JP4883277B2 - Porphyran-specific degrading enzymes produced by Cerrophaga bacteria - Google Patents

Description

  The present invention mainly produces a porphyran-specific degrading enzyme produced by a cellulophaga bacterium, a method for producing a porphyran degradation product using the enzyme, a porphyran degradation product produced by the method, and a porphyran-specific degrading enzyme It relates to the genus Cerrofaga.

  The Ariake Sea coast is one of the largest aquaculture production areas in the country, and some of the produced seaweeds contain low-priced pastes such as “color faded paste” and “lower paste”. It has been discarded. For this reason, there is an urgent need to reduce the producer cost burden and the environmental burden through the effective use of such waste glue.

  Under such circumstances, it has recently been reported that porphyran, which is a viscous polysaccharide contained in the seaweed of Amanori, has various physiological activities. For example, it has been reported that porphyran has physiological activities such as life-prolonging effect of Salmonella-infected mice (Patent Document 1), blood cholesterol lowering action (Non-Patent Document 1), antitumor activity (Non-Patent Document 2) and the like. Yes. The inventors have also found that porphyran has an immunostimulatory action.

  Porphyran is a mixture mainly containing D-galactose, 3,6-anhydro-L-galactose or L-galactose-6-sulfate, and a part of these D-galactoses in which C-6 is methylated. It is. Since porphyran is a mixture of a plurality of components, the component ratio and molecular weight differ depending on the starting material and the production method. For example, the molecular weight of porphyran extracted from the genus Amanori by conventional methods is often about 100,000 or more.

  Molecules having such a large molecular weight are usually difficult to be absorbed into the body when ingested and cannot exert sufficient effects in the body. Therefore, it is required to decompose (lower molecular weight) molecules.

  In general, as a method for decomposing a polysaccharide, a method using an acid or an alkali is currently known. However, in this method, since decomposition occurs randomly and frequently, it is difficult to control the decomposition. In addition, when alkali is used, the molecular structure may change and the desired action may be impaired or an undesirable action may be exerted. Therefore, the method using an acid or alkali is inappropriate for obtaining a decomposed product having stable properties and actions, and is difficult to use industrially. In addition, in the acid / alkali method, high temperature heat may be required for decomposition, and since a large amount of salt is generated as a by-product by neutralization, energy and labor for heating and salt removal are required, As a result, high manufacturing costs are required.

  As another method for degrading polysaccharides, a method using an enzyme is known. In the method using an enzyme, unlike the method using an acid or an alkali, degradation of the polysaccharide occurs gently. For this reason, it is easy to control the decomposition, and the properties and actions of the decomposition products are stable. Furthermore, since the decomposition reaction is performed under mild conditions, the structure of the decomposition product does not change, and the decomposition product is difficult to deteriorate and high quality can be maintained.

Therefore, there is a strong demand for a new enzyme that gently degrades porphyran and enables stable supply of high-quality porphyran degradation products.
JP-A-14-193828 Fisheries Sci 60 (1), 83-88 (1994) Biosci. Biotechnol. Biochem. 59 (10), 1993-1937 (1995)

  The main object of the present invention is to provide an enzyme that hydrolyzes porphyran and a microorganism that produces such an enzyme.

  As a result of earnest research for the novel enzyme that hydrolyzes porphyran, the present inventors have found that an enzyme having porphyran hydrolyzing activity has been produced by a cellulophaga bacterium, and completed the present invention. It came to.

That is, the present invention relates to the following matters.
[Item 1] Porphyran-specific degrading enzyme having the following characteristics:
(A) having specific hydrolysis activity for porphyran,
(B) produced by bacteria of the genus Cellulophaga,
(C) The molecular weight measured by SDS-PAGE is about 42 kDa,
(D) the optimum pH is 7.5,
(E) The optimum temperature is 30 ° C.
[Item 2] (f) substantially free of hydrolysis activity on agarose,
Item 2. A porphyran-specific degrading enzyme according to Item 1.
[Item 3] (g) The porphyran-specific degrading enzyme according to item 1 or 2, which has a hydrolytic activity for β-agarase-treated porphyran.
[Item 4] The porphyran-specific degrading enzyme according to any one of Items 1 to 3, wherein the porphyran is a porphyran produced in a seaweed of the genus Amanori.
[Item 5] The porphyran-specific decomposing enzyme according to any one of Items 1 to 4, wherein the Cerrophaga bacterium is Cellulophaga fucicola.
[Claim 6] The Porphyran according to Item 5, wherein the Cellulofarga fushicola is the Cellulofarga fushicola PDM-1 strain (consigned to the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology as deposit number FERM P-20648). Specific degrading enzyme.
[Item 7]
(I) a step of mixing the porphyran-specific decomposing enzyme according to any one of Items 1 to 6 and porphyran, and decomposing porphyran,
A process for producing a porphyran degradation product.
[Item 8]
(II) a step of mixing β-agarase and porphyran to decompose porphyran,
The manufacturing method of the porphyran degradation product of claim | item 7 which includes further (Here, process (I) and process (II) may be performed in arbitrary order).
[Item 9]
Item 9. The method according to Item 7 or 8, wherein the decomposition in the step (I) is performed under pH 6.0 to 8.0.
[Item 10]
Item 10. The method according to any one of Items 7 to 9, wherein the decomposition in the step (I) is performed at a temperature of 4 to 40 ° C.
[Item 11] The method according to any one of Items 7 to 10, wherein the porphyran is a porphyran contained in a water-soluble fraction of the seaweed genus Seaweed.
[Item 12] A porphyran degradation product obtained by the method according to any one of Items 7 to 11.
[Item 13] The porphyran degradation product according to Item 12, which has a molecular weight of 5,000 or less.
[Item 14] A bacterium belonging to the genus Cerrophaga having the ability to produce a porphyran-specific degrading enzyme.
[Item 15] The bacterium according to item 14, which is Cerulofaga fushicola.
[Item 16] The bacterium according to item 15, which is a Cellulofaga fushikora PDM-1 strain (consigned to the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under accession number FERM P-20648).

