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Production of alkaline protease from Streptomyces gulbargensis and its

application in removal of blood stains

Article  in  Indian Journal of Biotechnology · August 2009

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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 “Green Chemicals”, are proving extremely useful in

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
______________
*Author for correspondence:
Tel: 91 8472-263297; Fax: 91 8472-263206
E-mail: lingappak_micro@rediffmail.com
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
 VISHALAKSHI et al: BLOOD STAIN REMOVAL BY ALKALINE PROTEASE OF S. GULBARGENSIS
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
281
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
Tray with distilled water (100 mL) + blood stained
cloth + 1 mL of commercial detergent (7 mg/mL)
Tray with distilled water (100 mL) + blood stained
cloth + 1 mL of commercial detergent (7 mg/mL) +
partially purified enzyme
Tray with distilled water (100 mL) + blood stained
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.
282 INDIAN J BIOTECHNOL, JULY 2009

Comparative Evaluation of Washing Performance of Enzyme Effect of Temperature

Detergent with Commercial detergents The results of the studies on the effect of the
Washing performance of the enzyme with detergent temperature on enzyme activity are presented in
was compared with that of commercial branded Fig. 4. The temperature kinetics of the enzyme
detergents like Ariel, Henko, Nirma, Rin Advance, suggests that the enzyme activity increased sharply
Surf Excel and Tide9 . from 0 to 45°C, followed by a sudden decline
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 Fig. 2—Protease production under solid state condition using
wheat bran.
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.

Fig. 3—pH profile of alkaline protease

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

(b) Proteolytic activity of S. gulbargensis Fig. 4—Temperature profile of alkaline protease at pH 9.0
VISHALAKSHI et al: BLOOD STAIN REMOVAL BY ALKALINE PROTEASE OF S. GULBARGENSIS 283

Fig. 5—Evaluation of crude enzyme for washing of blood stains from cloths.
284 INDIAN J BIOTECHNOL, JULY 2009

thereafter. The study indicated that the enzyme from presented in Fig. 6. After an incubation of 20 min,
S. gulbargensis was maximally active at 45°C. stains could not be removed completely with
detergent alone, while the combination of the enzyme
Evaluation of Enzyme for Washing Performance
with commercial detergent removed the blood stains
Alkaline proteases are employed primarily as
from the surgical instruments very effectively.
cleansing additives. The results (Fig. 5) of evaluation
of enzyme for washing performance revealed that the Comparative Evaluation of Washing Performance of Enzyme
blood stains on the cloth pieces remained as it is even Detergent with Commercial Detergents
after 30 min of rinsing in the case of controls and Of all the branded detergents, Ariel and Tide
commercial detergents. Blood stains were completely removed blood stains after 20 min of rinsing which is
removed from the cloths after rinsing them with a at par with the commercial detergents supplemented
combination of detergent and partially purified with partially purified enzyme (Fig. 7).
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

Fig. 6—Evaluation of washing performance of the Fig. 7—Comparative evaluation of washing performance of the
enzyme+detergent on surgical instruments. enzyme detergent with commercial detergents on cloths.
 VISHALAKSHI et al: BLOOD STAIN REMOVAL BY ALKALINE PROTEASE OF S. GULBARGENSIS
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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