JP2000342278A - Alginate-degrading enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new alginate-degrading enzyme which is an enzyme produced by a microorganism and capable of degrading both a mannuronic acid unit of alginic acid and a guluronic acid unit thereof and providing a degradation product of the alginic acid useful as medicines, functional foods, etc. SOLUTION: This new alginate-degrading enzyme is produced by a microorganism belonging to the Alteromonas sp. such as a microorganism JSTN000272 (FERM P-17020) and is capable of degrading both a mannuronic acid unit of alginic acid and a guluronic acid unit thereof, contains amino acid sequences represented by formulae I and II and is useful for the production, etc., of a degradation product of the alginic acid expected of various uses as medicines, functional foods, etc., by physiological activities thereof. The enzyme is obtained by culturing the microorganism JSTN000272 (FERM P-17020) separated from submarine mud and belonging to the Alteromonas sp. and carrying out the purification of a culture supernatant by ion exchange and chromatography, etc., and concentration by ultrafiltration.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present application relates to an alginate-degrading enzyme. More specifically, the present application relates to a novel alginate-degrading enzyme extracted and purified from a marine microorganism, various alginic acid-derived degradants obtained by the action of the degrading enzyme, and a microorganism producing the enzyme.

[0002]

BACKGROUND OF THE INVENTION Alginic acid is a viscous polysaccharide that fills the cells of brown algae. It is a linear polysaccharide in which two uronic acid units, mannuronic acid (M) and guluronic acid (G), are linked by 1,4-glycosidic bonds. It is a molecule and is used as a food additive, a base material for preparations, a medical material (fiber), and the like.

[0003] On the other hand, the degradation product of this alginic acid has been expected to have an effect of excreting blood cholesterol and metals by a conjugation reaction caused by cation reactivity of a carboxyl group of alginic acid. However, it is known that when this alginic acid is made into a solution, the alginic acid forms a metal salt with calcium or the like in the solution and gels, so that it is very difficult to be decomposed. Attempts have been made to decompose alginic acid using an alginate-degrading enzyme produced by a microorganism.
No. 37783 discloses a method for producing potassium alginate oligosaccharides by allowing alginate-degrading enzyme produced by an Artenomonas genus bacterium isolated from the intestine of horseshoe crab to act on potassium alginate and / or sodium alginate, and a method for producing potassium alginate oligosaccharides. Potassium alginate oligosaccharides have been disclosed.

[0004]

Degraded products of alginic acid are expected to have various uses as pharmaceuticals and functional foods due to their physiological activities. However, in order to accurately specify their activities and define their uses. Requires a high-purity decomposed sample.

However, for example, Japanese Patent Application Laid-Open No.
In the case of the alginate-degrading enzyme disclosed in JP 7783, alginic acid is degraded at random, and it is difficult to control the degree of polymerization of the degradation product.
In addition, the ratio of the two sugar chain units (M unit and G unit) constituting alginic acid and their arrangement in the polymer are expected to affect biological effects.
Although an alginate degradation product consisting of each of the two units is regarded as important, an alginate degradation enzyme capable of decomposing both the M unit and the G unit to a desired length has not been known.

The invention of this application has been made in view of the above circumstances, and an object of the invention is to provide a novel alginate-degrading enzyme capable of decomposing both the M unit and the G unit of alginic acid. And

Another object of the present invention is to provide a degradation product of alginic acid or the like prepared using the alginate-degrading enzyme, and a microorganism producing the enzyme.

[0008]

