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.