JP2002045177A - NEW alpha-L-ARABINOFURANOSIDASE AND METHOD FOR PRODUCING THE SAME AND METHOD FOR PRODUCING L-ARABINOSE AND XYLITOL USING THE SAME - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly active α-L-arabinofuranosidase capable of specifically splitting the L-arabinose side chain of arabinoglucuronoxylan, and to provide a method for producing L-arabinose and xylitol using the above enzyme. SOLUTION: This new α-L-arabinofuranosidase has the physicochemical properties described below: (1) action and substrate specificity: acting on arabinoglucuronoxylan to specifically split the L-arabinose side chain thereof; (2) optimum pH and stable pH: the optimum pH is about 4.0 in a citrate buffer solution, and stable at about pH 7-10 under the treatment condition at 50 deg.C for 3 h; (3) optimum temperature and temperature stability: the optimum temperature is about 65 deg.C, substantially undergoing no deactivation at <=50 deg.C; (4) effect of metal ions: undergoing inhibition by Hg2+; (5) molecular weight: about 88,000 determined by gel filtration chromatography; and (6) isoelectric point pI: about 4.2.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel α-L-arabinofuranosidase having a high activity, which specifically cleaves the L-arabinose side chain of arabinoglucuronoxylan, a method for producing the same, and the novel α-L-arabinofuranosidase. The present invention relates to a method for producing L-arabinose and xylitol using L-arabinofuranosidase.

[0002]

2. Description of the Related Art In order to protect the global environment and global resources,
Effective use of plant biomass is essential. The organic components of plant waste and waste wood, including wood, which make up about 90% of plant biomass, are broadly classified into cellulose, hemicellulose and lignin. Of these, many studies have been made on cellulose and many examples have been put to practical use, and the fact that lignin has low utility due to non-uniform chemical structure and molecular weight, low production volume, and high cost of raw materials. There is. Therefore, in recent years, the effective use of hemicellulose, which is said to be present in a large amount after cellulose as a natural organic substance, has been attracting more and more attention. Hemicellulose is defined as a polysaccharide that constitutes the cell wall of land plants excluding cellulose and pectin-based polysaccharides.
It is roughly classified into glucuronoxylan contained in hardwood and arabino glucuronoxylan contained in softwood. Among hemicelluloses, particularly, xylose obtained by decomposing and saccharifying xylan has been actively studied, and is applied to, for example, ethanol fermentation by microorganisms. In addition, xylitol obtained from xylose has attracted attention as a low-calorie sweetener, and has recently been added to foods such as chewing gum and candies in anticipation of a caries prevention effect, and the demand has been rapidly increasing.

[0003] As a method for decomposing and saccharifying xylan, an acid saccharification method using sulfuric acid or hydrochloric acid, an explosion treatment method using a high-temperature and high-pressure saturated steam, and an enzymatic saccharification method using an enzyme have been known. Although the acid saccharification method has an advantage that the decomposition rate of xylan is high, it has a drawback in that by-products other than the intended sugar are generated due to lack of specificity. Although the explosion treatment method has the advantage of being able to decompose xylan in a short time without pollution, it consumes a large amount of energy because it requires high-temperature, high-pressure saturated steam, which is not preferable for the preservation of the global environment. It has the disadvantage of lacking in sex. In contrast, the enzymatic saccharification method has high specificity and does not consume a large amount of energy because decomposition proceeds under mild conditions.
It can be said that the xylan decomposition saccharification method is superior to the acid saccharification method and the explosion treatment method in that the sugar yield becomes almost the same as the theoretical value and there is little risk of environmental pollution.

[0004] Arabinoglucuronoxylan is a heteropolysaccharide in which an L-arabinose side chain is bonded to a main chain in which xylose is β-1,4 bonded. Therefore, in order to obtain xylose from arabinoglucuronoxylan by the above-described enzymatic saccharification method, the main chain and the side chain must be decomposed and cleaved. Since arabinoglucuronoxylan has a complex steric hindrance due to the L-arabinose side chain, when xylanase or xylosidase, which is an enzyme for decomposing the main chain of arabinoglucuronoxylan, is acted on, the side chain is cleaved and removed by the decomposing enzyme. It is known that this promotes the production of xylose by decomposition of the main chain, and it is important to cut and remove the side chain in the production of xylose. In recent years,
L-arabinose, when taken simultaneously with sucrose, has the function of reducing the digestion and absorption of energy derived from sucrose, and is therefore expected to have the effect of suppressing hyperglycemia due to sucrose ingestion due to diabetes. Cleavage and removal of side chains by enzymes that yield L-arabinose is important.

