JPH04237491A - New chitinase and its production - Google Patents

Abstract

PURPOSE:To establish a method for producing chitinase by separating bacteria without requiring colloidal chitin or powdery chitin in which its preparation as an induction substrate is troublesome and confirm effectiveness of the produced enzyme by separating the aforementioned bacteria in producing the chitinase. CONSTITUTION:A new microbial strain Bacillus.sp. 218C (FERM P-11914) is separated. The resultant strain is further cultured in a culture medium containing only deashed chitin as an induction substrate to produce a new chitinase, which is then separated from the culture medium. Furthermore, the aforementioned enzyme is reacted with chitosan to efficiently produce its low- molecular substance.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a novel chitinase and a method for producing the same.

0002

BACKGROUND OF THE INVENTION Chitin is the second most abundant biomass resource after cellulose, and its effective utilization has been strongly desired for some time. In addition, chitooligosaccharide, which is a partial decomposition product of chitin, is being used in the food field as a low-cariogenic, non-digestible sweetener, or as a texture improver, and in the pharmaceutical field, it has been used to strengthen cellular immunity. Expectations are high for its use as a bifidus factor. Chitooligosaccharides are usually produced by treating chitin with concentrated hydrochloric acid for a long period of time.
This requires complicated steps such as subsequent desalting operations. For this reason, decomposition products can be obtained with mild and simple operations.
The use of chitinase is desired. In addition, in recent years, research has been conducted to reduce the molecular weight of chitosan or to generate oligosaccharides by using chitinase to act on chitosan (Agric. Biol.
．． Chem. , vol. 52, p. 3181 (1988
); JP-A-63-98395), food shelf life improver,
It has also opened up the possibility of using it as a carrier for sustained-release pharmaceuticals. Chitinase is also produced by fungi, actinomycetes, animals and plants, but a method for producing it using bacteria is a method using bacteria of the genus Aeromonas (J. Gen. Appl. Microbiol., vo.
l. 32, p. 25 (1986)) and Serratia (Se
Method using bacteria of the genus Can.
icrobiol. , vol. 15, p. 689 (19
69) etc. are known. In addition, Bacillus (Bacillus)
Bacillus sp. P
I-7S strain (JP 1-247081), Bacillus circulans WL-1
2 strains (J. Gen. Appl. Microbiol.,
vol. 16, p. 39 (1970)), Bacillus licheniformis
) X-7U strain (Agric. Biol. Chem., v
ol. 53, p. 1537 (1989), etc. are known.

[0003] In chitinase production methods using these bacteria, it is generally necessary to colloidize or finely powder demineralized chitin and add it to the medium as an induction substrate. For this colloid formation, after the demineralized chitin is immersed in concentrated mineral acid and ground, the chitin must be thoroughly washed with a large amount of water to remove the remaining acid. In addition, pulverization is performed using a ball mill, etc., but the amount of processing is limited and it takes a long time, so there was a strong desire for a method for producing chitinase that could use demineralized chitin as it is as an induction substrate. .

0004

[Problems to be Solved by the Invention] The present inventors have conducted a wide search for various chitinase-producing bacteria from the natural world, and as a result, a new strain of the genus Bacillus was newly isolated.
The present invention was completed based on the knowledge that, without using colloidal chitin or finely powdered chitin as an induction substrate, by simply adding demineralized chitin, an enzyme that decomposes chitin efficiently can be produced outside the bacterial cell. reached. That is, the present invention provides a bacterial strain capable of efficiently producing chitinase from an enzyme production medium containing demineralized chitin without the need for a specially treated induction substrate, and a novel chitinase produced by the strain. The present invention also provides a method for producing chitinase using the strain, whereby
The production of chitinases on an industrial scale has now been significantly improved.

[0005] The chitinase-producing bacterium Bacillus sp of the present invention
．． The 218C strain was newly isolated from the soil of Soka City, Saitama Prefecture, and has the mycological properties shown below. A. Morphological properties This fungus grown on broth agar medium has a size of 0.5 to 0.
It is a bacillus with a size of 7 x 2.0 to 3.0 μm. It has mobility,
Gram stain is positive. Spores are very difficult to form, and on nutrient agar containing manganese ions, 30
Its formation is observed when cultured at ℃ for 3 weeks or more.

