Chapter 7
CHARACTERIZATION OF PURIFIED
ALKALINE
PROTEASE
FROM
MUTANT BACILLUS LICHENIFORMIS
(Bl8).
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The properties of purified extracelluar proteases of mutant B.licheniformis
were studied and the enzyme was characterized.
7.1 MATERIAL AND METHODS
7.1.1 Effect of pH on purified alkaline protease activity
The influence of pH on the alkaline protease activity was determined by
measuring the enzyme activity at varying pH values ranging form 7 to 12 at 400C for
15 min using different buffers viz., 0.05 M Potassium phosphate-NaOH buffer (pH
7.0, 8.0) and Glycine – NaOH buffer (pH 9.0, 10, 11, 12). The results were shown in
fig 20 and Table: XXIII.
7.1.2 Effect of pH on the stability of alkaline protease activity
To determine the stability of enzyme at different pH levels, the purified
enzyme was incubated in buffers with different pH for 24 h at 400C. The buffers used
were acetate (pH 5 and 6), phosphate (pH 7 and 8) Glycine NaOH (pH 9 and 10) and
phosphate NaOH (pH 11 and 12). The relative activity was determined before and
after incubation. The results were shown in fig: 21 and Table: XXIV. The percentage
of activity remaining was calculated.
7.1.3 Effect of temperature on the activity of alkaline protease
The influence of temperature on the activity of alkaline protease was
determined by measuring the enzyme activity at various temperatures ranging form
300 to 700C under the standard assay conditions, using Glycine NaOH buffer 0.2M
(pH 10.0) and using casein as substrate. The results were shown in fig: 22 and Table:
XXV.
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7.1.4 Effect of temperature on the stability of purified alkaline
protease
The effect of temperature on the stability of the enzyme was determined both
in the absence and presence of calcium chloride. The enzyme was incubated at
different temperatures for 30 min in 0.2M glycine NaOH buffer with and without
calcium chloride (5mM). After heat treatment the enzyme solutions were cooled
quickly. Enzyme activities were determined under the standard assay conditions.
Results were shown in fig: 23 and Table: XXVI.The percentage of activity remaining
after the heat treatments was calculated.
7.1.5 Effect of inhibitors on the activity of alkaline protease
The effects of inhibitors on the activity of the enzyme were studied. The
enzyme was incubated with various inhibitors at 5 mM concentrations for 15min at
400C and the residual activities were determined. Inhibitors used were PMSF, PCMB, Idoaceticacid and EDTA. The results were shown in fig: 24, Table-XXVII.
7.1.6 Effect of metal ions on the activity of alkaline protease
The effect of various metal ions on the enzyme activity was studied. The
enzyme was incubated with various metal ion sources.(10mM) viz, Ca2+, Mg2+, Co+2,
Cd+2, Fe+3, Na+, Zn2+ and Cu2+ for 30min at 400C and relative protease activities were
measured. The results are shown in fig: 25 and Table: XXVIII.
7.1.7 Effect of different substrates on the alkaline protease activity
Protease activity was measured with various substrates including bovine
serum albumin, casein, egg albumin and gelatin. 1ml enzyme was incubated with
different protein substrates as described earlier and the activities measured. The
results were shown in fig: 26 and Table: XXIX.
7.1.8 To determine the digestion of natural proteins
2ml of the enzyme was incubated with human blood clot, coagulated egg
white and chicken skin in glycine-NaOH buffer (pH 9.5) at 370C for 10h and the
results were shown in Plate 2.
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7.1.9 To determine the detergent stability of the enzyme
The compatibility of purified alkaline protease with local laundry detergents
was studied in the presence of 10mM CaCl2 and 1M glycine.The detergents used were
Ariel, Surf excel, Rin, Nirma and Wheal. The detergents were diluted in distilled
water (0.5% wt/Vol), incubated with protease for 3 h at 400C, and the residual activity
determined. The enzyme activity of a control sample was taken as 100%. The results
were shown in fig: 27 and Table: XXX.
7.1.10 Determination of destaining property of purified alkaline
protease:
The destaining property was studied by dipping two pieces of cloth artificially
stained with blood either in detergent solution or detergent solution supplemented
with enzyme followed by incubation for 10 min at 400C. Results were shown in
Plate 3.
7.1.11 Effect of substrate concentration on activity of the enzyme
Effect of substrate concentration on the activity of purified alkaline protease
was studied, by incubating the 1ml of purified protease with different concentrations
of casein (substrate) viz, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg respectively and the activity of
enzyme was measured. From this km results value of the enzyme was calculated.
Results were shown in fig: 28.
