Bioresource Technology 115 (2012) 228–236

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Bioresource Technology

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Purification and characterization of haloalkaline thermoactive, solvent stableand SDS-induced protease from Bacillus sp.: A potential additive for laundrydetergents

Deepti Jain, Imran Pancha, Sanjiv K. Mishra, Anupama Shrivastav, Sandhya Mishra ⇑Discipline of Salt Marine and Inorganic Chemicals, Central Salt and Marine Chemicals Research Institute, Council for Scientific and Industrial Research (CSIR), G.B. Marg,Bhavnagar 364021, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 24 August 2011Received in revised form 20 October 2011Accepted 22 October 2011Available online 29 October 2011

Keywords:HaloalkalineSerine proteaseSolvent stableThermotolerantWash performance

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.10.081

⇑ Corresponding author. Tel.: +91 278 2561354; faxE-mail address: smishra@csmcri.org (S. Mishra).

An extracellular haloalkaline, thermoactive, solvent stable, SDS-induced serine protease was purified andcharacterized from an alkali-thermo tolerant strain Bacillus sp. SM2014 isolated from reverse osmosisreject. The enzyme was purified to homogeneity with recovery of 54.4% and purity fold of 64. The purifiedenzyme was composed of single polypeptide of molecular mass about 71 kDa. The enzyme showed opti-mum activity at alkaline pH 10 and temperature 60 �C. The km and Vmax for the enzyme was 0.57 mg/mland 445.23 U/ml respectively. The enzyme showed novel catalytic ability at high pH (10), temperature(60 �C) and salinity (3 M). Moreover, the stability of enzyme in organic solvents (50% v/v) of logP P 2 sig-nified the prospective of this enzyme for peptide synthesis. The compatibility of the enzyme with surfac-tants and various detergent matrices together with wash performance test confirmed its potentialapplicability in laundry industry.

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1. Introduction

Global concern for the environment has attracted the research-ers to investigate enzymes as replacement of chemical catalysts invarious biochemical processes. The continual exploration of en-zymes and their utilities have expanded their industrial marketwith the growth of 7.6% per year (David et al., 2009). Of the variousindustrial enzymes, proteases alone contribute approximately 60%of the total sales in the world (Banik and Prakash, 2004) and bac-teria belonging to genus Bacillus produce most commercial prote-ases used today (Maurer, 2004). Proteases are the most versatileenzyme with a long history of catalytic applications in food andpharmaceutical industries. Recently, their roles in synthesis of bio-active peptides and as additive in commercial detergents are gain-ing attention. The composition and nature of the surfactants indetergents greatly manifest the stability of the enzyme. The otherprerequisites for the utilizing proteolytic enzymes in detergent for-mulations include their functional ability and stability at alkalinepH in presence of various surfactants and detergents. The naturalproteases in general are not stable under these conditions (Guptaet al., 2005). However several physiochemical methods such aschemical modification, immobilization, entrapment, proteinengineering and directed evolution have been employed for their

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stabilization at various extreme conditions (Ogino and Ishikawa,2001). Even the gene-shuffling techniques have not resulted in suc-cessful products. There have been extensive studies on screening ofmicrobes to obtain proteases with functional ability under the re-quired extreme conditions (Maurer, 2004) so that the overall pro-cess becomes environment friendly, feasible and economic. In thiscontext, alkaline proteases of microbial origin have been exten-sively studied however detergent stable proteases were character-ized from a few bacterial species (Deng et al., 2010). Since it isknown that extremophiles could be the potent producers of en-zymes with functional ability at extreme physico-chemical condi-tions, Shivanand and Jayaraman (2009) characterized salt stableprotease from halotolerant bacteria. Similarly organic solvent tol-erant bacteria were reported to produce solvent stable proteases(Xu et al., 2010; Shah et al., 2010). In this way it would be furtherinteresting to investigate polyextremotolerant microbes and theirextracellular enzymes with an assumption that they would havewider adaptability in various environmental conditions (Jainet al., 2010). However, the information on polyextremotolerantshas been less explored in comparison to extremotolerant microbes.

In our previous study, a strain belonging to genus Bacillus wasisolated from RO reject that showed characteristic features of sus-taining the three extremities such as pH (10), temperature (60 �C)and pressure tension of 450 psi under absolute oxygen atmosphere(Jain et al., 2010). The present study has been designed to extrap-olate the potential of this strain as a source of enzyme protease

D. Jain et al. / Bioresource Technology 115 (2012) 228–236 229

with distinct functional properties. Central to our study is theinvestigation of functional ability of protease under differentranges of pH, temperature, and salinity along with their combinedeffect. This study also investigated the possible utility of the en-zyme as an additive in detergent matrices.

2. Methods

2.1. Microorganism

The bacterial strain employed in this study was isolated fromthe reverse osmosis reject obtained after desalination of brackishwater. The 16S rDNA sequence of the isolated strain showed 94%similarity with Bacillus licheniformis and was characterized as athermoalkalitolerant strain (Jain et al., 2010).

