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Process Biochemistry 47 (2012) 1479–1487

Contents lists available at SciVerse ScienceDirect

Process Biochemistry

jo u rn al hom epage: www.elsev ier .com/ locate /procbio

urification, characterization and secondary structure elucidation of a detergenttable, halotolerant, thermoalkaline protease from Bacillus cereus SIU1

anjay Kumar Singh, Santosh Kumar Singh, Vinayak Ram Tripathi, Satyendra Kumar Garg ∗

enter of Excellence, Department of Microbiology, Dr. Ram Manohar Lohia Avadh University, Faizabad 224001, UP, India

r t i c l e i n f o

rticle history:eceived 31 March 2012eceived in revised form 12 May 2012ccepted 29 May 2012vailable online 8 June 2012

eywords:hermoalkaline proteaseurificationxidantsetergentsecondary structureD analysis

a b s t r a c t

A thermoalkaline protease with a molecular weight of 22 kDa was purified from the Bacillus cereus SIU1strain using a combination of Q-Sepharose and Sephadex G-75 chromatography. The kinetic analysesrevealed the Km, Vmax and kcat to be 1.09 mg ml−1, 0.909 mg ml−1 min−1 and 3.11 s−1, respectively, towardsa casein substrate. The protease was most active and stable at pH 9.0 and between a temperature rangeof 45–55 ◦C. It was fully stable at 0.0–2.0% and moderately stable at 2.5–10.0% (w/v) sodium chloride.Phenyl methyl sulfonyl fluoride, ethylene diamine tetra acetic acid and ascorbic acid were inhibitorywith regard to enzyme activity, whereas cysteine, �-mercaptoethanol, calcium, magnesium, manganeseand copper at concentration of 1.0 mM increased enzyme activity. Sodium dodecyl sulfate, Triton X-100,Tween 80, hydrogen peroxide and sodium perborate significantly enhanced protease activity at 0.1 and1.0% concentrations. In the presence of 0.1 and 1.0% (w/v) detergents, the protease was fairly stableand retained 50–76% activity. Therefore, it may have a possible application in laundry formulations. Aninitial analysis of the circular dichroism (CD) spectrum in the ultraviolet range revealed that the proteaseis predominantly a �-pleated structure and a detailed structural composition showed ∼50% �-sheets.The CD-based conformational evaluation of the protease after incubation with modulators, metal ions,

detergents and at different pH values, revealed that the change in the �-content directly correspondedto the altered enzyme activity. The protease combined with detergent was able to destain blood stainedcloth within 30 min.

. Introduction

Proteases are enzymes of utmost importance and are found inll cellular forms of life. Enzymes are utilized in various industries,nd proteases account for ∼66% of these industrial enzymes [1].mong these, alkaline proteases have vast applications, principally

n the food, detergent, leather and pharmaceutical industries [2].hermoalkaline proteases are the most commonly used of the alka-ine proteases because they can function at a pH range of 7.0–12.0nd a temperature range of 35–80 ◦C [1,3]. These properties of ther-oalkaline proteases make them useful for various commercial and

nvironmental applications. Proteases are commonly included inetergents for the removal of proteinaceous dirt, which was one ofhe early applications for these enzymes. Additionally, the presencef thermoalkaline proteases in detergents allows for easy washing,

ven at elevated temperatures [4].

Bacterial proteases are also known for their halotolerance andustained activity in the presence of high salt concentrations

∗ Corresponding author. Tel.: +91 9454755166/5278 245330;ax: +91 5278 246330.

E-mail address: sk garg001@yahoo.com (S.K. Garg).

359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.procbio.2012.05.021

© 2012 Elsevier Ltd. All rights reserved.

[5]. This property facilitates their use in saline conditions. Dur-ing industrial applications, the proteases are required to functionunder diverse environmental conditions where surfactants, oxi-dants, detergents and solvents are present. Therefore, the stabilityof alkaline proteases in the presence of metal ions, denatu-rants, surfactants, oxidants, detergents and organic solvents is ahighly desired characteristic for their use in industrial applications[6,7].

In general, the use of industrial proteases remains highly depen-dent on their stability during isolation, purification and storage, inaddition to their robustness in the presence of solvents, surfactantsand oxidants [8]. Therefore, it is necessary to determine the opti-mal physical and chemical conditions under which an enzyme ismost active and stable. Additionally, there is a growing awarenessthat structural studies need to be performed under the conditionsin which the proteins actually operate and perform their biologicalfunction [9]. Circular dichroism (CD) has become an increasinglyvaluable technique to elucidate insights into the structure of bio-logical molecules. The differential absorption (absorption of left-

and right-handed circularly polarized light due to the chiralitycenters nearby to the peptide bonds) of a peptide bond is depen-dent on its conformational relationship to neighboring peptidebonds. This characteristic has been exploited to obtain information

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n the number of secondary structural elements in a number ofroteins.

Secondary structure determination of several proteins has beenuccessfully achieved with CD [10]. This is a beneficial techniqueecause very little sample is used (<0.5 mg ml−1) and the technique

s non-destructive in nature, which makes it possible to re-usehe sample for other applications. In addition, relative changesn protein secondary structure due to environmental influences,uch as pH, temperature, and modulators, can be monitored veryccurately. Therefore, it is easy to establish the structure–functionelationship of an enzyme in very little time.

With this is mind, this study aimed to purify and characterizen alkaline protease. The effect of pH, surfactants, oxidants, deter-ents, modulators and metal ions on the secondary structure ofhe purified protease was also studied by CD in the far-ultravioletUV) range. The potential use of the protease as an enzyme for stainemoval was tested on blood stained cloth.

. Materials and methods

.1. Microorganism

Bacillus cereus SIU1 isolated in our laboratory was used in this study. The isolateas antibiotic and heavy metal resistant, halotolerant and produced a thermoalka-

ine protease [11]. The bacterial culture was maintained over nutrient agar slantspH 9.0) and stored at 4 ◦C.

