JP2000217580A - Arabinofuranosidase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new arabinofuranosidase which has a hydrolyzing activity against linear (1→5)-α-L-arabinan, and is useful as a reagent for research because of its high substrate specificity, for the production of arabinooligosaccharide and so on. SOLUTION: This new arabinofuranosidase has a hydrolyzing activity against linear (1→5)-α-L-arabinan, can hydrolyze efficiently arabinan and so on, and can be used as a reagent for research because of its high substrate specificity, for the production of arabinooligosaccharide and so on. This arabinofuranosidase is obtained by inoculating Streptomyces sp. strain GS-901 (FERM P-16916) which produces the arabinofuranosidase into a medium, culturing the strain to form or accumulate arabinofuranosidase intracellularly or into the medium, followed by collecting the enzyme from the broth.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a (1 → 5) -linked α-L-arabinofuranose residue which specifically acts on
Arabinofuranosidase having hydrolytic activity on linear (1 → 5) -α-L-arabinane, Arabinofuranosidase having glycosyl transfer activity, gene encoding the arabinofuranosidase, the arabinofuranosidase The present invention relates to a production strain and a method for producing the arabinofuranosidase.

[0002]

2. Description of the Prior Art L-arabinose is a naturally occurring pentasaccharide and has a moderate sweetness. In nature, it exists as arabinoxylan, arabinan, and arabinogalactan in the form of furanose. Arabinan containing arabinose as a main constituent sugar has been extracted from certain seeds, fruits, vegetables, bark or wood and subjected to structural analysis. Basically, the α-L-arabinofuranose residue is (1
→ 5) It is shown that α-L-arabinofuranose residues are bonded at the O-3 position or O-2 position in some parts of the bonded main chain.

[0003] In recent years, it has been shown by Seuri and others that the monosaccharide L-arabinose inhibits small intestinal sucrase and that oral administration suppresses an increase in blood glucose and insulin after sugar load (Seuri et al., Metabolism). , 45 volumes,
p. 1368 (1996); Seri et. a
l. , Metabolism, 45, p. 1368 (1
996)). Furthermore, Sanai et al. Have shown that L-arabinose, when taken with sucrose, suppresses digestion and absorption of sucrose and reduces its energy utilization (Sanui et al., Journal of Nutrition and Food Science, 50, p.13).
3 (1997)), and attention has been paid to the production method together with its physiological activity.

[0004] Arabinase is an enzyme that degrades arabinose-containing polysaccharide, and two types of endo- and exo-types have been known to date. The endo-type arabinanase is called endo- (1 → 5) -α-L-arabinanase (EC.3.2.1.99), and the exo-type is called arabinofuranosidase (EC.3.2.1.55). .

[0005] Arabinofuranosidases of various origins have been reported so far. At present, the following 2
(G. Beldman et al.,
Advansis in Macromolecular Carbohydrate Research, Volume 1, p. 1 (1997)). (1) α-L-arabinofuranosidase A (2) α-L-arabinofuranosidase B

[0006] This classification is based on the presence or absence of an action on arabinose-containing polysaccharide such as beet arabinan. However, the structure of arabinose-containing polysaccharide is still unclear, and at present, the classification of arabinofuranosidase is not clear. In arabinofuranosidase acting on arabinose-containing polysaccharide,
Many of the enzymes discovered so far preferentially hydrolyze (1 → 2) or (1 → 3) linked α-L-arabinose residues from beet arabinan, resulting in linear (1)
→ 5) -α-L-arabinane is generated and precipitates out of solution (McCleary, BV, Enzymes in Biomass Conversion, P. 422 (198)
4); Mc Cleary, B .; V. , Enzymes
In Biomass Conversion, p. 4
22 (1984)). The straight chain (1
→ 5) Since -α-L-arabinan is hardly decomposed, it was difficult to decompose beet arabinan to monosaccharide with high yield using these known enzymes.

The turbidity that often occurs in wine, fruit juice, etc. is based on this linear (1 → 5) -α-L-arabinane as a main component (MP Bellevil).
le et al., Fight Chemistry, Vol. 33, p. 227
(1993); P. Bellville et.
al. , Phytochemistry, 33, p. 2
27 (1993)). For the reasons described above, it has been difficult to prevent the occurrence of such turbidity or remove the turbidity by using known arabinofuranosidase.

[0008] Various glycosidases are widely used for structural analysis of polysaccharides, oligosaccharides and the like by utilizing their specificity. Enzymes with high substrate specificity are essential for structural studies of arabinose-containing polysaccharides and the like, but arabinofuranosidases whose substrate specificity has been clarified so far are few. We investigated the substrate specificity of several arabinofuranosidases using three methyl-α-L-arabinofuranobioside regioisomers (1 → 2, 1 → 3, 1 → 5) and so on. I checked. Until now, some enzymes that preferentially cleave (1 → 2) or (1 → 3) -bound α-L-arabinofuranose residues have been confirmed, but (1 → 5) -bound α-L -No enzyme specific to arabinofuranose residues has been obtained.

[0009] In recent years, various oligosaccharides and glycosides have been synthesized by the transglycosylation reaction of glycosidases, and are widely used mainly in the food field. However, there are few reports on the transamination reaction by arabinofuranosidase, and no application examples of arabino-oligosaccharide or arabinose glycoside are known.

