LWT - Food Science and Technology 65 (2016) 652e660

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LWT - Food Science and Technology

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Purication, physico-chemico-kinetic characterization and thermal

inactivation thermodynamics of milk clotting enzyme from Bacillus
subtilis MTCC 10422
Rajesh Kumari Narwal a, 1, Bharat Bhushan b, *, 1, Ajay Pal a, Anil Panwar c, Sarla Malhotra a
a
Department of Chemistry & Biochemistry, CCS Haryana Agricultural University, Hisar 125004, India
b
Processing Division, ICAR-Central Institute of Post-harvest Engineering & Technology, Abohar 152116, India
c
Department of Molecular Biology, Biotechnology & Bio-informatics, CCS Haryana Agricultural University, Hisar 125004, India

a r t i c l e i n f o a b s t r a c t

Article history: Extracellular milk clotting enzyme from Bacillus subtilis MTCC 10422, which has the capacity of forming
Received 28 February 2015 milk curds, was puried 32.9 fold with 20.82% recovery using sequential chromatographic techniques.
Received in revised form The molecular weight of the enzyme was found to be 27 kDa which is of monomeric nature. The opti-
23 July 2015
mum temperature of the enzyme was found to be 45
C and it was quite stable in the temperature range
Accepted 25 August 2015
Available online 31 August 2015
of 35e65
C. The enzyme showed the pH optima of 6.0, and quite stable in broad pH range of 5.0e8.0 and
showed a typical hyperbolic velocity saturation curve with Km value of 5 mg mL1 with skim milk as a
substrate. Calcium chloride at the concentration of 10 mM was found to be the most effective stimulator,
Keywords:
Milk clotting enzyme
accelerating the enzyme activity by about 2.8 folds. Various kinetic parameters towards thermo-
Purication inactivation of milk clotting enzyme were studied. Thermal inactivation behaviour of the puried
Characterization enzyme suggested its thermostability and also its sensitivity to duration of heat treatment. After puri-
Cheese cation, both yield and recovery of puried preparation were found appreciable. Since this enzyme has
wider pH stability and thermostability, usage of this high activity microbial enzyme for various cheese
preparations can be evaluated at commercial scale.
2015 Elsevier Ltd. All rights reserved.

1. Introduction
Rennet (E. C. 3.4.24.4) is the most exploited calf stomach pro-
teolytic enzyme in food processing for the manufacture of cheese.
But the commercial success of this enzyme has several hindrances
such as its limited availability, its cost and stability. Owing to rapid
growth and relatively inexpensive growth substrates, microbial
milk clotting enzymes (MCEs) have obtained great attention and so
have become popular rennet substitutes (Wilkinson & Kilcawley,
2005). At present, microbial rennet is used for one third of all the
cheese produced worldwide. Many microorganisms especially
Rhizopus spp. have been identied as producers of rennet which
can substitute the calf rennet (Sun, Wang, Yan, Chen, & Jiang, 2014).
From the last four decades, attempts have been made to isolate
coagulating enzyme from a number of microorganisms and have
* Corresponding author.
E-mail addresses: buddingbiochemist@gmail.com, bharat.bhushan@icar.gov.in
(B. Bhushan).
1 Bacillus subtilis is one of the vastly investigated microbial groups
Authors have contributed equally in this research work and manuscript
preparation.
been used to prepare cheeses of various types (Jacob, Jaros, & Rohm,
2011).
When rennet is added to milk, after a certain lag period, the milk
abruptly begins to coagulate and transforms into three dimensional
rm gel with the release of whey. Coagulants or other exogenous
enzymes not only clot the milk to curd but also play an important
role during the ripening of cheese, which is the most complex and
important process for the development of favourable avour and
texture.
Rennet is extensively or completely denatured during the
cheese preparation where the curd cooking temperature exceeds
55
C. In general, retention of residual coagulant activity in cheese
ranges from 0 to 15% and is inuenced by gel cutting temperature.
The partitioning of residual enzyme in curd and whey is also
inuenced by pH at whey drainage during cheese preparation
(Wilkinson & Kilcawley, 2005). Thus, there is enormous surge of
interest in the search for the stable coagulating enzymes of meso-
phillic or thermophillic microorganism.
as it is known to secrete several extracellular enzymes of industrial