B. Cultural Properties This strain is cultured in a normal bacterial culture medium at 25-45℃ for 1-3 hours.
It proliferates well when cultured for several days. 1. The color of the broth agar slant colony is white and the surface is smooth. No water-soluble or water-insoluble dyes are produced. 2. Colloidal chitin agar plate medium After 2 to 3 days, colloidal chitin is assimilated and the area around the colony becomes transparent. The colony surface is smooth with almost no bumps. Colony color is white.

C. Physiological properties 1. Nitrate reduction:
Negative 2. Generation of hydrogen sulfide:
Negative 3. Generation of indole:
Negative 4. V-P test:
Negative5. O-F test: Oxidative6.
Methyl red test: Negative7.
Catalase: Positive8. Phosphatase:
Positive9. Hydrolysis of starch:
Positive10. Hydrolysis of casein:
Negative 11. Deamination of phenylalanine:
Negative12. Assimilation of citric acid:
Negative13. Attitude towards oxygen: Aerobic14. Effect of salt concentration on growth: Grows at 2%, does not grow at 5%. 15. Sugar availability: a. The following sugars produce acid, but no gas. D-glucose, D-cellobiose, lactose, raffinose, sucrose. b. The following sugars do not generate acid or gas. L-arabinose, D-xylose, D-mannitol, salicin.

D. Cell wall composition Diaminopimelic acid in cell wall components includes meso form.

[0009] Based on the above properties, this bacterial strain is considered to belong to the genus Bacillus;
'sManual of Systematic
Bacillus, which does not match any species within the same genus and appears to be relatively closely related, is listed in Volume 2 of Bacteriology.
What is Bac.circulans?
In the production of acids from L-arabinose, D-xylose, and D-mannitol, Bacillus licheniformis also
-Different in P reaction. Furthermore, JP-A-1-
Bacillus sp. described in 247081.
Since it differs from PI-7S in the V-P reaction, this bacterium is recognized as a new species, and Bacillus sp. The strain was named 218C strain and deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and its accession number is Microtechnology Research Institute No. 11914. As seen in other common bacteria, the properties of this bacterial strain may change, and mutant strains can be generated by artificial mutation means such as irradiation with ultraviolet rays, X-rays, and treatment with chemicals. Such mutant strains can also be used for the purpose of the present invention as long as they produce enzymes with similar properties to those of the present strain.

[0010]E. Culture of microorganisms For culturing this bacterial strain, ordinary bacterial culture methods can be used, but as a carbon source, in order to induce chitinolytic activity, chitin, such as demineralized crab shell, is used as the main carbon source. It is desirable to use known carbon sources in appropriate combinations as necessary. Further, as the nitrogen source, organic or inorganic nitrogen compounds such as ammonium salts, nitrates, yeast extracts, and peptones are used alone or in combination. Furthermore, inorganic salts such as sodium, potassium, calcium, and magnesium are also added as necessary. The culture temperature is usually 25 to 37°C, preferably 27 to 32°C, and the initial pH is usually 6.0 to 7.5, but a particularly preferable value is around 7.0. The culture time is usually 24 to 96 hours, preferably 48 to 96 hours. When cultured by a method such as aerated agitation culture, chitinase is produced and accumulated in the culture.

[0011] In order to separate and purify chitinase from a culture, it is possible to use the means used for ordinary enzyme purification. Examples include various chromatography methods such as ion exchange chromatography, hydrophobic chromatography, and gel filtration methods, and fractional precipitation using inorganic salts such as ammonium sulfate or organic solvents. Chitinase purified by these operations has the physicochemical properties described below.

[0012]F. Measuring method of enzyme activity 1. Measuring method of chitinase activity: Chitin (manufactured by Wako Pure Chemical Industries),
Finely grind in a ball mill and dissolve in concentrated hydrochloric acid. This is poured into a large amount of water to reprecipitate it, and then finely dispersed with a homogenizer to prepare colloidal chitin. Add 1 ml of enzyme solution to 3 ml of 50 mM acetate buffer (pH 5.0) containing 0.33% colloidal chitin, and react at 37° C. for 10 minutes. The enzyme reaction was stopped by adding 1 ml of 0.5M sodium carbonate solution, and the reducing sugar produced in the reaction solution was quantified. Under these conditions, 1 micromole of N-acetyl-β- The amount of enzyme that releases reducing sugar corresponding to D-glucosamine is defined as one unit of chitinase activity. 2. Measuring method for demineralized chitin degrading activity: Decalcified chitinolytic activity was measured using Mikasion Brilliant Red 5BS.
Chitin EG (manufactured by Katokichi) combined with (manufactured by Mitsubishi Kasei)
Measured using Into 2.5 ml of 50 mM acetate buffer (pH 5.0) containing 20 mg of dye-bound demineralized chitin,
Add 0.5 ml of enzyme solution and react at 37°C for 16 hours. Then, insoluble matter was removed by centrifugation, and the supernatant was
Measure the absorption at 7 nm. Under these conditions, 537n
The amount of enzyme required to increase the absorbance of m by 1 is defined as 1 unit of demineralizing chitinolytic activity.

