JPH0789923B2 - Method for producing chitosan degrading enzyme - Google Patents

Description

Detailed Description of the Invention [Industrial applications]

The present invention relates to a novel method for producing a chitosan-degrading enzyme that acts on chitosan to decompose it to produce D-glucosamine and chitosan oligosaccharide.

2. Description of the Related Art D-glucosamine and chitosan oligosaccharides are known to have some physiological activities such as antibacterial activity and behavioral factor activity. Food additives, chemical components, pharmaceuticals, etc. Utilization research is underway as a raw material for These D-glucosamine and chitosan oligosaccharide are
It can be produced by degrading chitosan with a chitosan degrading enzyme. Regarding microorganisms that have produced chitosan degrading enzymes, Bacillus cereus (Japanese Patent Laid-Open No. 18058/1985)
5), Bacillus No. 7-M (Japanese Patent Publication No. 1-56755), Alcaligenes MHK-1 (JP-A-62-201571), Bacillus circulans (JP-A-63-94971), Bacillus pamilus (JP-A-SHO) 63-63382), etc .; Streptomyces in actinomycetes (JSPrice and R. Storck, (1975) Journal of Bact.
ericlogy, Vol.124 , 1574-1585), Nocardia orientalis (Agricultural Chemistry Society of Japan, 1989 Annual Conference, Abstracts p.608), etc. ), Journal
al of General Microbiology, Vol.126 , 151-165). Most of the chitosan degrading enzymes described in these reports have a liquefaction type (or Endo-type) activity that acts on chitosan to release a chitosan oligosaccharide, and D- which is a constituent monosaccharide of chitosan. It cannot be completely decomposed into glucosamine. In the above report, a glycated form (or Exo which acts on chitosan to release its constituent monosaccharide D-glucosamine)
The only report of a bacterium producing a chitosan-degrading enzyme having − type) activity is Nocardia orientalis, which is one of actinomycetes, and other than that, it has not yet been reported in filamentous fungi or bacteria. Absent.

[Problems to be Solved by the Invention]

In addition to the previously reported chitosan-degrading enzyme-producing microorganisms described above, if a microorganism that produces a chitosan-degrading enzyme having a liquefaction type or saccharification type activity can be found, it is possible to use this type of enzyme under mild conditions. At D-
Glucosamine or chitosan oligosaccharide can be produced. Therefore, the present invention has been made for the purpose of finding a new chitosan-degrading enzyme-producing microorganism and providing a novel method capable of efficiently producing a chitosan-degrading enzyme using this microorganism.

[Means for Solving the Problems]

