JPH0797987B2 - Novel β-agarase and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel β-agarase derived from a microorganism and a method for producing the same. More details, Pseudomonas ( Ps
The present invention relates to a novel β-agarase obtained by culturing a bacterium belonging to the genus Eudomonas ) and a method for producing the same.

[Conventional technology]

Agarose is a seaweed (red algae) such as Agarose japonica
It is a polysaccharide obtained from and is the main component of agar that has been used for food since ancient times. As shown in FIG. 1, its structure is a polysaccharide in which D-galactose and 3,6-anhydro-L-galactose are alternately β-1,4 linked and α-1,3 linked. The agar also contains agaropectin, which is obtained by partially esterifying agarose with sulfuric acid or pyruvic acid.

Although partial hydrolysis of agarose gives oligosaccharides, it is known that different oligosaccharides are produced depending on the hydrolysis method. To summarize the findings so far, the hydrolysis pattern of agarose is as shown in FIG. That is, when weakly hydrolyzed with a dilute acid, the α-1,3 bond is relatively selectively decomposed and the disaccharide hydrolyzed at each position of agarobiose (acid (1), acid (2), and acid (3)). ) And other agarooligosaccharides are obtained (C.Araki, Procee
dings of 4th Int. Congress of Biochemistry 1,15〜30
(1959) Pergamon Press Ltd.).

Separately, α-agarase which decomposes α-1,3 bond to mainly produce agarotetraose (which decomposes α-agarase (1) and (2)) is also known (KSYoung et al., Ca.
rbohydrate Research 66 207-212 (1978)).

On the other hand, the β-1,4 bond is selectively decomposed only by the enzyme β-agarase, and mainly neoagarotetraose (β-agarase (1) and (2), neo-agarohexaose (β-agarase (1 ) And (3)) (LM
Morrice et al., European Journal of Biochemistry 137 , 149
-154 (1983)).

In general, β-agarase cannot hydrolyze oligosaccharides of neo-agarotetraose or less, and it is known that these oligosaccharides are degraded by another enzyme (HJ van der Meul
en et al., Antonie van Leeuwenhoek 42 , 81-94 (1976)).

The main use of agar is for foods, it is widely used as a gelling agent and a thickening agent, and there are other reagents that use gel forming ability,
It is used as a raw material for media, a carrier for chromatography, a carrier for electrophoresis, as well as dental impression agents, aroma and deodorants. On the other hand, although the use of oligosaccharides obtained by enzymatic decomposition has not been developed, recently, starch aging prevention action, static tooth action, and indigestion have been revealed (Japanese Patent Laid-Open Nos. 62-210955 and 62-210).
965, 62-210974), new application development is expected.

[Problems to be Solved by the Invention]

In order to efficiently produce agar oligosaccharides, the enzymatic partial hydrolysis method is better than the acid method in terms of yield of oligosaccharides, ease of purification such as decolorization and desalting, workability, and simplicity of production equipment. Is clearly superior in terms of. However, most of the agarases known to date, agarase,
It is prepared from sea-derived microorganisms such as seawater, seaweed, and seaside soil. It is generally known that enzymes obtained from marine microorganisms have low thermostability, and agarase, which also has a high optimum temperature, is 40 to 45 ° C, and most are 40 ° C or lower. On the other hand, agar dissolves in water at a high temperature (80 ° C or higher), but if the temperature of the agar solution is lowered, it will gel at 50-40 ° C, making it very difficult to be decomposed by enzymes. In order to prevent gelation of agar, the concentration of agar, which is the substrate, should be 1
% Or less, and there is a problem that the productivity of oligosaccharides is significantly reduced. Therefore, a thermostable enzyme is needed to solve these problems.

[Means for Solving the Problems]

In order to solve the above problems, the present inventors have earnestly studied to obtain agarase with improved thermostability, as a result,
The inventors have found that a novel β-agarase produced by a microorganism belonging to the genus Pseudomonas has more excellent thermostability than ever before, and completed the present invention.