This application provides the following inventions for solving the above-mentioned problems. (1) An alginate-degrading enzyme produced by a microorganism, which degrades both a homomannuronic acid unit and a homoguluronic acid unit of alginic acid. (2) The alginate-degrading enzyme according to (1), wherein the microorganism is a microorganism belonging to the genus Alteromonas sp. Isolated from marine sediment. (3) The microorganism is JSTN000272 (FERM P-1702).
The alginate-degrading enzyme according to the above (2), which is 0). (4) The alginate-degrading enzyme according to (3), comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. (5) A peptide consisting of any amino sequence portion of SEQ ID NO: 2 or SEQ ID NO: 3. (6) A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 and encoding the alginate-degrading enzyme of (3). (7) An alginate hydrolyzate obtained by allowing any one of the above-mentioned (1) to (4) to act on alginic acid. (8) A mannuronic acid oligomer obtained by allowing any one of the above-mentioned (1) to (4) to act on the mannuronic acid polymer. (9) A guluronic acid oligomer obtained by allowing the alginate-degrading enzyme according to any of (1) to (4) to act on a guluronic acid polymer. (10) JSTN000272 (FERM P-17020), a microorganism belonging to the genus Alteromonas sp.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The alginate-degrading enzyme of the present invention is an enzyme capable of selectively decomposing M units and G units constituting alginic acid.
Such an enzyme can be extracted and purified from, for example, a microorganism belonging to the genus Artenomonas isolated from submarine mud. In the present invention, as a result of screening microorganisms isolated from marine sediment in Omura Bay, Nagasaki Prefecture, bacteria JSTN
000272 produced an enzyme having the above properties, and deposited it on October 9, 1998 with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (FERM P-17020). This JSTN000272 (hereinafter sometimes referred to as No. 272 bacterium) is a genus of Alteromonas ssp.
p.) It is clear from the results of the property tests shown in Table 1 that the bacteria are bacteria. In Table 1, No. 272 bacteria and literature (Bu
ll.Fac.Nagasaki Univ. 40: 35-38, 1975; Anal.Bioche
m. 9: 401, 1964; Nature 227: 685, 1970), other Alteromonas bacteria (A. haloplanktis, A. macleodii).
The results of the property tests were compared and shown.

[0010]

[Table 1]

The alginate-degrading enzyme of the present invention comprises a combination of means for culturing the above microorganism, separating the crude enzyme solution by centrifugation of the culture supernatant, purifying by ion exchange and chromatography, and concentrating by ultrafiltration and the like. It can be obtained by known methods.

The alginate-degrading enzyme of the present invention can also be used in the form of a peptide. Such a peptide can be prepared by digesting alginate-degrading enzyme with an appropriate protease, or can be prepared by chemically synthesizing a known peptide synthesis method based on the amino acid sequences of SEQ ID NOS: 2 and 3. it can.

Furthermore, the alginate-degrading enzyme of the present invention can be prepared by genetic engineering by expressing the gene in a suitable host-vector system.
The alginate-degrading enzyme gene can be prepared from N0.272 bacteria DNA using, for example, an oligonucleotide probe prepared based on the nucleotide sequence of SEQ ID NO: 1.

[0014]

EXAMPLES Hereinafter, the present invention will be described in more detail and specifically with reference to examples, but the present invention is not limited to the following examples.

The materials and methods used in the following Examples 1 to 5 are as follows. a) Microorganism JSTN000272 (FERM P-17020: No. 272) was used. b) Alginic acid Alginic acid extracted from kelp and sodium alginate (1,000 cps: manufactured by Nacalai Tex Corporation) were used. c) Activity measurement of alginate-degrading enzyme RAPID method (Bull. Fac. Nagasaki Univ. 40: 35-38,
1975). That is, the substrate (50 mM phosphate buffer containing 0.2% sodium alginate, pH 7.5)
Incubate 2 ml at 30 ° C for 10 minutes,
mixed with l. The absorbance at 235 nm, which is the specific absorption wavelength of the double bond obtained by
The measurement was performed every 30 seconds for 2 minutes after the second. The blank in that case was water. The amount of the enzyme at which the absorbance increased by 0.100 per minute was defined as 1 unit. d) Quantification of protein Using bovine serum albumin as a standard protein, the protein was measured by the microburette method (Anal. Biochem., 9: 401, 1964). e) Electrophoresis SDS-polyacrylamide gel electrophoresis (SDS-P
AGE) was performed by a slab method using a flat plate according to the method described in the literature (Nature, 227: 685, 1970). The concentrated gel used had a concentration of 5%, and the separation gel used had a concentration of 12%. Staining solution is isopropyl alcohol 25%, acetic acid 10%, purified water
Decolorization was performed in a 7% acetic acid solution using a 65% mixed solution.

Polyacrylamide gel electrophoresis (PAG)
E) shows that, except that SDS is not added to the running buffer,
Performed similarly to DS-PAGE. f) N-terminal amino acid sequence analysis of the enzyme High purity distilled water was added to 30 μl of the enzyme solution, placed ultra-free, and centrifuged at 10,000 rpm to 30 μl. Prepare the prepared sample with a 120 APTH analyzer (AppliedBiosyst.
ems) and a protein sequencer 477A linked to it
(Manufactured by Applied Biosystems), and each PTH-amino acid was determined. g) Determination of molecular weight by gel filtration Column chromatography using Sephadex G-100 standard protein (blue dextran 2,000: molecular weight 2,000; ribonuclease A: molecular weight 13,700; chymotrypsinogen A: molecular weight 25,000; ovalbumin:
(Molecular weight: 43,000; bovine serum albumin: molecular weight: 67,000) was eluted, respectively, to prepare a calibration curve. The enzyme was eluted under the same conditions as the standard protein elution, and the molecular weight was quantified from the calibration curve. Example 1 Purification of Alginic Acid Degrading Enzyme (1) Culture of Microorganism When the culture medium of No. 272 was examined, as shown in FIG. 1, the degree of growth and the enzyme activity were shown. .0), the enzyme activity was remarkable, and the activity was highest at 25 ° C. for 48 hours. Therefore, the culture condition was set at 25 ° C. for 48 hours.

