World Journal of Microbiology & Biotechnology 10, 33-35
Production of a thermophilic, extracellular alkaline protease by Bacillus stearothermophilus AP-4 R. Dhandapani* and R. Vijayaragavan A thermophilic Bacillus stearothermophilus strain AP-4 excreting a thermostable alkaline protease, was isolated from a local compost. Maximum activity of protease (250 U/ml) was after 36 h growth in broth at pH 9.0 and at 55°C. The protease was optimally active at pH 9.0 and 55°C and was stable in 5 mM CaCl 2. The enzyme was completely inactivated by PMSF, EDTA and ]/-mercaptoethanol. It is therefore a metal ion-dependent, alkaline, serine protease.
Key words: alkaline protease, Bacillus, thermophilic. Proteases from thermophilic bacteria have considerable commercial potential, especially as components in laundry detergents, where their ability to function at high pH values and temperatures is exploited (Anunstrup et al. 1972). The proteases, produced by different Bacillus species, vary not only in type but also in the pH and temperature necessary for optimal activity (Priest 1977). There have been many reports on thermophilic proteases produced by different strains of Bacillus spp., including the thermolysin from B. thermoproteolyticus (Endo 1962), and an alkaline protease from Bacillus sp. no A H I 0 I (Takami et al. 1989) and B. alcalophitus (Takii et al. 1990). A thermostable neutral protease has been reported frequently from B. stearothermophilus (Takii et al. 1987; Kubo et al. 1988; Fujio & Kume 1991). During a study on the microbial flora of a compost waste on the university farm, we found a highly-thermostable, alkaline protease produced by Bacillus stearothermophilus strain AP-4. We describe, in this paper, some of the properties of this protease.
in 10 ml sterile water and incubated at 55°C for 3 h. One ml of this suspension was then inoculated into 9 ml of LB broth, [containing (g/l): Bacto-tryptone, 10; yeast extract, 5; NaC1, 10; and casein, 10; pH adjusted to 9.0 with 5 M NaOH] and incubated at 55°C for 24 h. After two such enrichments, 50 #1 of the inoculum was plated on each of several LB agar plates (3% w/v agar) and kept at 55°C for 24 h. Based on caseinolytic activity on LB agar plates, a single colony of Bacillus sp., designated AP-4, was isolated from 2000 colonies by repeated streaking on the same agar media at 55°C.
Growth of Isolate AP-4 To study protease secretion and growth of the organism, a 250-ml side-arm flask containing 100 ml of LB medium at pH 9.0, was inoculated with 106 cells (initial A620= 0.07) and incubated at 55°C. Growth was assessed turbidometricalIy at 620 nm; a value of A6z0 = 1 corresponded to 6 x 109 cells/ml. One ml of the sample was taken at various intervals, centrifuged and the supematant used for assays of proteolytic activity, with casein as the substrate (see below). The pH of the medium was measured aseptically every 4 h using an electronic pH meter. All the experiments were repeated at least three times.
Partial Purification of Protease
The strain AP-4 was isolated from a compost heap of the Tamil Nadu Agricultural University's farm by culture enrichment techniques. About 100 mg of the sample was initially suspended
Strain AP-4 was cultivated in LB media as described above and incubated at 55°C with shaking at 200 rev/min for 40 h. The cell-flee supematant recovered by centrifugation (8000 x g, 20 rain) at 4°C was treated with (NH4)2SO 4 to 80% saturation. The precipitate was recovered by centrifugation (13,000 x g, 20 rain), dissolved in 10 mM Tris/HC1 (pH 9.0) and dialysed against the same buffer for 24 h at 4°C. The dialysate was used as enzyme solution.
R. Dhandapani and R. Vijayaragavan are with the Centre for Plant Molecular Biology & Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641 003, India; fax: 091-0422-41672. *Corresponding author.
