Studies on the Glucanase of Sclerotinia libertiana. EBATA and Yukio SATOMURA

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1 Studies on the Glucanase of Sclerotinia libertiana By Junko EBATA and Yukio SATOMURA Faculty of Science, Osaka City University, Osaka Received December 13, 1962 The digestion of yeast cells with the glucanase of Sclerotinia was found to be significantly increased by pretreating the cells with papaya lysozyme, as well as by pretreating with dilute sodium hydroxide solution. Defatted chlorella cells were digested to a certain extent with the glucanase alone. Pretreatment with lipolytic enzyme slightly stimulated the digestion of yeast cells by glucanase, but this effect was not found with the yeast cells treated by soaps or the defatted chlorella cells. Egg white lysozyme had no effect on digestion of yeast cells. The effect of papaya lysozyme seemed to have relation with the liberation of hexosamine compounds from the yeast-cell walls. It is suggested that, in normal cells, the glucan is present to form a further complex structure with certain other component, becoming insusceptible to the action of glucanase. INTRODUCTION Because of indigestibility of cell walls, yeast and chlorella have not been widely used as fodder or foods. Recently, it has been reported1) that a western barley diet mixed with a bacterial glucanase preparation markedly improved its feeding value in chick grown. It seems that digestion of the barley glucan is elaborated by the action of glucanase. On the other hand, the glucan present in yeastcell walls is difficult to be digested with glucanase alone, because yeast glucan is known to exist in combination with protein or lipid2,3). In the previous paper4), lipolytic enzyme or alkali-treatment was found to increase the digestion degree of native yeast cells by glucanase. In this paper, the digestibility of the yeastand chlorella cells by glucanase is described with special regard to the effect of various enzymic- or chemical pretreatments. The pretreatment effect with an extract of papaya latex was found to be noticeable in the digestion of yeast cells which had been grown in a synthetic medium. Thus, the effective factor in the extract was investigated, using purified preparations of papaya lysozyme and papain. The combined use of glucanase and papaya lysozyme resulted in almost complete digestion of yeast cells. Also, the experiment revealed that papaya lysozyme was similar to alkali in the effect, both liberating hexosamine compounds from yeast-cell walls to make the cells susceptible to the action of glucanase. EXPERIMENTAL Preparation and Partial Purification of Enzyme. The wheat bran culture of Sclerotinia libertiana was extracted with 10 volumes of water. Its extract was passed through an anion exchanger, Duolite A2, after salting out with 0.53 saturation of ammonium sulfate and dialysis. The break-through solution from the resin column was used as a crude glucanase solu-

$472 Junko EBATA and Yukio SATOMURA tion (G1) which was further purified by fractional precipitation with acetone followed by a weak cationic exchanger-chromatographic method.$

