AMERICAN JOURNAL BIOTECHNOLOGY AND MOLECULAR SCIENCES

ISSN Print: 2159-3698, ISSN Online: 2159-3701, doi:10.5251/ajbms.2013.3.1.8.23

© 2013, ScienceHuβ, http://www.scihub.org/AJBMS

Isolation of clt Genes Encoding milk clotting enzyme and characterization

of milk clotting enzyme from new isolate Streptomyces pseudogriseolus
NRC-15
El-Sayed E. Mostafa 1, Moataza M. Saad1, Mohsen H. Selim1 and D. E. El-Hadedy2*
1
Microbial Chemistry Department, National Research Centre (NRC), El-Bohouth Street,
Dokki, P.O. Box 12622, Cairo, Egypt.
2
Microbiology Department, National Center for Radiation Research and Technology, (NCRRT),
3
Ahmed Elzumor st., 8 th sector, Madinat Nasr, postal code 11371-cairo-Egypt
Accepted 16 Jan, 2013
*Corresponding author: dodyelhadedy@yahoo.com

ABSTRACT

Research focused on isolation and characterization on new strain of Streptomyces pseudogrisiolus

from Egyptian soil have proteinases which possesses activity (MCE) identified by 16S rRNA with
universal primer and submit in genbank with accession number AB739622. In this work, study
optimizes the extraction of milk-clotting enzyme (MCE) according to its adequacy to be employed
as substitute of rennin. The organism grew well in medium 3 after 4days of incubation at
temperatures of 35° ±2, pH 6.0. Cultural and environmental conditions for milk clotting enzyme
production by Streptomyces were studied. Glucose (3%, w/v) and ammonium sulphate (0.2%w/v),
were found to be the best C and N sources for milk clotting enzyme production respectively.
Partially purified and characterized. MCE was obtained by fractional precipitation with ammonium
sulphate, followed by the chromatography of the most active fraction on Sephadex G-100 with
20.39 purification fold and recovery about 80%. The maximum enzyme activity was at pH (6.5) and
45ºC. The clotting activity of the purified enzyme was stimulated with increasing CaCl2
concentration up to 10mM. However, a gradual reduction of the activity was observed by
+2 +2
increasing NaCl concentration between 4-8%. Whereas, Cu and Zn significant inhibition it.
Interestingly MnCl2 and MgSO4 were found to have stimulatory effect, which has been rarely
reported. Also study includes some kinetics calculation about MCE. Gene control MCE expression
was detected by specific primer design using DNA star program, sequence illustrate gene found clt
a and clt b, and submitted in genbank as new sequence with accession number AB739621. The
milk clotting enzyme (MCE) obtained from S. pseudogrisiolus has potential as calf rennet
substitutes in dairy products and it can be used in lypholyzed form as tablets in pharmaceutical
products for patients with digestive system.

Key words: S. pseudogrisiolus, phylogentic tree, Factors affecting the production of milk-clotting
enzyme (MCE), inhibitors factors, Kinetics MCE, Purification by chromatography and gene
isolation

INTRODUCTION as microbial rennet’s. At present, microbial rennet is

Animal rennet is conventionally used as a milk-
clotting agent in dairy industry for the manufacture
of quality cheeses with good flavor and texture.
Owing to an increase in demand for cheese
production world wide – i.e. 4% per annum over the
past 20 years, approximating 13.533 million tons–
coupled with reduced supply of calf rennet, has
therefore led to a search for rennet substitutes, such
used for one-third of all the cheese produced
worldwide.
Microorganisms like Bacillus subtilis Rhizomucor
pusillus: R. miehei, Endothia parasitica, Aspergillus
oryzae,; Bacillus sphaericus; and amylomyces roxii
are used extensively for rennet production in cheese
manufacture. Extensive research that has been
carried out so far on rennet substitutes has been