.2. Inoculum preparation and protease production

For inoculum preparation, the bacterial culture was grown in modified glucoseeast extract (GYE) broth containing the following (gl−1 distilled water): glucose,.0; peptone, 15.0; yeast extract, 4.0; CaCl2, 0.2; and NaCl, 5.0. A loopful of culturehat had been grown for 24 h was inoculated into 99.0 ml of the above medium, pH.0 (adjusted after autoclaving using a sterilized 1.0 M Na2CO3 solution in distilledater) in Erlenmeyer flasks and incubated at 50 ± 1 ◦C in a shaking incubator at

50 rpm. Briefly, 1.0 ml of the mother culture of B. cereus SIU1 isolate with an OD = 0.5A620; 1.0 cm cuvette) containing 3.4 × 107 cfu ml−1 was inoculated into 99 ml ofhe above modified GYE medium, pH 9.0 and incubated at 50 ± 1 ◦C in a shakingncubator at 150 rpm. After a 20 h incubation, the culture broth was centrifuged andhe cell-free supernatant was used for further studies.

.3. Protease purification

The extracellular protease produced by B. cereus SIU1 was fractionated by gradedrecipitation using ammonium sulfate. Initially, a 30% saturation step was per-ormed and the resulting precipitate was removed from the solution. Thereafter,he ammonium sulfate saturation was increased to 75%, and the resulting pre-ipitate was collected and dissolved in a minimum volume of 50.0 mM Tris–HCluffer, pH 9.0. This enzyme solution was dialyzed against the same buffer for 24 ht 4 ◦C and the buffer was changed at 6 h intervals. The dialyzed protease was con-entrated by lyophilization, loaded onto a Q-Sepharose column (2.0 cm × 10.0 cm)re-equilibrated with 50.0 mM Tris–HCl buffer, pH 9.0 and eluted with a linear gra-ient of 0.0–0.5 M NaCl in the same buffer at a flow rate of 0.2 ml min−1. The fractionsith protease activity were collected, pooled and dialyzed as described above. The

ractions were then loaded onto a Sephadex G-75 column (2.0 cm × 50.0 cm) pre-quilibrated with 50.0 mM Tris–HCl buffer, pH 9.0 and eluted with the same buffert a flow rate of 0.2 ml min−1. Fractions with activity were pooled, lyophilized andtored at −20 ◦C.

.4. Molecular weight determination

The molecular weight of the purified protease was determined by SDS-PAGEccording to the method of Laemmli [12] using a 12.5% resolving gel. Electrophoresisas performed at 200 V and the protein bands were visualized with Coomassierilliant Blue R-250 staining. The molecular weight of the protease was determinedy comparison with standard molecular weight markers of 97.4, 66, 43, 29, 20.1nd 14.3 kDa (Bangalore Genei Pvt. Ltd., Bangalore, Karnataka, India). The moleculareight was determined by software built into the Genei-UviTech gel documentation

ystem (Bangalore Genei Pvt. Ltd., Bangalore, Karnataka, India).Zymography was performed according to the method described by Singh et al.

13]. The gel was then incubated at 55 ◦C for 2 h in sodium carbonate–bicarbonateuffer, pH 9.0, followed by staining as described above to visualize the zone of gelatinydrolysis.

istry 47 (2012) 1479–1487

2.5. Characterization of purified protease

2.5.1. Kinetic analysesThe purified protease was used in kinetic studies for determination of the Km,

Vmax and kcat with Hammersten casein as a substrate. The enzyme was incubatedwith different concentrations of casein substrate, ranging from 0.25 to 4.0 mg andthe activity was assayed by standard assay method described in Section 2.8.1. TheKm, Vmax and kcat were calculated using a Lineweaver–Burk double reciprocal plotof the Michaelis–Menten equation.

2.5.2. Effect of temperature and pHThe effect of temperature on the activity of the purified protease was determined

by standard assay at 35, 45, 55, 65 and 75 ± 0.5 ◦C. The protease stability was assessedby incubation of the enzyme for 30 min at the above temperatures. The residualprotease activity was estimated under standard conditions according to the methoddescribed by Anson [14].

The effect of pH on enzyme activity was determined using casein as the sub-strate, which was dissolved in different buffers of pH 5.0–12.0. The enzyme stabilityat various pH values was determined by pre-incubating the enzyme with an equalvolume of each buffer for 30 min at 55 ± 1 ◦C. The residual protease activity wasassayed under standard conditions at an optimized temperature of 55 ± 1 ◦C.

2.5.3. Effect of NaClThe purified enzyme was diluted with an equal volume of NaCl solution with

concentrations ranging from 0.0 to 12.0% (w/v) and incubated for 30 min at 55 ± 1 ◦C.The residual protease activity was assayed under standard conditions.

2.5.4. Effect of modulators and divalent cationsPhenyl methyl sulfonyl fluoride (PMSF), EDTA, �-mercaptoethanol, ascorbic acid

(vitamin C) and cysteine were used to assess their effect on protease stability atdifferent concentrations. The solutions were prepared at concentrations rangingfrom 1.0 to 5.0 mM. The metal ions Ca2+ (calcium chloride), Mg2+ (magnesium sul-fate), Zn2+ (zinc chloride), Fe2+ (ferrous sulfate), Ni2+ (nickel chloride), Co2+ (cobaltchloride), Cu2+ (cupric chloride), Mn2+ (manganese chloride) and Hg2+ (mercuricchloride) were also tested for their effects on protease stability. The salt solutionswere used at 0.1, 1.0 and 10.0 mM. The enzyme was diluted separately in an equalvolume with each solution of modulator or metal ions and incubated for 30 min at55 ± 1 ◦C. Following incubation, the residual protease activity was assayed understandard conditions.

2.5.5. Effect of surfactants and oxidantsThe surfactants and oxidants Triton X-100, Tween 80, H2O2 (each in v/v) and

sodium dodecyl sulfate, sodium perborate (each in w/v) were used to assess theireffects on protease stability. The solutions were prepared at concentrations of 0.1,1.0, 5.0 and 10.0%, and the enzyme was incubated with each solution for 30 min at55 ± 1 ◦C. The residual protease activity was assayed.

2.5.6. Effect of commercial detergentsVarious commercially available detergents, including Rin, Surf, Ariel, Tide,

Wheel, Nirma, More and Ghari were used to determine their effects on proteasestability with regard to the potential use of the enzyme in industrial applications inthe detergent industry. Each detergent solution was prepared at concentrations of0.1, 1.0, 5.0 and 10.0% and the purified protease was mixed with an equal volume ofeach solution. After incubation for 30 min at 55 ± 1 ◦C, the residual protease activitywas assayed.

