Characterization of thermo- and detergent stable serine protease from isolated Bacillus circulans and evaluation of eco-friendly applications

Characterization of thermo- and detergent stable serine protease from isolatedBacillus circulans and evaluation of eco-friendly applications

Ch. Subba Rao, T. Sathish, P. Ravichandra, R.S. Prakasham *

Bioengineering and Environmental Centre, Indian Institute of Chemical Technology, Hyderabad 500607, India

Process Biochemistry 44 (2009) 262–268

A R T I C L E I N F O

Article history:

Received 13 May 2008

Received in revised form 17 September 2008

Accepted 21 October 2008

Keywords:

Alkaline protease

B. circulans

Detergent

Enzyme

Leather processing

Thermal kinetics

Serine protease

A B S T R A C T

Alkaline protease from Bacillus circulans has been purified and characterized in detail for its robustness

and its eco-friendly application potential at leather processing and detergent industries. The molecular

weight of the purified enzyme was estimated to be 39.5 kDa on SDS-PAGE. It exhibited optimum activity

at broad temperature range and maximum at 70 8C under alkaline pH environment, in the presence of

surfactants and oxidizing agents. It has revealed stain removal property and dehairing activity for animal

hide without chemical assistance and without hydrolyzing fibrous proteins. This enzyme showed

application potential in leather processing industry for production of better quality product in eco-

friendly process. In addition, the stability (pH, temperature and surfactants) and hydrolysis of blood stain

data also revealed its application in detergent industries.

� 2008 Published by Elsevier Ltd.

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Process Biochemistry

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1. Introduction

Proteases constitute one of the most important groups ofenzymes both industrially and academically. Their annual salesaccounts 60% of the total world enzyme market and estimated toreach 220 billion US$ by the year 2009 [1]. Compared to animal andfungal proteases, bacterial alkaline proteases have more commer-cial significance in laundry, food, leather and silk [3] due to theirhigh production capacity and catalytic activity [4–7]. However,proteases with high activity at different pH values and at hightemperatures have novel application potential in pharma, diag-nostic, detergent, tannery, amino acid production, contact-lenscleaning agents, effluent treatment, enzymic debridement, sup-porting the natural healing process in the skin ulcerations [5,6,8,9].They also hydrolyze peptide bonds in aqueous solutions andsynthesize them in non-aqueous conditions [10]. In addition, theirfunctional and thermal stability of protein chemistry and proteinengineering are the most important parameters to be investigatedto understand their utility in different sectors.

Although proteases producing microorganisms, plants andanimals are wide spread in nature, microbial community is

* Corresponding author. Tel.: +91 40 27193159; fax: +91 40 27193159.

E-mail address: [email protected] (R.S. Prakasham).

1359-5113/$ – see front matter � 2008 Published by Elsevier Ltd.

doi:10.1016/j.procbio.2008.10.022

preferred due to their growth and simplicity for generation of newrecombinant enzymes with desired properties. Physical, biochem-ical, molecular and catalytic properties of proteases varies with thenature of the organism [6,11,12]. In general, most of the industrialproteases have some limitations [13] and their use highly dependson their stability during isolation, purification and storage inaddition to their robustness against solvents, surfactants andoxidants [10,14–16]. Hence, in depth knowledge of kinetics andcatalytic behavior during protease production from any new strainis a prerequisite for evaluation of its biotechnological potential[17,18]. In this context, a potential alkaline protease producingbacterial strain was isolated in our laboratory [17,19] andevaluated in detail for fermentation parameters and the kineticsof enzyme production with respect to development of low cost andeasy available medium ingredients to fit for industrial use [2,17–19]. The present investigation reports the biochemical character-ization and potential application in leather processing anddetergent industries of the alkaline protease produced by isolatedB. circulans.

2. Materials and methods

2.1. Bacterial strain, media and growth conditions

Previously isolated B. circulans [19] which has potential to produce alkaline

protease was grown using fermentation medium according to Subba Rao et al. [2].

The culture filtrate was used for further studies as enzyme source.

Ch. Subba Rao et al. / Process Biochemistry 44 (2009) 262–268 263

2.2. Analytical methods

2.2.1. Measurement of protease activity and protein content

Protease activity was determined using modified Hagihara et al. method [20] and

according to Subba Rao et al. [2]. One unit of alkaline protease activity was defined

as 1 mg of tyrosine liberated min�1 under the assay conditions. The protein content

was determined by the Lowry method [21]. All experiments were conducted in

triplicate and average values were reported.