http://dx.doi.org/10.1016/j.lwt.2015.08.065
0023-6438/ 2015 Elsevier Ltd. All rights reserved.
 R. Kumari Narwal et al. / LWT - Food Science and Technology 65 (2016) 652e660
importance during the fermentation process (Wu, Chang, & Shih,
2013). We have exploited a soil-borne microorganism of this class
(unpublished work) which secretes the rennet-like coagulant with
appreciable milk clotting activity at high temperature, the
requirement for which in near future is bound to increase by leaps
and bounds, basically due to requirement of novel dairy products
prepared through stable coagulants, exogenous enzymes and
starter cultures worldwide.
2. Materials and methods
2.1. Materials
Column chromatography materials were purchased from Sigma
Chemicals Co. St. Louis, MO, USA. Commercial enzyme rennet of
Mucor miehei type II was also purchased from Sigma, USA. Other
chemicals used were of analytical grade.
2.2. Preparation of culture and crude enzyme production
Bacillus subtilis MTCC 10422 isolated from soil was one of the
best producers of milk clotting enzyme. This strain was grown
during the present investigation on modied yeast extract milk
agar medium sterilized at 120
C for 20 min (pH 6.0, temperature
40
C) supplemented with sucrose. The culture slants were main-
tained at 4
C; refreshing culture by transfer onto a fresh medium
after every 2e4 weeks. The enzyme was produced from this culture
when cultivated under liquid state fermentation (LSF) conditions in
yeast extract milk medium (pH 6.0, temperature 40
C). The bac-
terial growth was allowed for 48 h of incubation. The cell free broth
was separated through microltration. The ltrate obtained was
centrifuged at 10,000  g for 10 min at 4
C. The claried homog-
enate so obtained was referred as crude enzyme extract.
2.3. Purication of MCE through sequential chromatographies
The crude MCE was subjected to 20e60% (NH4)2SO4 saturation,
centrifuged at 10,000  g for 10 min and precipitates dissolved in
100 mM phosphate buffer (pH 6.0). The enzyme preparation ob-
tained from the above step was further passed through a column
(25  3 cm.) of activated DEAE-cellulose previously equilibrated
with 100 mM phosphate buffer (pH 6.0). Elution of bound proteins
was achieved through applying delayed linear gradient of 0e0.5 M
KCl in the same buffer at the ow rate of 15 mLh1. The chosen
strategy of delayed gradient application was based on pre-
standardized protocol to elute smaller proteins entrapped in
polymer matrix as larger proteins were eluted initially with
washing buffer. The active fractions of 3 mL each were collected and
analysed for protein (A280) and enzyme activity. The concentrated
enzyme preparation obtained after ion exchange chromatography
was carefully layered over the top of Sephadex G-100 column
(85  1.5 cm.) equilibrated with 100 mM phosphate buffer (pH 6.0)
and bound proteins were eluted through same buffer at a ow rate
of 12 mLh1. The active fractions showing milk clotting enzyme
activity were pooled, concentrated using sucrose and used for
further characterization. All steps of enzyme purication were
carried out at 0e4
C and stored at this temperature unless in use.
2.4. Coagulation and protease assays for various preparations
The coagulation procedure used was a slight modication of the
method described by Arima, Yu, and Iwasaki (1970). Five mL of the
assay milk (10% skim milk and 0.01 M CaCl2  2H2O in distilled
water) was taken in a test tube, contents brought to 37
C in a
constant temperature water bath and 0.5 mL of enzyme extract was
653
added. Curd formation was observed, while manually rotating the
test tube from time to time so as to form a thin lm on its inner
surface. The milk clotting enzyme units were expressed as Soxhlet
Units (SU) and calculated using the following formula:
SU 2400  5xD/Tx0.5; T time in seconds; D dilutions of
enzyme preparations One SU is dened as the amount of enzyme
which clots 1 mL of a solution containing 0.1 g skim milk powder
and 0.00111 g calcium chlorides in 40 min at 37
C. The procedure
used for estimating microbial proteolytic activity with casein was a
slight modication of the method described by Kunitz (1947). One
unit of protease activity was dened to release 1 mg tyrosine
equivalents under the assay conditions. The amount of protein in
different preparations was estimated by Lowry's method (1951)
using bovine serum albumen as the standard protein. The protein
content of individual fractions in both chromatographies was
monitored through absorbance at 280nm.
2.5. Molecular mass determination and purity of enzyme
preparation
The elution volume needed to elute proteins of known molec-
ular mass and puried MCE of unknown molecular mass was used
to prepare a calibration curve and then extrapolation was used to
deduce the molecular mass of MCE using Andrew plot. The
following markers (molecular mass given in parenthesis are in kDa)
were used: blue dextran [2000], apoferritin [443], alcohol dehy-
drogenase [150], bovine serum albumin [66], egg albumin [45],
pepsin [35], carbonic anhydrase [29] and cytochrome c [12]. The
molecular mass under denaturing condition was also determined
by sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) on 10% resolving slab gel system (Bio-Rad Labora-
tories). The SDS-PAGE was performed according to Laemmli's
protocol (1970). Molecular mass of the enzyme was calculated from
the relative mobility (Rm) of molecular mass markers run
simultaneously.
2.6. Effect of pH on the activity and stability of milk clotting enzyme
The results of this parameter were obtained by assaying with
the enzyme (appropriately diluted from puried pooled fraction; as
depicted in Table 1) and substrate (1%) mixture in different (4e10)
pH levels at 35
C. The stability experiment was performed with
pre-incubated buffers of different pH. The diluted enzyme prepa-
rations with those buffers (0.1 M) were incubated for time period
(0e24 h) at temperatures (35
C), and the remaining activity was
assayed in pH 6.0, at 40
C. The enzyme activity, as a function of pH,
was expressed as percentage of initial activity (initial activity in
pre-incubated buffer is regarded as 100%).
2.7. Effect of temperature on activity and stability of milk clotting
enzyme
Optimal temperature for milk clotting enzyme activity was
determined by incubating the enzyme solution with substrate in
100 mM phosphate buffer (pH 6.0) for 10 min at various temper-
atures. The thermal stability of milk clotting enzyme was investi-
gated at seven different temperatures between 35 and 70
C for
varying periods of time in a temperature controlled water bath. The
enzyme solution was placed in a pre-warmed tube at the specied
temperature and aliquots were withdrawn at 10 min time intervals,
cooled at ice bath and residual activity assayed according to pre-
viously mentioned assay. The enzyme activity, as a function of
temperature, was expressed as percentage of initial activity (initial
activity calculated at 37
C, is regarded as 100%). The stability of the
enzyme was expressed as per cent residual activity (%RA).
654 R. Kumari Narwal et al. / LWT - Food Science and Technology 65 (2016) 652e660