Hereinafter, the present invention will be described in more detail.
[I] Porphyran-specific degrading enzyme The present invention provides a porphyran-specific degrading enzyme having the following properties:
(A) having specific hydrolysis activity for porphyran,
(B) produced by Cerrofaga bacteria,
(C) The molecular weight measured by SDS-PAGE is about 42 kDa,
(D) the optimum pH is 7.5,
(E) The optimum temperature is 30 ° C.

  Furthermore, the porphyran-specific decomposing enzyme of the present invention usually has substantially no hydrolytic activity on (f) agarose. From this characteristic, the porphyran-specific decomposing enzyme of the present invention is clearly different from β-agarase having porphyran hydrolyzing activity.

  In addition, the porphyran-specific decomposing enzyme of the present invention usually has (g) hydrolysis activity for porphyran treated with β-agarase. In the hydrolysis of porphyran by β-agarase, it is considered that the portion rich in sulfate group in the porphyran structure remains without being decomposed. Since the porphyran-specific decomposing enzyme of the present invention can cleave such a portion that is difficult to cleave with β-agarase, the porphyran-decomposed product by β-agarase treatment can be further reduced in molecular weight. This means that the porphyran-specific decomposing enzyme of the present invention has a hydrolytic activity for porphyran treated with β-agarase.

  As described above, porphyran is obtained by methylation of D-6 galactose, 3,6-anhydro-L-galactose or L-galactose-6-sulfate, and a part of these D-galactoses. It is a mixture mainly containing. Since porphyran is a mixture of a plurality of components, its component ratio and molecular weight differ depending on the starting material and the production method, but the porphyran in the present invention includes any component ratio.

  The hydrolysis activity is an activity of reducing the molecular weight of porphyran by hydrolysis. The level of hydrolysis is not particularly limited as long as the molecular weight of the porphyran before hydrolysis is reduced compared to the molecular weight of the porphyran after hydrolysis.

  When the porphyran-specific decomposing enzyme of the present invention is used, for example, about 10% or more of porphyran having a molecular weight of 100,000 or more under the reaction conditions of 30 U, pH 7.5, about 72 hours and an enzyme concentration of 10 U / ml. More preferably 20% or more, more preferably about 30% or more, more preferably about 40% or more, more preferably about 50% or more, more preferably about 60% or more, particularly preferably about 70% or more. It can be hydrolyzed to 5,000 or less molecules.

  When the porphyran-specific decomposing enzyme of the present invention is used, for example, a porphyran having a molecular weight of 30,000 or more treated with β-agarase, for example, under reaction conditions of 30 U, pH 7.5, about 72 hours and an enzyme concentration of 10 U / ml. About 10% or more, more preferably 20% or more, more preferably about 30% or more, more preferably about 40% or more, more preferably about 50% or more, more preferably about 60% or more, particularly preferably about 70%. % Or more can be hydrolyzed into molecules having a molecular weight of 5,000 or less.

  When converted to a pure product, the porphyran-specific degrading enzyme of the present invention has a porphyran hydrolyzing activity of usually about 0.1 to 50 U, preferably about 1 to 30 U, more preferably about 5 to 15 U per mg. . Here, 1 U is the amount (μg) of reducing sugar increased in an enzyme reaction per unit time (hour) using 1 μg of enzyme at 30 ° C.

  The porphyran-specific degrading enzyme of the present invention shows hydrolytic activity specifically for porphyran treated with porphyran or β-agarase, but in most cases, agarose, agar, sodium alginate, chondroitin sulfate, sulfated dextran ( 500 kDa), ι-carrageenan, κ-carrageenan, λ-carrageenan and substantially no hydrolytic activity. Therefore, in this document, “porphyran-specific” indicates hydrolytic activity to porphyran or a degradation product derived from porphyran as described above, but agarose, agar, sodium alginate, chondroitin sulfate, sulfated dextran (500 kDa). , Ι-carrageenan, κ-carrageenan, and λ-carrageenan means substantially no hydrolytic activity.

  The origin of the porphyran-specific degrading enzyme is not particularly limited as long as it is a bacterium belonging to the genus Cellulophaga, but is preferably Cellulophaga ficcola, and particularly preferably an independent government as accession number FERM P-20648. It is Cerroferga Fushicola PDM-1 strain that has been entrusted to the National Institute of Advanced Industrial Science and Technology. The Cerroferga fushicola PDM-1 strain has bacteriological properties described below. Porphyran-specific degrading enzymes produced by these bacteria can degrade porphyran gently and efficiently.

  The molecular weight measured by SDS-PAGE of the porphyran-specific degradation enzyme of the present invention is about 42 kDa.