[0010]

The present invention relates to (1 → 5)
It can efficiently degrade linear (1 → 5) -α-L-arabinane with high specificity to the bound α-L-arabinofuranose residue, and can be used for research because of its high substrate specificity. It is intended to provide arabinofuranosidase which is highly useful as a reagent and arabinofuranosidase which has glycosyl transfer activity and can be used for production of various arabino-oligosaccharides and arabinose-containing glycosides. Further, the present invention provides a gene encoding the arabinofuranosidase.

[0011]

Means for Solving the Problems The present inventors have developed arabinofuranosidase having a novel property, in particular, a linear (1 →
5) an enzyme capable of efficiently decomposing -α-L-arabinane,
As a result of intensive studies to find a microorganism capable of producing an enzyme having a glycosyltransferase activity, α-L- (1 → 5) -linked arabinofuranosidase derived from actinomycetes belonging to the genus Streptomyces It has been found that arabinofuranose residues can efficiently degrade linear (1 → 5) -α-L-arabinan with high specificity, and that another enzyme derived from the fungus has glycosyltransferase activity The inventors have found out that the target enzyme has been extracted from the culture of this strain and the properties thereof have been elucidated, thereby completing the invention.

That is, the present invention provides a linear (1 → 5) -α
The present invention provides an arabinofuranosidase capable of efficiently degrading L-arabinan, a bacterium producing the arabinofuranosidase, and a gene encoding the arabinofuranosidase.

The present invention also provides an arabinofuranosidase having a glycosyl transfer activity, a strain producing the arabinofuranosidase, and a gene encoding the arabinofuranosidase.

Further, the present invention provides a method of inoculating a strain having these genes into a medium, culturing the strain to accumulate arabinofuranosidase in the cells or in the medium, and collecting the arabinofuranosidase. It is intended to provide a method for producing nofuranosidase.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION As a fungus producing the arabinofuranosidase of the present invention, there is a strain newly isolated by the present inventors and designated as GS-901. Has mycological properties.

1A. Morphological properties Vegetative mycelia develop well in synthetic agar and natural media and diverge irregularly. Spores are well formed on yeast / malt agar medium, oatmeal agar medium, starch / inorganic salt agar medium and the like. According to microscopic observation, the method of branching the sporulation hyphae is simple branching, and the spores are formed in a spiral. Spores usually have three or more linkages and show a long chain in the late stage of culture. The spores are spiny and cylindrical in shape.

1B. Table 1 shows the results of macroscopic observation when cultured on various media at 30 ° C for 14 days.

[0018]

[Table 1]

1C. Physiological properties (1) Optimal growth temperature: 25-37 ° C. It grows slightly even at 40 ° C. (2) Biochemical properties (a) Positive liquefaction of gelatin (b) Positive hydrolysis of starch (c) Positive melanin-like pigment formation (d) Reduction of nitrate positive (e) Resistance to salt up to 7% ( 3) Utilization of carbon source Utilization of carbon source of GS-901 strain is shown in Table 2. The medium was Prideham Gottlieb agar medium (ISP
The determination was made after culturing at 28 ° C. for 14 days using 9).

[0020]

[Table 2]

(Note) ++: Notably used, +: Used,
-: Not used

(4) Diaminopimelic acid (DA) on the cell wall
P) type, LL-DAP

From the above properties, it is clear that the present strain belongs to actinomycetes, and the taxonomic position is determined by referring to "Experimental method for identification of actinomycetes" (edited by the Japanese Society for Actinomycetes, 1985). As a result, it was identified as Streptomyces sp.

The actinomycetes are named as follows, and are deposited with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry. Streptomyces sp.GS-901 (Accession No. FERMP
-16916)

Next, a method of culturing the above microorganism and collecting a novel arabinofuranosidase from the culture will be described. The medium that can be used is not particularly limited as long as the strain of the present invention can grow on a medium used for culturing ordinary microorganisms.Examples of the organic carbon source and nitrogen source include glucose, starch, dextrin, and beet. Arabinan, peptone, yeast extract and the like are suitable, and as inorganic salts, addition of potassium phosphate, sodium phosphate, magnesium sulfate, salt and the like is effective. In addition, various organic substances and inorganic substances necessary for growth of bacteria and production of enzymes can be appropriately added as needed.

As the culture method in the above-mentioned medium, shaking culture or aeration and agitation culture using a fermenter or the like is suitable. The cultivation temperature is not particularly limited as long as it can grow,
C. is desirable.

After culturing as described above, the target substance, arabinofuranosidase, can be collected and purified from the culture according to a general method for collecting and purifying enzymes. That is, first, solids such as cells are removed from the culture solution by a method such as filtration or centrifugation to obtain a crude enzyme solution. The crude enzyme solution thus obtained can be used as it is, but the crude enzyme solution can be used in the usual enzyme purification method, that is, ammonium sulfate salting out, precipitation of an organic solvent with acetone, alcohol, etc., ultrafiltration, ion exchange resin. By using a technique such as gel filtration method alone or in combination, arabinofuranosidase with higher purity can be obtained.