2.2. Screening for protease enzyme

The screening for secretion of extracellular protease enzymewas performed by streaking the bacterial strain on milk agar platescontaining (g/l): yeast extract (10); agar (20); sodium chloride salt(35) as basal medium composites which was supplemented withskimmed milk (50% v/v). Milk was autoclaved separately andadded to the sterilized basal media. The inoculated plates wereincubated at 37 �C for 72 h. The protease production was con-firmed by the appearance of zone of clearance around the bacterialcolonies.

2.3. Enzyme production medium and culture condition

The enzyme production was carried out in the basal medium offollowing composition (% w/v): NaCl (3.5), casamino acid (0.5),yeast extract (0.5), glucose (0.5). The basal medium was sterilizedby autoclaving at 121 �C for 15 min in Erlenmeyer flasks (500 ml)containing 200 ml of culture medium. The sterilized media wasinoculated with 1 ml of 24 h old bacterial culture (3 � 104 cfu/ml)and incubated at 37 �C on an orbital shaker of speed 150 rpm for5 days. After incubation supernatant was collected by centrifuga-tion at 10,000 rpm for 10 min at 4 �C and was assayed for proteaseactivity.

2.4. Effect of nutritional parameters on bacterial growth and proteaseproduction

The effect of various carbon sources such as glucose, sucrose, lac-tose and starch at a concentration of 0.5% (w/v) was evaluated onbacterial growth. For evaluating nitrogen sources, basal mediumwas supplemented with either of yeast extract, beef extract, gelatin,casein, casamino acid, and peptone at a final concentration of 0.5%(w/v). The effect of various concentrations of NaCl (0–20% w/v)was also studied on bacterial growth and protease production.

2.5. Protease assay

Protease activity was measured by the method described byTakami et al. (1989). The enzyme (0.5 ml) was added to 2.5 mlcasein (0.65% w/v) buffered to pH 10 with 50 mM Na2CO3/NaHCO3.The reaction mixture was incubated at 37 �C for 30 min thereafter2.5 ml of TCA mixture (0.11 M trichloroacetic acid, 0.22 M sodiumacetate, 0.33 M acetic acid) was added to terminate the reaction.The terminated reaction mixture was incubated for 30 min at37 �C and the precipitates that were formed were removed by fil-tration through filter paper (Whatman-1). The absorbance of theclear filtrate was measured at 660 nm. One unit of alkaline prote-ase activity was defined as the amount of enzyme liberating 1 lg

of tyrosine per min under assay conditions. Enzyme units weremeasured against the standard curve of tyrosine plotted for con-centrations ranging from 0–100 lg. Protein concentration of thecell free supernatant was measured by Bradford method (1976)against the standard curve of bovine serum albumin (BSA).

2.6. Purification of protease

The cell free supernatant (100 ml) was precipitated overnight at80% saturation with (NH4)2SO4 at 4 �C and the pellet was recoveredafter centrifugation. The precipitate was dissolved in 40 ml of50 mM Na2CO3 /NaHCO3 (pH 10) and dialysed against the samebuffer for 2 days. The removal of ammonium sulphate was moni-tored with Nesseler’s reagent. The partially purified protease wassubjected to gel permeation chromatography. About 10 mL of thepartially purified enzyme was loaded to a Sephadex-G100(Sigma–Aldrich, USA) column (1.25 � 35 cm) pre-equilibrated with20 mM Tris-chloride buffer (pH 8.5). The fractions were elutedagainst the same buffer. Fractions of 2 ml each were collected ata flow rate of 2.5 ml/h. The eluates were monitored continuouslyat 280 nm for protein and also assayed individually for proteaseactivity as described above. Active fractions were lyophilized andstored at �20 �C. Fractions showing maximum activity were fur-ther analyzed for purity by electrophoresis.

2.7. Electrophoresis and zymography

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS–PAGE) as well as native PAGE was performed at 12% separat-ing gel to check the purity and molecular weight of the purified en-zyme. Protein bands were detected after staining the gel withsilver nitrate. For in vitro protease activity staining, the gel afterSDS–PAGE was submerged in 50 mM Na2CO3 /NaHCO3 buffer (pH10) containing 2.5% Triton X 100, with shaking for 30 min to re-move SDS. Triton X 100 was removed by washing the gel threetimes with 50 mM Na2CO3 /NaHCO3 buffer (pH 10). The gel wasthen incubated with 1% (w/v) casein in 100 mM Na2CO3 /NaHCO3

buffer (pH10) for 40 min at 50 �C. Finally, the gel was stained with0.25% Coomassie Brilliant Blue R250 in 45% ethanol-10% acetic acidand destained with 5% ethanol-7.5% acetic acid .The developmentof a clear zone on the blue background of the gel indicated thepresence of protease activity.