Table 1
Summary of purication of milk clotting enzyme from Bacillus subtilis MTCC 10422.

Purication step Total activity (U) Total protein (mg) Specic activity (U mg1 protein) Fold purication Yield (%)

Crude extract 39,320 2250 21 e 100

(NH4)2SO4 fractionation (30e70%) 29,600 69 429 20.42 75.27
DEAE-cellulose (Ion-exchange chromatography) 17,700 32 549 26.14 45.01
Sephadex G-100 (Gel ltration/molecular sieve/ 8190 12 691 32.90 20.82
exclusion chromatography)

Table 2a Table 3
Inactivation kinetic parameters of milk clotting enzyme (MCE) towards thermal Effect of different chemicals on milk clotting enzyme from
processes. Bacillus subtilis MTCC 10422.

Temperature (C) kd (min1) R2 t1/2 (min) D-value (min) Residual activity (%)

40 0.004 0.964 169.0 561.7 Chlorides (10 mM)

45 0.009 0.970 75.3 250.3 NaCl 106.1 5.6
50 0.015 0.955 43.9 145.7 KCl 109.6 6.8
55 0.021 0.964 32.2 107.1 MgCl2 193.8 5.6
60 0.033 0.967 21.0 69.6 CaCl2 280.6 6.6
65 0.047 0.974 14.5 48.2 CoCl2 13.4 1.3
70 0.131 0.907 5.2 17.5 MnCl2 166.8 5.0
CdCl2 15.9 1.6
z-value 22.32
C; Ea 5.5 kcal mol1; kd Thermal inactivation rate constant;
NiCl2 59.9 2.2
R2 Co-efcient of correlation; t1/2 Half-life; D-value Decimal reduction time.
Sulfates (10 mM)
MgSO4 206.3 5.6
FeSO4 88.7 2.5
Table 2b MnSO4 198.0 7.9
Thermodynamic parameters for thermal inactivation of milk clotting enzyme (MCE). AlSO4 94.8 6.3
ZnSO4 15.3 1.2
Temperature (C) DH (kJ mol1) DG (kJ mol1) DS (Jmol1 K1)
Selected data are presented as means of at least three in-
40 89.24 101.75 39.97
dependent experiments (n 3), each selected experiment
45 89.20 101.29 38.02
had a minimum of three replicates of each sample.
50 89.16 101.48 38.13
55 89.12 102.24 39.99
60 89.08 102.64 40.73
65 89.03 103.19 41.88 different concentrations (0e12 mg mL1) alone and in presence of
70 88.99 101.87 37.52 calcium chloride. Substrate equilibration was done at optimal
Ed (Activation energy for denaturation) 21.87 kcal mol1; DH Variations in conditions for 10 min. The kinetic constants, Vmax and Km for pu-
enthalpy; DG Variations in free energy; DS Variations in entropy. ried milk clotting enzyme were calculated from the plot of
Lineweaver and Burke (1934).