That is, the present invention makes it possible to industrially easily carry out the production of agar oligosaccharides by supplying a novel β-agarase having excellent heat resistance derived from microorganisms.

The present invention firstly relates to a novel β-agarase having the following physicochemical properties.

Action The β-1,4 bond of agarose is hydrolyzed to rapidly reduce the viscosity of the agarose solution, producing mainly neoagarotetraose and neoagarohexaose.

Substrate specificity It acts on galactan-based polysaccharides such as agarose, agaropectin, and agar having β-1,4 galactoside bond, and oligosaccharides of neo-agarooctaose or higher. It hardly acts on neo-agarohexaose, neo-agaro-tetraose, and neo-agarobiose, but it does not act on lactose.

Optimum pH and pH stability When agar is used as a substrate, the optimum pH is 7.0, 45 ° C, 30 minutes,
It is stable at pH 5-9 in the absence of substrate.

Optimum temperature and thermal stability When agar is used as the substrate, the optimum temperature is 50 ° C, pH 6.0,30
Min., Stable at 45 ° C in the absence of substrate, about 75 at 50 ° C
%, About 35% of the activity remains at 55 ° C.

Deactivation condition pH 6.0, 65 ℃, 30 minutes or more, or pH 6.0, 100 ℃, 10 minutes.

Molecular weight 360,000 (gel filtration chromatography method) Isoelectric point 4.9 (isoelectric focusing method) Effect of metal salt pH 6.0,45 ℃, Fe ⁺⁺⁺ ,
Hg ⁺⁺ , Ag ⁺⁺ , Al ⁺⁺⁺ 10% or less, Cu ⁺⁺ , Zn ⁺⁺ show residual activity of about 50-80%, Na ⁺ , K ⁺ , Mg ⁺⁺ , Co ⁺⁺ , It is stable in Mn ⁺⁺ and Pb ⁺⁺ . In addition, Ca ⁺⁺ activated EDTA (5 mM)
The residual activity becomes less than 10%.

The optimum pH of the β-agarase of the present invention (substrate: 0.5%, 0.5 ml,
Figure 2 shows the temperature stability: 45 ° C, reaction time 10 minutes, and the pH stability (45
The optimum temperature (substrate: 0.5%, 0.5 ml, pH 6, phosphate buffer, reaction time 10 minutes) is shown in Fig. 3 for the activity measurement at 45 ° C, pH 6 for 10 minutes. FIG. 4 shows the thermal stability (activity was measured at pH 6, 30 minutes, and at 45 ° C., pH 6, 10 minutes) in FIG. 5, respectively.

The β-agarase of the present invention is completely different from the conventional agarase in that it has a high molecular weight, an optimum temperature and a high thermostability,
Other properties such as isoelectric point, optimum pH, and substrate specificity are similar to agarase.

Secondly, the present invention is characterized by culturing a microorganism that belongs to the genus Pseudomonas and produces a novel β-agarase having the above-mentioned properties, and accumulating the enzyme in a culture solution for separation and collection. And a method for producing β-agarase. The novel β-agarase of the present invention is produced by using a microorganism, and as a producing microorganism thereof, a microorganism belonging to the genus Pseudomonas ( Pseudomonas ) and having an ability to produce an enzyme having the above-mentioned property may be used. Pseudomonas newly isolated from soil by the inventors
SP N-7 ( Pseudomonas sp. N-7) is mentioned. This strain has been deposited with the Institute of Microbial Science and Technology as Microindustrial Research Institute No. 9884, and its mycological properties are as follows.

(1) Morphology Cell shape and size Bacillus 0.2 to 0.4 μm × 1.0 to 2.0 μm Polymorphism None Motility Yes Flagella having one polar flagellum No sporulation No Gram stain Negative acid-free (2) Growth in each medium Condition Meat broth agar plate medium Growth is weak.