2.3. Purification

2.3.1. Ammonium sulphate precipitation

The cell free broth was obtained by centrifugation at 5000 � g for 10 min.

Solid ammonium sulphate was added to the culture supernatant to get 60%

saturation, stirred for 60 min and left overnight at 4 8C. The precipitate

was harvested by centrifugation at 10,000 � g for 10 min, dissolved in

50 mM glycine–NaOH buffer and dialyzed against the same buffer overnight

(4 8C). The dialyzed sample was then assayed for protease activity and protein

content.

2.3.2. Sephadex G-100 chromatography

Dialyzed enzyme was loaded on to a glycine–NaOH buffer (pH 11.0)

preequilibrated Sephadex G-100 column (1.5 cm � 90 cm). The same buffer

containing sodium chloride gradient (0.1–1 M) was used for elution of protein

with a flow rate of 10 ml h�1. The UV absorbance of each fraction was measured at

280 nm. Fractions were assayed for protease activity. Protease active fractions were

pooled and concentrated for further characterization.

2.3.3. Polyacrylamide gel electrophoresis

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was

carried out according to Laemmli [22] using a 10% crosslinked polyacrylamide gel.

Silver staining was performed to visualize protein bands. Native PAGE was

performed according to Davis [23] with Tris/glycine buffer, pH 8.3. Coomassie

Brilliant Blue (0.1%) staining was used to detect the protein bands.

2.3.4. Zymography with casein

Casein zymography was performed using 10% polyacrylamide slab gels

containing SDS and 1% casein in separating gel as described by Heussen and

Dowdel [24] at 4 8C. After electrophoresis, the gels were soaked thrice for 20 min in

2.5% (v/v) Triton X-100 at room temperature to remove the SDS. The gels were

stained with 0.1% Coomassie brilliant blue R-250 in methanol–glacial acetic acid–

water (40:10:60) followed by destaining with methanol–glacial acetic acid–water

(40:10:60). Enzyme activity was visualized by incubating the gel for 12 h in 50 mM

glycine–NaOH buffer at pH 11.0 at room temperature.

2.4. Determination of the pH optimum and stability

The pH optimum for purified protease was assayed by analyzing its activity in

the pH range of 5–12 using casein as a substrate and buffer systems of 0.05 mol l�1

phosphate buffer for pH 5.0–7.5, Tris–HCl for pH 8.0–9.0, glycine–NaOH for pH 9.5–

11.0, sodium phosphate for 11.5–12.0 and sodium carbonate for 12.5–13.0. pH

stability studies were performed by pre-incubating 5 ml of purified enzyme in

3.5 ml of selected pH buffer at 37 8C for 1 h and 48 h and subsequent analysis of

residual activities under standard assay.

2.5. Determination of optimum temperature and thermal stability

Optimum temperature of the enzyme was measured by incubating the

reaction mixture of the enzyme at different temperatures (35–75 8C) in glycine–

NaOH buffer (pH 11.0) and protease activity was measured. For determining

thermal stability, the enzyme was pre-incubated for 1.0 h at different

temperatures (35–90 8C) and the residual activity was measured under standard

assay conditions.

2.5.1. Thermo-inactivation studies

Thermo-inactivation assays were carried out by preheating 950 ml of standard

buffer at the corresponding temperature, then adding 1 mg protein in 50 ml of the

same buffer and pre-incubating the mixture at the same temperature. Samples

were collected every 1.0 h at 75, 80, 85 and 90 8C and cooled to 50 8C before

analyzing for protease activity.

2.6. Estimation of kinetic parameters

2.6.1. Determination of Vmax and Km values

The kinetic parameters, Vmax and Km, of the purified protease were determined

by measuring the enzyme activity at different substrate concentrations (0.2–1.6%).

The Km and Vmax values were determined using Michaelis–Menten equation using

Sigmaplot �10.0 enzyme kinetics module 1.3.