The activity of arabinofuranosidase is measured by the following method. That is, 0.25 ml of 2 mM p-nitrophenyl-α-L-arabinofuranoside, McIlvaine buffer (pH 7.
0) 0.05 ml of enzyme solution was added to 0.2 ml, and
Incubate for 0 minutes. The reaction is stopped by adding 0.5 ml of 0.5% sodium carbonate and the absorbance at 408 nm is measured. The enzyme activity was expressed in terms of the amount of enzyme that liberated 1 μmol of para-nitrophenol per minute under the above conditions as one unit.

Next, the physicochemical properties of the arabinofuranosidase obtained by the present invention will be described. For the sake of convenience, in the following description, arabinofuranosidase 1 having a glycosyltransferase activity is linked to arabinofuranosidase 1, (1 → 5) α
Arabinofuranosidase which specifically acts on -L-arabinofuranose residues to hydrolyze linear (1 → 5) -α-L-arabinane is referred to as arabinofuranosidase 2.

(1) Molecular weight Arabinofuranosidase 1: about 80 kilodalton (SD
Arabinofuranosidase 2: (by S electrophoresis): about 37
Kilo Dalton (by SDS electrophoresis) (2) Isoelectric point Arabinofuranosidase 1: around pH 6.6 (by isoelectric focusing) Arabinofuranosidase 2: pH 7.
Around 5 (by isoelectric focusing) (3) Working pH and pH stability The working pH of arabinofuranosidase 1 at 40 ° C. is optimally around pH 5.5. The stability of arabinofuranosidase action when left at 30 ° C. for 2 hours is pH 5.5.
It is stable in the range of ~ 8.5. The working pH of arabinofuranosidase 2 at 40 ° C. is optimally around pH 7. The stability of the arabinofuranosidase action when left at 30 ° C. for 2 hours is stable in the pH range of 5.0 to 9.0. (4) Optimum temperature and stability pH 5.5, arabinofuranosidase 1 at the time of reaction for 10 minutes
Is optimally around 55 ° C. pH 7, stability of arabinofuranosidase action after standing for 2 hours is 40 ° C
Up to. The working temperature of arabinofuranosidase 2 during the reaction at pH 7.0 for 10 minutes is optimally around 50 ° C. p
H7, the stability of the arabinofuranosidase action after standing for 2 hours is up to 40 ° C.

The arabinofuranosidase 1 of the present invention has the amino acid sequence shown in SEQ ID NO: 1 or has an amino acid sequence in which one or several amino acids are deleted, substituted or added. Things. Also,
The arabinofuranosidase 2 of the present invention has an amino acid sequence shown in SEQ ID NO: 2, or has an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted or added. . The degree of such deletion, substitution or addition is not limited as long as it has the arabinofuranosidase activity. In addition, in SEQ ID NO: 1, N
From the terminal Met to the 29th Ala, and in SEQ ID NO: 2 from the N-terminal Met to the 43rd Ala
Up to the leader sequence.

The gene encoding arabinofuranosidase 1 of the present invention has, for example, the nucleotide sequence shown in SEQ ID NO: 1, or has one or several bases deleted, substituted or added. The gene encoding arabinofuranosidase 2 has the following nucleotide sequence:
For example, it has the base sequence shown in SEQ ID NO: 2 or has a base sequence in which one or several bases have been deleted, substituted or added. The cloning of the arabinofuranosidase gene can be performed, for example, according to the following method.

That is, a genomic DNA library is prepared using, for example, Streptomyces genomic DNA. As the Streptomyces used here, Streptomyces sp. GS-901 or a mutant thereof is desirable. Next, the N of purified arabinofuranosidase
Colony hybridization is performed using the gene deduced from the terminal amino acid sequence, and a strongly hybridizing clone is obtained. From the obtained clones, clones having properties different from those of known arabinofuranosidase, that is, expressing the arabinofuranosidase of the present invention, are selected.

An example of the obtained clone is a plasmid (FIG. 1).

The arabinofuranosidase of the present invention has, for example, the nucleotide sequence shown in SEQ ID NOS: 1 or 2, or has the nucleotide sequence in which one or several bases are deleted, substituted or charged. It can also be produced by inoculating a strain into a medium, culturing it, accumulating arabinofuranosidase in the cell or in the medium, and collecting it.

FIG. 2 shows an HPLC chart of the reaction product of the arabinofuranosidase 1 of the present invention when it was allowed to act on methyl-α- (1 → 2) -L-arabinofuranobioside. . The HPLC analysis conditions were as follows. Column: Suger KS-801 manufactured by Showa Denko KK, eluent: water, temperature: 80
° C, flow rate: 1 ml / min, detection: differential refractometer.

By the action of arabinofuranosidase 1,
Hydrolysis products L-arabinose and methyl-α-L
-In addition to arabinofuranoside, a new peak indicated by an arrow was generated. After conducting a structural study on this substance,
It was confirmed that the enzyme was a trisaccharide composed of only L-arabinose as a constituent sugar, and it was revealed that the present enzyme had a glycosyltransferase activity.

With respect to the arabinofuranosidase 2 of the present invention, the time-dependent changes in the degradation of beet arabinan and linear (1 → 5) -α-L-arabinane are shown in FIG. FIG. 3 (b) shows the time-dependent changes in the decomposition of the -α-L-arabinofuranobioside regioisomer (1 → 2, 1 → 3, 1 → 5).

