JPH0353909B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for preparing healthy seaweed protoplasts, and more particularly, to a method for preparing seaweed protoplasts that can be effectively applied to cell fusion. In recent years, research on so-called cell fusion, the fusion between cells of different species as a genetic engineering method, has been actively conducted in various fields, and some studies have already reached the stage of practical application for land plants. It became like that. However, the polysaccharides that make up the cell walls of seaweeds, especially seaweed, are different from the polysaccharides (mainly cellulose) found in land plants, and are mainly indigestible polysaccharides such as xylan and mannan. , it was thought that it would be difficult to convert into protoplasts by enzymatic treatment. Incidentally, according to a report by Preston et al., mannan exists in granular form on the surface layer of seaweed, the cell wall of seaweed is composed of xylan in the form of microfibrils, and porphyrane exists as a cell filling substance. It is said that there are. Also, according to LAHanic et al., there is a thin coating of protein on the surface of seaweed. In view of the above-mentioned unique structure of seaweed, and from the viewpoint that seaweed cannot be transformed into protoplasts without physically cutting the tissue, in recent years, Pseudomonas has been added to leaf pieces obtained by cutting seaweed thallus by physical means. ) A method has been proposed in which protoplasts are prepared by allowing a crude enzyme solution obtained from a culture solution of SP strain (strain P-1) to react for about 4 hours.
(Japan Fisheries Society, Autumn Lecture Abstracts of 1981, Fujita et al., "Isolation of protoplasts of Nori and Aonori species by enzyme treatment and their generation," p. 23, 213). However, with this method, the enzyme treatment to obtain protoplasts takes a long time, and since the enzymes are applied to physically cut seaweed, there is a high possibility that the protoplasts obtained will be unhealthy.
Therefore, there is a possibility that cell fusion using these protoplasts will be hindered. However, in the cell fusion method, the health of the protoplasts used is a very important factor; if the protoplasts are unhealthy, the fusion rate during cell fusion will be low and the subsequent growth in culture will be affected. This is because it is thought that it will become inferior. In addition, in order to cut the seaweed thallus using the above method, it is recognized that the enzyme produced by the Pseudomonas SP strain does not act on the surface layer of the seaweed, but acts from the cut part, breaking down the cell wall of the seaweed and forming protoplasts. It is thought that it is based on. In view of the current situation as described above, the present inventor has taken the view that in order to obtain healthy protoplasts of seaweed suitable for cell fusion, it is necessary to disintegrate the seaweed from its surface layer without physically cutting it. As a result of studying the protoplast formation of seaweed, we found that by first treating the seaweed with an enzyme solution containing at least mannan hydrolase and xylan hydrolase, or furthermore, with an enzyme solution containing porphyrane hydrolase. obtained the knowledge that seaweed protoplasts can be prepared in a healthy state, and made an invention relating to a method for preparing seaweed protoplasts. [Patent Application No. 149378 (1982)
60-41485)]. That is, in the method according to the above invention, seaweed fronds are obtained by culturing a microorganism having the ability to hydrolyze indigestible polysaccharides belonging to the genus Pseudomonas in a medium containing seaweed or a seaweed-derived polysaccharide as an inducer. The method is characterized in that the treatment is performed with an enzyme solution containing at least mannan hydrolase and xylan hydrolase. As a result of subsequent research, the present inventor discovered that β-, which is a constituent unit of the structural polysaccharide of seaweed, was used instead of seaweed or seaweed-derived polysaccharides.
