STUDIES ON THE EXTRACELLULAR PROTEOLYTIC

ENZYMES OF RHIZOPUS OLIGOSPORUS

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HWA L. WANGA N D C. W. HESSELTINE

Northern Refional Resenrcl~Lnboralory,' Peorin, Illinois
Received January 4 , 1965

Abstract
T\vo proteolytic enzyme systems \yere observed in the culture filtrates o €
R h i z o p l ~ soliyn.vporz~s.One has an opti~nurnpII a t 3.0; the other, a t 5.5. Both
enzyme systems have n ~ a s i m ~ uactivities
n a t 50-55 "C and are f a ~ r ! ystable a t pI-I
3.0-6.0. hTasirn~improduction of the enzymes occurred after 72 to 96 hours of in-
cubation ancl then it decreased rapidly. Higher proteolytic activity was noted in
the cult~lrefiltrates oi the orgallism sro\vn in wheat flour nledium than in soy-
bean flour. Data suggest that formation of the enzyme systems appears t o be
inhibited by soybean c s t r x t s .
Introduction
Althougli considerable work has been done on the extracellular proteolytic
enzymes produced by molds, very little of it is concerned with the genus
Rhizopus. Sakoinoto and Shuzui (7, 8) found that strains of R. tonkinensis
Vuill., R . peka Takeda, and I?. javanicus Takeda produced mainly acid pro-
teases on rice, \vheat, or barley flour but both acid and neutral proteases,
with the neutral type predominating, on wheat bran. Pusltas and Elodi (6)
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reported that the proteolytic enzyme systems of R. nigricans Ehrenb. cul-

tured on solid wheat bran had a maximuin activity a t alltaline pH. This
species is actually a synonym of R. stolonifer (Ehrenb.) Vuill. iflore recently,
Hesseltine et al. (3) de~nonstratedthe production of proteolytic enzymes by
strains of R. oligosporus Saito, but noted no activity among the strains of R.
stolonifer that will produce tempell. Since R h i z o p ~ ~has
s been widely used
for fermentation of the Indoilesian food tempeh, it was considered worth-
while to study sonle of the characteristics of the proteolytic enzyme systems
released b ~ R. . oligosporus.
Materials and Methods
C~lltzlrcsc~ndI n o c ~ l l u m
All cultures \\-ere obtained fro111the ARS Culture Collection of this Labora-
tory and maintained on slants of potato-dextrose agar a t 4 OC. Before each
experiment, the organism was transferred to a potato-dextrose agar slant
and incubated a t 28 OC for 7 days. A spore suspension for inoculation was
then prepared by adding 4 1111 of sterilized distilled water to each slant and
shaking tlie culture vigorously for 1 minute.
Extracell~llarEnzyme Prefiaration
Fifty-milliliter portions of 2% whole wheat flour in water were placed in
250-ml Erlenmeyer flasks. After they were autoclaved a t 120 OC for 30 min-
utes, tlie flasks \\;ere inoculated with 0.1 ml of the spore suspension, prepared
as described, and then incubated on a reciprocating shaker a t 28 OC for 72
'This is a laboratory of the Northern Utilization Research and Development Division.
Agricultl~ralIiesearch Service, U.S. Department of Agriculture.
Canadian .lourilal of Microbiology. \rolume 11 (1965)
 728 C.Ah'ADI.IN JOURNAL O F MICROBIOLOGY. VOL. 11, 1965

hours or an otherwise indicated length of time. At the end of incubation,

the fermented medium was centrifuged a t 4 "C, and the supernatant was
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used for enzynle assay.

Determination of Proteolytic Activity
The supernatant was diluted with 0.05 JF citrate-phosphate buffer of
appropriate pH, and enzyme activity was measured according to the casein
digestion method described by I h n i t z (4). The reaction was carried out in
1 ml of 1% casein in 0.05 M buffer of desired pH with 1 ml of diluted culture
filtrate a t 40 "C for 10 minutes. I t was then stopped by the addition of 3 ml
5% trichloracetic acid. The alllount of solubilized protein was estimated by
use of the Folin-Ciocalteau reagent according to Lowry et al. (5). All readings
were corrected for the values of blanks, which were prepared by first mixing
casein solutions with trichloracetic acid and then adding enzyme solutions.