2.6. CD analysis for secondary structure elucidation of the protease

CD spectra were recorded in the far-UV range (190–240 nm) with a proteaseconcentration of 0.1 mg ml−1 in 50.0 mM sodium carbonate–bicarbonate buffer, pH9.0 at 25 ◦C using a Jasco J-815 spectropolarimeter. The protease was most active andstable at pH 9.0; therefore, the secondary structure at this pH was used as a reference.An average of 3 scans using a quartz cuvette of 0.1 cm length was recorded at a scanrate of 50 nm min−1. The bandwidth applied was 1.0 nm with a response time of 1 s.The ellipticity values (�) for every nanometer wavelength increase were obtained in‘mdeg’ directly from the instrument and were recorded online with a computer [15].The elements of the protease secondary structure were analyzed with the softwareSpectra Manager Version 1.00.00.

The effects of environmental factors on the secondary structure of the proteasewere also studied. The effect of various pH values was determined by pre-incubatingthe purified protease (0.2 mg ml−1) with an equal volume of different buffers(100 mM) for 30 min at 25 ± 1 ◦C. The buffers used were citric acid–sodium citratebuffer, pH 5.0, sodium phosphate buffer, pH 7.0 and sodium carbonate–bicarbonatebuffer pH 10.0 and 11.0.

To study the effects of EDTA, PMSF and �-mercaptoethanol on enzyme stabil-ity, the purified protease (0.2 mg ml−1) was pre-incubated with an equal volumeof 1.0 mM solutions of the above chemicals for 30 min at 25 ± 1 ◦C. The surfactantsand oxidants Triton X-100, Tween 80, sodium dodecyl sulfate, H2O2 and sodiumperborate were also studied with regard to their effects on protease structure. The

chemistry 47 (2012) 1479–1487 1481

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Fig. 1. SDS-PAGE analysis of the partially purified protease from strain SKG-1. Lane1: crude extract; Lane 2: ammonium sulfate precipitate of protease; Lane 3: pro-tease after Q-Sepharose chromatography; Lane 4: protease after Sephadex G-75chromatography; Lane 5: molecular weight markers (kDa): phosphorylase b – 97.4;

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S.K. Singh et al. / Process Bio

olutions were prepared at 1.0% concentrations and an equal volume of enzyme wasncubated with each solution for 30 min at 25 ± 1 ◦C.

After the 30 min incubations under various environmental conditions, the CDpectra were recorded as described above. Blank spectra of each suspension withoutrotease were used to correct the observed spectra. These studies were carried outt the National Institute of Pharmaceutical Education and Research (NIPER), Mohali,ndia.

.7. Washing efficiency of protease

To evaluate the stain removal capability of the protease, clean cotton clothieces (4.0 cm × 5.0 cm) were soiled with blood, dried for 30 min and then washedith water. The stained cloth pieces were incubated separately with 1.0 mg of puri-ed alkaline protease, 0.2% (w/v) of the commercial detergent Ariel and 0.2%, w/vetergent plus 1.0 mg of purified alkaline protease for 30 min, followed by rinsingith water for 2 min. The washed cloth pieces were then dried and the extent ofestaining was compared.

.8. Analytical determinations

.8.1. Enzyme assayThe alkaline protease activity was assayed using the casein digestion method of

nson [14]. One milliliter of enzyme was used for protease assay as described earlier11]. One unit of enzyme activity is defined as the amount of enzyme that liberates.0 �g of tyrosine min−1.

.8.2. Protein estimationThe method of Lowry et al. [16] was followed for protein concentration estima-

ion using BSA as the standard.

.9. Statistical analysis

The experiments were performed thrice, in triplicate each time. The standardeviation for each experimental result was calculated using Microsoft Excel. Thetandard deviation for each value was ≤5%.

. Results and discussion

.1. Protease purification and molecular weight determination

The isolate, B. cereus SIU1 produced 633 U of protease ml−1

uring a 20 h incubation in modified GYE broth. The pro-ease was purified by ammonium sulfate fractionation followedy Q-Sepharose and Sephadex G-75 chromatography (Table 1).mmonium sulfate precipitation was used to concentrate therotease, which resulted in a 2.4-fold purification with an 83%ecovery rate. Q-Sepharose chromatography resulted in a 7.1-foldurification with a 60% recovery rate. After Sephadex G-75 chro-atography, the purification was 9.5-fold with a final recovery of

8%. The molecular weight of this alkaline protease was determinedo be 22 kDa by SDS-PAGE (Fig. 1). An activity gel analysis confirmedhat the purified protease is a single monomeric protein (Fig. 1).ther researchers have also reported the isolation of proteasesith low molecular weights from Bacillus isolates. Adinarayana

t al. [17] purified an alkaline protease of 15 kDa from Bacillus sub-ilis PE-11 using ammonium sulfate precipitation and Sephadex

-200 chromatography. Gessesse et al. [18] purified an alkalinerotease of 24 kDa from Bacillus pseudofirmus AL-89 to 22.6-foldurity with an 18% recovery rate. They used ammonium sulfaterecipitation, DEAE-Sepharose ion exchange chromatography and

able 1teps involved in the purification of the protease produced by B. cereus SIU1.

Purification steps Total protein (mg) Total activity (U)

Crude extract 479 63 300

Ammonium sulfateprecipitation(30–75%)

165 52 470

Q-Sepharosechromatography

40.53 37 980

Sephadex G-75chromatography

19.18 24 054

bovine serum albumin – 66.0; ovalbumin – 43.0; carbonic anhydrase – 29.0; soya-bean trypsin inhibitor – 20.1 and lysozyme – 14.3; Lane 6: zymography of purifiedprotease.

Sephadex G-75 gel filtration chromatography. Gupta et al. [19] puri-fied an alkaline protease from B. pseudofirmus to 10-fold purity withan 82% yield using a single step method with a Phenyl Sepharose6 Fast Flow column. The apparent molecular mass of this protease,based on SDS-PAGE, was estimated to be 29 kDa. A halotolerantalkaline protease of 28 kDa was purified from Bacillus clausii I-52 using a combination of Diaion HPA75, phenyl-Sepharose andDEAE-Sepharose column chromatography [5]. Sareen and Mishra[7] purified a 55 kDa alkaline protease from the cell-free super-natant of Bacillus licheniformis RSP-09-37 using ammonium sulfateprecipitation and affinity chromatography with �-casein agarose.The last step of purification resulted in a 55% yield and an 85-foldpurification. Subba Rao et al. [8] purified a protease of 39.5 kDafrom Bacillus circulans using ammonium sulfate precipitation andSephadex G-100 chromatography.