[0005] As described above, an enzyme that cleaves the L-arabinose side chain of arabinoglucuronoxylan is called α-L-arabinofuranosidase, which is widely distributed in the animal and plant kingdoms and is mostly produced by microorganisms. As such α-L-arabinofuranosidase, Aspergillus niger (Asp.
ergillus niger, Penicillium capsulatum
tun), Trichoderma Resei (Trichoder)
maresei) etc. can be exemplified,
In addition, α-L-arabinofuranosidase produced by many microorganisms has been reported so far.

[0006]

However, most of the above-mentioned conventionally known α-L-arabinofuranosidases have an enzymatic activity in addition to an enzymatic activity for cleaving the L-arabinose side chain. In addition, various sugar by-products and the like are contaminated in the decomposition product of arabinoglucuronoxylan, and the work of separating and purifying the decomposition product is complicated, which makes it difficult to efficiently obtain xylose or L-arabinose. In addition, conventionally known α-L-arabinofuranosidases often have low enzyme activity, and xylanase from which L-arabinose side chains have been removed or xylo-oligosaccharides on which xylosidase acts can not be obtained in high yield. For this reason, there was a problem that xylose and also kyritol could not be produced in high yield. In addition, Risomucol Psilus (R
α-L-arabinofuranosidase produced by H. mucilor pusillus or a mutant thereof has not been reported so far.

The present invention has been made to solve the above-mentioned conventional problems. First, a highly active α-L which specifically decomposes and cleaves the L-arabinose side chain of arabinoglucuronoxylan. -To provide an arabinofuranosidase and a method for producing the same. A second object is to provide a method for producing L-arabinose efficiently and with high yield from arabinoglucuronoxylan. A third object of the present invention is to provide a method for producing xylitol efficiently and with high yield using arabinoglucuronoxylan as a starting material.

[0008]

Means for Solving the Problems The present inventors have proposed a xylanase-producing strain, Rhizomucor psilus (Rhiz).
Oxycor pucillus), a xylanase was produced in a crude enzyme solution.Since the productivity of xylanase gradually decreased, the xylanase of the present strain was intensively studied. With the decrease in productivity, it has been found that α-L-arabinofuranosidase is highly produced, and the present invention has been reached.

That is, the novel α-L-arabinofuranosidase of the present invention has the following physicochemical properties. 1) Action and substrate specificity It acts on arabinoglucuronoxylan and specifically cleaves L-arabinose side chains. 2) Optimum pH and stable pH The optimum pH is about pH 4.0 in citrate buffer.
It is stable at about pH 7 to 10 under the conditions of 50 ° C. for 3 hours. 3) Optimal temperature and temperature stability The optimal temperature is about 65 ° C. It hardly deactivates at 50 ° C. or less, and retains about 90% of the residual activity even after being left at 60 ° C. for 1 hour. 4) Influence of metal ions ^Inhibited by Hg ²⁺ . 5) Molecular weight It is about 88,000 by gel filtration chromatography. 6) Isoelectric point The isoelectric point pI is about pI4.2.

The α-L-arabinofuranosidase having the above-mentioned physicochemical properties specifically cleaves the L-arabinose side chain with high activity, so that the degradation product is free from contaminants such as complicated sugar by-products. A xylanase or a xylosidase easily acts on the xylo-oligosaccharide from which the side chain has been removed. Therefore, xylose can be efficiently obtained at a high yield, and xylitol can be efficiently obtained at a high yield. In addition, L-arabinose, which is attracting attention as a functional sweetener, can be efficiently obtained at a high yield.

