METHOD FOR PREPARING PROTOPLASTS OF LAVER THALLUS

Abstract: Protoplasts of laver thalus suitable for use in cell fusion can be obtained by treating a thallus of laver with a protease and then treating them with a combination of β-1,3-xylanase and β-1,4- mannanase or a combination of β-1,3-xylanase, β-1,4- mannanase and porphyranase.

Description: SPECIFICATION TITLE OF THE INVENTION Method for Preparing Protoplasts of Laver Thallus BACKGROUND OF THE INVENTION This invention relates to a method for preparing protoplasts of laver thallus which can be effectively used in cell fusion. DESCRIPTION OF THE PRIOR ART In recent years, very extensive studies on cell fusion have been being carried on and, with terrestrial plants, the stage of practical application has already been attained in some instances. However, research reports on the cell fusion of marine algae, especially of Porphyra species, are very few and no successful case has been reported as yet. Basically, the preparation of protoplasts of laver thallus has been considered to be difficult because, in contrast to terrestrial plants in which most of the polysaccharide constituting the cell wall is cellulose, thalli of laver have a cell wall composed principally of hardly digestible polysaccharides including xylan and mannan. At present, the only attempt to prepare protoplasts from Porphyra species is found in the report of Fujita et al. r entitled tSeparation of protoplasts of laver or green laver by enzyme treatment and their developmentg [Synopses of Lectures in the Autumn of 1982 (edited by the Japan Fisheries Society), p. 23, 213]. According to the method of Fujita et al., protoplasts are prepared by cutting a thallus of laver into pieces by physical means and then treating these pieces for about 4 hours with a crude enzyme solution obtained from a culture of a strain (strain P-l) of Pseudomonas species. However, this method requires a long period of time for the enzyme treatment in the preparation of protoplasts and, moreover, has a high possibility of producing unsound protoplasts and hence interfering with their cell fusion because a thallus of laver having been cut by physical means is treated with enzymes. In cell fusion techniques, the soundness of protoplasts used therefor is presumably a very important factor and the use of unsound protoplasts in cell fusion is considered to give a low rate of fusion and also hinder the growth of the resulting homokaryons in subsequent culture. Moreover, the reason why a thallus of laver is cut into pieces in the above-described method seems to be based on the recognition that the enzymes yielded by the strain of Pseudomonas species do not act on the superficial part of laver but on its cut ends to lyse the cell wall of laver and produce protoplasts. According to a report of Preston et al., the superficial layer of laver includes mannan distributed in the form of granules, its cell wall is composed of xylan having the form of microfibrils, and porphyran is present as an intercellular substance. Moreover, it has also been reported by L.A. Hanic that the superficial layer includes a thin coat composed of protein. Thus, in view of the above-described histology of laver, it has probably been thought that the preparation of protoplasts from a thallus of laver is impossible unless it is cut into pieces. The present inventor has investigated the preparation of laver protoplasts on the basis of the idea that, in order to prepare sound protoplasts of laver thallus suitable for use in cell fusion, it is necessary to disintegrate a thallus of laver from its superficial layer without cutting it by physical means, and has found that protoplasts of laver thallus can be obtained in a sound state by treating a thallus of laver with an enzyme solution containing a mannan hydrolase and a xylan hydrolase and, in certain cases, further containing a porphyran hydrolase. SUMMARY OF THE INVENTION As a result of further studies on the preparation of protoplasts of laver thallus, the present inventor has found that sound protoplasts of laver thallus can be prepared in a very short period of time by preliminarily treating a thallus of laver with a protease and then treating it with a combination of B-1,3-xylanase and B-1,4-mannanase or a combination of B-1,3-xylanase, B-1,4-mannanase and porphyranase, and has thereby completed this invention. Accordingly, it is the primary object of the present invention to provide a method for preparing protoplasts of laver thallus in a very short period of time in which the resulting protoplasts are sound and can be effectively used in cell fusion. Other objects of the present invention will be apparent from the following description. DETAILED DESCRIPTION OF THE INVENTION The characteristic feature of the present invention lies in the fact that sound protoplasts having no damage to the cell membrane can be prepared in a very short period of time by preliminarily treating a thallus of laver with a protease to hydrolyze a portion of the protein present in the superficial layer of its cell wall and then treating the thallus of laver with a combination of B-1,3- xylanase and B-1,4-mannanase in the case of a young thallus, or with a combination of the aforesaid enzymes and porphyranase in the case of an adult thallus. It is to be understood that the method of the present invention is also applicable to the preparation of protoplasts from the Conchocelis phase of Porphyra species and, therefore, the term 11thallus of laver" as used herein