Chapter a STUDIES ON SUITABILITY OF

PROTEASES OF BACILLUS SP. K 25 AND BACILLUS PWLCUS K 242

ASDETERGENTENZYMES

~ t h the objective of determining the suitability of proteases of Bacillus sp.

K 25 and Bacilluspumilus K 242 for use in commercial detergents, their w stability in presence of detergents and the ability to a d on insoluble substrate

in presence of detergents were studied. (The ability of proteases to a d on the

insoluble substrate is an indired measure of their ability to act on the

proteinaceous stains which are also insoluble in nature.) For a comparative

evaluation the predominantly used detergent protease, the protease of

Bacillus lichenifomis was also included in the study. Experiments were done

in triplicate.

MATERIALS AND METHODS

Stability in presence of commercial detergents

The proteases of Bacilfus sp. K 25 and Baci//us purniius K 242 were

produced providing the optimum conditions. For obtaining protease of

Bacillus lichenifomis, the strain NCIM 2042, procured from National

Collection of Industrial Microorganisms, NCL, Pune was cultured in peptone

yeast extract (PYE) medium as described under section A of Chapter 3. The

protease of Bacillus sp. K 25 obtained by ammonium sulphate precipitation at

75% saturation and those of Baciilus pumilus K 242 and Bacillus lichenifomis

obtained by ammonium sulphate precipitation at 80% saturation, were

dissolved in 0.02 M potassium phosphate buffer pH 7.5) and dialyzed against

the same buffer overnight. The dialysates were used as the partially purified

enzymes for the tests.

The enzymes were incubated with 1% solutions of commercial

detergents viz. Surf, White giant, Shudh, Ariel super soaker and Wheel,

at 45°C for 30 min. Enzyme activities were determined before and after

incubation.

Ability of proteases to act on insoluble substrate in presence of commercial detergents

The ability of proteases to act on insoluble substrate in presence of

commercial detergents was studied using blue casein-PAG, the protease

substrate obtained by the immobilization of dyed casein in the structure of

polyacrylamide gel.

Proteases and detergent solutions were prepared as described under

previous experiment.

Blue casein-PAG was prepared according to the method of Safarik

(1988). The preparation was as follows. Casein (10 g) was dissolved under

mixing and heating in 300 ml of 0.05 M trisodium orthophosphate solution.

After complete solubilization, the temperature of casein solution was adjusted

to 40-45°C (this temperature was maintained through out all operations) and

a freshly prepared solution of dye (5 g of Ostazin blue S-2G in 60 ml of water)

was added. After mixing for 10 min 20 g of sodium chloride was added. After

20 min of mixing, 5.5 g of sodium carbonate decahydrate was added and the

mixing was continued for another 60 min. After that the solution was allowed

to stand at ambient temperature overnight. The dyed casein was precipitated

from the solution by 10% hydrochloric acid. The precipitate was filtered out,

washed with water, acidified with hydrochloric acid to pH 4.5 and then

dissolved in 1% sodium hydroxide. The precipitation, washing and

dissolution of dyed casein was repeated four times. After the last precipitation

the dyed casein was washed with ethanol and acetone and dried at ambient

temperature. 5 g of dyed casein and 7 g of Na2HPO4.12 H20 were dissolved

in 70 ml of water and then 28.8 g of actylamide and 1.2 g of N,N'-methylene-

bis-aaylamide were added. After dissolution, the solution was deaerated

under vacuum and then 1.5 ml of 10% ammonium persulfate solution and

150 pl of TEMED were added. The volume of solution was adjusted to 100 rnl

with water. When the polyacrylamide gel had completely polymerized, it was

cut into smaller particles and homogenized in a knife homogenizer. The fine

particles of insoluble substrate were thoroughly washed with water until the

washings became colourless. Substrate was then washed with ethanol and

acetone, dried at ambient temperature and homogenized to obtain fine

particles of blue casein-PAG.

