JPH099962A - Alginic acid hydrolyzing enzyme and hydrolysis of alginic acid - Google Patents

Abstract

PURPOSE: To obtain an alginic acid hydrolyzing enzyme from a culture solution of a bacterium capable of producing an enzyme for strongly hydrolyzing alginic acid as a main component of a biofilm Pseudomonas aeruginosa. CONSTITUTION: A bacterium belonging to the genus Bacillus, capable of hydrolyzing alginic acid, is cultured. An alginic acid hydrolyzing enzyme is collected from the culture product to give the alginic acid hydrolyzing enzyme. Alginic acid is treated with the prepared crude or purified enzyme and hydrolyzed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an alginate-degrading enzyme produced by a microorganism belonging to the genus Bacillus, a method for producing the same, and a respiratory infection caused by dissolving a biofilm containing alginate as a main component using the enzyme. And a method for decomposing or reducing the molecular weight of alginic acid.

[0002]

2. Description of the Related Art In recent years, the rapid refractory / chronicization of respiratory tract infections centering on Pseudomonas aeruginosa has become an extremely serious problem in chemotherapy. The main body of this refractory treatment is formation of a biofilm composed of a complex of glycocalyx produced by an infecting bacterium and a biological component. The production of biofilms containing alginic acid as a main component creates a new situation of habitat formation of bacteria in the living body, which impedes the permeability of chemotherapeutic agents into the cells and causes drug resistance. . Intractable infections involving biofilms include infections caused by artificial medical materials, urinary tract infections, respiratory respiratory infections such as endocarditis and diffuse panbronchiolitis. Chemotherapeutic agents are less effective, and development of innovative therapeutic agents and treatment methods is desired. Although various chemotherapeutic agents have been developed so far, no attempts have been made to develop effective agents or enzyme preparations that directly act on biofilms or to treat them with therapeutic methods.

On the other hand, in this study, a method of utilizing alginic acid as a biomass resource by decomposing alginic acid into monomeric or oligomeric uronic acid can be considered. The aqueous solution of alginic acid is very viscous, and it is difficult to decompose alginic acid because it forms a metal salt with calcium and the like in the aqueous solution and gels. Therefore, attempts are being made to decompose alginic acid using microbial enzymes.

[0004]

The present inventors searched for a microorganism that produces an enzyme that more strongly decomposes alginic acid, which is the main component of Pseudomonas aeruginosa biofilm, and obtain it from the culture solution of the producing microorganism. The effect of degrading alginic acid by alginate-degrading enzyme was examined.

[0005]

In order to achieve the above object,
The present inventors actively search for an enzyme that decomposes alginic acid, which is the main body of glycocalyx, into a low molecular weight molecule, that is, an alginate lyase-producing bacterium in soil microorganisms, and is a strain belonging to the genus Bacillus sp. ATB-1015 strain. Succeeded in separating.

The present invention relates to an alginic acid degrading enzyme produced by Bacillus sp. ATB-1015 strain, a method for producing the same, and a method for degrading alginic acid by a microbial enzyme characterized by acting on alginic acid to decompose it.

The isolation method of this strain is to obtain a degrading enzyme producing bacterium capable of assimilating alginic acid as a carbon source from the soil by previously treating the soil with an aqueous sodium alginate solution, and effectively separating the desired bacterium. This is a useful method because it can be done.

The bacteria thus isolated have a medium composition containing alginic acid as a main carbon source, for example, sodium alginate (0.5%), peptone (0.5%), yeast extract (0.1%), Grows better in NaCl (0.5%), agar (1.5%). This producing bacterium is an aerobic rod that is positive for Gram stain. This bacterium forms spores and is thermostable.
Morphological observation with a scanning electron microscope (12,000 times) shows a conical shape with a length of 2.5 to 2.9 μm and a width of 0.5 to 0.9 μm and no flagella. From the above mycological properties, this bacterium belongs to the genus Bacillus.

This strain is treated with sodium alginate (0.5
%), Peptone (0.5%), yeast extract (0.1%),
_{NaCl (0.5%), MgSO 4} · 7H 2 O (0.1%) and KH ₂ PO
500m containing 100ml of medium consisting of ₄ (0.1%)
Inoculate a 1-liter Erlenmeyer flask and culture for 24 hours. This seed culture solution is inoculated into 3 L of the above-mentioned medium in a 5 L jar famentor, and aeration-agitation culture is performed at 27 ° C. for 36 hours. The culture broth thus obtained is separated into bacterial cells and culture supernatant to obtain a supernatant. The alginate-degrading enzyme was purified from this supernatant by the following method. The culture supernatant is subjected to ammonium sulfate precipitation and then dialyzed to obtain a crude enzyme solution. The crude enzyme solution is purified by DEAE-cellulose column chromatography to obtain a fraction having enzyme activity. Then, Sephacryl S-200 chromatography is performed to obtain an enzyme active fraction.