The enzyme reaction was carried out in a mixture containing 250 lal 2% (w/v)/J-casein (Sigma), 500/A 10 mM Tris/HC1 buffer, pH 9.0,
Materials and Methods Isolation of the Bacterium
Protease Assay
(~ 1994 Rapid Communications of Oxford Ltd World Journal of Microbiology & Biotechnology, Vol 10, 1994
33
R. Dhandapani and R. Vijayaragavan and 250/~l of the sample. After incubation for 30 min at 55°C, 500 #l of TCA solution (0.II M trichloroacetic acid, 0.22 M sodium acetate and 0.33 M acetic acid) was added to stop the reaction and the liberated tyrosine was assayed by absorbance at 275 nm against a standard tyrosine solution. One unit of protease activity was defined as the amount of enzyme required to produce I/~g tyrosine per min under the given conditions. Protein concentration was determined by the Lowry method, using BSA as the standard.
10C
o~o
-
.>
50 o
._>
0
Results and Discussion
I 20
I
40
30
50
60
70
80
90
Temperature (°C)
Characteristics of the Isolate Strain AP-4 was a Gram-positive, aerobic, catalase and oxidase negative, motile, terminal endospore-forming bacterium. It was an obligate thermophile (growing only at 45 to 65°C), forming long rods, often forming short chains after 21 h at 55°C. The culture grew exceptionally fast on LB agar plate at 55°C, covering the entire Petri dish (90-mm diam.) in 6 to 8h. The isolates did not grow at all at 28 -I- 5°C or in the media containing 7% or 10% (w/v) NaC1. However, the culture remained viable after storage at room temperature for 60 days. The strain hydrolysed casein, gelatin, bovine serum albumin and haemoglobin. It also weakly hydrolysed starch. This strain was identified as Bacillus stearothermophilus AP-4 (Claus & Berkeley 1986). The fatty acid profile of this microbes confirmed its species identification. The culture has been deposited in the Institute of Microbial Technology, Chandigarh, India.
Protease Production Maximum enzyme production occurred when Bacillus sp. AP-4 was grown at 55°C in LB medium with an initial pH of 9.0 (Figure 1). Protease production was growth associated and maximum activity was after 36 h.
Figure 2. Effect of temperature on the activity and stability of protease from Bacillus sp. AP-4. Activity (I-1) was determined at
different temperatures with an incubation of 30 min. The thermal stability of the enzyme in the presence of 5 mM CaCI2 (~) in 20 mM glycine/NaOH buffer, pH 9.0, or in the absence of CaCI2 (O) in 10 mM Tris/HCI buffer, pH 9.0, was determined by holding the enzymes for 30 min, at the desired temperature, then determining the residual activity by incubation at 55°C for 30 min. 100% activity = 288 U/ml.
after 30 min at 70°C. The enzyme did not lose activity when held for up to 10 days at 4°C. The protease was stable and active in CaC12 (Figure 2); optimum activity with 5 mM Ca2 + was at 65°C. The protease was active at pH 9.0 to 11.0, with optimum activity at pH 9.0 (Figure 3). At acidic pH values the enzyme lost activity rapidly. The enzyme was almost 100% stable over the pH range 8.0 to 12.0 in the presence of 5 mM CaC12 for 60 min at 55°C.
100
Physico-chemical Properties of the Protease Crude enzyme solution was active from 45 to 75°C. It was stable up to 65°C for 30 min but lost 20% of its activity
60-
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0 g
320
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160 ~
4" "="
0-~ e-.e-
.~"
~,
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...o-.--,I~'
Figure
1.
I
I
1 I
20 28 Cultivation time (h)
Extracellular
protease
I
I
36
z,z,
production
(O)
==
80 ,~ k0
by
B.
stearothermophilus AP-4 in LB medium (pH 9.0) at 55°C r T ~ r o w t h ; O---culture pH.
34
I
I
t
I
I
I
I
I
5
6
7
8
9
10
~1
12
pH
2-¢ 1,s
=
I
4
World Journal of Microbiology & Biotechnology, 1/oi I0, 1994
Figure 3. Effect of pH on the activity and stability of protease from Bacillus sp. AP-4. Protease activity (C)) was measured by incubation in the appropriate buffer (20 mM acetate/NaOH buffer at pH 4 to 6; 10mM Tris/HCI buffer at pH 7 to 8; 20mM glycine/NaOH buffer at pH 9 to 10; and 20 mM KCI/NaOH buffer at pH 11 to 12) at 55°C for 30 min. For pH stability (F3), the enzyme solution was kept at 4°C for 1 h at each pH value and the residual activity was then measured at pH 9.0. The stability of the enzyme (V) in the presence of 5 mM CaCI2 at each pH value was measured in the same way, except 50 mM sodium phosphate buffer was used for pH 7 to 8 instead of Tris/HCI buffer to avoid any precipitation of Ca 2+ ion. 100% activity = 288 U/ml.