[0011] The α-L-arabinofuranosidase of the present invention can be obtained from Rhizomuco.
r pusillus) or a mutant thereof. Rhizomucor
pusillus) is a mold isolated from jute fiber compost in Bangladesh and has the following mycological properties:

(1) Cultural and morphological properties Malt extract agar medium: A colorless to gray-brown colony having a diameter of about 70 mm and a height of about 3 mm is formed by culturing at 45 ° C. for 1 day. The sporangia stalks growing from the rhizoids and being multiply branched are about 10 μm wide, and the sporangia are about 80 μm in diameter. The ovoid or spherical pillar axis is about 45 μm in diameter, and the spherical or ovoid spore spores are 4.5-6 μm. No zygote is observed. Arabinose-Xylan agar medium: It grows well by culturing at 37 ° C. for 5 days, and forms black spores on the thick hypha moth. (2) Physiological properties (a) Growth temperature range: 30 ° C to 50 ° C (b) Optimal growth temperature: 30 ° C to 40 ° C (c) Growth pH range: 4.5 to 6.5 (d) Optimal growth p
H: 5.5-6.0 (e) Has the ability to produce α-L-arabinofuranosidase.

On the basis of the above mycological properties, the study in Mycology, No. 17 (Studie
s in Mycology, no. 17) (197)
8), the strain was determined to be a mutant of Rhizomucor pusillus, and the strain was identified as Rhizomucor pusillus (R).
hizomucor pusillus) HHT-1 and the accession number F
Deposited as ERM P-17973.

[0014] The novel α-L-arabinofuranosidase of the present invention is not limited to those having the above-mentioned physicochemical properties, but may be any of Rhizomucor.
pusillus) or a mutant thereof, a novel highly active α-L that specifically degrades and cleaves L-arabinose produced by
-Arabinofuranosidases.

Further, α-L- having the above physicochemical properties
The microorganism that produces arabinofuranosidase is not limited to the above microorganism, and may be any microorganism as long as it has the above physicochemical properties.

Further, the novel α-L-arabinofuranosidase of the present invention can be used to efficiently cut L-arabinose side chains from arabinoglucuronoxylan as a starting material and to convert L-arabinose efficiently. It can be produced in high yields. In addition, xylanase or xylosidase is allowed to act on the xylo-oligosaccharide obtained by cleavage of the L-arabinose side chain to obtain xylose, and xylitol can be efficiently produced at a high yield from the xylose.

[0017]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to embodiments. Rhizomucor psilus (Rhi) producing the novel α-L-arabinofuranosidase of the present invention
The culture conditions for increasing the productivity of α-L-arabinofuranosidase for H. zomucor pusillus) HTT-1 are as follows.

At a culture temperature of 30 ° C. to 40 ° C., α-L-arabinofuranosidase is highly produced. In particular, the highest production is obtained at about 40 ° C. The initial pH is 5.0 to 6.0.
At 0, α-L-arabinofuranosidase is highly produced.
In particular, it is most highly produced at about pH 5.5.

As the medium, an ordinary medium can be used. However, in order to increase the production of α-L-arabinofuranosidase, it is preferable to use the following carbon sources and nitrogen sources. That is, by using one or more of L-arabinose, L-arabitol, D-arabinose, and xylo-oligosaccharide as a carbon source, α-arabinose
L-arabinofuranosidase is highly produced. In particular, L
-High production with arabinose alone. The L-arabinose concentration is preferably 0.5% to 1.5%, more preferably 1.5%.

The use of any one or more of polypeptone, proteose peptone, bactokasciton, tryptone, bactopeptone, and meat extract as a nitrogen source allows α-L-arabinofuranosidase to be highly produced. In particular, α-L-arabinofuranosidase is highly produced by using polypeptone alone. The polypeptone concentration is preferably 0.5% to 1.5%, more preferably 1.
2%.

Α-L-arabinofuranosidase is separated and recovered from the culture cultured under the above culture conditions. The crude enzyme solution can be collected from the culture by a known method. Since α-L-arabinofuranosidase is an extracellular enzyme, the crude enzyme solution can be easily obtained by centrifugation or filtration of the culture. Can be. The obtained crude enzyme solution is purified by a known purification method. Known purification methods include salting out, fractional precipitation with an organic solvent, gel filtration chromatography, anion exchange chromatography, cation exchange chromatography, various other types of chromatography, electrophoresis, and the like. Alternatively, they can be used in combination.