2.8. Enzyme kinetics

Km and Vmax values of the enzyme were determined by mea-suring its activity with various concentrations of the substratecasein (0.1–1 g/100 ml). Kinetic constants were calculated fromLineweaver–Burke plot.

2.9. Effect of pH on activity and stability of protease

The pH optimum of the enzyme was determined by varying thepH of the assay reaction mixture using the following buffers(0.1 M): sodium acetate (pH 6), sodium phosphate (pH 7 and 8), so-dium bicarbonate (pH 9 and 10), NaHCO3/NaOH buffer (pH 11),and KCl/NaOH buffer (pH 12 and13). The stability of the proteasewas determined after pre-incubating the enzyme in various buffersolutions (pH 6–13) for 30 min. The residual enzyme activity wasthen determined under the standard assay condition.

2.10. Effect of temperature on activity and stability of protease

The temperature optimum of the enzyme was evaluated by mea-suring the protease activity at different temperatures (20–90 �C) in0.1 M sodium bicarbonate buffer (pH 10). The effect of temperature

230 D. Jain et al. / Bioresource Technology 115 (2012) 228–236

on protease stability was determined by measuring the residualactivity after 30 min of pre incubation in 0.1 M sodium bicarbonatebuffer (pH 10) at temperature ranging from 20 to 90 �C.

2.11. Effect of NaCl on activity and stability of protease

To determine the effect of NaCl on enzyme activity, the assaywas carried out at optimum conditions i.e. pH 10 and temperature60 �C with varying salt concentrations from 0% to 30%. For stability,the enzyme was pre-incubated for 30 min with different concen-trations of NaCl (w/v) in the range of 0–30%.

2.12. Effect of inhibitors and metal ions on activity of protease

The enzyme inhibitors studied include phenylmethyl sulfonyl-fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), and bmercaptoethanol each at concentration of 2 and 5 mM. The enzymewas pre incubated with inhibitors at 37 �C for 30 min and the resid-ual enzyme activity was determined under optimal assay conditionsagainst the control. The activity of the enzyme assayed in the ab-sence of inhibitors was taken as 100%. Effect of metal ions on en-zyme activity was determined by carrying out the enzymesubstrate reaction in the presence of various metal ions (Ca2+, -Cu2+,Mg2+,Mn2+,Ni2+,Zn2+) at concentration of 10 mM. Enzymeactivity was determined as percentage relative activity as comparedto control (without additives) which was considered as 100%.

2.13. Effect of organic solvents on stability of protease

The enzyme was mixed with different concentrations (15%, 25%and 50% v/v) of various organic solvents such as acetone, benzene,butanol, DMSO, hexane, iso-octane, n-decane, n-heptane, toluene,xylene and incubated at 37 �C on an orbital shaker at a speed of150 rpm. Residual protease activity with respect to control wasmeasured at different time intervals under optimized assayconditions.

2.14. Effect of bleaching agents, surfactants and detergents on stabilityof protease

The suitability of the enzyme as a detergent additive was deter-mined by examining its stability in the presence of surfactantssuch as SDS (Sodium dodecyl sulphate), Triton X-100, Tween 80and oxidizing agent (H2O2), and bleaching agent (sodium hypo-chlorite). The enzyme was incubated with these additives for60 min at 37 �C and then the residual enzyme activities weredetermined under the optimal assay conditions. The study furtherevaluated the compatibility of the enzyme with commercial soliddetergents. The solid detergents such as Ariel, Wheel and Surf excelwere diluted in tap water to give a final concentration of 7 mg/ml.The endogenous enzymes in the detergents were inactivated byheating the diluted detergents for 30 min at 100 �C prior to theaddition of the protease enzyme. The enzyme and detergent mix-ture was then incubated for 60 min and the residual enzyme activ-ity was determined under optimal assay conditions.

2.15. Analysis of casein hydrolysates

Casein was hydrolyzed by the purified protease enzyme at opti-mal conditions i.e. temperature 60 �C and pH 10 for 30 min. Thehydrolysate was centrifuged (10,000 rpm, 10 min, 4 �C) and thesupernatant was collected. The supernatant was freeze dried andused for FT-IR and gel permeation chromatography. Spectroscopicmeasurements were performed using approximately 25 mg freeze-dried hydrolysate. The hydrolysate was mixed with 225 mg driedKBr (10% w/w). The FTIR spectra were recorded between 4000

and 400 cm�1 on FTIR spectrometer (Perkin Elmer spectrum 4 X).The interferometer as well as the chamber that housed the detec-tor was purged with dry nitrogen to remove spectral interferencesresulting from water vapor and carbon dioxide. The spectral acqui-sition was carried out at room temperature at a resolution of 4 and10 cm�1. The single-beam spectrum of KBr was subtracted fromthe single-beam spectrum of each protein hydrolysate to obtainthe desired spectrum. All experiments were performed in dupli-cate. Prior to data analysis, the spectra were baseline correctedand normalized using mean normalization option.