The optimum pH for this enzyme activity is around 7.0. The stable pH of this enzyme is 6.0-8.0, and the optimum temperature is 37 ° C. Heat stability is 50% in 20 mM phosphate buffer (pH 7.0) at 60 ° C for 30 minutes.
The above alginate-degrading enzyme activity was retained. The substrate specificity of this enzyme shows a high decomposition activity for alginic acid derived from brown algae, while it also highly decomposes and dissolves alginic acid derived from Pseudomonas aeruginosa, that is, biofilm produced on the surface of Teflon disk. It showed activity. This purified enzyme is SDS
-Give a single band by polyacrylamide gel electrophoresis. Its molecular weight is 41,000.

Regarding alginate lyase, the marine bacterium Alteromonas sp. H-4 (Nippon Suisan) has been used so far.
Gakkaishi, 58 (3), 521-527 (1992)), Pseudomonas sp.OS-ALG-9 (J. Ferment. & Bioeng., 72 (2), 74-
78 (1991)), Flavobacterium sp. (J. Ferment. B
ioeng., 72 (3) 152-157 (1991)), Bacillus circulans (Appl. Environ. Microbiol., 47 (4), 704-7
09 (1984)), Klebziella Aloginas (Arch. Mic
robiol., 152, 302-308 (1989)). This time, the newly isolated Bacillus sp.A
The TB-1015 strain differs from the strains reported so far in its genus and morphological characteristics. Although belonging to the same genus as Bacillus circulans, sp. ATB-1015 strain is morphologically different in that it does not have flagella. The enzyme produced by Bacillus sp. ATB-1015 has a molecular weight of 41,000 on SDS-polyacrylamide gel electrophoresis, whereas the enzyme derived from Bacillus circulans has a molecular weight of 58,000. ing. In addition, the present enzyme has a different molecular weight from alginate-degrading enzymes produced by bacteria other than Bacillus.

[0012] As described above, the present enzyme is a new enzyme having different properties from the alginic acid degrading enzymes reported so far. This strain has already been deposited at the Institute of Biotechnology, Institute of Biotechnology, and is assigned the deposit number of FERM P-14946.

The microorganism used in the present invention can be grown in a suitable medium. For example, in addition to alginic acid as a carbon source, carbohydrates such as glucose, sucrose, and glycerin, inorganic sources such as ammonium sulfate, ammonium nitrate, and ammonium phosphate as nitrogen sources, or urea, peptone, casein, yeast extract, meat extract, etc. Any organic nitrogen can be used. Addition of a readily assimilated carbon source (glucose, glycerin, etc.) to the medium may inhibit the synthesis of enzymes involved in the decomposition of the slowly metabolized carbon source (in this case, alginic acid). As other inorganic salts, phosphates, magnesium salts, potassium salts, manganese salts, iron salts and the like are used. The culture method of this strain is a conventional method. Usually, the culture temperature is preferably 20-40 ° C. and pH 6.0-8.0, and the culture is aerobically cultivated by shaking or aeration and stirring for 1-5 days.

Extraction from the culture broth thus obtained,
By acting the purified alginic acid-degrading enzyme on seaweed-derived alginic acid or Pseudomonas aeruginosa-derived alginic acid, that is, biofilm, alginic acid can be decomposed or reduced in molecular weight, and further the biofilm can be dissolved. The selection of an alginate lyase-producing bacterium, the decomposition of alginic acid by this enzyme, the reduction of molecular weight, and the lytic ability of the Pseudomonas aeruginosa biofilm by this enzyme can be examined by the following methods.

1) Bacterial colonies were inoculated on an agar plate containing sodium alginate as the sole carbon source, and after culturing for 5 days, CaCl ₂ aqueous solution was added and allowed to stand, and the size of white halo and transparent zones formed around the colonies. Measure (J. Fer
ment. Bioeng., 76 (5) 427-437 (1993)). 2) Measurement of viscosity decrease due to alginic acid lyase treatment reducing the viscosity of highly viscous alginic acid (Nippon
Suisan Gakkaishi, 55 (4) 709-713 (1989)). 3) Degradation of alginic acid by enzymatic treatment and quantification of unsaturated uronides produced by depolymerization (Nippon Suisan Ga
kkaishi, 55 (4) 709-713 (1989)). 4) Measurement of the dissolution ability of alginate-derived biofilm produced by Pseudomonas aeruginosa (clinical isolate) by alginate lyase action.

