F o l i a Mierobiol.

24, 455--461 (1979)

Activation of Proteolytic Enzymes during Autolysis

of Disintegrated Baker's Yeast
B . B]~I~ALOVX a n d K . B E R A N

Department of Technical Microbiology, Institute of Microbiology,

Czechoslovak Academy of Sciences, 142 20 Prague 4

.Received October 10, 1978

ABSTIIACT. Disintegration substantially aeclerates autolysis of yeast cells. Three proteases (A, B, and C)
=ake part in the autolytie process, proteaso A being the activator of the other two enzymes. The role of
proteases B and C in the process depends on temperature. At 40 ~ both proteases are active while at 50 ~
t h e major role is played by protease C. At 40 ~ NaC1 acts as inhibitor while a t 50 ~ it activates the
process.

The proteolytie system of the yeast Saccharomyces cerevisiae is quite complex,

consisting of carboxypeptidases, aminopeptidases and proteinases and of several
specific inhibitors (Holzer and Saheki 1976). Three of the major proteolytic enzymes
have been described in some detail (Hata et al. 1972). They differ in their pH optima.
The acid protease A (EC 3.4.23.8) splits casein and denatured hemoglobin best at
pH 2--3. Protease B (EC 3.4.22.9) has the optimum for casein splitting at pH 9.
Both are endopeptidases. The third isolated enzyme is a neutral protease C, or
carboxypeptidase Y (EC 3.4.12.--), with an optimum at pH 5--6. Each of the three
proteases has its specific intracellular inhibitor. The proteolytic enzymes appear to
be localized in the vacuoles while their inhibitors are in the cytoplasm (Matile and
Wiemken 1967, Lenney et al. 1974).
The proteolytic enzymes of intact cells are activated during autolysis relatively
slowly, only after all the cell reserves have been used up. The autolytic process begins
after release of the enzymes from the vacuoles and after concersion of their inactive
complexes which are formed after the ]ysis of vacuoles with their respective inhibitors.
The mechanism of activation is the so-called autocatalytie limited proteolysis (Saheki
and Holzer 1975). At usual temperatures this process may take several days. Even
in technical practice when the conditions are optimal (temperature 45--50 ~
3--5 ~ NaC1, pH 5.5, organic solvents) (Peppler 1970) it will take 1 d at the least.
The release of proteases from the vacuoles may be speeded up by disintegration of
cells.
We developed a new technology of the autolysis process, cell disintegration being
the first step of the procedure. The present communication describes the mechanism
of autolysis under optimal conditions, proteolytic activity of the cell suspension dur-
ing autolysis serving as the indicator of the rate of the autolysis process.
4S6 B. B]~I-IALOVI and K. B E R A N Vol. 24

/,0
mg

30

20

10

.0 5 10 15 20 25
h

Fro. 1. Proteolytic activity of yeast cells during autolysis. Autolysis at 40 ~ pH 5.5, nondisintegrated ceils
plasmolyzed by 2 ~o chloroform. Proteolytie activity was determined b y the azoeasein test. In the dis-
integrated cells proteolytic activity was measured with enzymes released into solution. In nondisintegrated
cells it was measured in whole suspension, 1 disintegrated cells, 2 nondisintegrated cells; h hours of autolysis,
mg mg azocasein split per hour by 1 ml autolyzate.

I " I I i
~ J /.0 ~
~C
mg

30
25

20

10

o 65 o
1 1 F I I I
0 1 2 3 4. 5 . IfO I~5 20
h

Fzo. 2. Dependence of proteolytie activity of autolyzate on temperature of autolysis. The azocasein test
was used (pH 5.5). Figures at curves indicate the temperature of protease aetivatJo~ in ~ h hours of
autolysis; mg mg azoeasein split per hour by 1 ml autolyzate.

MATERIALS AND METHODS

Pressed baker's yeast was used. The yeast was disintegrated in a newly designed
mechanical disintegrator (l~ehgSek et al. 1969) for 20 min. A l0 ~o yeast suspension
was mixed with lead-free glass beads (0.45--0.50 ram) at a ratio of 1 : 1.7. The total
volume of the mixture was 1200 ml.
Proteolytic activity was followed by estimating the amount of split azocasein (Fa-
bign 1970; Hasen et al. 1965). 4 ml 1 ~ azocasein (pH 6) and 1 ml sample were
incubated for 30 min at 40 ~ 5 ml 5 % trichloracetic acid was added, the mixture
was stirred and filtered. Absorbance was measured in 2 ml filtrate with 2 m] 0.5
1979 P R O T E O L Y T I C ENZYMES D U R I N G AUTOLYSIS 457

