Process Biochemistry 40 (2005) 1701–1705

www.elsevier.com/locate/procbio

Extracellular acid protease from Rhizopus oryzae:

purification and characterization
Sushil Kumara,*, Neeru S. Sharmaa, Mukh R. Saharanb, Randhir Singhb
a
Division of Biochemistry, Sher-e-Kashmir University of Agricultural Sciences and Technology, R.S. Pura, Jammu, J&K, India
b
Division of Biochemistry, COBS&H, CCSHAU, Hissar, Haryana, India
Received 29 February 2004; accepted 15 June 2004

Abstract

Extracellular aspartate protease from Rhizopus oryzae was purified 91 times with 26% recovery using (NH4)2SO4 fractionation, ion-
exchange and size-exclusion chromatographic techniques. The enzyme was found to be monomeric in nature having a molecular mass of
34 kDa. The enzyme acts optimally at 60 8C with activation energy of 15.16 kcal/mol and was stable in the temperature range of 30–45 8C.
The purified enzyme is an acid protease with optimum pH of 5.5 and retained 96% of residual activity between pH 5.5 to 7.5. Ca2+ activation
(250 times) and varying substrate concentration gave an hyperbolic response. The Lineweaver–Burk plot showed Km value of 5 mg/ml, when
skim milk was used as substrate. The enzyme inhibition of 73 and 93% by pepstatin at 10 and 20 mM, respectively proved it to be an aspartate
protease; however, the additional requirement of histidine residue for enzyme activity has been indicated by differential spectra of diethyl
pyrocarbonate treated versus untreated enzyme.
# 2004 Published by Elsevier Ltd.

Keywords: Microbial enzyme; Aspartate protease; Milk clotting activity; Cheese; Rhizopus oryzae

1. Introduction lated from number of micro-organisms. However, several of

these could not produce good quality cheese with requisite
Enzymes have extensive applications in a range of indus- flavour and taste and even some of them produce bitter
trial processes. Proteases account for approximately 60% of peptides [9]. While screening a large number of bacteria
all enzyme sales because of their varied applications in food, and fungi for their capability to produce acid protease, a
pharmaceutical and number of other industries [1]. Rennet fungal strain was obtained which produced potential milk
(EC 3.4.23.4), an aspartate protease is the chief enzyme clotting protease (MCP), yielding better quality cheese when
employed in cheese production. Rennet not only clots the compared to commercialized enzymes. The fungal strain on
milk but also play an important role during cheese matura- identification from Institute of Microbial Technology, India
tion, which is a vital and complex process for the balanced was found to be Rhizopus oryzae Went and Prinsen-Geerlgs
development of flavour and texture [2–4]. The scarcity and (MTCC 3690). Some of the kinetic characteristics of the
high price of traditional calf rennet has promoted research purified enzyme preparation obtained from this fungus are
towards the alternate milk coagulants produced either by reported.
plant or by the native/genetically modified micro-organisms
[5]. A suitable rennet substitute must have high specific
caseinolytic activity (to promptly cleave the phe105–met106 2. Materials and methods
bond of k-casein) and small-generalized proteolytic activity
[6–7]. After initial reports of isolation of milk clotting 2.1. Materials
enzyme from fungi [8], analogous enzymes have been iso-
* Corresponding author. Fax: +91-1923-250639/250242. Column chromatography materials were purchased from
E-mail address: [email protected] (S. Kumar). Sigma Chemicals Co. St. Louis, MO, USA and molecular

0032-9592/$ – see front matter # 2004 Published by Elsevier Ltd.

doi:10.1016/j.procbio.2004.06.047
1702 S. Kumar et al. / Process Biochemistry 40 (2005) 1701–1705

markers for electrophoresis were obtained from Genei, 40 min i.e.

Bangalore, India. Other chemicals used were of analytical
Milk clotting ðU=mlÞ ¼ 2400=t
D:F:
grade.
where t is clotting time and D.F. is dilution factor.
2.2. Methods
2.2.6. Caseinolytic activity
2.2.1. Cultivation of fungal strain and enzyme production The proteolytic activity of the enzyme was assayed after
R. oryzae isolated from the soil of an effluent treatment Arima et al. [10]. To 2.5 ml of 1% (w/v) alkali soluble casein
plant was maintained by weekly transfer on potato dextrose in 0.02 M potassium phosphate buffer (pH 6.5) 0.5 ml of
agar medium fortified with 10% skim milk at 30 8C. The enzyme extract was added. The reaction mixture was incu-
extracellular acid protease was produced under solid-state bated at 37 8C in a water bath for 10 min and the reaction
fermentation conditions in wheat bran phosphate buffer was terminated by adding 2.5 ml of 0.44 M trichloroacetic
medium (50 mM and pH 6.0) at 30 8C. The enzyme was acid. The precipitates formed were removed by filtration
harvested after 48 h of incubation with distilled water. The through Whatman No. 1 filter paper. One milliliter of 1N
filtrate obtained was centrifuged at 10,000
g for 10 min at Folin-Ciocalteu reagent and 2.5 ml of 0.55 M sodium car-
4 8C. The supernatant so obtained was referred as crude bonate solutions was added to 1 ml of the above clear filtrate.
extract. All steps of enzyme purification were carried out at This was further incubated for 20 min at 37 8C for colour
0–4 8C. development. The optical density at 660 nm expresses activ-
ity in term of proteolytic units (PU).
2.2.2. (NH4)2SO4 fractionation
MCP in crude extract was precipitated between 20–60% 2.2.7. Protein estimation
saturation of (NH4)2SO4. The precipitate obtained after The protein content of individual fraction obtained after
centrifugation at 10,000
g for 30 min was suspended in different steps of chromatography was monitored by mea-
50 mM phosphate buffer, pH 6.0 and dialyzed overnight suring the extinction at 280 nm. Quantitative estimation of
against repeated changes of the same buffer. protein was done by the method of Lowry et al. [11].

