JPS63164885A - Production of polygalactosamine-decomposition enzyme - Google Patents

Abstract

PURPOSE:To obtain a polygalactosamine-decomposition enzyme useful for the production of physiologically active substances such as a substance having antitumor activity, etc., by culturing a microbial strain belonging to Pseudomonas genus and capable of producing polygalactosamine-decomposition enzyme in a medium. CONSTITUTION:Pseudomonas sp H881 strain separated from soil and having bacillar form, motility and asporogenous nature is cultured in a medium containing 0.25wt% polygalactosamine, 0.25% glucose, 0.05% yeast extract, etc., at pH of about 7.0 and 20-40 deg.C for 35-72hr under shaking to produce a polygalactosamine-decomposition enzyme in the culture liquid. The enzyme is separated and purified e.g. by chromatography to obtain a polygalactosamine- decomposition enzyme having the following properties. Action and substrate specificity, reacting to oligo- and polygalactosamines (alpha-1,4-polygalactosamine) having a polymerization degree of n>=5 (n=5 corresponds to pentagalactosamine) to form oligogalactosamine; non-reactive with starch and pullulan, etc., optimum pH range in citrate phosphate buffer solution, 4.5-7.0; stable pH range in the buffer solution, 4.5-8.0; molecular weight, 31,000; etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a novel polygalactosamine-degrading enzyme (polygalactosaminidase).

More specifically, the present invention relates to a method for producing a novel polygalactosamine-degrading enzyme that hydrolyzes polygalactosamine to produce oligogalactosamine.

In general, polygalactosamine (poly-alpha) produced by microorganisms is
-1,4 galactosaminogalactan), pFl
ol (Special Publication No. 56-12639) and PF102 (
Japanese Patent Application No. 61-134799) is only recognized.

In recent years, it has become known that polysaccharides or their oligosaccharides produced by microorganisms, plants, or animals have various physiological activities, and interest in polysaccharides or their oligones Δ is increasing.

It has also been discovered that chitin, chitosan, and their oligosaccharides have antitumor activity in polyglucosamine (chitosan), which is known as a polysaccharide similar to polygalactosamine. Furthermore, it has been discovered that polygalactosamine itself has a similar physiological activity (Yukibo Ishitani et al. Japanese Biochemical Society Abstracts 871.1986), and there is growing interest in the physiological activity of its oligosaccharides. Polygalactosamine and oligogalactosamine may be useful in fields other than bioactivity, and oligomers in particular are expected to exhibit properties that polymers do not have in terms of application and action.
Attention has been paid.

Although it is possible to obtain oligosaccharides by hydrolyzing polygalactosamine with acids or alkalis, the yield of oligomers is very poor.131 For example, when hydrolyzing polygalactosamine with hydrochloric acid, as a result of random decomposition, The amount of oligosaccharides obtained is in the order of heptanogalactosamine, di-galactosamine, trigalactosamine, tetra-galactosamine, and penta-galactosamine, and the higher the degree of polymerization, the sharply reduced the yield.

Therefore, there is a need for a polygalactosamine-degrading enzyme that can decompose polygalactosamine to obtain oligosaccharides with a large degree of polymerization and various degrees of polymerization.

The present inventors searched a wide range of microorganisms for polygalactosamine-degrading bacteria, and as a result found that bacteria belonging to the genus Pseudomonas produce a novel polygalactosamine-degrading enzyme, and completed the present invention.

The present invention relates to a novel method for producing a polygalactosamine degrading enzyme.

The bacteria used in the present invention belong to the genus Pseudomonas and may be any strain as long as it produces a polygalactosamine-degrading enzyme, but Pseudomonas sp H881, which the present inventors isolated from soil, is advantageously used. The mycological properties of Pseudomonas sp H881 are as follows.

(a) Morphological microscopic observation (cultured on broth agar medium at 30°C for 16 hours) (
1) Cell size: 0.3-0.6 x 1.0-2.
0 micron bacillus (2) Cell pleomorphism: Not observed (3) Motility: Possesses polar flagella and is motile (4) Presence or absence of spores: Not formed.

(5) Durham staining: Negative (6) Acid-fastness: Negative (b) Growth status in various media (1) Broth agar plate culture: Light yellow-brown colonies after 24 hours at 30°C, with smooth and glossy surface. There is no production of 6 pigments which are semi to opaque.

