JPH01228465A - Novel beta-agarase and production thereof - Google Patents

Abstract

PURPOSE:To obtain thermostable beta-agarase capable of readily producing agar oligosaccharide industrially by cultivating a bacterium belonging to the genus Pseudomonas, having specific physical and chemical properties, capable of producing beta-agarase. CONSTITUTION:A bacterium belonging to the genus Pseudomonas, having the following physical and chemical properties, capable of producing beta-agarase is cultivated and the beta-agarase is collected from the culture mixture. (a) Action: hydrolyzing beta-1, 4 bond of agarose, rapidly reducing viscosity of agarose substance and forming mainly neoagarotetraose and neoagarohexaose. (b) Substrate specificity: acting on galactan polysaccharide such as agarose having beta-1,4 galctoside bond and on oligosaccahrides of >=neoagarooctaose, slightly acting on neoagarobexaose and not acting on lactose. Having other given physical and chemical properties of (c) optimum pH and pH stability, (d) optimum temperature and thermostability, (e) condition of deactivation, (f) molecular weight, (g) isoelectric point and (h) influence of metallic salt.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel β-agarase derived from a microorganism and a method for producing the same. For more information, see Pseudomonas (P.
The present invention relates to a novel β-agarase obtained by culturing bacteria belonging to the genus Seudomonas and a method for producing the same.

[Conventional technology]

Agarose is a multi-vM compound obtained from red algae such as Amanita spp. As shown in FIG. 1, its structure is a polysaccharide in which D-galactose and 3,6-anhydro-galactose are alternately bonded by β-1,4 bonds and α-1,3 bonds.

Agar also contains agaropectin, which is a partial ester bond of sulfuric acid or pyruvic acid to agarose.

Oligosaccharides are obtained when agarose is partially hydrolyzed, but it is known that different oligosaccharides are produced depending on the hydrolysis method. Summarizing the knowledge so far, the hydrolysis pattern of agarose is shown in Figure 1. That is, when weakly hydrolyzed with dilute acid, α-1,3 bonds are relatively selectively decomposed and agarobiose (acid (1), acid (2)
, agarooligosaccharides including 2I-hydrolyzed at each position of acid (3) are obtained (C, Araki, P
roceedings of 4thInt, Con
Gress of Biochemistry 1+
15-30 (1959) Pergaa + on Pre
ss Ltd,).

Additionally, α-agarase is also known (K, S,
Young et al., Carbohydrate Re5
66207-212 (1978))
.

On the other hand, the β-1,4 bond is selectively decomposed only by the enzyme β-agarase, mainly neoagarotetraose (β-agarase (1) and (2)), neoagarohexaose (β-agarase ( 1) and (3)) (L
, M. Morris et al., European
Journal of Biochemistry 13
7.149-154 (1983), etc.).

Generally, β-agarase cannot hydrolyze oligosaccharides smaller than neoagarotetraose, and it is known that these oligosaccharides are degraded by other enzymes (TL, J, VA).
der Meulen et al., Antonie van
Leeuwenhoek 婬, 81-94 (1976
)Such).

The main use of agar is in food and as a gelling agent.

It is widely used as a thickener, and is also used as a reagent, medium raw material, and chromatography carrier that utilizes its gel-forming ability.

It can be used as a carrier for electrophoresis, as a dental impression agent, and as a fragrance/deodorizer. On the other hand, the uses of oligosaccharides obtained by enzymatic decomposition had not yet been developed, but recently their anti-starch, bacteriostatic, and indigestible properties have been revealed (JP-A-62-210955, JP-A-62-210965). .62-
210974), and the development of new applications is expected.

[Problem to be solved by the invention]

In order to efficiently produce oligosaccharides in agar, enzymatic partial hydrolysis methods have higher yields of oligosaccharides and easier purification through decolorization and desalting than acid methods.

It is clearly superior in terms of workability, simplicity of manufacturing equipment, etc. However, most of the agarases known to date have been prepared from microorganisms derived from the sea, such as seawater, seaweed, and seaside soil. It is generally known that enzymes obtained from marine microorganisms have low heat resistance, and agarase also has a high optimum temperature of 40-40°C.
On the other hand, agar dissolves in water at high temperatures (above 80°C), but when the temperature of the agar solution is lowered, it gels at 50-40°C. In order to prevent agar from gelling, the concentration of agar as a substrate must be kept below 1%, which poses the problem of significantly lowering oligosaccharide productivity. Therefore, thermostable enzymes are becoming necessary to solve these problems.

[Means to solve the problem]

The present inventors have conducted intensive studies to obtain agarase with improved heat resistance in order to solve the above problems.
The present inventors have discovered that a novel β-agarase produced by a microorganism belonging to the genus Pseudomonas has better heat resistance than conventional ones, and have completed the present invention.