2.2.3. Ion-exchange chromatography 2.2.8. Determination of purity and molecular mass

The enzyme preparation obtained from the above step The purity of the enzyme preparation was judged by
was further purified by passing through a column (25 cm
native PAGE and the molecular mass of the purified enzyme
2.6 cm) of activated DEAE-cellulose previously equili- was estimated by gel filtration through a column of Sepha-
brated with 50 mM phosphate buffer, pH 6.0. The fractions dex G-100, which has previously been calibrated with the
of 3 ml each were eluted at the flow rate of 35 ml/h with standard marker proteins viz. alcohol dehydrogenase
linear gradient of 0–0.4 M KCl and analyzed for enzyme (150 kDa), bovine serum albumin (66 kDa), carbonic anhy-
activity and protein content. The active fractions were drase (29 kDa) and Cyt. C (14.3 kDa). Subunit molecular
pooled and concentrated by osmosis. mass was determined by SDS-PAGE according to the
method of Laemmli [12]. The molecular weight markers
2.2.4. Size-exclusion chromatography used for electrophoresis were a-lactalbumin (14.3 kDa),
The concentrated enzyme was loaded on to Sephadex G- carbonic anhydrase (29 kDa), ovalbumin (43 kDa), bovine
100 column (65 cm
1.5 cm) pre-equilibrated with 50 mM serum albumin (68 kDa) and phosphorylase b (97.4 kDa).
phosphate buffer, pH 6.0. Enzyme fractions of 3 ml were
eluted at 12 ml/h flow rate with the same buffer and were
analyzed for enzyme activity and protein content. Active 3. Results and discussion
enzyme fractions were pooled and stored at 4 8C for further
studies. 3.1. Purification and molecular mass

2.2.5. Milk clotting activity The extent of purification and characteristic of an enzyme
The milk clotting activity of enzyme was determined as has a profound effect on product quality. The enzyme from
described elsewhere with modification [10]. Five milliliter R. oryzae was purified to electrophoretic homogeneity. The
of assay milk (10% skim milk and 0.01 M CaCl22H2O in enzyme was fractionated between 20 and 60% (NH4)2SO4
distilled water) was taken in a test tube and the contents were saturation; with 30-fold purification and 103% recovery.
brought to the temperature of 37 8C. 0.5 ml of enzyme Higher enzyme recovery was probably due to removal of
extract was then added and the curd formation was observed certain low molecular weight inhibitor. Passage from
while manually rotating the test tube from time to time. The DEAE-cellulose column further purifies the enzyme to
end point was recorded when discrete particles were dis- 79-fold with 32% recovery and finally on Sephadex G-
cernible. One milk clotting unit is defined as the amount of 100, the extent of purification was 91-fold with 26% re-
enzyme present in 1 ml of extract clotting 10 ml substrate in covery (specific activity 760 U/ml), having high specific
 S. Kumar et al. / Process Biochemistry 40 (2005) 1701–1705 1703

Table 1
Purification of milk clotting protease from Rhizopus oryzae
Purification steps Total Total Specific activity Purification Recovery
activity (U) protein (mg) (U/mg protein) fold (%)
Crude extract 32110 3850 8.34 – 100
(NH4)2SO4 fractionation (20–60%) 33178 132.6 250.21 30 103.3
DEAE-cellulose 10273 15.5 662.78 79.4 32
Sephadex G-100 8279 10.9 759.5 91 26