2.6.2. Determination of activation energy (Ea), enthalpy (DH), free energy (DG) and

Entropy (DS)

The activation energy of the purified enzyme was calculated using Arrhenius plot

by ploting ln v vs T�1 (K). The DH in kJ mol�1, DG in kJ mol�1 and DS in J mol�1 K�1

were calculated by using the following equations 1, 2 and 3, respectively.

DH ¼ Ea � RT (1)

DG ¼ �RT lnkða=dÞh

kB � T

� �(2)

DS ¼ DH �DG

T(3)

where Ea is the activation energy, T (K) is the corresponding absolute temperature, R

is the gas constant (8.314 J mol�1 K�1), h is the Planck constant

(11.04 � 10�36 J min), kB is the Boltzmann constant (1.38 � 10�23 J K�1) and k(a/d)

is the ‘a’ for activation and ‘d’ for deactivation.

First order reaction constant is the slope in the regression line obtained by

plotting ln v vs time at different temperatures (kJ mol�1).

2.6.3. Half life measurement

The half-life of the enzyme (t1/2, min�1) was calculated according to Cardoso and

Emery [25] using Eq. (4). The D (decimal reduction) value (h) is defined as the time

required to pre-incubate the enzyme at a given temperature to maintain 10%

residual activity and was calculated using Eq. (5):

t1=2 ¼A� 3:912

kd(4)

D ¼ A� 2:3026

kd(5)

where A is the intersect of the regression line, 3.912 and 2.3026 are the natural

logarithms values of 50 (for t1/2, min�1) and 10 (for decimal reduction), respectively.

2.7. Effect of various metal ions

Impact of various metal ions (Ca2+, Co2+, Cu2+, Mg2+, Mn2+, Fe2+, Hg2+, Na+ and

Zn2+) on the enzyme catalytic behavior was studied by pre-incubating at room

temperature purified enzyme in a specified ion (10 mM final concentration)

containing buffer solution. After 1 h of incubation, casein was added and residual

activity of the enzyme was measured.

2.8. Effect of protease inhibitors

The influence of different protease inhibitors, viz., phenylmethylsulphonyl

fluoride (PMSF), diisopropyl flurophosphate (DFP) (serine protease inhibitor),

ethylenediaminetetraacetic acid (EDTA) (metalloprotease inhibitor), p-chloro

mercuric benzoate (pCMB), and idoacetic acid (cysteine protease inhibitors) was

investigated by incubating the enzyme for 30 min at 30 8C in the selected protease

inhibitor containing reaction mixture in a final concentration of 1.0 and 5.0 mM and

the proteolytic activity was determined.

2.9. Effect of surfactants and oxidizing agents

The effect of 1.0% final concentration of different surfactants and oxidatives (SDS

in w/v, Tween-80, Triton X-100 in v/v and H2O2 in v/v) on proteolytic activity of the

purified protease was studied by pre-incubating it for 4 h in the above surfactant

solutions at 37 8C before testing for protease activity. A parallel control was kept

with enzyme and buffer with substrate and the value of the control activity was

considered as 100%.

2.10. Detergent stability

Different commercially available detergents like NirmaT1 (Nirma Chemical,

India); HenkoT1 (Henkel Spic, India); SurfT1, Surf ExcelT1, Super WheelT1, RinT1

(Hindustan Lever Ltd., India); and ArielT1 (Procter and Gamble, India) were used to

study the compatibility of the purified alkaline protease. The enzyme was incubated

in one percent of above detergent (w/v) solutions (in tap water) at pH 9.0 and at

room temperature for 1–2 h before measuring the enzyme activity. Enzyme activity

without any detergent was taken as 100%.

2.11. Blood stain removal studies

Clean cotton cloth pieces (5 cm � 5 cm) were soiled with blood, dried and soaked

in 2% formaldehyde for 30 min followed by washing with water to remove excess

formaldehyde. To evaluate the stain removal, stained cloth pieces were incubated

with 1 mg of purified alkaline protease for different time durations (10–40 min)

Table 1Summary of purification of alkaline protease produced B. circulans.

Total

activity

(U)

Total

protein

(mg)

Specific

activity

(U/mg)

Recovery

(%)

Purification

fold (%)

Crude 945000 1250 756 100 1

(NH4)2SO4 587790 350 1679 62.2 2.2

Sephadex G-100 189000 21 9000 20 11.9

Ch. Subba Rao et al. / Process Biochemistry 44 (2009) 262–268264

followed by rinsing with water for 2 min and then dried to compare with untreated

cloth piece stained with blood.