The enzymatic hydrolysis of casein was further analyzed by highperformance liquid chromatography (HPLC) with a gel permeationcolumn Ultrahydrogel 500 coupled with refractive index detector.Isocratic elution was carried out at 30 �C with a mobile phase (watercontaining 0.1 M sodium nitrate) at column flow rate of 0.8 mL/min.Molecular size markers ranging from 100 to 4,01,000 Da were usedto determine the size of the end product of the reaction.

2.16. Wash performance analysis

Clean cotton cloth pieces (4 cm � 6 cm) were stained withblood, tomato sauce, turmeric paste and then dried. The stainedclothes were subjected to wash treatments in flasks with 100 mleither of tap water, commercial solid detergent (Ariel) diluted intap water at 7 mg/ml, detergent solution supplemented with crudeenzyme. Each flask was incubated at temperatures 25, 50 and 60 �Cfor 30 min under agitation (200 rpm). After incubation, cloth pieceswere taken out, rinsed with water and dried. After drying, theresulting reflectance (%) of the cloth was measured on UV/Vis spec-trophotometer (Shimadzu, Japan) in range of wavelength from300–800 nm in reflectance mode. In this analysis baseline was cor-rected with a clean blank white cotton cloth. The optimization ofenzyme concentration and washing time was carried out after per-forming the wash analysis with different concentrations of enzyme(845,1690 and 5070 U), and for different washing times (5,15 and30 min). Visual examination of various pieces was also carried outto show the effect of enzyme in the removal of stains. The surfacecharacteristics of cotton fiber after each wash were examined withscanning electron microscopy (LEO 1430 VP).

3. Results and discussion

3.1. Characteristics of the microorganism

The isolated bacterium Bacillus SM2014 was a thermo-alkali-tolerant strain (Jain et al., 2010). This strain further showedtolerance to broad range of salt concentration from 0% to 15%representing its haloduric physiology (Supplementary Fig. 1). Thisis another unique property of the bacterium together with alreadyreported distinct characteristics.

3.2. Enzyme production medium and culture condition

The time course study on protease production in basal mediashowed an increment in protease activity with the growth of bac-terium and maximum protease production was found at the end ofthe stationary phase and it gradually decline (Fig. 1). The resultswere in accordance with other halotolerant bacterium Bacillusaquimaris strain VITP4 (Shivanand and Jayaraman, 2009 whereprotease production was also reported in stationary phase. Theprotease production was found highest when the basal mediawas supplemented with 3.5% (w/v) NaCl however the bacteriumcould tolerate salt up to 15% (w/v) (Table 1). Shivanand andJayaraman, 2009 reported optimum production of proteasefrom Bacillus sp. at 0.6 M NaCl. Therefore the bacterium and its

Fig. 1. Time-course studies on growth and protease production by Bacillus sp.

Fig. 2. SDS–PAGE analysis of purified protease. Lane 1 molecular mass markers,lane 2 purified protease, lane 3 in vitro activity staining.

D. Jain et al. / Bioresource Technology 115 (2012) 228–236 231

extracellular protease with salt tolerance signify their potentialapplicability in laundry industry in a better way than the otherreported surfactant and detergent stable proteases from Bacillussp. (Deng et al., 2010; Haddar et al., 2009).

3.3. Effect of nutritional parameters

The bacterium showed substantial growth in the presence of allthe carbon sources studied while protease production was highestin the presence of glucose and lowest in the media containing lac-tose (Table 1). There are several reports showing that different car-bon sources have different influences on extracellular enzymeproduction by different strains. The inducing effect of glucose onprotease production is similar to that observed for Bacillus sp.AR-009 (Gessesse, 1997), Bacillus licheniformis ATCC 21415(Mabrouk et al., 1999) and Bacillus cereus strain 146 (Shafeeet al., 2005). Of the organic nitrogen sources assessed for proteaseproduction, the maximum yield of protease was recorded withcasamino acids followed by casein and peptone whereas proteaseproduction was completely inhibited in the presence of beef ex-tract and gelatin (Table 1). Induction in protease enzyme produc-tion with casamino acid was also reported in Bacillus sp. (Joshiet al., 2008) and Bacillus mojavensis (Beg et al., 2002). Further pro-tease production was enhanced when the production media wassupplemented with 0.1% yeast extract together with casamino acid(Table 1) as also demonstrated by Kaur et al. (2001).

3.4. Enzyme purification and molecular weight

The purification of crude protease following ammonium sul-phate, dialysis and gel permeation chromatography resulted in ayield of 54% of pure enzyme with purity fold of 64. The purity ofthe enzyme was confirmed by obtaining a single band of molecularweight �71 kDa in both SDS–PAGE and native PAGE. Further, the

Table 1Effect of different nutritional components on protease production.