3.2. Characterization of purified protease

3.2.1. Kinetic analysesKinetic analysis of the protease using casein as a sub-

strate revealed the Km and Vmax to be 1.09 mg ml−1 and

Specific activity (U mg−1) Recovery (%) Fold purification

132 100 1318 83 2.4

937 60 7.1

1254 38 9.5

1482 S.K. Singh et al. / Process Biochemistry 47 (2012) 1479–1487

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0.0–2.0% (w/v) NaCl when incubated for 30 min. Further increases

ig. 2. Lineweaver–Burk plot for kinetics determinations of purified protease usingammersten casein as the substrate.

.909 mg ml−1 min−1, respectively (Fig. 2), as determined by aineweaver–Burk plot. The kcat value of 3.11 s−1 indicated both

high affinity and catalytic efficiency of this protease towardsasein. Additionally, the small Km value implies strong affinity;herefore, there is little substrate requirement for the enzymeatalyzed reaction velocity of Vmax/2. Gupta et al. [19] purifiedn alkaline protease from B. pseudofirmus with a Km and Vmax

o be 2.0 mg ml−1 and 289.8 �g min−1, respectively, towards aasein substrate. Fig. 2 reveals that the Km value of the SIU1nzyme is much less than that of the B. pseudofirmus protease,evealing a higher affinity for casein. A detergent stable, serinerotease from B. circulans exhibited a Km of 0.597 mg ml−1 andmax of 13 825 �mol min−1 towards a casein substrate [8]. Dengt al. [20] cloned and expressed the AprB protease from B. subtilisB600. This protease exhibited Km and Vmax values of 0.44 mM

nd 12.54 × 103 U mg−1 pure protein, respectively, towards a caseinubstrate. The kcat value for this enzyme was 250.86 × 103 min−1.herefore, it appears that the kinetic parameters vary from enzymeo enzyme and substrate to substrate.

.2.2. Effect of temperature and pHThe purified protease from strain SIU1 was active and stable

hroughout the temperature ranges used in this study. The temper-ture for maximum protease activity was in the range of 45–55 ◦CFig. 3A). At both 65 and 75 ◦C, the protease was quite active, with0 and 59% activity, respectively. Therefore, the results reveal thathe optimal temperature for SIU1 protease activity is 35–55 ◦C.

As far as protease stability is concerned, the enzyme retained00% activity in the temperature range of 35–55 ◦C. Even at 65 and5 ◦C, the protease was stable, as evidenced by residual activitiesf 61% and 48%, respectively (Fig. 3A). The broad, optimal tempera-ure range of 35–55 ◦C for maximum protease activity and stabilityeveals the thermostable nature of this protease from B. cereus SIU1.he activity of alkaline proteases in broad temperature ranges is aesired characteristic for their application in detergent formula-ions. Manachini et al. [21] have reported an alkaline protease fromacillus thermoruber that is active in a broad temperature rangef 10–80 ◦C, with an optimum of 45 ◦C. An alkaline protease from. clausii I-52 was observed to be stable in the temperature rangef 30–80 ◦C, with almost 100% activity in the temperature rangef 30–50 ◦C [6]. Sareen and Mishra [7] reported a thermoalkaline

rotease from B. licheniformis. The purified enzyme was active at

temperature range of 30–90 ◦C and the maximum activity of therotease was observed at 50 ◦C. Abusham et al. [22] also reported

pH

Fig. 3. Effects of temperature (A) and pH (B) on protease activity and stability.

an alkaline protease from B. subtilis strain Rand with 100% stabilityin the temperature range of 35–55 ◦C.

In the pH activity experiment, the protease was observed to be≥78% active in the pH range of 7.0–11.0, with 100% activity at pH9.0. At pH 5.0, 6.0 and 12.0, the protease activity was reduced to 42,76 and 55%, respectively (Fig. 3B). The pH stability studies revealedthat the protease was variably stable (18–100%) in the complete pHrange that was studied. However, it exhibited good stability (≥70%)in the pH range of 7.0–11.0, with 100% stability at pH 9.0 (Fig. 3B).Even at pH 6.0 and 12.0, the residual activity was ≥48%. The remark-able activity and stability over a wide pH range reveals the highlyalkaline nature of this protease, which makes it suitable for applica-tions in alkaline environments and with detergents. Several otherresearchers have also described alkaline proteases with broad pHactivities and stabilities. Manachini et al. [21] purified an alkalineprotease from B. thermoruber that is active in a broad pH range of7.5–11.0, with maximum activity at pH 9.0. An alkaline proteasefrom B. subtilis PE-11 was observed to be stable in the pH rangeof 8.0–11.0, with the highest activity at pH 10.0 [17]. Joo et al. [6]reported an alkaline protease from B. clausii I-52 that was stable inthe pH range of 4.0–12.0, with maximum activity at pH ∼ 12.0. Analkaline protease from B. licheniformis RSP-09-37 was observed tobe active in a broad pH range of 4.0–12.0, after a 20 min incubation.It was 100% active at pH 10.0 and exhibited 14, 28 and 40% residualprotease activities at pH 4.0, 5.0 and 12.0, respectively [7].

3.2.3. Effect of NaClThe protease stability was almost 100% at concentrations of

in salt concentration were inhibitory for protease stability. The pro-tease retained 98, 92, 87, 80, 67, 43 and 28% activity at 2.5, 3.0, 4.0,5.0, 7.0, 9.0 and 10.0% NaCl concentrations, respectively. Sodium

S.K. Singh et al. / Process Biochemistry 47 (2012) 1479–1487 1483

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Fig. 4. Effect of NaCl on protease stability after a 30 min incubation.

hloride at even higher concentrations further reduced the pro-ease stability, with only 12 and 3% residual activities retained at1.0 and 12.0% salt concentrations, respectively (Fig. 4).

It is well known that the presence of high salt concentra-ions destabilizes ion pairs and salt bridges and alters electrostaticnteractions between charged amino acids, leading to enzymeenaturation [23]. The stability of the SIU1 protease in the presencef high salt concentrations revealed its moderately to highly halo-olerant nature. This could be attributed to the halotolerant naturef our isolate [11]. Investigation of the protein molecular structureseveals that Na+ ions have a strong affinity for the side chain car-oxylates and backbone carbonyls, thereby weakening salt bridgesnd secondary structure hydrogen bonds [23].