Using arabinoglucuronoxylan as a starting material, L-arabinose and xylitol can be produced using the α-L-arabinofuranosidase of the present invention.
Since arabinoglucuronoxylan is used as a starting material, it is preferable to select one having a particularly high xylan content from plant waste and waste wood. The α-L-arabinofuranosidase of the present invention is allowed to act on hemicellulose obtained by delignification of plant waste and waste wood and then alkali treatment, thereby sufficiently cutting and removing the L-arabinose side chain.
The decomposition product of arabinoglucuronoxylan by the action of α-L-arabinofuranosidase is composed of L-arabinose and xylo-oligosaccharide, and there is no contamination such as complicated saccharide by-products. Therefore, separation of L-arabinose and xylo-oligosaccharide・
The purification operation is easy, and L-arabinose and xylo-oligosaccharide can be obtained in high yield. Then, L-
Xylose is obtained by allowing xylanase or xylosidase to act on the xylo-oligosaccharide from which the arabinose side chain has been removed. Then, xylose is hydrogenated with high-pressure hydrogen in the presence of a metal catalyst such as nickel, and then recovered and separated to produce xylitol. Alternatively, a microorganism that converts xylose into xylitol, for example, Candida tropicalis, may be allowed to act on xylose using a bioreactor, and xylitol may be recovered and separated. According to the method for producing xylitol of the present invention, L-arabinose and xylitol can be obtained efficiently because the operation of separating and purifying decomposition products in each step is not complicated. Further, L-arabinose and xylitol can be obtained in high yield relative to the starting material. In addition, xylitol can be efficiently produced at a high yield by the present production method using birch chips conventionally used in the production of xylitol as raw materials for arabinoglucuronoxylan.

[0023]

EXAMPLES Next, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.

Example 1 (Preparation of crude enzyme solution) L-arabinose 1.0%, polypeptone 0.6%,
(NH ₄ ) ₂ SO ₄ 0.12%, KH ₂ PO ₄ 1.5%
100 ml of a pH 5.5 medium consisting of (medium composition) was dispensed into a Sakaguchi flask and autoclaved. Risomucol Psilus (Rhizomu) stored on a preservation slant medium
cor pusillus) HHT-1 (FERM P
-17973) aseptically inoculated into a Sakaguchi flask into which the culture solution was dispensed, and a shaking incubator (MultiPurposeIncubator,
Using Takasaki Kagaku Kikai Co., Ltd.), preculture was performed at 40 ° C. and 150 rpm for 24 hours. The culture was aseptically inoculated into an Erlenmeyer flask into which 400 ml of the culture was dispensed,
Main culture was performed for 6 days under the same conditions as in the preculture. After completion of the culture, the culture was centrifuged at 10,000 rpm for 30 minutes to remove the cells. The supernatant was filtered through glass wool to remove mycelia, from which a crude enzyme solution was obtained.

Example 2 (Measurement of enzyme activity) From the above crude enzyme solution, an α-L-arabinofuranosidase activity of 4.0 U / ml was obtained. The measurement of enzyme activity is p
The crude enzyme solution is allowed to act on -nitrophenyl α-L-arabinofuranoside, sodium carbonate is added to p-nitrophenol obtained by the decomposition, and the absorbance is measured at a wavelength of 414 nm to obtain p-nitrophenol. The concentration was calculated and performed. In addition, 1 μmol of p-
The enzyme activity for producing nitrophenol was 1 U. Further, the protein content was determined to be 2.10 by the measurement using the Lowry method.
At 5 mg / ml, the specific activity was calculated to be 1.6 U / mg.
This value was higher than that of α-L-arabinofuranosidase reported so far. Result is,
It is shown in Table 1.

[0026]

[Table 1]

Example 3 (Purification of α-L-arabinofuranosidase) The crude enzyme solution obtained in Example 1 was purified by the following purification methods. Confirmation of α-L-arabinofuranosidase in each purification method was confirmed by SDS-PAGE and activity staining (Zy
mogram). SDS-PAGE is 1
Using a 2% polyacrylamide gel, myosin (MW 200,000), β-galactosidase (MW 116, 248), albumin (MW 66, 267), and aldolase (MW 42, MW) were used as protein molecular weight markers.
400), carbonic anhydrase (MW. 30,
000) and myoglobin (MW. 17, 201) (all manufactured by Daiichi Yakuhin Co., Ltd.). Confirmation by activity staining was performed as follows. First, Native-PA prepared using a solution of 4-methylumbelliphenyl-α-L-arabinofuranoside (manufactured by Nacalai Tesque Inc.)
Perform electrophoresis using a gel for GE. After completion of the electrophoresis, the α-L-arabinofuranosidase in the gel is reacted with 4-methylumbelliphenyl-α-L-arabinofuranoside, and the resulting 4-methylumbelliphenyl group is irradiated with ultraviolet light. Color was developed and the site where α-L-arabinofuranosidase was present was detected.

