Enzymes secreted from the bacteria isolated from cultured nori (Porphyra sp.) infected with Anaaki-disease
digested the crude fiber isolated from Porphyra. Bacteria, two strains of P4 and P5, were
identified as Vibrio fisheri by taxonomic studies.
The optimum temperatures for the growth of both strains were 25 to 30°C against 12 hour-culture,
20°C against 24 hour-culture, and 15°C against 48 hour-culture.
When the both strains were cultured in the presence of crude fiber in the cultural medium, the
enzyme activity increased by 2 to 9 times. The activity of the enzyme of P4 strain was the highest
in the presence of 4% crude fiber in the cultural medium. Optimum pHs of enzymes from P4 were
pH 6 and pH 8, whereas that from P5 was pH 8.departmental bulletin pape

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HAYASHI, Seiichi

KITO, Hitoshi

OOSHIRO, Zentaro

SAKATA, Taizo

坂田, 泰造

大城, 善太郎

林, 征一

鬼頭, 釣

English

Kagoshima University Repository

Mem. Fac. Fish., Kagoshima Univ.Vol. 33, No. 1, pp. 107-113 (1984)Enzymes Digesting the Crude Fiber Isolated fromCultured Nori (Porphyra sp.)*1Seiichi Hayashi*2, Taizo Sakata*3, Zentaro Ooshiro*2and Hitosi Kito*4AbstractEnzymes secreted from thebacteria isolated from cultured nori (Porphyra sp.) infected with Anaaki-disease digested thecrude fiber isolated from Porphyra. Bacteria, two strains of P4andP5, wereidentified as Vibrio fisheri by taxonomic studies.The optimum temperatures forthe growth ofboth strains were 25 to30°Cagainst 12 hour-culture,20 °C against 24 hour-culture, and 15 °C against 48 hour-culture.When thebothstrains were cultured in thepresence of crude fiber in the cultural medium, theenzyme activity increased by2 to 9 times. The activity of the enzyme of P4strain was thehighestinthepresence of 4% crude fiber in thecultural medium. Optimum pHs of enzymes from P4werepH 6 and pH 8, whereas that from P5 was pH 8.Recently theimprovement of plant breeding has been carried outbycell-fusion of protoplast.13>Although theattempt of theimprovement of marine algae bycell-fusion isexpected, it isdifficultto prepare a large quantity of protoplast from marine algae. Enzymatic method is very usefulfor the preparation ofa large quantity ofprotoplast. However, since thekinds ofpolysaccharidesconstructing thecell wall of red- or brown-algae is different from those of terrestrial plant, thecommercially available enzymes for thepreparation of protoplast of terrestrial plantisnot usefulfor the preparation ofprotoplast ofred- orbrown-algae. It is needed tofind out enzymes digestingthe cell walls of such algae.It is known thatthere are some bacteria which induce some diseases ofcultured nori (Porphyrasp.). Anaaki-disease isoneof diseases of Porphyra and thebacteria inducing Anaaki-disease attach to thethalli of Porphyra, digest their cells and make holes ontheir thalli. This suggests thatthebacteria secret some enzymes todigest thecell wall of thealgae. Then we investigated whetherthe medium after the culture of the bacteria digested the crude fiber prepared from Porphyra,and the enzymes digesting the crude fiber were found. In this paper the relationship between theconditions of growth of bacteria and the enzymes activity were described.Materials and MethodsReagents Polypepton and yeast extract were obtained from Daigo Eiyo Kagaku Co. Other*' This work was partly supported by the grant from Agriculture, Forestry, and Fisheries Research CouncilSecretariat.*2 Laboratory of Food Chemistry, Faculty of Fisheries, Kagoshima University.