APPLIED MICROBIOLOGY, Feb. 1969, p. 232-236 Vol. 17, No.

2
Copyright ( 1969 American Society for Microbiology Printed in U.S.A.

Separation of the Proteolytic and Streptolytic

Components of a Streptomyces albus Enzyme
Preparation and Their Effect on
Streptococcal Cells
JUANITA P. BRAY' AND JOSEPH A. BASS
Department of Microbiology, The University of Texas Medical Branch, and
The Shriners Burns Institute, Galveston, Texas 77550
Received for publication 4 December 1968

The complex of enzymes elaborated by a strain of Streptomyces albus was sepa-

rated by Sephadex G-75 column chromatography. Two major and one minor
proteolytic peaks and one streptolytic peak were obtained. The streptolytic fraction
alone did not cause lysis of the cells, but was highly effective in the presence of small
amounts of trypsin or of the proteolytic components of the enzyme complex. Treat-
ment of cells with the purified streptolytic fraction effected release of group carbo-
hydrate and of a trypsin and pepsin-resistant protein antigen which reacted with
type-specific antisera.

McCarty (6, 7) has described a complex of
enzymes elaborated by certain strains of Strepto-
myces albus which lyse the cell wall of group A
streptococci and release serologically active
group carbohydrate. Because of the presence of
strong protease(s) which could not be com-
pletely eliminated from the partially purified lytic
enzyme, M protein was not detectable in the
lysates. Recently, however, Schmidt (8) reported
that the proteolytic component could be inacti-
vated by treatment of the partially purified en-
zyme with diisopropyl fluorophosphate (DFP), a
trypsin inhibitor. By treatment of group A strep-
tococcal cells with this preparation, he obtained
a type-specific antigen which appeared to repre-
sent M protein. Because DFP is also a potent
cholinesterase inhibitor, this compound cannot be
safely handled by most laboratories. The present
report is concerned with the separation and par-
tial characterization of this complex of enzymes
by Sephadex G-75 gel filtration and describes the
optimal conditions for solubilizing cell wall
antigens.
MATERIALS AND METHODS
Organisms. A strain of S. albus known to produce
suitable amounts of lytic enzyme was kindly provided
by M. McCarty, Hospital of the Rockefeller Institute
for Medical Research, New York. A colony selected
to produce maximal amounts of lytic enzyme was
' James W. McLaughlin Fellow in Infection and Immunity.
232
inoculated with a minimal number of serial transfers
to a large number of Casamino agar slants which,
after growth for 5 days at 37 C, were stored at 5 C
and used as inocula for the preparation of lytic
enzyme (5).
Group A, type 1 (ATCC 12344) streptococci were
grown in supplemented Todd-Hewitt broth (1).
Cells were harvested by centrifugation after 18 hr
of growth at 37 C and washed three times with sterile
saline and stored at -70 C.
Preparation of S. albus lytic enzyme. The enzyme
was prepared by culturing S. albus on 100 ml of
Casamino Acids medium in Roux bottles (6). After
5 days of growth at 37 C, cultures were filtered
through coarse filter paper, pooled, and brought to
70% ammonium sulfate saturation. The precipitate
was recovered by filtration with Celite (1 g/liter;
Johns-Manville), redissolved in 0.066 M sodium-potas-
sium phosphate buffer, pH 8, and decolorized by the
addition of diethylaminoethyl (DEAE)-cellulose
chloride (about 1 g/100 ml). The enzyme was repre-
cipitated with ammonium sulfate, dissolved in a small
amount of buffer and dialyzed against buffer.
Molecular exclusion chromatography. Sephadex
G-75 (Pharmacia Fine Chemicals) was suspended in
phosphate buffer and used to pour a column 3 X
100 cm. The concentrated enzyme solution (10 to
15 ml volume) was placed on the column and eluted
with 800 ml of phosphate buffer in the cold. Frac-