(2) Broth agar slant culture: Grows well.

(3) Meat juice liquid culture: Growth in a thick film on the surface of the culture medium, and moderate growth within the liquid.

(4) Meat juice gelatin puncture culture Grows on the surface and liquefies in layers.

(5) Lidomus milk: alkaline, completely liquefied.

(c) Physiological properties (1) Nitrate reduction: negative (2) Denitrification reaction: negative (3) MR test: positive (4) VP test: negative (5) Indole formation: lta (6) Hydrogen sulfide Production: Negative (7) Starch hydrolysis: Negative (8) Utilization of citric acid: Used in both Koser and Christensen's media.

(9) Use of inorganic nitrogen: Nitrate and ammonia are also used.

(10) Pigment production: KingA; negative, King
B: produces a weak blue fluorescent dye, Fagar: produces a weak blue fluorescent dye, Pagar: negative (11) urease: positive (12) oxidase: positive (13) catalase: positive (14) Growth range: Raw￥fpH5-9, optimum temperature 30
~40℃ (15) Attitude towards oxygen: aerobic (16) O-F test dioxide (17) Casein degradation: positive (1 g) DNA degradation: positive (19) Salt tolerance: 2% salt; positive, 5% Salt; negative (2
0) Generation of acid and gas from sugars Generation of acid Generation of gas (1) L-arabinose 10 - (2) D
-xylose 10 -(3) D-glucose 10 -(4) D-mannose + -(5) D-fructose -- (6) D-galactose 10 -(7) Maltose - -(8) Sucrose 10 -( 9) Lactose
- - (10) Trehalose - (11) D-sorbitol - - (12) D-mannitol - - (13) Inositol - - (14) Glycerin - (15) Starch - (d) Other properties (1) ) Accumulate poly-β-hydroxybutyric acid ester (PH8,) within the bacterial cells in a nitrogen source-deficient medium.

(2) It grows using arginine and betaine as the sole carbon source and does not have arginine dehydrolase activity.

(3) Decompose fatty acids (Tween 80.60.20).

(4) Grows at 40℃. Unable to grow at 4℃.

The taxonomic properties of the above-mentioned novel polygalactosamine-degrading enzyme producing ability are summarized in the ``Burges Manual.''
of Deterministic Bacteriology” 8th edition (
1974) and Burgess Manual of Systematic Bacteriology, Volume 1 (1984).
In contrast to the classification of P) which accumulates IB and uses arginine and betaine as the sole carbon source, the Ministry does not require a gross factor (P), and the classification of arginine dehydrolase negative, denitrification reaction negative, 40 P, cep in section 2 (or RNA group 2) because it can grow at °C.
acia, P41adioli+P, margina
Although it is thought to be a related fungus to P. cepacia, it differs from P. cepacia in that it is positive for nitrate reduction and assimilates carbon sources in D(-)-1-levalose, maltose, lactose, and maleic acid. P. gladioli differs from P. gladioli in terms of assimilation of maltose, lactose, maleic acid, and m-hydroxybutyric acid ester.

The results for m-hydroxybutyric acid ester are different from p,IIIarginate. Also, P, cepac
ia.

P,marginate produces a non-fluorescent dye, but the Ministry of Health, Labor and Welfare has determined that when King B, Fagar, glutamic acid, L-arginine, L-threonine, and L-histidine are used as the sole carbon source, weak fluorescent dyes (blue-white fluorescence) are produced. ) is produced, but no non-fluorescent dye is observed under various culture conditions. Based on these results, the Ministry has decided to
pacia, P, gladioli, P, marg
5 pecies different from inate.

A characteristic feature of the physiological properties of this plant is that in the O-F test, acids are produced not only from monosaccharides but also from disaccharides such as flu-1-saccharide, sucrose, lactose, and cellbiose. This property is similar to Pseudomonas
Genus, psychrotrophic P, fragi, P.

taetrolens (both section 5) P
, 1undensis, but there are differences in growth temperature.

Based on the above results, the Ministry recognized this as a new species of Pseudomonas, named it Pseudomonas sp 888], and submitted it to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry.
It has been deposited as PERM P-8955.