TABT.E I. Comparison of autolysis procedures

Coils Dry weight yield Nitrogen yield Duration

% % h

Disintegrated 69 92 8-10
l~ondisintegrated 50 70 24 -- 48

I~aOH. Protease activity over the entire p H range was assayed b y the splitting of
denatured hemoglobin (Anson 1939). The absorbance of the split hemoglobin was
measured at 280 nm. Two ml 2 % hemoglobin, 2 ml universal B r i t t o n - - R o b i n s o n
buffer ( p H 2--11), 1 ml autolyzate were incubated for 60 min at 40 ~ addition of
5 ml 5 ~o ~richloroacetic acid was added, the filtrate diluted 1 : 10; absorbance was
measured at 280 nm. F o r each sample a special blank was prepared where TCA was
added before the sample. The activity of protease A was estimated according to
Lenney (1975). To inhibit proteases B and C the samples were preincubated for 15 rain
at 25 ~ with an equal volume of 5 m ~ citrate buffer of p H 6 containing 0.05 ~o Brij
35SP and 2 mM HgCI~ (Hata et al. 1967; Dot et al. 1967).
The assay was done as follows: 0.2 ml of the preincubatcd sample and 1.5 ml 1 ~o
hemoglobin (pH 3.4) were incubated for 30 min at 40 ~ 2.5 ml 5 ~o trichloroacetic
acid was added. The filtrate (1.5 ml) was mixed with 3 ml 0.3 ~t lqaOH containing
2.9 ~o Na2C03 and with 0.9 ml Folin--Cioca]tcau reagent diluted with water 1 : 1.
Absorbance was measured at 650 nm after 10 min. Blanks were prepared in the same
w a y b u t TCA was added before the enzyme. Tyrosine was estimated in the split
hemoglobin using the Folin--Ciocalteau reagent, b y measuring absorbance at 650 nm.
The unit of protease activity was set as the increase of absorbance b y 1.0 in 1 h.
The increase of ~-amino nitrogen was followed during autolysis potentiometrically
b y formaldehyde titration and b y the ninhydrin method (Iemm and Cocking 1955).
The autolysis experiments were done in 50 ml volumes of a disintegrated yeast
suspension while agitating it on a water b a t h at the temperature indicated.

RESULTS

Autolysis of disintegrated and intact cells

The rates and yields of autolysis of disintegrated and intact in the two t y p e s
of cells are summarized in Table I. The main reason of the differences appears to

TABLE II. Dependence of ~-amino nitrogen content (~o) in the autolyzate on the p H of autolysisa

pH Formaldehyde titration Ninhydrin method Proteolytic activity be

4.0 2.9 2.6 22

4.5 3.2 3.1 26.5
5.0 3.5 3.6 27.5
5.5 3.8 4.0 28
6.0 3.5 4.0 27.6

a Values in autolyzate dry weight after 3 h of autolysis.

b After 3 h of autolysis.
o Mg azocasoin split per 1 h by 1 ml autolyzate.
458 B. B ~ H A L O V f l _ a n d K . B E R A N Vol. 24

/60 , ....... i "

.mg
30

20
Fig. 3. :Effect of NACI o n t h e p r o t e o l y t i e a c t i v i t y
o f a u t o l y z a t e . T h e a z o e a s e i n t e s t w a s u s e d ( p H 5.5)
10 0 a t 40 ~ (curve 1) a n d 53 ~ (curve 2). Y a l u e s a f t e r
3 h (for 40 ~ a n d a f t e r 1 h (for 53 ~ are r e p o r t e d ;
m g m g a z o e a s e i n s p l i t per h o u r b y 1 m l a u t o l y z a t e ;
M m o l a r i t y of NACl.
o: 0.5 1
'M

| 'i ! |
0.8
A
t
I C
A28o 9 o t

0.6 7 ! B

0./.

0.2

FIG. 4. P r e s e n c e o f p r o t e o l y t i e f r a c t i o n s in a n a u t o -
l y z i n g y e a s t s u s p e n s i o n . A u t o l y s i s t o o k place a~
40 ~ a n d p H 5.5; p r o t e o l y t i v a c t i v i t y w a s e s t i m a t e d
b y t h e h e m o g l o b i n t e s t : 1 a f t e r 5 h of autolysis,
I I ! I
0 2 before a u t o l y s i s ; A2s0 a b s o r b a n c e at 280 nrn.
2 /, 5 8 10 A, B, C i n d i v i d u a l 'protease activities.
pH

lie in the rate of protease activation. The course of protease 'activation is shown
in Fig. 1. The proteolytie activity of disintegrated cells reaches its m a x i m u m within
5 h, and the autolysis is practically terminated before 10 h.