reviewed by several authors (Souse et al., 2001;
Ayhan et al., 2001; Libouga et al., 2008; Guiama et
al., 2010 and Ding et al., 2011).
Microbial rennets from various microorganisms
(marketed under the trade names such as
Rennilase, Fromase, Marzyme, Hanilase, etc.)
being marketed since the 1970s have proved
satisfactory for the production of different kinds of
cheese. The molecular and enzymatic properties of
chymosins have been studied extensively(Jonk and
Birkkjacer, 1985; Crawford, 1985; Ayhan et al.,
2001).In this study the production conditions of milk
clotting enzyme from new strain Streptomyces
pseudogrisiolus NRC 15 were optimized. In
addation the milk clotting enzyme was also purified
and characterized.
MATERIALS AND METHODS
Microorganism: The Streptomyces NRC-15.used
was isolated from Egyptian soil. The micro organism
was maintained at 35±2°C on Dox agar slants made
up of yeast extract 0.1% (w/v), agar 2.0% (w/v), and
identified by El-Sayed et al. (2012).
Medium and culture conditions: Each Erlenmeyer
flask (500 ml), containing 40 ml fermentation
medium was inoculated from the agar slant and
incubated at35 ± 0.5°C on a rotary shaker at 150
rpm. The fermentation medium contained wheat
-1 -1
bran (30 gL ), NaCl (5 g L ).
Identification: Strain was identified by 16S rRNA
sequencing data collection. A database containing
16S rRNA gene sequences of all validly published
filamentous actinomycetes (Euze´by, 2002) was
compiled from GenBank
(http://www.ncbi.nlm.nih.gov). All sequences used
were longer than 1400 bp. The sequences were
grouped by genus according to Stackebrandt et al.
(1997) and the NCBI Taxonomy Browser
(http://www.ncbi.nlm.nih.gov/Taxonomy/
Browser/wwwtax.cgi). Full length 16S rRNA (1500
bp) were amplified from isolates (Fc2) by PCR using
universal forward primer P1 and universal reverse
primer Forward: (5’-AGAGTTTGATCCTGGCTCAG-
3’), Reverse: (5’-CGGTTACCTTGTTACGACTT-3’)
ο
Optimum conditions (denaturation 94 C –1 min,
ο ο
annealing 45 C, 30 sec and extension 72 C, 2 min,
35 cycles). Amplified 16S rRNA was purified from
0.8% melting point agarose gel. Bands obtained
from PCR product were eluted and purify by (Qiagen
elution kit) PCR instructions, DNA band desired was
excised from ethidium bromide stained agarose gel
with a razor blade, transferred to Ependorf tube.
9
Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 8-23
DNA were sequenced directly using specific primer
with concentration 20 pmol in Promega company
lab. The sequence alignment was prepared with
DNA STAR software program. Nucleotides
sequences of the products were edited using Bioedit
version 5.0.6 [Hall, 1999] and phylogentic tree by
mega 4 program.
DNA sequenced at sequencer lab in labs of
Promega Company in USA: The sequence
alignment was prepared with DNASTAR software
programs (DNASTAR. INC. Madison, Wis.) and
manually edited with GeneDoc (www.NCBI /
blast.com), and determine translation encoded
regions at (www.expasy.org/cgi-bin/dna_aa).
Phylogenic tree established my mega program.
Assay for enzyme activity: Based on the
appearance of the first clotting flakes, MCA was
determined and expressed in terms of Soxhlet unite
(SU) according to the method of Arima et al. (1970).
The milk clotting enzyme as described, 5ml
substrate (10% skim milk in 10mM CaCl2) was
incubated for five minutes at 35°C and then 0.5 ml
of enzyme extracted was added. The length of time
starting from the addition of the enzyme extract to
the formation of the first particles was recorded, and
the milk-clotting activity was calculated as:
SU=2400×5×D/T×0.5
Where T is milk-clotting time (s), and D is dilution of
the enzyme. One Soxhlet unit (SU) of milk-clotting
activity was defined as the amount of enzyme
required to clot 1mL of substrate within 40min at 35
ºC (Arima et al. 1970 and Shieh et al., 2009).
Protein concentration: Protein concentration was
determined by the method of (Lowry et al. 1951)
with bovine serum albumin as standard.
Biomass determination: The biomass
concentration was determined as mycelia dry weight
after centrifugation (5 000 g for 5min) of 10 ml of
culture broth in duplicate, and dried at 105ºC
overnight until constant weight.
Optimization of the fermentation process: Five
different types of broth media were used in this
study for primary evaluation for medium optimization
process. All these media were reported before for
their high support of protease production. The
compositions of these media were as follows: a
medium 1:800 ml of solution (A):KH 2PO4 3g; Na
2HPO4 3g; NH4Cl 2g and NaCl 50 mg and 200 ml of
solution (B):8 g glucose and 1 g MgSO4. The