also comprehends filaments of the Conchocelis phase of Porphyra species. The present inventor isolated and purified the xylan, mannan and porphyran constituting thalli of laver, analyzed their structures by the periodate oxidation method, and determined their monosaccharide compositions by subjecting their hydrolyzates to liquid chromatography. As a result, it was that the xylan is a monopolymer of xylose having ss-1,3-linkages, the mannan is a monopolymer of mannose having 8-1,4- linkages, and the porphyran is a polymer of galactose. Furthermore, it was also found that, among thalli of laver, young thalli (1-2 mm) are very low in porphyran content, while adult thalli (5 mm or thereabouts) are considerably high in porphyran content. In this connection, some terrestrial plants rarely include, in addition to cellulose a small amount of xylan as a constituent polysaccharide of the cell wall, but most of it has ss-1,4-linkages. On the other hand, in plants (such as devil's-tongue) having mannan as the principal constituent, its monosaccharide components are glucose and mannose joined by f3-l,4-linkages. Thus, on the basis of the above-described findings, the present invention makes it possible to prepare sound protoplasts of laver thallus bn a short period of time by preliminarily treating a thallus of laver with a protease to hydrolyze a portion of the protein present in the superficial layer of its cell wall and then treating the thallus of laver with a combination of ss-1,3-xylanase and B-1,4- mannanase (in the case of a young thallus) or a combination of these two enzymes and porphyranase (in the case of an adult thallus or the Conchocelis phase). It should be mentioned in this connection that xylan hydrolases are classified into ss-1,3- xylanase and B-1,4-xylanase and mannan hydrolases are classified into 8-1,3-mannanase and ss-1,4-mannanase, and that the treatment of a thallus of laver with a combination of 8-1,4-xylanase and B-1,3-mannanase cannot cause hydrolysis of the polysaccharides constituting its cell wall. As described above, the method of the present invention includes the step of preliminarily treating a thallus of laver with a protease to hydrolyze a portion of the protein present in the superficial layer of its cell wall. This is due to the fact that, if a thallus of laver is directly treated with a combination of B-1,3-xylanase and B-1,4-mannanase, the thallus of laver will be gradually disintegrated from its marginal portions as if individual cells were separated one by one, and sound protoplasts cannot always be obtained because their cell membrane itself may suffer damage and, in the worst case, allow the cell contents to flow out. More specifically, when a thallus of laver is preliminarily treated with a protease to hydrolyze the protein present in the superficial layer of its cell wall and then treated with a combination of B-1,3-xylanase and 6-1,4-mannanase, the thallus of laver is disintegrated not from its marginal portions but over its whole body, so that the thallus is broken up and its cell wall is ysed in a short period of time (about 30 minutes) to produce protoplasts. The treatment of the thallus of laver with the protease is preferably carried out at room temperature for a period of about 10 minutes. It is also preferable to use papain as the protease The thallus which has been treated with the protease is washed and then treated at room temperature for a period of about 20 minutes with a mixed enzyme solution containing B-1,3-xylanase and B-1,4- mannanase in the case of a young thallus, or treated in the same manner with a mixed enzyme solution containing the aforesaid two enzymes and porphyranase in the case of an adult thallus or the Conchocelis phase. The enzyme solution used for this purpose can be a mixture of crude enzyme solutions containing 6-1,3-xylanase and B-1,4-mannanase and, if necessary, further containing porphyranase. Such enzyme solutions may be obtained, for example, by isolating a microorganism of the genus Pseudomonas having the ability to hydrolyze hardly digestible polysaccharides, culturing it in a medium containing laver or a laverderived polysaccharide as an inducer, and removing solid matter from the resulting culture. However, in order to prepare protoplasts in a short period of time, it is preferable to use a mixture of solutions containing a purified preparation of each enzyme. It has been observed that, when the protoplasts of laver thallus prepared as above in accordance with the present invention are placed in a Petri dish and suspended in artificial sea water (Asp. 12 for algae) having the composition given below, they become attached to the bottom of the Petri dish and grow in a very good condition. Composition of Artificial Sea Water (Asp. 12): NaCl 28 g MgSO4t7H2O 7 g MgC12 6H2O 4 g KC1 700 mg CaCl2.2uU2O 1.47 g NaNO NaN03 100 mg K2HPO4 10 mg Sodium glycerophosphate 10 mg Vitamin B12 0.2 g Biotin 1 pg Thiamine 100 g P II Metal 10 ml S II Metal 10 ml Trisaminomethane 1 g Distilled water 1,000 ml pH 8.0-8.1 Composition of P II Metal Composition of S II Metal EDTA 1 mg NaBr 1.2 mg H3BO3 1 mg AlCl36H2O 1.2 mg MnC12 4H2O 0.14 mg SrCI2-6H2O 0.6 mg FeC126H2O 0.05 mg NaMoO42H2O 0.12 mg ZnCl2 0.01 mg PbCl 0.03 mg CoC12-6H2O 4 <RTI ID=10.7> ug KI 1.5 g CuSO4.5H2O 0.5 ijg Distilled water 1 ml Distilled water 1 ml As described above, the method of the present invention makes it possible to prepare sound protoplasts of laver thallus suitable for purposes of cell fusion in a very short