a protein having a molecular weight of 38 kDa and having an optimum temperature of 50 ° C. and an optimum pH of 8.0. According to these reports, xylanase X produces xylobiose (a dimer of D-xylose) and xylotetraose (a tetramer of D-xylose) from birchwood xylan. Moreover, although xylanase X is an intracellular localization type xylanase, it is also reported that it is released from the cytoplasm into the periplasm by a shock of lowering osmotic pressure. Furthermore, xylanase X has been confirmed to belong to family 10 of glycosyl hydrolases by X-ray crystal structure analysis.
Usui, and three others, “Xylanase X, a possible exo-xylanase of Aeromonas caviae ME, produces xylobiose and xylotetraose exclusively from xylan. -1 that produces exclusively xylobiose and xylotetraose from xylan.) ", Biosci. Biotechnol. Biochem. , 63 (8), 1346-1352 (1999) Usui, 3 others, “Aeroplasmas xylanase (XynX) of intracellularly localized xylanase (xylanase X) in the strain of ME-1 is released from the cytoplasm into the periplasm by osmotic pressure shock (A cytoplasmic xylanase (XynX) of) Aeromonas caviae ME-1 is released from the cytoplasm to the periplasm by osmotic downshock. Biosci. Bioeng. , 95, 488-495 (2003) Fujimoto, 5 others, “Crystallization and preliminary X-ray crystallographic studies of XynX, a. Crystallization and preliminary X-ray crystallographic studies of XynX, a family 10 xylanase from Aeromonas punctata ME-1.) ", Acta Crystallographica Section F, 61, 255-256 (2005)

  In the non-patent document 1, the present inventors have reported that xylanase X has produced xylobiose and xylotetraose from birchwood xylan. As a result of earnest studies, xylanase X has a specific structure. It has been found that there is an action of generating xylooligosaccharides. And based on these knowledge, it came to complete this invention. An object of the present invention is to provide a method for producing a xylooligosaccharide capable of easily producing a xylooligosaccharide having a specific structure.

  In order to achieve the above object, the invention described in claim 1 is a method for producing a xylooligosaccharide, which comprises a decomposition step of decomposing a xylan derivative with an exo-type xylanase, The main point is to have a structure in which a side chain is bonded to a main chain composed of β1,4-xylan.

The method for producing xylo-oligosaccharide according to claim 2 is the invention according to claim 1, wherein the exo-type xylanase belongs to family 10 of glycosyl hydrolase and is from the N-terminal to the seventh β-sheet-α. The gist is that a (β / α) _8- barrel structure having a loop structure composed of 13 or more amino acid residues is formed between the ribs.

  The method for producing xylo-oligosaccharides according to claim 3 is the method according to claim 1 or 2, wherein the exo-type xylanase reacts with methylglucuronoxylan as a substrate to produce xylobiose and 4-O-methyl. The gist is to have an enzyme activity that produces glucuronic acid-α1,2-xylotriose.

  The method for producing xylo-oligosaccharide according to claim 4 is the invention according to any one of claims 1 to 3, wherein the exo-type xylanase is Aeromonas pantata ME-1 strain (received by the Patent Microbial Deposit Center). No .: Xylanase X derived from NITE AP-127).

  According to a fifth aspect of the present invention, there is provided a method for producing a xylo-oligosaccharide according to any one of the first to fourth aspects, wherein the xylan derivative is methyl glucuronoxylan.

  The method for producing xylooligosaccharide according to claim 6 is the invention according to any one of claims 1 to 5, wherein the xylan derivative comprises 4-O-methylglucuronic acid as the side chain. Is the gist.

  The xylo-oligosaccharide production method according to claim 7 is the invention according to any one of claims 1 to 6, wherein the production method further separates the xylo-oligosaccharide produced through the decomposition step. The gist is to provide a process.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the xylooligosaccharide which can manufacture the xylooligosaccharide which has a specific structure easily can be provided.

Hereinafter, an embodiment embodying the production method of the xylooligosaccharide of the present invention will be described.
The method for producing xylo-oligosaccharide of the present embodiment includes a decomposition step of decomposing a xylan derivative having a side chain bonded to a main chain composed of β1,4-xylan with an exo-type xylanase. In the decomposition step, the xylan derivative is cut out sequentially from the end in oligosaccharide units by exo-type xylanase. For this reason, in the manufacturing method of the xylooligosaccharide of this embodiment, the xylooligosaccharide which has a specific structure depending on the kind of said side chain, a coupling | bonding position, etc. is manufactured.

  Specifically, the xylan derivative used in the production method of the present embodiment has a main chain composed of β1,4-xylan, which is a linear polymer of D-xylose residues, an L-arabinofuranosyl residue, It has a structure in which side chains such as D-glucuronic acid residue and 4-O-methylglucuronic acid residue are bonded. Examples of such xylan derivatives include arabinoxylan, glucuronoxylan, arabinoglucuronoxylan, and methylglucuronoxylan. In addition, as a xylan derivative, what was equipped with all kinds of side chains other than the side chain mentioned above including fructose etc. can be used.