  The optimum pH of the porphyran-specific degrading enzyme of the present invention is usually about pH 6 to 8, preferably about pH 7 to 8, and more preferably about pH 7.5. The pH stable region is usually about pH 6.5 to 8, preferably about pH 7.0 to 8.0, and more preferably about pH 7.5.

  The optimum temperature of the porphyran-specific degradation enzyme of the present invention is usually about 40 ° C. or lower, preferably about 35 ° C. or lower, more preferably about 30 ° C. The temperature stable region is usually about 35 ° C. or lower, preferably about 32 ° C. or lower, more preferably 30 ° C. or lower.

  The optimum temperature will be further described. The porphyran-specific degrading enzyme of the present invention can exhibit activity even at a low temperature as long as the solution is not frozen. For example, in the refrigerated state (about 4 ° C.), about 80% Activity can be retained. As described above, since the porphyran-specific degrading enzyme of the present invention has a wide temperature range that retains high activity, the temperature appropriately selected according to the type, use, desired properties, etc. of the temperature control device provided in the facility Can be accompanied by decomposition below.

  For example, when the porphyran degradation product of the present invention is used in the field of foods, cosmetics, hair care products, pharmaceuticals, etc., the enzyme of the present invention has a temperature that can suppress the growth of microorganisms in the enzyme reaction solution, for example, about It is desirable to be subjected to decomposition at a low temperature of 30 ° C. or lower, preferably about 20 ° C. or lower, more preferably about 10 ° C. or lower, more preferably about 4 ° C. or lower. Moreover, when it decomposes | disassembles for a long time at the temperature of about 50-60 degreeC, the oxidation deterioration odor by a heating odor, the coexisting acid, a metal ion, etc. may generate | occur | produce. Therefore, when the porphyran degradation product of the present invention is used in the fields of foods, cosmetics, hair care products, pharmaceuticals, etc., the enzyme of the present invention is less likely to generate an odor caused by heating or oxidative degradation due to coexisting acids or metal ions. It is desirable to undergo decomposition at a temperature, for example about 30 ° C. or less, preferably about 20 ° C. or less, more preferably about 10 ° C. or less, more preferably about 4 ° C. or less. From the viewpoint of energy saving, it is also desirable to be subjected to decomposition at room temperature, for example, about 4 to 35 ° C., often about 15 to 25 ° C.

The porphyran-specific decomposing enzyme of the present invention can be produced by a conventional method. For example, a bacterium producing porphyran-specific decomposing enzyme is purely cultured in a liquid medium containing seawater, and after a predetermined period, the bacterial cells are obtained. It can be produced by removing by centrifugation and roughly separating by molecular weight fractionation with an ultrafiltration membrane. If further purification is required, for example, after salting out with ammonium sulfate, purify the enzyme by gel filtration chromatography using molecular weight fractionation or ion exchange chromatography utilizing ionic interaction characteristics. Can do. It is not limited to these methods.
[II] Bacteria producing porphyran-specific degrading enzymes The present invention further provides bacteria producing the porphyran-specific decomposing enzymes of the present invention. The bacterium of the present invention is not particularly limited as long as it is a bacterium belonging to the genus Cellulophaga, but is preferably Cellulophaga fucicola. As Cellulofarga fushikora that produces the porphyran-specific degrading enzyme of the present invention, the present invention specifically includes the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1-1-1 East Tsukuba, Tsukuba City, Ibaraki Pref.) 6) in the form of “Ceruloferga Fushikora PDM-1”, deposited on August 30, 2005, deposited on September 1, 2005, and confirmed to survive on September 29, -20648 strains are provided.

  In addition to having porphyran resolution, the cellulofarga fushicola PDM-1 strain further has the following bacteriological properties.

(A) Morphological properties (1) Shape and size of cells: rod-shaped, 0.6-0.7 × 2.0-5.0 μm
(2) Existence of motility: None (3) Presence of spore: None
(B) Culture characteristics (1) Colony properties (cultured on MB2216 agar medium for 48 hours):
Diameter: 1.0 mm
Color: Yellow Shape: Circular Protruding state: Lens shape Periphery: Root shape Surface shape, etc .: Smooth Transparency: Opaque Consistency: Butter shape (2) Growth temperature test:
20 ℃: can grow
30 ℃: Can grow
35 ° C: Unable to grow (3) Growth using seawater: Good
(C) Physiological properties (1) Gram staining: Negative (2) Catalase reaction: Positive (3) Oxidase reaction: Positive (4) Acid and gas production from glucose: Negative (5) Nitrate reduction: Negative (6) Indole production: negative (7) glucose acidification: negative (8) arginine dihydrolase: negative (9) urease: negative (10) esculin degradation: positive (11) gelatin degradation: positive (12) β-galactosidase activity: positive ( 13) Cytochrome oxidase: positive (14) starch hydrolysis: negative (15) agar hydrolysis: positive (16) glucose utilization: negative (17) L-arabinose utilization: negative (18) D-mannose utilization: negative (19) D-mannitol utilization: negative (20) N-acetyl-D-glucosamine utilization: negative (21) maltose utilization: negative (22) potassium gluconate N-capric acid utilization: negative (24) adipic acid utilization: negative (25) dl-malic acid utilization: negative (26) sodium citrate utilization: negative (27) acetic acid Phenyl assimilation: Negative (28) Attitude toward oxygen: Aerobic
(D) Chemical taxonomic properties (1) DNA GC content (high performance liquid chromatography): 32.4 mol%
In addition, the cellulofarga fushicola PDM-1 strain has the 16S rDNA base sequence described in SEQ ID NO: 1.