As a culture medium for polygalactosamine-degrading enzyme-producing bacteria, either a synthetic medium or a natural medium can be used as long as it contains a suitable amount of carbon sources, nitrogen sources, inorganic substances, and other nutrients. Suitable examples of the culture medium include:
Polygalactosamine 0.25%, glucose 0.25%
, yeast extract 0.05%.

An example is polypeptone 0.05%, pH 7.0.

The culture temperature is in the range of 20-40°C, preferably 30-38°C, and the culture starting pH is 6-8, preferably around 7 and 35-38°C.
If cultured with shaking or deep stirring for 72 hours, a polygalactosamine-degrading enzyme can be obtained in the culture solution. and.

Polygalactosamine degrading enzymes are isolated and purified as necessary. For example, the crude enzyme is separated from the culture filtrate by ethanol precipitation, dissolved in an aqueous medium, subjected to Sephadex G-50 gel filtration, CM-Sephadex C-25 ion exchange chromatography, and phenyl-Sepharose CL-4B. A purified polygalactosamine-degrading enzyme is obtained by treatment such as hydrophobic chromatography.

The physicochemical properties of the polygalactosamine degrading enzyme obtained in Example 1 are as follows.

(1) Action and substrate specificity This enzyme acts on oligo and polygalactosamine (α-1,4 polygalactosamine) with a degree of polymerization n=5 (penta-galactosamine) or higher to produce oligogalactosamine.

Other polysaccharides, starch (α-1,4 glucan), glycogen (α-1,4 glucan), pullulan (α-1,4
glucan), dextran (α-1,6 glucan), laminaran (β-1,3 glucan), carboxylcellulose (β-1,4-glucan), chitosan (β-1,4
gelcosaminoglucan), ethylene glycol chitin (
β-1,4N-acetylgelcosaminoglucan), Ps
poly N-acetylgalactosaminogalactan (poly β-1
, 3N-acetylgalactosaminogalactan) (Y, A
kiyama,, et, al.

Agric, Biol, Chem, +50 (3)
747.1986) etc. has no effect at all.

Moreover, it does not act on α-1,4 galactosaminooligosaccharide having a degree of polymerization n=4 (tetra-galactosamine) or less.

(2) Optimal pH and stable pH range When using citrate phosphate buffer, the optimal pH is 4.5
~7.0. (Figure 1) Also, the stable pH range is
As shown in the figure, it is <pi (4.5 to 8.0). This measurement shows the residual activity of the enzyme after being left at 37° C. for 1 hour as a relative value.

(3) Enzyme activity measurement method Enzyme activity was determined using PFIOI or PF102 produced by Paecilomyces I-1 bacteria (the main constituent sugar being α-1,4 galactosaminogalactan). This 0.5% 10.1M acetate buffer pH 6.0 solution.
Add enzyme solution 0 and 5+nQ to 5+nQ, and incubate at 37℃ for 1
React for 0 minutes, and reduce the resulting reducing power with Somogyi-Ne
It was measured by the lson method. The enzyme unit is 1 per minute.
The activity that increases the reducing power corresponding to μmol of galactosamine was defined as one unit.

(4) Range of suitable operating temperature and temperature stability The results of measurements in the range of 20 to 70°C are shown in Figure 3.

The optimum temperature for this enzyme is 55°C, and the temperature decreases rapidly above that temperature.

Next, Figure 4 shows the temperature stability. p
The residual activity when kept at each temperature for 0 to 80 minutes under H6.0 conditions is shown. 70% activity remains after 1 hour at 50°C.

(5) Effects of metal ions, etc. Various metal ions and inhibitors LmM (PCMB only 0.
After standing in a solution containing 1mM) at 37°C for 1 hour, the residual enzyme activity was measured and expressed as a relative value. (Table-1) Table-1,
Inhibitors such as metals Residual activity (%) Inhibitors Residual activity (%)
) No additives 100 to C196NaC197 CaC1,98LiC1100 BaC1□' 99 MnC1,103
CoC1□88 NiC1,90CdC1
z 98 5nC1226FeC
1,5FeCl36 ZnC1292HgC1,0 Pb(CH,Coo)295 NH4Cl
98 (Ni+4), So, 100
CuSO429tris 1)
97 SDS 2) 4NBS
3) 88 EDTA 4
) 99MIA 5) 100
PCMB 6) 93■) Tris(hydroxyl)aminomethane 2) Sodium dodecyl sulfate 3) N-bromosuccinimide 4) Disodium ethylenediaminetetraacetate 5) Monoiodoacetic acid 6) From the above results, it was found that this poly Galactosamine degrading enzymes are inhibited by tin, iron, copper, inorganic mercury and SO5.