[0013]G. Properties of enzymes 1. Action: Demineralized chitin, colloidal chitin, N
-Acts on acetylchito-oligosaccharides, chitosan, etc. and hydrolyzes β-glycosidic bonds. Paranitrophenyl-N
-It has no effect on acetyl-β-D-glucosaminide. 2. Optimal pH and stable pH range: When the reaction was performed at 37° C. using colloidal chitin as a substrate, the optimal pH was around 5.0. After treatment at 37° C. for 1 hour, residual activity of 90% or more was shown in the pH range of 4.0 to 9.5. The relationship between pH and enzyme activity is shown in Figure 1, and the pH stability is shown in Figure 2. Good stability has been shown over a wide range of pH 4.5 to 9.5. 3. Optimal temperature and thermal stability: When colloidal chitin was used as a substrate, the optimal temperature was around 60°C at pH 5.0. Furthermore, it was stable at 45° C. or lower when treated at pH 5.0 for 60 minutes. The relationship between temperature and enzyme activity is shown in Figure 3, and the thermal stability is shown in Figure 4. 4. Molecular weight: 56,000±2,00 as measured by gel filtration using Sephacryl S-200
It was 0.

[0014] The above-mentioned enzyme chemical properties are in contrast to those of previously reported chitinases, such as those of other species of the genus Serratia, Streptomyces, or Bacillus, and the degradability of powder or colloidal chitin; pH
The chitinases of the present invention are novel enzymes that differ in stability and range of action.

[0015]

[Example] Example 1 Liquid medium 1 containing 0.5% decalcified chitin (Kimitsukitin C: Kimitsu Chemical), 0.1% peptone, 0.1% yeast extract, 0.1% monopotassium phosphate
Bacillus sp. 218C
(No. 11914, hereinafter abbreviated as 218C strain) was inoculated. After culturing with aeration at 30° C. for 48 hours, the culture solution was centrifuged to remove bacteria, and 90 ml of chitinase-containing supernatant was obtained. The chitinase activity of the supernatant was 0.
It was 5 units/ml.

Example 2 Demineralized chitin, colloidal chitin, and powdered chitin were used as induction substrates, and 2
A chitinase production test was conducted using the 18C strain, and the results are shown in Table 1. 100 ml of a liquid medium (pH 7.0) with a composition of 0.5% chitin, 0.1% peptone, 0.1% yeast extract and 0.1% monopotassium phosphate was placed in a 500 ml Erlenmeyer flask, and heated at 30°C. Culture was carried out with aeration and stirring for 48 hours. As shown in Table 1, the highest enzyme activity was obtained when decalcified chitin was used as the inducing substrate.

[0017]

[Table 1]

Example 3 100 ml of preculture medium (pH 7.0) containing 0.5% peptone, 0.5% yeast extract, and 0.068% monopotassium phosphate was placed in a 500 ml Erlenmeyer flask, and the mixture was heated at 218C. Inoculate the strain at 30℃,
It was cultured with shaking for 1 day. This culture solution is used as the primary culture solution. 1 L of the culture solution with the same composition was taken into a 5 L Erlenmeyer flask, inoculated with 1% of the primary culture solution, and cultured with shaking at 30° C. for 1 day. This culture solution is used as a secondary culture solution. Next, decalcified chitin (Kimitsukitin C: manufactured by Kimitsu Chemical) 0.
5% peptone, 0.1% yeast extract, 0.2% monopotassium phosphate, and 0.01% antifoaming agent. prepared, inoculated with the secondary culture solution, and rotated at a rotation speed of 2.
The cells were cultured at 30° C. for 62 hours at 20 rpm and an aeration rate of 1 vvm. The production status of chitinase activity during culture,
Figure 5 shows the change in pH. The chitinase activity in the medium after completion of the culture was 0.94 units/ml. The culture solution was centrifuged at 10,000 xg for 20 minutes to remove insoluble matter such as bacterial cells, and 80% saturated ammonium sulfate was added to the supernatant to perform salting out. The precipitate fraction was 10,000
It was collected by centrifugation at ×g for 20 minutes, dissolved in 25 mM phosphate buffer (pH 7.0), and dialyzed against the same buffer. The dialysis solution was equilibrated with the same buffer,
The solution was passed through a DEAE-Toyopearl column, and the non-adsorbed fraction was collected to obtain a crude enzyme solution. The recovery rate of chitinase from the culture solution was 80%.