2 472 Junko EBATA and Yukio SATOMURA tion (G1) which was further purified by fractional precipitation with acetone followed by a weak cationic exchanger-chromatographic method. The precipitate obtained between the concentration of acetone of 30 and 70 vol.% was suspended in 0.1M acetate buffer (ph 3.5). The supernatant solution was passed through an ion exchange column, Duolite CS 101 which had been equilibrated with 0.1M acetate buffer (ph 3.5), whereby the enzyme was adsorbed onto the resin. After having been washed with the same buffer solution, the resin column was eluted with 0.1M acetate buffer of increased ph. The glucanase was eluted at nearly ph 5, and this glucanase fraction was used as a partially purified glucanase preparation (G2). was passed through the cation exchanger, Amberlite IR 120. This break-through solution was used as lipase after dialysis. The extract obtained by directly extracting fresh bran culture with N/20 ammonium hydroxide was found to contain glucanase and lipase, after precipitation with 0.45 saturation of ammonium sulfate and dialysis; the solution was employed as the two enzymes mixture. Papaya Lysozyme, Papain and Other Enzymes. Purified papaya lysozyme and papain preparations were courteously provided by Department of Biochemistry and Biophysics of the University of Hawaii. The papain was, before use, dissolved in 0.1M KH2PO4 and activated by the addition of NaBH4. As the other proteinases, the extract of dried papaya latex, trypsin (Yashima Co.) and Nagarse (Nagase Co.) were used. Egg white lysozyme was purified and crystallized in our laobratory according to the method of Alderton5). The activity of egg white lysozyme was compared with papaya lysozyme, using Micrococcus lysodeikticus under the condition reported by Smith et al.6) In order to obtain the same degree of lysis with M. lysodeikticus, the required amount of egg white lysozyme was, as reported by Smith et al.6), less than one eighth of that of papaya lysozyme. Yeast- and Chlorella Cells Used. The dry yeast produced by Oriental Yeast Co. was used. The moist cells of Saccharomyces cerevisiae were grown in our laboratory. The defatted chlorella cells were provided by Nippon Chlorella Cooperation. Autolysed Yeast Residue. To 100g of dry yeast was added 500ml of water 60% of yeast cellular substance by weight was lost. After having been washed twice with water by centrifugation at 3000r.p.m. for 15min, the residue was employed. Enzyme Activity Assay. Glucanase-The activity was measured according to the method reported previously4). One unit of the enzyme activity was defined as the enzyme quantity Lipase-The hydrolytic activity on Tween 20 was measured by the same method as in the previous paper4), and its enzyme activity was expressed by the volume, ml, required to neutralize the reaction mixture with N/20 sodium hydroxide solution under the 5) G. Alderton, Biochemical Preparations, 1, 67 (1950). 6) E. L. Smith, J. R. Kimmel, D. M. Brown and E. O. P. Thompson, J. Biol. Chem., 215, 67 (1955). Enzymic- or Chemical Pretreatment of Cells. One ml of cell suspension containing 100mg of dry Digestion of Cells by Glucanase. The cells treated as above were centrifuged and washed thoroughly, with water and then resuspended control with heat inactivated enzyme was performed. After having been centrifuged and washed twice with water, the precipitate was brought to dryness till its constant weight was obtained. The digestibility of cells was expressed by the following equation; The residue after autolysis of dry yeast was also examined under the same condition. However, its digestibility was expressed as % of decrease in weight based on the dry matter of the above residue.

3 Studies on the Glucanase of Sclerotinia libertiana. Part III 473 RESULTS Effect of Enzymic Pretreatment on Digestion of Yeastand Chlorella Cells by Glucanase. Table I shows that the dry yeast cells are not attacked by glucanase or lipase alone, but are slightly digested with the combination of the two enzymes. On the other hand, defatted chlorella cells were attacked to a certain extent with glucanase alone. A similar result TABLE II. EFFECT OF LIPASE ON DIGESTION OF DRY YEAST AND DEFATTED CHLORELLA BY GLUCANASE (G1) * Incubated for 2hr. at each step. L: lipase, G: glucanase TABLE I. EFFECT OF COMBINATION OF GLUCANASE AND LIPASE ON DRY YEAST AND DEFATTED CHLORELLA G: glucanase, L: lipase TABLE III. EFFECT OF PROTEINASE ON DIGESTION. OF FRESH YEAST BY GLUCANASE (G2) * 2.5% reducing sugar as glucose. The wort was supplied with 7.5% sucrose before use.

4 474 Junko EBATA and Yukio SATOMURA was also found when the cells were digested successively with lipase and glucanase (Table II). In Table III, the effect of various proteinases on the fresh yeast cells of Saccharomyces cerevisiae and the digestibility of the treated cells by glucanase are shown. The result indicates that the moist yeast cells are not digested by proteinase or glucanase alone, but the proteinase treated-cells are easily digested by glucanase. The best digestion was found when the cells grown in the synthetic medium (2) were pretreated with papaya extract. Papain was also effective on the yeast cells grown in a synthetic medium, but was ineffective on the yeast cells cultured in a wort medium (Table IV). Whereas, Table V shows that pretreatment with papaya lysozyme greatly increases the digestion of yeast cells even when the cells were grown in a wort medium. However, such marked effect was TABLE VI. EFFECT OF PAPAYA LYSOZYME, EGG WHITE LYSOZYME AND PAPAIN ON DIGESTIBILITY OF DRY YEAST CELLS BY GLUCANASE (G2) TABLE IV. EFFECT OF PAPAIN ON DIGESTIBILITY OF MOIST YEAST CELLS BY GLUCANASE (G2) * The same as in Table V. ** Papain and glucanase were mixed. * Papaya lysozyme and glucanase were mixed. ** Papain and glucanase were mixed. TABLE V. EFFECT OF PAPAYA LYSOZYME AND EGG WHITE LYSOZYME ON DIGESTIBILITY OF MOIST YEAST CELLS BY GLUCANASE (G2)