The medium used had a composition in which sodium alginate was added instead of glucose and magnesium sulfate was removed because seawater was used (Table 2). In addition, as shown in FIG. 2, the pre-cultured Zo showed the best growth of the bacteria.
Bell2216E (pH7.5) was used.

[0018]

[Table 2]

(2) Preparation of Crude Enzyme Solution The obtained culture solution was centrifuged (6,000 G, 20 minutes) to remove cells. Ammonium sulfate was added to the sterilized culture solution so as to be 100% saturated, and the mixture was allowed to stand at 4 ° C. overnight, followed by centrifugation (6,000 G, 20 minutes) to obtain a target protein. After desalting by dialysis, the solution was concentrated by ultrafiltration to obtain a crude enzyme solution. (3) Anion exchange (batch method) Since the crude enzyme solution was high in clay and difficult to perform column chromatography, first, anion exchange was performed by the batch method. The sample was added to the ion exchanger equilibrated with 50 mM Tris buffer (pH 7.5), stirred for several minutes, allowed to stand for about 1 hour to adsorb the protein, transferred to a glass filter, and suction filtered. Was done. The concentration of NaCl and Tris buffer was increased stepwise until the enzyme activity of the filtrate disappeared, and this operation was repeated. Fractions having enzyme activity were collected, concentrated by ultrafiltration, and used as a sample. (4) Anion exchange (second time) The obtained active fraction was concentrated by dialysis, and then subjected to column chromatography using DEAE-Cellulofine. The active fraction was adsorbed by adding the sample to a column equilibrated with 0.1 M Tris buffer (pH 6.5), and then eluted by a linear gradient method from 0 to 0.5 M NaCl (FIG. 3). . The obtained active fractions were collected and concentrated by ultrafiltration. (5) Gel Filtration with Sephadex G-100 Gel filtration of the enzyme preparation previously obtained by DEAE-chromatography was performed by column chromatography using Sephadex G-100 (FIG. 4). Active fractions were collected and concentrated by ultrafiltration. (6) Cation exchange column chromatography The active fraction obtained in the above (5) was further subjected to cation exchange column chromatography using CM-Cellulofine. The sample was added to the column equilibrated with 20 mM phosphate buffer (pH 6.0), and a pH gradient from pH 6.0 to 8.0 was applied.
As shown in FIG. 5, the activity was observed only in the flow-through fraction. This active fraction was collected and concentrated by ultrafiltration.

Furthermore, DEA was added to the filtered concentrate.
Anion exchange (third time) was performed by column chromatography using E-Cellulofine. The active fraction was eluted by a 0.1 to 0.3 NaCl linear concentration gradient method. The active fraction was collected, concentrated by ultrafiltration, and used as an enzyme preparation (FIG. 6). (7) Purification of enzyme The enzyme was purified according to the steps shown in FIG. The yield of the protein at the time of purification is as shown in Table 3. The total protein amount was changed from 446.7 mg at the start of purification to 6.3 mg, and the specific activity of the enzyme at the final stage was 487.4 units / mg. The specific activity was 8.8 times, and the activity recovery was 12.4%.

[0021]

[Table 3]

Example 2 Confirmation of Characteristics of Purified Enzyme (1) Purity of Purified Enzyme The enzyme preparation obtained in Example 1 was subjected to polyacrylamide gel electrophoresis in the presence and absence of SDS. As shown in FIG. 8, the number of enzyme bands was one in each case.

Further, the primary structure of the amino acid of the enzyme protein was examined, and it was found that the N-terminal sequence was as shown in SEQ ID NO: 3.