Carbon Source Glucose Fructose Starch

Protease activity (U/ml) 1269 683 455.5Biomass (Absorbance at 600 nm) 1 1.1 1.13

Nitrogen Source Peptone Gelatin Casein

Protease activity (U/ml) 715.73 ND 943.5Biomass (Absorbance at 600 nm) 1 0.21 2.1

NaCl% (w/v) 0 3.5 5

Protease activity (U/ml) 1194 1623.4 1139Biomass (Absorbance at 600 nm) 1.2 0.91 0.79

ND: Not detected.

in vitro activity staining showed zone of clearance at the same po-sition as observed in the PAGE analysis confirmed the purity andmolecular weight of the enzyme (Fig. 2). This result is unique asonly a few reports are available on the haloalkaliphilic thermosta-ble protease having such a high molecular weight. Sookkheo et al.(2000) reported the production of thermostable proteases ofmolecular mass of 71 kDa from Bacillus stearothermophilus strainTLS33. The results of the purification and recovery are summarizedin Table 2.

3.5. Determination of km and Vmax

The kinetic study of protease towards different concentrationsof casein revealed km and Vmax as 0.57 mg/mL and 445.2 U/mLrespectively. The affinity of the enzyme towards casein was highas compared to the other alkaline protease characterized fromBacillus sp. (Dodia et al., 2008; Kazan et al., 2005) as its value ofkm was lower than others.

3.6. Effect of pH on activity and stability of protease

The protease characterized herein was found to be functionallyactive at broad pH ranges from 8 to 12. The purified enzymeshowed optimum activity at pH 10 (Fig. 3A). These findings arein accordance with several earlier reports on alkaline proteasesthose have pH optima of 10–10.5 (Banik and Prakash, 2006; Begand Gupta, 2003). In general, all detergent compatible enzymesshould tolerate alkaline conditions (Sana et al., 2006) because thepH of laundry detergents is generally in the range of 9–12 whichis an important criterion for a detergent based protease (Joo

Sucrose Arabinose Maltose Lactose

390.4 651 553 1631.15 1.16 1.16 1.13

Casamino acid Beef extract Yeast extract

1041.07 ND 1138.672.04 0.22 1.8

10 15 20

683.2 ND ND0.67 0.5 0.3

Table 2Purification of extracellular protease by bacterium Bacillus sp. SM2014.

Purification step Total activity (U) Total protein (mg) Specific activity (U/mg) Purification fold Recovery (%)

Crude enzyme 1690 164.87 10.25 1 100Ammonium sulphate precipitation 1421 25.23 56.32 5.49 84.08Dialysis 1290 6.3 204.7 19.97 76.33Sephadex gel-filtration 920 1.4 657.14 64.11 54.4

Fig. 3. Characterization of protease describing the effect of (A) pH, (B) temperatureand (C) salt on enzyme activity and stability.

232 D. Jain et al. / Bioresource Technology 115 (2012) 228–236

et al., 2005). The enzyme characterized herein accomplished thiscondition as it was found to be active and stable over broad rangeof pH (7–12) thereby signify its potential applicability in laundryindustry (Fig. 3A).

3.7. Effect of temperature on activity and stability of protease

The enzyme showed optimum proteolytic activity at tempera-ture 60 �C. Interestingly the enzyme was active over a wide rangeof temperature from 40 to 80 �C (Fig. 3B). Together with high pHtolerance, proteases with thermal stability are more suited for theirapplicability in laundry industry (Sana et al., 2006). Li et al. (2009)characterized an alkaline protease from B. licheniformis YP1A whichhad optimum activity at pH 9.5 and temperature 60 �C. The prote-ase in this study was also found to be stable at broad range of tem-peratures from 40 to 80 �C (Fig. 3B). The activity and stabilityprofile of the enzyme was better or comparable with otherproteases which were reported compatible with detergent formu-

lations (Deng et al., 2010; Haddar et al., 2009; Beg and Gupta,2003).

3.8. Effect of salinity on activity and stability of protease

The enzyme showed optimum proteolytic activity at salt con-centration 1% (w/v) but it could retain about 93% of its activity atsalt concentration as high as 9% (w/v). Moreover, enzyme retained50% of its optimum activity even at a very high salt concentrationof 24% (w/v) (Fig. 3C). Besides being pH and temperature stable, aprotease with functional activity and stability in the presence ofsaline conditions will surely effective in ground water having highsalt content. Although the optimum proteolytic activity is at 1% saltconcentration but the enzyme is highly active as it could retain itsactivity at very high salt concentration when incubated at pH 10and temperature 60 �C. The enzyme was stable even at salt concen-tration as high as 15% (w/v) (Fig 3C). To the best of our knowledgeno such protease is reported that can catalyze the reaction underthree extreme conditions i.e. pH, temperature and salinity alltogether.