Prepared from a seaweed containing a polysaccharide having a 1,3 xyloside bond and a β-1,4 mannoside bond as a structural polysaccharide component, or a culture solution obtained by culturing in a medium containing a polysaccharide derived from the seaweed as an inducer. The present invention was made based on the knowledge that protoplast formation of seaweed can be effectively carried out by treating the seaweed fronds with an enzyme solution containing at least mannan hydrolase and xylan hydrolase. I've reached it. The present invention will be explained in detail below. The main structural feature of the present invention is to convert microorganisms having the ability to hydrolyze indigestible polysaccharides belonging to the genus Pseudomonas into polysaccharides having β-1,3 xyloside bonds and β-1,4 mannoside bonds. At least a xylan hydrolase prepared from a culture solution obtained by culturing in a seaweed containing as a structural polysaccharide component (excluding seaweed thallus) or the above-mentioned polysaccharide derived from the seaweed as an inducer. The purpose of the present invention is to treat seaweed fronds with an enzyme solution containing mannan hydrolase and mannan hydrolase. Note that other configurations of the present invention will become clear from the following description. In the present invention, the microorganisms having the ability to hydrolyze indigestible polysaccharides used in the preparation of the enzyme solution used for the protoplastization of seaweed belong to the genus Pseudomonas isolated from seawater, and are Pseudomonas sp. It is designated as PT-5 and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under the accession number FERM BP-330. The microorganism has the ability to hydrolyze the indigestible polysaccharides such as xylan, mannan, and porphyrane, and has the following mycological properties. Mycological properties: () Morphology Regarding the cells grown in Zo BELL 2216E medium, (a) The morphology of the cells is culm, and the size is 0.5 to 1 μm ×
1.5 to 2.5 μm (b) Motile, flagella are monopolar (c) Gram staining is negative () Growth condition Zo BELL 2216E growth condition in slant medium, (b) At a temperature of 20 to 27℃ Grows well (b) Deposit pale yellow pigment (c) Forms filiform colonies () Physiological properties (b) Catalase test positive (b) Oxidase test positive (c) Acids from glucose Formation Positive (d) 0-F test 0 (by Hugh Leifson method) (e) Vibro-Static Agent (0/129) Negative (f) Agar liquefaction ability + (g) Xylan resolution + (h) Mannan resolution + (resolution) ) Aerobic In the present invention, the above-mentioned microorganism is used as a seaweed containing β-1,3 xyloside bonds and β-1,4 mannoside bonds as structural polysaccharide components (however,
(excluding seaweed), for example, Iwatsuda (a type of seaweed), Amanita cactus, etc., and cultured in a medium containing seaweed or a polysaccharide derived from the seaweed as an inducer to produce mannan hydrolase and xylan hydrolase. causes porphyrane hydrolase to be produced in the medium. Incidentally, the polysaccharides in the seaweed thallus are mainly composed of xylan and mannan, and the binding mode and composition of these polysaccharides were determined by periodic acid oxidation method and liquid chromatography analysis of hydrolysates. As a result of investigation, xylan is a monopolymer of xylose with β-1,3 bonds, and mannan is a monopolymer of xylose with β-1,3 bonds.
Although it was confirmed that the polysaccharide of the seaweed and the polysaccharide derived from the seaweed contained in the medium as an inducer in the present invention do not necessarily have β-1,3 xyloside bonds, It does not have to be a monopolymer of xylose and a monopolymer of β-1,4-linked mannose;
Any disaccharide or higher containing a portion of 1,3 xyloside bonds and β-1,4 mannoside bonds can be used. The above-mentioned "seaweed-derived polysaccharides" used in the present invention refer to seaweed containing polysaccharides having β-1,3 xyloside bonds and β-1,4 mannoside bonds as described above, which are extracted with hot water and made soluble. A residue mainly consisting of polysaccharides obtained by removing components,
Alternatively, it refers to the residue that has been further purified to increase the polysaccharide content. For example, the above seaweed is soaked in 10 times the volume of water, heated in an autoclave at 120°C for 30 minutes, the soluble fraction is removed using a filter cloth, and the resulting residue is heated in the same manner as above. Repeat the operation to remove the soluble fraction (about 5 times), then soak the resulting residue in 100% ethanol,