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

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

0 action It hydrolyzes the β-1,4 bonds of agarose and rapidly reduces the viscosity of the agarose solution, producing mainly neoagalotetraose and neoagarohexaose.

■Substrate specificity Agarose with β-1,4-force lactosidic bonds.

It acts on polysaccharides such as agaropectin and agar, as well as oligosaccharides greater than neoagarooctaose.

It has little effect on neoagarohexaose, neoagarotetraose, and neoagarobiose, and has no effect on lactose.

■Optimal ρ■ and pH stability When using agar as a substrate, the optimal pn is 7.0, and 45°
Stable at C930 min, p) 15-9 under conditions in the absence of substrate.

■Optimal temperature and thermal stability When using agar as a substrate, the optimal temperature is 50°C, and p1
16.0.30 minutes, stable at 45°C in the absence of substrate, approximately 75% activity remaining at 50°C, and approximately 35% activity remaining at 55°C.

■Deactivation conditions pH 6.0, 65'C, 30 minutes or more or pH 6.0°1
It is almost completely inactivated at 00°C for 110 minutes.

■Molecular weight 360.000 (gel filtration chromatography) ■Isoelectric point 4.9 (isoelectric focusing) ■Effect of metal salts Treatment at pH 6, o, 45°C, 1mM metal salt concentration for 3 hours So Fe-", Hg"", Ag", A/2"" is 1
0% or less, Cu", Zn'+ shows residual activity of about 50-80%, Na", Ni', Mg", Co".

It is stable in Mn'' and pb''.It is also activated by Ca'', and the residual activity is 10% in EDTA (5mM).
% or less.

Optimum pH of β-agarase of the present invention (substrate; 0.5%,
0.5d, temperature: 45°C, 1 reaction time 10 minutes) is shown in Figure 2, p) 1 Stability (standing at 45°C for 130 minutes, 45'C,
Activity measurement at pH 6, 10 minutes) is shown in Figure 3, and the optimum temperature (
Substrate = 0.5%, 0.5%, pH 6, IJ acid buffer,
The reaction time (10 minutes) is shown in Figure 4, and the thermal stability (p) is 16.3.
The activity was measured at 45° C., pH 6, 10 minutes after standing for 0 minutes) and is shown in FIG.

The β-agarase of the present invention has a molecular weight and an optimum temperature.

Although it is completely different from conventional agarase in that it has high heat resistance, it has other properties such as isoelectric point.

There are agarases that have similar optimal pl+ and substrate specificity.

Second, the present invention is directed to Pseudomonas (Pseudomonas).
A method for producing β-agarase, which comprises culturing a microorganism that produces a novel β-agarase belonging to the genus As) and having the above-mentioned properties, and accumulating the enzyme in the culture solution, separating and collecting the enzyme. . The novel β-agarase of the present invention is produced using a microorganism, and any microorganism that belongs to the genus Pseudomonas and has the ability to produce an enzyme having the above-mentioned properties may be used. Pseudomonas sp. N-7 (P
seudomonas sp, N-7). This strain has been deposited with the National Institute of Microbial Technology as Microbiological Research Institute No. 9884, and its mycological properties are as follows.

(1) Morphology ■Cell shape and size Bacillus 0.2-0.4 μm x 1.0-2.0 p
m ■ Pleomorphism absent ■ Motility present ■ Flagellum One polar flagellum ■ Sporulation
None ■ Durham stain negative ■ Acid-fast None 2) Growth status in each medium ■ Broth agar plate growth is weak.

■Liquid culture of meat juice (using artificial seawater) Normal growth, no color change, no surface growth, and sediment.

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

(3) Physiological properties ■ Reduction of nitrate: Positive ■ Formation of indole: Negative ■ Formation of hydrogen sulfide: Negative ■ Hydrolysis of starch: Positive ■ Use of inorganic nitrogen sources: Nitrate and ammonium salts are used as nitrogen sources.

■Pigment production: Produces a yellowish white water-insoluble pigment.

■Oxidase: Negative ■Catalase: Positive ■Growth range: Grows at a temperature of 10 to 45°C, 28 to
37°C is optimal.

A pH around neutrality is suitable for growth. There is.

[Phase] Attitude towards oxygen 2Grows only aerobically.