Liquid culture of broth (using artificial seawater) Normal growth, no detachment color, no surface growth, and sedimentation.

Marine agar plate medium (Difco) Growth is good and the shape is circular, but it grows by perforating agar and adhering to the surface of the hole. The cloudiness of the agar around the colony becomes transparent. The color is yellowish white.

(3) Physiological properties Nitrate reduction: Positive Indole formation: Negative Hydrogen sulfide formation: Negative Starch hydrolysis: Positive Use of inorganic nitrogen source: Use nitrate or ammonium salt as nitrogen source.

Dye formation: A yellowish white water-insoluble dye is formed.

Oxidase: Negative Catalase: Positive Growth range: Grows at a temperature of 10 to 45 ° C, optimally 28 to 37 ° C. Growing
A pH around neutral is suitable.

Attitude toward oxygen: Only grows aerobically.

OF test: acid generation from O-type sugars sugar acid formation L-arabinose + D-xylose + D-glucose + D-mannose + D-fructose-D-galactose-maltose + sucrose-lactose + trehalose + D-torbitol-D-mannitol-inositol-glycerin-starch + adonitol-neoagarotetrarose + neoagarohexaose + agarose + agar + (4) Other properties DNase production: negative gelatinolysis test : Negative arginine decomposition test: Negative Esculin degradability: Positive Saltiness test: Saltiness (Comparison in the same medium using artificial seawater and tap water) About this strain having the above-mentioned mycological properties, Bergey's Manual of Systema tic Bacteriolog
y) (1986), Marine Microbiology Research Method (Japan Society Press,
1985) and found that Pseudomonas ( Pseu
It was identified as a strain belonging to the genus domonas ). Further, when the present strain was compared in detail, it was recognized that the present strain is highly likely to be a new strain belonging to the genus Pseudomonas, and the strain was named Pseudomonas sp. N-7.

The microorganism used in the present invention is not limited to the strain of the present invention and its variants and mutants, and may be any microorganism having a novel β-agarase-producing ability.

The novel β-agarase-producing bacterium of the present invention can be cultured by a known conventional method. As a medium to be used, either a synthetic medium or a natural medium can be used as long as it contains an appropriate amount of carbon source, nitrogen source, inorganic compound and other nutrients, and culture should be performed using a liquid or solid medium. You can Specifically, since agarase is an inducible enzyme as a carbon source, agar, agarose, agaropectin, a partial hydrolyzate of agar, and red algae such as agar, which is a raw material of agar, are used alone or in combination. Can be used. It is also possible to use other carbon sources such as glucose together.

As a nitrogen source, meat extract, peptone, yeast extract, dry yeast, soybean powder, azein, casamino acid, various amino acids, corn steep liquor, fish meal,
Urea and other proteins derived from animals, plants and microorganisms, organic nitrogen sources such as their hydrolysates and inorganic nitrogen compounds such as various inorganic ammonium salts and nitrates are used. One or more species in consideration of assimilability of microorganisms. Is appropriately selected and used.

As inorganic salts, sodium, magnesium, calcium,
When one or more of phosphates such as iron, zinc, manganese, and copper, hydrochlorides, sulfates, carbonates, acetates, etc. are appropriately added, or when halophilic bacteria are used, The salt concentration using artificial seawater can be set and used in an appropriate range up to the concentration of seawater. If necessary, a defoaming agent such as vegetable oil or a surfactant may be added.

The culture is shaking culture in a liquid medium containing the medium components,
It can be carried out using an ordinary culture method such as aeration and agitation culture or continuous culture. The culture conditions may be appropriately selected depending on the type of culture medium and the culture method, and there is no particular limitation as long as the agarase-producing bacterium can grow and produce β-agarase. Usually, it is preferable to culture by aeration and stirring at the initial pH of culture of 6.5 to 8.5 and 25 to 37 ° C. The appropriate number of days for culturing is usually 1 to 2 days.

