Agr. Biol. Chem., 40 (1), 27 `32, 1976 Purification and Some Properties of Red Yeast Cell Wall Lytic Enzyme õ Motoo ARAI, Ryoohei YAMAMOTO and Sawao MURAO Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka Received May 28, 1975 The red yeast cell wall lytic enzyme of Penicillium lilacinum No. 2093 was purified about 220 fold with a yield of 22% from the culture filtrate by ammonium sulfate fractionation, DEAE-cellulose and CM-cellulose column chromatography and Bio-Gel P-60 gel filtration. The enzyme was most active at ph 4.0 `4.5 and 50 Ž. The enzyme was stable at ph value from 3.5 to 7.0 on 20hr incubation at 37 Ž. The enzyme released reducing sugar from the red yeast cell walls, but Saccharomyces yeasts did not show any susceptibility to the enzyme. The knowledge of the cell walls of Saccharo myces yeast has been expanded by chemical and enzymatic analyses.1 `4) However, the structures of red yeast cell walls whose chemi cal compositions differ from those of Sac charomyces5,6) are still unknown. This lag of the studies on them may be partly due to the fact that enzymes that degrade specifically the red yeast cell walls have not been found. In the previous paper,7) we reported the isolation of a microorganism which produced the red yeast cell wall lytic enzyme. This paper de scribes the purification of the lytic enzyme and some properties of the enzyme. and 0.5ml of 0.3% yeast cell wall suspension. It was incubated at 37 Ž for 10min with gentle shaking. After the reaction was stopped by the addition of copper reagent, reducing groups released was measured by Somogyi-Nelson method. One unit of the lytic ac tivity was defined as the enzyme quantity which liberated reducing sugars equivalent to 10ƒÊg glucose per min under the above condition. Assay of the laminarinase and the dextranase activity. The laminarinase and the dextranase were assayed in the same manners as the cell wall lytic enzyme assay system replacing 0.3% cell wall suspension by 0.5 laminarin or dextran solution. Protein determination. The protein concentration was determined spectrophotometrically by measuring its absorbancy at 280nm using Hitachi spectrophoto MATERIALS AND METHODS Microorganisms. Penicillium lilacinum No. 2093 isolated from soil was used for the enzyme preparation. The cell wall preparation of Rhodotorula glutinis K-24 was used for the substrate of the enzyme. The yeast strains used were from the stock cultures in our laboratory. The yeast cell wall preparation. The yeast cell walls were prepared as described in the previous paper.7) Assay of the cell wall lytic enzyme activity. The cell walls of Rh. glutinis, 0.3g were suspended in 100ml water and sonicated gently. The reaction mixture, in a total volume of 1.5ml, was composed of 0.5ml of enzyme solution, 0.5ml of 0.1M acetate buffer (ph 4.5) meter 124. Disc electrophoresis. Disc electrophoresis of the purified enzyme preparation was carried out by the method described by Reisfed et al.8) at ph 4.3 with an apparatus of M. S. Instrument Co. Polysaccharide for enzyme substrate. Yeast glucan and colloidal chitin were prepared as described in the previous paper.7) Yeast mannan was obtained from Sigma Chemical Co., Ltd. Laminarin was obtained from K. K. Laboratories, Inc. Luteose and barley glucan presented by Amano Pharm., Ltd. Other poly saccharides were supplied commercially. Chemicals. DEAE and CM-cellulose were pur chased from Seikagaku Kogyo Co. Bio-Gel P-60 (Bio Rad Laboratories) was purchased from Muromachi Kagaku Kogyo Co. Other chemicals were of reagent õ Studies on the Red Yeast Cell Wall. Part II. See reference 7). grade and used without further purification.