3.9. Effect of inhibitors and metal ions on protease activity

Among the metal ions tested, enzyme activity was found to beinduced by Ni2+ , Ca2+ and Mn2+ by 101%, 109%, and 198% respec-tively (Fig. 4A). However, Mn2+ and Ni2+ ions at 10 mM concentra-tion were reported as strong inhibitor of alkaline proteases of someBacillus sp. (Ozturk et al., 2009; Beg and Gupta, 2003). These twometal ions were even reported to inhibit the activity of organic sol-vent, detergent, and thermal stable alkaline protease of B. licheni-formis (Rachadech et al., 2010). For most of the proteasecharacterized from Bacillus sp. Ca2+ ions were reported to inducethe proteolytic activity of the enzyme (Haddar et al., 2009) as wellas enhances its thermal stability (Deng et al., 2010). On the otherhand other divalent ions such as Cu2+, Mg2+ and Zn2+ decreasedthe activity to 37%, 7% and 5% respectively. These results were inaccordance to earlier reports where bacterial serine proteases wererepressed by Zn2+ and Cu2+ (Sana et al., 2006). The enzyme activitywas completely inhibited by 5 mM PMSF suggesting that the en-zyme is a serine-protease (Fig. 4B). Many of the Bacillus-derivedalkaline proteases reported so far, belong to the class of serine pro-teases (Gessesse, 1997). Slight inhibition of about 7% in the enzymeactivity was observed with EDTA (2 mM) while activity inhibited to51% when incubated with 5 mM EDTA indicating that the enzymeis not a metallo-type protease. However, the slight stabilityshowed by the enzyme in the presence of EDTA (2 mM) is advan-tageous for its utilization as a detergent additive. Further, incre-ment in enzyme activity in presence of 2 mM mercaptoethanolsuggested this enzyme as a thiol dependent serine protease(Fig. 4B) (Beg and Gupta, 2003).

3.10. Effect of solvent on protease stability

The effect of various organic solvents at their different concen-trations (15%, 25% and 50% v/v) on enzyme stability is summarizedin Table 3. The protease activity was found stable in solvent

Fig. 4. Effect of various additives (A) metal ions, (B) inhibitors and (C) solvents on enzyme activity.

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concentration 15% and 25% (v/v) even after an incubation of15 days. However, the enzyme retained more than 50% of its activ-ity up to 192 h in solvents (50% v/v) except iso-octane, butanol andacetone (Fig. 4C). Enzyme could retain 70% of its activity in acetoneand butanol after a pre-incubation of 96 h and a significant declinein its activity was observed after 196 h of incubation (Fig. 4C). Fur-ther enzyme was found to be activated 2.4, 1.77, 1.59, 1.53, 1.13and 1.10-fold in the solvents (15% v/v) DMSO (logP = �1.3), Ace-tone (logP = �0.2), Xylene (logP = 3.1), Benzene (logP = 2.0), Hex-ane (logP = 3.5) and Butanol (logP = 0.8) respectively. At highersolvent concentration (25% v/v) enzyme was activated 1.18, 1.16,

1.09, 1.08, 1.07 and 1.04-fold in Xylene (logP = 3.1), n-Decane(logP = 5.6), DMSO (logP = �1.3), n-Heptane (logP = 4.0), Benzene(logP = 2.0) and Hexane (logP = 3.5) respectively. Therefore theseresults revealed that protease was activated by the solvents whichwere having logP < 4.0. Solvents with logP < 4.0 are considered tobe extremely toxic because of their higher degree of partitioninginto the aqueous layer and enzyme loose its activity due to reduc-tion in structural flexibility because of the disruption of hydrogenbonding and hydrophobic interactions that deprive the waterhydration shell of the protein. The possible reason for enzyme sta-bility in presence of such organic solvents could be the presence of

Table 3Protease stability in different concentration of organic solvents after 15 days.

Solvents logP Stability at solvent concentrations

15% (v/v) 25% (v/v) 50% (v/v)*

Acetone �0.2 177.69 ± 1.22 85.83 ± 1.20 22.12 ± 1.42Butanol 0.8 110.79 ± 1.54 62.92 ± 0.92 26.24 ± 1.28Benzene 2.0 153.02 ± 1.44 107.53 ± 1.50 81.20 ± 1.86DMSO �1.3 246.04 ± 3.25 109.05 ± 1.62 83.08 ± 1.43Hexane 3.5 113.66 ± 2.22 104.35 ± 1.84 105.86 ± 1.29Isooctane 4.5 73.38 ± 2.12 95.67 ± 1.77 58.00 ± 1.71n-Decane 5.6 67.62 ± 1.32 116.08 ± 2.35 101.51 ± 1.85n-Heptane 4.0 69.06 ± 0.85 108.82 ± 3.72 85.00 ± 1.69Toulene 2.5 84.89 ± 1.52 78.35 ± 1.82 69.21 ± 1.05Xylene 3.1 159.71 ± 2.65 118.52 ± 2.28 90.03 ± 1.80

* Stability of enzyme after 192 h of incubation.