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7.2 RESULTS AND DISCUSSION
7.2.1 Effect of pH on purified enzyme activity:
For the determination of the pH optimum, phosphate (pH 7.0, 8.0) and glycine
NaOH (pH 9.0-12.0) buffers were used. The highest protease activity was found to be
at pH10 using glycine NaOH buffer. The results are shown in Table: XXIII and fig:
24. The maximum activity shown has been taken as 100%. Durham et al., (1987)
reported that alkaline proteases generally have broad pH optima in the range of
pH 8-12. These findings are also in accordance with several earlier reports showing
pH optima of 10.0 – 10.5 for protease from Bacillus sp. Thermos aquaticus,
Xanthomonas maltophila and Vibrio metscnikovii (Durham, 1987 and Kwon
et a1.1994).
The important detergent enzymes subtilisin Carlsberg and subtilisn NOVO or
BPN also showed maximum activity at pH 10.5 as was reported by Horikoshi
et al., (1995, 1996).
7.2.2 Effect of pH on the stability of enzyme:
The stability of purified protease was also determined by pre incubation of the
enzyme in various buffers with different pH values. The enzyme was stable over a
broad range of pH 7 to 10. The results were shown in fig: 21 and Table: XXIV. In the
present study enzyme could retain 70% of its original activity after incubation in
Glycine NaOH buffer with pH 12.0 at 400C for 24h. This was comparable with the
stability shown by the highly alkali stable proteases such as those obtained from
Bacillus sp. GX 6638 (Durham et al., 1987). Bacillus subtilis RM 615 (Moon et al.,
1994) and Vibrio metschnikovil RH530 (Kwon et al., 1994). Alkaline protease from
these bacteria was retaining 88% 85% and 80% activity after incubation under more
or less similar condition. The pH stability was also shown by subtilisin
Carlsberg ( Durham et al., 1987).
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Table XXIII
Effect of pH on purified alkaline protease activity
pH range
Relative alkaline protease activity
(%)
7.0
70
8.0
75
9.0
85
10.0
100
11.0
90
12.0
86
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Fig 20
Relative alkaline protase activity
(%)
Effect of pH on the activity of purified alkaline protease
120
100
80
60
40
20
0
7
8
9
10
pH
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11
12
Table XXIV
Effect of pH on the stability of alkaline protease activity
pH range
Relative alkaline protease activity (%)
5.0
40
6.0
60
7.0
98
8.0
100
9.0
100
10.0
100
11.0
86
12.0
70
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Fig 21
Relative alkaline protase activity (%)
Effect of pH on the stability of purified alkaline protease
120
100
80
60
40
20
0
5
6
7
8
9
pH
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10
11
12
7.2.3 Effect of temperature on activity of alkaline protease
The activity of the purified enzyme was determined at different temperatures
ranging from 300C to 700C using Glycine NaOH buffer (pH: 10.0) for 1 hr. The
optimum temperature for caseinolysis was recorded was at 500C.Results were shown
in fig: 22 and Table: XXV.
Alkaline proteases of Bacillus with similar temperature optima have been
reported by Durham et al.,(1987), Takil et al.,(1990), Kobayashi et al.,(1995,1996)
and Ferrero et al(1996).
7.2.4 Effect of temperature on stability of purified alkaline protease
The thermal stability of the purified protease was tested at different
temperatures ranging from 300C to 700C for 30 min in Glycine NaOH buffer, with
and without 5mM calcium chloride, was studied. The percentage of activity remaining
after heat treatment at different temperatures is shown in fig 23 and Table: XXVI.
The enzyme was stable with about 100% activity at 300, 400 & 500C for 30
min. Reduction in activity could be observed after incubation at 60C and above. The
presence of calcium chloride was found to improve the thermo stability of the
enzyme. The thermo stability shown by this enzyme was comparable with or better
than that reported for the alkaline proteases from different Bacillus species
(Tobe et al., 1975; Durham et al., 1987; Takil et al., 1990); Kobayashi et al., (1995,
1996).
7.2.5 Effect of inhibitors on the activity of alkaline Protease
Effect of different inhibitors on the activity of the enzyme was shown in
Table: XXVIII and fig: 24.The enzyme was strongly inhibited by PMSF, and slightly
by EDTA. A complete loss of enzyme activity in the presence of PMSF was reported
by Gold et al. (1964). Slight inhibition of enzyme activity by EDTA has been reported
in alkaline serine protease from bacterial sources such as Bacillus licheniformis
(strongin et al., 1979; Ferrero et al., 1996). Bacillus thermoruber (Manchini et al.,
1985). Bacillus sp. (Tobe et al., 1983). Halobacterium halobium (Izotova et al.,
1983), Pseudomonas sp. (Kato et al., 1974) Myxococcus viriscens (Gnoss pelius,
1978) and Streptonyces sp. (Yum et al., 1994). In the present study also the enzyme
purified from mutant B.licheniformis (Bl8) showed similar inhibition by PMSF and
EDTA as reported by various researchers. Among the other inhibitors studied, slight
inhibition was observed with iodoacetate, PCMB.