Salt tolerance of alkaline proteases makes their applicationor washing under saline conditions possible. Joo and Chang [5]eported an alkaline protease from halotolerant B. clausii I-52 hav-ng good activity in the range of 0.0–10.0% (w/v) NaCl. B. cereus

TCC 6840 produced an alkaline protease with activity in NaCloncentrations up to 5.0%. At an even higher NaCl concentrationf 10.0% (w/v), the protease activity only decreased to 40% [24].rotease stability at high salt concentrations is a desirable charac-eristic because NaCl is used as a core component in granulationf the protease prior to its addition to detergents [25]. Addition-lly, the ground water from different Indian geoclimatic regionss saline, which may be detrimental for the cleaning potential ofetergents; therefore, the presence of a halotolerant alkaline pro-ease in detergents will make efficient washing less difficult.

.2.4. Effect of modulators and divalent cationsPMSF, EDTA and ascorbic acid had adverse effects on pro-

ease stability and activity. PMSF was completely inhibitory for thenzyme at all concentrations tested. On the other hand, the extentf protease inhibition increased with increasing concentrationsf EDTA and ascorbic acid (Fig. 5A). In contrast, 1.0–3.0 mM �-ercaptoethanol and 1.0–5.0 mM cysteine did not have any effect

n protease activity, indicating its stability in the presence of reduc-ng agents. However, �-mercaptoethanol at higher concentrationsf 4.0 and 5.0 mM decreased the protease activity by ∼20% (Fig. 5A).omplete inhibition of protease activity by PMSF, even at 1.0 mM,

ndicates that the enzyme is a serine alkaline protease with a serineesidue in its active site. PMSF blocks the active site of proteases byulfonating the essential serine residue, resulting in complete inhi-ition of protease activity [17]. The decrease in activity by EDTAeveals the requirement of metal ion(s) for this protease activity

ecause EDTA removes metal ion(s) through chelation. A serinelkaline protease from B. licheniformis RSP-09-37 was inhibitedompletely by 10.0 mM PMSF within 20 min. Similar to our find-ngs, this protease was also inhibited by EDTA and exhibited only 55

Fig. 5. Effects of modulators (A) and divalent cations (B) on protease stability aftera 30 min incubation.

and 50% residual activity in the presence of 1.0 and 10.0 mM EDTA,respectively, after a 20 min incubation, indicating the requirementof metal ions for the activity of this enzyme [7]. In our study, thedecreased activity of the SIU1 protease in the presence of ascorbicacid may be due to the acidic environment. The presence of ascorbicacid is known to create a highly acidic environment (pH ∼ 4.0) [26].In the presence of 1.0–3.0 mM �-mercaptoethanol and 1.0–5.0 mMcysteine, protease activity remained unaffected, indicating that

SH groups are not essential for catalytic activity, but are nec-essary for the maintenance of the three-dimensional structure ofthe enzyme [3,27]. The stability of the SIU1 protease in the pres-ence of reducing agents is also demonstrated by SDS-PAGE analysis,where it was observed to be a monomeric polypeptide. Further-more, zymography analysis confirmed the monomeric structureof this protease (Fig. 1). Correa et al. [3] also reported the activ-ity of a keratinase produced by Bacillus sp. P7 that was only slightlyinhibited by the reducing agent, �-mercaptoethanol. The residualprotease activities of the keratinase were found to be 88.0 and 72.2%in the presence of 1.0 and 5.0 mM �-mercaptoethanol, respectively.

Among metal ions, calcium, manganese and copper increasedthe protease activity up to 112, 105 and 102%, respectively, at aconcentration of 0.1 mM, while magnesium and zinc had no effect.Mercury, cobalt, iron and nickel reduced the protease activity to68, 81, 94 and 98%, respectively (Fig. 5B). Furthermore, at a con-centration of 1.0 mM, calcium, magnesium, copper and manganese

increased the protease activity up to 120, 115, 115 and 110%,respectively. Zinc had no effect on protease activity, even at a con-centration of 1.0 mM, while other metal ions inhibited the proteaseactivity to a variable extent (Fig. 5B). At the highest concentration

1484 S.K. Singh et al. / Process Biochemistry 47 (2012) 1479–1487

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ig. 6. Effects of oxidants and surfactants (0.1–10.0%) on protease stability after a0 min incubation.

f 10.0 mM, each metal ion exerted inhibitory effects except cal-ium. In the presence of 10.0 mM calcium, the protease activityas slightly increased to 105%. Mercury drastically reduced therotease activity and only 5% of the residual activity remainedfter a 30 min incubation with 10.0 mM mercury. The proteaseetained 30–98% activity at a 10.0 mM concentration of other metalons tested (Fig. 5B). Metal ions are well known to induce bothnzyme activity and thermostability. Calcium, magnesium, coppernd manganese exhibited positive effects on protease activity, indi-ating that such metal ion(s) are required for increased activity ofhis protease. Several other researchers have also demonstratedncreased protease activity in the presence of different metal ion(s)17]. Joshi et al. [24] reported the positive effects of iron and cobaltn the protease activity of B. cereus MTCC 6840. Sareen and Mishra7] reported a metal-dependent serine alkaline protease from B.icheniformis RSP-09-37. The protease activity increased in the pres-nce of Ca2+, Mg2+ and Mn2+ metal ions, while Zn2+ and Cu2+ hadnhibitory effects. This protease exhibited only 55% activity in pres-nce of 1.0 mM EDTA, indicating that removal of metal ions throughhelation by EDTA was responsible for the decreased proteasectivity.

.2.5. Effect of oxidants and surfactantsThe protease demonstrated significant stability in the presence

f surfactants, detergents and oxidants. Sodium dodecyl sulfateSDS), Triton X-100, Tween 80, H2O2 and sodium perborate at con-entration of 0.1 and 1.0% (w/v) increased the protease activity to

maximum of 129%. Furthermore, higher concentrations of Tri-on X-100, Tween 80, H2O2 and sodium perborate did not affecthe protease activity significantly and the residual protease activ-ty was in the range of 65–100%. In contrast, higher concentrationsf SDS were inhibitory to protease stability and the protease activ-ty decreased to 58 and 16% at 5.0 and 10.0% (w/v) concentrations,espectively (Fig. 6). The action of the detergents on the protein cane correlated to their hydrophilic/lipophilic balance (HLB), which isefined as how a detergent distributes between polar and nonpo-

ar phases. Triton X-100, with a HLB of 13.5, has a less detrimentalffect compared to SDS, with a HLB of 40 at a 0.5% concentration [7].t is interesting to observe that the protease activity significantlyncreased, up to 129%, in the presence of surfactants, detergentsnd oxidants at concentrations of 0.1 and 1.0% (w/v), making ituitable as a detergent additive. Commercially available proteases,uch as Subtilisin Carlsberg, Subtilisin BPN’, Alcalase, Esparase andavinase, exhibit great stability in the presence of detergents; how-

ver, most are unstable in the presence of oxidants and bleaches [2].herefore, the stability of the B. cereus SIU1 protease suggests that its a better candidate for a detergent additive. Proteases from Bacil-us sp. that are stable in the presence of surfactants, detergents and