2.8. Thermodynamic determinants and thermal inactivation 2.11. Casein hydrolysis

kinetics
The data obtained from the thermal stability prole was used to
determine inactivation temperature as described by Hayaloglu,
Karatekin, & Gurkan, 2014). Afterwards, the inactivation tempera-
ture was used to analyse corresponding thermodynamics de-
terminants (DG, DS and DH) and thermal inactivation kinetics as
reported in our previous work (Pal & Khanum, 2011). D-value is the
time at a specied temperature for the enzyme activity to decrease
by one log cycle (90%). Z-value is the change in temperature needed
to alter the D-value by one log cycle.
2.9. Impact of additives
The effect of various monovalent and divalent ions such as
chlorides and sulfates on the enzyme activity was assessed by
including them in the enzymeesubstrate reaction mixtures at a
nal concentration of 10 mM for 30 min and proceeded for enzyme
assay. For comparison, the control (without addition of CaCl2 or any
metal ion; as shown in Table 3) was taken or set as 100% and the
effect of various metal ions was expressed as residual or relative
activity.
2.10. Determination of kinetic constants
To determine the maximum velocity (Vmax) of the enzyme,
initial reaction rates were measured by using casein substrate at
Extraction of cheese proteins was performed using the proce-
dure of Kumar, Sharma, Saharan, and Singh (2005). Twenty mg of
cheese samples were extracted with 500 mL of 0.05 M Tris {Tris
(hydroxymethyl) aminomethane} buffer (pH 8.0) containing 0.01 M
b-mercaptoethanol (b-ME) for 1 h with vortexing every 10 min.
Samples were then centrifuged at room temperature for 20 min at
11,000  g; the supernatant contained the total casein proteins.
Samples using different enzyme preparations were analysed
through urea e polyacrylamide gel electrophoresis (De Jong, 1975).
2.12. Statistical analysis
The selected data are presented as means of at least three in-
dependent experiments (n 3), each selected experiment had a
minimum of three replicates of each sample. The selected experi-
ments were repeated thrice for reproducibility. Statistical signi-
cance among levels was evaluated using analysis of variance
(ANOVA). SPS (version 9.4) was used for the statistical analyses.
3. Results and discussion
3.1. Purication of milk clotting enzyme
A summary of purication steps and process parameters has
been listed in Table 1. The crude enzyme extract which had total
activity of 39,320 U and specic activity of 20.80 Umg1 protein was
 R. Kumari Narwal et al. / LWT - Food Science and Technology 65 (2016) 652e660
subjected to (NH4)2SO4 precipitation (30e70%) at 4
C. During this
step, enzyme was puried 20.42 fold with 75.27% recovery. Selec-
tion of ion exchange chromatography ahead of gel ltration was
based on the permitting the enzyme to bind even when a large
volume of buffer is applied to elute at rapid ow rate. Elution was
carried out rst with the same buffer and then by using 0e0.5 M
KCl gradient (gradient was applied after 200 mL of elution volume)
(Fig. 1a). This purication step resulted in 26.14 fold purication
with 45.01% recovery of the total activity as compared to the crude
extract. The concentrated enzyme was further loaded on to
Sephadex G-100 column, previously equilibrated with 0.1 M
phosphate buffer (pH 6.0) for gel ltration chromatography. Elution
prole of the protein and enzyme activity of fractions obtained
from Sephadex G-100 column is presented in Fig. 1b, which shows
that MCE was present in a single narrow peak between fractions
41e51 which coincided with one moderate protein peak. This pu-
rication scheme resulted in the enzyme preparation puried to
32.9 fold with 20.82% recovery of the total activity as compared to
the crude extract.
The study provided better results in terms of purication fold as
well as recovery in comparison to some of the earlier published
reports from various microorganisms. The MCE has been puried
from Enterococcus faecalis TUA2495L (Sato, Tokuda, Koizumi, &
Nakanishi, 2004), Capra hircus (Kumar, Sharma, Mohanty, Grover,
& Batish, 2006), Rhizopus oryzae (Kumar et al., 2005), Cyanara
scolymus (Llorente, Brutti, & Cafni, 2004) and from Centaurea
calcitrapa (Raposo & Domingos, 2008). MCE from Bacillus
Fig. 1. Elution prole of milk clotting enzyme (MCE) from Bacillus subtilis MTCC 10422 on DEAE-cellulose column (a) and on Sephadex G-100 (b).
655
sphaericus has been puried 48 fold with specic activity of
648,148 Umg1 protein by fractional precipitation with acetone,
followed by the chromatography of most active fraction on DEAE-
Sephadex A-25 and nally on Sephadex G-100 (El-Bendary,
Moharam, & Ali, 2007). MCE from B. subtilis K-26 was puried
24-fold with an 80% yield (Rao & Mathur, 1979), 107 fold from
Pseudomonas uorescences M3/6 (Kohlmann, Nielsen, & Ladisch,
1991) and from Mucor pusillus 18 fold with 7.56% recovery by ion-
exchange chromatography and gel ltration (Nouani et al., 2009).
Vishwanatha, Rao, and Singh (2010) puried the milk clotting
enzyme with specic activity of 3500 Umg1 from the culture of
Aspergillus oryzae MTCC5341 using afnity precipitation with
alginate and subsequent elution with 0.5 M sodium chloride con-
taining 0.2 M CaCl2.
3.2. Purity and molecular mass determination
Crude bacterial broth or claried homogenate may contain a
mixture of extracellular proteins which can affect the process and
product quality. The enzyme from B. subtilis MTCC 10422 was pu-
ried to electrophoretic homogeneity (Plate 1). The step-wise
reduction of protein bands in slab polyacrylamide gel has
revealed the ascending purity of nal preparation with sequential
purication. The determination of molecular mass was estimated
through elution volume (Fig. 2a) and relative mobility of our pro-
tein with standard proteins (Fig. 2b). The purity and sub-unit
composition of MCE was checked through SDS-PAGE prole
656 R. Kumari Narwal et al. / LWT - Food Science and Technology 65 (2016) 652e660

