production of alkaline protease from streptomyces...

Indian Journal of Biotechnology

Vol 8, July 2009, pp 280-285

Production of alkaline protease from Streptomyces gulbargensis and its

application in removal of blood stains

N Vishalakshi, K Lingappa*, S Amena, M Prabhakar and A Dayanand

Department of Microbiology, Gulbarga University Gulbarga 585 106, India

Received 1 August 2008; revised 12 January 2009; accepted 15 March 2009

The alkaline protease obtained from a newly isolated strain of Streptomyces gulbargensis was used for the washing of

surgical instruments. The isolate showed β-haemolysis. Therefore, the isolate was employed for the production of thermo

stable alkaline protease enzyme using wheat bran as the substrate under solid state fermentation. The characterization

studies of the enzyme showed that it is active at 45°C and pH 9.0 with casein as the substrate. The wash performance

analysis of blood stains on cotton fabrics and on surgical instruments showed an increase in the reflectance as the time

increased with the enzyme treatment. The removal of blood stains completely was observed at 20 min incubation of cotton

cloths and surgical instruments.

Keywords: Alkaline protease, blood stains, surgical instruments, S. gulbargensis, wheat bran

Introduction Surgery is a complex process performed by

employing various surgical instruments. During

surgery, the surgical instruments invariably come in

contact with blood of the patients. If such instruments

are not properly washed, it leads to contamination and

foul smelling due to microbial degradation of blood

and finally paving way to transmission of diseases to

other patients and health care personnel. Hence, the

surgical instruments and other medical devices, for

reuse, must be cleaned with proper solutions.

Cleaning not only avoids the transmission of diseases,

but also forms an important aspect for the

maintenance of hygiene and safety of surgical

instruments.

Usually the surgical instruments are washed or

cleaned by sterilization or by using chemical

steriliants. However, chemical steriliants can not

remove microbes that usually get trapped behind the

bioburden that is encrusted on or within surgical

instruments. Therefore, this has spurred us to expand

our efforts to identify a product that can be employed

for the cleaning of surgical instruments. However, the

recent cleaning technologies include enzyme-

containing formulations and zeolite based detergents.

Of these, the enzyme detergents often referred to as

“Green Chemicals”, are proving extremely useful in

keeping a check on the environmental pollution1.

Addition of alkaline protease to detergents

considerably increases (35-40%) the cleaning effect

(particularly in removing stains containing proteins,

e.g., blood, cocoa, milk, eggs, and sauces) and

increases the consumption of surface-active

substances, thereby improving the ecological

situation.

Out of the vast pool of enzymes, proteolytic

enzymes from microorganisms are the most widely

exploited enzymes in the detergent industries world

wide2,3

. Proteases are present in all living beings and

play an important role in normal and abnormal

physiological conditions. Looking into the depth of

microbial diversity, there is always a chance of

finding microorganisms producing alkaline enzymes,

which are suitable for the manufacture of

“biocleaners”. Therefore, an attempt has been made to

produce alkaline proteases, which can hydrolyse

specifically haem as the substrate by employing a

novel strain of Streptomyces gulbargensis. The

enzyme can be used as an ingredient in the

preparation of biocleaning agents especially for

washing surgical instruments.

Materials and Methods Microorganism and Growth Conditions

Streptomyces gulbargensis sp. nov., was isolated

and identified by 16S rRNA gene sequence and

______________

*Author for correspondence:

Tel: 91 8472-263297; Fax: 91 8472-263206

E-mail: [email protected]

VISHALAKSHI et al: BLOOD STAIN REMOVAL BY ALKALINE PROTEASE OF S. GULBARGENSIS

281

deposited in GenBank database under the accession

number DQ3174114. The type culture/strain (DAS

131) was deposited at =CCTCC Ac No 206001;

=KCTC Ac No 19179. The organism was

collected from the Department of Microbiology,

Gulbarga University and then studied for its

ability to grow on skim milk agar (SMA) (HiMedia,

Mumbai) medium and on blood agar plates, incubated

at 37°C to determine potential protease enzyme5

and

its caseinolytic and heamolytic activites. The strain

was preserved at 4°C on starch casein agar (SCA)

slants. The isolate was maintained as a spore

suspension6.