2.12. Dehairing studies

Dehairing property of the enzyme was studied using fresh goat-skin pieces

measuring 4 cm2 with hair. The skin pieces were dipped in 50 ml of 50 mM glycine–

NaOH buffer (pH 11.0) supplemented with 2 mg of purified protease and incubated

at 35 8C for 6–12 h before analyzing for dehairing property.

2.13. Histochemical studies

Histochemical characterization of dehaired goat samples was carried out

according to official methods of analysis [26]. Dehaired pelts and soaked skin (as

control) samples were washed thoroughly before fixing in 10% formal saline and

dehydrated using ethanol series. Sections of 4 mm were obtained using microtome

after embedding in paraffin block and they were stained using hematoxylin and

eosin to examine the histological features.

3. Results and discussion

3.1. Enzyme purification and molecular weight

This enzyme was purified 11.9-fold starting from the culturefiltrate and achieved near homogeneity by ammonium sulfateprecipitation (60%), and gel filtration using Sephadex G-100(Table 1). The specific activity of the purified enzyme was9000 U/mg protein, indicating 20% recovery. Appearance of singleband in SDS-PAGE and native PAGE as well as zymographyindicated that the purified alkaline protease was a monomer withmolecular mass of 39.5 kDa (Fig. 1a and b). These results are in

Fig. 1. (a) SDS-PAGE analysis of the purified protease. Lane M1, molecular mass markers

35.0 – lactate dehydrogenase; 25.0 – REase BSP981; 18.4 – b-lactoglobulin; 14.4 – lysozym

purified protease by Sephadex G-100. (b) Native-PAGE M, molecular mass markers (in

carbonic anhydrase – 29.0; soyabean trypsin inhibitor – 20.1. L1, purified protease; L2

accordance with the literature reports where most of themolecular masses of proteases from Bacillus genus are less than50 kDa [27].

3.2. Effect of pH on the protease activity and stability

In general, bacteria belonging to Bacillus genus are known tosecrete mostly two types of extracellular proteases, a neutral ormetalloprotease which exhibits optimum activity at pH 7.0 and analkaline protease having pH optima between 9.0 and 11.0 [12].The enzyme produced by Bacillus circulans showed its optimumactivity at pH 11.0 indicating that this enzyme belonged toalkaline protease group. Any further variation of the pH of thereaction mixture caused reduction in catalytic activity (Fig. 2insert). This activity variation was more and drastic with increaseof pH of the reaction mixture towards alkalinity. The relativeactivities of the purified enzyme were 50 and 15% at pH 11.5 and12.0, respectively. However, a progressive reduction wasobserved with change of pH of the reaction mixture towardsacidic side indicating its robust nature in pH range of 5–11.0(Fig. 2). Even though it is active from pH 5.0 it showed only 40–50%of its activity below pH 7.0 with respect to its activity at pH 11.0indicating its alkaline nature. Nilegaonkar et al. [28] reportedprotease with broad pH range from 6.0 to 12.0 having optimumactivity at pH 9.0 and drastic reduction with the change of pH oneither side of pH optima. pH dependent enzyme stability studiesat broad range of pH solutions from 5.0 to 13.0 for 1–48 h at roomtemperature denoted that the enzyme activity varied withincubation time and storage pH (Fig. 2). Incubation of proteasefor 1 h in 10.5–11.5 pH solution did not show any reduction in theactivity profile whereas approximately 10% reduction in activitywas noticed when incubated in the pH range of 5.0–10.0indicating its stable nature (Fig. 2). Whereas, increase inincubation time to 48 h revealed that enzyme activity wasaffected and showed only 50% and 60% of its activity at pH 5.0 and12.0, respectively, with maximum stability in the pH range of 9.0–11.5. Similar kind of pH stability for protease produced by Bacillus

sp., was observed [14,29].

(in kDa): 116.0 – b-galactosidase; 66.2 – bovine serum albumin; 45.0 – ovalbumin;

e. L1, crude enzyme; L2, ammonium sulphate precipitated and dialyzed sample. L3,

kDa): phosphorylase b – 97.4; bovine serum albumin – 66.0; ovalbumin – 43.0;

, zymography of purified protease.