Fig. 3. Effect of pH on activity (a) and stability (b) of puried milk clotting enzyme
(MCE) from Bacillus subtilis MTCC 10422.

enzymes which are stable over a wide range of pH but optimal pH is

Fig. 2. Estimation of molecular weight of puried milk clotting enzyme (MCE) from
Bacillus subtilis MTCC-10422 by gel ltration through Sephadex G-100 (a) and on SDS-
PAGE (b).
(Plate 2). The molecular mass of puried MCE, as determined by the
gel ltration and electrophoresis, was found to be monomeric of
27 kDa.
MCE(s) of almost similar molecular weight have been reported
from B. subtilis K-26 (Rao & Mathur, 1979) and Aureobasidium pul-
lulans (Donaghy & McKay, 1993). MCEs are generally exclusively
single subunit proteins, however, some MCE, especially those
having large molecular weight are found to possess more than one
subunit.
kept in consideration for cheese preparation (Jacob et al., 2011). The
titrable acidity of milk should be between 0.19 and 0.25% lactic acid
equivalents which correspond to a pH range of 5.0e6.5 at the time
of addition of enzyme. Therefore, the milk clotting enzyme should
be stable between this pH range. The results show that MCE re-
mains stable in acidic as well as basic pH (Fig. 3b). The instability in
extreme pH suggested irreversible inactivation of enzyme.
The enzyme isolated from actinomycetes was found to be stable
between pH 3.0 to 8.0 (Laxer, Pinsky, & Bartoov, 1981), from Irpex
lacteus between pH 3.0e6.0 (Kobayashi, Kusakabe, & Murakami,
105

3.3. pH optima and pH stability 90

Relative activity (%)

The enzyme shows optimal activity at pH 6.0 (Fig. 3a). The ac-
tivity slightly declined on either side of this pH. Except for pH 4.0 75
and 9.0, all the pH levels alters the enzyme activity signicantly
(P < 0.0001).
2.04
This value agrees well with the values reported for bacilli 60 1.98
Log V

1.92
enzyme produced by Bacillus subtilis K-26 (Rao & Mathur, 1979), 1.86
1.8
Bacillus licheniformis 5A1 (Esawy & Blanc, 2006), Bacillus mega- 1.74
3.1 3.1 3.2 3.2 3.2 3.3
terium (Shah & Mathur, 1990), Bacillus polymyxa B-17 (Matta & 45 2 6 4 8 2
1000/T (K)
Punj, 1998) and B. subtilis natto (Wu et al., 2013). Milk clotting
enzyme from E. faecalis TUA 2495 L (Sato et al., 2004), A. oryzae
MTCC 5341 (Vishwanatha et al., 2010) and from Nocardiopsis sp. 30
(Cavalcanti, Teixeira, Fillo, & Porto, 2004) showed the pH optima of 25 30 35 40 45 50 55 60 65
6.0, 6.2 and 8.0, respectively.
Temperature (C)
Since ionic stability of enzyme in differently acidied colloidal
solutions determine its usage and storage, the puried enzyme was Fig. 4. Effect of temperature on milk clotting activity. Inset is the Arrhenius plot to
pre-incubated with various acidic and basic buffers. Milk clotting calculate activation energy (Ea) of catalysis.
 R. Kumari Narwal et al. / LWT - Food Science and Technology 65 (2016) 652e660 657

Fig. 5. First order thermal deactivation of the puried milk clotting enzyme (MCE).
Inset is the Arrhenius plot to calculate activation energy (Ed) for thermal denaturation.