Results and Discussion

E j e c t of PI3 o n Enzyme Activity
The influence of pH on the hydrolysis of casein by the extracellular enzyme
preparation is shown in Fig. 1. Two peaks of activity were noted, one near
pH 3.0 and a smaller peak near pH 5.5. The fall in enzyme activity, particu-
larly a t pH 4-5, is not due to the limited solubility of casein in that pH range.
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This drop was ascertained by comparing the results obtained from increasing
enzyine concentrations: the degree of hydrolysis was proportional to the
amount of enzyme.
Inczrbation Temperature and T i m e
The rate of casein hydrolysis a t pH 3.0 by the enzyme system increased
rapidly as the reaction temperature was increased from 20 to 50 "C (Fig. 2).
R4aximum activity was observed a t 50-55 "C, after which a sharp decrease in
activity occurred. Enzyme activity a t pH 5.5 was similar except that the
increased rate of hydrolysis with increasing temperature was not as great as
that noted a t p H 3.0.
As shown in Fig. 3, hydrolysis of casein a t 40 "C was linear up to 30 minutes
a t pH 3.0. At pH 5.5, hydrolysis took place a t a very slow rate after the first
10 minutes.
Stability of E n z y m e Preparation at Various p H Solutions
Portions of culture filtrates obtained after the cultures were incubated for
72 hours a t 28 "C were adjusted to pH values ranging from 1.5 to 8.0. After
16 hours a t 28 "C, the solutions were readjusted to pH 3.0 or 5.5. The enzynle
activities a t p H 3.0 and 5.5 were then measured as usual. As shown in Table I,
the enzyme system having maximum activity a t p H 3.0 was stable between
pH 3.0-6.0. The enzyine system having optimal activity a t pH 5.5 was also
stable between pH 4.0-6.0, but only 77.5% of its activity remained a t pH 3.0.
This system, however, appeared more stable a t neutral pH than is that with
an optimum a t pH 3.0.
On the basis of differences in behavior of the enzyme systems a t different
pH values, it appears that a t least two acid-proteolytic enzyme systems are
present in culture filtrates of R. oligosporz~s.
 WANG AND IIESSELTIXE: PROTEOLYTIC ENZYIMES O F Rl-IIZOPUS 729
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I 1 1 1 1
20 30 40 50 60 70 10 20 30 40 !
PH Temperature O C
Time in minutes
FIG. 1. Effect of pH of reaction mixture on hydrolysis of casein by proteolytic en7y1nes
rcleasecl by R. oli,oosporr~s.( 1 1111 of d i l ~ ~ t eculture
d supernatant incubated with 1 nll sub-
strate a t 40 "C for 10 minutes; 0.05 111 citrate-PO, buffers were uscrl.)
FIG. 2. Effect of reaction temperature on hydrolysis of casein (reaction time = 10
minutes).
FIG. 3. Effect of length of incubation 011 hydrolysis of casein (reaction tenlperature
40 "C).
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TABLE I
Percentage of proteolytic activity renlaining after storage a t
28 "C for 16 hours a t various pH's

pI-I of
euzylne Activity Activity
meparation a t 01-1 3.0. % a t pH 5.5. '?&

Inhibitors
Natural inhibitors, such as extracts from potato, barley, and soybeans,
reportedly inhibit the activities of neutral and alkaline microbial proteases
but not the activities of acid proteases (1). Our investigations also indicated
that the activities of proteases released by R. oligosporus were not inactivated
either by boiled extracts from soybean or by crystalline soybean trypsin
inhibitors.
Production of Extracellz~lar E n z y m e s
The effect of time of growth on the production of proteolytic enzymes b y
R. oligosporus was established b y determining the proteolytic activities of
the culture filtrates after the cultures were incubated for 24 to 112 hours.
Gradual increases in activities noted during the first 64 hours of incubation
were thereafter followed by a sharp rise (Fig. 4). Enzyme activities reached
a maximuin between 72 to 96 hours of incubation, after which they rapidly
 C \S.\DIXi\: J O L K S X L OF blICROBIOLOGY. VOL. 11, I')05
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Hours

FIG. 4. Eftect of time of growth on the lor~liationof cnrylne and pI1 of LJIV cl~lture
filtrate.

disappeared. Large shifts in culture PI-I values were also noted. Initial pH
of 6.5 fell below 4 during the first 48 hours of incubatioil and then rose to
above 7 presunlably because of the brealcdown of protein. Since the enzyme
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s)rstems were unstable a t pH 7 or above, the corresponding decreases in en-

zyme levels are not surprising.
Table I1 presents data indicating that the ability of RIzi~opiisto prod~ice
proteolytic enzyines varied greatly between different strains of the same
species as well as between different species. Among the cultures tested, R.
oligospor~~s strains N R R L 2710 and N R R L 2549 produced the highest amount
of proteolytic enzymes. R. stolonifer N R R L 2233 produced negligible amounts
of enzymes. Black aspergilli, Aspergilllis z ~ s a m i i Sakaguchi, Iizuka, and
Yanlazaki (R 1031) N R R L A-12940 and -1. saitoi Sal<aguchi. Iizulca, and
Yamazaki ( R 3812) N R R L A-12941 received frorn Dr. H. Iizulca of the
University of Tokyo have previously been reported (9) to produce mainly
acid protease. The present data indicate t h a t S R R L A-12940 a~lcl.A-12941
grown in 2y0 whole wheat flour in water produced considerabl>- less acicl
protease than some strains of Rhizopris.
Higher proteolytic activity was found in the culture filtrates of lihizop~is
grown in wheat flour medium than in filtrates from soybean flour medium.
These differences are probably partly due to the C/N ratio of the medium
or pIH of the culture filtrates. As indicated in Table 11, most of the so>-bean
c u l t ~ ~ rfiltrates
e had a final pIH value above 7. I t is likely that the enzyme
produced had become inactive. On the other hand, the lo\\-er arnounts of
enzymes released by R h i z o p l ~ sgrown in soybean medium coulcl be due to
the presence of inhibitors in the soybean meal.
Eflect of Soybean Extracts on the E n z y m e Formation by Rhizoplls nrltl :lsfier-
gillus
Although Hesseltine et al. (2) observed the presence of a mold gro~v-th
inhibitor from soybean extracts, no difference in growth of the or-ganis~nsin
inediuin containing 2% whole wheat flour or whole wheat flour supple~nented
with soybean extracts was noted in this study. Data from Table I11 also
 \VI\KG .ASD IIESSL?LTISE: PROTEOL'I'TIC ESZYIMI<S O F RIIIZOPUS 73 1

