JPS6257306B2 - - 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 into so-called cell fusion, the fusion between cells of different organisms, has become very active as a genetic engineering method, and some cases have already reached the stage of practical application in land plants. However, there are few research reports on cell fusion in seaweeds, especially in seaweeds, and no successful example has yet been found. Incidentally, although protoplasts of land plants such as wheat, barley, and soybeans can be easily prepared using commercially available enzymes (e.g., cellulase, macerozyme, etc.), enzymes that can convert linseed plants into protoplasts are not currently available. This is thought to be one of the reasons why cell fusion technology in laver species lags behind that of land plants. So far, as an attempt to convert laver to protoplasts, Fujita et al. reported on ``Isolation of protoplasts from laver and laver by enzyme treatment and their generation'' (Japan Fisheries Society, 1981). Autumn lecture abstracts, page 23,
213) can only be seen. In this method by Fujita et al., a crude enzyme solution obtained from a culture of Pseudomonas SP strain (strain P-1) is applied to leaf pieces obtained by physically shredding seaweed thallus for about 4 hours. However, in this method, the enzyme treatment to obtain protoplasts takes a long time, and the protoplasts obtained are unhealthy because the enzymes are applied to physically cut seaweed. 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, the reason for cutting the seaweed thallus using the above method is 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. According to a report by Preston et al., mannan exists in the form of granules on the surface of seaweed, the cell wall of seaweed is composed of xylan in the form of microfibrils, and porphyranes are thought to exist as a cell filling substance. Ru. Also, according to LAHanic et al., there is a thin coating of protein on the surface of seaweed. In other words, from the viewpoint of the structure of seaweed, it was thought that seaweed could not be transformed into protoplasts unless it was cut. The present inventor investigated the conversion of seaweed into protoplasts from the viewpoint that in order to obtain healthy seaweed protoplasts suitable for cell fusion, it is necessary to disintegrate the seaweed from its surface layer without physically cutting it. As a result, by treating seaweed with an enzyme solution containing at least mannan hydrolase and xylan hydrolase, or further containing polyphyllane hydrolase,
The present invention was made based on the knowledge that seaweed protoplasts can be prepared in a healthy state. That is, an object of the present invention is to provide a method for preparing healthy seaweed protoplasts that can be effectively applied to cell fusion. The present invention will be explained in detail below. The main structural features of the present invention are that seaweed fronds are
At least mannan hydrolase and xylan prepared from a culture solution obtained by culturing a microorganism capable of hydrolyzing indigestible polysaccharides belonging to the genus Pseudomonas in a medium containing seaweed or a seaweed-derived polysaccharide as an inducer. The process consists of treatment with an enzyme solution containing hydrolytic enzymes. Note that other configurations of the present invention will become clear from the following description. The microorganisms that have the ability to hydrolyze indigestible polysaccharides used in the preparation of the enzyme solution used in the protoplastization of seaweed in the present invention are those that are isolated from seawater and are used to It has the ability to hydrolyze porphyrane and has the following mycological properties. Mycological properties: Morphology Regarding cells grown in ZoBELL2216E medium, (a) The cell morphology is 0.5 to 1μ in size for rods.
m x 1.5 to 2.5 μm (B) Motile, flagella are monopolar (C) Gram staining is negative Growth condition Growth condition in ZoBELL2216E slant medium, (B) Good at temperature of 20℃ to 27℃ (b) Deposit pale yellow pigment (c) Form filiform colonies Physiological properties (b) Catalase test positive (b) Oxidase test positive (c) Production of acid from glucose Positive ( d) O-F test (based on Hugh Leifson method)
0 (e) Vibro-Static Agent (0/129) Negative (f) Capacity to liquefy + (g) Xylan decomposition ability + (ch) Mannan decomposition ability + (li) Aerobicity In view of the above mycological properties, the present invention The microorganism utilized can be identified as a strain belonging to the genus Pseudomonas. This strain of Pseudomonas sp. PT-5 has been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology under accession number 330. In the present invention, the above-mentioned microorganism is cultured in a medium containing seaweed or seaweed-derived polysaccharide as an inducer to produce mannan hydrolase, xylan hydrolase, and porphyrane hydrolase in the medium. The term "polysaccharide derived from seaweed" as used herein refers to a residue mainly consisting of polysaccharides obtained by hot water extraction of seaweed to remove soluble components, or a residue obtained by further purification to increase the polysaccharide content. means something For example, seaweed is soaked in 10 times the amount of water, heated in an autoclave at 120°C for 30 minutes, then the soluble fraction is removed using a filter cloth, and the resulting residue is heated in the same manner as above to dissolve the soluble fraction. Repeat the operation of removing the segment (about 5 times), then immerse the resulting residue in 100% ethanol, leave it at room temperature overnight, remove the soluble segment, and dry it as a residue consisting of polysaccharides. Alternatively, the residue obtained by repeating the above heating and removal of the soluble fraction (not immersed in ethanol) is immersed in a 1N-NaOH solution, left overnight at room temperature, and then the soluble fraction is removed. The residue was diluted with NaOH
