characterization of alkaline proteasesshodhganga.inflibnet.ac.in/bitstream/10603/256/13/13_chapter...
TRANSCRIPT
Chapter
CHARACTERIZATION OF ALKALINE PROTEASES
h e properties of purified extracellular proteases of Bacillus sp. K 25 and T Bacillus pumilus K 242, referred to as protease K 25 and protease
K 242, respectively were studied and the enzymes were characterized.
Experiments were done in triplicate.
MATERIALS AND METHODS
Effect of pH on activity
The effect of pH on the enzymatic activity of protease K 25
and protease K 242 was studied by measuring weinolysis in buffers (0.2 M)
with different pH. The buffers used were Tris-HCI (pH 7.59.0),
carbonate-bicarbonate (9.0-10.5) and phosphate-NaOH (10.512.0).
Reactions were carried out at 40°C for 10 min.
Effect of temperature on activity
The effect of temperature on the activity of proteases was studied in
glycine-NaOH buffer 0.2 M (pH 10.0), using casein as substrate. Proteases
K 25 and K 242 were tested at different temperatures ranging from 30-70°C
and 30-65" respectively. Reactions were carried out for 10 min.
Effect of inhibitors on activity
The effects of inhibitors on the activity of the enzymes were studied.
The enzymes were incubated with various inhibitors (Sigma) at 1 mM and
10 mM concentrations for 15 min at 37"C, and the residual activities were
determined. Activity of the controls not containing any inhibitors was also
determined.
Effect of metal ions on activity
The effect of various metal ions on the enzyme activity was studied.
The enzymes were incubated with various metal ion sources (1 mM) in 20 mM
Tris-HCI buffer (pH 9.0) at 37°C for 15 min and the enzyme activity was
determined. The enzyme activity in buffer without any of these metal ion
sources (control) was also determined.
Effect of pH on stability
pH stability of proteases K 25 and K 242 were studied. The enzymes
were incubated in buffers (0.2 M) with different pH, for 24 h at 25°C. 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).
Activities were determined before and after incubation. The percentage of
activities remaining was calculated.
Effect of temperature on stability
The effect of temperature on the stability of the enzymes were studied
both in the absence and presence of calcium chloride. The enzymes were
incubated at different temperatures for 30 min in 0.2 M glycine-NaOH buffer
with and without calcium chloride (5 mM). After heat treatment, the enzyme
solutions were cooled quickly. Enzyme activities were determined before and
after heat treatments. The percentage of activities remaining after the heat
treatments were calculated.
RESULTS
Effect of pH on activity
Relative activities of protease K 25 and protease K 242 at different pH
are shown in Figures 9a and 9b respectively. The maximum activity shown
by each enzyme has been taken as 100%.
-0- Carbonate-bicarbonate buffer
100 - + Phosphate-NaOH buffer - - 90 -- 2' > .- +
4 3 70 2
60 7 I
7 7.5 8 8.5 9 9.5 10 10. 11 11. 12 5 5
Figure 9a. Effed of pH on caseinolysis by protease K 25
-c- Carbonate-bicarbonate buffer
Figure 9b. Effed of pH on caseinolysis by protease K 242
The optimum pH for caseinolysis by proteases K 25 and K 242 were
found to be 11.0 and 10.5 respectively.
Effect of temperature on activity
The relative activities of the purified enzymes at different temperatures
are shown in Figure 10. The maximum activity shown by each enzyme has
been taken as 100%.
I -t Protease K 25 + Protease K 242
Temperature ("C)
Figure 10. Effect of temperature on caseinolysis by protease K 25 and protease K 242
The optimum temperatures for caseinolysis by protease K 25 and
protease K 242 were 60°C and 55°C respectively.
Effect of inhibitors
Effects of different inhibitors on the activity of the enzymes are shown
in Table 28. Activities after incubation with different inhibitors have been
expressed relative to the controls. Activities shown by the controls are taken
as 100%.
Table 28
Effects of inhibitors on the activity of proteases K 25 and protease K 242
- Concentration Relative activity (%)
Inhibitor of the inhibitor Protease K 25 Protease K 242 (mM)
PMSF 1 18.4 7.9
10 0 0
p-CMB 1 102.8 100.2
10 97.8 98.3
lodoacetic acid 1 100.4 101.0
10 98.3 100.5
EDTA 1 98.8 99.1
10 103.2 94.5 >
Both the enzymes were strongly inhibited by PMSF. A slight inhibition
of activity of protease K 242 by EDTA at 10 mM concentration could also be
noticed.
Effect of metal ions on activity
Effects of various metal ions on the activity of the enzymes are shown
in Table 29. Activities are expressed relative to the controls. Activities of the
controls are taken as 100%.