  The strains producing the porphyran-specific degrading enzyme of the present invention include natural and artificial mutants of the strains as well as the Cerroferga fushicola PDM-1 strain.

The strain producing the porphyran-specific degrading enzyme of the present invention includes closely related species of Cellulofarga fusicola PDM-1. The close relative of Cellulofarga fushikora PDM-1 has porphyran resolution and has a 16S rDNA base sequence of SEQ ID NO: 1, for example, 90% or more, preferably 93% or more, more preferably 95% or more, more Preferably 97% or more, particularly preferably 99% or more identity, and among the above bacteriological properties, for example, 5 or less, preferably 3 or less, more preferably 2 or less, particularly preferably 1 Are included.
[III] Method for Producing Porphyran Degradation Product The present invention provides a method for producing a porphyran degradation product comprising the step of (I) mixing the porphyran-specific degradation enzyme of the present invention and porphyran to decompose porphyran.

In addition to the step (I), the present invention also includes (II) a method for producing a porphyran degradation product, further comprising a step of decomposing porphyran by mixing β-agarase and porphyran (here, the step (I ) And step (II) may be performed in any order).
That is, the method for producing a porphyran degradation product of the present invention basically includes any of the following steps (1) to (4):
(1) a step of degrading porphyran with a porphyran-specific degrading enzyme;
(2) A step of further decomposing the obtained product with β-agarase after degrading porphyran with a porphyran-specific degrading enzyme,
(3) Decomposing porphyran with β-agarase and then further decomposing the resulting product with porphyran-specific decomposing enzyme, or
(4) A step of simultaneously degrading porphyran with a porphyran-specific degrading enzyme and β-agarase.

  The origin of porphyran as a raw material is not particularly limited. However, since porphyran is contained in a large amount in the water-soluble fraction of Amanori seaweeds, it is preferably derived from the water-soluble fraction of Amanori seaweeds.

  The seaweed genus seaweed is an algae belonging to the primordial red algae class, Bovidaceae, Bovidae family, Amano genus (scientific name: Porphyra). , Ononori, Danshakusai etc. are included in the seaweed genus seaweed. In particular, it is preferable to be derived from the water-soluble fraction of Susavinori because it contains a large amount of porphyran and has good production efficiency and can be stably supplied.

  In addition, it is classified into several grades (standards) according to differences in quality, color, flavor, form, etc., and the types of proteins, amino acids, flavor components, etc. There are differences in content. Porphyran is contained in almost the same amount in all grades of paste, but there is a slight difference in the sugar composition. Porphyran obtained from a high grade paste (higher paste) tends to have a higher ratio of galactose-6-sulfate than a porphyran derived from a low grade paste (lower paste). From the viewpoint of effective utilization of waste paste, porphyran derived from waste paste, which is usually lower paste, is preferable.

  Porphyran can be purified by a conventional method. For example, after extracting an aqueous solution fraction from a seaweed seaweed using a batch type extractor, an alcohol precipitation purification method, a precipitation purification method using a quaternary ammonium salt, an ion It can be purified by a purification method using exchange chromatography, a membrane separation method or the like. It is not limited to this method.

  Porphyran may be a crude sample containing contaminants (for example, a water-soluble fraction extracted from the seaweed genus seaweed), or a single product that is completely or almost completely purified. It is desirable to use a purified product of about 50% or more, preferably about 70% or more, more preferably about 85% or more, more preferably about 95% or more. Further, as described above, the porphyran may be a porphyran that has been treated with β-agarase and purified as necessary.

  (I) In the step of mixing the porphyran-specific decomposing enzyme of the present invention and the porphyran to decompose porphyran, the decomposition is, for example, about pH 6.0 to 8.0, preferably about pH 7.0 to 8.0, More preferably, the reaction is performed at a pH of about 7.5, for example, about 40 ° C. or less, preferably about 20 to 35 ° C., more preferably about 20 to 30 ° C. Under such conditions, the activity of the enzyme is high and the enzyme can exist stably, so that porphyran can be efficiently decomposed. However, the porphyran-specific degrading enzyme of the present invention exhibits sufficient hydrolytic activity and can exist stably if the solution does not freeze and is at a temperature of 40 ° C. or lower. Decomposition can be suitably performed at 40 ° C, preferably at a temperature at which the solution does not freeze to about 35 ° C, more preferably at a temperature at which the solution does not freeze to about 30 ° C. The reaction time is usually 0.5 to 72 hours, but may be longer or shorter than this range as long as the desired effect is obtained. During decomposition, the solution may be stirred or shaken as necessary.

  The concentration of porphyran subjected to decomposition is not particularly limited, but is usually about 0.5 to 100 mg / ml, preferably about 0.5 to 75 mg / ml, more preferably about 0.5 to 50 mg / ml. Usually, since the enzyme reaction is more likely to proceed at a higher concentration, the reaction time can be shortened, and further, the energy required for temperature control and the like can be reduced. Porphyran is usually subjected to a decomposition reaction in an aqueous solution.

