J. Biochem. 83, 893-903 (1978)

Purification and Characterization of Lysozyme Produced by Streptomyces erythraeus Takashi MORITA, Saburo HARA, and Yoshio MATSUSHIMA Department of Chemistry, College of Science, Osaka University, Toyonaka, Osaka 560 Received for publication, October 24, 1977

A species of lysozyme (SE lysozyme) was purified from culture filtrate of Streptomyces erythraeus. The enzyme has a molecular weight of 18,500 as determined by ultracentrifugation. Its isoelectric point is 9.5, and it shows optimal activity at pH 4.0 with an optimal ionic strength of 0.1. Investigation of the substrate specificity showed SE lysozyme to be an N-acetylmuramidase. The simplest product in the digest of cell walls of Micrococcus lysodeikticus was identified as a disaccharide, [GlcNAcjS(l—>4)MurNAc]. While S. aureus as well as M. lysodeikticus was lysed by this lysozyme, chitin and its derivatives were not.

Since Fleming found a lysozyme [EC 3.2.1.17] in 1922, similar enzymes have been shown to be widespread in animals, plants, and microorganisms (/). Lysozymes of vertebrates are chemically and structurally related to HEW lysozyme, which is the most extensively investigated lysozyme. While invertebrate lysozymes have similar lytic activity, lysozymes of other origins show somewhat different enzymic activity (2). Some bacteriolytic enzymes produced by microorganisms are active against the cell walls of Staphylococcus aureus, which are not hydrolyzed by lysozyme (5). These enzymes have been useful tools for elucidating the fine structures of bacterial cell walls (4). We have found a species of lysozyme in the culture broth of Streptomyces erythraeus which can Abbreviations: GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid; HEW lysozyme, hen egg-white lysozyme; SE lysozyme, lysozyme produced by Streptomyces eryihraeus; Ch lysozyme, lysozyme produced by Chalaropsis sp.

Vol. 83, No. 3, 1978

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lyse S. aureus cell walls as well as M. lysodeikticus cell walls. This paper describes the purification and characterization of SE lysozyme. MATERIALS AND METHODS Materials—HEW lysozyme ( x 6 crystallized) was purchased from Seikagaku Kogyo Co. The culture filtrate of Streptomyces erythraeus was kindly supplied by Drs. H. Ohtsuka and N. Yoshida of the Central Institute of Shionogi Pharmaceutical Co. Dried cells of Micrococcus lysodeikticus NRLL 384 were provided by Prof. James T. Park of Tufts University, School of Medicine. > Cell walls of M. lysodeikticus were prepared according to Salton and Home (5). N-Acetylated hydrazinolysates of M. lysodeikticus (backbone sugar moiety of peptidoglycan) were prepared according to Hara and Matsuhima (6). General Methods—Baceteriolytic activity was measured at 25°C in terms of the reduction in turbidity of a suspension of M. lysodeikticus. The

894

cells of M. lysodeikticus (17.3 mg) were suspended in 0.1 M acetate buffer of pH4.5 (100 ml) to give an absorbance of 0.8 at 450 nm. To the suspension (3 ml) was added the enzyme solution (0.1 ml), and the reduction of absorbance at 450 nm was recorded using Beckman DB spectrophotometer. The initial velocity of the enzyme reaction was calculated from the linear part of the curve. The enzyme unit was defined as the amount required to cause a reduction in absorbance of the cell suspension of 0.001 per min. The enzyme activity on cell walls and Nacetylated hydrazinolysates of M. lysodeikticus was also mesasured in terms of the increase of the reducing power of the reaction mixture. The reducing power was measured by the Park-Johnson method (7). Disc electrophoresis was performed on 15% (w/v) polyacrylamide gel, pH 4.0, at room temperature in apparatus from M.S. Instrument Co. (8). Electrophoresis was carried out for 2 h with a current of 5 mA per tube. The gels were stained with Coomassie brilliant blue (9), and destained by washing in methanol-acetic acid-water ( 2 : 3 : 35, v/v). The molecular weight of SE lysozyme was estimated by the sedimentation equilibrium method. A Spinco model E analytical ultracentrifuge equipped with a Rayleigh interference system was used. High-speed sedimentation equilibrium experiments were performed at 25°C. A double sector cell (not a synthetic boundary cell) was used for the experiments. Based on the following equation of Nazarian (70), a graph of ln^cj/ versus r 1 (=q) yields the weight-average molecular weight (Mw):