28 M. ARAI, R. YAMAMOTO and S. MURAO RESULTS 1. Purification of the cell wall lytic enzyme The various stages in the purification of the red yeast cell wall lytic enzyme of Pen. lilaci num are detailed in the following procedure. Step 1. Culture filtrate The culture broth was filtered through a celite layer to remove cells and insoluble materials. The clear filtrate was adjusted to ph 6.0 with 1N acetic acid. Step 2. Ammonium sulfate. fractionation To the clear filtrate was added solid ammonium sulfate to give a final concentration of 0.8 saturation. After standing overnight in the cold, the resulting precipitate was collected by filtration and dissolved in a small volume of 1mM phosphate buffer, ph 6.0, and then dialyzed against the same buffer for 2 days in the cold during which the outer liquid was changed every 12hr. Step 3. DEAF-cellulose column chromato graphy. After removal of the insoluble materials by centrifugation, the clear super natant was passed through a DEAE-cellulose column (10 ~20cm) equilibrated with 1mM phosphate buffer, ph 6.0. A further 2 liters of the same buffer was allowed to pass through the column. The red yeast cell wall lytic enzyme and the dextranase were eluted in the void volume. The laminarinase, pigmented matter and some protein were retained on the column. Step 4. CM-cellulose column chromato graphy. The effluent was concentrated under the reduced pressure and dialyzed against 1mM acetate buffer, ph 5.0, for 2 days during which the outer liquid was changed every 12hr. The dialyzate was loaded on a column (5 ~ 40cm) of CM-cellulose which had been equilibrated with 1mM acetate buffer, ph 5.0, and linear gradient elution was carried out in the same buffer between 1.5 liters each of 0 and 0.6M NaCl. The elution pattern is shown in Fig. 1. The red yeast cell wall lytic enzyme was separated from the dextranase. FIG. 1. Elution Pattern of Enzymes from a Column of CM-Cellulose. The crude enzyme was applied to a CM-cellulose column (44 ~450nm) equilibrated with 0.01M acetate buffer, ph 5.0. The enzyme was eluted with 0.7M NaCl linear gradient. \, cell wall lytic activity; \, laminarinase activity; œ \ œ, dextranase activity; ------, protein; \, NaCl. FIG. 2. Gel Filtration on a Column of Bio-Gel P-160. Five ml of concentrated enzyme solution was applied to a Bio-Gel P-60 column (25 ~370mm) equilibrated with 0.01M acetate buffer, ph 5.0, and 5ml fractions were collected. \, cell wall lytic activity; œ \ œ, protein. However, this lytic enzyme fraction was con taminated with trace amount of the dextranase and the laminarinase. These contaminants were removed by the following gel filtration procedure. The active fractions (Nos. 70 `85) were concentrated under the reduced pressure and dialyzed against 1mM acetate buffer, ph 5.0.
Red Yeast Cell Wall Lytic Enzyme 29 TABLE 1. SUMMARY OF PURIFICATION PROCEDURE Step 5. Bio-Gel P-60 gel filtration. The dialyzate was subjected to gel filtration with Bio-Gel P-60 column equilibrated with 1mM acetate buffer, ph 5.0. Figure 2 shows that in the peak fractions of eluted enzyme there was a coincidence of protein concentration and lytic activity and a constancy of specific activity though there was no longer any gain in specific activity. The purification steps summarized in Table I show 220 fold purifica tion over the culture filtrate with 22% recovery. Homogeneity of purified enzyme. Homoge neity of the purified enzyme was examined by disc electrophoresis at ph 4.3. In Fig. 3, disc electrophoretic pattern of the lytic enzyme at ph 4.3 is shown, indicating that the prepara tion is electrophoretically homogeneous. FIG. 4. Optimum ph of the Lytic Enzyme. The reaction mixture contained 0.5ml of enzyme solution, 0.5ml of 0.3% cell wall suspension and 0.5m1 of individual buffer solution, was incubated at 37 Ž for 10min. ph 3.5 `5.5: 0.1M acetate buffer. ph 5.5 `8.0: 0.1M phosphate buffer. 2. Properties of the enzyme Effect of ph and temperature on enzyme activity. The effect of ph on enzyme activity is shown in Fig. 4. The enzyme was most active at ph 4.0 to 4.5. The effect of tem perature on enzyme activity at ph 4.5 is shown in Fig. 5. The optimum temperature for the enzyme reaction was 50 Ž, and 50% of the maximal activities were found at 25 Ž and 60 Ž. Effect of temperature and ph on the enzyme stability. To elucidate the thermal stability of the enzyme, the enzyme solution was in cubated at various temperatures for 30min and then the activity remained was assayed. The FIG. 3. Polyacrylamide Gel Disc Electrophoresis of the Purified Enzyme. Fifty tag of purified enzyme were subjected to electro phoresis. results are shown in Fig. 6, indicating the enzyme to be stable at temperature below 40 Ž. On the other hand, to elucidate the effect of ph on the stability of the enzyme, the
30 M. ARAI, R.. YAMAMOTO and S. MURAO FIG. 5. Optimum Temperature of the Lytic Enzyme. The reaction mixture contained 0.5ml of enzyme solution, 0.5ml of 0.3% cell wall suspension and 0.1mm acetate buffer ph 4.5, was incubated at various temperatures for 10min. FIG. 7. ph Stability of the Lytic Enzyme. Two-tenthsml of enzyme solution were incubated with 0.2ml individual buffer solution at 37 Ž for 20hr. Then the mixture were diluted and the remaining activities were assayed under the standard assay condition. ph 3.0 `5.5: acetate buffer. ph 5.5 `8.5: phosphate buffer. TABLE II. HYDROLYSIS OF VARIOUS POLYSACCHARIDES BY LYTIC ENZYME The reaction mixture contained 0.5ml of enzyme solution, 0.5ml of polysaccharide solution and 0.5ml of acetate buffer, (ph 4.5), was incubated at 37 Ž for 30min. FIG. 6. Thermal Stability of the Lytic Enzyme. 0.5ml of enzyme solution were incubated with 0.5ml of 0.1M acetate buffer, ph 4.5, at various temperatures for 30min and then the remaining activities were assayed under the standard assay condition. enzyme solution was incubated in 0.05M buffer of various ph values at 37 Ž for 20hr and then the activity remained was assayed at ph 4.5. As shown in Fig. 7, the enzyme was stable in the ph range between 3.5 to 7.0, but not at ph lower than 3.5 or higher than 7.0. Substrate specificity. Table II shows that the substrate specificity of the enzyme. By this experiment it was proved that only Rh. glutinis cell walls are cleaved by the enzyme. ƒ -1,4, ƒ -1,6, ƒà-1,3, ƒà-1,4 glucanase which were contained in the culture filtrate7) were removed in the purification procedure. It was also evident that the purified enzyme had no activity of ƒ -mannanase or chitinase. Lysis of various yeast cell walls and living yeasts by the enzyme The lytic activity of the purified enzyme on various yeasts cell walls and living cells was examined. The results are shown in Table III. Among the yeasts tested, good lysis was ob served for Rhodotorula and Sporobolomyces except Rh. peneous IFO 0930, while Sacch.
