JPH03280877A - Production of saccharification-type chitosan liase - Google Patents

Abstract

PURPOSE:To obtain the subject liase having a high productivity with low denaturation thereof in a mild condition by culturing a saccharification-type chitosan liase-producing bacterium belonging to the genus Penicillium and collecting the subject liase from the cultured material. CONSTITUTION:A saccharification type chitosan liase-producing bacterium belonging to the genus Penicillium is cultured and the objective enzyme is collected from the cultured material. In addition, the above-mentioned liase- producing bacterium is preferably Penicillium AF9-P-112 (FERM 11374), Penicillium AF9-P-115 (FERM 113375) or Penicillium AF9-P-128 (FERM 11376). The bacterium is preferably cultured, e.g. at pH5.0-7.0 and at 25 deg.C for 1-7 day.

Description

[Detailed description of the invention] [Industrial application field]

This invention relates to a novel method for producing a saccharified chitosan-degrading enzyme that acts on chitosan and directly decomposes it into its constituent monosaccharide, D-glucosamine.

[Conventional technology]

D-glucosamine can be used as a food additive, chemical component, pharmaceutical, etc., or as a raw material thereof, and has traditionally been produced by thermally decomposing chitosan or chitin with mineral acids such as hydrochloric acid. On the other hand, the method of producing D-glucosamine by allowing a chitosan-degrading enzyme to act on chitosan without mineral acid treatment allows the production of natural D-glucosamine with less denaturation because chitosan can be degraded under fairly mild conditions. become. Until now, the microorganisms that produce chitosan-degrading enzymes have been Bacillus cereus (Japanese Unexamined Patent Application Publication No. 80-188).
0585), Bacillus No. 7-M (Special Publication No. 1
-56755), Alcaligenes MIIK-1 (Japanese Patent Publication No. 62-201.571), Bacillus circulans (Japanese Patent Publication No. 63-94971), Bacillus bamilus (Japanese Patent Publication No. 63-63382), etc.; Seth (J, S, Pr1ce and R
, 5torck, (1975) Journal o
f Bacteriology, Vol, 124.
1574-1585), Nocardia orientalis (Japan Society of Agricultural Chemistry, 1989 Annual Conference, Collection of Lecture Abstracts, p.
809) etc.: Among filamentous fungi, Penicillium islandicum (D, M, Fenton and D, E, Ev
elite. (1981), Journal of Genera
l Old crobiology. Vol. 1213.151-185). Most of the chitosan-degrading enzymes described in these reports are liquefaction-type (or Endo-type) that act on chitosan to release oligosaccharides, and completely degrade D-glucosamine, the constituent monosaccharide of chitosan. It cannot be disassembled. Among the above reports, there are reports on bacteria that produce saccharifying (or Exo-type) chitosan-degrading enzymes that act on chitosan to directly or indirectly release the constituent monosaccharide D-glucosamine. The only report has been on one species, Nocardia orientalis, and there have been no other reports in either filamentous fungi or bacteria.

[Problem to be solved by the invention]

The present inventors focused on an enzymatic decomposition method that can produce D-glucosamine under milder conditions than the currently used acid decomposition method. Therefore, the present invention was made with the object of providing a method that can efficiently produce a saccharified chitosan-degrading enzyme that is essential for the enzymatic decomposition method described above.

[Means to solve the problem]

