JPH01291793A - Production of chitinase - Google Patents

Abstract

PURPOSE:To perform the decomposition of chitin at a low cost, by culturing AF6-T8 strain of genus Trichoderma in a medium. CONSTITUTION:Trichoderma AF6-T8 strain (A) having the microbiological properties such as capability of generating conidiophore from aerial mycelium is separated from cultured product of the soil of plowed land. The strain A is inoculated on a liquid medium (B) containing colloidal chitin or fine particles of chitin and peptone, etc., and cultured under aeration and agitation at about 25 deg.C for 2-3 days to obtain a cultured product (C) having a chitinase activity of 3-5 unit/ml. The component C is centrifuged, salted out and purified by chromatography, etc., to obtain a chitinase having the following properties. Action, exerting decomposition action to chitin; optimum pH, 4-6.5; optimum temperature, about 37 deg.C (at pH5.2); molecular weight, about 40,000; ultraviolet absorption spectrum of aqueous solution, exhibits a peak at 280nm; specific activity, about 300 unit/mg-protein (measured by turbidimetry using colloidal chitin as a substrate); etc.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a method for producing chitinase, which is a chitin-degrading enzyme, using a new strain of the genus Trichoderma.

<Prior Art> Chitin and chitin substances are widely distributed in nature as components contained in crustaceans such as shrimp, crabs, and krill, and insects, and are also known as constituent components of filamentous fungal cell walls.

Chitinases, which are chitin-degrading enzymes, are known to exist in animals and plants, but in research on their use as enzymes, those derived from microorganisms are often used.

Many types of microorganisms that produce chitinases have been reported; for example, the genus Streptomyces has been reported in Japanese Patent Publication No. 48-35476 and Japanese Patent Application Laid-Open No. 61-40790, and bacteria have been reported in Japanese Patent Application Publication No. 60-54 [1989]. It is described in No. 381, etc.

Furthermore, collecting chitinases from cultures of Trichoderma bacteria has been described, for example, by Toyama et al. (J.

Fern+ent, Tachnol,, 45.6
63 (1967)) and Kitamoto et al. (Trans, Hyco
1, Soc, Japan, 26.69 (1984)). The method of Toyama et al.
Viride (TrichoderrBa viride)
Kitamoto et al.'s method produces chitinase by culturing Trichoderma harzianum (T, harzianun+) in a liquid using basidiomycete mycelia or fruiting bodies as a carbon source. This is a method of producing it.

<Problems to be Solved by the Invention> In recent years, chitinases produced by these microorganisms have come to be used as effective cell wall lytic enzymes for preparing protoplasts for protoplast fusion, which is a method of microbial breeding. The microorganisms targeted for protoplast fusion are mainly fungi such as Aspergillus aspergillus, yeast, and basidiomycete. These fungi are widely used for brewing, fermentation, and food, and there are many types of them. Since the components are different, a variety of effective cell wall lytic enzymes are desired.

In addition, research is underway to use chitinase not only as a cell wall lytic enzyme, but also as an enzyme that acts on chitin, chitin-containing natural polymers, and chitooligosaccharides as food materials and medical materials. From this point of view as well, the emergence of a variety of chitin-degrading enzymes is desired.

Therefore, an object of the present invention is to provide a new method for producing chitinase, which can advantageously decompose chitin and, when used in combination with glucanase, can advantageously convert various fungi into protoplasts.

Means for Solving the Problem> In the process of searching and researching antagonistic microorganisms for soil pathogenic bacteria, the present inventor found that among the bacteria isolated from soil, there were some bacteria that were able to dissolve the cell walls of soil pathogenic bacteria. The beginning,
Trichoderma AF6-, a strain belonging to the genus Trichoderma
It passed 78 stocks.

This fungus is a soil pathogen, Particillium (Vert.
icilliu+++) and Fusarium (Fusar i
We have discovered that not only does it effectively dissolve cell walls such as um), but also that its culture produces chitinase that decomposes colloidal chitin and powdered chitin.

This invention is based on the above findings.

That is, the method for producing chitinase according to the present invention is characterized by culturing AFL-78 strain belonging to the genus Trichoderma and collecting chitinase from the culture.

The following sections will be described in detail in the order of (1) the microorganism used, (2) culture of the microorganism and collection of chitinase, and (3) properties of chitinase.