Fig. 2. Effect of pH on activity (a) and stability (b) of the purified enzyme.

Fig. 3. Effect of temperature on purified protease activity from 35 to 80 8C.

Fig. 5. Temperature dependence of the decimal reduction of purified protease.

Fig. 4. Temperature dependence of the thermoinactivation constant of purified

protease with an insert of ln v is the natural logarithm of the relative percent activity

for kDa values calculation.

Ch. Subba Rao et al. / Process Biochemistry 44 (2009) 262–268 265

3.3. Effect of temperature on protease activity and stability

3.3.1. Temperature mediated activity profile

Analysis of the temperature dependent protease activityrevealed that the enzyme catalytic behavior was similar in thetemperature range of 35–45 8C (Fig. 3). However, further increasein the incubation temperature influenced the protease activity.Maximum enzyme activity was noticed at 70 8C and furtherincrease in incubation temperature drastically reduced theenzyme activity. The rate of enzyme activity was observed to be315 U per increase of 1 8C in the temperature range of 45–70 8C.

3.3.2. Activation energy

The Arrhenius plot of protease activity exhibited two ‘‘breakpoints’’ one at 45 8C and another at 60 8C (Fig. 3) with meanactivation energies of 4.04, 59.5 and 21.8 kJ mol�1 in the tempera-ture range of 35–45 8C, 45–60 8C and 60–70 8C, respectively. Similartype of break point was observed by Lee and Anstee [30] forendopeptidases from Spodoptera littoralis. Such variation of activa-tion energies indicates the conformational changes especially at thecatalytic site which improves affinity towards substrate binding. The

protease was further characterized for its Km and Vmax towardscasein as a substrate at 70 8C. It was noticed that this proteaseshowed Km of 0.597 mg ml�1 and Vmax of 13825 mmol min�1

indicating its high affinity and efficient catalytic role compared toliterature reported alkaline proteases from Bacillus clausii GMBAE 42(Km of 1.8 mg ml�1 and Vmax of 11.50 mM) [31], haloalkaliphilicBacillus sp. (Km of 2 mg ml�1 and Vmax of 289 mg min�1) [32],Haloalkaliphilic bacterium sp. AH-6 (Km of 2.5 mg ml�1 and Vmax of625 U min�1) [33] and Pseudomonas aeruginosa PseA (Km of2.69 mg ml�1 and Vmax of 3.03 mmol min�1) [10].

3.3.3. Thermoinactivation studies

Thermal inactivation studies indicated a high correlationcoefficient (0.98) suggesting the first order deactivation kineticsin the temperature range of 75–90 8C (Fig. 4A) indicating itsirreversible inactivation at higher temperatures. Further, theobserved high conformation deactivation energy (Ead = 201.44 kJ)(Fig. 4) is uncharacteristic of a covalent reaction, and agrees withthe existence of protein unfolding followed by refolding into newthermodynamically stable structure but catalytically inactive asreported by Klibanov [34]. Analysis of the thermal inactivationcurve traversing one log cycle (z) according to Lopez and Burgos[35] depicted a variation of approximately 8 8C was essential for a

Table 2Deactivation kinetics parameters enthalpy (DH), free energy (DG), entropy (DS) and

half-life t1/2 of the purified protease from B. circulans.

Temperature (8C)

75 80 85 90

DG (kJ mol�1) 100.3433 98.80537 98.31786 97.37604

DH (kJ mol�1) 198.5549 198.5134 198.4718 198.4302

DS (J mol�1 K�1) 282.2173 282.4589 279.7596 278.3862

t1/2 (half-life) 150.7031 68.23302 49.54128 26.37584

Table 4Relative activity of purified alkaline protease at different concentrations of

inhibitors, surfactants and stability towards the detergents at different time

intervals.