The crude enzyme solution obtained in Example 1 was treated with 35-70
% Ammonium sulfate, and the obtained precipitate was dialyzed in a 20 mM phosphate buffer using a dialysis membrane (manufactured by Sanko Junyaku). As a result, the activity was 270 U / ml, and the protein amount was 21.
An enzyme solution having a specific activity of 13 U / mg was obtained.

Next, the enzyme solution obtained by salting out with 35 to 70% ammonium sulfate was purified by gel filtration chromatography. 20 mM phosphate buffer (pH
Superdex 200pg equilibrated in 6.8)
(Manufactured by Pharmacia), and the column obtained by the salting-out was added to the column.
ml of the sample and a flow rate of 0.2 ml / min. Was eluted. As a result, the activity was 410 U / ml and the protein content was 7.
An enzyme solution having a concentration of 5 mg / ml and a specific activity of 55 U / mg was obtained.

Further, the enzyme solution obtained by the above gel filtration chromatography was purified by cation exchange chromatography. 20 mM phosphate buffer (pH
6.0) CM-TOYOPEARL65 equilibrated in)
Apply 3 ml of the enzyme solution to a 0M (manufactured by Tosoh Corporation) column,
Flow rate 0.15 ml / min. Eluted at 0-0.1
The adsorbed protein was eluted with a linear concentration gradient of MNaCl. As a result, the activity was 98 U / ml, and the protein mass was 1.4 mg.
/ Ml, an enzyme solution having a specific activity of 68 U / mg.

Next, the enzyme solution obtained by the above cation exchange chromatography was purified by anion exchange chromatography using HPLC. An anion exchange column, PorosQE / M (perSep), was installed on a SMART System (manufactured by Pharmacia) as an HPLC system.
active Biosystem Co. And equilibrated with a phosphate buffer (pH 6.8) as a developing solvent. The enzyme solution was injected into an injection valve, and the flow rate was 150 μl / min. To 0-0.5M NaC
The adsorbed protein was eluted by a linear concentration gradient. As a result, the activity was 22 U / ml and the protein content was 0.25 mg / m
1. The most purified enzyme solution having a specific activity of 86 U / mg was obtained. As a result of performing a purity test by SDS-PAGE on this purified enzyme solution, α-L-arabinofuranosidase was confirmed as a single band, and the molecular weight was 85,000 to 9
It was estimated to be 0000. The purification results of this enzyme are shown in Table 2.

[0032]

[Table 2]

Example 4 (Properties of α-L-arabinofuranosidase) The properties of α-L-arabinofuranosidase finally purified in Example 3 were examined.

Substrate Specificity A substrate solvent was prepared using 12 types of low-molecular synthetic substrates, and the enzyme was allowed to act at 50 ° C. to measure the activity. The results are shown in Table 3. Relative activity (%) was defined as the enzyme activity for pNPα-L-arabinofuranoside, which is an action substrate of the present enzyme, taken as 100. Also,
The crude enzyme solution was also examined. From Table 3, it was found that the present enzyme did not have any other contaminating enzyme activity acting on arabinoglucuronoxylan other than the L-arabinose side chain. In the figures, pNP means p-nitrophenyl.

[0035]

[Table 3]

Optimum pH and pH stability The activity of each substrate solution in a 50 mM citrate buffer at pH 3 to 8 was measured to determine the optimum pH.
H4.0. The results are shown in FIG. The activity was expressed as a relative activity (%) with the maximum value of the activity being 100. In addition, pH3
-8 is citrate buffer, pH 8-9 is Tris buffer, p
For H9-11, glycine buffer was used, the enzyme solution was added to each buffer, and the pH stability was examined by measuring the residual activity at 50 ° C. after incubating at room temperature for 3 hours. It was stable. The results are shown in FIG. The activity was expressed as a relative activity (%), with the activity before incubation as 100.