  For example, in the production method of the present embodiment, when a xylan derivative having 4-O-methylglucuronic acid as a side chain, such as methylglucuronoxylan, is used, the xylo-oligosaccharide to be produced includes aldtriouronic acid (4- Aldooligos such as O-methylglucuronylxylobiose), aldotetrauronic acid (4-O-methylglucuronylxylotriose), aldohexauronic acid (4-O-methylglucuronylxylopentose), etc. Contains uronic acid. Birchwood xylan (white xylan) is particularly preferably used as the xylan derivative capable of producing such aldo-oligouronic acid.

The exo-xylanase used in the production method of the present embodiment belongs to the glycosyl hydrolase family 10 and has a loop structure longer than usual between the seventh β sheet-α helix from the N-terminus (so-called loop 7). (Β / α) has a structural feature of forming an _8- barrel structure. Further, the exo-type xylanase has a functional characteristic that it has an enzymatic activity to produce xylobiose and 4-O-methylglucuronic acid-α1,2-xylotriose when reacted with methylglucuronoxylan as a substrate. . That is, the exo-type xylanase of the present embodiment has at least one of the above-mentioned structural characteristics and functional characteristics, preferably both characteristics. Since such a xylanase is an exo type that cleaves the main chain of xylan with an oligosaccharide unit having a specific degree of polymerization, only a xylo-oligosaccharide having a specific structure is specifically generated in the decomposition step.

  Examples of such exo-type xylanases include cells derived from Aeromonas pantata ME-1 strain (patent microorganism deposit center receipt number: NITE AP-127), xylanase X (XynX), and Geobacillus stearothermophilus. Examples thereof include xylanase (1N82A) and xylanase T6 (1HIZA) derived from Bacillus stearothermophilus. XynX is a protein consisting of an amino acid sequence represented by SEQ ID NO: 2 encoded by a gene consisting of the base sequence represented by SEQ ID NO: 1, and has a three-dimensional structure schematically shown in FIG. XynX is described in detail in Non-Patent Documents 1 to 3. 1N82A is a protein encoded by a gene consisting of a base sequence represented by SEQ ID NO: 3 and consisting of an amino acid sequence represented by SEQ ID NO: 4. 1HIZA is a protein consisting of an amino acid sequence represented by SEQ ID NO: 6 and encoded by a gene consisting of a base sequence represented by SEQ ID NO: 5.

In the structural characteristics of exo-type xylanase, the (β / α) ₈ barrel structure has a barrel shape with eight repeating structures composed of β sheet-α helices, as schematically shown in FIG. It is a three-dimensional structure. This barrel structure is often found in sugar hydrolase and forms a catalytic domain that plays a central role in the catalytic activity of the enzyme. Loop 7 is a loop structure existing between the seventh β sheet from the N-terminal of the exo-type xylanase and the seventh α helix. In addition, in the case of the loop 7, the loop structure longer than the usual means that it consists of 13 or more amino acid residues. Incidentally, the loop 7 of the exo-type xylanase of this embodiment consists of 19 to 20 amino acid residues (see FIG. 1). Incidentally, FIG. 1 is a diagram in which the amino acid sequences of the exo-type xylanases are arranged side by side so that they can be easily compared, and the position corresponding to the loop 7 is also shown in the same drawing.

  In the functional characteristics of exo-type xylanases, these exo-xylanases all generate xylobiose and 4-O-methylglucuronic acid-α1,2-xylotriose when reacted with methylglucuronoxylan as a substrate. It has enzymatic activity and may produce other xylo-oligosaccharides in some cases. Incidentally, XynX produces only xylobiose and 4-O-methylglucuronic acid-α1,2-xylotriose when reacted with methylglucuronoxylan as a substrate.

  As the xylooligosaccharides produced by the production method of the present embodiment, those consisting of only a main chain in which about 2 to 7 xyloses are linearly bonded by β1,4-glycoside bonds, or the main chain The thing which the side chain couple | bonded is mentioned. Examples of the xylo-oligosaccharide consisting only of the main chain include xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentose (X5), xylohexaose (X6) or xyloheptaose (X7). It is done. Examples of the xylo-oligosaccharide having a side chain include those in which a monosaccharide or oligosaccharide other than xylose is bound to the sugar constituting X2 to X7. The side chain is a side chain derived from a xylan derivative, and specifically includes an L-arabinofuranose residue, a D-glucuronic acid residue, a 4-O-methylglucuronic acid residue, and the like.