-1
medium 2: (gl ): 20 starch; 3 yeast; 1 K2HP4; 3
-1
CaCO3 and 0.01 g FeSO4. The medium 3: (g l ): 30
sucrose; 0.5 KCl; 0.01 Fe SO4; 0.5 Mg SO4; 1
K2HPO4 and 3 KNO3. A medium 4: (%) 2 starch; 1.2
K2HPO4; 0.05 Mg SO4. A medium 5: (%) 1 glucose;
peptone 0.5; Yeast extract 0.5; NaCl 0.5 and CaCl
0.2. There are four main factors affecting MCA
under submerged conditions, such as glucose
concentration, fermentation time and medium pH
with full-factorial central composite design, a
response surface methodology (RSM) was applied
for improving MCA. Samples were withdrawn and
centrifuged at5900×g for 5 min with supernatant
used for MCA assay. The fermentation broth was
filtered and the crude enzyme powder was stored in
4°C.
Partial purification of the protease
Ammonium sulphate precipitation: The culture
supernatant containing the extracellular enzyme
was first subjected to ammonium sulphate
precipitation. Ammonium sulphate fractions of 0–
30%, 30–50% ,50–75% and 75-100%(v/v) were
collected by centrifugation at 10,000 g and the pellet
obtained in each fraction was suspended in a
minimal volume of 100 phosphate buffer, pH 6.5.
Sephadex G-100 gel filtration: The 50–75% (v/v)
Ammonium sulphate fraction was subjected to gel
filtration on a Sephadex G-100 column (3 cm ×100
cm) equilibrated with 25 mM phosphate buffer pH
6.5 . Enzyme fractions of 5 ml were collected at a
flow rate of 25 ml/h with the same buffer. Protein
content (Abs 280 nm) and protease activity were
measured. Fractions showing protease activities
were pooled.
SDS-PAGE and Protein Quantification: The
molecular mass of the purified enzyme was
determined by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDSPAGE) as
described (Laemmli,1970) using molecular mass
markers of 14.4, 20.0, 26.0, 35.0, 45.0, 66.2 and
94.0 kDa. The total protein content was measured
by the Bradford method( Bradford, 1976) with
bovine serum albumin (BSA) as the standard.
Effect of pH and temperature on enzyme activity
and stability: To assay optimum pH, milk clotting
activity was determined at 35ºC, at different pH
values using following 0.1M buffer solutions.
Optimum temperature for milk clotting activity was
determined by incubating the reaction mixture at
different temperatures ranging from 25ºC to 80ºC,
with 5ºC intervals, and assaying the activity at the
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Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 8-23
pH determined as optimum. For stability, the
enzyme was dispersed (1;1) in the following 0.1M
buffer solutions ,the following buffer systems were
used: phosphate buffer, pH 6.0–7.5; Tris–HCl buffer,
pH 8.0–8.5; carbonate-bicarbonate buffer, pH 9.0–
11.0. , and maintained at 28±2 for 24h. After wards,
residual milk clotting was determined under
optimum conditions of pH and temperatures.
Thermal stability was assayed by incubating the
enzyme at different temperatures ranging from 25ºC
to 80ºC, with 5ºC intervals for 1h. Afterward,
residual milk clotting was determined under
optimum conditions of pH and temperature.
Effect of CaCl2 concentrations on milk clotting
enzyme activity: The effect of CaCl2 concentrations
on milk clotting enzyme activity was determined,
however using increasing concentrations of the
CaCl2 solution (0, 0,1 0, 15, 20, 25, 30, and 35mM).
Effect of inhibitors and metal ion on enzymatic
activity: To 0.2 ml 0f enzymatic extract , different
inhibitors were added including serine protease
inhibitor (PMSF, EDTA, iodoacetic) in a
concentrations,0.01 and 0.001 µ M. Samples were
incubated at 35°C for 10 min and residual milk-
clotting was determined and compared to the
control, which was incubated without the inhibitors
and corresponds to 100% activity.
-1 -3
The effect of various metal ions (10 M and 10 M)
on enzyme activity was investigated using CaCl 2,
MnCl2, ZnSO4, CoSO4, CuSO4, BaCl2, NaCl, KCl,
FeSO4 and MgSO4. The effects of some surfactants
(Tween 80 and SDS) on gated using CaCl2, MnCl2,
ZnSO4, CoSO4, CuSO4, BaCl2, NaCl, KCl, FeSO4
and MgSO4. The effects of some surfactants (Tween
80 and SDS) on enzyme stability were also studied
by pre-incubating enzyme for 1 h at 4 °C. The
residual activity was measured at pH6.5 and 50 °C.
The activity of the enzyme (without any surfactants)
was considered as 100%.
Gene isolation: DNA extraction, polymerase chain
reaction (PCR) and DNA sequencing Genomic DNA
was extracted by a modification of the method of
Engelke et al. (1992). The polymerase chain
reaction analysis followed procedure described by
Horn et al. (1991) with some modifications. The PCR
amplification was carried out in a 50-Al mixture in a
DNA thermo Forward and reverse primers were as
followed: primer F 5 cgc cct ttc tag agt cga aac g
3) primer R 5 gtt gct cga gcc tgg ttc gac t 3) design
by PCR fast program. The amplified PCR products
were purified from low melting point agarose and the