From the above results, it was confirmed that the enzyme preparation obtained in Example 1 was highly purified. (2) Molecular weight Using a protein of known molecular weight as a standard and measuring by the Whitaker method using Sephadex-100, as shown in FIG. 9, the molecular weight of this enzyme was about 25,000.
Was estimated. (3) Properties of Enzyme As shown in FIG. 10, this enzyme had a higher relative activity in a temperature range of 30 to 70 ° C., and it was confirmed that 60 ° C. at which the relative activity was maximum was the optimum temperature. . In addition, as shown in FIG. 11, since it is stable up to around 40 ° C., it is considered preferable to react at 30 to 40 ° C. when producing alginate oligo-oligosaccharide using this enzyme. .

As shown in FIG. 12, the relative activity of this enzyme increased around pH 6.0 to 9.0, and the optimum pH range was pH 7.5 to 8.0. Also, as shown in FIG.
Since this enzyme is stable in the pH range of 6.0 to 11.0, it is considered that cultivation of bacteria and storage of the enzyme are preferably performed in this pH range. (4) Substrate Specificity As shown in FIG. 14, this enzyme showed activity on both mannuronic acid polymer (PM) and guluronic acid polymer (PG). Example 3: Preparation of mannuronic acid oligomer PM previously prepared according to the method of Haug et al. Shown in FIG.
Was enzymatically degraded and fractionated as follows.

20 ml of a 50 mM phosphate buffer in which 4 g of PM has been dissolved
Add 12 units of alginate-degrading enzyme to
After reacting for 3.5 hours, the same amount of enzyme was further added and reacted for a total of 5.5 hours. The reaction was stopped by adjusting the pH to 4.2 using HCl and then neutralized (pH 7.0) to obtain a digest. This digest was separated by column chromatography using Biogel P-6 (mobile phase: 50 mM phosphate buffer) (FIG. 16). In order to remove the phosphate in each of the obtained fractions, column chromatography using Biogel P-6 was performed using pure water. For those which seemed to have a large overlap in the elution pattern, column chromatography was performed again with Biogel P-6 to obtain each fraction of mannuronic acid oligomer, which was lyophilized.

From the elution position of column chromatography using Biogel P-6, galacturonic acid (M
The molecular weight was determined by Whitaker's method on the basis of (equivalent to the elution position of 1, G1) and the degree of polymerization of each obtained fraction was determined. Table 4 shows the yield (per 4 g of raw material) of each digested product having a degree of polymerization of 1 to 11. Example 4: Preparation of guluronic acid oligomer 4 g of PG prepared in advance as in Example 3 was dissolved.
100 units of alginate-degrading enzyme was added to 20 ml of 50 mM phosphate buffer, and the mixture was reacted at 30 ° C. for 3 hours. Then, the same amount of the enzyme was further added and reacted for a total of 5 hours. After neutralization with HCl, a guluronic acid oligomer was obtained in the same manner as in Example 3 (FIG. 17), which was freeze-dried. Further, the degree of polymerization of each fraction was determined in the same manner as in Example 3, and the yield of each digest was shown in Table 4.

[0028]

[Table 4]

Example 5: Preparation of alginate hydrolyzate 27 units of alginate-degrading enzyme was added to 2 ml of 50 mM phosphate buffer in which 0.4 g of alginate extracted from kelp was dissolved, and reacted at 30 ° C. for 2.5 hours. Further, the same amount of enzyme was added and reacted for a total of 5 hours. After removing the phosphoric acid, it was freeze-dried. Example 6: Identification of alginate-degrading enzyme gene After culturing No.272 bacteria at 25 ° C. for 24 hours with shaking, DNA was extracted by known phenol / ethanol extraction, and the alginate-degrading enzyme gene was amplified by PCR using this as a template. . Alginate degrading enzymes were lysyl endopeptidase and endoproteinase as PCR primers.
Based on the overlap of the peptides obtained by digestion with Asp-N (FIG. 18), SEQ ID NO: 4 (forward primer AAL-VP-1) and SEQ ID NO: 5 (reverse primer
An oligonucleotide consisting of the nucleotide sequence of AAL-CP-1) was synthesized. Table 5 shows the composition of the PCR reaction solution.
The PCR was performed at (94 ° C.-45 ° C.-72 ° C.) × 40 cycles.

[0030]

[Table 5]

A DNA fragment of about 450 bp was obtained by the above PCR. As a result of analyzing the nucleotide sequence with a DNA sequencer, it was found that the nucleotide sequence of this DNA fragment was as shown in SEQ ID NO: 1, and the amino acid sequence of the polypeptide encoded by this DNA fragment was SEQ ID NO: 2.
The polynucleotide of SEQ ID NO: 1 still encodes the 3 ′ side of the alginate gene, and the polypeptide of SEQ ID NO: 2 is the C-terminal side of the alginate enzyme.