After leaving it at room temperature overnight, remove the soluble fraction and use the dried product as a residue consisting of polysaccharides, or repeat the above heating and removal of the soluble fraction to obtain a residue (soaked in ethanol). (not included) in 1N-NaOH solution,
After standing overnight at room temperature, the residue from which the soluble fraction was removed was extracted with a 20% hot aqueous solution of NaOH, the resulting extract was centrifuged, Fehling's solution was added to the supernatant to form a precipitate, and this precipitate was Add HCl to the polysaccharide (having β-1,4 mannoside bond) obtained by washing the product with water to remove Cu ions or the supernatant liquid obtained by adding Fehling's solution to form a precipitate to adjust the pH. The polysaccharides (having β-1,3 xyloside bonds) formed from the precipitates obtained by adjusting the polysaccharides prepared in Example 3 are used as purified polysaccharides. The appropriate amount of seaweed or seaweed-derived polysaccharide to be added to the medium as the inducer is 1 to 2% by weight. Next, the medium used for _culturing the microorganisms used in the present invention contains the above-mentioned inducer as a carbon source, peptone and yeast extract as a nitrogen source, and _K2HPO4 , _FeCl3, etc. as an inorganic substance in seawater or artificial Dissolved in seawater and buffered (e.g. Tris buffer)
Preferably, the pH is adjusted to around 7.5. Examples of medium compositions are as follows. Medium composition: The above seaweed or polysaccharide derived from the seaweed 1.0 (wt%) Peptone 1.0 Yeast extract 0.1 K ₂ HPO ₄ 0.01 FeCl ₃ 0.6mg / Tris buffer 0.1 (wt%) Dissolve in seawater at the above ratio to adjust the pH. Adjust to 7.5. The culture conditions for the microorganisms used in the present invention in the medium are as follows: aeration at a temperature of 25°C for 4 days with agitation (aeration rate 1000-2000ml/min, number of stirring 100-
300r.pm). To prepare an enzyme solution from the culture solution obtained by culturing as described above, the culture solution is incubated at a temperature of 4°C.
Centrifuge for 30 minutes (10,000 rpm) and use the supernatant as the enzyme solution. The enzyme solution thus obtained contains mannan hydrolase, xylan hydrolase and porphyrane hydrolase. In the present invention, in the above culture, the plant-derived polysaccharide mainly consisting of mannan or xylan is contained in the medium as an inducer to mainly produce mannan hydrolase or xylan hydrolase, respectively, in the medium. It is also possible to prepare an enzyme solution mainly containing each of these enzymes from the culture solution obtained, and to combine both enzyme solutions and use it for protoplast formation of seaweed thallus. In addition, in the present invention, mannan hydrolase and xylan hydrolase are mainly used to convert the seaweed thallus into protoplasts. This is based on facilitating the latter action on the microfibrillar form of xylan that forms the cell wall. It can be said that it is working. In addition, since porphyrane hydrolase acts on and decomposes porphyrane as a cell-filling substance of the seaweed thallus, protoplast formation is further promoted when an enzyme solution containing this enzyme is used. The appropriate amount of the enzyme solution to act on the seaweed leaves is about 10 ml of the enzyme solution concentrated 5 to 10 times for 4 to 5 pieces of seaweed leaves of about 5 cm. Seaweed protoplasts can be prepared as a function of time. In addition, in the present invention, when the above-mentioned enzyme solution is applied to the seaweed thallus, it is possible to degrade the thin layer of protein covering the surface layer of the seaweed by allowing the protease to act on the thallus, which is useful for preparing protoplasts. It is even more effective. As the protease, papain is particularly preferred, and the thin layer of the above protein can be effectively decomposed and removed by acting on the thallus as a 10% papain solution for about 15 to 30 minutes. The action of protease on the seaweed fronds may be carried out prior to the above-mentioned treatment with the enzyme solution for protoplast formation, or may be carried out in parallel with the treatment with the enzyme solution. The seaweed protoplasts obtained as described above are prepared only by enzymatic treatment without physically cutting the seaweed, so they are in a healthy state and suitable for use in cell fusion. There is. For example, seaweed protoplasts prepared by the method of the present invention are placed in artificial seawater (for algae) in a Petri dish.