Thermophilic alkaline protease from Bacillus Table
1. Effect of inhibitors Bacillus sp. AP-4.
on the protease
activity of
Inhibitor
Activity (%)
None PMSF (10 mM)t PMSF (20 mM)t Iodoacetamide (10 mM) EDTA (10 mM)l" EDTA (20 mM)t Iodoacetic acid (5 mM)l" Mercaptoethanol (10 mM) DMSO (5 mM) SDS (0.1%) SDS (0.2%) Urea (5 M) Acetonitrile (2 mM) Acetonitrile (5 mM) H202 (2 raM)l" Aniline (5 raM) Ethylmethyl ketone (2 mM) Acetone (5 mM)
100" 23 19 21 10 4 45 3 98 63 47 42 100 92 37 47 86 84
* 100% activity = 288 U/ml. l-The reaction mixture was that described in Materials & Methods except 20 mM glycine/NaOH buffer, pH 9.0, was used to avoid the interaction of Tris base with the inhibitors.
Of various potential inhibitors of protease activity (Table 1), EDTA was particularly effective, indicating a requirement for metal ions for activation or stabilization (Sigma & Mooser 1975; North 1982). As enzyme activity was totally inhibited by the reducing agent, ~-mercaptoethanol, there might be a sulphydryl (--SH) group present at the active site. In addition, as the enzyme was completely inactivated by PMSF, it is probably a metal ion-dependent, serine protease.
References Anunstrup, K., Outtrup, H., Anderson, O. & Dambmann, C. 1972 Protease from alkalophilic Bacillus species. In Proceedings of the 4th International Symposium on Fermentation Technology, Osaka, Japan, pp. 299-305. Claus, D. & Berkeley, R.C.W. 1986 Genus Bacillus Cohn 1872. In Bergey's Manual of Systematic Bacteriology, Vol. 2, 9th edn, ed Sneath, P.H.A. pp. 1105-1141. Baltimore MD: Williams & Wilkins. Endo, S., 1962 Studies on protease by thermophilic bacteria. Hekko Kogaku Zasshi 40, 346--353 (in Japanese). Fujio, Y. & Kume, S., 1991 Characteristics of a highly thermostable neutral protease produced from Bacillus stearothermophilus. World Journal of Microbiology and Biotechnology 7, 12-16. Kubo, M., Maruyama, K., Seto, A. & Imanaka, T. 1988 Highly thermostable neutral protease from Bacillus stearothermophilus. Journal of Fermentation Technology 66, 13-17. North, J.M. 1982 Comparative biochemistry of proteinase of eukaryotic microorganisms. Microbiological Reviews 46, 308-340. Priest, F.G. 1977 Extracellular enzyme synthesis in the genus Bacillus. Bacteriological Reviews, 41, 711-753. Sigma, D. S. & Mooser, G. 1975 Chemical studies of enzyme active sites. Annual Review of Biochemistry 44, 889-931. Takami, H., Akiba, T. & Horikoshi, K. 1989 Production of extremely thermostable alkaline protease from Bacillus sp. no. AH 101. Applied Microbiology and Biotechnology 30, 120-124. Takii, Y., Kuriyama, N. & Suzuki, Y. 1990 Alkaline serine protease produced from citric acid by Bacillus atcalophilus sub-sp. halodurans KP 1239. Applied Microbiology and Biotechnology 34, 57-02. Takii, Y., Taguchi, H., Schimoto, H. & Suzuki, Y. 1987 Bacillus stearothermophilus KP 1236 neutral protease with strong thermostability comparable to thermolysin. Applied Microbiology and Biotechnology 27, 186-191.