1983), from Thermomucor sp. between 4.0 and 4.5 for 24 h (Silva,
Geraldes, Murari, Gomes, & Da-Silva, 2014) and from B. subtilis K-
26, it was most stable at pH 7.5 (Rao & Mathur, 1979). The bacillus
enzyme completely retained its milk clotting activity (MCA) even
after incubation for more than 2 h at pH 6, and temperature at 40
C
and 50
C (Wu et al., 2013). Some neutral and alkali proteases have
been isolated from Bacillus amyloliquifaciens FSE-68 which were
100% stable for 60 min at 30
C in the alkali range (Cho, Oh,
Pridmore, Juillerat, & Lee, 2003). Whereas, MCE with stability
over a broad range has also been reported in Bacillus shaericus (El-
Bendary et al., 2007), Aspergillus niger (Fazouane-Naimi et al., 2010)
and Rhizopus microsporus (Sun et al., 2014).
3.4. Temperature optima and thermostability
The activity of puried enzyme from B. subtilis MTCC 10422 start
increasing with increase in temperature and get maxima at 45
C.
After that, the enzyme activity starts decreasing gradually (Fig. 4).
Microbial coagulants have intrinsic ability to execute the tech-
nological process of specic cheese preparation which entails milk
clotting at higher temperature just after pasteurization (Nouani
et al., 2009). The optimum temperature of majority of MCEs lies
in the range of 30e75
C (Sun et al., 2014; Fazouane-Naimi et al.,
2010).
Fig. 7. Lineweaver-Burk plot and substrate saturation curve (inset) of puried milk
clotting enzyme (MCE) from Bacillus subtilis MTCC 10422 for skim milk.
The value reported for the enzyme from B. licheniformis (Esawy
& Blanc, 2006), E. faecalis TUA2495 L (Sato et al., 2004), Penicillum
oxalicum (Hashem, 2000) and C. albidus (Alessandro & Federico,
1980) lies in range of commercial utility. The Arrhenius plot-
calculated activation energy (Ea), 5.5 kcal mol1 (22.99 kJ mol1),
is in the range that is characteristic of a typical enzymatic reaction
(Fig. 4 inset). It is also obvious from the Arrhenius plot that the
enzyme had a single conformation up to the transition state.
Thermostability studies have revealed MCE from B. subtilis MTCC
10422 retained 99.9% of its original activity after 50 min of incu-
bation at 35
C. At 40
C, activity decreased to 82% after 50 min.
Similarly at 50
C, the enzyme retained 80 and 53% of its original
activity after 20 and 40 min, respectively. At 60
C, the enzyme
retained 58%, 46% and 29% of its activity after 20, 30 and 40 min,
respectively. There was minute activity at 60
C after 50 min and at
70
C, after 30 min (Fig. 5).
Similarly, Wu et al. (2013) observed that MCA decreased
dramatically when temperature increased from 40 to 50
C, the
enzyme lost 50% of MCA after it was incubated at 60
C for 20 min

2.8

2.6

2.4

2.2

2
Log D

1.8

1.6

1.4

1.2 Z-value
1
35 40 45 50 55 60 65 70 75

Temperature (C)