tions (8 ml) were collected with an automatic frac-
tion collector. Protein was estimated by absorption at
280 nm in a Beckman DU spectrophotometer. Indi-
vidual fractions were assayed for proteolytic and lytic
activity by the methods described below. Pooled
VOL. 17, 1969 STREPTOMYCES ALBUS ENZYME PREPARATION
fractions containing the desired activity were con-
centrated by dialysis against a concentrated solation
of Carbowax 6000 (Union Carbide) in a refrigerator
and then dialyzed against the phosphate buffer.
Assay of enzymes. Fractions were assayed for
proteolytic activity by the casein hydrolysis method of
Kunitz (4). Lytic activity was determined by the
method of McCarty (6). A suspension of heat-killed,
type 1 streptococci (ATCC 12344) was prepared in
0.066 M phosphate buffer to a density of 400 as read
with a Klett-Summerson photoelectric calorimeter
(green filter, 500 to 570 nm). To tubes containing
2.5 ml of the diluted enzyme were added 2.5 ml of the
cell suspension. The tubes were incubated at 37 C in a
water bath and readings were taken at 5-min inter-
vals. Under these conditions, decrease in turbidity
was essentially linear with time. One unit of lytic
activity was defined as that amount of enzyme which
caused a linear rate of fall of 25 turbidity units per
min.
Analytical procedures. Dialyzed samples were dried
in aluminum planchets for 2 hr at 105 C and weighed.
The procedure was repeated when necessary until
constant weights were obtained. Rhamnose was
determined by the method of Dische and Shettles (3)
and pentoses by the method of Dische (2). Protein
concentrations were measured by the biuret method.
RESULTS
Preliminary experiments were performed to de-
termine whether the proteolytic component could
be selectively inhibited by changes in pH or tem-
perature. Proteolytic activity could be demon-
strated over a broad pH range (pH 6 to 11), with
maximum activity at pH 11. The lytic activity was
demonstrable from pH 7 to 10 with maximum
activity at pH 10. Over the range of temperatures
tested, both enzymes showed maximum activity
.20-
a
z
w -1
J
0* .10'
0
TUBE
FIG. 1. Separation of S. albus enzymes by Sephadex G-75 column chromatography.
233
at 50 C. However, stability tests demonstrated
that activity was rapidly lost at this temperature.
At room temperature, both enzymes were stable
for 2 to 3 days.
A group of trypsin inhibitors was tested in an
attempt to inhibit the proteolytic activity while
retaining lytic activity. Sodium azide, sodium
fluoride, mercuric chloride, ethylenediaminetetra-
acetate (EDTA), Wyamine 1622, and phenyl-
methyl sulfonylfluoride were added to the casein
hydrolysis assay system at concentrations of
2 Mmoles/ml. All of these inhibitors were inactive
as was soy bean trypsin inhibitor. DFP was not
tested because of its cholinesterase activity.
Separation of enzymes by Sephadex. Columns
were packed with Sephadex G-25, G-50, G-75,
and G-100 and Bio-Gel P-30, an acrylamide gel.
Sephadex G-75 effected the best separation
(Fig. 1). There were two major and one minor
proteolytic peaks and one streptolytic peak, par-
tially separated from the major proteolytic peak.
A summary of the results of a typical fractiona-
tion procedure is given in Table 1. The initial
steps of ammonium sulfate precipitation and
DEAE absorption accomplished little more than
simple concentration of the crude filtrate and re-
moval of nonspecific materials such as pigments.
The final fraction C, however, showed a substan-
tial degree of purification, as evidenced by the
240-fold increase in specific activity and the
70-fold increase in the lytic to proteolytic ratio.
However, lysis with this fraction required the
addition of a proteolytic enzyme. C fractions
from four batches were pooled and recycled
through Sephadex G-75. The resultant product,
a,
NUMBER
 ~ ~-
234 BRAY AND BASS APPL. MICROBIOL.