(1) Microorganisms used The chitinase-producing bacterium used in this invention is the AF6-78 strain belonging to the genus Trichoderma, which was isolated from field soil and was transferred to the Institute of Microbial Technology, Agency of Industrial Science and Technology in 1985.
On the 17th of May, “FER
NP-10019).

The mycological properties of this strain are as follows.

杵゛・F and - The growth conditions were observed on malt extract agar medium and potato dextrose agar medium.The growth conditions were observed to be the same on both media, and the growth was fast, and there was little aerial idleness in the colony at first. The vegetative hyphae appear white. Gradually, fine, woolly-like, fluff-like aerial hyphae are produced, and the color of the hexaconidia, which produce conidia (asexual spores) in a ring pattern, becomes dark green over time. . The underside is planar, no pigment production is observed, and older cultures produce a coconut odor.

Growth at 37°C is not observed; the optimum temperature for growth is around 25°C. Growth is slow at temperatures below 20°C and above 30''C.

It grows poorly at pH 2, but grows at pH 2-10.

This strain was grown in a conventional manner on malt extract agar plates, slanted media, etc., slide specimens were prepared, stained with lactophenol cotton blue, and shape B was observed under a side microscope.

■It arises from conidiophore vaporized hyphae and hardens into a fluffy shape, about 4 μm wide, colorless, and each branch is divided almost at right angles, with a phialide at the tip.

■An average of 3 whorls grow below the phialide at the tip of the phialide branch, and they occur at nearly right angles, with 1 lateral growth or 2 opposite growths.

The average size is 10 x 2.8 μm, and it is straight or slightly curved. It has a YK shape, and the proximal end is MA.

■Conidia Phialo-type conidia, exhibiting a spherical shape. It has a smooth surface, an average diameter of 3 x 2.8 μm (length-to-width ratio is 1.25 or less), and a dark green color.

From the above morphological observation results, according to the classification of l1ifai, this seedling morphologically matches Trichoderma harzianum Rifai (■, harzianun Rifai). Furthermore, among the examples of chitinase production by Trichoderma mentioned above, the report by Kitamoto et al. uses Trichoderma harzianum.

However, these documents only describe the morphological properties of the bacterial strains, so other mycological properties cannot be compared.Furthermore, the source of the bacterial strain reported by Kitamoto et al. is shiitake logs. On the other hand, AF1 used in this invention
The source of energy for strain -78 is field soil. Therefore AFL-78
It cannot be immediately determined that the strain is the same as Trichoderma harzianum of Kitamoto et al.

(2) Cultivation of microorganisms and collection of chitinase J For culturing and preserving this strain, a medium normally used for filamentous fungi, such as a potato extract-glucose medium, etc., can be used, and a medium containing chitin can also be used. good.

As a medium for chitinase production, a liquid medium containing chitin (powdered chitin, colloidal chitin, particulate chitin, mycelia and fruiting bodies of mushrooms, mycelia of filamentous fungi, etc.), a nitrogen source, and inorganic salts is preferable. Specifically, for example, 2.0 g of peptone, 0.5 g of yeast extract, 1.09 g of potassium dihydrogen phosphate, 0.3 g of magnesium sulfate, and 1.0 g of colloidal chitin were dissolved and suspended in 1 j of water, and the pH was adjusted to 5.0. Liquid media can be preferably used.

In slant culture, for example, after inoculating a potato extract-glucose agar slant medium, culture at 10 to 35°C, preferably around 25°C for 7 to 10 days, and store at f & 5°C for a short period of time. I can do it.

For chitinase production, a liquid medium such as the one described above is used, and shaking culture in a flask or jar fermenter is used.
Aerated agitation culture can be performed using a tank or a tank.

For example, if the above liquid medium 3j is placed in a 5j volume jar fermenter and cultured with aeration at 25°C, chitinase will be produced and accumulated as the bacteria proliferate, and the accumulated amount will reach its maximum in 2 to 3 days. , the chitinase activity at that time is 3~
It reaches 5 units/111 (according to turbidity method). This value is
Compared to the above-mentioned report by Kitamoto et al. (production of chitinase by liquid culture of T. harzianul using basidiomycete hyphae or fruiting bodies as a carbon source), Kitamoto et al.'s method for measuring chitinase activity (by colorimetry) and use of Although a strict comparison cannot be made because the substrate is not the same as that of the method of this invention, the chitinase productivity (accumulated amount of chitinase per unit volume of medium) by the method of this invention is thought to be 5 to 10 times that of Kitamoto et al. It will be done.