Fig. 6. Temperature dependence of the decimal reduction of puried milk clotting Fig. 8. Lineweaver-Burk plot and substrate saturation curve (inset) of puried milk
enzyme (MCE) to calculate z-value. clotting enzyme (MCE)from Bacillus subtilis MTCC 10422 for CaCl2.
658 R. Kumari Narwal et al. / LWT - Food Science and Technology 65 (2016) 652e660
Plate 1. Electrophoretic pattern of different enzyme preparations from sequential
purication displayed on native-polyacrylamide gel electrophoresis. Lane 1: Crude
extract. Lane 2: Ammonium sulphate fraction (20e60%). Lane 3: DEAE e cellulose
fraction. Lane 4: Puried enzyme. Lane 5: Standard markers.
and it was deactivated completely at temperature more than 70
C
within 10 min of incubation.
3.5. Thermal inactivation kinetics and thermodynamic
determinants
The rst order temperature inactivation kinetics in the tem-
perature range of 35e70
C has been observed here. The rst-order
kinetic model is based on the assumption that the disruption of a
single bond or structure is sufcient to inactivate the enzyme.
Considering the complexity of the structure of an enzyme and the
numerous factors involved in the inactivation, this explanation
Plate 2. Electrophoretic pattern of puried MCE on denaturating gel electrophoresis.
Lane 1: Standard markers. Lane 2: Puried enzyme.
seems to be exceedingly simple.
Half-lives (t1/2) of enzyme preparations at different tempera-
tures clearly depicted the enzyme stability as a function of tem-
perature. With increasing temperature, the t1/2 and D-value
decreased and the rst order thermal deactivation rate constants
(kd) increased (Table 2a). The z-value of puried enzyme prepara-
tion indicates that this enzyme is more sensitive to duration rather
than intensity of temperature (Fig. 6). In general, high and low z-
values mean more sensitivity to the duration and intensity of the
temperature. The higher value found for the activation energy of
denaturation,Ed (91.41 kJ mol1) in comparison to activation en-
ergy, Ea (22.99 kJ mol1) suggesting two facets, (1) enhanced
catalysis with minimal activation energy and (2) higher energy
requirement to destabilize the enzyme. The increasing high values
of DH
, the change in enthalpy during denaturation process with
increasing temperature indicate that enzyme undergoes a number
of transient changes in conformation before getting thermo-
inactivated. The inverse relationship between DH
and tempera-
ture conrmed that less energy is required to denature enzyme at
high temperature (Table 2b).
The negative values of DS
(entropy change during thermo-
inactivation) at each temperature are presented here, have also
been observed by us earlier also (Pal & Khanum, 2011) indicate that
there are signicant processes of protein aggregation, since had this
not happened, the values would have been positive. It is also worth
mentioning that higher DG
>DH
values are due to the negative
entropic contribution in total free energy change of the process.
Thermal inactivation kinetics is important here because residual
enzyme activity is exponentially related to activation energy (Ea)
and inactivation rate constant (k). Thermal inactivation and kinetic
studies revealed that enzyme undergoes a number of transient
phases with increase in temperature and ultimately leads to ag-
gregation at intense temperature for longer duration. The energy
requirement for enzyme catalysis is much less than required for
enzyme inactivation.
Thermostable enzymes including proteases, offer major
biotechnological advantages over mesophillic enzymes (McMohan,
Kelly, & Fogarty, 1999). Stability of enzymes at higher temperatures
for prolonged periods is of great concern for their suitability in
cheese making industries. It has been reported earlier that MCE
from B. sphaericus was quite stable at 40
C for more than 30 min
while it lost 35% and 70% of its activity after 10 and 20 min incu-
bation at 60
C, respectively (El-Bendary et al., 2007). Silva et al.
(2014) and Hashem (2000) reported that MCE from Thermomucor
sp. and P. oxalicum possessed good activity at 55 and 75
C indi-
cating thermostability of the enzyme. At 40
C after incubation for
10, 20 and 40 min, it possessed 100, 90 and 65% of its original ac-
tivity. At 50
C also, the enzyme retained 80% activity after 20 min
but at 55
C there was a dramatic loss in the activity. Nouani et al.
(2009) reported that MCE from M. pusillus was relatively stable in
the temperature range of 30e50
C and was inactivated after
30 min at 65
C. Similarly, the MCE from two different microor-
ganisms was also stable at 40
C for 30 min, whereas at 50e60
C, it
retained only 10e20% of its activity after 30 min (Rao & Mathur,
1979; Sun et al., 2014). No changes were observed in the activity
of Rhizopus miehei protease between 55 and 62
C where it retained
~80% activity at this temperature and the rst decrease in its ac-
tivity was recorded after 62e65
C (Hayaloglu et al., 2014).
The storage studies of puried MCE indicate towards negligible
loss of its activity after 28 days of storage at 4
C suggesting that the
enzyme had appreciable storage stability which is an essential and
important aspect for industrial applications (storage studies data
not shown).
Similarly Laxer et al. (1981) also reported the maintenance of
100% activity of the actinomycete enzyme during storage at 4
C.
 R. Kumari Narwal et al. / LWT - Food Science and Technology 65 (2016) 652e660 659

Esawy and Blanc (2006) reported that the puried enzyme from Boscolo, and Silva (2010).
B. licheniformis was highly tolerant to repeated freezing and
thawing and the activity remained as such for six months. 3.8. Extent of proteolysis