  The concentration of the porphyran-specific degrading enzyme to be subjected to degradation is not particularly limited, but is usually about 0.1 to 50 U / ml, preferably about 1 to 30 U / ml, more preferably about 5 to 15 U / ml. Here, 1 U is the amount (μg) of reducing sugar increased in an enzyme reaction per unit time (hour) using 1 μg of enzyme at 30 ° C.

  A person skilled in the art can obtain the desired porphyran decomposition product by appropriately adjusting the decomposition conditions (temperature, time, composition, etc.).

  Furthermore, if necessary, the enzyme may be deactivated by heat, or the enzyme may be removed by ultrafiltration to stop the decomposition reaction.

(II) In the step of mixing β-agarase and porphyran and decomposing porphyran, the decomposition can be performed by a conventional method, for example, temperature 20 to 40 ° C., pH 6.0 to 8.0, 12 to 96. The incubation can be performed for about an hour. As β-agarase, commercially available β-agarase can be preferably used. For example, β-agarase manufactured by Sigma or Yakult Pharmaceutical Co., Ltd. can be preferably used. Degradation conditions (temperature, pH, time, composition, etc.) may follow the instructions attached to the commercially available β-agarase. Those skilled in the art can appropriately change these decomposition conditions.
[IV] Porphyran degradation product The present invention provides a porphyran degradation product obtained by the above production method.

  The degradation product of the porphyran of the present invention is a product itself obtained by the above production method, or a product obtained by purification, concentration, dilution, drying (powdering), freeze-drying, or the like as necessary.

  The molecular weight of the porphyran decomposition product is not particularly limited as long as it is smaller than the porphyran before decomposition. The molecular weight of the porphyran degradation product varies depending on the use, but for example, the molecular weight is about 45,000 or less, preferably about 30,000 or less, more preferably about 15,000 or less, and particularly preferably about 5,000 or less. . In particular, a porphyran degradation product having a molecular weight of about 15,000 or less is desirable because it is easily absorbed by the body and can sufficiently exhibit the action of the porphyran degradation product in the body.

  In this book, unless otherwise indicated, the molecular weight of porphyran or a porphyran degradation product is the value measured by the high performance liquid chromatography method (HPLC method) or the high performance liquid chromatography mass spectrometry (HPLC-MS method).

  In this book, "porphyran degradation product" means by hydrolyzing porphyran (or porphyran, which is the degradation product in the case of degradation with other enzymes such as β-agarase in advance) by the porphyran-specific degradation enzyme of the present invention. It means the product obtained. The porphyran degradation product hydrolyzed by a plurality of enzymes including the porphyran-specific degradation enzyme of the present invention is also included in the porphyran degradation product of the present invention.

  According to the present invention, there are provided a novel porphyran-specific degrading enzyme, a novel method for producing a porphyran degradation product using the enzyme, a novel porphyran degradation product obtained by the method, and a bacterium that produces a porphyran-specific degrading enzyme.

  According to the present invention, porphyran can be decomposed gently, so that the control of the decomposition is easy. Further, unlike a method using an acid or an alkali, random decomposition does not occur and structural change does not occur, so that a porphyran decomposition product having desired properties and actions can be stably produced.

  Moreover, the porphyran-specific decomposing enzyme of the present invention maintains high activity even at low temperatures and can be efficiently hydrolyzed. By hydrolyzing porphyran at a low temperature, it is possible to obtain a high-quality porphyran degradation product without generating a heating odor or an oxidative degradation odor due to coexisting acids or metal ions. In addition, by hydrolyzing porphyran at a low temperature, it is possible to suppress the growth of microorganisms and to produce a porphyran degradation product hygienically. Therefore, the porphyran degradation product produced at a low temperature according to the present invention is suitable as a raw material for foods, pharmaceuticals, hair care products, cosmetics and the like.

  In addition, since the enzyme of the present invention can be decomposed at room temperature, a special temperature control device or temperature control is not necessarily required, and labor-saving, resource-saving, energy-saving in manufacturing, and thus manufacturing cost. Leads to reduction.

  Since the porphyran degradation product of the present invention has a molecular weight smaller than that of the porphyran before degradation, the absorption efficiency into the body is good, and the action of the porphyran degradation product can be sufficiently exerted when absorbed into the body.

  Examples of the present invention will be described below. This example is for explaining the present invention more easily and does not limit the present invention.

<Rough separation of microorganisms from the surface of paste>
An appropriate amount of laver algae was washed twice with sterile seawater (collected in the Genkai Sea, Saga Prefecture) and placed in a liquid medium mainly composed of agar (a medium in which 1 g of agar and 1 g of potassium nitrate were dissolved in 1 liter of seawater). Pre-cultured at 7 ° C. for 7 days. The culture medium is a flat medium mainly composed of agar (a medium in which 25 g of agar and 1 g of potassium nitrate are dissolved in 1 L of seawater) and a Zobell flat medium (25 g of agar, 1 g of polypeptone, 1 g of yeast extract, and iron phosphate (III) in 1 L of seawater. The medium was applied to a medium in which 0.1 g was dissolved, and main culture was performed at 22 ° C. for 7 days. A group of microorganisms whose surfaces were softened or liquefied was roughly separated.
<Screening for microorganisms having hydrolytic activity>
Next, the roughly separated microorganism group was inoculated into a liquid medium mainly composed of porphyran (POR) (medium in which 1 g of POR and 1 g of potassium nitrate were dissolved in 1 L of seawater) and cultured at 22 ° C. for 7 days. The culture solution was separated into cells and culture supernatant by centrifugation. The sugar composition contained in the culture supernatant was analyzed by thin layer chromatography, and a group of microorganisms having a degrading activity was selected. Among these microorganism groups, six kinds of groups that showed strong POR degradation reaction were further subjected to pure culture using a Zobel flat medium to obtain 25 kinds of purified microorganisms. These microorganisms were inoculated into a liquid medium containing POR as a main component as described above, cultured at 24 ° C. for 7 days, and then the cells and the culture supernatant were separated by centrifugation. The molecular weight distribution of the sugar present in the culture supernatant is measured by ultrafiltration (exclusion molecular weight: 5,000 or 30,000) of the culture supernatant by centrifugation, and about 65% of the molecular weight is 5,000 or less. A strain having about 10% degraded to a molecular weight of 5,000 to 30,000 was selected and used for further experiments.