  These xylooligosaccharides can be a nutrient source for useful enteric bacteria such as bifidobacteria (so-called good bacteria) and cannot be a nutrient source for so-called enteric bacteria. Highly effective intestinal regulation. In addition, since these xylooligosaccharides do not serve as a nutrient source for caries fungi such as mutans, they can also be used as sweeteners having a caries-preventing effect. Furthermore, these xylooligosaccharides can also exert an immunostimulatory effect. For example, X2 as a xylo-oligosaccharide has been reported to have a proliferative action against bifidobacteria. X2 has a structure in which two D-xylose residues are linked by β1 → 4 bond as shown in Chemical Formula 1, but useful enteric bacteria such as bifidobacteria cleave the β1 → 4 bond. It is possible to decompose and assimilate the cleaved D-xylose.

In addition, 4-O-methylglucuronic acid 1-α1,2-xylotriose as a xylooligosaccharide is linearly linked with three D-xylose residues by β1 → 4 bonds as shown in Chemical Formula 2. It consists of a bonded xylotriose residue and a 4-O-methylglucuronic acid residue bonded to the D-xylose residue located at the end of the xylotriose residue by an α1 → 2 bond. Among useful enteric bacteria, an enzyme that cleaves β1 → 4 bond between D-xylose residues and a1 → 2 bond between D-xylose residues and 4-O-methylglucuronic acid residues. What is provided with an enzyme is known.

The production method of the present embodiment preferably further includes a separation step of separating a plurality of types of xylo-oligosaccharides produced through the decomposition step. In the separation step, a known separation method capable of separating xylo-oligosaccharides having different structures from each other is used. That is, in this separation step, a plurality of types of xylo-oligosaccharides obtained in the decomposition step are classified (isolated) into various xylo-oligosaccharides.

  Such separation methods include gel filtration chromatography, cation exchange resins (Na type, Ca type, K type, H type, etc.) or ions using anion exchange resins (Cl type, OH type, etc.) as carriers. Exchange chromatography, affinity chromatography in which lectin or antibody is bound to a carrier, activated carbon, silica gel, chromatography using octadecylsilyl group (ODS) resin or gel, etc. are appropriately selected and used. The A batch-type separation method using the same carrier instead of the chromatography described above can also be carried out. Separation methods include fixed bed method (one-pass method), continuous method (pseudo moving bed method) or semi-continuous method (a combination of fixed bed method and continuous method), cation exchange resin, anion Desalination using ion exchange resin, electrodialysis membrane, etc., ultrafiltration, membrane filtration, diatomaceous earth (or hyflosupercell) filtration, filtration by reverse osmosis membrane filtration, etc., crystallization by cooling crystallization method or sucrose method , Known yeast treatment, decolorization using activated carbon or anion exchange resin, etc. are appropriately selected and employed.

  When separating a xylo-oligosaccharide having no charge such as X2 to X7 and a xylo-oligosaccharide having a positive charge such as 4-O-methylglucuronic acid-α1,2-xylotriose, the negative It is most convenient and effective to use ion exchange chromatography. Incidentally, the xylooligosaccharide produced by the production method of the present embodiment may be a mixture of a plurality of types of xylooligosaccharides or a single type of xylooligosaccharide.

The effects exhibited by the embodiment will be described below.
(1) In the method for producing xylooligosaccharide of the present embodiment, exo-type xylanase is used. The endo-type xylanase generally has a property of generating a continuous xylo-oligosaccharide having a polymerization degree such as xylobiose, xylotriose, and xylotetraose. However, the exo-type xylanase of this embodiment has a discontinuous polymerization degree. It has a very unique property of producing a xylo-oligosaccharide having a specific structure. Therefore, by using the exo-xylanase of the present embodiment to hydrolyze a xylan derivative having a specific structure, a xylo-oligosaccharide having a specific structure depending on the structure of the side chain of the xylan derivative is easily obtained. be able to.

(2) In the method for producing xylooligosaccharides of this embodiment, a structure that belongs to the glycosyl hydrolase family 10 and has a (β / α) _8- barrel structure having a loop 7 composed of 13 or more amino acid residues. An exo-xylanase with the above characteristics is used. Further, in the method for producing xylo-oligosaccharide of the present embodiment, the function of having an enzyme activity to produce xylobiose and 4-O-methylglucuronic acid-α1,2-xylotriose when methylglucuronoxylan is reacted as a substrate. An exo-xylanase having the above characteristics is used. The exo-type xylanase of the present embodiment only needs to have at least one of the structural characteristics and functional characteristics. That is, in the present embodiment, there is only a functional feature without the structural feature, or only a structural feature without the functional feature. It doesn't matter. When such a xylanase is used, a xylo-oligosaccharide having a specific structure can be easily obtained as in (1) above. In particular, as in the production method of the present embodiment, a technique capable of producing a large amount and low cost of a xylooligosaccharide having a specific structure such as 4-O-methylglucuronic acid-α1,2-xylotriose is provided. is important.