*3 Laboratory of Microbiology, Faculty of Fisheries, Kagoshima University.*4 Seikai Regional Fisheries Research Laboratory, Nagasaki.108 Mem. Fac. Fish., Kagoshima Univ. Vol. 33, No.l (1984)reagents were purchased from Wako Pure Chemical Industries Ltd.Bacteria Marine bacteria, two strains of P4 and P5, were isolated from cultured nori (Porphyra sp.) infected with Anaaki-disease. Thesestrains werecultured in Zobell 2216E medium,composed of 0.5 g polypeptonand 0.1 g yeastextractin 100 ml of artificialsea water (pH 7.6).The artificial sea water was the following composition: 20.1 mM KC1, 10.9 mM CaCl22H20, 24.3mM MgS047H20, 29.5 mM MgCl26H20, and 462 mM NaCl.Taxonomic Studies Taxonomic studies were done by investigating morphological andphysiological properties according to the usual method.Preparation ofCrude FiberfromPorphyra Thecrudefiberof Porphyra waspreparedaccordingto the method of Iriki et al*>. 500 g of dried Porphyra was cut into small pieces with scissorsand dipped into 3/ of 196 HCl-1096 ethanol at roomtemperature for 3 hours. The material wasfiltered with cotton cloth and washed with water. The residue was added into 5/ of 1.25^ H2S04and heated at 100 °C for 30 min. After the material was filtered and washed with water, the residuewas added into 51of 1.2596 NaOH and heated at 100 °C for 30 min. Then the material was filteredand washed with water. The residue was added into 51 of 196 sodium hypochlorite and allowedto stand for about 30 min. The material was filtered with Buchner type-funnel (0 30 cm) layingfilter paper (Toyo No.2) by suction. After washing with water the residue was added into 3/ of5 96 sodium sulfite and allowed to stand for 30 min. The material was filtered with Buchner type-funnel by suctionand washed withwater adequately. The residue was added into 2/ of ethanoland allowed to stand overnight. Then the material was filtered and washed with acetone anddiethylether. The residue wasdriedunderthe reduced pressure in a desicator. The average yieldof crude fiber was 55 g.Preparation ofCrude Enzymes Cell- suspension afterculture was centrifuged to remove cellsat 6,000 rpm for 30min. Thesupernatant was dialyzed against 10mM (NHLOHCOaCpH 7.7). Thendialyzed solution was centrifuged at 10,000 rpm for 30min. Thesupernatant was used as crudeenzymes.Enzyme Assay The reaction mixture contained, in a final volume of 3 ml, 1.5 ml of 0.1mTris-Cl buffer (pH 8.0), 60 mg of crude fiber, and 1.5 ml of crude enzymes.The reaction was carried out at 30°C and stopped by heating at 100°C for 3 min. The reactionmixturewasfiltered with filterpaper (ToyoNo.2)and the reducing sugarin the filtrate wasdetermined by the method of Somogyi-Nelson5*.One unit of the enzyme was defined as the amount of enzyme which producedone fimole ofreducing sugar being equivalent to glucose for one hour under the condition described above.Protein concentration was determined spectrophotometrically by Bio-Rad Protein Assay Kit.Electron Microscope Copper grids (150 mesh) were coated with0.196 collodion in amylacetateand carbon stabilized. Grids were inverted on drops of bacterial cell-suspension for 20 to 30 secand then 196 phosphotungstic acid solution on ammonium acetate for 15 to 20 sec. After theprescribed time hadpassed, theexcess solution was blotted with filter paper. Samples thus preparedwere observed with a Hitachi H-300 electron microscope at 75 kV.ResultsTaxonomic Studies of the Isolated BacteriaThe isolatedbacteria, two strainsof P4 and P5, wereoxidasepositive, catalasepositive,fermentative forglucose