As a result of searching for microorganisms that produce chitosan-degrading enzymes from various soils, the present inventors discovered that filamentous fungi belonging to the genus Penicillium produce saccharified chitosan-degrading enzymes outside of their bodies, and have developed the present invention. It has been completed. That is, the method for producing a saccharified chitosan degrading enzyme according to the present invention is characterized by culturing a saccharified chitosan degrading enzyme producing bacterium belonging to the genus Penicillium, and collecting the saccharified chitosan degrading enzyme from this culture. Below, (1) the microorganisms used, (2) cultivation of the microorganisms and collection of the saccharified chitosan degrading enzyme, and (3) properties of the saccharified chitosan degrading enzyme will be described in detail. (1) Microorganisms used The saccharifying chitosan-degrading enzyme-producing bacteria belonging to the genus Penicillium used in the present invention is, for example, Penicillium 8F9-P-112, which the present inventors isolated from soil. No. 11374, FERNP-11,374)
, Penicillium 8F9-P-115 (Feikoken Bacillus 11
．． No. 375, FERN P-11375), Penicillium AP9-P-128 (, FERN P-11376)
No., FERM P-11376). As can be seen from the mycological properties shown below, these strains all belong to the genus Penicillium, which is a filamentous fungus, but there has been no previous report that filamentous fungi belonging to the genus Penicillium produce saccharified chitosan-degrading enzymes. Therefore, it is thought to be a new bacterial strain belonging to the genus Penicillium, and both have been deposited at the National Institute of Microbial Technology, Agency of Industrial Science and Technology. Mycological properties of Penicillium AF9-P-112: Culture findings and properties It grows well on malt extract agar medium, and colonies are white at the early stage of development, but turn gray-green as conidia form and mature. . It is an aerobic bacterium. It grows well in a temperature range of about 25°C, and no growth is observed at 37°C. Growth is slow at temperatures above 30°C and below 20°C. The pH at which growth is possible is pH2~pl! 8, but the optimal p
H has a pH of 5 to IIH8.・Morphological properties The morphology of the cells grown on malt extract agar medium was observed under a microscope according to a conventional method. The conidiophore is colorless, and its tip forms a broom-like structure, a benicillus. At this tip, a bead-like chain of conidia is produced. As a result of the above observations, this bacterial strain was determined to be a filamentous fungus belonging to the genus Penicillium. Properties of various media are as follows. Malt extract agar medium Growth status: 25°C, 4 to 5 am Colony color: gray-green, periphery white. The underside is colorless to light yellowish brown. Oatmeal agar medium growth condition = 4 to 50 I colonies for 7 days at 25°C, one color tone:
Gray-green, white margin. Growth status on PDA medium: 4-5 cm colony size in 7 days at 25°C Colony color: pale gray-green with white periphery. The underside is pale yellow to colorless. Growth status on Zapek agar medium: 2.5 to 3.5 co+ colonies in 7 days at 25°C Color tone: pale gray-green, periphery white. Mycological properties of Penicillium AF9-P-115: Culture findings and properties It grows well on malt extract agar medium, and colonies are white at the early stage of development, but turn gray-green as conidia form and mature. . It is an aerobic bacterium. It grows well in a temperature range of about 25°C, and no growth is observed at 37°C. Growth is slow at temperatures above 30°C and below 20°C. The pH at which growth is possible is between pH 2 and pH 8, but the optimum pH is between pH 5 and pH 6.