  (3) The method for producing xylo-oligosaccharides of this embodiment includes a separation step for separating a plurality of types of xylo-oligosaccharides produced through the decomposition step, so that useful xylo-oligosaccharides having a specific structure can be easily isolated. can do. In particular, when anion exchange chromatography is used in the separation step, xylooligosaccharides having a specific structure such as 4-O-methylglucuronic acid-α1,2-xylotriose can be isolated very easily. It is possible and convenient.

  (4) The exo-xylanase of this embodiment decomposes a xylan derivative into a plurality of xylo-oligosaccharides. For example, xylanase X derived from Aeromonas pantata ME-1 strain decomposes xylan derived from birch into two xylo-oligosaccharides of xylobiose and 4-O-methylglucuronic acid-α1,2-xylotriose. In this case, in this embodiment, by further performing a separation step of separating the plurality of xylo-oligosaccharides produced by the decomposition step by ion exchange chromatography, xylobiose and 4-O-methylglucuronic acid-α1,2- Each can be divided into xylotriose. As a result, xylobiose and 4-O-methylglucuronic acid-α1,2-xylotriose can be produced in large quantities with high purity. Incidentally, the method for producing xylo-oligosaccharide of the present embodiment can be applied to the production of only xylobiose, and also can produce only 4-O-methylglucuronic acid-α1,2-xylotriose. It is also possible to apply.

<Preparation of crude enzyme solution of XynX>
After preparing the plasmid pXOL1 (3.7 kbp) in which the xynX gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 is inserted by the method of Non-Patent Document 1, the E. coli recombinant JM109 / pXOL1 is 1 was used. In addition, pXOL1 is deposited with the receipt number: NITE AP-126 at the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation. Next, the recombinant was precultured by the method of Non-Patent Document 1 (LB medium containing 100 μg / ml ampicillin 5 ml, 37 ° C., 12 hours, 120 rpm shaking culture) and main culture (100 μg / ml ampicillin, 0.1 mM). Of IPTG-containing LB medium at 37 ° C. for 5 hours at 120 rpm with shaking). Subsequently, the recombinant was harvested by centrifuging the culture solution after the main culture at 6000 × g for 10 minutes. The recombinant after collection was washed with 100 ml of water and then ultrasonically crushed with a French press (Otake Co., Ltd.) to obtain a crude enzyme solution of XynX.

<Examination of Birchwood Xylan Hydrolyzate 1>
In order to examine the hydrolysis product of xylan by a crude enzyme solution of XynX, a crude solution of XynX was added to 50 mM phosphate buffer (pH 6.8) containing 0.5% birchwood xylan (SIGMA) and 0.02% sodium azide. An equal amount of the enzyme solution was added and reacted at 37 ° C. for 12 hours or 24 hours. After completion of the reaction, the enzyme was inactivated by boiling at 100 ° C. for 5 minutes, and then centrifuged at 12000 rpm for 5 minutes in order to remove unnecessary substances such as undegraded xylan. The supernatant after centrifugation was collected and lyophilized with a centrifugal evaporator. The hydrolyzate after drying was dissolved in 40 μl of methanol, and then centrifuged again at 12000 rpm for 5 minutes in order to remove unnecessary substances. Thin layer chromatography (TLC) was performed using each supernatant after centrifugation.

  To perform TLC, first, 10 μl of the supernatant was spotted on a TLC plate (TLC aluminum sheets Silica gel 60F (MERCK)), and then developed with a developing solvent 1 of chloroform: acetic acid: water = 5: 7: 1. . After the developed TLC plate was sufficiently dried, the coloring reagent 1 of sulfuric acid: methanol = 1: 1 was sprayed onto the TLC plate and heated on the hot plate to visualize the developed hydrolyzate. . The results are shown in FIG.

  In FIG. 3 (a), lane 1 is a standard xylo-oligosaccharide marker that is a mixture of X1, X2, X3, and X4. In lane 2, a hydrolyzate after reaction for 12 hours is developed. The hydrolyzate when reacted for 24 hours is developed. From FIG. 3 (a), it was confirmed that XynX specifically produces only xylobiose (hereinafter referred to as product A) and xylobitetraose (hereinafter referred to as product B) from birchwood xylan. In general, endo-type xylanases produce xylo-oligosaccharides having a continuous polymerization degree such as xylobiose, xylotriose, and xylotetraose, and thus XynX xylanase activity is very unique.

<Preparation of XynX separation preparation (purified preparation)>
The xynX gene inserted into the plasmid pXOL1 was excised with XbaI and HindIII, and then subcloned into the pTTQ18 vector to construct a plasmid pXLIQ1 (5.6 kbp) that was expressed under the control of the tac promoter and lacIq. In addition, pXLIQ1 is deposited with the receipt number: NITE AP-128 in the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation. Plasmid pXLIQ1 was introduced into E. coli BL21 (DE3) and transformed to produce E. coli recombinant BL21 (DE3) / pXLIQ1 (see Non-Patent Document 3).