nucleotide sequences in promega lab, biotechnology
company. The sequence alignment was prepared
with DNA STAR software program (DNASTAR.
INC., Madison, Wis.) and manually edited with
GeneDoc [www.NCBI/blast.com], and determine
translation encoded regions at [www.expasy.org/cgi-
bin/dna_aa].
Statistical analysis: Results are expressed as the
mean ± S.E. The statistical analyses were carried
out using (one way anova). All analyses were
performed with GraphPad Prism 4.02 for Windows
(GraphPad Software)
RESULTS AND DISCUSSIONS
The partial 16S rRNA sequence followed by
construction of phylogenetic tree by neighbor joining
Fig 1. Distance neighbor-joining phylogenetic tree of isolates Egyptian isolate NRC15 Consensus bootstrap.
11
Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 8-23
method has revealed that the isolate was
Streptomyces pseudogrisiolus (similarity 94%) as
indicated in Fig 1. Strain submitted in genebank as
new sequence with accession number AB739621.
The 16S rRNA sequences for Streptomyces
pseudogrisiolus and the relevant sequences were
downloaded and phylogenetic analysis has been
carried out. Figure 1 show phylogenic tree of
Streptomyces pseudogrisiolus.
PCR-based methods have provided a rapid and
accurate way to identify bacteria (Gurtler et al.,
1991; Kohler et al., 1991; Beyazova and
Lechevalier, 1993; Telenti et al., 1993; Soini et al.,
1994; Mehling et al., 1995; Steingrube et al., 1995a,
1997; Wilson et al., 1998 and Laurent et al., 1999).
 Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 8-23

Small rRNA gene sequencing, particularly 16S rRNA
sequencing in bacteria, has led to advances on
multiple fronts in microbiology. First, the construction
of a universal phylogenetic tree classifies organisms
into three domains of life: bacteria, Archaea, and
Eucarya. (Olsen et al., 1992), (Olsen and Woese,
1993) and (Thompson et al., 1994).
Second, it revolutionizes the classification of
microorganisms, and makes the classification of
non-cultivable microorganisms possible (Relman et
al., 1990) and (Relman et al., 1992).
Third, it helps to elucidate the relation of unknown
bacterial species to known ones. 16S rRNA gene
sequencing will continue to be the gold standard for
the identification of bacteria, and the automation of
the technique could enable it to be used routinely in
clinical microbiology laboratories, as a replacement
of the traditional phenotypic tests. Modern
technologies have made it possible to construct a
high density of oligonucleotide arrays on a chip with
oligonucleotides representing the 16S rRNA gene
sequence of various bacteria. Such a design will
facilitate automation of the annealing and detection
of the PCR products of 16S rRNA gene
amplification, and hence routine identification of
most clinical isolates will be possible (Woo et al.,
2001). The phylogenetic tree generated from partial
16S rRNA gene sequences, including the
sequences of (Streptomyces pseudogrisiolus and
other sequences from the database of gene bank
showed that the isolate formed one clusters. Our
data are in agreement with the division of the 16s
rRNA topology into major clusters as described.
Data obtained in this study illustrate primer design
will be useful to identify many bacterial genera.
Effect of composition of medium on milk clotting
enzyme production: In order to evaluate the
synthesis level of milk clotting proteolytic activity of
Streptomyces pseudogrisiolus NRC-15. It was used
several media, the milk clotting activity was
determined in the supernatant of each medium after
4 days of growth . Results of (Fig. 2) show that the
medium No. 3 gave highest enzyme production
(1714.3 units/ ml with 519.48 units/mg protein
specific activity respectively).This results related in
this paper suggest the importance of induction of the
Streptomyces pseudogrisiolus NRC-15. The
synthesis and secretion of milk clotting is induced by
peptides or other proteinaceous substrates.
Depending on the nature and level of peptides, they
may induce or repress proteases synthesis and
secretion (Porto et. al., 1996).
Effect of culture time on milk clotting enzyme:
The production of extracellular milk clotting enzyme
changes according to the age of the culture (data
not shown). The studies showed that the optimum
incubation periods for milk clotting enzyme
production were 4 days of fermentation. These
results are agreement with that obtained by Porto et.
al.,(1996) and Moreira et al (2001)which that the
synthesis of milk clotting starts in the post
exponential phase of growth of St. clavuligerus. It
has been reported higher production of milk clotting
enzyme between 72h and 192h of growth (Escobar
and Barnet, 1993; Areces et. al., 1992; Hashem,
1999).
Production of milk clotting activity affected by
initial pH of medium: Fig (3) shows the MCE
production by St. pseudogrisiolus , initial pH values
(pH from 5 to 10) and cultivated at 35±2°C with
shaking at 150rpm for four days. It was observed
that MCE production reached their maximum when
the initial pH of medium was adjusted to 6.0. Further
decrese or increase of the initial pH values resulted
in the reduced MCE production by St. The effect of
initial pH of the medium on the MCE production has
been illustrated, the MCE production by B.
licheniformis (D'souza and Pereira, 1982; Bacillus
subtilis (Jenshich et al., 2009) and A. rouxii (Chou
and 1997).