The present inventors have conducted a search for microorganisms that produce chitosan-degrading enzymes from various soils, and found that filamentous fungi belonging to the genus Verticillium produce chitosan-degrading enzymes outside the cells, and It has been completed. That is, the method for producing a chitosan-degrading enzyme according to the present invention is characterized by culturing a chitosan-degrading enzyme-producing bacterium belonging to the genus Verticillium and collecting the chitosan-degrading enzyme from the culture. Hereinafter, (1) microorganisms used, (2) culture of microorganisms and collection of chitosan-degrading enzymes, and (3) properties of chitosan-degrading enzymes will be sequentially described in detail. (1) Microorganisms used Examples of the chitosan-degrading enzyme-producing bacterium belonging to the genus Verticillium used in the present invention include, for example, Verticillium AF9-V-156 (microorganisms-derived microorganism isolated from soil by the present inventors. No. 11377, FERMP-11377). This strain belongs to the genus Verticillium, which is a filamentous fungus as can be seen from the mycological properties shown below, but there is no conventional report that filamentous fungi belonging to the genus Verticillium produce chitosan-degrading enzymes. , Which is considered to be a new strain belonging to the genus Verticillium, and has been deposited at the Institute of Microbial Technology, Institute of Industrial Science and Technology. -Cultural findings and properties Growth on malt extract agar and Tuapeck agar is good, and pure white and velvety-like hypha mats are formed. 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 above 30 ° C and below 20 ° C. The pH that can grow is pH 3 to pH 9, but the optimum pH is pH 5 to pH 6.・ Morphological properties Phialides are formed from conidia peduncle in a singular or ring-like form, and the tip is thin. Conidia originated from the phialide, and one laterally colonized at the tip of the phialide, exhibiting a half-convex, half-concave or plano-convex lens type. From these observation results, this strain was determined to be a filamentous fungus belonging to the genus Verticillium. The properties of various media are as follows. Malt extract agar medium Growth condition: 4.5-5.5 cm colonies: 5 mm or more over 7 days at 25 ° C. It produces a pale magenta to magenta pigment in the medium. Oatmeal agar medium Growth condition: 4.5-5.5 cm colonies: 5 mm or more over 7 days at 25 ° C. It produces a pale magenta to magenta pigment in the medium. PDA medium Growth condition: 4.5-5.5 cm colonies: 5 mm or more over 7 days at 25 ° C. Tuapec agar medium Growth condition: 2.5-3.5 cm colonies: 5 mm or more over 7 days at 25 ° C. (2) Culture of microorganism and collection of enzyme As a medium for culturing the above-mentioned filamentous fungus to produce chitosan-degrading enzyme, for example, a medium having the following composition (hereinafter referred to as "basic medium") can be used. The chitosan 5.0g peptone 2.0g Yeast extract _{0.5g KH 2 PO 4 1.0g MgSO 4} · 7H 2 O 0.3g chitosan to be added to the deionized water 1 pH 6.5 medium, a what kind of chitosan deacetylation degree Further, it may be in the form of flakes, crushed ones, or a solution dissolved in a dilute acid (for example, an inorganic acid such as hydrochloric acid or sulfuric acid, or an organic acid such as citric acid or acetic acid). The molecular weight of these chitosans may be any, and further those containing chitosan (eg cell walls of zygomycetes) may be added to the medium as they are. Other medium components can be used as long as they are used for usual culture of filamentous fungi. The pH of the medium may be any pH as long as the strain used can grow, but it is preferably adjusted to pH 5.0 to 7.0 and cultivated. The culturing temperature is usually around 25 ° C., the number of culturing days is 1 to 7 days, and the culturing is carried out by aeration and stirring culturing or the like. Chitosan degrading enzyme is produced and accumulated in the medium. After the completion of the culture, the crude enzyme solution is obtained by removing the bacterial cells from the culture solution by centrifugation or filtration. The crude enzyme solution may be subjected to a treatment such as ultrafiltration concentration, ammonium sulfate salting-out and extraction, if necessary, to give a concentrated crude enzyme solution. It can be a powder. (3) Properties of chitosan degrading enzyme Action on chitosan The chitosan degrading enzyme produced according to the present invention acts on chitosan to decompose it, and by changing the amount of enzyme and the reaction time, the monosaccharides D-glucosamine and chitosan. Releases oligosaccharides, or mixtures of these. Therefore, this chitosan degrading enzyme has both a saccharification type activity that produces D-glucosamine as a degradation product and a liquefaction type activity that produces chitosan oligosaccharide. The activity against the substrate chitosan was examined in the optimum pH range from pH3.6 to pH10.0. The relationship between the buffer solution used and the pH is as follows. pH3.6-6.0 Acetate buffer pH5.8-7.8 Phosphate buffer pH8.0-1.0 Sodium carbonate / borate buffer 100% deacetylated chitosan "Chitosan 100L" (Wako Pure Chemical Industries, Ltd.) as the substrate chitosan (Product name)
When used, it is insoluble and precipitates in the buffer solution in the neutral to alkaline range. Therefore, when examining the optimum pH in a wide pH range, glycol chitosan soluble in a wide pH range was used as a substrate. The enzyme activity was measured when the enzyme was reacted with the substrate in buffer solutions of various pH (reaction temperature 37 ° C). The results are shown in the graph of FIG. As can be seen from these graphs, the chitosan degrading enzyme produced by the present invention has an optimum pH in the acidic range (pH 4 to 5.5 for 100 L of chitosan and pH 4 to 6 for glycol chitosan). Optimum temperature and temperature stability Optimum temperature: The enzyme activity was measured when the enzyme was reacted with the substrate at various temperatures (reaction solution pH 5.0, substrate chitosan 1
00L). Temperature stability: Diluted and diluted enzyme with acetate buffer of pH 5.0 for 30 minutes at various temperatures, then cooled and left after reacting with the substrate at 37 ℃ using these enzyme solutions. The activity was measured (pH of reaction solution was 5.0, substrate was chitosan 100)
L). The results are shown in the graph of FIG. As can be seen from this graph, the optimum reaction temperature of the chitosan degrading enzyme produced by the present invention is about 50 ° C, and the temperature stability is 40 ° C, 30 ° C.
It was stable until heat treatment for 1 minute. Chitosan degrading enzyme activity measurement method 0.1M acetate buffer (pH 5.0) with 100L chitosan concentration 0.5%
To obtain a chitosan solution. This chitosan solution 200μ is added to 0.1M acetate buffer (pH 5.0) 70
Add 0μ and 100μ of enzyme solution, mix well, and incubate at 37 ℃ for 30 minutes. After the reaction was completed, the produced free reducing sugar was treated with the Rondle-Morgan method (Rondle, CJMand Morgan, WTJ (1955), Biochemic
al Journal, Vol. 61 , 586-589). Glucosamine hydrochloride is used as a control. Under this reaction condition, the amount of enzyme that releases 1 μmol of glucosamine per minute is 1 unit.