19-2-pronged and 1 As described above, chitinase-producing bacteria are cultured by the method of the present invention, and chitinase is accumulated in the culture solution. After the culture is completed, a crude enzyme solution is obtained by removing the bacterial cells by centrifugation, r. filtration, or the like. A concentrated crude enzyme solution can be obtained by subjecting this crude enzyme solution to treatments such as ultra-superconcentration, ammonium sulfate salting out, and extraction as necessary. If this is dried by freeze-drying or the like, enzyme powder can be obtained.

These crude enzyme solutions and crude enzyme powder solutions are: &
It can be used for the dissolution of chitin substances such as colloidal chitin, the protogelastization of Manae Aki by using in combination with glucanase, etc.

Note that the above-mentioned crude enzyme contains chitobiase, which is an enzyme that decomposes chitooligosaccharide into N-acetylglucosamine. If necessary, this chitobiase can be separated from chitinase by a known method such as affinity chromatography using chitin or ion exchange chromatography. Chitinase activity can be measured by a known colorimetric method using p-nitrophenyl-N-acetyl-β-D-glucosaminide.

In this invention, the crude enzyme obtained when colloidal chitin is used as a culture substrate is chitinase produced by Trichoderma harzianum as reported by Kitamoto et al.
) does not contain proteases such as those in

The chitinase in the crude enzyme obtained by the method of the present invention described above is purified by known methods such as affinity chromatography or ion exchange chromatography using chitin, and a single band is isolated by 5O8-polyacrylamide gel electrophoresis. The protein can be isolated as shown below.

(3) Properties of chitinase The chitinase obtained by this invention is characterized by the so-called end
It is an o-type chitinase and has the following properties.

■Action: Acts on kidneys and breaks them down.

■To 3i! ! pH: p when using colloidal chitin as a substrate
H4~6.5 ■Optimal temperature: When colloidal chitin is used as a substrate, around 37℃ (pH 5,2) ■pH stability: When treated at 30℃ for 30 minutes, DH4~
7 shows residual activity of 60% or more.

■Temperature stability: at pH 5.2, 0-40℃, 3
Shows residual activity of 95% or more after 0 minute treatment.

■Molecular weight: Approximately 40,000 (according to SO3-polyacrylamide gel electrophoresis.) ■Ultraviolet absorption spectrum of aqueous solution + Shows shuttle-sized absorption at 280 nn.

■Extinction coefficient ([1% IC[l 280r+n+):
12.6 ■Specific activity: Approximately 300 units/rng protein (Activity measurement is by turbidity method using colloidal chitin as a substrate, protein quantification is by the method of Lovry et al.) The measurement of chitinase activity and the display of activity units are as follows. I did it like this.

Reaction buffer for activity measurement 3.7 nj Substrate suspension 0.2 iJ! Enzyme solution 0.1 ifI Here, the buffer is Mac Ilva with pH 5.2.
i ne [Using street liquor, the substrate suspension was colloidal chitin (
For example, Fermentation Engineering Journal, Vol. 42, 212 (1964
) prepared by a method similar to the method described in ) was suspended in hot water, and the concentration was adjusted to the turbidity of the reaction solution (0, 0, 66
0 nl) was adjusted to approximately 1.0.

After mixing the above reaction solution, heat the mixture at 37°C for 30 minutes to react the enzyme and substrate, and immediately reduce the turbidity (0.0°660n).
I) was measured to determine the turbidity reduction rate relative to the control. As a control, a buffer solution of 0.10 was used instead of the enzyme solution.
Those treated in the same manner were used.

The chitinase activity unit was expressed as one unit, which was the amount of enzyme that reduced the turbidity of the reaction solution by 1% per minute.

In addition, when comparing the activity of chitinase in this invention with the activity of known enzymes, reducing sugars produced in the same reaction solution system as above, for example,
The activity value can also be determined by colorimetric determination using the n method or the like.

<Example> This invention will be explained below by giving examples.