  Next, the recombinant was precultured by the method of Non-Patent Document 3 (5 ml of LB medium containing 100 μg / ml ampicillin, 37 ° C., 12 hours, shaking culture at 120 rpm) and main culture (100 μg / ml ampicillin, 0. 1 ml IPTG-containing LB medium 100 ml, 37 ° C., shaking culture at 120 rpm). Subsequently, using the osmotic shock method described in Non-Patent Document 2, a crude enzyme solution containing the XynX protein was recovered from the recombinant periplasm obtained in the main culture.

  The obtained crude enzyme solution was separated by the method of Non-Patent Document 3 (Hi TrapQ anion exchange chromatography → (HiPrep Sephacryl HR gel filtration → POROS HQ anion exchange high performance liquid chromatography). The xylanase activity at the time of separation was dinitro. The presence of the protein was confirmed by SDS-PAGE as measured by producing a reducing sugar by the salicylic acid (DNS) method.

[Hi Trap Q]
Column; Q Sepharose Fast Flow anion exchange column (Amersham Biosciences)
Eluent: Buffer A = 20 mM Tris-HCl (pH 8.0)
Buffer B = 20 mM Tris-HCl (pH 8.0) / 1M NaCl
Gradient: Buffer B concentration 0% → 100% (0 min → 30 min)
Flow rate: 5 ml / min Detection wavelength: 280 nm
Column temperature: 4 ° C
Fraction volume: 5 ml
[Hi Prep 26/60]
Column; Hi Prep 26/60 gel filtration column (Amersham Biosciences)
Eluent: 20 mM Tris-HCl (pH 8.0)
Flow rate: 1 ml / min Detection wavelength: 280 nm
Column temperature: 4 ° C
Fraction volume: 5 ml
[POROS HQ]
Column: POROS HQ anion exchange column (PerSeptive Biosystems)
Eluate: Buffer a = 20 mM Tris-HCl (pH 8.0)
Buffer b = 20 mM Tris-HCl (pH 8.0) / 1M NaCl
Gradient: Buffer b concentration 0% (0 min → 5 min)
0% → 30% (5 minutes → 15 minutes)
30% → 100% (15 minutes → 20 minutes)
Flow rate: 4 ml / min Detection wavelength: 280 nm
Column temperature: Room temperature Fraction volume: 1 ml
<Characterization of XynX separation standard>
The active fraction after separation was dialyzed against 20 mM Tris-HCl (pH 8.0). XynX after dialysis (hereinafter referred to as XynX separation preparation) exists as a monomer having a molecular weight of 38 kDa, has an optimum temperature of 50 ° C. and an optimum pH of 8.0, and is 40 ° C. or less. The protein was stable at temperature. Substrate specificity was 195% for autospertoxylan and 0% for carboxymethylcellulose and soluble starch, assuming that the xylanase activity for birchwood xylan was 100%.

  When the amino acid sequence of the XynX isolated sample was determined by a protein sequencer, it was confirmed to be a protein consisting of amino acid sequences 2 to 333 in SEQ ID NO: 2. Further, as shown in FIG. 1, it was also found that XynX has a high homology with family 10 endo-xylanase having a known structure and a high proportion of amino acids in the active site.

Next, when the three-dimensional structure prediction was performed by the homology modeling method using the obtained amino acid sequence, it was suggested that a (β / α) _8- barrel structure could be formed, and a longer amino acid residue than usual. The existence of the following loop 7 was suggested. Subsequently, when a three-dimensional structure analysis was performed by X-ray crystal diffraction, it was found that a three-dimensional structure schematically shown in FIG. 2 was formed. In addition, the code | symbol 20 shown in FIG. 2 shows XynX. In XynX, the spiral ribbon 21 that is darkly colored represents an α helix, the ribbon ribbon 22 that is lightly colored represents a β sheet, and the belt-shaped region indicated by hatching represents a loop 7.

  Next, in order to determine the nucleophilic catalytic residue and the acid-base catalytic residue at the active center of XynX, E133A and E240A (in the amino acid sequence represented by SEQ ID NO: 2, the 134th and 241st E are respectively represented. The azide test was conducted on two types of amino acid substitution mutants (substituted by A). As a result, it was found that E240 is a nucleophilic catalyst residue and E133 is an acid-base catalyst residue (see FIG. 2).