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Table XXV
Effect of Temperature on activity of alkaline protease
Temperature 0C
Relative enzyme activity (%)
30
30
40
70
50
100
60
90
70
65
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Fig 22
Relative alkaline protease activity (%)
Effect of temperature on the activity of purified alkaline protease
120
100
80
60
40
20
0
30
40
50
Temperature0c
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60
70
Table XXVI
Effective of temperature on the stability of enzyme activity
Temperature 0C
Relative alkaline protease activity
(%)
with CaCl2
without CaCl2
30
40
50
60
70
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100
100
100
90
100
85
60
50
30
20
Fig 23
Effect of temperature on the stbility of purified alkaline protease
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Table XXVII
Effect of protease inhibitors on the alkaline protease activity
Inhibitor (5mM)
(%) Relative enzyme activity
Control
100
PMSF
(Phenyl methyl sulphonyl fluoride)
00
PCMB
(Para chloro mercuric benzoate)
95
90
Idoacetic acid
EDTA
(Ethelene Diamine tetra acetic acid)
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93
Fig 24
Relative alkaline protease activity (%)
Effect of inhibitors on the activity of purified alkaline protease
120
100
80
60
40
20
0
Control
PMSF
PCMB
Inhibitors
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Idoacetic
acid
EDTA
7.2.6 Effect of metal ions on activity of alkaline protease
Effect of various metal ions on the activity of the enzyme is shown in Table
XXVIII and: Fig: 25. Activities are expressed relative to the control. Activity of the
control was taken as 100%.
None of the metal ions showed considerable enhancing effect on the activity
of the protease. Ca2+ & Mn+2 had a slight enhancing effect on the activity of the
enzyme. Of the different metal ions tested Zn+2 showed the maximum inhibition.
Mn+2 has been reported as enhancing the activity of alkaline serine proteases from
some bacterial sources such as Bacillus sterothermophilus F1 (Rahman et al., 1994)
and Nocardiopsis derssonvillei (Tsujibo et al., 1990). Its mechanism of action is not
well understood.
The inhibition of bacterial alkaline proteases by cations such as
Fe2+ (Chang et al., 1988, Rattray et al., 1995) Co2+ (Rahman et al., 1994),
Cu 2+ (Tsujibo et al., 1990) and Zn2+ (Rattray et al., 1995) have also been reported.
7.2.7 Effect of different substrates on the alkaline proteases activity
Effect of different substrates on enzyme activity was studied. High level the
hydrolytic activity was shown by casein and with poor to moderated hydrolysis of
BSA and egg albumin. However the hydrolysis was hardly observed with gelatin. The
results were shown in Table: XXIX and. Fig: 26.
7.2.8 To determine the digestion of natural proteins
The ability of purified protease to digest some natural proteins was tested. The
results were shown in Plate 2. These results showed that enzyme can convert the
insoluble forms of human clot and coagulated white egg to soluble form. The enzyme
was also able to digest chicken skin after incubation for a long time with it.
The results suggest usefulness of this enzyme for different application such as,
extraction of collagen from skin for collagen replacement therapy, waste treatment
and others.
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Table XXVIII
Effect of metal ions on the activity of alkaline protease
Metal ion Source
Relative activity (%)
(10mM)
Control
100
CaCl2 (Ca2+)
102.1
MgCl2 (Mg2+)
101
CoCl2 (Co2+)
90
CdCl2 (Cd2+)
90
FeCl3 (Fe3+)
80
NaCl (Na+)
98
ZnCl2 (Zn+2)
82
CuCl2 (Cu+2)
90
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Fig 25
Effect of metal ions on the activity of purified alkaline protease
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Table XXIX
Effect of different substrates on the alkaline protease activity
Substrate (1 ml)
Relative enzyme activity (%)
Casein
100
Bovine serum albumin
54
Egg albumin
32
Gelatin
12
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Fig 26
Relative alkaline protease activity (%)
Effect of substrates on the activity of purified alkaline protease
120
100
80
60
40
20
0
Casein
Bovine serum
albumin
Egg albumin
Substrates
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Gelatin
Plate-2
A
C
B
T
T
C
C
Control
Test
Digestion of natural proteins:
A. Blood clot
B. White egg
C. Chicken skin
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7.2.9 To determine detergent stability of alkaline protease activity
Enzyme activity and stability in the presence of some available commercial
detergents was studied with a view to exploit the enzyme in detergent industry. A
good protease is expected to be stable in the presence of commercial detergents. In the
present study, the protease showed excellent stability and compatibility in the
presence of locally available detergents (wheel, Ariel, Surf excel, Rin, Nirma) at 400C
in the presence of CaCl2 and glycine as stabilisers. The proteases showed good
stability and compatibility in the presence of Nirma, Surf excel and wheel. The results
were shown in Fig: 27 and Table:XXX. Enzyme activity and stability in the presence
of detergents was reported by Madan et al. (2002). They observed that enzyme retains
84.5% of activity in the presence of Vim and more than 40% in the presence of Nirma
super, Wheel and Nirma.