Arabinofuranosidase 2 hardly acts on beet arabinan, and has no effect on linear (1 → 5) -α-L-
It has been shown to work well for Arabinans. Also,
This enzyme hardly acts on methyl-α- (1 → 2) -L-arabinofuranobioside or methyl-α- (1 → 3) -L-arabinofuranobioside, and methyl-α- ( 1 →
5) It has been shown to work well on -L-arabinofuranobioside. From these facts, it was confirmed that arabinofuranosidase 2 is a novel arabinofuranosidase that specifically acts on the (1 → 5) -bound α-L-arabinofuranose residue.

[0040]

EXAMPLES The present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Streptomyces sp. For the GS-901 strain, the following medium was prepared and cultured. That is, beet arabinan 1.0%, diammonium hydrogen phosphate 0.3%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, and yeast extract 0.3%. One loopful of platinum loop was inoculated into a 100 ml Erlenmeyer flask containing 20 ml of the above medium, and cultured with shaking at 30 ° C. and 210 rpm for 3 days to obtain a seed culture. Then, 2 ml of the seed culture was inoculated into a 500 ml Erlenmeyer flask containing 100 ml of the same medium, and the mixture was inoculated at 30 ° C. and 210 rp.
Shaking culture was performed at m for 4 days. The arabinofuranosidase activity at the end of the culture was about 0.03 unit / ml.

Example 2 The supernatant obtained in Example 1 was concentrated by ammonium sulfate precipitation, and purified to a single protein electrophoretically by ion exchange chromatography and gel filtration chromatography. The specific activity of the purified arabinofuranosidase 1 is about 10 units /
mg, the specific activity of arabinofuranosidase 2 is about 3 units / mg
Met. These arabinofuranosidases had the above substrate specificities and enzymatic properties.

Example 3 Cloning The N-terminals of the purified arabinofuranosidases 1 and 2 and the N-terminal of the internal peptide which was cleaved with V8-protease.
Using primers synthesized based on the terminal amino acid sequence, the method of Saito and Miura (Bio Kimika Bioffice Acta, 7
2, p. 619 (1963); Biochim. Bio
phys. Acta, 72, p. 619 (1963)), and the DNA was amplified by PCR using the genomic DNA extracted as a template. As a result, an amplified fragment containing a region encoding the N-terminal and internal amino acid sequences of the purified enzyme was obtained. Next, genomic southern hybridization was performed using this fragment as a probe. As a result, arabinofuranosidase 1 was converted to BamH1 and Bg
A single band was obtained when the genomic DNA was co-cleaved with II and when arabinofuranosidase 2 was cut with Sal I.

Based on the above results, cloning of the arabinofuranosidase 1 and 2 genes was attempted. First, Trig
lia et al.'s method (Nucleic Acid Research,
16, p. 8186 (1988); Nucleic A
cid Research, 16, p.8186 (198
8)), a DNA amplified fragment of about 3.5 kbp was obtained for arabinofuranosidase 1 and about 2.3 kbp for arabinofuranosidase 2. After confirming these sequences, DNAs containing the respective full lengths were amplified by PCR.

[0045]

The arabinofuranosidase 1 of the present invention comprises:
Since it has glycosyltransferase activity, it is useful for preparing various arabinose-containing glycosides and arabino-oligosaccharides. The arabinofuranosidase 2 of the present invention has a linear (1 → 5) -α-
Since it acts well on L-arabinan, it is effective for enzymatic degradation of arabinan and production of monosaccharide L-arabinose.
Furthermore, because of its high substrate specificity, it is also useful as a reagent for structural studies of arabinose-containing polysaccharides.

[0046]