Table 4Wash performance analysis of protease at different temperatures.

Reflectance (%) at temperatures

25 �C 50 �C 60 �C

Blood stainTap water 29.75 ± 0.23 32.64 ± 1.10 23.19 ± 0.54Detergent (7 mg/ml) 55.05 ± 1.21 37.48 ± 1.25 23.77 ± 0.42Protease + detergent 58.24 ± 0.84 54.01 ± 2.21 36.38 ± 0.74Tomato sauce stainTap water 52.58 ± 1.25 58.79 ± 1.82 65.90 ± 1.29Detergent (7 mg/ml) 60.3 ± 1.32 64.07 ± 1.23 88.43 ± 1.89Protease + detergent 57.85 ± 0.35 97.84 ± 1.37 87.99 ± 0.23Turmeric stainTap water 10.17 ± 0.12 10.89 ± 0.27 11.57 ± 0.18Detergent (7 mg/ml) 18.99 ± 0.11 14.8 ± 0.12 17.53 ± 0.19Protease + detergent 20.16 ± 0.13 31.4 ± 0.16 54.1 ± 2.75

234 D. Jain et al. / Bioresource Technology 115 (2012) 228–236

greater number of negatively charged acidic amino acids comparedto few basic amino acids on their surface. These negative chargesare thought to keep the protein soluble either by forming a hy-drated ion network with cations or by preventing the proteinaggregation through electrostatic repulsive charges at the proteinsurface. Thus the stability of the purified enzyme in the presenceof organic solvents of logP < 4.0 even at their higher concentration(50% v/v) is novel and unique. The stability of protease was foundto be superior compared to the alkaline protease characterizedfrom Streptomyces clavuligerus strain Mit-1 which showed toler-ance towards 2% solvent for 120 min (Thumar and Singh, 2009),from Bacillus subtilis strain Rand which showed stability with 25%of solvent after incubation of 30 min (Abusham et al., 2009) andfrom B. cereus which showed stability in 33% of solvents after144 h of incubation (Shah et al., 2010). Rahman et al. (2006) re-ported solvent stability of a protease from Pseudomonas aeruginosastrain K even after an incubation of 14 days with 25% (v/v) sol-vents. However, in this study the effect of different concentrationsof solvents were demonstrated and the results revealed that theenzyme was active and showed enhanced activity in the presenceof solvents of logP < 4 compared to improved stability of proteasein presence of solvents of logP > 4 as reported by Rahman et al.(2006). The superior protease stability in water immiscible sol-vents over water miscible solvents studied here in substantiateits utility in kinetic- and equilibrium- controlled synthesis particu-larly peptide synthesis, where homogenous aqueous-organic mix-tures of high solvating organic solvent media is preferentiallyemployed.

3.11. Effect of bleaching agents, surfactants and detergents on proteasestability

The potential of the protease enzyme purified was assessed forits compatibility with detergent formulations after analyzing itsstability with different non-ionic, anionic surfactants and oxidizingagents. The protease showed excellent stability towards non-ionic,anionic surfactants and oxidizing agents (Supplementary Fig. 2A).The detergents were found to induce the proteolytic activity as en-zyme activity increased to 1.12-fold with Triton X-100, 1.75-foldwith Tween 80 and 3.35-fold with SDS (Supplementary Fig. 2A).Generally, the enzymes are less stable in strong anionic surfactantsuch as SDS. Haddar et al. (2009) reported detergent stable alkalineprotease which was stable in non-ionic surfactants but enzymeactivity was inhibited by SDS. An increment in protease residualactivity after incubation with these surfactants has been reportedfrom the protease of Bacillus sp. B001 (Deng et al., 2010). Thehigher stability of the protease characterized in this study indeedpreferred this enzyme for detergent formulations. The residualactivity of protease after incubation of 1 h with H2O2 and sodium

hypochlorite (1% v/v) was comparable to the same reported fromthe protease of Bacillus sp. (Haddar et al., 2009) when studied ata higher temperature (50 �C).

Moreover the increase in the proteolytic activity in the presenceof commercial laundry detergents after pre incubation of 60 min at50 �C suggested its eventual application in detergent industry(Supplementary Fig. 2B). The enzyme retained 100% of its initialactivity with Ariel and Surf excel followed by Wheel (67%). Thesefindings are superior to the earlier report of (Singh et al., 2001)where alkaline protease from Bacillus sp. SSR1 lost 63% of its initialactivity when incubated in detergent (Ariel) at a lower concentra-tion of 5 mg/ml for 1 h at 40 �C. However similar trend of stabilitywith different detergent formulations was reported from proteasesprojected as potential candidate for laundry industry (Deng et al.,2010; Haddar et al., 2009).