5 Studies on the Glucanase of Sclerotinia libertiana. Part III 475 not found with egg white lysozyme. For digestion of dry yeast cells, papaya lysozyme was most effective, reaching a digestibility more than 70%. Papain was not so effective as papaya lysozyme. Egg white lysozyme, on the other hand, was quite ineffective, as given in Table VI. the most effective in decreasing the weight of dry yeast cells, but this effect was not TABLE IX. ALKALI-TREATMENT OF AUTOLYSED YEAST RESIDUE AND ITS DIGESTIBILITY WITH GLUCANASE (G2) Effect of Alkali- and Soap-pretreatment on Digestibility of Yeast and Chlorella Cells by Glucanase. The treatment of dry yeast or defatted chlorella cells with dilute sodium hydroxide solution slightly decreased the weight of cellular substance (Table VII), and the treated cells became susceptible to the action of glucanase (Table VIII). Alkali-treatment of the autolysed yeast residue also resulted in a good digestion on incubation with glucanase, as shown in Table IX. This effect was remarkable when the alkali-treatment was carried TABLE X. TREATMENT OF DRY YEAST AND DEthat the autolysed yeast residue became susceptible to glucanase by only FATTED CHLORELLA WITH VARIOUS SOAPS heating. The effect of soaps on the digestibility of cells by glucanase was also investigated. As will be seen in Table X, caprylic acid salt was * To 1g of the residue as dry wt. was added 10ml of sodium hydroxide solution. TABLE VII. ALKALI-TREATMENT OF DRY YEAST AND DEFATTED CHLORELLA TABLE VIII. EFFECT OF ALKALI-PRETREATMENT ON DIGESTIBILITY OF DRY YEAST AND DE- FATTED CHLORELLA BY GLUCANASE (G2) TABLE XI. DIGESTIBILITY BY GLUCANASE (G1) OF DRY YEAST PRETREATED WITH CAPRYLIC ACID SALT