According to the results of observing the growth status of protoplasts suspended in Asp.12) and attached to the bottom of a Petri dish, the growth status was very good. As mentioned above, according to the present invention, seaweed protoplasts can be obtained in a healthy state only by enzymatic treatment, and therefore it can be said to be beneficial for the future advancement of seaweed cell fusion technology. EXAMPLES The present invention will be explained in more detail with reference to Examples below. Example 1 Preparation of medium Peptone 1.0 (wt%) Yeast extract 0.1 (〃) K ₂ HPO ₄ 0.01 (〃) FeCl ₃ 0.6 mg/Tris buffer (tris aminomethane)
0.1 (wt%) Hot water of Iwatsuda (a type of seaweed) 1.0 (wt%) Extraction residue The above composition was added to seawater and adjusted to pH 7.5, which was used as a culture medium. Preparation of enzyme solution Add Pseudomonas to the above medium.
Inoculated with sp.PT-5 Kaikoken Joyori No. 330, 300 r.p.
While aerating under stirring of m. (aeration volume 2000ml/
min) Culture was performed at 25°C for 4 days. The obtained culture solution was centrifuged (10,000 rpm) at a temperature of 4° C. for 30 minutes, and the supernatant was concentrated 10 times to obtain an enzyme solution. The results of examining the enzyme activity of the enzyme solution thus prepared against indigestible raw polysaccharides are shown in the table below.

[Table] (Note) ...Very strong activity
...strongly active
+...Slightly strong activity As seen in the table, the enzyme solution used in the present invention has excellent activity against mannan and xylan. Preparation of protoplasts: Place 4 to 5 pieces of seaweed fronds of about 5cm in an L-shaped test tube, add 10ml of the above enzyme solution, and while immersing the nori fronds in the enzyme solution, incubate at 20°C. - Shake for 4 hours using a type shaker (70 strokes/
minutes) to obtain seaweed protoplasts. Example 2 This example shows an example in which papain was applied to seaweed thallus during protoplast formation. In addition, in addition to making papain act on the thallus,
Protoplasts were prepared in the same manner as in Example 1. Treatment with papain Place 4 to 5 sheets of seaweed thallus of about 5 cm in an L-shaped test tube, add 10 ml of papain solution (dissolved in acetate buffer to pH 6.0), was immersed in the papain solution while being shaken at a temperature of 25° C. for 20 minutes (mono-type shaker 70 strokes/min). Preparation of protoplasts The seaweed thallus treated with papain as described above was treated with an enzyme solution according to the procedure described in Example 1.
Protoplasts were prepared by treating for 60 minutes.

Claims (1)

[Scope of Claims] 1. A microorganism having the ability to hydrolyze indigestible polysaccharides belonging to the genus Pseudomonas is used as a structural polysaccharide, which is a polysaccharide having β-1,3 xyloside bonds and β-1,4 mannoside bonds. At least xylan hydrolase and mannan hydrolase prepared from a culture solution obtained by culturing in a medium containing seaweed as an ingredient (excluding seaweed thallus) or the above-mentioned polysaccharide derived from the seaweed as an inducer. A method for preparing seaweed protoplasts, which comprises treating seaweed thallus with an enzyme solution containing. 2. The preparation method according to claim 1, wherein the seaweed thallus is treated with protease prior to the treatment with the enzyme solution. 3. The preparation method according to claim 1, wherein the seaweed thallus is treated with protease in parallel with the treatment with the enzyme solution. 4. Claim 1, wherein the polysaccharide derived from seaweed is a polysaccharide-containing residue obtained by hot water extraction of the seaweed.
The preparation method according to any one of Items 3 to 3. 5. The preparation method according to any one of claims 1 to 3, wherein the polysaccharide derived from seaweed is obtained by purifying a residue obtained by hot water extraction of the seaweed. 6 Claims 1 to 3, wherein the enzyme solution also contains porphyrane hydrolase.
Preparation method according to any one of paragraphs. 7. The enzyme solution according to any one of claims 1 to 3, wherein the enzyme solution is a combination of an enzyme solution containing a mannan hydrolase and an enzyme solution containing a xylan hydrolase. Preparation method. 8. The preparation method according to any one of claims 1 to 3, wherein the medium contains peptone and yeast extract as nitrogen sources. 9. The preparation method according to claim 2 or 3, wherein the protease is papain.