■0-F test: Type 0 ■Generation of acid from sugar I! Acid-generated L-arabinose + D-xylose + D-glucose + D-mannose + D-fructose - D-galactose Maltose Mosucrose - Lactose
Tentrehalose + D-Sorbitol - D-Mannitol - Inositol - Glycerin - Starch + Adonitol - Neoagalotetralose Neoagarohexaose + Agarose Shiagar + (4) Other properties■DNase production: Negative■ Gelatin decomposition test: Negative ■Arginine decomposition test: Negative ■Degradability of Aesculin: Positive ■Halophilic test: Halophilic (compared with the same medium using artificial seawater and tap water) A book with the above mycological properties For bacterial strains, see Bergey's Manual or Syst.
Stematic Bacteriology) (19
1986), Marine Microbial Research Methods (Gakkai Publishing Center, 1)
As a result of the search based on Pseudomonas (P. 985),
The strain was identified as belonging to the genus Seudomonas. Furthermore, when comparing this strain in detail, it was recognized that this strain is highly likely to be a new strain belonging to the genus Pseudomonas, and it was found to be Pseudomonas sp. N-7 (Pseudo+wonas s.
p, N-7).

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

The novel β-agarase-producing bacteria of the present invention can be cultured by known conventional methods. The medium to be used can be either a synthetic medium or a natural medium as long as it contains appropriate amounts of carbon sources, nitrogen sources, inorganic compounds, and other nutrients, and culture can be performed using liquid or solid media. Can be done. Specifically, as a carbon source, since agarase is an inducible enzyme, agar, agarose, agaropectin, a partial hydrolyzate of agar, and even red algae such as Amanita spp. It can be used. Note that it is also possible to use other carbon sources such as glucose.

Nitrogen sources include meat extract, peptone, yeast extract, dried yeast, soy flour, casein, casamino acids, various amino acids, and cornstarch liquor.

Uses organic nitrogen sources such as Fitzmeal, urea, and other proteins derived from animals, plants, and microorganisms, and their hydrolysates, as well as inorganic nitrogen compounds such as various inorganic ammonium salts and nitrates. Alternatively, two or more types can be appropriately selected and used.

Inorganic salts include sodium, magnesium, calcium,
Phosphates such as iron, zinc, manganese, and copper.

Add one or more of hydrochloride, sulfate, carbonate, acetate, etc. as appropriate, or if halophilic bacteria are used, use artificial seawater or reduce the salt concentration to the same level as seawater. It can be set and used within an appropriate range. Further, an antifoaming agent such as vegetable oil or a three-surface active agent may be added as necessary.

Culture is performed by shaking culture 2 in a liquid medium containing the above-mentioned medium components.
This can be carried out using normal culture methods such as aerated agitation culture and continuous culture. Culture conditions depend on the type of medium.

It may be selected as appropriate depending on the culture method, and there are no particular restrictions as long as the conditions are such that agarase-producing bacteria can proliferate and produce β-agarase. Usually, the initial culture pH is 6.5 to 8.5.25.
It is preferable to culture at ~37°C with aeration and agitation. The appropriate number of days for culturing is usually 1 to 2 days.

The β-agarase produced during the culture as described above can be separated and recovered by the following method. Since wood β-agarase is mainly accumulated outside the bacterial cells, after the cultivation is completed, the bacterial cells are removed by filtration, centrifugation, etc. to obtain a culture filtrate. The bacterial enzyme can be used by crushing the bacterial cell and extracting β-agarase by a commonly used means.

The obtained enzyme-containing liquid can be used as it is by concentrating it using a vacuum-awl or ultrafiltration membrane to obtain a liquid enzyme, or by pulverizing it by a freeze-drying method or a spray-drying method. Other methods include commonly used purification methods such as ammonium sulfate salting out, solvent precipitation to precipitate β-agarase, ion exchange chromatography, gel filtration chromatography, agarose affinity adsorption, chromatography, It is also possible to use one or a combination of two or more purification methods such as isoelectric focusing to achieve high purity.

The method for measuring β-agarase activity in the present invention is as follows. 0.5 d of enzyme diluted and dissolved in pH 6, 0, 0, 1M acetate buffer was kept at 45°C, and 0.
Start the reaction by adding 0.5 me of 5% agar solution (preheated to 45°C). The reaction is carried out at 45°C for 10 minutes, the reaction is stopped by adding Somogyi solution, and the reducing sugar is determined by the Somogyi-Fulson method. Activity is 1μmo
The amount of enzyme that generates the reducing power equivalent to le galactose is expressed as one unit.

[Example] Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Pseudomonas sp. N7 (Pseudo
-monas sp, N-7)
No. 4) with 1.5% Amakusa cut material, 0.5% polypeptone,
Suspend and dissolve 0.5% yeast extract in artificial seawater, pH 6,
One platinum loop was inoculated into a sterilized medium 40d (200d Erlenmeyer flask) adjusted to a temperature of
When enzyme activity was measured, β-agarase was 0.1
9 units/unit were produced.

Example 2 Pseudomonas sp.
No. 84), powdered agar 0.7%, glucose 0.1%, sodium nitrate 0.5%, and yeast extract 0.5%.

Magnesium sulfate 0.2%, calcium chloride 0.2%.