(Received in revised form 7 ]une 1993; accepted 7 ]une 1993)
World]ournalof Microbiology& Biotechnology,Vot I0, 1994
3~
Production of a thermophilic, extracellular alkaline protease by Bacillus stearothermophilus AP-4 R. Dhandapani* and R. Vijayaragavan A thermophilic Bacillus stearothermophilus strain AP-4 excreting a thermostable alkaline protease, was isolated from a local compost. Maximum activity of protease (250 U/ml) was after 36 h growth in broth at pH 9.0 and at 55°C. The protease was optimally active at pH 9.0 and 55°C and was stable in 5 mM CaCl 2. The enzyme was completely inactivated by PMSF, EDTA and ]/-mercaptoethanol. It is therefore a metal ion-dependent, alkaline, serine protease.
Key words: alkaline protease, Bacillus, thermophilic. Proteases from thermophilic bacteria have considerable commercial potential, especially as components in laundry detergents, where their ability to function at high pH values and temperatures is exploited (Anunstrup et al. 1972). The proteases, produced by different Bacillus species, vary not only in type but also in the pH and temperature necessary for optimal activity (Priest 1977). There have been many reports on thermophilic proteases produced by different strains of Bacillus spp., including the thermolysin from B. thermoproteolyticus (Endo 1962), and an alkaline protease from Bacillus sp. no A H I 0 I (Takami et al. 1989) and B. alcalophitus (Takii et al. 1990). A thermostable neutral protease has been reported frequently from B. stearothermophilus (Takii et al. 1987; Kubo et al. 1988; Fujio & Kume 1991). During a study on the microbial flora of a compost waste on the university farm, we found a highly-thermostable, alkaline protease produced by Bacillus stearothermophilus strain AP-4. We describe, in this paper, some of the properties of this protease.
in 10 ml sterile water and incubated at 55°C for 3 h. One ml of this suspension was then inoculated into 9 ml of LB broth, [containing (g/l): Bacto-tryptone, 10; yeast extract, 5; NaC1, 10; and casein, 10; pH adjusted to 9.0 with 5 M NaOH] and incubated at 55°C for 24 h. After two such enrichments, 50 #1 of the inoculum was plated on each of several LB agar plates (3% w/v agar) and kept at 55°C for 24 h. Based on caseinolytic activity on LB agar plates, a single colony of Bacillus sp., designated AP-4, was isolated from 2000 colonies by repeated streaking on the same agar media at 55°C.
Growth of Isolate AP-4 To study protease secretion and growth of the organism, a 250-ml side-arm flask containing 100 ml of LB medium at pH 9.0, was inoculated with 106 cells (initial A620= 0.07) and incubated at 55°C. Growth was assessed turbidometricalIy at 620 nm; a value of A6z0 = 1 corresponded to 6 x 109 cells/ml. One ml of the sample was taken at various intervals, centrifuged and the supematant used for assays of proteolytic activity, with casein as the substrate (see below). The pH of the medium was measured aseptically every 4 h using an electronic pH meter. All the experiments were repeated at least three times.
Partial Purification of Protease
The strain AP-4 was isolated from a compost heap of the Tamil Nadu Agricultural University's farm by culture enrichment techniques. About 100 mg of the sample was initially suspended
Strain AP-4 was cultivated in LB media as described above and incubated at 55°C with shaking at 200 rev/min for 40 h. The cell-flee supematant recovered by centrifugation (8000 x g, 20 rain) at 4°C was treated with (NH4)2SO 4 to 80% saturation. The precipitate was recovered by centrifugation (13,000 x g, 20 rain), dissolved in 10 mM Tris/HC1 (pH 9.0) and dialysed against the same buffer for 24 h at 4°C. The dialysate was used as enzyme solution.
R. Dhandapani and R. Vijayaragavan are with the Centre for Plant Molecular Biology & Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641 003, India; fax: 091-0422-41672. *Corresponding author.