The assay was performed as follows. 2 ml of blue casein-PAG

suspension in water (35 mg ml-') previously swelled for 30 min at room

temperature was mixed with equal volume of detergent solution containing

proteases (0.05 u ml-I). The mixture was shaken vigorously and the enzyme

was allowed to act on blue casein-PAG for 30 min at 40°C. After thorough

mixing the reaction was stopped by filtration of the reaction mixture through a

paper filter. The filtrate was centrifuged and the absorbance of supernatant

was measured at 620 nm in a Shimadzu UV-visible spectrophotometer

W-1601 (path length: 1 an). The controls were containing heat inactivated

(100°C x 10 min) proteases in place of active proteases.

RESULTS

Results of the studies on stability of proteases of Bacillus sp. K 25,

Bacillus pumilus K 242 and Bacillus lichenifomis NCIM 2042, in commercial

detergents are shown in Table 30. Residual activities (%) of the proteases

after incubation with different commercial detergents are given.

Table 30 Stability of proteases of Bau'IIus sp. K 25, BacilluspumiIus K 242 and

Badus lichenifonnis NCIM 2042 in commercial detergents

Residual activities (%) of the proteases of different bacteria after incubation with different detergents

Detergent Bacilus sp. B. pumilus B. licheniformis K 25 K 242 NClM 2042

Surf 78 82 85

White giant 93 82 77

Shudh 91 88 88

Ariel Super soaker 79 66 75

Wheel 74 90 85

The residual activities of the proteases of Bacillus sp. K 25 and Bacillus

pumilus K 242 after incubation with different detergent solutions were in the

ranges 7493% and 66-90% respectively. The residual activities shown by the

protease of Bacillus lichenifonnis NCIM 2042 after incubation under similar

conditions were ranging from 75 to 88%.

The activities of the proteases on blue casein-PAG in presence of

different detergents are shown in Table 31. Absorbances (A620) of the filtrates

of reaction mixtures resulted due to the cleavage of blue casein-PAG are

given.

Table 31

Activity of proteases of Bacillussp. K 25, Baciluspumilus K 242 and Bacillus lichenifomis NCIM 2042 on blue casein-PAG

in presence of different commercial detergents

Absorbances (bO + SEM) of the filtrates of reaction mixtures due to the activity on blue casein-PAG by

Detergent the proteases of different bacteria BaciUus sp. B. pumilus 5. Lichenifomis

K 25 K 242 NCIM 2042

Surf 1.220 1.992 0.928 k 0.026 + 0.091 k 0.022

White giant 1.239 1.093 0.718 + 0.061 + 0.042 + 0.027

Shudh 1.376 1.684 1.057 + 0.055 + 0.093 + 0.030

Ariel super soaker 0.651 0.564 0.884 + 0.024 + 0.038 + 0.048

Wheel 0.798 1.059 0.785 + 0.029 + 0.042 _+ 0.016

In the presence of Surf, Shudh and Wheel, protease of Bacilluspumilus

K 242 was better than the other two proteases in cleaving blue casein-PAG.

In the presence of White giant, protease of Bacihssp. K 25 was the best. The

highest activity on blue casein-PAG in presence of Ariel super soaker was

shown by the protease of 5. Iichenifomis NClM 2042. In general the

proteases of Bacillus sp. K 25 and Baciluspumilus K 242 could be observed

to be better than the protease of B. lichenifomis NCIM 2042 in cleaving the

insoluble substrate.

DISCUSSION

Results of studies on the properties of proteases of Bacihs sp. K 25 and

Bacillus pumilus K 242 were indicative of their possible usefulness as

detergent enzymes. Taking this into consideration some other properties of

the enzymes, such as the stability in detergents and the activity on the

insoluble substrates in presence of detergents were studied. The activity on

the insoluble substrate was studied, since it could be taken as a measure of

ability of proteases to a d on proteinaceous stains which are insoluble in

nature. For a comparative evaluation of results, protease of Bacillus

/ichenifomis, the predominantly used detergent protease, was also included in

the study. The studies were canied out with the partially purified enzymes.