[0032]

As described above in detail, according to the present application, a novel alginate degrading enzyme capable of decomposing both the M unit and the G unit of alginic acid, and alginic acid and the like prepared by using the alginate degrading enzyme. A degradation product, a microorganism producing the enzyme, and a polynucleotide encoding the alginate-degrading enzyme are provided.

[0033]

[Sequence List] SEQUENCE LISTING <110> Choko Soy Sauce Miso Cooperative Science and Technology Promotion Agency <120> Alginate Degrading Enzyme <130> <140> <141> <150> JP 11-093826 <151> 1999-03-31 <160> 5 <170> PatentIn Ver. 2.1 <210> 1 <211> 468 <212> DNA <213> Alteromonas sp. <220> <221> CDS <222> (1) .. (468) <400> 1 gta gaa atg agt gat gga gga aag aca att ata tcg cag cac cat gct 48 Val Glu Met Ser Asp Gly Gly Lys Thr Ile Ile Ser Gln His His Ala 1 5 10 15 agc gat acc ggg act ata tct aaa gtg tat gtg tcg gat act gat gaa 96 Ser Asp Thr Gly Thr Ile Ser Lys Val Tyr Val Ser Asp Thr Asp Glu 20 25 30 tcg ggc ttt aat gat agc gta gcg aac aac gga att ttt gat gtg tac 144 Ser Gly Phe Asn Asp Ser Val Ala Asn Asn Gly Ile Phe Asp Val Tyr 35 40 45 gta cgt tta cgt aat acc agc ggt aat gaa gaa aaa ttt gct ttg ggt 192 Val Arg Leu Arg Asn Thr Ser Gly Asn Glu Glu Lys Phe Ala Leu Gly 50 55 60 aca atg acc agc ggt gag aca ttt aac ttg cgg gta gtt aat aac tac 240 Thr Met Thr Ser Gly Glu Thr Phe Asn Leu Arg Val Val Asn Asn Tyr 65 70 75 80 ggc gat gta gag gtt acg gca ttc ggt aac tcg ttc ggt ata ccg gta 288 Gly Asp Val Glu Val Thr Ala Phe Gly Asn Ser Phe Gly Ile Pro Val 85 90 95 gag gat gat tcg caa tca tac ttt aag ttt ggt aac tac ctg caa tcg 336 Glu Asp Asp Ser Gln Ser Tyr Phe Lys Phe Gly Asn Tyr Leu Gln Ser 100 105 110 caa gac cca tac aca tta gat aaa tgt ggt gag gcc gga aac tct aac 384 Gln Asp Pro Tyr Thr Leu Asp Lys Cys Gly Glu Ala Gly Asn Ser Asn 115 120 125 tcg ttt aaa aac tgt ttt gag gat tta ggc att aca gag tca aaa gtg 432 Ser Phe Lys Asn Cys Phe Glu Asp Leu Gly Ile Thr Glu Ser Lys Val 130 135 140 acg atg acc aat gtt act tat act agt gaa aca aac 468 Thr Met Thr Asn Val Thr Tyr Thr Ser Glu Thr Asn 145 150 155 <210> 2 <211> 156 <212> PRT <213> Alteromonas sp. <400> 2 Val Glu Met Ser Asp Gly Gly Lys Thr Ile Ile Ser Gln His His Ala 1 5 10 15 Ser Asp Thr Gly Thr Ile Ser Lys Val Tyr Val Ser Asp Thr Asp Glu 20 25 30 Ser Gly Phe Asn Asp Ser Val Ala Asn Asn Gly Ile Phe Asp Val Tyr 35 40 45 Val Arg Leu Arg Asn T hr Ser Gly Asn Glu Glu Lys Phe Ala Leu Gly 50 55 60 Thr Met Thr Ser Gly Glu Thr Phe Asn Leu Arg Val Val Asn Asn Tyr 65 70 75 80 Gly Asp Val Glu Val Thr Ala Phe Gly Asn Ser Phe Gly Ile Pro Val 85 90 95 Glu Asp Asp Ser Gln Ser Tyr Phe Lys Phe Gly Asn Tyr Leu Gln Ser 100 105 110 Gln Asp Pro Tyr Thr Leu Asp Lys Cys Gly Glu Ala Gly Asn Ser Asn 115 120 125 Ser Phe Lys Asn Cys Phe Glu Asp Leu Gly Ile Thr Glu Ser Lys Val 130 135 140 Thr Met Thr Asn Val Thr Tyr Thr Ser Glu Thr Asn 145 150 155 <210> 3 <211> 10 <211> PRT <213> Alteromonas sp. <400> 1 Asp Gly Ser Gln Ile Pro Ser Thr Ile Thr 1 5 10 <210> 4 <211> 24 <211> DNA <213> Artificial Sequence <220> <213> Artificial Sequence: Synthesized Oligonucleotide <400> 4 gtagaaatga gtgatggagg aaag 24 <210> 5 <211> 27 <211> DNA <213> Artificial Sequence <220> <213> Artificial Sequence: Synthesized Oligonucleotide <400> 5 aatgttactt atactagtga aacaaac 27