<Identification of strain>
For isolated strains exhibiting hydrolytic activity, 16S rDNA molecular phylogenetic analysis, physiological and biochemical property tests, bacterial fatty acid composition (CFA) analysis, quinone analysis, DNA base composition measurement (GC content measurement) and DNA-DNA Taxonomic position was identified by hybridization test. These tests were consigned to NCIMB Japan providing a microbiological test contract service with ID number: SIID3681.

  As a result of 16S rDNA molecular phylogenetic analysis, the 16S rDNA base sequence of this strain shows a high homology of 99.8% with respect to the 16S rDNA base sequence of Cellulofarga fushicola NN015860 strain (reference strain) in the international base sequence database. It was. In addition, most of the sequences searched for at the top were occupied by 16S rDNA of bacteria derived from the genus Cerrophaga.

  As a result of the physiological and biochemical property test, this strain was found to be a cellulophaga bacterium in terms of positive catalase reaction with non-motile Gram-negative bacilli, oxidation of glucose, and yellow colonies on MB2216 agar plates. Consistent with the properties of Fushikora. However, the positive point of the oxidase reaction did not coincide with the properties of Cellulofaga fushicola. In addition, it hydrolyzes esculin and gelatin, has positive β-galactosidase activity, does not grow under anaerobic conditions, does not grow at 35 ° C., does not produce Flexirubin type dye, and hydrolyzes agar It was consistent with the properties of Cellulofaga fushikora. However, it did not agree with the properties of Cellulofaga fushicola in that it did not hydrolyze starch.

  As a result of cell fatty acid composition (CFA) analysis, C15: 0 ISO 3OH (17.68%), C15: 0 ISO (16.15%), C17: 0 ISO 3OH (12.03%) are recognized as main fatty acids. It was.

  The quinone analysis revealed that the main quinone composition of this strain was menaquinone MK-6. Incidentally, the main quinone of Cellulophaga pacifica, another species of the genus Cellulophaga, is also MK-6.

  As a result of measuring the DNA base composition, the GC content of this strain measured by high performance liquid chromatography was 32.4%. By the way, it is reported that the cellulophaga fusicola has a GC content of 32.4%, and that of another species of the cellulophaga genus Cellulophaga lytica is 32%.

  From the results of the three DNA-DNA hybridization tests, the average value of DNA-DNA homology between the present strain and the Cellulofarga fushicola reference strain was 72%. At present, bacterial species are defined as homologous strains showing DNA-DNA homology of 70% or more (Wayne L. G et al., Int. J. Syst. Bacteriol., 1987, 37, 463- 464).

  From the above results, the present inventors thought that the present strain was classified as a bacterium belonging to the genus Cerrophaga, and more likely to belong to Cerulophaga fushicola or its related species.

This strain is displayed at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (1-1-1 Tsukuba Center, Tsukuba City, Ibaraki Prefecture) on the label of “Cellulofaga Fushikora PDM-1” August 30, 2005 Deposited by date, it was deposited on September 1 of the same year (Accession Number FERM P-20648), and its survival was confirmed on September 29 of the same year.
<Separation and purification of POR-specific degradation enzyme>
Cellulofaga fushikora PDM-1 was inoculated into a liquid medium containing POR as a main component (medium in which 1 g of POR and 1 g of potassium nitrate were dissolved in 1 L of seawater) and precultured at 24 ° C. for 14 days. About 10 L of a culture medium having a Zobell medium composition in which POR is dissolved instead of agar (1 g of POR, 5 g of polypeptone, 1 g of yeast extract and 0.1 g of iron (III) phosphate dissolved in 1 L of seawater) The main culture was performed at 24 ° C. for 21 days. After culturing, the cells and the culture solution were separated by centrifugation. The obtained culture broth was subjected to ultrafiltration in an ice water bath, desalted and concentrated. In order to salt out the protein contained in the obtained concentrated liquid, it was gradually added so as to be saturated with ammonium sulfate 90%, and after sufficiently dissolving, it was left at 4 ° C. overnight. The precipitate from salting out was collected by centrifugation and redissolved in 50 mM phosphate buffer containing 0.2 M sodium chloride to obtain a crude protein solution. The crude protein solution was subjected to gel filtration chromatography (Sephaacryl S-200 High Resolution, 75 cm × 2.5 cm id, resin bed volume 340 mL, manufactured by GE Healthcare Biosciences), and 50 mM phosphorus containing 0.2 M sodium chloride. The protein was roughly separated with an acid buffer. Each eluted fraction was subjected to fractionation of the eluted protein with a fraction collector. An arbitrary fraction was evaluated for degradability by an enzyme reaction using POR as a substrate, and a fraction in which degradability was observed was collected. The collected fractions were desalted and concentrated by centrifugal ultrafiltration to obtain a concentrated solution. This concentrated solution is subjected to ion exchange chromatography (TOYOPEARL DEAE-650S, 10 cm × 1.5 cm id, resin bed volume 10 mL, manufactured by Tosoh Corporation), and sodium chloride is added so as to be 0, 100, 150, 200, 250 mM. Proteins were separated by a stepwise gradient with a dissolved 50 mM phosphate buffer. Similar to gel filtration chromatography, the eluted fraction was fractionated with a fraction collector. When an arbitrary fraction was evaluated for degradability by an enzyme reaction using POR as a substrate, strong activity against POR was confirmed in the 0 mM elution fraction. Fractions with recognized activity were collected, and desalted and concentrated by centrifugal ultrafiltration. This concentrated solution is further subjected to HPLC using an ion exchange column (SHODEX QA-825, 75 mm × 8 mm id manufactured by Showa Denko KK), and the sodium chloride concentration is linearly changed (0 to 200 mM, linear gradient). The protein was eluted with 20 mM Tris / HCl buffer. The eluted fraction was fractionated by a fraction collector.