indicate that soybean extracts had no effect on the amount of enzymes

released by Aspergillzls strain XRRL X-12940. However, t h e extracts greatly
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depressed the protease contents of R. oligospor~~sculture filtrates. Since

soybean extracts do not inhibit the activities of proteolytic enzymes released
by R. oligospor~~s,our results suggested t h a t soybean extracts probably
inhibit the formation of proteolytic enzymes by R. oligosporr~s.
TABLE I1
Extracellular proteolytic enzymes of RAhopzcs and ilspergill~es

Enzyme activities*
measured at:
Strains Medium Final pH pH 3 . 0 pH 5 . 5
R . oligosporzcs
XRRL A-9868 \Vheat
Soybean
NRRL 2710 \Vheat
Soybean
N R R L 2549 M.heat
Soybean
R . oryeae
N R R L A-9847 \\Theat
Soybean
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\\'heat
Soybean
R . stolonifer
NRRI. 2233 \Vhrat
Soybean

\Vhea t
Soybean
:I. snitoi
N R R L -4-12941 \Vheat
Soybean
*Enzyme activity is expressed in terrns of pmoles of tyrosine formed per inilliliter of filtrate per hour a t 40 "C

TABLE 111
Effect of soybean extract on the production of proteolytic enzyrncs by Rl~izopllsand Aspe~gillzcs

Enzyme activities*
NRRL mea-;ured a t :
culture
No. Medium Final ~ 1 1 DH 3 . 0 DH 5.5
2710 Soybean
2710 Wheat
2710 Wheat + soybean
extract?
2710 Residue:
A-12940 Soybean
X-12940 \\fhe;lt
.%-I2940 \\'heat + soybean
extract?
A-12940 Residuef
*Enzyme activity is expressed in terms of p~nolesof tyrosine formed per milliliter of filtrate per hour a t 40 "C.
tolie gram of soybean meal was extracted with 40 ml of H2O a t room temperature and centrifuged. The soluble
soybean protein was removed by adjusting pH of the supernatant to 1.5. T h e supernatant was again centrifuged,
adjusted to pH 6.5, and made u p to 50 ml with Ii?O. The extracts were then added to 1 g wheat flour.
$Soybean residue from water extraction.
 732 CAXXDIAN JOURN.-\L OF MICROBIOLOGY. VOL. 11, 1965

References
1. HAGIHARA, H. Bacterial and mold proteases. I?z P. D. Boyer, H. Lardy, and I<. Myrbacl;.
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The enzymes. Vol. IV. Academic Press, New Yorlc. 1960. pp. 193-213.
2. IIESSELTINE,C. W., DECAMARGO, I<., and I<:\cI<Is, J. J. A mold inhibitor in soybeans.
NaLure, 200. 1226-1227 (1963).
3. HESSEI.TISE,C. It7.,SMITII, M., BRADLE,B., and D j r n ~ I<. , S. Investigations of tempeh.
a n Indonesian food. Develop. Ind. i\/Iicrohiol. 4, 275-287 (1963).
4. I < u ~ r ~ M.
z , Crystalline soybean trypsin inhibitor. J . Gen. Physiol. 30, 291-310 (1947).
5. Lownu, 0. H., ~ \ O S E I ~ R O N. U GJ.,~ ~F ,~ R RA., C., and RANDALL, R. J. Protein measurement
with the Folin phenol reagent. J. Bio!. Chem. 193, 265-275 (1951).
6. PUSI~AS, A. and ELODI, Z. Examination of mold proteases. Buclapesti hluszalci Egyet.
?vIezogazd. Icemi. Technol. Taosz. I<ozlemen. 26, 31 (1958); Chem. Abstr. 55,
26104j (1961j.
7. S ~ ~ t o h ~ w r o ,and SRUZUI,I<. Studies on the enzymes produced by microorganisms and
their utilization (VI). Ha1;lio I<ogaku Zasshi, 35, 238-242 (1957).
8. S n r t o ~ o ~M.
o , and S ~ n z u r I<.
, Studies on the enzymes produced by microorganis~nsand
their ~itilization(VII). Halclio I<ogal;~~ Zasshi, 35, 278-282 (1957).
9. Y o s a r ~ h ,F. Studies on the proteolytic enzymes of Blaclc Aspergilli. Bull. Agr. Chem.
Soc. Japan, 20, 252-256 (1956).
For personal use only.