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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, P-

CMB, 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

Effect of pH on the activity of purified alkaline protease

0

20

40

60

80

100

120

7 8 9 10 11 12

pH

Relative alkaline protase activity

(%)

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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

Effect of pH on the stability of purified alkaline protease

0

20

40

60

80

100

120

5 6 7 8 9 10 11 12

pH

Relative alkaline protase activity (%)

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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

Effect of temperature on the activity of purified alkaline protease

Temperature0c

0

20

40

60

80

100

120

30 40 50 60 70

Relative alkaline protease activity (%)

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Table XXVI

Effective of temperature on the stability of enzyme activity

Temperature 0C

Relative alkaline protease activity (%) with CaCl2 without CaCl2

30

100 100

40

100

90

50

100

85

60

60

50

70

30

20

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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

Idoacetic acid

90

EDTA

(Ethelene Diamine tetra acetic acid) 93

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Fig 24

Effect of inhibitors on the activity of purified alkaline protease

0

20

40

60

80

100

120

Control PMSF PCMB Idoaceticacid

EDTA

Inhibitors

Relative alkaline protease activity (%)

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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

Effect of substrates on the activity of purified alkaline protease

0

20

40

60

80

100

120

Casein Bovine serumalbumin

Egg albumin Gelatin

Substrates

Relative alkaline protease activity (%)

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Plate-2

A B C 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

Detergent

(%) 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

Effect of detergents on the stability of purified alkaline protease

0

20

40

60

80

100

120

0 0.5 1 1.5 2 2.5 3

Time (min)

(%)Retention of enzyme activity

Control

Wheel

Arieal

Surfexcel

Rin

Nirma

- 153 -

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 Michalis-

Menten 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)