[Sequence List] SEQUENCE LISTING <110> GODO Shusei co, Ltd. <120> Arabinofuranosidase <130> P00361101 <160> 2 <210> 1 <211> 2478 <212> DNA <213> Streptomyces sp. <220> <221 > CDS <222> (1) .. (2478) <400> 1 atg tca cgc atc cgc tgg aga tac ggc aca gcc gcc acc gcc ctc ctg 48 Met Ser Arg Ile Arg Trp Arg Tyr Gly Thr Ala Ala Thr Ala Leu Leu 1 5 10 15 gtc gcg gcc ggc ctc gtc ccc acc gcc acc gcg cac gcc gag gac gtc 96 Val Ala Ala Gly Leu Val Pro Thr Ala Thr Ala His Ala Glu Asp Val 20 25 30 acc gac tac tcg atc acc gtc gac ccg gcc gcc aag ggc gcc gcc atc 144 Thr Asp Tyr Ser Ile Thr Val Asp Pro Ala Ala Lys Gly Ala Ala Ile 35 40 45 gac gac acg atg tac ggc gtc ttc ttc gag gac atc aac cgg gcc gcg 192 Asp Asp Thr Met Tyr Gly Val Phe Phe Glu Asp Ile Asn Arg Ala Ala 50 55 60 gac ggc ggt ctg tac gcc gag ctc gtg cag aac cgg tcc ttc gag tac 240 Asp Gly Gly Leu Tyr Ala Glu Leu Val Gln Asn Arg Ser Phe Glu Tyr 65 70 75 80 tcc acg gac gac aac cgg tcc tac acg ccc ctc acc tcc tgg atc gtc 288 Ser Thr Asp Asp Asn Ar g Ser Tyr Thr Pro Leu Thr Ser Trp Ile Val 85 90 95 gac ggc acc ggc gag gtc gtg aac gac gcc ggc cga ctg aac gag cgc 336 Asp Gly Thr Gly Glu Val Val Asn Asp Ala Gly Arg Leu Asn Glu Arg 100 105 110 aac cgc aac tac ctc tcc ctg ggc gcc ggt tcg tcc gtc acg aac gcc 384 Asn Arg Asn Tyr Leu Ser Leu Gly Ala Gly Ser Ser Val Thr Asn Ala 115 120 125 ggc tac aac acc ggc atc cgg gtc gag cag ggc aag gac ttc 432 Gly Tyr Asn Thr Gly Ile Arg Val Glu Gln Gly Lys Arg Tyr Asp Phe 130 135 140 tcg gtg tgg gcc cgc gcc ggc agc gcc agc acg ctc acc gtc gcc ctg 480 Ser Val Trp Ala Arg Ala Gly Ser A Ser Leu Thr Val Ala Leu 145 150 155 160 aag gac gcc gcc ggc acg ctg gcg acc gcc cgc cag gtg gcc gtc gag 528 Lys Asp Ala Ala Gly Thr Leu Ala Thr Ala Arg Gln Val Ala Val Glu 165 170 175 ggc ggc tgg gcc ag tac agg gcc acg ttc acc gcg acc cgc acc agc 576 Gly Gly Trp Ala Lys Tyr Arg Ala Thr Phe Thr Ala Thr Arg Thr Ser 180 185 190 aac cgc ggc cgc ctc gcc gtc gcc gcc aac gac gcg gcg gcc ctc gac 624 Gly A rg Leu Ala Val Ala Ala Asn Asp Ala Ala Ala Leu Asp 195 200 205 atg gtg tcg ctg ttc ccg cgc gac acc tac cgg aac cag cag aac ggc 672 Met Val Ser Leu Phe Pro Arg Asp Thr Tyr Arg Asn Gln Gln Asn G210 215 220 ctg cgc aag gac ctc gcc gag aag atc gcg gcc ttg cac ccg ggc ttc 720 Leu Arg Lys Asp Leu Ala Glu Lys Ile Ala Ala Leu His Pro Gly Phe 225 230 235 240 gtg cgc ttc ccg ggc gcg cg gcc gcc gcc gcc gcc gcc gcc ggc tcc atg gag gac 768 Val Arg Phe Pro Gly Gly Cys Leu Val Asn Thr Gly Ser Met Glu Asp 245 250 255 tac agc gcg gcc tct ggc tgg cag cgc aag cgc tcc tac cag tgg aag 816 Tyr Ser Ala Ala Ser Gly Trp Gln Arg Lys Arg Ser Tyr Gln Trp Lys 260 265 270 gac acc gtc ggc ccg gtc gag gag cgc gcc acc aac gcc aac ttc tgg 864 Asp Thr Val Val Gly Pro Val Glu Glu Arg Ala Thr Asn Ala Asn Phe Trp 275 280 285 gt g tac aac cag agt tac ggc ctc ggc tac tac gag tac ttc cgc ttc 912 Gly Tyr Asn Gln Ser Tyr Gly Leu Gly Tyr Tyr Glu Tyr Phe Arg Phe 290 295 300 tcc gag gac atc ggc gcc atg ccg ctg ccc gtc gtc gtc gtg S er Glu Asp Ile Gly Ala Met Pro Leu Pro Val Val Pro Ala Leu Val 305 310 315 320 acg gga tgc ggc cag aac aag gcc gtc gac gac gag gcg ctg ctc aag 1008 Thr Gly Cys Gly Gln Asn Lys Ala Val Asp Asp Glu Ala Leu Leu Lys 325 330 335 agg cac atc cag gac act ctc gac ctg atc gag ttc gcg aac ggc ccg 1056 Arg His Ile Gln Asp Thr Leu Asp Leu Ile Glu Phe Ala Asn Gly Pro 340 345 350 gcg acc tcg aag tgg gg cgt gcc gag atg ggc cat ccg cgg 1104 Ala Thr Ser Lys Trp Gly Lys Val Arg Ala Glu Met Gly His Pro Arg 355 360 365 ccc ttc cgc ctc acg cac ctc gag gtc ggc aac gag gag aac ctc ccc 1152 Pro Phe Arg Le His Leu Glu Val Gly Asn Glu Glu Asn Leu Pro 370 375 380 gac gag ttc ttc gac cgc ttc aag cag ttc cgt gcc gcc atc gag gcc 1200 Asp Glu Phe Phe Asp Arg Phe Lys Gln Phe Arg Ala Ala Ile Glu Ala 385 400 gag tat ccg gac atc