Medium 401 with pH 8.5 containing 2.0% sodium chloride
11! (200 mf Erlenmeyer flask) was inoculated with 1 platinum loop,
After pre-culturing at 37°C with one mouth of shaking, this pre-culture solution was mixed with 1.5 I of the same medium! 31 jar fermenters including
was inoculated. 24 hours at 37°C, air flow 1.5 l/
Culture was performed at a minimum stirring speed of 400 rpm.

After the culture is completed, the bacterial cells are removed by centrifugation and the culture filtrate 1.4
I got j2. Agarase activity in the culture filtrate was 1.0 units/d. When this culture filtrate was concentrated approximately 20 times using an ultrafiltration membrane with a molecular weight cutoff of 10,000, 60 d of enzyme solution with a concentration of 21.3 units/relative was obtained.

Example 3 Pseudomonas sp.
No. 84) with 0.5% agarose and 0.5% sodium nitrate.
%, yeast extract 0.5% dissolved in artificial seawater p) 17
．． The cells were precultured in No. 5 medium. The preculture solution was mixed with medium 15 of the same composition.
I! , inoculated into a 302 jar fermenter containing
The cells were cultured at 30°C for 30 hours.

During cultivation, the aeration rate was 15 i! ,/min, 200r
pm stirring was performed.

After the culture was completed, the bacterial cells were removed by filtration to obtain a culture filtrate. The agarase activity in the culture filtrate was 1.1 units/ml. The culture filtrate was added to CL-Sepharose 6B (Pharmacia) 50mj! The solution was passed through the column at a flow rate of 500 ml/hr for affinity adsorption, and after washing with water, 500 d7 volumes of 2% neoagarooligo tJH solution were poured out to elute the agarase. Since then, DEAE has been used as a routine method.
l-Jovar (Tosoh Corporation).

Toyobar HW55 S was purified by chromatography to obtain a purified enzyme that was almost pure (22 units/
mg protein). Note that the activity yield was about 15%.

[Effects of the Invention] According to the present invention, a novel β-agarase can be obtained by culturing an explant belonging to the genus Pseudomonas. Since this enzyme has excellent heat resistance, it can be used for industrial production of agar oligosaccharides. Agar oligosaccharide has properties such as starch anti-aging action, bacteriostatic action, and indigestibility, and is expected to be used in a variety of fields.

[Brief explanation of the drawing]

Figure 1 shows the structure and hydrolysis mode of agarose. FIG. 2 shows the optimum pl+ of the 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. Patent Applicant Food Industry Bioreactor System Figure 2 pH 3r! 1J 4) e+/a 9

Claims (1)

[Claims] (1) A novel β-agarase having the following physical and chemical properties. [1] Effect: Hydrolyzes the β-1,4 bond of agarose to rapidly lower the viscosity of the agarose solution, producing mainly neoagalotetraose and neoagalohexaose. [2] Substrate specificity Acts on galactan polysaccharides such as agarose, agaropectin, and agar, which have β-1,4 galactoside bonds, and oligosaccharides of neoagarooctaose and higher. It has little effect on neoagarohexaose, neoagarotetraose, and neoagarobiose, and has no effect on lactose. [3] Optimal pH and pH stability When using agar as a substrate, the optimal pH is 7.0, and at 45°C
, 30 minutes, and is stable at pH 5-9 in the absence of substrate. [4] Optimal temperature and thermostability When using agar as a substrate, the optimal temperature is 50°C, and the pH is 6.
．． Under the conditions of 0 and 30 minutes in the absence of substrate, it is stable at 45°C, approximately 75% activity remains at 50°C, and approximately 35% activity remains at 55°C. [5] Inactivation conditions pH 6.0, 65°C, 30 minutes or more or pH 6.0, 10
It is almost completely deactivated in 10 minutes at 0°C. [6] Molecular weight 360,000 (gel filtration chromatography) [7] Isoelectric point 4.9 (isoelectric focusing) [8] Effect of metal salts pH 6.0, 45°C, 1mM metal salt concentration Bottom, after 3 hours of treatment, Fe^+^+^+, Hg^+^+, Ag^+,
Al^+^+^+ is 10% or less, Cu^+^+, Zn^
+^+ shows residual activity of about 50-80%, Na^+,
K^+, Mg^+^+, Co^+^+, Mn^+^+,
It is stable at Pb^+^+. It is also activated by Ca^+^+, and the residual activity is 10% with EDTA (5mM).
% or less. (2) Pseudomonas￥
A method for producing a novel β-agarase, which comprises culturing a novel β-agarase-producing bacterium having the physicochemical properties according to claim 1 belonging to the genus P. agarina and collecting the β-agarase from the culture. (3) Pseudomonas￥
A new β-agarase producing bacterium belonging to the genus Pseudomonas sp.
．． Claim 2 which is N-7) (Feikoken Bibori No. 9884)
Manufacturing method described.