Example 4 Comparison of demineralized chitinolytic activity was carried out as follows. When chitinase derived from the genus Aeromonas (“Chitinase GODO” joint sake refinery), chitinase derived from the genus Trichoderma (“Chitinase T-1” manufactured by Asahi Kogyo), and the chitinase of the present invention were added to deionized water, the chitinase activity values were the same. The strength of the degrading activity against pigment-bound demineralized chitin was compared. As the results are shown in FIG. 5, the chitinase of the present invention has a higher
It was revealed that the demineralizing power of demineralized chitin is strong.

Example 5 The following results were obtained by allowing the chitinase of the present invention to act on chitosan. 1. Chitosan with a degree of deacetylation of 99% (manufactured by Katokichi)
After adding 400 ml of distilled water to 3 g and 3 ml of glacial acetic acid, the mixture was thoroughly stirred and dissolved. After adjusting the pH to 5.0 by adding 2N sodium hydroxide solution, the total amount was adjusted to 5.0.
A 0.6% chitosan solution was prepared. To this was added 37.5 units of the enzyme of the present invention as a chitinase activity, and after reacting for 16 hours in an incubator at 37°C, the reaction was stopped by heating at 100°C for 10 minutes to obtain chitosan decomposition product A. 2. The enzyme of the present invention was added so that the chitinase activity was 150 units, and the same treatment as in 1 was performed to prepare chitosan decomposition product B. 3. Chitosan with a degree of deacetylation of 99% in 1 was replaced with 90% chitosan (manufactured by Katokichi), and the enzyme of the present invention was added thereto so that the chitinase activity was 37.5 units. Chitosan decomposition product C was prepared by reacting in the same manner as above. 4. Chitosan decomposition product D was prepared by the same treatment as in 3 using the enzyme of the present invention so that the chitinase activity was 150 units.

For the above chitosan decomposition products A, B, C, and D, the detector was an RI detector, the eluent was 0.4 M acetate buffer (pH 5.0), and the column was Asahi Pack GS-3.
20, pullulan (SHODEX STAN
DARD P-82) was used as a molecular weight marker, and the molecular weight distribution was investigated by high performance liquid chromatography. Chitosan decomposition products A, B, C, and D each have a molecular weight of 3.
2,000; 17,000; 12,000; 1,000
The molecular weight distribution was centered around , and no accumulation of monosaccharides was observed in any of the decomposition products.

[0022]

[Effects of the Invention] As explained above, a new strain of the genus Bacillus that produces a new chitinase was isolated by using only demineralized chitin as an inducing substrate, and the enzyme produced by this strain can be converted into chitosan. Works efficiently,
It has been confirmed that this can be reduced in molecular weight to produce industrially useful decomposition products.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram showing the relationship between pH and enzyme activity of the enzyme of the present invention.

FIG. 2 is an explanatory diagram showing the pH stability of the enzyme of the present invention.

FIG. 3 is an explanatory diagram showing the relationship between temperature and enzyme activity of the enzyme of the present invention.

FIG. 4 is an explanatory diagram showing the thermostability of the enzyme of the present invention.

FIG. 5 is an explanatory diagram showing the production status of chitinase activity and changes in pH during culturing of the strain of the present invention.

FIG. 6 is an explanatory diagram showing a comparison of the demineralizing chitinolytic activity of the enzyme of the present invention and other chitinases.

[Explanation of symbols]

Curve 1 shows the degrading activity of the enzyme of the invention. Curve 2 is the enzyme “chitinase T-” derived from the Trichoderma genus.
1". Curve 3 is the enzyme “chitinase GO” derived from the genus Aeromonas.
Degradation activity by ``DO'' is shown.

Claims (3)

[Claims] Claim 1: New strain Bacillus sp. 218C strain. Claim 2: Bacillus sp. A novel chitinase produced by the 218C strain. Claim 3: Bacillus sp. 218C strain (Feikoken Kyoiku No. 11914) or its mutant strain is cultured in a medium containing demineralized chitin, the chitinase according to claim 2 is produced in the medium, and the chitinase is collected from the medium. A method for producing chitinase characterized by the following.