A polysaccharide obtained by extracting with a 20% hot aqueous solution, centrifuging the resulting extract, adding Fehling's solution to the supernatant to form a precipitate, and washing the precipitate with water to remove Cu ions. (mannan) or a polysaccharide (xylan) that is obtained by purifying the polysaccharide (xylan) that is obtained by adding HC1 to the supernatant liquid obtained by adding Fehling's solution to form a precipitate and adjusting the pH to 3. used as The appropriate amount of seaweed or seaweed-derived polysaccharide to be added to the medium as the inducer is 1 to 2% by weight. The medium used for culturing the above-mentioned microorganisms used in the present invention is prepared by dissolving the above-mentioned inducer as a carbon source, peptone and yeast extract as a nitrogen source, and K ₂ HPO ₄ , FeCl ₃ and the like as inorganic substances in seawater or artificial seawater. , a buffer solution (for example, a Tris buffer with pH adjusted to around 7.5 is preferable. Examples of the culture medium composition are as follows.Medium composition: seaweed or seaweed-derived polysaccharide 1.0 (wt%) peptone 1.0 yeast extract 0.1 K ₂ HPO ₄ 0.01 FeCl ₃ 0.6 mg/1 Tris buffer 0.1 (wt%) Dissolve in seawater at the above ratio and adjust the pH to 7.5. The culture conditions in the above medium for the above microorganisms used in the present invention are as follows: Aeration for 4 days at a temperature of 25℃, under stirring aeration (aeration rate 1000-2000ml/min, number of stirrings 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, a polysaccharide derived from seaweed, 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 use the combination of both enzyme solutions 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.
The growth status of protoplasts suspended in Asp.12) and attached to the bottom of a Petri dish was observed, and the growth status was found to be 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/1 Tris buffer (tris aminomethane)
0.1 (wt%) Hot water extraction residue of seaweed 1.0 (wt%) The above composition was added to seawater and adjusted to pH 7.5 and used as a culture medium. Preparation of enzyme solution Add Pseudomonas to the above medium.
25℃ while aerating with 300rpm stirring (air flow rate 2000ml/min)
Culture was carried out 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 polysaccharides are shown in the table below. For reference, the preparation method of the enzyme solution used in the method of Fujita et al. mentioned above is shown below, and the results of investigating the enzyme activity are also displayed. Preparation method of enzyme solution: Medium composition NH ₄ NO ₃ 0.1 (wt%) K ₂ HPO ₄ 0.01 (〃 ) FeCl ₃ 0.6 mg/1 Tris-aminomethane 0.1 (wt%) Asakusanori powder 0.2 (wt%) The above composition was mixed with seawater and adjust the pH to 7.5. Asakusanori powder was sterilized and added separately. Cultured in the above medium for 2 days in advance.
A strain of Pseudomonas sp was inoculated and cultured at 25°C for 5 days under aeration, followed by static culture for 2 days, and the resulting culture solution was filtered through Celite (Standard Super Cell) to remove bacteria. was used as the enzyme solution.

[Table] As seen in the table, the enzyme solution used in the present invention has superior activity against mannan and xylan compared to the enzyme solution used in the method of Fujita et al. Preparation of protoplasts: Place 4 to 5 pieces of seaweed fronds of approximately 5cm in an L-shaped test tube, add 10ml of the above enzyme solution to this, and while immersing the nori fronds in the enzyme solution, incubate at a temperature of 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 nori fronds approximately 5 cm in length in an L-shaped test tube, add 10 ml of 10% papain solution (dissolved in acetate buffer to pH 6.0), While immersing the leaf in the papain solution,
It was shaken for 20 minutes (70 strokes/min on a monoshaft shaker) at a temperature of .degree. 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 seaweed thallus is cultured in a medium containing seaweed or seaweed-derived polysaccharide as an inducer using a microorganism having the ability to hydrolyze indigestible polysaccharides belonging to the genus Pseudomonas. A method for preparing seaweed protoplasts, which comprises treating with an enzyme solution containing at least mannan hydrolase and xylan hydrolase prepared from the obtained culture solution. 2. The method according to claim 1, wherein the seaweed thallus is treated with protease prior to the treatment with the enzyme solution. 3. The method according to claim 1, wherein the seaweed thallus is treated with protease in parallel with the treatment with the enzyme solution. 4. The method according to any one of claims 1 to 3, wherein the polysaccharide derived from seaweed is a polysaccharide-containing residue obtained by hot water extraction of seaweed. 5. The 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 seaweed. 6 Claims 1 to 3, wherein the enzyme solution also contains porphyrane hydrolase.
The method described in 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. Method. 8. The method according to any one of claims 1 to 3, wherein the medium contains peptone and yeast extract as nitrogen sources. 9. The method according to claim 2 or 3, wherein the protease is papain.