  Subsequently, each fraction was evaluated for POR degradability by an enzyme reaction using POR as a substrate, and a fraction containing a target enzyme exhibiting degradability was obtained.

Degradability was calculated based on the content of reducing sugar using 2-Cyanoacetamide. That is, 1 μL of each fraction was added to a 0.1% POR aqueous solution (100 μL), and a decomposition reaction was performed at 30 ° C. for a fixed time. After a predetermined time, 100 μL of 1% 2-Cyanoacetamide aqueous solution was added to 100 μL of the reaction solution and mixed well, and then 200 μL of 0.5 M boric acid / sodium hydroxide / potassium chloride buffer (pH 9.0) was added, and The mixture was heated and reacted at 100 ° C. for 5 minutes. After the reaction, the mixture was cooled and the absorbance at 274 nm was measured. The content of terminal reducing sugar was calculated from a calibration curve prepared using galactose as a standard substance. The method for measuring reducing sugar is described in Anal. Biochem. 203 (2), 335-339 (1992) was appropriately modified. In addition, 1U of the porphyran-specific decomposing enzyme of the present invention was defined as the amount (μg) of reducing sugar increased in an enzyme reaction per unit time (hour) using 1 μg of enzyme at 30 ° C.
<Molecular weight measurement>
A fraction containing the target enzyme exhibiting POR hydrolysis ability was pretreated using Pro260 kit manufactured by Bio-Rad Laboratories according to the attached instructions. Next, SDS-PAGE was performed on the pretreated sample using Bio-Rad Laboratories fully automated electrophoresis system Experion according to the attached instructions.

The result of SDS-PAGE is shown in FIG. From FIG. 1, it is clear that the molecular weight of the porphyran-specific degrading enzyme measured by SDS-PAGE is about 42 kDa.
<Substrate specificity>
Agarose, agar, sodium alginate, chondroitin sulfate, sulfated dextran (500 kDa), ι-carrageenan, κ-carrageenan, and λ-carrageenan were used as substrates, and the hydrolysis activity of the enzyme of the present invention for each substrate was measured. The activity was measured by the same method as POR. The content measurement of the terminal reducing sugar is as described above. The unit (U) is calculated by the same method as POR. When U is 0 or less (that is, when hydrolysis activity is not shown), “−” is obtained, and when U is greater than 0 (ie, hydrolysis activity is determined). (When indicated) was evaluated as “+”.

  The results of substrate specificity are shown in Table 1 below.

<Measurement method of optimum pH>
1 μL of the enzyme solution (enzyme concentration at the time of addition: 9.64 μg / mL) was added to 100 μL of the substrate solution (0.1% POR solution), and reacted at 30 ° C. for 24 hours. The pH of the substrate solution was adjusted so that the buffer solution shown below was added to a 0.5% POR aqueous solution immediately before use so that the final substrate concentration was 0.1%. It should be noted that citrate buffer in the range of pH 3.0 to 6.0, phosphate buffer in the range of pH 6.0 to 8.0, and boric acid / potassium chloride / hydroxylated in the range of pH 8.0 to 10.0. Sodium buffer was used.

  The reaction solution was measured by a reducing sugar determination method using 2-Cyanoacetamide. The reducing sugar quantification method is as described above.

The measurement result of the optimum pH is shown in FIG.
<Measurement method of pH stability>
80 μL of the aforementioned buffer solution was added to 1 μL of the enzyme solution (enzyme concentration at the time of addition: 9.64 μg / mL) and held at 30 ° C. for 6 hours or 24 hours.

  20 μL of 0.5% POR aqueous solution was added to the above solution to carry out an enzyme reaction (30 ° C., 22 hours). After the reaction, it was measured by a reducing sugar quantitative method using 2-Cyanoacetamide. The reducing sugar quantification method is as described above.

The measurement result of pH stability is shown in FIG.
<Measurement method of optimum temperature>
1 μL of the enzyme solution (enzyme concentration at the time of addition: 9.64 μg / mL) was added to 100 μL of the substrate solution (0.1% POR solution (using phosphate buffer of pH 7.5)), and the mixture was reacted at a predetermined temperature for 21 hours. . The reaction temperature was set to 20, 25, 30, 35, 40, 45, 50 and 60 ° C.