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 Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 8-23

specific activity (u/mg protein)

1000 3000

800 2500
Specific
2000

activity ( u/ml)
600 activity(U/mg
1500 protein)
400
1000 Enzyme
200 activity
500
(Units/ml)
0 0
5 6 7 8 9 10
pH values
Fig.3 Effect of different pH on the production of milk clotting
enzyme produced by Streptomyces pseudogriseolusNRC-
15

Effect of carbon sources on the production of
milk clotting enzyme by Streptomyces
pseudogrisiolus NRC-15. To investigate the effect of
carbon sources on the MCE production,
experiments were carried out with seven different
carbon sources, namely glucose, fructose, lactose,
maltose, sucrose, galactose and starch. Medium NO
3 with the presence absence of (30 g/L) was further
supplemented with various carbon sources at (3 %
level). The Streptomyces pseudogrisiolus NRC-15
was incubated at 35±2ºC with shaking at 150 rpm
for 4days. The results are shown in (Fig 4 and Fig.
5). Accordingly, it is seem that glucose was the best
carbon sources for the production (1714 units / ml)
with specific activity (428.58 units/ mg protein). On
the other hand, when use glucose at 3%
concentration gave the highest enzyme production
(2000 units/ml). The results are not in agreement
with those obtained by Hashem, (1999); Areces et.
al. (1992); Cavalcanti et. al.(2004) and Jenshieh et.
al. (2009), They found for many microorganismsin
that sucrose (0.05%) was the most favorable carbon
sources for Penicillium oxalicum; Aspergillus
versicolor; M. baciliformis ,Nocardiopsis sp. and
Bacillus subtilus.

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 Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 8-23

1000 2500
800 2000 Specific
activity(U/mg

specific activity (u/mg)

protein)

Activity (u/ml)
600 1500
400 1000
Enzyme
200 500 activity
(Units/ml)
0 0
1 2 3 4 5
Carbon concentration %
Fig.5. Effect of different concentration of galactose on a milk clotting
enzyme production produce by Streptomyces pseudogriseolusNRC-
15

Effect of nitrogen sources on the production of not agreement with that obtained by Jenshieh et. al.
milk clotting enzyme by: Results in fig (6) show (2009); Thakur et. al.(1990); Areces et. al.(1992)
that, the induction of milk clotting protease was and Porto et. al. (1996).They reported that inorganic
maximum on ammonium sulphate (2181.8U/ml).The nitrogen gave poor production of MCE, but organic
effect of different concentration of nitrogen were nitrogen sources favored good enzyme yield by
studied. Data in Fig.(7) Showed that 2% Bacillus subtilis, Mucormiehei ; Streptomyces
concentrations gave the highest milk clotting clavuligerus.
enzyme production, it gave (2400U/ml). Results are

1000 2500

900
specific activity (u/mg protein)

Specific
800 2000
activity(U/mg
700 protein)

600 1500
Enzyme
activity
Activity (u/ml)

500
(Units/ml)
400 1000

300

200 500

100

0 0

Nitrogen sources
Fig.6 Effect of different nitrogen source on the production of milk
clotting enzyme produced by Streptomyces pseudogriseolusNRC-
15

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 Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 8-23

Specific activity(U/mg protein)

1200 2450

Enzyme activity (Units/ml)

1000 2400
2350 Specific
800 2300
activity(U/m
g protein)
600 2250
400 2200 Enzyme
activity
2150 (Units/ml)
200 2100
0 2050
1 2 3 4 5
nitrogen concentration %
Fig.7 Effect of different concentration of Amm.sulphate on milk
clotting enzyme production produced by Streptomyces
pseudogriseolusNRC-15