Fig. 7. Effect of commercial detergents (0.1–10.0%, w/v) on protease stability ofstrain SIU1 after a 30 min incubation.

oxidants have been studied and reported by other researchers[6]. Sana et al. [28] observed that a protease from gamma-Proteobacteria was completely stable in the presence of thelaboratory detergents Tween 80 and Triton X-100, oxidizing agents,reducing agents, commercial detergents and bleaches, includinghydrogen peroxide and sodium perborate. Beena et al. [29] reportedan alkaline protease from a B. cereus isolate. The protease activ-ity increased in the presence of 10.0 mM SDS and Tween 80 aftera 30 min incubation. Even in the presence of 10.0 mM H2O2, theactivity was almost completely (99%) retained.

3.2.6. Effect of commercial detergentsThe protease from B. cereus SIU1 was remarkably stable

(50–93%) in the presence of 0.1 and 1.0% (w/v) commercialdetergents. Furthermore, at a 5.0% concentration of commercialdetergent, the protease retained residual activity in the range of15–28% (Fig. 7). Even at a concentration of 10.0%, the residual pro-tease activity was 7, 10 and 5% in Rin, Ariel and Wheel detergents,respectively. Such a remarkable stability of our protease in the pres-ence of higher detergent concentrations reveals its usefulness as adetergent additive. Detergent stability of a protease is an importanttrait for its industrial application. Generally, detergent powders areused at <1.0% (w/v) concentrations for washing clothes. Because ourprotease exhibited 50–76% stability in the presence of 1.0% (w/v)commercial detergents, it is highly suitable for use in detergents.Detergent-stable proteases with variable stability in the presence ofdifferent detergents have been studied by several other researchers[5,8,28]. Adinarayana et al. [17] reported an alkaline protease fromB. subtilis PE-11 with 87–96% residual activity when incubated with0.7% (w/v) commercial detergents for 30 min.

3.3. CD analysis for secondary structure of protease

The secondary structure of the purified protein at pH 9.0 wasused as a reference structure. An initial analysis of the far-UV CDspectrum (Fig. 8A) revealed that the native protein at pH 9.0 is an�-, �-polypeptide with a majority of �-structure. Although nega-tive ellipticity was present, no clear negative peak, characteristic of�-helical structure, was evident at 222 nm. These results stronglysuggested that the protease from B. cereus SIU1 is predominantly a�-rich protein (Fig. 8A). The detailed structural composition of theprotease indicated a large fraction (∼50%) of �-sheets (Table 2). CD-

based conformational evaluation of the protease after incubationat different pH values and with modulators, metal ions and deter-gents, demonstrated that deviation in the �-content was directly

S.K. Singh et al. / Process Biochemistry 47 (2012) 1479–1487 1485

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ig. 8. Far-UV CD spectra of (A) purified protease (0.1 mg ml−1) in 50.0 mM sodiumrotease incubated at pH 11.0 for 30 min; and (D) protease incubated with H2O2 fo

orrelated with the altered (increased/decreased) protease activityTable 2).

At an acidic pH of 5.0, the protease lost its secondary struc-ure. The �-structures were greatly reduced and the percentage

f unordered structures were dominant (Fig. 8B). There was a con-omitant reduction in protease activity to only 28%. Similarly, athe highly alkaline pH of 11.0, the percentage of �-structures wereeduced, while the percentage of unordered structure increased

able 2D secondary structure and residual activity of purified protease (0.1 mg ml−1) at pH 9.0

Environmental condition CD secondary structure content

�-Helix �-Sheets

pH 9.0 (reference) 13.1 49.8

pH 5.0 4.1 15.1

pH 7.0 8.0 40.0

pH 10.0 12.7 44.1

pH 11.0 15.2 33.8

Protease + Ca2+ (1.0 mM) 12.6 52.4

Protease + EDTA (1.0 mM) 18.6 39.2

Protease + PMSF (1.0 mM) 13.9 48.2

Protease + �-mercaptoethanol (1.0 mM) 16.1 51.6

Protease + SDS (1.0%) 11.5 53.0

Protease + Triton X-100 (1.0%) 12.1 52.6

Protease + Tween 80 (1.0%) 10.4 52.2

Protease + H2O2 (1.0%) 13.5 53.4

Protease + Sodium perborate (1.0%) 12.7 54.0

onate–bicarbonate buffer, pH 9.0; (B) protease incubated at pH 5.0 for 30 min; (C)in.

(Fig. 8C), compared to the native protein at pH 9.0. The proteaseactivity also corresponded in a similar manner. Therefore, it wasclear that with a reduction in �-content, the protease activity wassignificantly reduced (Table 2). The extent of �-structures at other

pH values also corresponded with their residual protease activities.The presence of EDTA reduced the percentage of �-structures andthe protease activity also decreased. Although PMSF had no signif-icant effect on the secondary structure of the protein, its presence

and all other environmental exposures after a 30 min incubation.

(%) Protease activity (%)

Turns Unordered

17.4 19.7 10027.6 53.2 2821.2 30.8 9120.4 22.8 9219.8 31.2 7018.5 16.5 12012.4 29.8 9116.6 21.3 013.3 19.0 10618.7 16.8 12617.3 18.0 10518.1 19.3 11810.7 22.4 12415.9 17.4 105

1486 S.K. Singh et al. / Process Biochemistry 47 (2012) 1479–1487

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ig. 9. Effect of protease on blood stain removal: (A) untreated; (B) treated withrotease.

ompletely inhibited the protease activity. In the presence of Ca2+,-mercaptoethanol, surfactants, oxidants and detergents, the per-entage of �-structures increased. Accordingly, these treatmentsncreased the protease activity to variable extents (Table 2). There-ore, the findings indicated that the purified protease from B. cereusIU1 is predominantly a �-protein. In the presence of H2O2, theercentage of �- and unordered structures increased more than

n the native protease at pH 9.0 (Fig. 8D and Table 2). However,he protease activity increased to 124%, indicating that the activ-ty is affected more by the �-structure, and once again confirminghat this protease is predominantly a �-protein. Tornvall et al. [30]eported that hydrogen peroxide has been shown to oxidize theethionine, cysteine, tyrosine and tryptophan residues in various

roteins. However, the number of oxidized residues, and the effecthereof, differs widely from protein to protein, depending on theosition of the sensitive amino acid and its role in the stability orctivity of the enzyme.