Table 29
Effect of metal ions on the activity of proteases K 25 and K 242
Metal ion Relative activity (%) source Protease K 25 Protease K 242
Barium chloride 95.2 96.4
Calcium chloride 99.4 102.1
Cobalt chloride 75.8 90.4
Cupric chloride 82.0 90.9
Ferrous chloride 94.3 99.6
Magnesium chloride 98.3 100.7
Manganese chloride 105.2 95.5
Mercuric chloride 36.1 54.7
Nickel chloride 88.8 87.3
Potassium chloride 98.6 96.4
Sodium chloride 101.5 98.2
Strontium chloride 90.0 92.2
Zinc chloride 78.7 82.6 -
None of the metal ions tested showed considerable enhancing effed on
the activity of the protease K 25. P4n2+ had a slight enhancing effed on the
activity of the enzyme. Of the different metal ions tested' ~ d ' was the most
inhibitory one. The inhibitory effects of Ba2+, co2+, cu2+, ~ e ~ + , Ni2+, s?+ and zn2+ were lesser compared to that of H$+. The other ions, ca2+, M$+,
K' and Na+ had little or no effect on the activity of the enzyme.
Of the different metal ions tested none showed a noticeable enhancing
effect on the activity of protease K 242. The ions which showed the inhibitory
effects were Ba2+, Co2+, cu2+, H$+, Mn2+, Ni2+, s?+, K+ and zn2+.
Of these the most inhibitory one was H$+. The other ions, ca2+, ~e" , Mg2+
and Nai had only negligible effects on the activity of the enzyme.
pH stability
The pH stability of the proteases was studied. The percentage of
activity remaining after incubation of enzymes in buffers with different pH are
shown in Figure 11.
-D- Protease K 242
Figure 11. Effect of pH on the stability of protease K 25 and protease K 242
The enzymes protease K 25 and protease K 242 were found to be
stable in the pH ranges 6-11 and 6-10 respectively.
Thermostability
Thermostability of the proteases in the absence and presence of
calcium chloride was studied. The percentage of activity remaining after heat
treatment at different temperatures are shown in Figure 12.
+Protease K 242 (without Calcium chloride) -e- Pmtease K 25 (with Calcium chloride) t Protease K 242 (with Calcium chloride)
45 50 55 60 65 70
Temperature ("C)
Figure 12. Effect of temperature on the stability of protease K 25 and protease K 242
Both the enzymes were retaining almost complete activity after heat
treatment at 450C for 30 min. Reduction in activity could be observed after
incubation at 50°C and above. The thermostability exhibited by protease
K 25 was better than that shown by protease K 242. In the absence of calcium
chloride, both the enzymes lost their activity completely after incubation at
70°C for 30 min. The presence of calcium chloride was found to be improving
the thermostability of both the enzymes. In the presence of calcium chloride,
the thermostability exhibited by protease K 242 was slightly better than that
shown by protease K 25.
DISCUSSION
Some properties of the proteases purified from the culture supernatant
of Bacillus sp. K 25 (protease K 25) and bacterial bran extract of Bacillus
pumilus K 242 (protease K 242) were studied.
The optimum pH for caseinolysis at 40°C were 11.0 for protease K 25
and 10.5 for protease K 242. From a survey of literature it can be seen that
the bacterial alkaline proteases with pH optima higher than that observed in
this study are rare. Such alkaline proteases have been isolated only from a
few bacteria such as &cillus alcdophhilus sub sp. halodurans K 1239 (Takii
et af., 1990) and Baciflussp. KSM-K 16 (Kobayashi ef af., 1995).
The optimum temperatures for caseinolysis by protease K 25 and
protease K 242 were found to be 60°C and 55°C respectively, when tested at
pH 10.0. Alkaline proteases of Bacillus species with similar temperature
optima have been reported by Durham et a/. (1987), Takii et af. (1990),
Kobayashi eta/. (1995, 1996) and Ferrero eta/. (1996).
The effects of various inhibitors on the activity of the proteases were
studied. Both the enzymes were inhibited by PMSF strongly. This indicated
the presence of active serine residue in the catalytic site of the enzymes. So
these enzymes could be classified under serine proteases. p-CMB and
iodoacetic acid, the inhibitors of sulphydyl proteases were having no
inhibitoy effect on the activity of the proteases. While EDTA, the metal
chelator was not having any inhibitoy effect on the activity of protease K 25,
it was slightly inhibitoy on protease K 242 at a concentration 10 mM. At the
lower concentration, i.e. 1 rnM its effect was negligible. EDTA which has
been shown to be a general inhibitor of neutral proteases is not so inhibitoy
on alkaline proteases. However slight inhibition of activity by EDTA has been
reported in alkaline serine proteases from bacterial sources, such as Bacillus
lichenifonnis (Strongin et al., 1979; Ferrero et d., 1996), Bacillus thennoruber
(Manachini et al., 1988), Bacillus sp. (Tobe et al., 1975), Halobacterium
halobium (Izotova et al., 1983), Pseudomonas sp. (Kato et d., 1974),
Myxococcus viriscens (Gnosspelius, 1978) and Streptomyces sp. (Yum et al.,
1994).