3.6. Effect of additives
Milk coagulation takes place in two stages. In the rst stage, the
enzyme converts k-casein in milk to para-casein, while in the
second stage, para-casein in the presence of calcium ions gives a
rm clot. Therefore, it is a common practice to add CaCl2 in milk to
get rm clot in cheese manufacture. Other divalent cations such as
magnesium are also known to cause coagulation. Hence, the effect
of different monovalent and divalent ions on enzyme activity was
investigated under standard assay conditions in the presence of
chlorides and sulfates at a concentration of 10 mM each (Table 3).
As per our expectations, Ca2 increased the activity by two folds,
reecting signicant effect on second phase of milk clot formation
(ANOVA: P 0.04). Similarly, Mg2 and Mn2 also had stimulatory
effect and increased the activity by about 206 and 198 per cent,
respectively. Fe2, Ni2 and Al3 had not such signicant effect on
milk clotting activity whereas Co2, Cd2 and Zn2 were found to
have inhibitory effect (ANOVA: P 0.003). Monovalent salts such as
potassium and sodium chloride were slightly stimulatory. Addition
of EDTA (10 mM) also completely inhibited the enzyme indicating
that calcium ions are essential for the formation of milk clot.
Calcium not only creates iso-electric conditions but also creates
ion bridges between phosphate moieties of casein micelles (Sun
et al., 2014). These results are in accordance with results reported
by Kumar et al. (2005) in R. oryzae, by El-Bendary et al. (2007) in
B. sphaericus and by Rao and Mathur (1979) in B. subtilis K-26.
Hashem (2000) reported that metal ions like Mg2, Ca2, Ba2,
Mn2, Al3, Fe2 and Co2 had very clear function to accelerate milk
coagulation whereas Na decelerated the activity slightly.
3.7. Kinetic studies
The major utility of exogenous enzyme in cheese manufacturing
is its proteolytic mode of action. The commercial success of mi-
crobial rennet not only depends upon milk clotting activity but also
on limited proteolysis associated with protease. In the present
investigation, most appropriate substrate of protease viz. cheese
casein was hydrolysed by the different enzyme preparations
including the commercial one viz. R. miehei. The different enzyme
preparations have demonstrated different degrees of protein hy-
drolysis with passage of time as they have differential protease
activity. Proteolysis of casein revealed that comparable extensive
hydrolysis was achieved with the puried as well as semi-puried
preparations (Plate 3). The crude enzyme preparation have more
protease activity as shown by more caseinopeptides produced
(Lane 5) whereas the puried enzyme have comparable protease
activity to commercial enzyme. Results of this study suggested the
potential of puried enzyme in cheese preparation.
4. Conclusion
The highly versatile milk clotting enzyme from B. subtilis MTCC
10422 was extracted with high yield and recovery. The enzyme was
puried to absolute homogeneity using combinatorial fractionation
and chromatographies. Biochemical techniques viz. molecular
exclusion and denaturing gel electrophoresis estimated the mo-
lecular mass of monomeric MCE ~27 kDa. Thermal inactivation and
kinetic studies revealed that this enzyme has moderate thermo-
stability. The presence of least non-specic protease activity or loss
of protease activity after milk clotting is always appreciated. Both
the characters have been found in this enzyme. Enzyme showed the
excellent half-lives of 169 and 43.9 min at 40 and 50
C, respectively

MCE from B. subtilis MTCC-10422 followed a typical Michae-

liseMenten kinetics with increasing concentration of substrate in
an otherwise standard assay mixture. The enzyme was fully satu-
rated at a concentration of 8 mg mL1 (Fig. 7 inset). However, at
higher concentration of substrate, the enzyme activity remained
constant or was slightly inhibited due to the phenomenon named
substrate inhibition in which the enzyme molecules get fully
saturated with higher number of substrate and all the active sites
were occupied so that no longer increase in velocity took place.
It is slight deviation from classical milk clotting kinetics of Ba-
cillus enzyme reported by Ageitos, Vallejo, Sestelo, Poza, and Villa
(2007) in which no enzyme inhibition effect detected at high
concentration of substrate. The Km as determined by double
reciprocal plot was 5 mg mL1 (Fig. 7). The present ndings are in
agreement with those reported for B. sphaericus (El-Bendary et al.
2007), B. subtilis K-26 (Rao & Mathur, 1979) and R. oryzae (Kumar
et al., 2005) while Shah and Mathur (1990) reported lower value
of 1.5 mg mL1 for B. megaterium PM24-1 enzyme.
As Ca2 was the most effective stimulator, its effect was studied
in detail by taking different concentrations of CaCl2 (5e50 mM).
Substrate saturation curve depicts steep hyperbolic response of
milk clotting enzyme in presence of CaCl2 (Fig. 8 inset). The enzyme
activity increased with increase in CaCl2 concentration, until it
attained maximum value at 30 mM of this additive, above which
the enzyme activity remained almost constant suggesting that the
enzyme got fully saturated at this concentration. From the double
reciprocal plot, Km was found to be to 9.09 mM (Fig. 8). Plate 3. Urea-polyacrylamide gel electrophoretograms of casein protein (Lane 1) and
Similar results of effective calcium concentration as stimulator hydrolysed peptides from cheddar cheese proteins with the action of commercial (Lane
of enzyme activity have been reported by Merheb-Dini, Gomes, 2), puried (Lane 3), partial puried (Lane 4) and crude (Lane 5) enzyme.
660 R. Kumari Narwal et al. / LWT - Food Science and Technology 65 (2016) 652e660

and high value of DG
of thermal denaturation (103.19 kJ mol1 at and characterization of milk clotting enzyme from goat (Capra hircus).
Comparative Biochemistry and Physiology, 145, 108e113.
65
C) indicated that thermostable MCE exhibits some resistance
Kumar, S., Sharma, N. S., Saharan, M. R., & Singh, R. (2005). Extra cellular acid
against thermal unfolding at higher temperatures. protease from Rhizopus oryzae: purication and characterization. Process
Biochemistry, 40, 1701e1705.
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