Purification of milk clotting enzyme produced by

new isolated Streptomyces pseudogrisiolus
NRC-15: A summary of purification steps for milk-
clotting enzyme is given in table ( 1 ).The
purification of milk –clotting resulted in 4.59 fold
purification with 95.2%recovery by ammonium
sulfate precipitation. The purification of crude
enzyme through sephadex G100 column
chromatography gave 20.39 folds increase in purity
with 80% recovery of milk clotting enzyme from
Streptomyces pseudogrisiolus NRC-15.Similar
observation was reported by Somkuti and Bobel
(1968); Lenoir et. al. (1979) in Penecillum
caseicolumthey precipitated enzyme with
ammonium sulphate 80% saturation Nouani et. al
.(2011), in Mucorpusillus, who used the salt
fractionation principle. In fact in a similar study on
Bacillus subtilis milk-clotting enzyme, Matoub (2000)
noted a 93.75% performance activity. Moreover,
Cavalcanti et. al. (2004), recorded 55% performance
and a seven fold purification by fractioning the crude
extract of fungal coagulant from Nocardiapsis sp.
The works of Khan et .al. (1979) concluded that,
fractional ammonium sulphate precipitation (50 to
80% saturation). The purified enzyme has a
molecular mass of 66.2 kDa as determined by SDS- Fig.8 SDS-PAGE electrophoretogram of the
PAGE (Fig.8), which is higher than those in the Streptomyces pseudogriseolus NRC-15
milk-clotting enzyme after purification step.
literature (34-49 kDa) for other microbial milk-
Marker lane, standard molecular mass
clotting enzymes(Kumar et.al.,2005; Nouani et., markers
al.,2009 and Vishwanatha, et., al.,2009).

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 Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 8-23

Table (1):Steps of purification and yield recovery of milk clotting enzyme

Purification Total Total Specific Enzyme Purification
step protein(mg) activity(Units) activity (U/mg recovery fold
protein) %
Crude enzyme 135 1250 9.25 100 1
Amm. Sulfate 28 1190 42.5 95.2 4.59
fraction(50-
70%)
Sephadex G100 5.3 1000 188.67 80 20.39
fraction (6-23)

Enzyme characterization: of the colloidal calcium phosphate that is an integral

part of casein micelles under acid conditions.
pH: The effect of pH on the activity of milk clotting
enzyme from Streptomyces pseudogrisiolus NRC- The stability of the purified enzyme at different pH
15 was studied with various pH from5.0-11 (Fig. 9 ). values is shown in Fig. (9).The enzyme was stable
The optimum pH of milk clotting enzyme was in a relatively wide range of pH 5-7.5 with maximum
determined as 6.5 for purified MCE. At lower milk stability at pH 6.0. Outside either end of this range,
pH, it formed bad and non-firmed clots therefore, the activity of the enzyme decreased drastically,
and their results were excluded. It was reported that which showed that the Streptomyces
gels made at low pH (6.0-6.3) appeared to have a pseudogrisiolus NRC-15 milk clotting enzyme was
denser or more interconnected structure than gels stable in the acid or neutral range. The low pH
made at pH 6.7 by using plant chymosin (EL- sensitivity is very important for the coagulant
Bendary et. al.2007 and Esteves et. al.,2003). because the use of highly pH sensitive rennet can
These findings are in accordance with earlier reports led to reduced yields and defective cheese due to a
for milk clotting enzyme for Rhizopusorizae (Kumar soft coagulant at cutting (Harboe and Budtz,1999 )
et.al.2005). Like rennet, the purified enzyme from The low pH- sensitivity of the Streptomyces
Streptomyces pseudogrisiolus NRC-15 had a pseudogrisiolus NRC-15 enzyme is useful for
higher level of milk clotting enzyme in the acidic cheese making.
range. This is attributed in part to the solubilization

1050
1000
Specific activity (U/ mg protein)

950
900
850
800
750
700
650
600
550
500
450
400
350
300
250
200
150
100
50
0
30 35 40 45 50 55 60 65 70 75 80
Temperature°C
Fig. 9 : Effect of incubation temperature on milk clotting enzyme
activity produce by Streptomyces pseudogriseolusNRC-15

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 Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 8-23

Temperature activity and stability profile: The (Hashem,2000) and Bacillus amyloliquefaciens D4
milk-clotting enzyme demonstrated activity at 45⁰C (Xiaoling et al.2011) , but different calf rennet, which
(Fig. 10). Different enzymes have different optimum has an optimum temperature in the range 40-45⁰C.
temperatures, mainly depending on the enzyme This substantial difference in optimum temperature
structure. This result was in accord with the milk suggests strongly that they are suitable for use
clotting enzyme from Penicillum oxalicum under different conditions.

120
0
100 30
35
Relative activity %

80 40
45
60 50
55
40 60
65
70
20
75
80
0
0 10 20 30 40 50 60 70
Time(min.)