<Examination of birchwood xylan hydrolysis products 2>
The xylan hydrolyzate by the XynX isolated sample was examined in detail. First, the XynX separation sample was added to 20 mM Tris-HCl buffer (pH 8.0) containing 0.5% birchwood xylan so as to be 10 μg / ml, and reacted at 37 ° C. for 12 hours. After completion of the reaction, the enzyme was inactivated by boiling at 100 ° C. for 5 minutes, and then centrifuged at 12000 rpm for 5 minutes in order to remove unnecessary substances such as undegraded xylan. The supernatant after centrifugation was collected and lyophilized with a centrifugal evaporator. The hydrolyzate after drying was dissolved in methanol, and then centrifuged again at 12000 rpm for 5 minutes to remove unnecessary substances. The supernatant after centrifugation was collected and lyophilized with a centrifugal evaporator.

  Next, sugar was hydrolyzed using a part of the hydrolyzed product after freeze-drying. First, 500 ml of 0.5 M sulfuric acid was added to 5 mg of the hydrolyzate, incubated at 105 ° C. for 2 hours, neutralized with sodium hydroxide, and then lyophilized. The acid hydrolyzate after drying was dissolved in methanol.

  TLC was performed using the remaining hydrolyzate after lyophilization and the acid hydrolyzate. For the TLC, first, 10 μl of each sample solution of hydrolyzate and acid hydrolyzate was spotted on four TLC plates. The two TLC plates were developed with the developing solvent 1, and the remaining two TLC plates were developed with developing solvent 2 of 1-butanol: acetic acid: water = 2: 1: 1. After each developed TLC plate is sufficiently dried, the coloring reagent 1 is sprayed on one TLC plate developed with the developing solvent 1 and one TLC plate developed with the developing solvent 2, respectively. Heated on plate. Further, the coloring reagent 2 composed of diphenylamine-aniline reagent (1 g of diphenylamine, 1 ml of aniline, 50 ml of acetone and 80% phosphoric acid) was sprayed on the remaining TLC plate and heated on a hot plate. The results are shown in FIGS.

  4 (a) and 4 (b) are both TLC plates developed with a chloroform-based developing solvent 1, and FIG. 4 (a) is visualized with a coloring reagent 1 of sulfuric acid / methanol. FIG. 4 (b) is visualized with a coloring reagent 2 comprising a diphenylamine-aniline reagent. 4A and 4B, a standard xylo-oligosaccharide marker is developed in lane 1, a hydrolyzate is developed in lane 2, an acid hydrolyzate is developed in lane 3, and lanes 4-7. Are developed glucuronic acid, glucose, fructose and arabinose, respectively. 5 (a) and 5 (b) are TLC plates developed with a butanol-based developing solvent 2, and FIG. 5 (a) is visualized with the coloring reagent 1, and FIG. ) Is visualized with the coloring reagent 2. The samples developed in each lane 1 in FIGS. 5A and 5B are the same as those in FIG.

  As a result, the spot (product B) detected at substantially the same position as X4 in lane 2 of FIG. 4A was detected at a position different from marker X4 in lane 2 of FIG. 5A. Further, in both lanes 3 of FIGS. 4A and 5A, spots were detected at the position of X2 in addition to X1. When only X2 and X4 are produced specifically from birchwood xylan, only X1 should be detected if the hydrolyzate is acid hydrolyzed. Therefore, it was suggested that the hydrolyzed products when birchwood xylan was hydrolyzed with XynX, that is, products A and B were not X2 and X4.

  On the other hand, when a TLC plate is visualized with a coloring reagent 2 comprising a diphenylamine-aniline reagent, it is known to develop an aldohexose blue-violet, a pentose brown-violet, a ketohexose yellow-red, and a uronic acid purple-brown. . The spot of X2 (product A) and the spot near X4 (product B) shown in lane 2 of FIGS. 4 (b) and 5 (b) are both colored in different colors, and product A is brown brown of xylose. It resembles purple and product B resembles the purple brown of glucuronic acid. Further, the X1 spot and the spot near X2 shown in lane 3 (acid hydrolyzate) in FIGS. 4B and 5B are both colored in different colors, and the X1 spot is a xylose spot. It resembles a brown-purple color, and the spot near X2 resembles a purple-brown color of glucuronic acid. Therefore, it was suggested that the spot near X2 shown in lane 3 of FIGS. 4B and 5B is not an incomplete acid hydrolyzate but a uronic acid substance. Therefore, it is strongly suggested that when birchwood xylan is hydrolyzed by XynX, product A composed of X2 and product B composed of a substance containing uronic acid are specifically generated.

  Birchwood xylan consists of a β1,4-linked xylose main chain (Xn) and a side chain consisting of 4-O-methyl-D-glucuronic acid α1,2 bonded to the xylose residue constituting the main chain. It is known that it contains a high proportion of xylan derivatives comprising Further, the side chain is considered to be randomly distributed at a ratio of about 1 to 10 xylose residues constituting the main chain. Assuming that XynX hydrolyzes such a xylan derivative, the spot (product B) detected in the vicinity of X4 in each lane 2 in FIGS. 4 and 5 is 4-O-methyl-D-glucuron in Xn. It is presumed to have a structure in which an acid residue is bound.