caseinolytic activity desirable for rennet substitutes that of calf rennet i.e. 14 kcal/mol [23]. The thermal inacti-
(Table 1). MCP from different sources has been purified vation experiments indicated that the enzyme was quite
using a range of chromatographic techniques [13–18], but stable up to 45 8C, however, the activity declined linearly
only limited numbers can be used as calf rennet substitutes. with further increase in temperature. The enzyme at 40 8C
The purified enzyme has molecular mass of 34 kDa as remains fully active even after 60 min of incubation but at
determined by gel filtration (Sephadex G-100) and gel 60 8C it losses 62% of its initial activity (inset Fig. 2).
electrophoresis, which suggested the monomeric nature of
enzyme (Fig. 1). The molecular mass of 34 kDa for MCP has 3.3. pH optima and pH stability
also been reported for Endothia parasitica [19] and Mucor
bacilliformis [20] like ours. The acid protease from R. oryzae exhibited the maximal
rate of reaction at milk pH of 5.5 (Fig. 3). The activity
3.2. Temperature optima and thermostability declined with further increase in the pH. The acidity of milk
at the time of enzyme use should be 0.19–0.20%, which fall
The enzyme acted optimally at 60 8C and afterward, it with in the pH range of 5.5–6.5. So the enzyme should be
starts losing activity quite rapidly with complete inactivation steady in this range. The present enzyme retained 96% of its
at 70 8C (Fig. 2). MCP from Cryptococcus albidus [21] and activity from pH 5.5 to 7.5, resembling other aspartate
Penicillium oxalicum [22] also act optimally at 60 8C. The protease (inset Fig. 3). The pH optima in the same pH range
activation energy as calculated from Arrhenius plot was have also been reported for MCP from Mucor miehei [9] and
found to be 15.16 kcal/mol, which is quite comparable with C. albidus [21]. Calf rennet also acts strongly in the acidic
range.

3.4. Effect of substrate concentration

The enzyme showed an hyperbolic response with increas-

ing concentration of skim milk in an otherwise standard

Fig. 1. Electrophoretogram of protein after various steps of purification on Fig. 2. Temperature optima () and stability (^) of aspartate protease. The
SDS-PAGE. Lane 1: crude extract; lane 2: (NH4)2SO4 fractionation; lane 3: milk clotting activity of enzyme was assayed at indicated temperatures
DEAE-cellulose. Lane 4: Sephadex G-100; lane 5: standard molecular under standard conditions. Inset is the curve depicting the thermostability of
weight markers. enzyme at 40 8C (~) and 60 8C (&).
1704 S. Kumar et al. / Process Biochemistry 40 (2005) 1701–1705

Table 2
Relative inhibition of fungal milk clotting protease by various selective
protease inhibitors
Reagent Concentration Relative
(mM) activity (%)
Iodoacetamide 2 93
4 94
p-Chloromercury benzoate 2 94
4 94
Ethylenediaminetetraacetic acid 5 100
10 100
Diethylpyrocarbonate 0.15 48
0.3 18
Phenylmethane sulphonyl fluoride 1 97
2 94
Benzamidine 1 98
Fig. 3. Influence of milk pH on milk clotting activity of enzyme (^). The
2 98
enzyme activity was measured by ranging milk pH from 5.5 to 7.5 under
standard conditions. Inset is the curve showing pH stability of R. oryzae Pepstatin 0.01 27
protease (&) incubated for 1 h at 30 8C in buffers of various pH 0.02 7
values.
Enzyme was incubated with different concentrations of specified reagents at
30 8C for 30 min and relative enzyme activity as compared to control was
assay mixture (Fig. 4). However, at higher concentration of determined as mentioned in Section 2.
the substrate the enzyme activity declined slightly. The
Michaelis constant as determined by double reciprocal plot Ca2+ accelerated milk clotting activity at all the concentra-
was found to be 5 mg/ml. tions with hyperbolic response using skim milk as substrate.

3.5. Effect of CaCl2 concentration 3.6. Effect of inhibitors

Ca2+ was found to be potent activator with 250% increase Protease inhibitors were employed to identify group at
in milk clotting activity compared to control (in absence of the active site of this enzyme (Table 2). The enzyme retained
metal ion). Ca2+ combines with para casein to form firm clot 93–94% of relative activity in presence of iodoacetamide
during second phase of clotting process. So the activation of and p-chloromercurybenzoate respectively, precluding the
250% by Ca2+ is important in this case. The similar response role of –SH group in enzyme activity. Similarly, phenyl-
has also been observed in MCP from Irpex lacteus [24], C. methane sulphonyl fluoride and benzimidine did not affect
albidus [21], Rhizomucor miehei [25] and P. oxalicum [18]. the enzyme activity; hence serine participation at the active
Mg2+ and Mn2+ also had stimulatory affects. Addition of site of the enzyme has also been ruled out. Ineffectiveness of

Fig. 5. Inhibition of milk clotting activity of enzyme by diethyl pyrocar-

bonate. The enzyme was assayed for milk clotting activity after incubating
for different durations of time at specified concentrations of diethyl
Fig. 4. Substrate saturation profile of an aspartate protease from R. oryzae. pyrocarbonate [0.15 (^), 0.30 (&) and 0.45 mM (~)].
 S. Kumar et al. / Process Biochemistry 40 (2005) 1701–1705 1705

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