Commercial detergents used in this study were selected only after confirming

the absence of proteolytic enzymes in them. Those enzymes not containing

proteolytic enzymes viz. Surf, White giant, Shudh, Ariel super soaker and

Wheel were used in the study.

In detergent powders proteases are generally used in the granulated

form. They are incorporated into the inner core of granules. The core also

contains inorganic salts, sugars and reinforcing fibres of carboxymethyl

cellulose which are protedive in nature and is coated with an inert waxy

material made from paraffin oil or polyethylene glycol plus various hydrophilic

binders. So the proteases in the granules can be supposed to be stable.

But as the detergent powders are dissolved in water the waxy protective

coating dissolves to release the proteases. In the detergent solution they are

vulnerable to the action of many deleterious agents. So the stability in

presence of detergents is an important factor determining the performance of

proteases during the time of washing especially when washing is performed at

an elevated temperature. So in this study the stability of the proteases in

presence of detergents was examined at 45°C. There was no considerable

difference in the stability shown by the proteases of Bacillus sp. K 25, Bacillus

pumi/us K 242 and aaci//us kchenifomis NCIM 2042, in the presence of four

out of the five detergents tested. The residual activities of the proteases of

Bacil/us sp. K 25 and Bacillus pumilus K 242 after incubation in the different

detergent solutions were in the ranges 74-93% and 66-90% respectively.

Under similar conditions, the residual activities shown by the protease of

Bacillus Iichenifotmis NCIM 2042 were ranging from 7588%. All these

proteases can be expected to be more stable in the detergents specially

formulated with the intention of incorporating proteases.

The ability of proteases to remove proteinaceous stains m be

measured either by performing wash tests (Durham, 1987) or by testing their

ability to a d on insoluble subsh-ates under conditions mimicking the wash

conditions (Safarik and Safarikova, 1994). Since the wash tests are laborious

and time consuming, it is better to study the ability to ad on proteinaceous

stains, by determining their activity on insoluble substrates.

In a typical washing process the reaction system leading to the removal

of proteinaceous stains is a heterogenous one. The protease being soluble and

substrate for it (proteinaceous stain) being insoluble, the kinetics of the

reaction will be different from the kinetics of reaction where both the enzyme

and the substrate are soluble. So,for the evaluation of ability of proteases to

a d on proteinaceous stains under wash conditions their activity on insoluble

substrate in presence of detergents had to be studied. In this study the

activities of the proteases of Bacillus sp. K 25, Bacillus pumilus K 242 and

Baa'I/us kchenifomis NCIM 2042 on insoluble substrate in presence of

different commercial detergents were assayed using blue casein-PAG, a

substrate obtained by the immobilization of dyed casein in the sixudure of

polyacylamide gel. The proteases were tested in five different commercial

detergents viz. Surf, White giant, Shudh, Ariel super soaker and Wheel. In the

presence of Surf, Shudh and Wheel the protease of Bacillus pumilus K 242

was the best among the three proteases in cleaving blue casein-PAG. In the

presence of white giant, the protease of Bacillus sp. K 2 5 was the best. The

highest activity in presence of Ariel was shown by the protease of Bacillus

lichenifomis NCIM 2042. Since the general performance of the protease of

Bacillus pumilus K 242 was better than the protease from the other two

sources, it can be suggested as the most suitable one for the use in detergents.

Protease of Bacillus sp. K 25 also can be suggested to be utilizable as

detergent protease because, its ability to act on the insoluble substrate was

generally better than that of the protease of B. lichenifomisNCIM 2042.

Since the ability of proteases to cleave blue casein-PAG can be taken

as an indirect measure of their ability to a d on proteinaceous stains, these

proteases can be presumed to be performing better than the protease of

Bacillus lichenifomis during the wash process also. However before

recommending these enzymes for application in detergent industry, their

performances under field trials also have to be evaluated.