In particular, 4) is the method newly invented in this patent. This enzyme activity measurement method is an effective method for evaluating the therapeutic effect of this enzyme on intractable respiratory infections associated with biofilms. In this method, as shown in Example 4, a Teflon disk was statically cultured with Pseudomonas aeruginosa for 4 to 7 days, and the biofilm attached to the surface of the Teflon disk piece was incubated with an alginate lyase. It is a method of examining the solubility of erythrocyte by the toluidine blue staining method.

Extraction and purification of this enzyme from the culture solution of the alginate lyase-producing microorganism are generally carried out by ammonium sulfate precipitation, dialysis,
It can be performed by effectively combining gel filtration such as Sephacryl S-200 HR and various ion exchange resin chromatography such as DEAE-cellulose, DEAE-Sephadex and CM-cellulose. The microorganism found by the present invention, Bacillus sp. ATB-1015, and the alginic acid-degrading enzyme purified from the culture solution thereof are expected to be used in the medical and food fields. This enzyme is a diffuse panbronchi disease that is a respiratory infection of Pseudomonas aeruginosa.
olitis) and a genetic respiratory disease, cystic fibrosis (Cyst
ic fibrosis) and respiratory infections can be treated by spray inhalation. On the other hand, this enzyme efficiently decomposes alginic acid derived from seaweed such as kelp and lowers its molecular weight.Therefore, these are used as biomass resources, and those decomposed to an appropriate size are processed into foods of plants and insects. It is used as a raw material. From this, it is possible to increase the yield of agricultural crops by treating the soil with an alginic acid-degrading enzyme-producing bacterium together with an organic material containing alginic acid, and it can be considered to be used as a useful microorganism in a soil improving material.

[0018]

The present invention will be described below in detail with reference to examples. Example 1 A method for selectively separating alginate-degrading microorganisms was performed as follows. About 3% of sodium alginate was added to a predetermined soil for every 2-3 months for about 2-3 days. About 1 g of this treated soil was diluted with 2 ml of sterilized water.
1 ml of the pour solution sample was mixed with the medium composition shown in Table 1 in which sodium alginate was the only carbon source, and an agar plate was prepared on a petri dish. This plate at 27 ° C for 7 days
Incubated. The colonies formed on the plate were further inoculated on a plate having the same composition, incubated at 27 ° C. for 5 days, and then a 1M CaCl ₂ solution was added to the plate, and after standing, a bacterium that formed white halo and a transparent zone around the colony. Was isolated as an alginate lyase producing bacterium.

[0019]

[Table 1]

This strain was inoculated into a large test tube containing 10 ml of the liquid medium shown in Table 2 containing sodium alginate as a carbon source. The culture broth cultivated at 37 ° C. for 24 hours was transferred to a 500 ml Erlenmeyer flask containing 100 ml of a medium of the same composition, shake-cultured at 27 ° C., and the culture was stopped at 48 hours. The extracellular alginate lyase activity produced in the culture broth was measured by using a substrate solution (5 ml) of kelp-derived sodium alginate and a culture supernatant (0.5 ml) at 37 ° C.
After reacting for 0 minutes, the viscosity was measured by a viscosity measurement using an Ostwald viscometer. The enzyme activity of the 48-hour culture broth determined by the viscosity method was 2.0 Units / ml, and this enzyme showed an activity of reducing the viscosity of alginic acid to 96% in 10 minutes.

[0021]

[Table 2]

Example 2 2 obtained by Erlenmeyer flask culture in Example 1
Preliminarily use 90 ml of 4-hour culture solution in an autoclave for 12
A 5 L jar fermenter containing 3 L of sterilized medium of the same composition was inoculated at 0 ° C. for 15 minutes. To examine the progress of the culture, aerobically agitate at 27 ° C for 72 hours (2
(00 rpm) culture was performed. During the culture, an antifoaming agent, adecanol, was used to prevent the foaming of the culture solution. The enzyme activity was determined by the viscosity method described in Example 1. For the growth of the bacterium, 10 ml of the culture solution was centrifuged at 3,000 rpm for 10 minutes,
It was shown by the amount of remaining cells. The bacteria started to grow from the 10th hour, and reached the maximum at the 24th hour. The enzyme production increased with the growth of the fungus, reaching a maximum at 36 hours.
After that, the enzyme production is in a steady state, and the enzyme activity remains even after 72 hours. The pH in the culture solution slightly increased from 7.0, increased sharply after 48 hours, and reached pH 9.0 at 72 hours. The method for purifying alginate lyase from the 36-hour culture was shown in Example 3.