Optimum conditions for autolysis of disintegrated yeast

The effect of temperature on protease activation at p H 5.5 is shown in Fig. 2.
Temperatures above 50 ~ are seen to be detrimental to the activity while tempera-
tures above 40 ~ permit, after a slnwer start, to obtain highest yields.
 1979 P R O T E O L Y T I C ENZYMES D U R I N G AUTOLYSIS 459

~o
mg
3O
a.-NH 2
20

10

0,9
U FIG. 5. Activation of proteaso A and total proteolytic
0.8 activity during the autolysis process. For total
proteolytic activity the azocasein test was used
at p H 5.5; mg mg azocasein split per hour by 1 ml
0.? autolyzate (upper cu~es); ~-NH2 mg ~-amino
nitrogen per ml autolyzed suspension (middle
0.6
curves); U activity of protease A in specific units
I l I ! I I
(lower curves); h hours of catalysis. I n all cases,
0 2 .4 6 8 10 open circles stand for 40 ~ full circles for 53 ~
h during autolysis.

30

mg

20

Fig. 6. Dependence of protease activity on temperature

during the azocasein test. Proteases wore activated by a
3-h autolysis at 40 ~ and p H 5.5. The azocasein test
was done at p H 5.5. (curve 1) and a t p H 9 (curve 2); nag
nag split azocasein per hour per 1 ml autolyzate.
30 ~0 50 oc 60

The p H optimum determined at 40 ~ was at 5--6, both with respect to maximum

proteolytic activity and to the amount of ~-amino nitrogen released (Table II).
Since sodium chloride was reported to support yeast autolysis (Sugimoto 1974)
it was used here at two temperatures. While it speeds up protease activation at 53 ~
it inhibits it at 40 ~ (Fig. 3).
Various divalent metal cations and cysteine were applied b u t no effect on protease
activation could be observed.
450 B. B~I-IALOV~- and K . B E R A N Vol. 24

Role of proteases A, B, and C during autolysis at gO and 50 ~

The roles of the individual proteases could be studied separately by making use
of their different temperature and pH optima and of the inhibitability of proteases
B and C with mercuric chloride (Hata et al. 1967; Doi et al. 1967; l~Shr et at. 197l).
Fig. 4 indicates that both before autolysis and after 5 h of autolysis, all the three
proteases A, B, and C are present but that all their activities are markedly increased
by the autolysis.
Protease A is assumed to act as an activator of protease B and C (Saheki and
ttolzer 1975; Hayashi et al. 1968). This was eheeked and confirmed here (Fig. 5).
The maximum of proteolytic activity of protease A was observed at 50 ~ after
2--3 h of autolysis whereafter the activity dropped while the overall proteolytie
activity reached its maximum after 4--5 h of autolysis. At 40 ~ the maximum
of protease A activity was reached after 4 h of autolysis while the overall proteolytic
activity kept rising for the 9 h of the experiment. Protease A is thus an important
link in the system of proteases but is not itself a major protein-splitting enzyme.
The activities of proteases B and C were determined at different pH values -- at
pH 5.5--6 the total activity of the suspension is obtained (Meyer et al. 1974) while
at pH 9 the activity corresponds mainly to protease B (RShr et al. 1971). The activity
of proteases B and C (after a 3-h autolysis of cells at 40 ~ and pH 5.5) was measured
at different temperatures (Fig. 6). It is clear that at higher temperatures the activity
is mainly due to protease C.
Holzer and Saheki (I976) invoke the role of small amounts of protease B for the
activation of protease A in yeast. This fact was again confirmed here. I f HgCI~ was
added before autolysis (to block proteases 13 and C) protease A did not reach its
full activity. When it was added after 30 and 60 min of autolysis there was a 20 ~
drop of activity of protease A. 1

DISCUSSION

It appears that the activation of proteases proceeds as suggested by Holzer (1975).