The β-agarase produced in the culture as described above can be separated and collected by the following method. Since the β-agarase of the present invention is mainly accumulated outside the cells, after the completion of the culture, the cells are removed by a method such as filtration or centrifugation to obtain a culture filtrate. Regarding the bacterial enzyme, β-agarase can be extracted by crushing the bacterial cells by a commonly used means and used.

The obtained liquid containing the enzyme can be used as it is as a liquid enzyme by concentrating it in a vacuum or using an ultrafiltration membrane, or by pulverizing it by a freeze-drying method or a spray-drying method.
As another method, a purification method usually used, for example, a method of precipitating and purifying β-agarase by salting out with ammonium sulfate, a solvent precipitation method, ion exchange chromatography, gel filtration chromatography, agarose affinity adsorption method, chromatofocusing, etc. Purification method such as electrofocusing 1
It is also possible to use a method of purifying to high purity by combining two or more species.

The method for measuring β-agarase activity in the present invention is as follows. 0.5 ml of enzyme diluted and dissolved in pH 6.0, 0.1M acetate buffer
Is kept at 45 ° C, and 0.5 ml of 0.5% agar solution (preheated to 45 ° C) is added thereto to start the reaction. Reaction is 45 ℃
The reaction is stopped for 10 minutes, and the reaction is stopped by adding a Somogy solution, and the reducing sugar is quantified by the Somogy Nelson method.
The activity is expressed as 1 unit of the amount of enzyme that produces a reducing power corresponding to 1 μm mole of galactose.

〔Example〕

 Next, the present invention will be described in more detail with reference to examples.

EXAMPLE 1 Pseudomonas sp. N-7 (Pseudomonas sp.N
-7) (Microtechnological Research Institute No. 9884) Amakusa cut product 1.5%, polypeptone 0.5%, yeast extract 0.5% suspended and dissolved in artificial seawater, adjusted to pH 6.5 and sterilized medium 40 ml (200 ml triangle) (Plasma) was inoculated with 1 platinum loop and cultured at 25 ° C. for 45 hours with shaking. After culturing, remove the bacterial cells and Amakusa residue by centrifugation,
When the enzyme activity was measured, β-agarase contained 0.19 units.
/ ml was produced.

Example 2 Judemonas SP N-7
No.) powder agar 0.7%, glucose 0.1%, sodium nitrate 0.5%, yeast extract 0.5%, magnesium sulfate 0.2%,
PH containing calcium chloride 0.2% and sodium chloride 2.0% 8.
Inoculate 1 platinum loop into 40 ml of 5 medium (200 ml Erlenmeyer flask),
After preculturing by shaking at 37 ° C for 1 day, this preculture solution was inoculated into a 3 jar fermenter containing the same medium 1.5. The cells were cultured at 37 ° C for 24 hours under the conditions of an aeration rate of 1.5 / min and a stirring rate of 400 rpm.

After the culture was completed, the bacterial cells were removed by centrifugation to remove the culture filtrate 1.4.
Got The agarase activity in the culture filtrate was 1.0 unit / ml. This culture filtrate was passed through an ultrafiltration membrane with a molecular weight cut-off of 10,000.
When concentrated to 20 cultures, 60 ml of an enzyme solution containing 21.3 units / ml was obtained.

Example 3 Pseudomonas sp. N-7
No.) was pre-cultured in a medium of pH 7.5, in which 0.5% agarose, 0.5% sodium nitrate and 0.5% yeast extract were dissolved in artificial seawater. The preculture liquid was inoculated into a 30 jar fermenter containing medium 15 having the same composition, and cultured at 30 ° C for 30 hours. During the culture, the aeration rate was set to 15 / min and stirring was carried out at 200 rpm.