<Isolation of products A and B>
100 ml of 1.0% birchwood xylan solution was added to 100 ml of the crude enzyme solution of XynX prepared in Example 1 and reacted at 37 ° C. for 12 hours, and then the hydrolyzate containing products A and B was extracted with methanol. Next, an anion exchange resin Dowex (Cl ⁻ ) (Muromachi Technos) was added to the methanol extract containing the hydrolyzate and stirred overnight, and then packed in an 1.5 cm × 15 cm Econo column (Bio-Rad). The non-adsorbed fraction was eluted by washing the inside of the column with 300 ml of water. Next, the adsorbed fraction was eluted by flowing 100 ml of 0.5 M sodium chloride through the column. The non-adsorbed fraction was evaporated and then subjected to the next test. The adsorbed fraction was evaporated, desalted in an Econo column packed with gel filtration resin Sephadex G-10 (Amersham Biosciences), and subjected to the next test after the solvent was replaced with water.

  Finally, 25 mg of the non-adsorbed fraction and 12 mg of the adsorbed fraction were obtained. Further, the hydrolyzate, the non-adsorbed fraction and the adsorbed fraction before being subjected to the anion exchange resin were spotted on a TLC plate, and TLC was performed under the same conditions as in Example 1. The results are shown in FIG. In FIG. 3 (b), the standard xylo-oligosaccharide marker is developed in lane 1, the hydrolyzate is developed in lane 2, the non-adsorbed fraction is developed in lane 3, and the adsorbed fraction is developed in lane 4. Has been. As shown in the figure, products A and B could be isolated by a separation method using an anion exchange resin.

<Identification of products A and B>
¹ H-NMR was measured for the non-adsorbed fraction (product A) and the adsorbed fraction (product B), respectively. As a result, product A was identified as X2. On the other hand, product B is a complex compound, and a lot of noise was detected during measurement, so analysis that led to identification could not be performed.

  Next, the product B was subjected to mass spectrometry by MALDI-TOF MS. As a result, it was found that the molecular weight of product B was about 627. This molecular weight coincided with the molecular weight obtained by adding 4-O-methyl-D-glucuronic acid and sodium salt to X3. Therefore, it was found that product B was 4-O-methyl-D-glucuronyl xylotriose (aldohexauronic acid).

  Next, methylation analysis was performed on product B in order to examine at which position of X3 the 4-O-methyl-D-glucuronic acid residue was bound. In the methylation analysis, product B was first methylated, acid hydrolyzed, and then acetylated, and then subjected to gas chromatography (GC). As a result, the results of alditol acetate and GC predicted to be detected at the binding position of 4-O-methyl-D-glucuronic acid residue indicate that 4-O-methyl-D-glucuronic acid is located at the non-reducing end position. Consistent with the pattern when the residues were bound.

  In order to confirm this, product B was reduced, methylated, acid hydrolyzed and acetylated, and then subjected to GC. The detected alditol acetate was 4-O-methyl-D- It was consistent with the alditol acetate presumed when glucuronyl xylotriose was reduced. Furthermore, after reducing product B, methylation, acid hydrolysis and acetylation were followed by analysis by GC-MS analysis. When 4-O-methyl-D-glucuronylxylotriose was reduced It was consistent with the alditol acetate speculated. Therefore, it is concluded that product B has a structure in which a 4-O-methyl-D-glucuronic acid residue is bonded to the 2-position of xylopyranose on the non-reducing terminal side of X3, that is, the structure shown in Chemical Formula 2 above. It was. And according to the manufacturing method of the xylo-oligosaccharide of a present Example, it was shown that xylobiose and 4-O-methylglucuronic acid- (alpha) 1, 2- xylotriose can be manufactured in high purity and in large quantities.

<Hydrolysis of autospertoxylan>
To a 20 mM Tris-HCl buffer solution (pH 8.0) containing the XynX isolated preparation prepared in Example 2 at a concentration of 10 μg / ml, 500 μl of a 0.5% autospertoxylan solution as a substrate was added, and the mixture was added at 37 ° C. for 6 minutes. The hydrolyzate was obtained by reacting for 12 hours. Moreover, the acid hydrolyzate was obtained by carrying out the acid hydrolysis of each obtained hydrolysis product similarly to the said Example 2. FIG. TLC was performed on these hydrolysates and acid hydrolysates under the same conditions as in Example 2 above.

  As a result, spots having almost the same position, size, and darkness were confirmed in the hydrolyzate when reacted for 6 hours and 12 hours. Incidentally, the confirmed spots included those located in the vicinity of X1, X2, X3, X4, and X5. That is, the pattern of spots generated using autospertoxylan (arabinoglucuronoxylan) as a substrate was different from the pattern when birchwood xylan of each of the above examples was used as a substrate. Therefore, it was confirmed that XynX generates different xylooligosaccharides depending on the structure of the side chain of the xylan derivative.