The enzyme reaction was carried out in a mixture containing 250 lal 2% (w/v)/J-casein (Sigma), 500/A 10 mM Tris/HC1 buffer, pH 9.0,
Materials and Methods Isolation of the Bacterium
Protease Assay
(~ 1994 Rapid Communications of Oxford Ltd World Journal of Microbiology & Biotechnology, Vol 10, 1994
33
R. Dhandapani and R. Vijayaragavan and 250/~l of the sample. After incubation for 30 min at 55°C, 500 #l of TCA solution (0.II M trichloroacetic acid, 0.22 M sodium acetate and 0.33 M acetic acid) was added to stop the reaction and the liberated tyrosine was assayed by absorbance at 275 nm against a standard tyrosine solution. One unit of protease activity was defined as the amount of enzyme required to produce I/~g tyrosine per min under the given conditions. Protein concentration was determined by the Lowry method, using BSA as the standard.
10C
o~o
-
.>
50 o
._>
0
Results and Discussion
I 20
I
40
30
50
60
70
80
90
Temperature (°C)
Characteristics of the Isolate Strain AP-4 was a Gram-positive, aerobic, catalase and oxidase negative, motile, terminal endospore-forming bacterium. It was an obligate thermophile (growing only at 45 to 65°C), forming long rods, often forming short chains after 21 h at 55°C. The culture grew exceptionally fast on LB agar plate at 55°C, covering the entire Petri dish (90-mm diam.) in 6 to 8h. The isolates did not grow at all at 28 -I- 5°C or in the media containing 7% or 10% (w/v) NaC1. However, the culture remained viable after storage at room temperature for 60 days. The strain hydrolysed casein, gelatin, bovine serum albumin and haemoglobin. It also weakly hydrolysed starch. This strain was identified as Bacillus stearothermophilus AP-4 (Claus & Berkeley 1986). The fatty acid profile of this microbes confirmed its species identification. The culture has been deposited in the Institute of Microbial Technology, Chandigarh, India.
Protease Production Maximum enzyme production occurred when Bacillus sp. AP-4 was grown at 55°C in LB medium with an initial pH of 9.0 (Figure 1). Protease production was growth associated and maximum activity was after 36 h.
Figure 2. Effect of temperature on the activity and stability of protease from Bacillus sp. AP-4. Activity (I-1) was determined at
different temperatures with an incubation of 30 min. The thermal stability of the enzyme in the presence of 5 mM CaCI2 (~) in 20 mM glycine/NaOH buffer, pH 9.0, or in the absence of CaCI2 (O) in 10 mM Tris/HCI buffer, pH 9.0, was determined by holding the enzymes for 30 min, at the desired temperature, then determining the residual activity by incubation at 55°C for 30 min. 100% activity = 288 U/ml.
after 30 min at 70°C. The enzyme did not lose activity when held for up to 10 days at 4°C. The protease was stable and active in CaC12 (Figure 2); optimum activity with 5 mM Ca2 + was at 65°C. The protease was active at pH 9.0 to 11.0, with optimum activity at pH 9.0 (Figure 3). At acidic pH values the enzyme lost activity rapidly. The enzyme was almost 100% stable over the pH range 8.0 to 12.0 in the presence of 5 mM CaC12 for 60 min at 55°C.
100
Physico-chemical Properties of the Protease Crude enzyme solution was active from 45 to 75°C. It was stable up to 65°C for 30 min but lost 20% of its activity
60-
"~
20-
d
"t
11 '
>
111
13C
0 g
320
0.8
160 ~
4" "="
0-~ e-.e-
.~"
~,
12
...o-.--,I~'
Figure
1.
I
I
1 I
20 28 Cultivation time (h)
Extracellular
protease
I
I
36
z,z,
production
(O)
==
80 ,~ k0
by
B.
stearothermophilus AP-4 in LB medium (pH 9.0) at 55°C r T ~ r o w t h ; O---culture pH.
34
I
I
t
I
I
I
I
I
5
6
7
8
9
10
~1
12
pH
2-¢ 1,s
=
I
4
World Journal of Microbiology & Biotechnology, 1/oi I0, 1994
Figure 3. Effect of pH on the activity and stability of protease from Bacillus sp. AP-4. Protease activity (C)) was measured by incubation in the appropriate buffer (20 mM acetate/NaOH buffer at pH 4 to 6; 10mM Tris/HCI buffer at pH 7 to 8; 20mM glycine/NaOH buffer at pH 9 to 10; and 20 mM KCI/NaOH buffer at pH 11 to 12) at 55°C for 30 min. For pH stability (F3), the enzyme solution was kept at 4°C for 1 h at each pH value and the residual activity was then measured at pH 9.0. The stability of the enzyme (V) in the presence of 5 mM CaCI2 at each pH value was measured in the same way, except 50 mM sodium phosphate buffer was used for pH 7 to 8 instead of Tris/HCI buffer to avoid any precipitation of Ca 2+ ion. 100% activity = 288 U/ml.