acg gtc gtc tcc aac tcc ggc ccc gac gac gcc 1248 Glu Tyr Pro Asp Ile Thr Val Val Ser Asn Ser Gly Pro Asp Asp Ala 405 410 415 ggc acc acc ttc gac acc gcc tgg aag ctc aac cgc gag gcg aac gtc 1296 Gly Thr Thr Phe Asp Thr Ala Trp Lys Leu Asn Arg Glu Ala Asn Val 420 425 430 gag atg gtc gac gag cac tac tac aac agc ccc aac tgg ttc ctc cag 1344 Glu Met Val Asp Glu His Tyr Tyr Ser Pro Asn Trp Phe Leu Gln 435 440 445 aac aac gac cga tac gac tcc tac gac cgg ggc ggc ccg aag gtc ttc 1392 Asn Asn Asp Arg Tyr Asp Ser Tyr Asp Arg Gly Gly Pro Lys Val Phe 450 455 460 ctc ggc gcc tcc cag ggc aac gcc tgg aag aac ggc ctc tcc 1440 Leu Gly Glu Tyr Ala Ser Gln Gly Asn Ala Trp Lys Asn Gly Leu Ser 465 470 475 475 480 gaa gcc gcg ttc atg acc ggc ctg gag cgc aac gc gc gac gc gc gac gc gac gc gc g Glu Ala Ala Phe Met Thr Gly Leu Glu Arg Asn Ala Asp Val Val Lys 485 490 495 ctg gcc tcg tac gcc ccg ctt ctc gcc aac gag gac tac gtc cag tgg 1536 Leu Ala Ser Tyr Ala Pro Leu Leu Ala Asn Glu Asp Tyr Gln Trp 500 505 510 cgt ccg gac ctg gtc tgg ttc aac aac cgc gcc tcc tgg aac tcg gcg 1584 Arg Pro Asp Leu Val Trp Phe Asn Asn Arg Ala Ser Trp Asn Ser Ala 515 520 525 525 aac tac gag gtc cag aaa ttc atg aac aac gtc ggc gac cgg gtc 1632 Asn Tyr Glu Val Gln Lys Leu Phe Met Asn Asn Val Gly Asp Arg Val 530 535 540 gtc ccc tcg aag gcc acc acc acg ccg gac gtc agc ggc ccg Atc Acc 1680 Ala Thr Thr Thr Pro Asp Val Ser Gly Pro Ile Thr 545 550 555 560 ggt gcc gtc ggc ctc tcg acc tgg gcg acc ggc acc gcg tac gac gac 1728 Gly Ala Val Gly Leu Ser Thr Trp Ala Thr Gly Thr Ala Tyr Asp Asp Asp 565 570 575 gtg aag gtc acc gcg gcg gac ggg gcc acg ctg ctg agc gac gac ttc 1776 Val Lys Val Thr Ala Ala Asp Gly Ala Thr Leu Leu Ser Asp Asp Phe 580 585 590 tcc ggt gac gcc tcg aag tgg acgac acc ggc agc tgg agc 1824 Ser Gly Asp Ala Ser Lys Trp Thr His Thr Gly Ala Gly Ser Trp Ser 595 600 605 gtc cag gac ggc cag tac gtc cag acg gac gcg gcg gcg gag aac acc 1872 Val Gln Asp Gly Gln Tyr Val Gln Thr Asp Ala Ala Ala Glu Asn Thr 610 615 620 atg gtc cag gcc ggc gac ccg tcc tgg cac gac tac gac ctg cat gtg 1920 Met Val Gln Ala Gly Asp Pro Ser Trp His Asp Tyr Asp Leu His Val 625 630 635 635 640 aag g cc acc aag aag tcc ggc aag gag ggc ttc ctc gtc gcc ttc ggc 1968 Lys Ala Thr Lys Lys Ser Gly Lys Glu Gly Phe Leu Val Ala Phe Gly 645 650 655 gtc aag gac acc ggc aac tac tac tgg tgg aac aac 2016 Val Lys Asp Thr Gly Asn Tyr Tyr Trp Trp Asn Leu Gly Gly Trp Asn 660 665 670 aac acc cag tcc gcc gtc gag cag gcc gtg gac ggc ggc aag ggc acg 2064 Asn Thr Gln Ser Ala Val Glu Gln Ala Val Asp Gly Gly Lys Gly Thr 675 680 685 ctg ctc acc aag gcc ggc tcg atc gag acg ggc cgc gcc tac gac atc 2112 Leu Leu Thr Lys Ala Gly Ser Ile Glu Thr Gly Arg Ala Tyr Asp Ile 690 695 700 gac gtc ag ggcg cag gtc acc ctg tac ctc gac ggc cag 2160 Asp Val Lys Val Arg Gly Arg Gln Val Thr Leu Tyr Leu Asp Gly Gln 705 710 710 720 gag tgg ggc ggc ttc acg gac gac aag ccg gcc gag ccg ttc cgt cag 2208 Gly Phe Thr Asp Asp Lys Pro Ala Glu Pro Phe Arg Gln 725 730 735 gtc gtc acc aag gac gcc cgg acc ggt gac ctg atc gtc aag gtc gtc 2256 Val Val Thr Lys Asp Ala Arg Thr Gly Asp Leu Ile Val Lys Val V al 740 745 750 aac gcc cag ccg gcc gag gcc cgc acc gcg atc gac ctg ggc ggc gcc 2304 Asn Ala Gln Pro Ala Glu Ala Arg Thr Ala Ile Asp Leu Gly Gly Ala 755 760 765 agg gtc gcc tcc acg gcc accg ctc gcc gcc gac cag gac 2352 Arg Val Ala Ser Thr Ala Arg Val Thr Thr Leu Ala Ala Asp Gln Asp 770 775 780 gcg gtg aac acc gag acg gac gca ccg gtc acc ccg gcg acg tcc acc 2400 Ala Val Asn Thr Glu Thr Asp Ala Pro Val Thr Pro Ala Thr Ser Thr 785 790 795 800 ttc tcg ggg gtc acg gac agg ttc acg tac acc ttc ccg gcg aac tcc 2448 Phe Ser Gly Val Thr Asp Arg Phe Thr Tyr Thr Phe Pro Ala Asn Ser 805 810 815 815 gtg acg ttc ctg cgg ctc aag cag agg tag 2478 Val Thr Phe Leu Arg Leu Lys Gln Arg 820 825