The SIU1 protease is a �-rich protein and the change in proteasectivity follows the pattern of change in �-sheets (Fig. 8). Another-rich protein with ∼30% �-pleated structure is �-chymotrypsin

31]. In contrast, our protease differs from the Subtilisin novo andubtilisin BPN’ from Bacillus amyloliquefaciens because both of theseroteins are �/� proteins as demonstrated by their secondary andertiary structures [10].

At an acidic pH of 5.0, the loss of �-structures and increase innordered structures occurred, leading to a dramatic loss of pro-

ease activity. Other environmental factors did not exert such arofound effect on the �-content of the protease. The factors underhich the �-structure of the protease increased also exerted a pos-

tive effect on the alkaline protease activity (Table 2). Our findings

gent alone; (C) treated with protease alone; and (D) treated with detergent plus

are in accordance with Bhattacharyya and Babu [32], who reporteda trypsin protease inhibitor to be a �-protein (∼40% �-sheets).Similar to our findings, they also observed that under extremeheat and acid-alkali conditions, deviation occurred between the �-helix and �-sheet content. This deviation was in accordance withthe protease inhibitory activity. The increase in �-sheet contentcorrelated with the increase in the protease inhibitory activity. Sim-ilarly, the far-UV (200–260 nm) CD spectra of Wrightin, a serineprotease from the plant Wrightia tinctoria, revealed informationregarding the secondary structure of the protein. The native spectraat pH 7.0 showed a negative peak at 215 nm, suggesting that the sec-ondary structure was predominantly �-sheets [33]. The secondarystructure of an alkaline protease, AL-20, from Nesterenkonia sp. wasstudied by Bakhtiar et al. [15]. The far-UV CD spectrum of the AL-20 protease at pH 10.0 (10.0 mM glycine–NaOH buffer) revealed itssecondary structure to be a combination of �-helix and �-sheetstructures. The secondary structure was found unaffected evenafter a prolonged storage of 24 h at 50 ◦C. Zhang et al. [34] studiedthe secondary structure of the Ca2+-binding domain (RTX) of a Pseu-domonas aeruginosa alkaline protease with far-UV CD spectroscopy.At low Ca2+ levels, the RTX domain exhibited fewer �-structures.Calcium addition shifted the absorbance minimum to 217 nm, con-sistent with a �-rich structure. At and above 50.0 mM Ca2+, theabsorbance shifted to a single minimum at 217 nm, revealing thecomplete dominance of �-structures.

3.4. Washing efficiency of protease

The results in Fig. 9 indicate that the purified protease from B.cereus SIU1 has good blood stain removal capability. Incubation of

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he protease with blood stained cotton cloth destained the clothithin 30 min without application of any detergent (Fig. 9C). How-

ver, enhanced removal of the blood stain by the alkaline proteaseas observed when supplemented with commercially availableetergent (Fig. 9D).

The purified protease from B. cereus SIU1 in combination withetergent removed the blood stain rapidly and efficiently within a0 min incubation. Our findings are in accordance with the resultsf Wolff et al. [35]. They studied the effects of Subtilisin on laundryerformance and demonstrated that the protease acted syner-istically with the detergent to efficiently remove the stain byydrolyzing large insoluble protein fragments strongly adhered tohe fabric. Similar results were observed with the proteases from B.ubtilis PE-11 (2003). An alkaline protease (100 U) from Bacillus sp.as found effective for removal of blood stains when the stained

loth was incubated with the enzyme plus detergent (1.0%, w/v)or 30 min at 45 ◦C [36]. Subba Rao et al. [8] purified an alkalinerotease from B. circulans that was capable of blood stain removaloth alone and with detergents during a 30 min incubation. Ourndings revealed the suitability of the B. cereus SIU1 protease forse in industrial applications, especially in laundry formulations.

. Conclusions

The molecular weight of the purified protease was 22 kDa basedn SDS-PAGE and zymography analysis. The protease was activend stable in a broad range of pH values and temperatures, withn optimum pH of 9.0 and temperature range of 35–55 ◦C. It wasemarkably stable in the presence of NaCl, surfactants, oxidantsnd detergents. These properties make this protease suitable forpplications in the detergent, food, pharmaceutical, leather andgriculture industries. Determination of the secondary structuref the protease by CD analysis in the far-UV range revealed that �-tructures were predominant and were responsible for the activityf the enzyme. Efficient removal of a blood stain within 30 minroved the potential use of this protease as a laundry additive.

cknowledgements

The senior author, Sanjay Kumar Singh, is thankful to the Univer-ity Grants Commission for providing a research fellowship underhe scheme “Research Fellowships in Science for Meritorious Stu-ents”. The financial assistance provided by the Government ofttar Pradesh and Department of Science and Technology, Gov-rnment of India, under the schemes of Center of Excellence andST-FIST, respectively, are duly acknowledged. The assistance withD analysis by Dr. Vikas Grover, NIPER, Mohali, India, and Dr. Suman. Jha, NIT, Rourkela, Odisha, India is gratefully acknowledged.

eferences

[1] Rao MB, Tanksale AM, Ghatge MS, Deshpande VV. Molecular and biotechnolog-ical aspects of microbial proteases. Microbiol Mol Biol Rev 1998;62:597–635.

[2] Gupta R, Beg QK, Lorenz P. Bacterial alkaline proteases: molecular approachesand industrial applications. Appl Microbiol Biotechnol 2002;59:15–32.

[3] Correa APF, Daroit DJ, Brandelli A. Characterization of a keratinase produced byBacillus sp. P7 isolated from an Amazonian environment. Int Biodeter Biodegr2010;64:1–6.

[4] Garg SK, Johri BN. Proteolytic enzymes. In: Johri BN, Satyanarayana T, Olsen J,editors. Thermophilic moulds in biotechnology. Dordrecht, The Netherlands:Kluwer Academic Publishers; 1999. p. 191–218.

[5] Joo HS, Chang CS. Oxidant and SDS-stable alkaline protease from a halo-tolerantBacillus clausii I-52: enhanced production and simple purification. J Appl Micro-biol 2005;98:491–7.