Red Yeast Cell Wall Lytic Enzyme 31 TABLE III. DEGRADATION OF VARIOUS YEAST CELL WALL AND LIVING CELL WITH LYTIC ENZYME degradated specifically the cell walls of red yeast. Generally, ƒà-1,3 glucanase which hydro lyzed laminarin degraded the cell walls of Saccharomyces yeasts, but did not degrade those of Rhodotorula or Sporobolomyces yeasts.9,10) On the contrary, the enzyme of Pen. lilacinum did not hydrolyze laminarin, but degraded the cell walls of red yeasts. These facts might reflect the remarkable dif ference of the cell wall structures between Rhodotorula and Saccharomyces. Bartnicki- Garcia reported that the cell walls of Rhodo torula yeasts do not contain glucan which was contained in the walls of Saccharomyces but are composed of chitin and mannan.6) The lytic enzyme of Pen. lilacinum released reducing sugars from the cell walls of red yeasts. These reducing sugars were composed of glucose and mannose. This result might indicate that the cell walls of red yeasts composed by copoly saccharide of glucose and mannose. Therefore it might be evident that the lytic enzyme of Pen. lilacinum was a new type lytic enzyme different from ƒà-glucanase. a) Liberation of reducing sugar from 3mg of cell wall, after incubation with lytic enzyme for 60min at 37 Ž. b) Decrease in turbidity of living cell suspension, after incubation with lytic enzyme for 120min at 37 Ž. c) Not determined. cerevisiae, C. japonica and P. mogi seemed to be unsusceptible to the enzyme. DISCUSSION The lytic enzyme of Pen. lilacinum degraded not only the cell walls of Rhodotorula glutinis K-24, but also those of various Rhodotorula and Sporobolomyces yeasts. In particular, the cell walls of Rh. glutinis IFO 0892, Rh. slooffii CBS 5706, Sp. pararoseus CBS 2637, Sp. sal monicolor CBS 490 and Sp. odorus CBS 483 showed high susceptibility to the enzyme. While, some yeasts such as Saccharomyces and Pichia were unsusceptible to the enzyme. Namely, the lytic enzyme of Pen. lilacinum On the other hand, susceptibility of living cells of Rhodotorula to the enzyme differed from each other. When Rh. glutinis IFO 0695 was compared with Rh. rubra IFO 0890, the susceptibilities of the cell walls were similar, but the living cells of the former degraded more than those of the latter. These results indicated that the surface structure of the cell walls differed delicate with each other. Mode of action of the enzyme is now under the investigation and will be described else where. REFERENCES 1) A. Misaki, J. Johnson, Jr., S. Kirkwood, J. V. Scaletti and F.Smith, Carbohyd. Res., 6,150 (1968). 2) D. J. Manners, A. J. Masson and J. C. Patterson, Biochem. J., 135, 19, 31 (1973). 3) T. S. Stewart and C. E. Ballou, Biochemistry, 7, 1843, 1855 (1968). 4) G. H. Jones and C. E. Ballou, J. Biol. Chem., 244, 1043 (1969). 5) H. J. Phaff, Ann. Rev. Microbiol., 17, 15 (1963). 6) S. Bartinicki-Garcia, ibid., 22, 86 (1968).
32 M. ARAI, R. YAMAMOTO and S. MURAO 7) S. Murao, R. Yamamoto and M. Arai, Agr. Biol. Chem., 40, 23 (1975). 8) R. A. Reisfed, U. J. Lewis _??_ and D. E. Williams, Nature, 195, 281 (1962). 9) H. Tanaka and H. J. Phaff, J. Bacteriol., 89, 1570 (1965). 10) T. Kaneko, K. Kitamura and Y. Yamamoto, Agr. Biol. Chem., 37, 2295 (1973).