After completion of the culture, the bacterial cells were removed by filtration to obtain a culture filtrate. The agarase activity in the culture filtrate was 1.1 units of 1 ml. The culture filtrate was passed through a CL-Sepharose 6B (Pharmacia) 50 ml column at a flow rate of 500 ml / hr for affinity adsorption, washed with water, and then eluted with 500 ml of a 2% neo-agarooligosaccharide solution to elute agarase. After that, DEAE Toyopearl (Tosoh Corporation) and Toyopearl HW55, which are used as usual methods
S, Purified by chromatofocusing to obtain a purified enzyme that was almost blunt (22 units / mg protein). The activity yield was about 15%.

〔The invention's effect〕

According to the present invention, a novel β-agarase can be obtained by culturing a bacterium belonging to the genus Judemonas. Since this enzyme has excellent thermostability, it can be used for industrial production of agar oligosaccharides. Agar oligosaccharides have properties such as starch aging-preventing action, bacteriostatic action, and indigestion, and are expected to be used in various fields.

[Brief description of drawings]

FIG. 1 shows the structure and hydrolysis mode of agarose. FIG. 2 shows the optimum pH of β-agarase of the present invention. FIG. 3 shows the pH stability of β-agarase of the present invention. FIG. 4 shows the optimum temperature of β-agarase of the present invention. FIG. 5 shows the thermostability of β-agarase of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hironori Kitagawa 3-5-1, Asahimachi, Machida-shi, Tokyo Inside Denka Kagaku Kogyo Co., Ltd. (72) Inventor Tetsuo Hiraga Nishinokyo Kuwabara, Nakagyo-ku, Kyoto-shi, Kyoto Prefecture Machi 1 Stock Company Shimadzu Central Research Institute

Claims (3)

[Claims] 1. A novel β-agarase having the following physicochemical properties. Action The β-1,4 bond of agarose is hydrolyzed to rapidly reduce the viscosity of the agarose solution, producing mainly neoagarotetraose and neoagarohexaose. Substrate specificity It acts on galactan-based polysaccharides such as agarose, agaropectin, and agar having β-1,4 galactoside bond, and oligosaccharides of neo-agarooctaose or higher. It hardly acts on neo-agarohexaose, neo-agaro-tetraose, and neo-agarobiose, but it does not act on lactose. Optimum pH and pH stability When agar is used as a substrate, the optimum pH is 7.0, 45 ° C, 30 minutes,
It is stable at pH 5-9 in the absence of substrate. Optimum temperature and thermal stability When agar is used as the substrate, the optimum temperature is 50 ° C, pH 6.0,30
Min., Stable at 45 ° C in the absence of substrate, about 75 at 50 ° C
%, About 35% of the activity remains at 55 ° C. Deactivation condition pH 6.0, 65 ℃, 30 minutes or more, or pH 6.0, 100 ℃, 10 minutes. Molecular weight 360,000 (gel filtration chromatography method) Isoelectric point 4.9 (isoelectric focusing method) Influence of metal salt pH 6.0,45 ℃, Fe ⁺⁺⁺ , by treatment for 3 hours at 1mM metal salt concentration
Hg ⁺⁺ , Ag ⁺ , Al ⁺⁺⁺ show 10% or less, Cu ⁺⁺ , Zn ⁺⁺ show residual activity of about 50-80%, Na ⁺ , K ⁺ , Mg ⁺⁺ , Co ⁺⁺ , Mn It is stable in ⁺⁺ and Pb ⁺⁺ , and activated by Ca ⁺⁺ , and the residual activity becomes less than 10% with EDTA (5 mM). 2. A novel β-agarase-producing bacterium having a physicochemical property according to claim 1, which belongs to the genus Pseudomonas , is cultured, and the β-agarase is collected from the culture. Method for producing β-agarase. 3. A novel β-agarase-producing bacterium belonging to the genus Pseudomonas is Pseudomonas sp. N-7 ( Pseudomonas sp.
No. 9884).