T. MORITA, S. HARA, and Y. MATSUSHIMA containing a sucrose density gradient (0-50%). Electrophoresis was continued until the current had decreased to 2.5 mA at constant voltage (900 V, 40 h) at 4°C. The protein content was estimated by the method of Lowry et al. (12). Bovine serum albumin was used as a standard protein. Amino acids were analyzed according to Spackman et al. (13) with a Hitachi KLA-3B automatic amino acid analyzer. Amino sugars (glucosamine, glucosaminitol, muramic acid, and muramicitol) were analyzed according to Hara and Matsushima (14) using an automatic amino acid analyzer. The amino-terminal amino acid sequence of SE lysozyme was determined with a JEOL JAS47K automatic protein-peptide sequencer. The thiazolinone derivatives of the amino acids released in the sequencer were converted to phenylthiohydantoins by heating the thiazolinone derivatives in 1 N HCI. The PTH-derivatives were identified by thin layer chromatography (IS). RESULTS

Purification of SE Lysozyme—Step 1. Adsorption on Amberlite IRC-50: The pH of the dark brown crude filtrate of S. erythraeus (12.4 liters) was adjusted to 6.0 with glacial acetic acid. Amberlite IRC-50 (0.5 liter) which had been equilibrated with 0.01 M potassium phosphate buffer, pH6.0, was added to the culture filtrate. The mixture was stirred for an hour and left to stand for 30 min to precipitate the resin. The supernatant was decanted and the resin was washed with distilled water until the washings showed no color. The washed resin was packed in a column dlnC(r) dln4 Q / (l-Sp)w% (10 cm in diameter) and washed again with water Mw dq dr« 2RT until the absorbance at 280 nm of the eluate fell below 0.1. Then SE lysozyme was eluted with where 4 Q / is the number of fringes crossed in 0.7 M potassium phosphate buffer, pH 6.0 (1.2 traversing the interference pattern from q to q+Q: liters). Further concentration and purification was r is the distance from the center of rotation; C(r) accomplished by bringing the eluate to 0.8 saturais the concentration of sample at the distance r; T tion with solid ammonium sulfate. The salt was is the absolute temperature; 8 is the partial specific added to the eluate with stirring, and precipitation volume; p is the buffer density; a> is the angular occurred above 0.3 saturation. After standing overnight at 4°C, the precipitate was collected by velocity. centrifugation, dissolved in the least possible Isoelectric focusing was carried out according amount of water and dialyzed against deionized to Vesterberg and Svensson (11) using a 110 ml water. column. The purified enzyme (4 mg) was applied Step 2. Chromatography on CM-cellulose: to a 1% carrier ampholyte column (pH 3.5-10) J. Biochem.

LYSOZYME PRODUCED BY S. erythraeus

895

5.0

C-50: The enzyme preparation was applied to a CM Sephadex C-50 column (3.0x20 cm) which had been equilibrated with 0.3 M ammonium acetate buffer, pH 6.0. The column was eluted with the

5.0

1 *2

4.0

ffi cc

;/ IIli /^"^

0.3 o

100

200

FRACTION NUMBER

Fig. 1. Chromatography of SE lysozyme on CMcellulose. The crude enzyme preparation (2.39 g) was applied to a column (3 x 50 cm) which had been equilibrated with 0.01 M potassium phosphate buffer, pH 6.0. After washing with the same buffer (500 ml), the column was eluted with a linear gradient of potassium phosphate concentration (0.01-0.6 M, pH 6.0); ten ml fractions were collected, o , Absorbance at 280 nm; • , enzyme activity.