  The reaction solution was measured by a reducing sugar determination method using 2-Cyanoacetamide. The reducing sugar quantification method is as described above.

The measurement result of the optimum temperature is shown in FIG.
<Temperature stability measurement method>
80 μL of pH 7.5 phosphate buffer solution was added to 1 μL of enzyme solution (enzyme concentration at the time of addition: 9.64 μg / mL), and the mixture was held at a predetermined temperature for 18 hours. The holding temperature was set to 20, 25, 30, 35, 40, 45, 50 and 60 ° C.

  After a predetermined time, 20 μL of 0.5% POR aqueous solution was added to carry out an enzyme reaction (30 ° C., 26 hours). The reaction solution was measured by a reducing sugar determination method using 2-Cyanoacetamide. The reducing sugar quantification method is as described above.

The measurement results of temperature stability are shown in FIG.
<Measurement of Hydrolytic Activity for Porphyran Treated with β-Agarase>
3 g of POR was dissolved in 50 mM acetate buffer (pH 6.0), 1000 U of commercially available β-agarase (derived from Pseudomonas atlantica) was added, and the mixture was decomposed at 40 ° C. for 72 hours. After heating in hot water for 15 minutes to stop the enzyme reaction, insolubles were removed. The obtained solution was subjected to ultrafiltration with an apparatus having an ultrafiltration membrane with an exclusion limit of 30 kDa, and the retained fraction was pulverized by freeze-drying. The weight of the 30 kDa retentate at that time was 1.2 g. This β-agarase-treated product was re-dissolved ultrapure to obtain a substrate solution. 1 μl of the enzyme solution was added to 100 μl of the substrate solution (0.1% aqueous solution) (enzyme concentration at the time of addition was 38.6 μg / ml), and the mixture was reacted at 30 ° C. for 15 hours. The reaction solution was measured by a reducing sugar quantitative method using 2-Cyanoacetamide. As a result, an increase in reducing sugar of 9.19 μg / ml (galactose conversion value) was observed in the reaction solution.

FIG. 1 shows the result of SDS-PAGE. It is clear that the porphyran-specific degradation enzyme of the present invention has a molecular weight of about 42 kDa. FIG. 2 shows the results of measuring the residual activity of the enzyme of the present invention at 30 ° C. for each pH. From this result, the enzyme of the present invention showed the highest residual activity at a pH of about 7.5. FIG. 3 shows that the enzyme of the present invention is kept in a solution of each pH at 30 ° C. for 6 hours or 24 hours, and then this enzyme is mixed with porphyran and reacted at 30 ° C. for 22 hours. It is the result of measuring the residual activity of an enzyme. From this result, the enzyme of the present invention showed the highest residual activity at pH of about 7.5 when held for 6 hours, and the highest residual activity at about pH of 7 when held for 21 hours. FIG. 4 shows the results of measuring the residual activity of the enzyme of the present invention at pH 7.5 for each temperature. From this result, the enzyme of the present invention showed the highest residual activity at about 30 ° C. FIG. 5 shows that the enzyme of the present invention was held in a phosphate buffer solution (pH 7.5) at each temperature for 18 hours, then mixed with porphyran and reacted at 30 ° C. for 26 hours. It is the result of measuring the residual activity of an enzyme. From this result, the enzyme of the present invention showed a residual activity at a temperature of about 40 ° C. or lower, and a particularly high residual activity at a temperature of about 30 ° C. or lower.

Claims (10)

Porphyran-specific degrading enzymes with the following properties:
(A) having specific hydrolysis activity for porphyran,
(B) produced by Cellulophaga fucicola PDM-1 strain (Accession No. FERM P-20648, National Institute of Advanced Industrial Science and Technology Patent Organism Depositary)
(C) The molecular weight measured by SDS-PAGE is about 42 kDa,
(D) the optimum pH is 7.5,
(E) The optimum temperature is 30 ° C. (F) substantially free of hydrolysis activity on agarose,
The porphyran-specific degrading enzyme according to claim 1. (G) The porphyran-specific decomposing enzyme according to claim 1 or 2, which has a hydrolytic activity for β-agarase-treated porphyran. The porphyran-specific decomposing enzyme according to any one of claims 1 to 3, wherein the porphyran is a porphyran produced in a seaweed of the genus Amanori. (I) a step of mixing the porphyran-specific decomposing enzyme according to any one of claims 1 to 4 and porphyran, and decomposing porphyran;
A process for producing a porphyran degradation product. (II) a step of mixing β-agarase and porphyran to decompose porphyran,
The process for producing a porphyran degradation product according to claim 5 , wherein step (I) and step (II) may be performed in any order. The process according to claim 5 or 6 , wherein the decomposition in step (I) is carried out under pH 6.0 to 8.0. The method according to any one of claims 5 to 7 , wherein the decomposition in the step (I) is carried out at a temperature of 4 to 40 ° C. The method according to any one of claims 5 to 8 , wherein the porphyran is a porphyran contained in a water-soluble fraction of the seaweed seaweed. Cellulofaga fushikora PDM-1 strain (accession number FERM P-20648, National Institute of Advanced Industrial Science and Technology patent biological deposit center) having the ability to produce a porphyran-specific degrading enzyme.

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