Small amounts of protease B activate protease A which, in its turn, activates pro-
teases B and C which are the major lytic proteases during autolysis. In our hands,
it is likely that at 40 ~ both B and C are active while above 50 ~ 9rotease C plays
the major role. The activity of this enzyme rapidly decreases as the amount of
cleavable proteins in the cell drops.
The different effects of NaC1 at the two temperatures can apparently be attributed
to the distinct action of the salt on proteases B and C, on the one hand, and on
protease A, on the other (cf. Sugimoto 1974). Since protease A regulates mainly
protease B which is known to form a stable enzyme--inhibitor complex (Lenney
and Dalbec 1969; Saheki and Holzer 1975) it may so happen that at 40 ~ most of
the proteolytic activity is due to protease B which, in the presence of NaC1, cannot
be readily released from the inactive complex and thus is prevented from activating
protease A. At 50 ~ on the other hand, protease C plays a major role. Its low-
stability complex with the unstable inhibitor is split directly during autolysis (Lenney
1975) without intervention of protease A. NaC1 thus may stimulate its activation
(either through its ionic strength or denaturation of the peptide inhibitor) which
leads to faster autolysis of disintegrated cells at 50 ~
It is of interest to note that autolyzed disintegrated cells possess much higher
proteolytic activities than cells plasmolyzed by chloroform.
1979 P R O T E O L Y T I C ENZYMES D U R I N ~ AUTOLYSIS 45|

REFERENCES

A~SON M. L.: The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J.Gen.Physiol. 22,
79 (1939).
Dot E., H.~YASHI R., HATA T.: Purification of yeast proteinases. Part II. Purification and some properties
of yeast proteinase C. Agr.Biol.Chera. 31, 160 (1967).
FABIAN J.: Synthesis of the extracellular protease by Bacillus pumilus. Folia Microbiol. 15, 160 (1970).
HASEN G. G., HAZ~SE J. A., HUBmXY J. A.: An automated system for the quantitative determination of
proteolytic enzymes using azoeasein. Ann.N.Y.Acad.Sci. 130, 761 (1965).
]:IAWAT., HAYAS~I R., Do1 E. : Purification of yeast proteinases. Part I. Fractionation and some properties
of the proteinases. Agr.Biol.Chem. 31, 150 (1967).
HATA T., HAYASm R., Doi E., MIAMI I., AIBA~.~ S.: Yeast proteinase. Prec. IV. IFS Ferment. Teehnol.
Today, p. 279 (1972).
HAYASHI R., OKAY., D o I E . , HATA T." Activation of intracellular proteases of yeast. P a r t II. Activation
and some properties of proteinase C. Agr.Biol.Chem. 32, 367 (1968).
Hor.zE]~ H.: Regulatory role of proteinases and proteinase inhibitors in yeast. Prec. 9th tZEBS Meet.
Mechanism of Action and Regulation of Enzymes 32, p. 181 (1975).
HOLZ~R H., SAH]~I T.: Mechanism and regulation of proteolytic processes in yeast. To~ai J.Exp.Clin.Med.
1, 115 (1976).
IEM~ E. W., COOKING E. E.: The determination of aminoaeids with ninhydrin. Analyst 89, 209 (1955).
LENNEu J. F., DALBEC J. M. : Yeast proteinase B: Identification of th einaetive form as an enzyme-inhibitor
complex. Arch.Bioehem.Biophys. 129, 407 (1969).
LENN~-Y J. F., MATILE Pro, WIEMKEZ~A., SCHELLENBERG M., MEYER J. : Activities and cellular localisation
of yeast proteases and their inhibitors. Bioehem.Biophys.Res.Commun. 60, 1378 (1974).
L~Z~NE:Z J. F.: Three yeast proteins that specifically inhibit yeast proteascs A, B and C, J.Bacteriol. 122,
1265 (1975).
MATILE PH., WIEMKEN A.: The vacuole as the lysosome of the yeast cell. Areh.Mikrobiol. 56, 148 (1967).
MEYER J., SCEELLENBE~G M., LENN~Y J. F. : Activities and cellular localisation of yeast proteases and their
inhibitors. Biochem.Biophys.Res.Comm. 60, 1378 (1974).
P~PPLE~t H. J.: Food yeast, p. 442 in The Yeasts, A. H. Rose, J. S. Harrison (Eds), Vol. 3. Academic Press,
New York-- London 1970.
R()HR H., DAUBER D., HELLER L.: Autolytic enzymes of baker's yeast. (In Hungarian.) Szeszipar 19, 17
(1971).
I~EHK~EK J., BE~A~ K., B~Si~z V.: Desintegration of microorganisms and preparation of yeast cell walls
in a new type of disintegrator. Appl.Microbiol. 17, 462 (1969).
S A ~ x ] T., Ho~zE~ H. : Proteolytic activities in yeast. Biochim.Biophys.Acta 384, 203 (1975).
SUC~MOTO :H.: Synergistic effect of ethanol and sodium chloride on autolysis of baker's yeast for preparing
foodgrade yeast extracts. J.2eood Sci. 39, 939 (1974).