6 476 J unko EBATA and Yukio SATOMURA found on chlorella cells. The yeast cells pretreated with caprylic acid salt were, however, still insusceptible to the action of glucanase alone and, by further treatment with alkali, they became somewhat digestible with glucanase (Table XI). Hexosamine in the Supernatant of the Autolysed Yeast Residue after Incubation with Papaya Lysozyme or Alkali. The supernatant solution, after treating the autolysed yeast residue with lysozyme or alkali, was subjected to analysis of hexosamine according to the method of Belcher7). The result shown in Table XII reveals that certain TABLE XII. HEXOSAMINE IN THE SUPERNATANT OF THE AUTOLYSED YEAST RESIDUE AFTER INCUBATION WITH. PAPAYA LYSOZYME AND EGG WHITE LYSOZYME TABLE XIII. ANALYSIS OF THE SUPERNATANT OF THE AUTOLYSED YEAST RESIDUE TREATED WITH ALKALI 7) R. Belcher, et al., Analyst, 79, 201 (1954) 8) Q. H. Lowry, N. J. Rosenbrough, A. L. Forr, and T. J. Randall, J. Biol. Chem., 193, 265 (1951). hexosamine compound (s) was liberated from autolysed yeast residue by the action of papaya lysozyme. Similar result was also obtained in the case with alkali-treatment. As seen in Table XIII, there was some parallel relation between the improvement of digestibility by alkali-treatment and the dissolution degree of hexosamine, polysaccharide and protein for the autolysed yeast residue on treating with alkali. DISCUSSION The enzyme of snails gut has been known to be markedly effective on digestion of a high lipase activity on Tween 2012). This lipase seems to be effective on the digestion of yeast cells, attacking the lipid in the cell wall. In the present study, the lipase preparation possessing a substantial activity on Tween 2013) was used for pretreatment of cells of yeast and chlorella. The effect of the lipase was, however, found to be insufficient to make the cells susceptible to the glucanase. Soaps were found to be effective to dissolve the cellular substance, and among the soaps used, caprylic acid salt was the most effective on yeast cells, but the cells treated by soaps were insusceptible to the action of glucanase. The effect of lipase and soaps may be attributed to the disorganization of permeability barrier of the cells. It is likely that the glucan-protein complex in the cell wall of yeast becomes a further complicated structure with certain components other than lipid, and this structure prevents the action of glucanase and proteinase. Also it is certain that the alkalitreatment of yeast cells not only dissolves the mannan-protein, but also possibly dissociates the complicated structure. Although the 9) J. Giaja, Compt. Rend. Soc. biol., 86, 708 (1922). 10) A. A. Eddy and D. Williamson, Nature, 179, 1252 (1957). 11) G. Svihla, F. Schlenk and J. L. Dainko, J. Bact., 82, 808 (1961). 12) F. L. Meyers and D. H. Northcote, Biochem. J., 69, 54 (1958). 13) Y. Satomura, S. Oi and M. Ono, Agr. Biol. Chem. 25, 15 (1961).

7 Studies on the Glucanase of Sclerotinia libertiana. Part III 477 complexity of the cell wall structure may vary with the growth condition of yeast cells, a certain pattern for polysaccharide and protein to link together seems to exist in yeastcell walls. Korn and Northcote14) have reported that several kinds of glycoproteins obtained from yeast-cell walls contain glucosamine and have suggested that this glucosamine serves as the connecting link between the polysaccharide and protein. If so, it follows that the liberation of glucosamine by some means from yeast-cell walls causes breakdown of glucoprotein and the polysaccharide, glucan thereby produced, become susceptible to glucanase. In the present study, papaya lysozyme may have acted in that way, thus resulting in a good digestion of yeast cells on incubation with glucanase. Although the action of egg white lysozyme on the mucopolysaccharide prepared from Micrococcus lisodeikticus has recently became quite clear, the action of papaya lysozyme has not been so clarified. In our experiment, it was found that the snail enzyme also showed a bacteriolytic activity on M. lysodeikticus. The activities of those enzymes which were determined by the bacteriolytic activity did not run parallel to the glucanase effect on improving the digestibility of yeast-cell walls. As described above, papaya lysozyme was greatly effective to dissolve yeast-cell walls, in spite of being less in activity of bacteriolysis compared with egg white lysozyme. Hence, further investigation will be necessary to clarify the mechanism involved in improving enzymatically the digestibility of yeast-cell walls by glucanase. The digestion degree by glucanase of chlorella differed from that found in the yeast cell. However, from the fact that the glucanase is effective for digestion of chlorella, it may be certain that chlorella cells also con- Acknowledgements. The authors wish to express their sincere gratitude to Prof. J. Fukumoto for his valuable suggestion. They are also indebted to Mr. K. Nakanishi for his assistance in part of this work. 14) E. D. Korn and D. H. Northcote, Biochem. J., 75, 12 (1960).

BIOCHEMICAL STUDIES ON CARBOHYDRATES. XL. Preparation of Mucoitin* from Umbilical Cords.

The Journal of Biochemistry, Vol. 28, No. 3. BIOCHEMICAL STUDIES ON CARBOHYDRATES. XL. Preparation of Mucoitin* from Umbilical Cords. MASAMI BY SUZUKI. (From the Medico-Chemical Institute, Hokkaido Imperial