<Influence of loop 7 on xylanase activity>
A xynX loop 7 deletion gene (mL7) was prepared by removing nucleotides 919 to 975 of the base sequence represented by SEQ ID NO: 1. Next, TA7 was cloned into pT7 vector and then subcloned into pTTQ18 vector to construct plasmid pXLIQ1 (mL7) (5.6 kbp) expressed under the control of tac promoter and lacIq. Plasmid pXLIQ1 (mL7) is introduced into E. coli BL21 (DE3) and transformed to produce E. coli recombinant BL21 (DE3) / pXLIQ1 (mL7), and then preculture and main culture in the same manner as in Example 1. did. Subsequently, the culture solution after the main culture was ultrasonically disrupted in the same manner as in Example 1 to obtain a crude enzyme solution containing a XynX loop 7 deletion protein (XynX mL7).

  The obtained crude enzyme solution was reacted with birchwood xylan in the same manner as in Example 1 except that the reaction temperature was 40 ° C. and the reaction time was 2 hours, and then analyzed by TLC. The wild-type XynX crude enzyme solution obtained in Example 1 was similarly reacted and then analyzed by TLC. As a result, in the wild type XynX, X2 (product A) and 4-O-methylglucuronylxylotriose (product B) were clearly detected on the TLC plate, whereas in XynX mL7, No xylo-oligosaccharide was detected.

Therefore, it can be expected that the presence of the loop 7 composed of 13 or more amino acid residues is important for the expression of the unique xylanase activity (activity for generating products A and B from birchwood xylan) by XynX. In addition, assuming a three-dimensional structure, the loop 7 longer than usual found in XynX holds the end of the main chain of the xylan derivative so as to cover the end of the xylan derivative in an oligosaccharide unit in order from the end. It can be suggested that it contributes directly to the type of enzyme activity. Therefore, using an exo-xylanase belonging to the family 10 of glycosyl hydrolase and having a (β / α) _8- barrel structure having a loop 7 consisting of 13 or more amino acid residues, xylan is used as the main chain. It can be strongly suggested that it is possible to produce a xylo-oligosaccharide having a specific structure by decomposing the polysaccharide.

The figure which compared the amino acid sequence of the some xylanase. The figure which shows the three-dimensional structure of xylanase X typically. (A) shows the TLC plate which developed the hydrolysis product which hydrolyzed birch wood xylan with XynX in Example 1, (b) shows the TLC which developed after separating the hydrolysis product with the ion exchange resin in Example 3. The plate is shown. In Example 2, a TLC plate in which a hydrolyzed product was developed with a chloroform-based developing solvent, (a) was visualized with sulfuric acid / methanol, and (b) was visualized with a diphenylamine-aniline reagent. Is. In Example 2, a TLC plate in which a hydrolyzed product was developed with a butanol-based developing solvent, (a) was visualized with sulfuric acid / methanol, and (b) was visualized with a diphenylamine-aniline reagent. Is.

Explanation of symbols

  20: Xylanase X.

Claims (7)

A method for producing a xylo-oligosaccharide comprising:
The method comprises a decomposition step of decomposing a xylan derivative with an exo-type xylanase,
The method for producing a xylooligosaccharide, wherein the xylan derivative has a structure in which a side chain is bonded to a main chain composed of β1,4-xylan. The exo-xylanase belongs to the family 10 of glycosyl hydrolases, and has a loop structure consisting of 13 or more amino acid residues between the 7th β sheet-α helix from the N terminus (β / α) ₈ barrels The method for producing a xylo-oligosaccharide according to claim 1, wherein a structure is formed.   The exo-type xylanase has an enzyme activity for producing xylobiose and 4-O-methylglucuronic acid-α1,2-xylotriose when reacted with methylglucuronoxylan as a substrate. A method for producing the xylo-oligosaccharide according to claim 2.   The exo-type xylanase is xylanase X derived from Aeromonas pantata ME-1 strain (patent microorganism deposit center receipt number: NITE AP-127), according to any one of claims 1 to 3. The manufacturing method of xylo-oligosaccharide of description.   The method for producing a xylo-oligosaccharide according to any one of claims 1 to 4, wherein the xylan derivative is methyl glucuronoxylan.   The said xylan derivative is a manufacturing method of the xylooligosaccharide as described in any one of Claims 1-5 provided with 4-O-methyl glucuronic acid as the said side chain.   The said manufacturing method is further equipped with the process of isolate | separating the said xylooligosaccharide produced | generated through the said decomposition | disassembly process, The manufacturing method of the xylooligosaccharide as described in any one of Claims 1-6 characterized by the above-mentioned.