・Morphological properties The morphology of the cells grown on malt extract agar medium was observed under a microscope according to a conventional method. The conidiophore is colorless, and its tip forms a broom-like structure, a benicillus. At this tip, a bead-like chain of conidia is produced. As a result of the above observations, this bacterial strain was determined to be a filamentous fungus belonging to the genus Penicillium. Properties of various media are as follows. Malt extract agar medium Growth condition: 3.5 to 4 cn+ colonies in 7 days at 25°C Color tone: Gray-green, periphery white. The underside is colorless to light yellowish brown. Growth status on oatmeal agar medium: 4-5 cm colonies at 25°C for 7 days Colony color: gray-green, periphery white. PDA medium growth condition: 2.5-3.5 cm at 25°C for 7 days Colony color: gray-green, white periphery. Growth status on Czapek agar medium: 2-3 c+n colonies in 7 days at 25°C Color tone: white to light yellow. Mycological properties of Penicillium AP9-P-128: Culture findings and properties It grows well on malt extract agar medium, and colonies are white at the early stage of development, but turn gray-green as conidia form and mature. . It is an aerobic bacterium. It grows well in a temperature range of around 25°C. Slight growth is observed at 37°C, but no growth occurs at higher temperatures. Growth is slow at temperatures above 30°C and below 20°C. The pH at which growth is possible is between pH 3 and pH 8, but the optimum pH is between pH 5 and pH 6.・Morphological properties The morphology of the cells grown on malt extract agar medium was observed under a microscope according to a conventional method. The conidiophore is colorless, and its tip forms a broom-like structure, a benicillus. At this tip, a bead-like chain of conidia is produced. As a result of the above observations, this bacterial strain was determined to be a filamentous fungus belonging to the genus Penicillium. Properties of various media are as follows. Growth status on malt extract agar medium: 4.5-5.5 CO+ after 7 days at 25°C Colony color: gray-green, with white periphery. The underside is colorless to light yellowish brown. Growth status on oatmeal agar medium: 4.5 to 5.50 rrl colonies in 7 days at 25°C.Single color tone: gray-green, periphery white. PDA medium growth condition: 4.5-5.5 cm at 25°C for 7 days Colony color: gray-green, periphery white. Czapek agar medium Growth status: 2.5 to 3.5 c+n colonies at 25°C for 7 days Colony color: White. The underside is light yellow to light yellowish brown.As can be seen from the above, all three strains belong to the genus Penicillium, but there are slight differences in growth conditions in various media, temperature and pH range at which they can grow. Therefore, they were determined to be different strains. (2) @ Cultivation of living organisms and collection of enzymes As a medium for culturing the filamentous fungi described above to produce saccharified chitosan-degrading enzymes, for example, a medium having the following composition (hereinafter referred to as "basic medium") may be used. I can do it. Chitosan 5. Og Peptone 2・Og Yeast extract 0.5g K112PO4 1.0g MgSO4・7H200.3g Deionized water Mouth pH H6.5 Even if chitosan is not added to the medium, a small amount of enzyme is produced. However, enzyme productivity is higher when chitosan is added to the medium. The chitosan to be added to the culture medium may be chitosan of any degree of deacetylation, and may be in the form of flakes or pulverized, or may be added with dilute acids (for example, inorganic acids such as hydrochloric acid, sulfuric acid, citric acid, etc.). or an organic acid such as acetic acid). Moreover, these chitosan may have any molecular weight, and further, chitosan-containing materials (for example, cell walls of zygomycetes, etc.) may be added to the medium as they are. Other medium components can be used as long as they are commonly used for culturing filamentous fungi. The pH of the culture medium can be set at any PL as long as the strain used can grow. However, preferably the pH is 5.0 to 7.
．． Adjust to 0 and culture. The culture temperature is usually around 25℃, and the number of culture days is 1 to 7.