Effect of inhibitors Relative activity (%)

1 mM 5 mM

Control 100 100

PMSF 15 2

Idoacetic acid 94 90

EDTA 98 98

PCMB 92 89

DFP 20 8

Surfactants (1%) 4 h

Control 100

Triton X-100 115

Tween-20 120

SDS 75

H2O2 105

Effect of detergents Relative activity at different incu-

bation times

1 h 2 h

Nirma 93 89

Henko 105 98

Surf 85 82

Surf excel 94 90

Super wheel 95 89

Rin 115 108

Ariel 94 91

Ch. Subba Rao et al. / Process Biochemistry 44 (2009) 262–268266

decimal reduction of enzyme activity per hour (Fig. 5). Such low D

values suggested that this enzyme was stable at higher tempera-tures similar to the reports of Cobos and Estrada [36] revealing itspotential importance in detergent industry. Evaluation of DH

(enthalpy), DG (free energy) and DS (entropy) values at 75, 80, 85and 90 8C revealed little variation in enthalpy values indicating thestability in enzyme heat capacity as noticed for xylanase producedby Trichoderma reesei QM9414 [36]. DG values, however, decreasedgradually from 100 to 97 kJ mol�1 with increase in temperaturefrom 75 to 90 8C (Table 2). Whereas, entropy (DS) values showed adifferent trend with respect to DH and DG and was almost constantin the temperature range of 75 and 80 8C and decreased withincrease in temperature from 80 to 90 8C suggesting thedestruction of ordered structure of alkaline protease at 80 8C orabove. The half-life of the protease enzyme at 75 and 90 8C wasobserved to be 150 and 26 min suggesting the thermo stablenature of enzyme (Table 2). This data shows the higher half-life atthis temperatures compared to proteases reported from Bacillus

licheniformis NH1 by El-Hadi-Ali et al. [14] and Bacillus subtilis PE11by Adinarayana et al. [37] indicating its for potential industrialapplication.

3.4. Effect of metal ions

Ca2+, Mg2+ and Mn2+ ions positively regulated the enzymeactivity and other tested metal ions did not show much influenceexcept Cu2+ compared to control (Table 3). The Ca2+ ion dependentactivity improvement indicated that the enzyme required calciumions for its optimal activity this phenomena might be attributed tocalcium ion involvement in stabilization of the enzyme molecularstructure as reported in some of the proteases derived from Bacillus

sp. [38–42]. In fact, calcium ions are known as inducers andstabilizers of many enzymes and protect them from conforma-tional changes [27].

3.5. Effect of enzyme inhibitors

North [42] has classified proteases based on their sensitivity tovarious inhibitors. To know the nature of the alkaline proteaseproduced by B. circulans, the enzyme activity in presence of 1.0 and

Table 3Effect of different metal ions on the purified protease activity.

Metal ion Residual activity (%)

Control 100

Ca2+ 130

Zn2+ 96

Cu2+ 84

Mg2+ 115

Mn2+ 110

Hg2+ 92

Co2+ 90

Na+ 102

Cd+ 94

Al3+ 96

5.0 mM concentrations of different protease inhibitors wasanalyzed. The results revealed that EDTA (metalloproteaseinhibitor), iodoactetic acid and p-chloromercuribenzoate (cysteineprotease inhibitor) showed no or very small effect on proteaseactivity (Table 4). However, PMSF (serine protease inhibitor)completely inhibited the enzyme activity even at very lowconcentration suggesting that the protease produced by B.

circulans belongs to serine proteases group.

3.6. Effect of surfactants and oxidizing agents

The purified enzyme showed stability in the presence of all thestudied compounds. In fact the non-ionic detergents, Triton X-100and Tween-20, enhanced its residual activity to 15 and 20%respectively. In the presence of 1% strong anionic surfactant SDSthe enzyme retained 75% of its initial activity. In general and as perthe literature, the proteases belonging to Bacillus genus areunstable against the oxidants and bleaching agents [8]. However,the enzyme under investigation did not show any inhibition inpresence of 1% hydrogen peroxide (Table 4). This experimentaldata suggested that the purified enzyme was stable to all testedcationic, anionic, non-ionic and to the different commerciallyavailable detergents (Table 4). The compatibility studies of thepurified enzyme with detergents revealed that the activity of theenzyme decreased slightly with increasing of incubation time. A 3–7% decrease in protease activity was evidenced with increase ofincubation time from 1 to 2 h indicating its compatibility withmost of the branded detergents except Rin indicating its suitabilityfor formulation of commercial detergents.

3.7. Evaluation of industrial application

3.7.1. Removal of blood stains

Incubation of protease with blood stained cotton cloth pieceshowed removal of the stains without usage of any detergents

Fig. 6. Effect of protease on the blood stain removal. (a) Untreated; (b) treated with detergent; (c) treated with protease; (d) treated with detergent and protease.