Fig ( 10 ) Effect of thermal stabilty on milk clotting enzyme activity

Thermal stability profile (Fig. 10), showed that the
enzyme was optimally stable at 40⁰C for 60 min. but
the activity gradually decreased with time at 50⁰C
compared with enzyme of different microorganisms
(El-Bendary et al.,2007) in B. shaericus, which
completely lost its activity after 20 min. at 70⁰C,
(Nouani et. al., 2009) in Mucorpusillus; (Preetha and
Boopathy,1997) in Rhizomuco rmiehei, which lost
activity after 30 min. at 65⁰C. and (Xiaoling et. al.,
2012) in B. amyloliquefaciens D4, the enzyme lost
after 30min. at 60⁰C.
Effect of substrate concentration: The enzyme
showed an increase in its activity with increasing
skim milk concentration up to 6 µg/ml (Fig.
11).Misallies constant as determined by double
reciprocal plot was found to be 1.8 /µg/ml, the Km
value is compared with other microbial milk clotting
enzyme, whose km values for casein were
0.388±0.002g % (Areces et. al.,1992) and 0.371±
0.067% (Fernandez-Lahore,1999).

17
 Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 8-23

140

Enzyme activity (Units/ml)

120
100
80
60
40
20
0
0 5 10 15

Substrate concentration ( µg/ml)

Fig. 11: Determination of Km and V max for purified milk clotting enzyme
through Michaelis-Menten equation

Effect of calcium chloride concentration: CaCl2

Specific Enzyme activity (U/ mg protein)

accelerated milk-clotting activity at all the tested 1100

+2
concentrations (Fig.12). It is known that Ca 1000
combines with Para casein to form firm clot during 900
+2 800
second phase of clotting process Ca was found to
700
be potent activator when use 25mM increase in 600
milk-clotting activity compared to control .It known 500
that Calcium has important function on casein 400
aggregation during the second step(non enzymatic ) 300
200
of milk clotting, caused by naturalization of casein 100
micelles negative residues (Phosphosrineand 0
+2
carboxylic group) by Ca and Calcium phosphate 0 10 15 20 25 30 35
complex(Pires et al .,1999) and so that an increase Concentration (mM)
in its concentration led to an increase in the Fig. 12: Effect of CaCl2 concentration on milk
coagulation rate(El-Bendaryet al., 2007 ; Abdel clotting enzyme activity produce by
Malaket al.,1996 and Kumar, et. al.,2005). Milk Streptomyces pseudogriseolusNRC-15
clotting activity decrease at concentration 35mM,
probably due to the increase of ionic force or to the
Effect of sodium chloride concentration: Sodium
saturation of negative residues of micelles at
+2 chloride is usually used during the process of
increasingCa concentration(Vairo-Cavalli
et.al.,2005). cheese manufactured in Egypt because it protects
milk against spoilage by various microorganisms.
Result in Fig.(13) showed that a gradual reduction in

18
 Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 8-23

milk clotting activity was observed when increased Table 2. Effect of metal ion on milk clotting activity of
the NaCl concentration . the purified enzyme from Streptomyces
pseudogrisiolus NRC-15
The amount of salt required to reduce water activity
to prevent microbial growth is 4-5%.The sensitivity Substance Final Conc. Of Relative
of milk clotting enzyme from various sources to added substance (mol) activity %
sodium chloride is not the same addition of NaCl to Control - 100
milk from (1-8%) Fig. (13). Resulted in a marked CuSO4 0.01 61
decrease in clotting activity with rennet used St. In 0.001 73
the presence of 8% Streptomyces rennet retained ZnSO4 0.01 55
4% of its activity. This results agree with those 0.001 68
reported by El-sayed (2012), El-Bendary et. al., CoCl2 0.01 99
0.001 100
(2007) and Ahmed & Helmy, (2012).
NaCl 0.01 82
0.001 97
KCl 0.01 89
0.001 95
MgSO4 0.01 101
0.001 105
FeSO4 0.01 83
0.001 97
MnCl2 0.01 108
0.001 115.6

Effect of inhibitor: Protease inhibitors were used to

.Fig 13 Effect of NaCl concentration on milk clotting
enzyme activity
Effect of metal ion on enzyme activity: In
investigation on the effect of metal ions on the milk
clotting activity of the enzyme, the enzyme was
treated with each metal ion in 0.1M phosphate
buffer (pH 6.5) at 50°C for 30 min. and residual milk
clotting activity was assayed. Data in table (2)
shows that significant inhibition was observed in the
case of CuSO4, ZnSO4,.In contrast MnCl2 and
MgSO4 had a significant stimulatory effect on milk
clotting activity. These results were similar to those
reported by (Xiaoling et al., 2012).
identify the group at the active site of the enzyme
table (3). Inhibition studies showed the sensitivity of
the purified enzyme to a serine protease inhibitor
(PMSF) a cysteine protease inhibitor
(Iodoacetamide), ametalloprotease inhibitor (EDTA).
The fact that PMSF and Iodoacetamide did not
inhibit the enzyme activity, the results showed that
the purified enzyme not a serine protease or
cysteine protease. The strong inhibition 97.3% and
98.1% at 0.001µM EDTA concentration showed that
the enzyme belongs to the metalloprotease group.
The present work describes the isolation clt A and B
gene from Streptomyces pseudogrisiolus. Gene
was detected at 2 kbp sequence PCR product direct
about 1190 bp. The sequences were assembled
and analyzed using both DNAstar (Lasergene) and
the BLAST 2.0 application (1) available on the Web
site of the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/BLAST/).
Sequence translated to amino acids using
http://web.expasy.org/cgi-bin/translate/dna_aa as in
dicated in fig (14).