The culture is carried out by aerated agitation culture, etc. Saccharified chitozanase is produced and accumulated in the medium. After the culture is completed, a crude enzyme solution is obtained by removing bacterial cells from the culture solution by centrifugation, filtration, or the like. This crude enzyme solution can be processed as necessary by ultrafiltration concentration, ammonium sulfate salting out, extraction, etc. to obtain a concentrated crude enzyme solution. It can be made into powder. (3) Properties of saccharified chitosan-degrading enzyme ■ Effect on chitosan The saccharified chitosan-degrading enzyme produced by this invention is as follows:
It acts on chitosan to decompose it and liberate its constituent monosaccharide, D-glucosamine. (2) Optimal pn The activity towards the substrate chitosan was investigated in the range from pH 3.8 to pH 1O.O. The relationship between the buffer used and pi is as follows. pH3,6~6.0 Acetate buffer pl+5.8~7.8 Phosphate buffer pH8,0~I
O30 Sodium carbonate/Boric acid buffer
When 00LJ (trade name manufactured by Wako Pure Chemical Industries, Ltd.) is used, it is insoluble in buffer solutions in the neutral to alkaline range and precipitates. Therefore, when investigating the optimal pH in a wide range of pH ranges, glycol chitosan, which is soluble in a wide range of pH ranges, was used as a substrate. The enzyme activity was measured when the enzyme was reacted with the substrate in various I)H buffers (reaction temperature: 37°C). Results first
This is shown in the graphs of Figs. The optimal pl (which can be read from these graphs) is as follows: Chitosan Glycol 100L Chitosan AF9-P-112 produces around 5 4-6 enzymes (Figure 1) 8 F9-P-115 produces 4. Around 5 5~
6 enzyme (Figure 2) Produced by AF9-P-128 4-5.5 4-6
Enzymes (Figure 3) As can be seen from the table above, all saccharified chitosan degrading enzymes have an optimum pH in the acidic range. (2) Optimal Temperature and Temperature Stability The enzyme activity was measured when the enzyme was reacted with the substrate at two optimal temperatures (pH of the reaction solution was 5.0, and the substrate was 100 L of chitosan). Temperature stability: Diluted enzyme with pH 5.0 acetate buffer, mixed, heat treated at various temperatures for 30 minutes, cooled, and reacted with substrate at 37°C using these enzyme solutions. The activity was measured (reaction solution p11 was 5.0, substrate was 100 L of chitosan). These results are shown in the graphs of FIGS. 4 to 6. The optimum temperature and temperature stability that can be read from these graphs are as follows. 8F9-P-112 Production of enzyme 8F9-P-1,15 Production of enzyme 8F9-P-128 Production at 60℃ (Figure 4) Production at 60℃ (Figure 5) 60℃ 60℃, 30 60℃ for 30 minutes, 50℃ for 30 minutes, Penicillium AP9-P-11
The optimum temperature for the reaction of the saccharified chitosan degrading enzyme produced by 2 and AF9-P-115 is around 60°C;
It remained stable without being deactivated even after heat treatment for 0 minutes. In addition, the optimum temperature for the reaction of the saccharified chitosan-degrading enzyme produced by Penicillium 8F9-P-128 is around 60°C, but 50°C
, was stable up to 30 minutes of heat treatment. ■Activity measurement method for saccharified chitosan degrading enzyme Add 100L of chitosan to 0.1M acetate buffer (all 5.0) at a concentration of 0.
．． A chitosan solution is prepared by dissolving it to a concentration of 5%. Add 200 μg of this chitosan solution to 0.1 M acetate buffer (p
Add 700 μg of H5,0) and 100 μg of enzyme solution, mix well, and react at 37° C. for 30 minutes. After the reaction is complete, the generated free reducing sugars are treated with RondIeM.
organ method (Rondle, C, J, M, and
Morgan, W., T.J., (1955), Bio
Chemical Journal, Vol. 61.
588-589). Note that glucosamine hydrochloride is used as a control. The amount of enzyme that releases 1 μmol of glucosamine per minute under these reaction conditions is defined as 1 unit.