【Example】

The present invention will be described below with reference to examples. Example 1: Enzyme production in a flask with Verticillium AF9-V-156 100 ml of the basic medium having the above-mentioned composition containing chitosan was dispensed into a Sakaguchi flask, and autoclaved at 120 ° C for 20 minutes. Verticillium AF grown on Tuapec agar
A mycelial mat piece (about 5 mm × 5 mm) of 9-V-156 was inoculated into the medium in this flask, and reciprocal shaking culture was carried out at 25 ° C. for 4 days. After culturing, the culture solution was subjected to sterilization filtration using a paper filter (5C) to obtain an enzyme solution. The chitosan-degrading enzyme activity of this enzyme solution was 137 milliunits per ml of the culture filtrate. Example 2: Enzyme production in a small fermenter with Verticillium AF9-V-156 5 basal medium 3 of the composition described above containing chitosan.
It was dispensed into a small capacity fermenter and sterilized by autoclaving at 120 ° C for 20 minutes. Erlenmeyer flasks containing 100 ml of the same basic medium were inoculated with mycelial mat pieces (about 5 mm x 5 mm) of Verticillium AF9-P-156 and cultivated at 25 ° C for 2 days with rotary shaking to prepare seed cultures in advance. This was inoculated into the small fermentor and cultured. The culture conditions in the fermenter were as follows. Temperature 25 ° C Aeration rate 3 / min Rotation speed 300 rpm Number of culture days 2 days After culturing, the culture solution was sterilized and filtered using a filter paper (5C) to obtain an enzyme solution. The chitosan-degrading enzyme activity of this enzyme solution was 300 milliunits per ml of the culture filtrate. Example 3: Action of the obtained enzyme on the substrate 10μ of the chitosan-degrading enzyme solution obtained in the above example,
Chitosan solution (100 L of chitosan is adjusted to pH 5.
40 μm (dissolved in 0 acetate buffer), mixed and reacted at 37 ° C. for 1 hour and 24 hours under static conditions. Next, this reaction solution was heated at 100 ° C. for 5 minutes to stop the reaction, and then subjected to thin layer chromatography (TL
The enzymatic degradation products in the reaction solution were analyzed by C). TLC plate is "Kieselgel 60F" (product name manufactured by Merck)
Is used as the standard, and the chitosan oligosaccharide mixture (20 mg / m
l) 2.5 μ was used. N-Butanol-acetic acid-water = 2: 1: 2 was used as a developing solvent, the solvent was developed according to a conventional method, and spots were detected by coloring with sulfuric acid. The TLC analysis pattern obtained is shown in FIG. As you can see from this pattern, Verticillium AF9
-V-156 produced chitosan degrading enzyme is chitosan 100
When L is allowed to act, chitosan oligosaccharides are produced in the early stage of the enzymatic reaction, and D-glucosamine is produced when the enzymatic reaction is completed. From this, the chitosan-degrading enzyme obtained by the present invention contains both saccharifying activity that acts on chitosan to produce D-glucosamine as a degradation product and liquefying activity that produces chitosan oligosaccharide. It became clear that it is.