19
 Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 8-23

Table 3. Effect of inhibitors on milk clotting activity of the purified enzyme from Streptomyces pseudogrisiolus NRC-15
Inhibitors Concentration(µM) Relative activity %
Iodo acetate 0.01 91
0.001 98
0.01 2.7
EDTA 0.001 1.9

PEMS 0.01 87
0.001 92

cgccctttctagagtcgaaacgcccgaccatatccatggaacccgaagcaatcacgcgggtt
P F L E S K R P T I S M E P E A I T R V
tgcggcgactggcgtcaactctatttacgcgcccgtgacgcgggcctggagcgcagatac
C G D W R Q L Y L R A R D A G L E R R Y
tccacgctagtcaactattccttgtttctctctctgcgggactcattggcctaccatttg
S T L V N Y S L F L S L R D S L A Y H L
ctcaagggccgggacgcgtctcgccgcggctggtggataccaggcggacggcgcgtcccc
L K G R D A S R R G W W I P G G R R V P
ccgcagtcaaaacggacgaggaatctccgatgcccatgttcaagaagatgttcaagggag
P Q S K R T R N L R C P C S R R C S R E
ctgcagtcctcgcggtgaccggtagcatgatgctgacgggctgcggcactgacgtgcagg
L Q S S R - P V A - C - R A A A L T C R
ccgattcgcagcacgagcaggatgagatcatctccaacctggtcgaggccggcttcccgg
P I R S T S R M R S S P T W S R P A S R
cggacgacctcctgatctccgacggccaggtgtacgtgggtcgcgacgcgcacgtgacgc
R T T S - S P T A R C T W V A T R T - R
tcgaggcgtcccgcgagatgctccagacggacctgaagacgcaggagcagtaccgcacga
S R R P A R C S R R T - R R R S S T A R
cgaacctcgtcgccgcctccatcacccgcatctgcatcgtcccgaactccgcgtacgccg
R T S S P P P S P A S A S S R T P R T P
ccaacaccacgctgatgtccgccctggaccgggcgatcgccaactacaacgcgctgggcc
P T P R - C P P W T G R S P T T T R W A
tgtcgttcaccatgcagcgcggcggctccggctgcagcgcgaccatctccgcacgcacca
C R S P C S A A A P A A A R P S P H A P
tgtccggcgctggcggctcggcgggcttcccctccggcggccgtccctacggcatcatca
C P A L A A R R A S P P A A V P T A S S
acatcggcaccggcacggccacctacggcgtggccgtggtcgagcacgtcgtgacccacg
T S A P A R P P T A W P W S S T S - P T
agctgggccacgccgtgggcctgcgccactctgactactacaaccgcagcatcagctgcg
S W A T P W A C A T L T T T T A A S A A
gcggcgcggcccccaacgaaggcaccgcgggcgttggcgccatccacatcccgggcacgc
A A R P P T K A P R A L A P S T S R A R
cgaccacggccacccgcggcggctcgctcatgaactcctgcttcggctcgtccgagacgg
R P R P P A A A R S - T P A S A R P R R
gtaacttcaagagctccgacgtcacggcgctccagtacctgtactgagtcatggcgcgac
V T S R A P T S R R S S T C T E S W R D
accccacagacttccacctgacgctgtggggtccgctcctcgctgggaccgtcttcctgg
T P Q T S T - R C G V R S S L G P S S W
gcctcaccgcctgcggaggacggtccgagccggccgagcctgcggtct
A S P P A E D G P S R P S L R S

Fig. 14. Nucleotide sequence and deduced amino acid sequence of the clt a and clt b gene isolated from
Streptomyces pseudogrisiolus . The amino acid sequence is shown below the coding sequence.

20

CONCLUSION
The milk clotting enzyme (MCE) obtained from
Egyptian isolate S. pseudogrisiolus has potential as
calf rennet substitutes. in dairy products and it can
be used in lypholyzed form as tablets in
pharmaceutical products for patients with digestive
system.
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