Fig. 7. Hematoxylin and eosin staining of sections from: (a) soaked skin; (b) partially dehaired pelts of enzymatic process after 6 h; (c) dehaired pelts after enzymatic process

of 12 h. ED, epidermis; BV, blood vessel; GS, glandular structures; HS, hair shaft; HF, hair follicles.

Ch. Subba Rao et al. / Process Biochemistry 44 (2009) 262–268 267

within 30 min. Rapid blood stain removal was noticed withsupplementation of commercially available detergents (Fig. 6).Similar results were noticed with proteases from B. subtilis PE-11and Pseudomonas aeruginosa [15,37] indicating the role of B.

circulans protease in industrial application especially in detergent.

3.7.2. Dehairing of skin

Enzymatic dehairing process has been gaining importance as analternative chemical methodology in present day scenario as thisprocess is significant in reduction of toxicity in addition toimprovement of leather quality [16]. Several microbial proteaseswere evaluated for their unhairing character [16,43,44] and it wasnoticed that only those enzymes with pH stability under alkalineconditions especially between 9.0 and 11.0 and with non-keratinase and non-collagenolytic properties were having edgeover others [28,45,46]. The enzyme produced by B. circulans

revealed robustness towards alkaline pH, detergent and bloodstain removal. Therefore application of this enzyme in termsdehairing character was investigated using goat skin withoutapplication of sodium sulfide. The experimental dehaired pelts ofgoat skin showed complete removal of fine hairs (Fig. 7c) withincreased brightness may be due to elimination of sulfide in theprocess (data not shown). Similar experimental observations werenoticed with alkaline protease produced by B. subtilis (MTCC 6537)by [16]. Histological sections of dehaired pelts upon staining withhematoxylin and eosin revealed the removal of epidermis,glandular structures, hair shafts and follicles (Fig. 7b and c).Complete absence of the above structural features along withopening up of collagen fiber structure was seen with samplestreated for >12 h (Fig. 7c). On the other hand, incomplete andmoderate removal was observed with 6 h incubation (Fig. 7b). Thedata depicted that there was no apparent damage to the collagenfibres in dehaired pelts (Fig. 7c). B. circulans protease has

advantages in dehairing process as this enzyme effectivelyunhaired the goat skin within 12 h compared to literature reportswhere alkaline proteases from B. subtilis, B. cereus, A. tamarii,dehaired the goat skin in 18, 21 and 24 h, respectively [28,44,45]indicating its potential application in leather industry foreconomizing the process.

4. Conclusion

Characterization and environmental friendly potential applica-tion of alkaline protease produced by isolated B. circulans wasstudied. The enzyme was purified using (NH4)2SO4 precipitationand Sephadex G-100 column chromatography. SDS-PAGE, nativegel and zymography analyses revealed that this protease ismonomeric in nature and has a molecular weight of 39.5 kDa.The protease belongs to serine-type with more substratespecificity for casein compared to BSA, egg-albumin and gelatinand influenced by divalent ion presence. The enzyme is thermo-stable with Km and Vmax values of 0.597 mmol min�1 and13,825 mg ml�1, respectively and retained 100% activity up to10 h at 70 8C. Thermal activation studies depicted two break points(at 45 and 60 8C). The enzyme showed half-life of 150 and 26 minat 75 and 90 8C respectively. Optimum catalytic activity wasobserved 9–11.5 pH range with effective stability in 5–12 pH. Theenzyme revealed excellent stability and compatibility towardsdetergents, oxidizing, reducing, and bleaching agents. Studiesindicated its utility for blood stain removal and detergent anddehairing properties. The physical properties of the experimentalgoat pelt revealed effective dehairing of fine hairs completelywithin 12 h without sodium sulfate indicating its eco-friendlynature in dehairing. Hematoxylin and eosin staining revealed theremoval of epidermis, glandular structures, hair shafts and follicleswith complete opening of collagen fiber structure.

Ch. Subba Rao et al. / Process Biochemistry 44 (2009) 262–268268

Acknowledgements

Two of the authors, viz., Ch. Subba Rao and T. Sathish arethankful to Council of Scientific and Industrial Research, New Delhifor financial support in the form of Senior Research Fellowship.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.procbio.2008.10.022

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