TABLE 1. Purification of S. albus lytic enzyme

Lytic activity Proteolytic activity
Fraction Protein Volume_____(ml)___ Specific L/Pb
(mg/ml) Volume (ml) activitya
(units/ml) Total (units/ml) Total

Crude filtrate 0.73 4400 0.32 1,408 0.38 1,672 0.44 0.84
Precipitate (0.7) 2.98 184 5.3 975 6.7 1,232 1.9 0.80
DEAE absorbance 3.85 252 3.8 970 4.9 1,230 3.0 0.80
Concentration 16.72 111.2 61.3 687 59.6 668 3.7 1.0
A 3.0 8.4 2.0 17 20 168 6.6 0.7
B 2.8 5.6 8.2 46 10 56 3.0 0.8
BC 4.6 7.0 48 326 19 135 9.9 2.5
C 0.78 9.0 84e 756e 1.4 12 105 60
n Lytic units per milligram of protein.
b Lytic/proteolytic activity.
c Required addition of trypsin for complete lysis.

which exhibited a further increase of the specific sin. Trypsin or the lytic enzyme alone did not
activity and of the lytic to proteolytic ratio, was produce significant lysis (Fig. 3, lines A and E).
used in the actual antigen extractions described. Trypsin-treated cells were lysed somewhat faster
The apparent increase in total lytic activity re- than untreated cells but less rapidly than when
covered from the column is probably an artifact
in the assay due to factors such as the addition of
trypsin to the assay system to replace the S. albus
proteases removed. However, it may have repre- 9'- _ o = _ o o~~~~~~~~~- A
sented the removal of an inhibitory factor present w E 200 -
in the crude concentrate as purification pro- I-
- -

t,
C
D
ceeded. This phenomenon was frequently ob-
served in the purification of different lots of
enzyme but has not been further investigated.
Effect of trypsin on rate of lytic reaction. 5 o1 15 20 25 30

Schmidt (8) has stated that DFP-treated lytic en-
zyme produced visible lysis of streptococci in the
absence of detectable proteolytic activity. As
shown in Fig. 2, the purified lytic enzyme pro-
duced no lysis unless trypsin was added to the
assay system. The reaction apparently occurs in
two stages, with the trypsin producing final lysis
after preliminary change in the structure of the
cell wall by the lytic enzyme. Full activity of the
lytic enzyme could be restored also by the addi-
tion of varying amounts of either of the two pro-
TIME (Min)
FIG. 2. Effect of trypsin on disruption of cells by
lytic enzyme. (A) lytic enzyme, (B) lytic enzyme +
0.05 mg of trypsin, (C) lytic enzyme + 0.10 mg of
trypsin, (D) lytic enzyme + 0.20 mg of trypsin.
300-
. oE
Z 200- . ....