[Brief description of the drawings]

FIG. 1 is a graph showing changes in the degree of growth and activity of No. 272 bacteria in Davis medium.

FIG. 2 is a graph showing changes in the degree of growth and activity of No. 272 bacteria in ZoBell2216E medium.

FIG. 3 is an elution curve of an enzyme by column chromatography using DEAE-Cellulofine.

FIG. 4 is an elution curve of an enzyme by gel filtration using Sephadex G-100.

FIG. 5 is an elution curve of an enzyme by column chromatography using CM-Cellulofine.

FIG. 6 is an elution curve of an enzyme by column chromatography (the third time) using DEAE-Cellulofine.

FIG. 7 is a purification process chart of an enzyme.

FIG. 8 is a gel electrophoresis diagram of the enzyme of the present invention.

FIG. 9 is a graph showing the estimated molecular weight of the enzyme of the present invention.

FIG. 10 is a graph showing the relationship between the temperature and the activity of the enzyme of the present invention.

FIG. 11 is a graph showing the temperature stability of the enzyme of the present invention.

FIG. 12 is a graph showing the relationship between the pH and the activity of the enzyme of the present invention.

FIG. 13 is a graph showing the pH stability of the enzyme of the present invention.

FIG. 14 is a graph showing the relationship between the enzyme of the present invention and a substrate.

FIG. 15: Haug used to prepare PM and PG
FIG. 4 is a process chart showing these methods.

FIG. 16 shows P by gel filtration using Biogel P-6.
M is an elution curve.

FIG. 17 shows P by gel filtration using Biogel P-6.
G is an elution curve.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:01) (C12N 1/20 C12R 1:01) (C12N 9/42 C12R 1:01) (C12P 19/14 C12R 1:01) (72) Inventor Shinjiro Hayashida 13-40, Tsutomachi, Isahaya-shi, Nagasaki Prefecture (72) Inventor Hisataka 1258, Inheicho-cho, Omura-shi, Nagasaki F-term (reference) 4B024 AA01 AA05 BA12 CA02 GA19 HA01 HA14 4B050 CC01 DD02 LL01 LL02 LL05 4B064 AF04 AH19 CA21 CB07 CE17 DA01 DA10 4B065 AA01X AC14 BA22 BB18 CA21 CA31 CA41 CA44

Claims (10)

[Claims] 1. An alginate-degrading enzyme produced by a microorganism, which degrades both a mannuronic acid unit and a guluronic acid unit of alginic acid. 2. The alginolytic enzyme according to claim 1, wherein the microorganism is a microorganism belonging to the genus Alteromonas sp. Isolated from marine sediment. 3. The method according to claim 1, wherein the microorganism is JSTN000272 (FERM).
P-17020). The alginate-degrading enzyme according to claim 2, which is P-17020). 4. The alginolytic enzyme according to claim 3, which comprises the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3. 5. A peptide consisting of any amino sequence portion of SEQ ID NO: 2 or SEQ ID NO: 3. 6. The method according to claim 3, which comprises the nucleotide sequence of SEQ ID NO: 1.
A polynucleotide that encodes an alginate-degrading enzyme. 7. An alginate hydrolyzate obtained by allowing the alginate hydrolyzing enzyme according to claim 1 to act on alginic acid. 8. A mannuronic acid oligomer obtained by allowing the alginate-degrading enzyme according to claim 1 to act on a mannuronic acid polymer. 9. A guluronic acid oligomer obtained by allowing the alginate-degrading enzyme according to any one of claims 1 to 4 to act on a guluronic acid polymer. 10. The genus Alteromonas s.
p.) belonging to the microorganism JSTN000272 (FERM P-1702).
0).