Fermentation Studies

The production of proteases was carried out by

using 25 g of wheat bran as the substrate under solid

state fermentation. The contents of the flask were

inoculated with 1 mL of inoculum (1×108 spores/mL)

after autoclaving. The contents were mixed

thoroughly by gently beating the flasks on the palm of

the hand and incubated in a slanting position at 37°C

for 7 d. After incubation, the substrates were analyzed

for the production of proteases as described by

Raimbult et al7.

Extraction and Assay of Enzyme Activity

After the fermentation, culture mass of the solid

medium was extracted with 1:10 volumes of distilled

water with shaking at 160 rpm (Remi) for 1 h at

25-30°C, centrifuged and supernatant was used as the

crude enzyme extract for the assay. Protease activity

of the culture mass extract was determined by

modified method of Anson8. The reaction mixture

contained 1 mL of 1% casein in an M 0.1 NaH2PO4

phosphate buffer (pH 7.5) and 1 mL of the crude

enzyme solution. It was incubated at 37°C for 20 min

and the reaction was stopped by adding 3 mL of 10%

trichloroacetic acid. The absorbance of the liberated

tyrosine in the filtrate was measured at 280 nm using

a UV-Visible spectrophotometer (GENESYS 10 UV)

against a standard tyrosine solution. One unit of

protease activity was defined as the amount of

enzyme which releases 1 µM of tyrosine per min

under assay conditions.

Ammonium Sulfate Precipitation

Salt precipitation was carried out using 100 mL of

the crude enzyme extract upto 70%. All the

subsequent steps were carried out at 4°C. The

resulting precipitate was collected by centrifugation at

10,000 rpm for 10 min at 4°C. The precipitate was

dissolved in 50 mM Tris-HCl (pH 8.5) for further use.

Characterization of Enzyme Activity

Effect of pH

Effect of pH on the stability of protease was studied

as per the method of Adinarayana et al9 by incubating

the enzyme with buffers covering the range of

4.0-10.0, different buffers (0.1 M) used were

potassium phosphate (pH 4.0-7.5), Tris-HCl (pH 8.0-

8.5) and sodium bicarbonate (pH 9.0-10.0). Enzyme

samples were added to different buffers and incubated

at 45°C for 20 min and assay was carried out as

described by Adinarayana et al9.

Effect of Temperature

The enzyme samples were incubated at various

temperatures ranging from 0-80°C, at pH 9.0. After

20 min, the assay was carried out as described by

Adinarayana et al9.

Evaluation of Washing Performance of Enzyme

Application of protease (5000 µ/mL) as a detergent

additive was studied on white cotton cloth pieces

(15×15 cm) stained with blood9,10

. The stained cloth

pieces were taken in separate trays. The following sets

were prepared and studied:

Tray with distilled water (100 mL) + blood stained

cloth


cloth + 1 mL of commercial detergent (7 mg/mL)


cloth + 1 mL of commercial detergent (7 mg/mL) +

partially purified enzyme


cloth + partially purified enzyme.

The above trays were incubated at 40°C for 25 min.

The cloth pieces were taken out from each set at

regular intervals of 5 min, rinsed with water, dried

and visually examined. Untreated cloth pieces stained

with blood were taken as control. Evaluation of Washing Performance of Enzyme with

Detergent on Surgical Instruments

Various surgical instruments stained with blood

were taken and subjected to washing performance for

20 min at 40°C by commercial detergents and the

enzyme7. Visual observation was made for the extent

of blood stain removal from the instruments after

20 min.