teolytic fractions (A or B). Addition of sterile

uninoculated culture medium did not restore
lytic activity. Inasmuch as no ions, except those
in the original culture medium, were added, this 100- \
finding infers that ions present in the original
crude preparations did not serve as cofactors.
In order to determine whether trypsin acted
upon the cells or upon the lytic enzyme, a series
of assays was set up as follows: tube A, cells and
lytic enzyme; tube B, cells, lytic enzyme, and
trypsin; tube C, cells treated with trypsin subse- TIME (Min)
quently inactivated by heating before the lytic FIG. 3. Effect of pretreatment and simultaneous
enzyme was added; tube D, lytic enzyme first treatment of cells with trypsin and lytic enzyme. (A)
treated with trypsin which was then inhibited by Cells + lytic enzyme, (B) cells + lytic enzyme +
the addition of soybean inhibitor before the cells trypsin, (C) trypsin-ireated cells + lytic enzyme, (D)
were added; tube E, cells treated only with tryp- cells + trypsin-treated enzyme, (E) cells + trypsin.
VOL. 17, 1969 STREPTOMYCES ALBUS ENZYME PREPARATION 235
the trypsin was added simultaneously with the pension (w/v) of group A, type 1 streptococci in
lytic enzyme. Finally, treatment of the lytic en- 0.066 M phosphate buffer, pH 8, was heated at
zyme with trypsin prior to assay did not ac- 56 C for 30 min. The cooled suspension was
celerate lysis (Fig. 3, line D). treated with purified lytic enzyme (1 lytic unit
Effect of pH on rate of lytic reaction. The ac- per ml) for 4 hr at room temperature with stirring.
tivity of the enzymes isolated by Sephadex G-75 At the end of this time, the enzyme was inacti-
chromatography was tested over a wide pH range. vated by heating at 70 C for 3 min. The yield of
Both proteolytic enzymes exhibited optimal ac- antigens could be increased by 10 min of sonic
tivity at pH 11. The lytic component (Fig. 1, treatment in a Raytheon sonic oscillator. Cell
fraction C) was assayed both for lytic activity in residue was removed by centrifugation, and the
the presence of trypsin and for release of rham- supernatant fluid was tested for protein, rham-
nose in the absence of trypsin (Table 2). Maximal nose, and pentose, before and after dialysis.
lysis occurred at pH 8. Tryptic activity is optimal Dialyzed samples were tested against anti-group
at pH 8; therefore, observeable lysis is probably A and anti-type 1 antisera (Table 3).
attributable to this, since maximal release of
rhamnose, presumably due to the lytic enzyme, DISCUSSION
occurred at pH 10. However, in an effort to mini-
mize the effect of any residual proteolytic activity The initial studies of the complex of enzymes
in the lytic enzyme preparation, treatment of cells produced by S. albus demonstrated that they were
with the latter was conducted at pH 8, since, at capable of dissolving isolated cell walls and that
this level, activity of the proteolytic enzyme was they release soluble, serologically active group A
low. carbohydrate (6, 7). Because the proteolytic en-
Extraction of cells by lytic enzyme. A 25 % sus- zymes alone were incapable of dissolving the cell
wall, McCarty concluded that the "lytic" enzyme
was directed toward the carbohydrate component
TABLE 2. Effect of pH on activity of of the cell wall. More recently, Schmidt (8) used
purified lytic enzyme DFP to inactivate the proteases, and he was able
pHa Lytic activity Rhamnose released to lyse the walls with the lytic enzyme and to ob-
tain serologically active group carbohydrate. He
units/mnl gimi concluded that proteolysis did not contribute to
6 0.44 116 the streptolytic activity of the Streptomyces en-
7 0.70 201 zyme. On closer examination of his data, however,
8 0.87 306 it can be seen that the rate of lysis was inversely
9 0.82 420 proportional to the amount of inhibitor added,
10 0.80 475 and that addition of sufficient DFP to inhibit
11 0.17 41 proteolysis resulted in complete loss of lytic
a Equal volumes (2.5 ml) of a standardized activity.
suspension of heat-killed cells and enzyme prepa- The purified lytic enzyme obtained in our
ration (0.008 mg/ml) in 0.066 M phosphate buffer, studies was not capable of lysing cells. If, on the
pH 8, adjusted to pH 6 to 11 by addition of 0.1 M other hand, a small amount of trypsin was added
HCl or NaOH. Use of the purified enzyme prepa- to the system, lysis could readily be demon-
ration required addition of 0.05 mg of trypsin for strated. These findings suggest that the lysis ob-
complete lysis. served by Schmidt was attributable to small
amounts of residual proteolytic activity acting in
TABLE 3. Extraction of antigens by purified conjunction with the lytic factor.
lytic enzyme It is evident from our data that the purified
lytic factor was capable of releasing various cell
Antigen Total Nondialyzable wall components; these included a protein anti-
gen which reacted with type-specific antisera. In
Protein, mg/ml 6.4 2.7 this respect, Schmidt (8) reported that a protein
Pentose, ,g/ml 454 110 which reacted with type-specific antisera could be
Rhamnose, ,Ag/ml 93 60 demonstrated in enzyme lysates of type 19
Solids, mg/ml 3.7 streptococci. However, the protein antigen ob-
Precipitinogensa tained in the present investigations, after treat-
Group 8
Type 8 ment of type 1 streptococci, was resistant to both
trypsin and pepsin digestion and, therefore, did
a Per milligram of protein
expressed as re- not conform to the characteristics of M protein.
ciprocal of dilution of antigen containing super- The implications of these findings will be dis-
natant fluid. cussed in a separate communication.
236 BRAY AND BASS
LITERATURE CITED
1. Barkulis, S. S., and M. F. Jones. 1957. Studies of streptococcal
cell walls. I. Isolation, chemical composition, and prepara-
tion of M protein. J. Bacteriol. 74:207-216.
2. Dische, Z. 1949. Spectrophotometric method for the deter-
mination of free pentose and pentose in nucleotides. J. Biol.
Chem. 181:379.
3. Dische, Z., and L. B. Shettles. 1948. A specific color reaction
of methylpentoses and a spectrophotometric micromethod
for their determination. J. Biol. Chem. 175:595.
4. Kunitz, M. 1947. Crystalline soybean trypsin inhibitor. R.
General properties. J. Gen Physiol. 30:291-310.
APPL. MICROBIOL.
5. Lancefield, R. C. 1962. Current knowledge of type-specific M
antigens of Group A streptococci. J. Immunol. 89:307-313.
6. McCarty, M. 1952. The lysis of group A hemolytic streptococci
by extracellular enzymes of Streptomyces albus. I. Production
and fractionation of the lytic enzymes. J. Exptl. Med.
96:555-568.
7. McCarty, M. 1952. The lysis of group A hemolytic streptococci
by extracellular enzymes of Streptomyces albus. II. Nature
of the cellular substrate attacked by the lytic enzymes. J.
Exptl. Med. 96:569-580.
8. Schmidt, W. C. 1965. The lysis of cell walls of group A strepto-
cocci by Streptomyces albus enzyme treated with diisopropyl
fluorophosphate. Characteristics of the lytic reaction and the
soluble cell wall fragments. J. Exptl. Med. 121:771-792.