(6) Enzyme purification method Isolation and purification of this enzyme can be performed according to conventional methods.

The ethanol precipitate was subjected to Sephadex G-50 column chromatography (Figure 5), Sephadex C-25 column chromatography (Figure 6), and phenyl-Sepharose 4B column chromatography (Figure 7).
fi! 'Purified by means A or a combination thereof.

(7) Molecular Weight The molecular weight of this enzyme is calculated to be 31,000 when measured by polyacrylamide gel slab electrophoresis. The results are shown in Figure 8.

(8) Polyacrylamide gel electrophoresis The purified enzyme was purified using a 7.5% polyacrylamide gel (pt(
8,6) Subjected to electrophoresis. As shown in FIG. 9, a single band was observed.

(9) Isoelectric focusing of a sucrose density gradient was performed using a conventional isoelectric focusing method. As shown in FIG. 10, the isoelectric point of this enzyme is pI=6.7.

This enzyme is a novel enzyme whose action and site specificity are completely unknown.

Next, examples of the present invention will be shown.

Example 1 Pseudomonas sp 11881, FERM P-89
55 in a 500 mΩ Erlenmeyer flask with 0.5 glucose
%, yeast extract 0.05%, and polypeptone 0.05%.
Incubate for hours.

The obtained seed culture solution was mixed with 0.25% polygalactosamine (PF102) in a 3[)Q jar fermenter.
Inoculated into enzyme production medium 18111 containing 0.25% glucose, 0.05% yeast extract, and 0.05% polypeptone,
Aeration (18Q/min) stirring (200r) at 30℃ for 48 hours
pm) Cultivate.

The obtained culture solution was centrifuged (14,000 rpm) to remove bacterial cells, and the obtained culture filtrate (enzyme activity 0.00
The proteins are precipitated by adding chilled ethanol to 60% concentration (35 U/ml, total concentration 63 U/1.8 Q), and the precipitated proteins are separated from the solution by centrifugation.

The obtained protein was dissolved in 0.1 molar acetate buffer (ρ115
．． The sample is adsorbed onto a C-tocephadex C-25 column (2.5 x 60 cm) equilibrated with 0) and eluted using the same buffer with a concentration gradient of 0 to 0.5 molar saline.

The eluted enzyme activity fraction was collected and filtered using an ultrafiltration device (molecular weight 1
Concentrate using 1,000,000 ml of water. Next, 0 containing 2 molar salt
．． A Sephadex G-50 column (5X 9
0 cm) chromatography. The salt concentration in the active section was then increased to 4 molar, and a phenyl-Sepharose CL-/IB column (2,
50 mg of purified polygalactosamine-degrading enzyme (yield: 23.1%, specific activity: 52).
μg galN+/min/mH protein) was obtained.

[Brief explanation of the drawing]

Figure 1 shows the optimum pi for this enzyme, Figure 2 shows the stable pH, Figure 3 shows the optimum temperature for action, Figure 4 shows the temperature stability, and Figure 5 shows Sephadex G-50. Figure 6 shows the pattern of C-tosephadex C-25, Figure 7 shows the column chromatography pattern of Phenyl-Sepharose CL-4B, and Figures 8 and 8 show the results of polyacrylamide gel slab electrophoresis. , Figure 9 shows the results of polyacrylamide gel desk electrophoresis, and the first
Figure 0 shows the results of isoelectric focusing, respectively. Agent Patent Attorney Door 1) Parent Male Diagram 1 x 10' 3 to pH Diagram 2. L ---m= Temperature (0C) jifr Pit (min) Fig. 5 7 comb length (818mQ) Fig. 6 Warakushikorei (618mλ) Fig. 7 Fraction U (each 10mffi) Fig. 8 Rm Fig. 9 10 Figure

Claims (1)

[Claims] (1) A method for producing a polygalactosamine-degrading enzyme, which comprises culturing a polygalactosamine-degrading enzyme-producing bacterium belonging to the genus Pseudomonas, and collecting the polygalactosamine-degrading enzyme from the culture.