Optimum temperature and temperature stability: 20 ° C. to 90 ° C.
, The activity was measured to determine the optimum temperature, which was about 65 ° C. In addition, 20 ° C-80
After incubation at each temperature of 50 ° C. for 1 hour, the remaining enzyme activity was measured at 50 ° C. to examine the temperature stability.
Approximately 90% of the residual activity was retained after 1 hour incubation at 0 ° C. From these results, it was found that the optimum temperature was higher than that of the known α-L-arabinofuranosidase derived from mold. In addition, since most known α-L-arabinofuranosidases are inactivated at a temperature of 60 ° C. or higher,
The temperature stability of this enzyme was found to be high. The results are shown in FIGS. In FIG. 3, the relative activity (%) is represented by the relative activity (%) when the highest value of the activity is 100, and in FIG. 4, the activity before the incubation is represented by 100.

Influence of Metal Ions Various metal ions were dissolved in an enzyme solution to a concentration of 5 mM, left at room temperature for 15 minutes, and the residual activity was measured. Most known α-L-arabinofuranosidases are completely inhibited in the presence of 2 mM Hg ²⁺ , but the enzyme retained about 10% activity even at 5 mM. It was also inhibited by EDTA.
The results are shown in Table 4. The activity when no metal ion was added was defined as a relative activity (%), where the activity was defined as 100.

[0039]

[Table 4]

Molecular weight The molecular weight was estimated by gel filtration chromatography using HPLC.
It was estimated to be 8,000. This molecular weight was consistent with the molecular weight estimated by SDS-PAGE described above, and it was estimated that this enzyme was a monomeric enzyme. Known α-L-arabinofuranosidase derived from mold has a low molecular weight,
This enzyme was found to have a large molecular weight as a mold-derived monomeric α-L-arabinofuranosidase.

Isoelectric point As a result of measurement by the isoelectric focusing method, the isoelectric point was about pI4.2.

N-terminal amino acid sequence analysis and homology search Peptide sequencer (ProciseProtei)
As a result of N-terminal amino acid sequence analysis using nSequenching System (manufactured by Applied Biosystems), Met-Ile-Gly-Arg-Gln-Leu
-Pro-Thr-Thr-Ser-Gly-Gly-
Pro-Leu-Asp-Asn-Tyr-Ile-T
22 residues of hr-Leu-Asn-Leu (see SEQ ID NO: 1 in the Sequence Listing) could be analyzed. This N-terminal amino acid sequence was subjected to homology search using an amino acid sequence database on the Internet. As a result, 22 residues of the N-terminal amino acid were Chlamydomonas reinhardti.
It showed 60% homology with i) -derived dynein. However, dynein is present in eukaryotic flagella and cilia, and ATP
It is a protein that has an ase activity, and has low similarity to this enzyme in nature. The molecular weight of dynein is generally about 1,
It is about 200,000, and the molecular weight of this enzyme is about 88,0
It is significantly different from 00. From these, it was determined that the present enzyme and dynein were different proteins.

Further, as a result of comparing this enzyme with another α-L-arabinofuranosidase using a database different from the above on the Internet, no homology with this enzyme was found.

Therefore, from the results of the homology search described above, it has been reported that Rhizomucor psilus (Rhi
zomucor pusillus) or a mutant thereof, because there is no report of α-L-arabinofuranosidase, and further, there are many differences from known mold-derived α-L-arabinofuranosidase. Was determined to be a novel α-L-arabinofuranosidase.

[0045]

According to the present invention, the novel α-L-arabinofuranosidase is an arabinoglucuronoxylan L-
As the arabinose side chain is specifically degraded and cleaved, there is no contamination such as complicated sugar by-products in the degraded product,
It is easy to purify and can efficiently obtain arabinose, xylose and eventually xylitol. Also, a new α
-L-arabinofuranosidase has a high enzyme activity,
In combination with the substrate specificity, arabinose, xylose, and thus xylitol can be obtained in high yield.