fermentation, sensitive to Vibrio static compound (o/129), grown in396 NaCl,Hayashi et al.: Enzymes Digesting the Crude Fiber Isolated from Nori 109Table 1. Taxonomic Studies of P4 and P5 StrainsTest P4 P5 Vibrio fisheriMotility + + +Gram strainingCell form Rod Rod RodSporulation - - -Oxidase + + +Catalase + + +Hugh & Leifson test F F FNovobiocin sensitivity + + +o/129 sensitivity + + +Hydrolysis ofCasein + + +Starch + + dChitin + +Agar + +Salts requirementGrowth withH HO^NaCl - - -3 96 NaCl + + +196 NaCl + - +\096 NaCl - - -M.R. test + + +V.P. test - - -BTB-Teepol Agar - - -Swarming - - -Growth at 37°C - - -Flagella M MF: fermentative, H: halophilic, M: monofrichousnot grown at 37°C, and Gram-negative rod with monofrichous (Table 1). From these results bothstrains were identified as Vibrio fisheri comparing the description in Bergey's Manual.Optimum Temperature for the Growth of Bacteria and the Enzyme ActivityAs shown in Fig. 2 the optimum temperature was changed according to the period of culture.The optimum temperature for the growth of both strainswere 25 to 30°C against 12 hour-culture,20°C against 24 hour-culture, 15°C against 48 hour-culture. The activities of the enzymes obtained from P4 strain cultured at 15°C for 48 hours and P5 strain cultured at 20°C for 24 hourswere the highest (Table 2).The Effect of Crude Fiber in the Cultural Medium on the Enzyme ActivityAs shown in Table 3-A the addition of 2.996 crude fiber into the cultural medium increasedthe enzyme activity of P4 and P5 strainscultured at 20°C for 24 hours by 7 and 9 times, respectively.When the amount of the crude fiber added into the cultural medium was changed, the additionof 4'96 of crude fiber induced the highest activity of the enzyme of P4 strain (Table 3-B).The absence of polypepton affected the enzyme activity less than the absence of yeast extract110 Mem. Fac. Fish., Kagoshima Univ. Vol. 33, No.l (1984)P5Fig. 1. Electron Micrograph of P4 and P5 Strains. Magnification: 10,00020 25 30Temperature °C20 25 30Temperature °CFig. 2. Effect of Temperature on Growth. The composition of the medium was 0.5 g of polypep-ton and 0.1 g of yeast extract in 100 m/ of artificial sea water (pH 7.6). Indicated hourin the figure represents the cultured time.Hayashi et al.: Enzymes Digesting the Crude Fiber Isolated from Nori 111Table 2. Effects of Temperatures and Cultures Time for the Bacterial Growth on the Activities ofEnzymes. P4 and P5 strains were cultured in 30m/ of medium at the optimum temperaturefor each cultured time. Enzyme assay was carriedout at 30°C for 2, 4, and 6 h as describedin "Materials and Methods".Temperature &Cultured TimeP4 P5Activity(U/m/)Protein(mg/m/)S.A.(U/mg)Activity(U/m/)Protein(mg/m/)S.A.(U/mg)25°C 12 h20°C 24 h15°C 48 h0.0260.0480.1610.0200.0480.0851.281.001.8900.0380.0190.0230.0110.0703.450.27Table 3. Effect of the Addition of CrudeFiber into the CultureMedium on the Activity of Enzymes.The culture mediums of A and B werecomposed 0.596 polypepton and 0.196 yeast extract.Polypepton or yeast extract or both of them were removed from the culture medium of C.P4 and P5 strains were cultured in 30m/ of the medium.CultureP4 P5AdditionActivity(U/m/)Protein(mg/m/)S.A.(U/mg)Activity(U/m/)Protein(mg/m/)S.A.