Add 11 parts of water to 2.0 g of peptone, 0.5 g of yeast extract, 0.3 g of KM magnesium, 1.0 g of colloidal chitin, 2.0 g of peptone, 0.5 g of yeast extract, 1.0 g of colloidal chitin, stir well, and add with caustic soda water. 1) Prepare a liquid medium by adjusting 11 to 5.0. Transfer this medium to a 5001 volume Sakaro flask at 100 l.
! The mixture was dispensed into Jl portions and sterilized at 120°C for 20 minutes.

Potato extract - A obtained by glucose agar slant culture
Spores of F6-T8 strain were inoculated into the above medium and incubated at 25°C.
Culture was performed with reciprocating shaking. The chitinase activity of the cultured P solution obtained by separating bacterial cells using two pieces of paper reaches its maximum between 48 and 72 hours, and the activity value (turbidity method) is 4.2 units/
It was lj.

The same culture medium 31 as in Example 1 was placed in a 51-volume jar fermentor, and after sterilization at 120°C for 20 minutes, AFL
A spore suspension of -T8 strain was inoculated and cultured with aeration and stirring at 25°C. After 65 hours, the chitinase activity (turbidity method) of the culture P solution obtained by separating the bacterial cells from P was 5.2 units/n+
It was jl.

When this culture P solution was concentrated by ultrafiltration (using "Labo Module" manufactured by Asahi Kasei Industries, Ltd.), a concentrated enzyme solution of 50 units/11 (turbidity method) was obtained.

In addition, when this concentrated enzyme solution is freeze-dried and smoked, 20 units/l
A crude enzyme powder of lCl (turbidity method) was obtained.

Further, this crude enzyme solution was adsorbed onto colloidal chitin, coexisting chitobiase and other substances were removed as a non-adsorbed fraction, and the colloidal chitin adsorbed with chitinase was suspended in a pH 5.2 buffer solution and heated at 25°C. When left overnight, a chitinase solution in which most of the colloidal chitin had been digested was obtained. After removing impurities by centrifugation, D
The mixture was adsorbed onto an EAE-5P14 column (manufactured by Tosoh Corporation), washed with a buffer solution, and eluted with a buffer solution containing sodium chloride to obtain a chitinase active fraction. The obtained active fraction was subjected to SO3-polyacrylamide gel electrophoresis.
It was shown as a single band at a molecular weight of 40,000, and the chitinase activity (turbidity method) was about 300 units per 110 of the protein.

<Effects of the Invention> As can be seen from the above, according to the present invention, chitinase can be provided as a crude enzyme or a purified enzyme.

The chitinase obtained in this invention can advantageously decompose chitin. Furthermore, since the crude enzyme obtained in this invention contains chitobiase, it decomposes chitin and N-
Acetylglucosamine can be advantageously obtained.

Furthermore, by using the chitinase obtained in this invention in combination with a commercially available glucanase, it is possible to more advantageously convert koji molds, basidiomycetes, phytopathogenic filamentous fungi, etc. into protoplasts. - An example will be explained below as an experimental example.

Fire Wataruyo The enzyme solution used is as follows.

Enzyme solution A: Commercially available glucanase Z (2111g/IIJ
) only Enzyme solution B: City version glucanase N (5 ng#aJ) only Enzyme solution C: Enzyme solution A or B to which the chitinase obtained in this invention was added Take about 0.5 g of live mycelia of the test bacteria and Add 5 lj of each of the above enzyme solutions prepared with 2011+HHES slow-moving solution (pH 5,2) containing .68 mannitol, shake gently at 37°C, and after about 60 minutes, count the number of protoplasts produced using a sequential microscope. It was measured. The results are shown in the table below.

+ in the table indicates the number of 10 deplasts from 1 to 10 per field of view (X100), ++ indicates the number of 10 deplasts from 1 to 4 per field of view (X400), +
++: 5 or more in 1 field of view (X400), each rg1
Indicates that something has been noticed.

As can be seen from the above table, when the chitinase obtained by the present invention is added to a commercially available glucanase when producing a protoplast from Aspergillus, Basidiomycota, or filamentous fungi, it significantly accelerates the production of the protoplast. increase the number of

Claims (1)

[Claims] 1. Cultivating the AF6-T8 strain belonging to the genus Trichoderma,
A method for producing chitinase, which comprises collecting chitinase from the culture. 2. The method according to claim 1, wherein a liquid medium containing colloidal chitin or particulate chitin is used as a culture substrate when culturing the AF6-T8 strain.