Interestingly, in the present study, the enzyme retained around 20-40% activity
with most of the detergents tested even after 3 hours incubation. Phadatare et al.
(1993) studied the compatibility of alkaline protease of Condiobolus cornatus (Ncl
86.8.20) with commercial detergents. They observed the enzyme protease retains
more than 80 percent of its activity in the presence of detergents viz, snow white,
Nirma, Revel and more than 56 percent of its activity in the presence of wheel and
surf when incubated for 1h. Similarly among the three proteases isolated from
Tritirachium album limber, proteinase R and T were reported to retain 90 and 89
percent activity respectively up to 1h in the presence of detergents like ERA plus and
Dyname. BPN was highly unstable in all the detergents and retained just 4 percent
activity even after 10 min as reported by samal et al., (1990).
7.2.10 Determination of destaining property of purified alkaline
protease
The results of distaining experiment showed the complete removal of stain in
detergent solution supplemented with enzyme where as blood stain was not
completely removal from cloth dipped in detergent solution alone (Plate 3).This
suggested usefulness of this enzyme in detergent industry. From this study the
protease could be recommended as an additive in detergents to improve their washing
performance of the detergent.
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Table XXX
Detergent stability of the enzyme
Detergen
t
(%) Retention of enzyme activity
Time
0 hr
0.5 hr
1
1.5
2
2.5
3.0
Control
100
100
100
100
100
100
100
Wheel
100
90
80
70
60
50
46
Ariel
100
74
62
51
43
36
20
Surf excel
100
84
82
64
62
48
35
Rin
100
65
59
47
36
30
22
Nirma
100
90
86
74
50
48
40
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Fig 27
(%)Retention of enzyme activity
Effect of detergents on the stability of purified alkaline protease
120
100
Control
Wheel
80
Arieal
60
Surfexcel
Rin
40
Nirma
20
0
0
0.5
1
1.5
Time (min)
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2
2.5
3
Plate - 3
(A)
(B)
(C)
Slide 3:- Washing performance of alkaline protease from mutant
Bacillus licheniformis (Bl8)
(A):- Cloth stained with Blood.
(B):- Blood stained cloth washed with detergent only.
(C):- Blood stained cloth washed with detergent and enzyme.
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The above results showed that the enzyme isolated from mutant strain of
Bacillus licheniformis (Bl8) was stable at high pH and temperature and in the
presence of detergents and may be exploited as an additive in the detergent industry.
7.2.11 Effect of substrate concentration on alkaline protease activity
Effect of substrate concentration on enzyme activity was studied. Results were
shown in fig 28.The results showed that the enzyme followed a typical MichalisMenten kinetics from a double reciprocal plot. The apparent Km of the enzyme for
casein was found to be 3.2 mg/ml.The km values of 0.4 and 1.3 towards casein (mg
ml-1)
have been reported for alkaline proteases of Bacillus alcalophilus var.
halodurans (Takil et al., 1990) and Brevibacterium linens (Juhasz and Skarka, 1996)
the higher values 3.7, 7.4 and 9.4 mg ml-1 have been reported for the proteases of
Bacillus polymyxa
( kaur et al., 1998) Halomonas sp. Es10 (Kim et al., 1992) and
Bacillus licheniformis N3 (Sinha and Satyanarayana, 1991). respectively. The lower
km value of 3.2 mg ml-1 in the present study indicating lower affinity of the enzyme
from mutant B.licheniformis(Bl8). Menu madan et al.,(2002) reported that apparent
km of the enzyme for casein was found to be 2.9 mg ml-1 for mutant Bacillus
polymyxa. It has lower km value than the wild B.polymyxa protease.
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Fig 28
Effect of substrate concentration on purified alkaline protease
(Line weaver-Burk plot)
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