Thermophilic alkaline protease from Bacillus Table
1. Effect of inhibitors Bacillus sp. AP-4.
on the protease
activity of
Inhibitor
Activity (%)
None PMSF (10 mM)t PMSF (20 mM)t Iodoacetamide (10 mM) EDTA (10 mM)l" EDTA (20 mM)t Iodoacetic acid (5 mM)l" Mercaptoethanol (10 mM) DMSO (5 mM) SDS (0.1%) SDS (0.2%) Urea (5 M) Acetonitrile (2 mM) Acetonitrile (5 mM) H202 (2 raM)l" Aniline (5 raM) Ethylmethyl ketone (2 mM) Acetone (5 mM)
100" 23 19 21 10 4 45 3 98 63 47 42 100 92 37 47 86 84
* 100% activity = 288 U/ml. l-The reaction mixture was that described in Materials & Methods except 20 mM glycine/NaOH buffer, pH 9.0, was used to avoid the interaction of Tris base with the inhibitors.
Of various potential inhibitors of protease activity (Table 1), EDTA was particularly effective, indicating a requirement for metal ions for activation or stabilization (Sigma & Mooser 1975; North 1982). As enzyme activity was totally inhibited by the reducing agent, ~-mercaptoethanol, there might be a sulphydryl (--SH) group present at the active site. In addition, as the enzyme was completely inactivated by PMSF, it is probably a metal ion-dependent, serine protease.
References Anunstrup, K., Outtrup, H., Anderson, O. & Dambmann, C. 1972 Protease from alkalophilic Bacillus species. In Proceedings of the 4th International Symposium on Fermentation Technology, Osaka, Japan, pp. 299-305. Claus, D. & Berkeley, R.C.W. 1986 Genus Bacillus Cohn 1872. In Bergey's Manual of Systematic Bacteriology, Vol. 2, 9th edn, ed Sneath, P.H.A. pp. 1105-1141. Baltimore MD: Williams & Wilkins. Endo, S., 1962 Studies on protease by thermophilic bacteria. Hekko Kogaku Zasshi 40, 346--353 (in Japanese). Fujio, Y. & Kume, S., 1991 Characteristics of a highly thermostable neutral protease produced from Bacillus stearothermophilus. World Journal of Microbiology and Biotechnology 7, 12-16. Kubo, M., Maruyama, K., Seto, A. & Imanaka, T. 1988 Highly thermostable neutral protease from Bacillus stearothermophilus. Journal of Fermentation Technology 66, 13-17. North, J.M. 1982 Comparative biochemistry of proteinase of eukaryotic microorganisms. Microbiological Reviews 46, 308-340. Priest, F.G. 1977 Extracellular enzyme synthesis in the genus Bacillus. Bacteriological Reviews, 41, 711-753. Sigma, D. S. & Mooser, G. 1975 Chemical studies of enzyme active sites. Annual Review of Biochemistry 44, 889-931. Takami, H., Akiba, T. & Horikoshi, K. 1989 Production of extremely thermostable alkaline protease from Bacillus sp. no. AH 101. Applied Microbiology and Biotechnology 30, 120-124. Takii, Y., Kuriyama, N. & Suzuki, Y. 1990 Alkaline serine protease produced from citric acid by Bacillus atcalophilus sub-sp. halodurans KP 1239. Applied Microbiology and Biotechnology 34, 57-02. Takii, Y., Taguchi, H., Schimoto, H. & Suzuki, Y. 1987 Bacillus stearothermophilus KP 1236 neutral protease with strong thermostability comparable to thermolysin. Applied Microbiology and Biotechnology 27, 186-191.
(Received in revised form 7 ]une 1993; accepted 7 ]une 1993)
World]ournalof Microbiology& Biotechnology,Vot I0, 1994
3~