(U/mg)A None 20°C, 24h 0.072 0.046 0.63 0.038 0.073 0.5215°C, 48 h 0.038 0.018 2.11 0.019 0.068 0.28Crude Fiber(2.996)20°C, 24h 0.144 0.034 4.20 0.194 0.044 4.4015°C, 48 h 0.144 0.050 2.88 0.048 0.060 0.80B Crude Fiber 20°C, 24h096 0.019 0.020 0.971 0.048 0.037 1.302 0.34 0.043 7.914 0.36 0.030 12.08 0.30 0.032 9.37C Crude Fiberalone20°C, 24h 0.038 0.019 2.00Crude Fiber +Yeast Extract0.24 0.027 8.99Crude Fiber +Polypepton0.26 0.058 4.48(Table 3-C). P4 strain could grow in the medium containingonly crude fiber as carbon source,but the growth and the enzyme activity was very low.Optimum pH of the EnzymesEnzyme of P4 strain had two optimum pHs, pH 6 and pH 8 (Fig. 3). Whereas optimum pHof the enzyme of P5 strain was pH 8.112 Mem. Fac. Fish., Kagoshima Univ. Vol. 33, No.l (1984)Fig. 3. Optimum pH of the Enzymes. Buffers used were as follows: pH 3,4, and 5, 0.1 m Citrate-phosphate buffer; pH 4 and 5, 0.1 m Acetate buffer; pH 6, 7, and 8, 0.1 m Phosphatebuffer; pH 8 and 9,0.1 m Tris-Cl buffer. Enzyme assay was carried out at 30°C for 6 h.DiscussionTwo strains isolated from Porphyra infected with Anaaki-disease were identified as Vibriofisheri.Both strains could digest agar, chitin, and starch. The crude fiber isolated from Porphyrawasdigested by enzymes secreted from these two strains. These enzymes had their optimum pH atpH 8, which is close to pH of the sea water. These results suggest that the bacteria attached tothe thalli of Porphyra secret enzymes, and then the enzymes produced by the bacteria digest thecell wall of the algae in the sea water.When P4 and P5 strains were cultured, the addition of crude fiber of Porphyra in the culturalmedium increased the enzyme activity to digest crude fiber by 2 to 9 times. This suggests thatthe enzymes were one of the inducible enzymes. The activity of the enzyme of P4 strain was thehighest in the presence of 496 crude fiber in the cultural medium. P4 strain could grow in themedium containing only crude fiber as carbon source, but the growth and the enzyme activitywas very low. Optimum condition for the growth and the enzyme production needs the presenceof polypepton and yeast extract besides crude fiber in the medium.It is under the investigation whether the enzymes were available to prepare the protoplast ofPorphyra. It is reported that the protoplast of Porphyriodium, one of the red algae, was preparedby using cellulase6*. However, the protoplast of Porphyra could not be prepared by cellulase.Recently the isolation of protoplast from Laminaria and Porphyra has been tried by using seaurchin gut juice7*. However, the yield of them were less than 103cells/m/ and the viabilities ofthem were less than 10%.References1) Melchers, G., M.D. Sacristan and A.A. Holder (1978): Somatic hybrid plants of potato and tomatoregenerated from fused protoplasts. Carlsberg Res. Commun., 43, 203-2182) Dudits, D., O. Fejer, G. Hadlaczky, C. Koncz, G.B. Lazar and B. Hervath (1980): Intergenetic genetransfer mediated by plant protoplast fusion. Molec gen. Genet., 179, 283-2883) Schieder, O. and I.K. Vasil (1980): Protoplast fusion and somatic hybridization. Intern. Rev. Cytol.,Supplement 11B, 21-46Hayashi et al.: Enzymes Digesting the Crude Fiber Isolated from Nori 1134) Iriki, Y., T. Suzuki, K. Nishizawa and T. Miwa (1960): Xylan of siphonaceous green algae. Nature, 187,82-835) Sonogyi, M. (1952): Notes on sugar determination. J. Biol. Chem., 195, 19-236) Clement-Metral, J.D. (1976): Preparation and some properties of protoplasts from the red alga Por-phyridium cruentum. J. Microscopie Biol. Cell, 26, 167-1727) Saga, N. and Y. Sakai (1984): Isolation of protoplast from Laminaria and Porphyra. Bull. Japan. Soc.Sci. Fish., 50, 1085

アサクサノリの粗繊維分解酵素

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アサクサノリの粗繊維分解酵素

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