•

-3

3.0

|

2.0

(I 0.30M I I

. 0.27M

100

200

o 1

9

300

FRACTION NUMBER

Fig. 2. Chromatography of SE lysozyme on Amberlite IRC-50. The enzyme preparation (221 mg) was applied to a column (2.5x70 cm) which had been equilibrated with 0.01 M potassium phosphate buffer, pH 6.0. The column was eluted with stepwise changes of concentration of potassium phosphate buffer, pH 6.0. Ten milliliter fractions were collected. O, Absorbance at 280 nm; • , enzyme activity. Vol. 83, No. 3, 1978

< O

1.0

4.0

1.0

C

y 3 f:3 IVI

o 3.01- • 0.6

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The crude enzyme preparation (50 ml) was applied to a CM-cellulose column (3x50 cm) which had been equilibrated with 0.01 M potassium phosphate buffer, pH 6.0. After washing with 0.01 M potassium phosphate buffer, pH 6.0 (500 ml), the column was eluted with a linear concentration gradient of potassium phosphate buffer, pH 6.0 (from 0.01 M to 0.6 M). Ten milliliter fractions were collected. SE lysozyme was eluted at about 0.3 M concentration (Fig. 1) of the eluent. The fractions (110155) were combined and the protein was precipitated with ammonium sulfate (0.8 saturation). The precipitate was collected by centrifugation and dialyzed against 3 liters of deionized water (three changes) over a 24 h period. Step 3. Chromatography on Amberlite IRC50: The dialyzed enzyme solution was chromatographed on an Amberlite IRC-50 column (2.5 x 70 cm) which had been equilibrated with 0 . 0 1 M potassium phosphate buffer, pH 6.0. The column was eluted stepwise with 0.20 M, 0.27 M, and 0.30 M potassium phosphate buffer, pH 6.0. The lytic activity was eluted with 0.30 M buffer (fractions 226-245, Fig. 2). The fractions were combined and the solution was brought to 0.8 saturation of ammonium sulfate. The precipitate was collected by centrifugation, dialyzed against deionized water (three changes) and then lyophilized. Step 4. Chromatography on CM Sephadex

T. MORITA, S. HARA, and Y. MATSUSHFMA

896

same buffer. The fractions (65-120) were collected (Fig. 3), lyophilized and dialyzed against deionized water to remove remaining ammonium acetate. The sample was lyophilized and stored at —20°C. Step 5. Crystallization of SE lysozyme: The lyophilized enzyme preparation (100 mg) was dissolved in 6 ml lots of ammonium sulfate solution at 0.3, 0.4, and 0.5 saturation. The insoluble material was removed by centrifugation. Saturated ammonium sulfate solution was added dropwise to each enzyme solution until faint turbidity was observed. Each mixture was kept overnight at 4°C. Rod-shaped crystals formed in the 0.3 saturation sample (Fig. 4). In the case of 0.4 and 0.5 saturation crystals also formed, but the size and amount of the crystals were smaller than those of the 0.3 saturation sample. The crystals were centrifuged and washed three times with 0.3 saturated ammonium sulfate solution (3 ml). The crystalline enzyme was dissolved in 0.3 saturated ammonium sulfate solution and recrystallized in the manner described above. A summary of the purification procedure is

50 100 FRACTION NUMBER

150

Fig. 3. Chromatography of SE lysozyme on CM Sephadex C-50. The enzyme preparation (176 mg) obtained at Step 3 was applied to a CM Sephadex C-50 column (3.0x20 cm) which had been equilibrated with 0.3 M ammonium acetate buffer, pH 6.0. The column was eluted with the same buffer. Fractions of 5.5 ml were collected. O, Absorbance at 280 nm; • , enzyme activity.