INDIAN J BIOTECHNOL, JULY 2009

282

Comparative Evaluation of Washing Performance of Enzyme

Detergent with Commercial detergents

Washing performance of the enzyme with detergent

was compared with that of commercial branded

detergents like Ariel, Henko, Nirma, Rin Advance,

Surf Excel and Tide9 .

Results and Discussion

The strain of S. gulbargensis was selected for the

production of protease on the basis of formation of

clear zone near the vicinity of the colony, visible to

naked eyes (Figs 1a & b). The fermentation studies

indicated that the enzyme production was maximum

at 120 h of fermentation (Fig. 2). The enzyme was

extracted from fermented bran and partially purified

by 70% ammonium sulphate and used for

further studies.

Enzyme Activity

Effect of pH

The results on the effect of pH on the enzyme

activity are presented in Fig. 3. The pH kinetics of the

enzyme activity revealed that the activity increased

from pH 5 to 10. The optimum pH recorded was

9.0 for maximum activity.

Effect of Temperature

The results of the studies on the effect of the

temperature on enzyme activity are presented in

Fig. 4. The temperature kinetics of the enzyme

suggests that the enzyme activity increased sharply

from 0 to 45°C, followed by a sudden decline

Fig. 1—(a) β-haemolysis shown by S. gulbargensis;

(b) Proteolytic activity of S. gulbargensis

Fig. 2—Protease production under solid state condition using

wheat bran.

Fig. 3—pH profile of alkaline protease

Fig. 4—Temperature profile of alkaline protease at pH 9.0


283

Fig. 5—Evaluation of crude enzyme for washing of blood stains from cloths.

INDIAN J BIOTECHNOL, JULY 2009

284

thereafter. The study indicated that the enzyme from

S. gulbargensis was maximally active at 45°C.

Evaluation of Enzyme for Washing Performance

Alkaline proteases are employed primarily as

cleansing additives. The results (Fig. 5) of evaluation

of enzyme for washing performance revealed that the

blood stains on the cloth pieces remained as it is even

after 30 min of rinsing in the case of controls and

commercial detergents. Blood stains were completely

removed from the cloths after rinsing them with a

combination of detergent and partially purified

enzyme for a period of 20 min, whereas it was

removed after 25 min when rinsed with partially

purified enzyme alone. These results clearly indicated

that the enzyme is stable as an ingredient in the

presence of detergents.

Evaluation of Washing Performance of Enzyme Detergent on

Surgical Instruments

The evaluation of the washing performance of the

enzyme detergent on the surgical instruments is

presented in Fig. 6. After an incubation of 20 min,

stains could not be removed completely with

detergent alone, while the combination of the enzyme

with commercial detergent removed the blood stains

from the surgical instruments very effectively.

Comparative Evaluation of Washing Performance of Enzyme

Detergent with Commercial Detergents

Of all the branded detergents, Ariel and Tide

removed blood stains after 20 min of rinsing which is

at par with the commercial detergents supplemented

with partially purified enzyme (Fig. 7).

Fig. 6—Evaluation of washing performance of the

enzyme+detergent on surgical instruments.

Fig. 7—Comparative evaluation of washing performance of the

enzyme detergent with commercial detergents on cloths.


285

The enzyme obtained in the present study is less

expensive as it is produced on the agricultural wastes,

however, its economics for commercial exploitation

has to be worked out. The enzyme is stable at alkaline

pH. In the present study, the enzyme showed

promising results in the removal of blood stains from

cloths and surgical instruments and indicated that it is

substrate specific to heam. Therefore, these properties

of the enzyme indicated the possibilities of its

use in the manufacture of surgical cleaning

detergent industry.

Acknowledgement

The authors would like to thank The Chairman,

Department of Microbiology, Gulbarga University,

Gulbarga, for kindly providing the strain

Streptomyces gulbargensis and laboratory facilities.

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production of alkaline protease from streptomyces...

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