【Example】

This invention will be explained below with reference to Examples. 100 m of basal medium of the above composition containing chitosan
1 was dispensed into a Sakaro flask and autoclaved at 120°C for 20 minutes. Penicillium 8F9 grown on Czapek agar medium
-P-112 mycelial mat pieces (approximately 5 squares x 5 orders) were inoculated into the medium in this flask, and cultured with reciprocating shaking at 25°C for 6 days. After culturing, the culture solution was sterilized and filtered using filter paper (No. 2) to obtain an enzyme solution. The saccharified chitosan degrading enzyme activity of this enzyme solution was 139 milliunits per ml of culture filtrate. Basic medium 311 containing chitosan and having the above-mentioned composition was added to 5
The mixture was dispensed into a small-sized fermentation device with a capacity of 1.5 g and autoclaved at 120° C. for 20 minutes. Into an Erlenmeyer flask containing 100 ml of the same basic medium, add a piece of mycelial mat of Penicillium AF9-P-112 (approximately 5 mm
A seed culture was prepared in advance by inoculating the seed culture with 50% of the seed culture and culturing with rotational shaking at 25° C. for 2 days, and the seed culture was inoculated into the small-sized fermentation apparatus and cultured. The culture conditions in the fermentation apparatus were as follows. Temperature: 25℃ Controlled with hydrochloric acid so as not to exceed medium pH Hall 7 Aeration rate: 311/min Rotation speed: 300 rpm Number of culture days: After culturing for 4 days, the culture solution was sterilized and filtered using filter paper (No. 2), and the enzyme solution was removed. Obtained. The saccharified chitosan degrading enzyme activity of this enzyme solution was 123 milliunits per culture filtrate 1a+I. An enzyme solution was obtained in the same manner as in Example 1 except that Penicillium 8F9-P-115 was used in place of Penicillium AP9-P-112. The saccharified chitosan degrading enzyme activity of this enzyme solution was 248 milliunits per 11 culture filtrate. An enzyme solution was obtained in the same manner as in Example 2, except that Penicillium AP9-P-115 was used in place of Penicillium AF9-P-112, and the number of days for culturing in the fermentation apparatus was 8 days. The saccharified chitosan degrading enzyme activity of this enzyme solution was 289 milliunits per 11 culture filtrates. An enzyme solution was obtained in the same manner as in Example 1, except that Penicillium AF9-P-128 was used in place of Penicillium 8 F9-P-112, and the number of days of culture with reciprocating shaking in the flask was 4 days. The saccharified chitosan degrading enzyme activity of this enzyme solution was 253 milliunits per 11 culture filtrate. Example 6: Enzyme production using Penicillium 8 F9-P-128 in a small fermentation apparatus Penicillium AP9-P-128 was used in place of Penicillium AP9-P-112, and the number of days of culture in the fermentation apparatus was set to 7 days. An enzyme solution was obtained in the same manner as in Example 2 except for this. The saccharified chitosan degrading enzyme activity of this enzyme solution was 559 milliunits per 111 culture filtrate. Example 7: Effect of the obtained enzyme on the substrate 10 μg of each saccharified chitosan degrading enzyme obtained in Examples 1 to 6 above
, chitosan solution (chitosan 100L 511g/ml
(dissolved in acetate buffer at pH 5.0)
They were added to 40 μg of each, mixed, and allowed to react at 37° C. for 1 hour and 24 hours under standing conditions. Next, this reaction solution was heat-treated at 100° C. for 5 minutes to stop the reaction, and then enzymatic degradation products in the reaction solution were analyzed by thin layer chromatography (TLC). TLC plate is rKieselgel 60F J
(trade name, manufactured by Merck & Co., Ltd.), and 2.5 μg of chitosan oligosaccharide mixture (20 B/ml) was used as a standard product. Using n-butanol-acetic acid-water (2:1:2) as a developing solvent, development was carried out according to a conventional method, and spots were detected by coloring with sulfuric acid. The obtained TLC analysis pattern is shown in FIG. As you can see from this pattern, Penicillium 8F9-
P-112, 8F9-P-115 and 8F9-P-1
The saccharified chitosan-degrading enzyme produced by No. 28 acted on chitosan to produce only the D-glucosamine monosaccharide in both 1-hour and 24-hour reactions, and no chitosan oligosaccharides were detected. Ta.

【Effect of the invention】

As can be seen from the above, according to the present invention, a saccharified chitosan-degrading enzyme can be produced by culturing a novel filamentous fungus belonging to the genus Penicillium. The saccharified chitosan-degrading enzyme obtained in this invention can decompose chitosan by acting on it to produce D-glucosamine, which is its constituent monosaccharide. This enzymatic decomposition acts on chitosan under milder conditions than conventional thermal decomposition using mineral acids and decomposes it into D-glucosamine, making it possible to produce natural D-glucosamine with less denaturation. .

[Brief explanation of drawings]

Figures 1, 2 and 3 show Penicillium 8 F9-P-112, Ar1-P-115 and Ar1, respectively.
- A graph showing the relationship between the enzymatic activity of the saccharified chitosan degrading enzyme produced by P-128 and p++; Figures 4, 5 and 6 are respectively
2, 8 A graph showing the relationship between the enzyme activity and temperature of the saccharified chitosan degrading enzyme produced by F9-P-115 and Ar1-P-128; and FIG.
-P-112, Ar1-P-115 and Ar9-P-
128 is a TLC analysis pattern showing the analysis results of decomposition products when each of the saccharified chitosan-degrading enzymes produced by No. 128 were allowed to act on chitosan. 5 Figure H Figure 2 ■ Figure H Figure 4 Temperature ('C)

Claims (1)

[Scope of Claims] 1. A method for producing a saccharified chitosan degrading enzyme, which comprises culturing a saccharified chitosan degrading enzyme-producing bacterium belonging to the genus Penicillium, and collecting the saccharified chitosan degrading enzyme from this culture. 2. The saccharified chitosan-degrading enzyme-producing bacteria are Penicillium AF9-P-112 (Feikoken Bibori No. 11374), Penicillium AF9-P-115 (Feibokuken Bibori No. 1137).
Claim 1 characterized in that it is Penicillium AF9-P-128 (Feikoken Bibori No. 11376).
The method for producing the saccharified chitosan degrading enzyme described above.