Example 3 The culture solution obtained in Example 2 was separated into bacterial cells and culture supernatant by centrifugation. Purification of alginate lyase from the culture supernatant was performed by the method shown in Table 3. The alginate lyase activity at each purification step was determined from the absorbance of uronate, which is a decomposition product, at UV absorption at λ235 nm. The amount of enzyme protein was determined by the Lowry method using absorption at λ280 nm and bovine serum albumin as a standard. After alginate supernatant was salted out with 75% saturated ammonium sulfate, a small amount of 20 mM phosphate buffer (pH 7.
It was dissolved in 0) and dialyzed in the same buffer overnight. This crude enzyme solution was passed through a DEAE-cellulose column chromatography, which is an ion exchange resin equilibrated with a 20 mM phosphate buffer solution (pH 7.0). The crude enzyme solution obtained by collecting the active fractions and concentrating by ultrafiltration was equilibrated with Sephacryl S-200 in 50 mM phosphate buffer (pH 7.0).
Gel filtration was performed with HR. The active fraction was concentrated and freeze-dried to obtain a white powder. This powder was purified again by the DEAE-cellulose column chromatography described above. The yield from the culture supernatant was 5.8%. The purified enzyme had a specific activity about 25 times higher than that of the sample after dialysis.
The purified enzyme solution was subjected to 12.5% SDS-polyacrylamide gel electrophoresis and then stained with CBB. As shown in FIG. 1, this enzyme gave a single band consisting of a protein having a molecular weight of 41,000 on SDS-polyacrylamide gel as compared with existing molecular weight markers.

[0024]

[Table 3]

Example 4 A clinical isolate of Pseudomonas aeruginosa, M-3 mucoid strain, was subjected to liquid culture in a large test tube for 18 hours using 10 ml of Mueller Hinton medium, and this was used as a seed culture. This seed culture is 1.
Medium composition consisting of 5% NaCl and 0.2% glucose (5m
The petri dish containing 1) was inoculated. At this time, a piece of Teflon sheet disk having a diameter of 6 mm was put together in advance in the petri dish, and static culture was carried out at 37 ° C. for 5 days. 400 µl of a culture solution of the Teflon disk piece produced and attached to the biofilm composed of alginic acid polysaccharide and the alginic acid-degrading enzyme-producing bacterium was incubated at 37 ° C for 20 minutes. After the reaction, the Teflon disk was taken out and stained with a toluidine blue solution for 30 minutes. Both the culture supernatant and the purified enzyme solution decompose and dissolve the biofilm adhering to the surface of the Teflon disk. As a result, the surface of the disk is hardly stained, and the ability to dissolve Pseudomonas aeruginosa biofilm is visually observed. I was able to. In addition, this method is extremely useful for in vivo evaluation because it is possible to examine the solubility of biofilm under conditions closer to that in vivo by adding proteins such as serum and glycoproteins together with Teflon disk. It is a test method.

[0026]

The alginate lyase produced by a new strain belonging to the genus Bacillus found by the method of the present invention can efficiently decompose and reduce the molecular weight of alginate produced by a clinical isolate of Pseudomonas aeruginosa. It can be a useful therapeutic tool for diseases such as chronic refractory respiratory infections, urinary tract infections and cystic fibrosis caused by formation. The enzyme is relatively stable against acid and heat, and can be supplied in a large amount because it is accumulated in the culture supernatant. Since this enzyme can also decompose alginic acid derived from seaweed and reduce its molecular weight, it can be used for the treatment of waste liquid containing alginic acid and food products.In addition, uronic acid, a decomposition product of alginic acid, is a biomass resource. It is also useful as a soil improving material as a useful microorganism for the purpose of increasing the yield of agricultural products.

[Brief description of drawings]

FIG. 1 shows the results of molecular weight measurement by SDS-PAGE of alginate lyase derived from Bacillus sp. ATB-1015.

Claims (3)

[Claims] 1. An alginic acid-degrading enzyme which is produced by a microorganism belonging to the genus Bacillus and capable of degrading alginic acid to decompose alginic acid. 2. A method for producing an alginate degrading enzyme, which comprises culturing a microorganism belonging to the genus Bacillus and capable of degrading alginate and collecting the alginate degrading enzyme from the culture. 3. A method of culturing a microorganism of the genus Bacillus capable of degrading alginic acid, and degrading the alginic acid by causing the culture, crude enzyme or purified enzyme obtained from the culture to act on the alginic acid. Decomposition method.