<210> 2 <211> 987 <212> DNA <213> Streptomyces sp. <220> <221> CDS <222> (1) .. (987) <400> 2 atg tgt acc cga gag gca gtc cgt atg agc cgc gaa cac gac ctg ccc 48 Met Cys Thr Arg Glu Ala Val Arg Met Ser Arg Glu His Asp Leu Pro 1 5 10 15 gaa atc ccc agc cgc aga ctg ctg ttg aag ggt gcc gcg gcc gcc ggc 96 Glu Ser Arg Arg Leu Leu Leu Lys Gly Ala Ala Ala Ala Gly 20 25 30 gcc ctc acg gcc gtc ccc ggc gtc gcc cat gcc gcc ccc agg ccg gcg 144 Ala Leu Thr Ala Val Pro Gly Val Ala His Ala Ala Pro Arg Pro Ala 35 40 45 ccc tac gag aac ccc ctc gtc cgg cag cgc gcc gac ccc cac atc cac 192 Pro Tyr Glu Asn Pro Leu Val Arg Gln Arg Ala Asp Pro His Ile His 50 55 60 cgc cac acg gac ggc cgt tac tac ttc acg gcc acc gct ccc gag tac 240 Arg His Thr Asp Gly Arg Tyr Tyr Phe Thr Ala Thr Ala Pro Glu Tyr 65 70 75 80 gac cgg atc gtc ctg cgc cgc tcc cgc acc ctg ggc ggc ctg tcc acg 288 Asp Arg Ile Val Leu Arg Arg Ser Arg Thr Leu Gly Gly Leu Ser Thr 85 90 95 gcg gcc gag tcg gtc atc tgg cgg gcc cac c cc acc ggc gac atg gcc 336 Ala Ala Glu Ser Val Ile Trp Arg Ala His Pro Thr Gly Asp Met Ala 100 105 110 gcc cac atc tgg gcg ccg gag ctg cac cgc atc ggc ggc aag tgg tac 384 Ala His Ile Trp Ala Pro Glu Leu His Arg Ile Gly Gly Lys Trp Tyr 115 120 125 gtc tac ttc gcc gcc gcg ccc gcc gaa gac gtg tgg cgc atc cgt atc 432 Val Tyr Phe Ala Ala Ala Pro Ala Glu Asp Val Trp Arg Ile Arg Ile 130 135 140 tgg gtc ctg gag aac tcc cac ccc gac ccg ttc aag ggc acc tgg gag 480 Trp Val Leu Glu Asn Ser His Pro Asp Pro Phe Lys Gly Thr Trp Glu 145 150 155 160 gag aag ggg cag gtc agg acg gcc tgg gag acc ttc tcc ctc gac gcc 528 Glu Lys Gly Gln Val Arg Thr Ala Trp Glu Thr Phe Ser Leu Asp Ala 165 170 175 acc acc ttc acc cac cgg ggc gcc cgc tac ctc tgc tgg gcg cag cac 576 Thr Thr Phe Thr His Arg Gly Ala Arg Tyr Leu Cys Trp Ala Gln His 180 185 190 gag ccc ggg gcg gac aac aac acc ggc ctg ttc ctg tcc gag atg gcg 624 Glu Pro Gly Ala Asp Asn Asn Thr Gly Leu Phe Leu Ser Glu Met Ala 195 200 205 aac ccc tgg acg ctg acg c ct cag atc cgg ctg tcc aca ccg gag 672 Asn Pro Trp Thr Leu Thr Gly Pro Gln Ile Arg Leu Ser Thr Pro Glu 210 215 220 tac gac tgg gag tgc gtc ggc tac aag gtc aac gag ggc ccc tac gcc 720 Tyr Asp Trp Glu Cys Val Gly Tyr Lys Val Asn Glu Gly Pro Tyr Ala 225 230 235 240 ctc aag cgc aac ggc cgc atc ttc ctc act tac tcg gcc tcc gcc acc 768 Leu Lys Arg Asn Gly Arg Ile Phe Leu Thr Tyr Ser Ala Ser Ala Thr 245 250 255 gac cac cac tat tgc gtc ggc atg ttc acc gcc gac gcc ggc ggc aac 816 Asp His His Tyr Cys Val Gly Met Phe Thr Ala Asp Ala Gly Gly Asn 260 265 270 ctc atg gac ccg ggc aac tgg tcc aag tcc ccg gtc ttc acc 864 Leu Met Asp Pro Gly Asn Trp Ser Lys Ser Pro Ile Pro Val Phe Thr 275 280 285 ggc aac gag acc acg aag cag tac ggc ccc ggc cac aac tgc ttc acc 912 Gly Asn Glu Thr Thr Lys Gln Tyr Gly Pro Gly His Asn Cys Phe Thr 290 295 300 gtc gcc gag gac ggc cgc agc gac gtg ctc gtc tac cac gcc cgt cag 960 Val Ala Glu Asp Gly Arg Ser Asp Val Leu Val Tyr His Ala Arg Gln 305 310 315 320 tac aag gag a tc gtc ggc gac ccc tga 987 Tyr Lys Glu Ile Val Gly Asp Pro 325