period of time, and is believed to serve for further promotion of cell fusion technology for'laver thallus. To further illustrate the present invention, the following examples are given. However, these examples are intended to be illustrative only and are not to be construed to limit the scope of the invention. Example 1 About 0.1 g (wet weight) of young thalli (1-2 mm in length) of laver were placed in an L-shaped test tube. After the addition of 10 ml of a 0.5% papain solution (in M/15 Tris-HCl buffer, pH 7.4), the test tube was incubated, with shaking (at 100 strokes per minute), at a temperature of 25"C for a period of 10 minutes. The thalli so treated were thoroughly washed with sea water and then with M/15 phosphate buffer (pH 7.0), and transferred to another L-shaped test tube. After the addition of 5 ml of a B-1,3-xylanase solution and 5 ml of a 6-1,4-mannanase solution, the test tube was incubates, with shaking (at 100 strokes per minute), at a temperature of 250C for a period of 20 minutes. When the resulting fluid was examined under the microscope, it was confirmed that there were obtained 5.9 x 104 protoplasts. The B-1,3-xylanase and B-1,4-mannanase solutions used in this example were prepared in the following manner. B-1,3-xylanase solution: Among the bacteria attached to thalli of laver being cultivated, ones having the ability to hydrolyze agar were isolated and cultured in a medium comprising sea water to which peptone, yeast extract and the like were added and, moreover, ss-1,3-xylan was added as an inducer so as to give a concentration of 1%. The resulting culture was centrifuged to collect the supernatant, which was purified with ammonium sulfate and then dialyzed against M/15 phosphate buffer (pH 7.0). The B-1,3-xylanase solution thus obtained had an activity of 23 units/ml. B-1,4-mannanase solution: The same procedure as described above was repeated except that there was used the aforesaid medium to which, in place of the B-1,3-xylan, S-1,4- mannan was added as the inducer so as to give a concentration of 1%. The B-1,4-mannanase solution thus obtained had an activity of 18 units/ml. The papain used was a product t10 units/mg) of Wako Junyaku Kogyo K.K. Then, the protoplasts of laver thallus obtained as above were cultured according to the following procedure. Using artificial sea water Asp. 12 as the 4 medium, 8 x 10 protoplasts were cultured at a temperature of 170C under illumination at a light intensity of 5,000 lux (with alternation of 9-hour light periods and 15-hour dark periods). The results of observations made under the microscope in the course of the culture are given in the following table. EMI13.1 <tb> <SEP> After <SEP> 3 <SEP> days <SEP> After <SEP> 4 <SEP> days <SEP> After <SEP> 5 <SEP> days <tb> <SEP> of <SEP> culture <SEP> of <SEP> culture <SEP> of <SEP> culture <tb> <SEP> Formation <SEP> of <SEP> Formation <SEP> of <SEP> Formation <SEP> of <tb> Findings <SEP> cell <SEP> wall <SEP> in <SEP> cell <SEP> wall <SEP> in <SEP> cell <SEP> wall <SEP> and <tb> <SEP> 10-20% <SEP> 60-70% <SEP> cell <SEP> division <tb> <SEP> in <SEP> 60-70% <tb> Example 2 Protoplasts were prepared according to the same procedure as described in Example 1, except that an adult thallus of laver (about 5 cm in length) was used in place of the young thalli and 5 ml of a porphyranase solution was used in addition to the B-1,3-xylanase solution (5 ml) and the ss-1,4-mannanase solution (5 ml). When the resulting fluid was examined under the microscope, it was confirmed that there were obtained 4.0 x 10 protoplasts. The porphyranase solution used in this example was prepared in the same manner as described above for the 8-1,3-xylanase solution, except that, in place of the B"1,3-xylan, porphyran was added to the medium so as to give a concentration of 1%.

Claims: CLAIMS: 1. A method for preparing protoplasts of laver thallus suitable for use in cell fusion which comprises treating a thallus of laver with a protease and then treating said thallus of laver with a combination of B-1,3-xylanase and B-1,4-mannanase. 2. A method for preparing protoplasts of laver thallus suitable for use in cell fusion which comprises treating a thallus of laver with a protease and then treating said thallus of laver with a combination of a-1,3-xylanase, ss-1,4-mannanase and porphyranase. 3. A method as claimed in claim 1 wherein said protease is papain. 4. A method as claimed in claim 2 wherein said protease is papain. 5. A method as claimed in claim 1 wherein said thallus of laver is a young thallus. 6. A method as claimed in claim 2 wherein said thallus of laver is an adult thallus or the Conchocelis phase of Porphyre species. 7. A method as claimed in claim 1 wherein said thallus of laver is treated with a mixed enzyme solution containing B-1,3-xylanase and B-1,4-mannanase at room temperature for a period of about 20 minutes. 8. A method as claimed in claim 2 wherein said thallus of laver is treated with a mixed enzyme solution containing B-1,3-xylanase, B-1,4-mannanase and porphyranase at room temperature for a period of about 20 minutes. 9. A method as claimed in claim 3 wherein the treatment with papain is carried out at room temperature for a period of about 10 minutes. 10. A method as claimed in claim 4 wherein the treatment with papain is carried out at room temperature for a period of about 10 minutes.

Inventors: SHIBATA, TERUHIKO
Applicants: KOASA TRADING CO.,LTD.
Application year: 1984
Main classification: C12N15
IPC codes: C12N5, C12N1, C12R1, C12N15