0.1 wm Fig. 4. Photomicrographs of crystalline SE lysozyme. TABLE I. Summary of the purification procedure. Step

Culture supernatant Adsorption on Amberlite IRC-50 CM-cellulose chromatography Amberlite IRC-50 chromatography CM-Sephadex C-50 chromatography

Vol. (ml)

Total activity (units)

Total protein ()

12,400 1,180

546 479

99,300 2,390

450

317

221

185

259

176

300

168

97

Specific activity

(units/mg of protein)

Yield

5. 45(1)

100

(37)

88

1,440 (264) 1,470 (270) 1,750 (321)

58

201

47 31

Biochcm.

LYSOZYME PRODUCED BY S. erythraeus

897

0.50

3

I S0.25

A

B

Fig. 5. Polyacrylamide gel electrophoresis. Disc electrophoresis was carried out at a current of 5 mA per tube for 2 h on 15% (w/v) polyacrylamide gel, pH 4.0. The gels were stained with Coomassie brilliant blue. (A) Original culture filtrate, (B) Step 3 (Amberlite IRC-50), ( Q Step 4 (CM Sephadex C-50).

shown in Table I. The results of disc polyacrylamide gel electrophoreses at various purification steps are shown in Fig. 5. pH-Activity Profile—The effect of pH on the enzyme activity was examined using whole cells or N-acetylated hydrazinolysates of M. lysodeikticus cell walls as substrates. Britton-Robinson's buffer (pH 2.0-8.0) (76) was used in the experiments. In the turbidimetric method, the purified enzyme solution (0.1 ml, 48 ^g/ml in water) was added to the cell suspension (3 ml, 0.173 mg/ml in buffer) and the initial velocity of decrease in turbidity at 450 nm was measured. In the determination of reducing power released by the enzyme, an aliquot of 0.3 ml of N-acetylated hydrazinolysate solution (21 mg/ml in water), the enzyme solution (0.2 ml, 1.2 mg/ml in water) and buffer at various pH's (3 ml) were mixed and the mixture was incubated at 37°C for 1.5 h (because of poor activity toward this substrate, a longer incubation period was adopted). Then the reducing power was determined by the Park-Johnson method. The results are shown in Fig. 6.

Fig. 6. pH-Activity profile of SE lysozyme. BrittonRobinson's buffer (pH 2.0-8.0) was used. Experimental details are given in the text. O, Decrease in turbidity of a whole cell suspension of M. lysodeikticus; • , reducing power released from N-acetylated hydrazinolysates of M. lysodeikticus. 0.10 Q

m tr

- go.05

n UJ

0.10

0.20 0.30 0.40 IONIC STRENGTH

0.50

Fig. 7. Effect of ionic strength on SE lysozyme activity. Whole cell suspension (2 ml, 0.23 mg/ml in buffer, pH 4.0) of M. lysodeikticus, NaCl solution (1 ml, 0.031.5 M), and SE lysozyme solution (0.05 ml, 65//g/ml) were mixed, and the decrease in turbidity in 1 min was

measured.

strength on bacteriolytic activity was investigated by the turbidimetric method. Whole cells of M. lysodeikticus (23 mg) were suspended in sodium acetate buffer (0.0015 M, pH4.0, 100 ml). An SE lysozyme catalyzed the lysis of M. lyso- aliquot of 1 ml of NaCl solution (0.03-1.50 M) was deikticus cells over a pH range of 3.0-5.0 with added to the bacterial suspension (2 ml). The maximum activity at pH4.0. The pH-activity enzyme solution (0.05 ml, 65 pg/ml) was added to profile in the hydrolysis of N-acetylated hydra- the suspension and the decrease in turbidity at zinolysates of M. lysodeikticus was similar to that 450 nm was measured. Optimal ionic strength of in the lysis of whole cells. SE lysozyme for the lysis of M. lysodeikticus whole Effect of Ionic Strength—The effect of ionic cells was about 0.10 (Fig. 7). Vol. 83, No. 3, 1978