[0046]

[Sequence List] <110> Nagoya Industrial Science Research Institute <120> New α-L-arabinofuranosidase and its production, furthermore production of L-arabinose and xylitol using new α-L-arabinofura nosidase <130> PTL01 <160> 1 <210 > 1 <211> 22 <212> PRT <213> Rhizomucor pusillus HHT-1 <400> 1 Met Ile Gly Arg Gln Leu Pro Thr Thr Ser Gly Gly Pro Leu Asp Asn 1 5 10 15 Tyr Ile Thr Leu Asn Leu 20

[Brief description of the drawings]

FIG. 1 is a graph showing the optimum pH of the novel α-L-arabinofuranosidase of the present invention.

FIG. 2 is a graph showing the pH stability of the novel α-L-arabinofuranosidase of the present invention.

FIG. 3 is a graph showing the optimum temperature of the novel α-L-arabinofuranosidase of the present invention.

FIG. 4 is a graph showing the temperature stability of the novel α-L-arabinofuranosidase of the present invention.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1: 645) C12R 1: 645) (C12N 1/14 (C12N 1/14 A C12R 1: 645) C12R 1: 645) F term (reference) 4B050 CC01 DD03 FF03E FF05E FF11E FF12E LL02 LL05 4B064 AC05 AF02 AG01 CA05 CB07 CB16 CD19 DA01 DA10 4B065 AA58X AC14 BA16 CA31 CA41 CA44

Claims (13)

[Claims] 1. A novel α-L-arabinofuranosidase having the following physicochemical properties: 1) Action and substrate specificity It acts on arabinoglucuronoxylan and specifically cleaves L-arabinose side chains. 2) Optimum pH and stable pH The optimum pH is about pH 4.0 in citrate buffer.
It is stable at about pH 7 to 10 under the conditions of 50 ° C. for 3 hours. 3) Optimal temperature and temperature stability The optimal temperature is about 65 ° C. It hardly deactivates at 50 ° C. or less, and retains about 90% of the residual activity even after being left at 60 ° C. for 1 hour. 4) Influence of metal ions ^Inhibited by Hg ²⁺ . 5) Molecular weight It is about 88,000 by gel filtration chromatography. 6) Isoelectric point The isoelectric point pI is about pI4.2. 2. Rhizomu
(cor pusillus) or a mutant strain thereof, which is a novel α-L-arabinofuranosidase. (3) Rhizomu (Rhizomu)
(cor pusillus) or a mutant thereof. The novel α-L-arabinofuranosidase according to claim 1, wherein the novel α-L-arabinofuranosidase is produced. 4. Rhizomu
cor pusillus) HHT-1FERM P-
A novel α-L-albinofuranosidase produced by 17973. 5. Rhizomu
cor pusillus) HHT-1FERM P-
17973 produces the novel α-L- according to claim 1.
Albinofuranosidase. 6. Rhizomu (Rhizomu)
cor pusillus) or its mutant strain,
A method for producing α-L-arabinofuranosidase, comprising separating and recovering a novel α-L-arabinofuranosidase from the culture. 7. Rhizomu Psilus (Rhizomu)
cor pusillus) or its mutant strain,
The α-L-arabinofuranosidase according to claim 1, which is separated and recovered from the culture.
A method for producing L-arabinofuranosidase. 8. Rhizomu Psilus (Rhizomu)
cor pusillus) HHT-1FERM P-
A method for producing α-L-arabinofuranosidase, comprising culturing 17973 and separating and recovering a novel α-L-arabinofuranosidase from the culture. 9. Rhizomu (Rhizomu)
cor pusillus) HHT-1FERM P-
A method for producing α-L-arabinofuranosidase, comprising culturing 17973 and separating and recovering the novel α-L-arabinofuranosidase according to claim 1 from the culture. 10. A method for producing the novel α-L-arabinofuranosidase according to any one of claims 1 and 2, wherein Rhizomucor psilus (Rhiz) is produced.
omucor pusillus) or a mutant thereof. 11. The method of claim 1, wherein the accession number is FERM P-17973.
zomucor pusillus) HHT-1. 12. An L-arabinose comprising the novel α-L-arabinofuranosidase according to any one of claims 1 to 5 using arabinoglucuronoxylan as a starting material. Manufacturing method. 13. A process for producing xylitol, which comprises using the novel α-L-arabinofuranosidase according to any one of claims 1 to 5 using arabinoglucuronoxylan as a starting material. Method.