The effects of different metal ions on the activity of the enzymes were
studied. None of them was found to be having considemble enhancing effect
on the activity of these enzymes. ~ n ~ + was increasing the activity of protease
K 25 slightly. Though ~ n ~ + has been reported as enhancing the activity of
alkaline serine proteases from some bacterial sources such as Bacillus
stearothennophilus F1 (Rahman et al., 1994) and Nocardiopsis dassonvillei
(Tsujibo et a/., 1990), its mechanism of action is not well understood. Of the
different metal ions inhibiting the activity of protease K 25, H$+ was the most
inhibitoy one. The inhibitoy effects of other ions such as I3a2+, CO", cu2+,
~ e ' + , ~ i* ' , s?+ and zn2+ were comparatively lesser. The ions such as ca2+,
M ~ * + , K+ and Na+ were having only negligible effect on the activity of the
enzyme. Fig2+ was the ion having the greatest inhibitory effect on the activity
of protease K 242 also. The other ions inhibiting the activity of this enzyme
were Ba2+, coZ+, cu2+, Mn2+, Ni2+, K+, s?+ and 2n2+. The effects of ~ e ~ +
and Na+ were negligible. From the review of literature it could be seen that
is the most commonly reported cation inhibitor of bacterial alkaline
serine proteases (Rahman et al., 1994; Yum et a/., 1994; Kobayashi et a/.,
1995, 1996; Rattray et d, 1995). The inhibition of bacterial alkaline serine
proteases by cations such as ~ e ' + (Chang et d., 1988; Rattray et a/., 1995)
co2+ (Rahman et d., 1994), CU" (Tsujibo et d., 1990) and 2n2+ (Rattray
et a/., 1995) has also been reported by the earlier workers.
The effect of pH on the stability of proteases was studied. Protease
K 25 was found to be stable in the pH range 6-11 after incubation for 24 h
at 25°C. Under similar conditions protease K 242 was showing stability in the
pH range 6-10. The stability shown by protease K 25 at high alkaline pH was
very high. It could retain 76% of its original activity after incubation in buffer
with pH 12.0 at 25°C for 24 h. This was comparable with the stability shown
by the highly alkali stable proteases such as those obtained from Bacillus sp.
GX 6638 (Durham et a/., 1987), Bacil/us subtilis RM 615 (Moon et a/., 1994)
and Kbrio metschnikovii RH 530 (Kwon et dl., 1994). Alkaline proteases
from these bacteria were retaining 88%, 85% and 80% activity after
incubation under more or less similar conditions. The pH stability shown by
subtilisin Carlsberg (Durham eta/., 1987), the widely used detergent protease
was much lesser compared to the pH stability exhibited by protease K 25.
The effect of temperature on the stability of the enzymes was studied.
Both the enzymes were retaining almost full activity after heat treatment at
45°C for 30 min. Reduction in activity was seen after incubating the enzymes
at 50°C or above. Both the enzymes lost their activity completely, after heat
treatment at 70°C for 30 min. Results show that themostability of protease
K 25 was better than that of protease K 242. The themostability shown by
both the proteases was comparable with or better than that reported for the
alkaline proteases from different Bacillus species (Tobe et a/., 1975; Durham
eta/., 1987; Takii eta/., 1990; Kobayashi et al., 1995, 1996).
Addition of calcium chloride was found to be enhancing the
thermostability of both the proteases. In the presence of calcium chloride, the
thermostability exhibited by protease K 242 was slightly better than that
shown by protease K 25. Though Ca2+ has been reported to be enhancing
the thermostability of alkaline serine proteases from many bacterial sources
(Tobe eta/., 1975; Manachini et a/., 1988; Yun eta/., 1989; Tsuchiya eta/.,
1992; Dhandapani and Vijayaragavan, 1994; Kwon et a/., 1994; Rahman
et a/., 1994; Kobayashi eta/., 1995), the mechanism behind it has not been
studied in detail.
Due to the possession of desirable properties such as activity at high pH
and temperature, high pH stability and thermostability and insensitivity to
chelating agents, the proteases K 25 and K 242 can be supposed to be
suitable for commercial applications.