【The invention's effect】

As described above, according to the present invention, a chitosan-degrading enzyme can be produced by culturing a novel filamentous fungus belonging to the genus Verticillium. The chitosan-degrading enzyme obtained in this invention has both a saccharification type activity that acts on chitosan to produce D-glucosamine as a degradation product and a liquefaction type activity that produces chitosan oligosaccharide. Therefore, by changing the amount of the enzyme that acts on chitosan and the reaction time, it is possible to generate a degradation product composed of monosaccharide D-glucosamine, chitosan oligosaccharide, or a mixture thereof. Furthermore, the chitosan degrading enzyme obtained in this invention alone,
Alternatively, by acting on filamentous fungi, especially zygomycetes, in the presence of glucanase or the like, these can easily be converted into protoplasts. An example will be described below as an experimental example. Experimental Example Spore spores of Rhizopus oligosporus NRRL 2549, which is a zygote, were suspended in potato leachate and sucrose liquid medium, and germinated at 30 ° C. to about 10 μm. This was centrifuged to separate the germinated spores of spores of 0.6 M mannitol.
Suspended in MES buffer. The concentrated enzyme solution (chitosan degrading enzyme activity: about 1 unit / ml) obtained by subjecting the crude enzyme solution of the chitosan-degrading enzyme according to the present invention obtained in the above-mentioned example to ultrafiltration is treated with the above-mentioned germinated spore suspension. And incubated at 30 ° C. with gentle shaking. In addition, in addition to the concentrated enzyme solution of chitosan-degrading enzyme according to the present invention, "Novozyme 234" (trade name of glucanase manufactured by Novo Co.) was used in combination, and even when only "Novozyme 234" was used as a control, no enzyme was used. The addition case was also tested. The results are summarized in the table below. As can be seen from the table below, it was observed that the chitosan-degrading enzyme according to the present invention, or by using it in combination with glucanase, easily converted the germinated spores of spores of Lysopath oligosporus into protoplasts in a short time, and that the protoplast yield was high. .

[Brief description of drawings]

Fig. 1 is a graph showing the relationship between the pH of the chitosan-degrading enzyme produced by Verticillium AF9-V-156 and pH; Fig. 2 is the chitosan-degrading enzyme produced by Verticillium AF9-V-156. FIG. 3 is a graph showing the relationship between the enzyme activity of Escherichia coli and temperature; and FIG. 3 is a TLC analysis pattern showing the analysis results of the degradation products when the chitosan degrading enzyme produced by Verticillium AF9-V-156 was allowed to act on chitosan. Is.

Claims (2)

[Claims] 1. A method for producing a chitosan-degrading enzyme, which comprises culturing a chitosan-degrading enzyme-producing bacterium belonging to the genus Verticillium and collecting the chitosan-degrading enzyme from the culture. 2. The method for producing a chitosan-degrading enzyme according to claim 1, wherein the chitosan-degrading enzyme-producing bacterium is Verticillium AF9-V-156 (Microtechnical Laboratory No. 11377).