3.12. Washing performance analysis

Stain removal ability of crude enzyme was analyzed using cot-ton cloths stained with blood, tomato sauce and turmeric. Thetreatment of stained cloth with detergent supplemented withcrude protease gave a better stain removal over the wash perfor-mance employing detergent alone (Supplementary Fig. 2C). Thereflectance (%) of the stained cloth washed with detergent supple-mented with crude protease was higher than the cloth washedwith detergent alone (Table 4). The reflectance was monitored atwavelength 420 nm, 430 nm and 433 nm for blood, turmeric andtomato sauce respectively. The effect of temperature revealedmaximum reflectance (%) at 50 �C for blood and tomato saucestained cloth, and 60 �C for turmeric stained cloth (Table 4). Thestain removal by various protease enzymes were reported to beoptimum at temperature 45–60 �C (Oberoi et al., 2001; Beg andGupta, 2003). The enzyme concentration of 1690 U resulted inmaximum reflectance for all the staining agents (Table 5). Howeverno substantial difference was observed in reflectance with incuba-tion time for blood and tomato sauce therefore 5 min of incubationwas sufficient to remove these stains (Table 5). For the clothstained with turmeric paste, maximum reflectance was observedafter an incubation of 30 min. This study further involved theexamination of cloth fiber after treatment with detergent, deter-gent supplemented with protease and protease alone (in case ofblood stain) using scanning electron microscopy (SEM). The SEMimages of cotton cloth revealed characteristic parallel ridges anda very smooth surface after washing (Supplementary Fig. 2D). Nodegradation of cloth fiber can be seen thus the enzyme can be con-sidered for utilization as detergent additive. All the visual examin-ations of wash performance analysis are supplied asSupplementary Figs. 3–5. The enzyme was found to be a potentialdigester of blood stain even in the absence of detergent (Supple-mentary Table 1) therefore substantiated its proteolytic activity.

Table 5Effect of protease concentration and washing time on wash performance.

Reflectance (%)

Blood Tomato sauce Turmeric

Enzyme concentration (U)845 70.48 ± 1.64 86.76 ± 1.42 75.99 ± 1.221690 73.42 ± 2.23 96.85 ± 1.76 74.69 ± 1.315070 69.82 ± 0.21 93.64 ± 1.53 73.99 ± 1.10Incubation time (Min)5 51.55 ± 1.09 97.84 ± 1.35 31.4 ± 1.1515 52.44 ± 1.11 94.29 ± 1.54 67.7 ± 1.6430 54.53 ± 0.89 – 71.6 ± 1.32

D. Jain et al. / Bioresource Technology 115 (2012) 228–236 235

Similar results were also discussed by Beg and Gupta (2003) for theprotease enzyme isolated from B. mojavensis. Detergent proteasehydrolyzes large insoluble proteinaceous stain into small frag-ments which were removed from the fabric by detergent compo-nents or by water alone. Thus the removal of blood stain by theenzyme without detergent made it suitable for utilization inremoving the stains especially the hospital stains.

3.13. Analysis of casein hydrolysate

Comparison of the FTIR spectra of casein and its hydrolysate re-vealed lowering of absorbencies in the range of 1000–1500 cm�1

that confirmed the hydrolysis of casein (Supplementary Fig. 6A).The fingerprint region of the IR spectrum from 1800 to 800 cm�1

is a very useful part for analysis of proteinaceous material sincein this range the bonds forming the amide group (C@O, NAH,and CAN) absorb the frequency. The two most important vibra-tional modes of amides are the amide I vibration, caused primarilyby the stretching of the C@O bonds, and the amide II vibration,caused by deformation of the NAH bonds and stretching of theCAN bonds. The amide I vibration is measured in the range from1700–1600 cm�1 and the amide II region from 1600 to1500 cm�1 (Curley et al., 1998).

GPC analysis further confirmed the hydrolysis of casein as therewas decline in both molecular weight and polydisparsity was re-corded after an incubation of 12 h (Supplementary Fig. 6B).

4. Conclusion

This study extrapolate the potential of a polyextremotolerantstrain after characterization of alkaline, halothermoactive, surfac-tant stable, SDS induced serine type protease as its extracellularsecretion which have functional activity at broad pH ranges (8–12), temperatures (20–80 �C) and salinity up to 20%. Moreoverthe stability of the enzyme in the presence of various surfactants,bleach, oxidizing agents, solvents and in saline conditions togetherwith wash performance analysis confirmed the potential prospectsof this enzyme for detergent industry.

Acknowledgements

We gratefully acknowledge Dr. P. K. Ghosh (Director, CSMCRI)for his valuable suggestions. Deepti Jain and Sanjiv K. Mishra wishto acknowledge CSIR for awarding Senior Research Fellowship. Im-ran Pancha and Anupama Shrivastav acknowledge CSIR for thefinancial support. We would like to thanks Analytical Section forusing FT-IR and HPLC.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biortech.2011.10.081.

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