[Brief description of the drawings]

FIG. 1 shows a plasmid, p, carrying the enzyme gene of the present invention.
It is a figure which shows the structure of CR-2.1. The molecular weight of the plasmid is about 6.3 kb. In the figure, the arrow marked AFase1 is the enzyme gene of the present invention, and the arrow indicates the transcription direction of the gene. In addition, the leader line in the figure indicates a typical restriction enzyme site in the present plasmid.

FIG. 2 shows arabinofuranosidase 1 of the present invention.
It is an HPLC chart of a reaction product when made to act on methyl-α- (1 → 2) -L-arabinofuranobioside.

FIG. 3 shows arabinofuranosidase 2 of the present invention;
(A) is beet arabinan and linear (1 → 5) -α-L
(B) shows the time course of decomposition for three kinds of methyl-α-L-arabinofuranobioside regioisomers (1 → 2, 1 → 3, 1 → 5). FIG. The vertical axis indicates the decomposition rate and the horizontal axis indicates the reaction time.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) (C12N 1/21 C12R 1: 465) F term (reference) 4B024 AA01 AA05 BA12 CA02 DA05 EA04 GA11 HA01 HA14 4B050 CC03 DD02 LL01 LL02 4B065 AA50Y AB01 AC14 BA02 CA31 CA41 CA44

Claims (14)

[Claims] 1. An arabinofuranosidase having an activity of hydrolyzing linear (1 → 5) -α-L-arabinane. 2. The arabinofuranosidase according to claim 1, which specifically acts on (1 → 5) bound α-L-arabinofuranose residue. 3. The arabinofuranosidase according to claim 1, which has the following enzymatic properties. (1) Molecular weight about 37 kilodalton (by SDS electrophoresis) (2) Isoelectric point pH around 7.5 (by isoelectric focusing) (3) Working pH and pH stability Arabinofuranosidase at 40 ° C Working pH is pH
There is an optimum near 7.0. The stability of the arabinofuranosidase action when left at 30 ° C. for 2 hours is pH 5.0 to 5.0.
It is stable in the range of 9.0. (4) Optimum temperature and stability The arabinofuranosidase action temperature during the reaction at pH 7.0 for 10 minutes is optimally around 50 ° C. pH 7.0 Stability of arabinofuranosidase action after standing for 2 hours is 40 ° C
Up to. 4. An arabinofuranosidase having a sugar translocation activity 5. The arabinofuranosidase according to claim 4, which has the following enzymatic properties: (1) a molecular weight of about 80 kDa (by SDS electrophoresis); and (2) an isoelectric point of around pH 6.6. (By isoelectric focusing) (3) Working pH and pH stability The working pH of arabinofuranosidase at 40 ° C. is pH
There is an optimum near 5.5. The stability of arabinofuranosidase action when left at 30 ° C. for 2 hours is pH 5.5 to 5.5.
It is stable in the range of 8.5. (4) Optimum temperature and stability The arabinofuranosidase action temperature at the time of reaction at pH 5.5 for 10 minutes is optimally around 55 ° C. pH 7.0 Stability of arabinofuranosidase action after standing for 2 hours is 40 ° C
Up to. 6. An amino acid sequence having the amino acid sequence shown in SEQ ID NO: 1, or having an amino acid sequence in which one or several amino acids of the amino acid sequence have been deleted, substituted or added. The arabinofuranosidase described. 7. An amino acid sequence having the amino acid sequence shown in SEQ ID NO: 2, or having an amino acid sequence in which one or several amino acids of the amino acid sequence have been deleted, substituted or added. The arabinofuranosidase according to any one of the above. 8. A Streptomyces sp. GS-901 strain which produces arabinofuranosidase having the properties described in any one of claims 1 to 7 (Streptomy).
ces sp. GS-901) 9. Streptomyces sp. GS-90
1 strain (Streptomyces sp. GS-90)
The arabinofuranosidase according to any one of claims 1 to 7, which is derived from 1) or a mutant thereof. A gene encoding the arabinofuranosidase according to any one of claims 1 to 7. 11. It has the nucleotide sequence shown in SEQ ID NO: 1 or one or several nucleotides of the nucleotide sequence are deleted,
The gene according to claim 10, which has a substitution or an added base sequence. 12. It has the nucleotide sequence shown in SEQ ID NO: 2, or one or several nucleotides of the nucleotide sequence are deleted,
The gene according to claim 10, which has a substitution or an added base sequence. 13. Streptomyces sp. GS-9
01 strain (Streptomyces sp. GS-90)
13. The gene according to claim 11, which is derived from 1) or a mutant thereof. 14. A strain having the gene according to any one of claims 10 to 13 is inoculated in a medium, cultured, and arabinofuranosidase is produced and accumulated in the cells or in the medium. The method for producing arabinofuranosidase according to any one of claims 1 to 7, wherein the enzyme is collected.