[6] Joo HS, Kumar CG, Park GC, Paik SR, Chang CS. Oxidant and SDS-stable alka-

line protease from Bacillus clausii I-52: production and some properties. J ApplMicrobiol 2003;95:267–72.

[7] Sareen R, Mishra P. Purification and characterization of organic solvent sta-ble protease from Bacillus licheniformis RSP-09-37. Appl Microbiol Biotechnol2008;79:399–405.

[

istry 47 (2012) 1479–1487 1487

[8] Subba Rao Ch, Satish T, Ravichandra P, Prakasham RS. Characterization ofthermo- and detergent stable serine protease from isolated Bacillus circu-lans and evaluation of eco-friendly applications. Process Biochem 2009;44:262–8.

[9] Kelly SM, Jess TJ, Price NC. How to study proteins by circular dichroism. BiochimBiophys Acta 2005;1751:119–39.

10] Venyaminov SY, Vassilenko KS. Determination of protein tertiary structureclass from circular dichroism spectra. Anal Biochem 1994;222:176–84.

11] Singh SK, Tripathi VR, Jain RK, Vikram S, Garg SK. An antibiotic, heavy metalresistant and halotolerant Bacillus cereus SIU1 and its thermoalkaline protease.Microb Cell Fact 2010;9:59.

12] Laemmli UK. Cleavage of structural proteins during assembly of head of bacte-riophage T4. Nature (London) 1970;227:680–5.

13] Singh SK, Singh SK, Tripathi VR, Khare SK, Garg SK. A novel psychrotrophic,solvent tolerant Pseudomonas putida SKG-1 and solvent stability of its psychro-thermoalkalistable protease. Process Biochem 2011;46:1430–5.

14] Anson ML. The estimation of pepsin, trypsin, papain, and cathepsin withhemoglobin. J Gen Physiol 1938;22:79–89.

15] Bakhtiar S, Andersson MM, Gessesse A, Mattiasson B, Hatti-Kaul R. Stabilitycharacteristics of a calcium-independent alkaline protease from Nesterenkoniasp. Enzyme Microb Technol 2002;32:525–31.

16] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with thefolin phenol reagent. J Biol Chem 1951;193:265–75.

17] Adinarayana K, Ellaiah P, Prasad DS. Purification and partial characterizationof thermostable serine alkaline protease from a newly isolated Bacillus subtilisPE-11. AAPS PharmSciTech 2003;4:E56.

18] Gessesse A, Hatti-Kaul R, Gashe BA, Mattiasson B. Novel alkaline proteasesfrom alkaliphilic bacteria grown on chicken feather. Enzyme Microb Technol2003;32:519–24.

19] Gupta A, Roy I, Patel RK, Singh SP, Khare SK, Gupta MN. One-step purificationand characterization of an alkaline protease from haloalkaliphilic Bacillus sp. JChromatogr A 2005;1075:103–8.

20] Deng A, Wu J, Zhang G, Wen T. Molecular and structural characterization of asurfactant-stable high-alkaline protease AprB with a novel structural featureunique to subtilisin family. Biochimie 2011;93:783–91.

21] Manachini PL, Fortina NG, Parini C. Thermostable alkaline protease producedby Bacillus thermoruber – a new species of Bacillus. Appl Microbiol Biotechnol1988;28:409–13.

22] Abusham RA, Rahman RNZRA, Salleh AB, Basri M. Optimization of physicalfactors affecting the production of thermo-stable organic solvent-tolerant pro-tease from a newly isolated halo tolerant Bacillus subtilis strain Rand. MicrobCell Fact 2009;8:20.

23] Dzubiella J. Salt-specific stability and denaturation of a short salt-bridge-forming �-helix. J Am Chem Soc 2008;130:14000–7.

24] Joshi GK, Kumar S, Sharma V. Production of moderately halotolerant, SDS stablealkaline protease from Bacillus cereus MTCC 6840 isolated from lake Nainital,Uttaranchal state, India. Braz J Microbiol 2007;38:773–9.

25] Jaouadi B, Ellouz-Chaabouni S, Rhimi M, Bejar S. Biochemical and molec-ular characterization of a detergent-stable serine alkaline protease fromBacillus pumilus CBS with high catalytic efficiency. Biochimie 2008;90:1291–305.

26] Chen P, Hwang Y, Kuo T, Liu F, Lai J, Hsieh H. Improvement in the propertiesof chitosan membranes using natural organic acid solutions as solvents forchitosan dissolution. J Med Biol Eng 2007;27:23–8.

27] Riffel A, Brandelli A, Bellato CM, Souza GHMF, Eberlin MN, Tavares FCA.Purification and characterization of a keratinolytic metalloprotease from Chry-seobacterium sp. kr6. J Biotechnol 2007;128:693–703.

28] Sana B, Ghosh D, Saha M, Mukherjee J. Purification and characterization ofa salt, solvent, detergent and bleach tolerant protease from a new gamma-Proteobacterium isolated from the marine environment of the Sundarbans.Process Biochem 2006;41:208–15.

29] Beena AK, Geevarghese PI, Jayavardanan KK. Detergent potential of a spoilageprotease enzyme liberated by a psychrotrophic spore former isolated fromsterilized skim milk. Am J Food Technol 2012;7:89–95.

30] Tornvall U, Hedstrom M, Schillen K, Hatti-Kaul R. Structural, functional andchemical changes in Pseudozyma antarctica lipase B on exposure to hydrogenperoxide. Biochimie 2010;92:1867–75.

31] Ogino H, Gemba Y, Yutori Y, Doukyu N, Ishimi K, Ishikawa H. Stabilities andconformational transitions of various proteases in the presence of an organicsolvent. Biotechnol Prog 2007;23:155–61.

32] Bhattacharyya A, Babu CR. Purification and biochemical characterization of aserine protease inhibitor from Derris trifoliata Lour. Seeds: insight into struc-tural and antimalarial features. Phytochemistry 2009;70:703–12.

33] Tomar R, Dubey VK, Jagannadham MV. Biophysical characterization and foldingstudies of plant protease, wrightin: identification of folding intermediate underdifferent conditions. Protein J 2009;28:213–23.

34] Zhang L, Conway JF, Thibodeau PH. Calcium-induced folding and stabiliza-tion of the Pseudomonas aeruginosa alkaline protease. J Biol Chem 2012;287:4311–22.

35] Wolff AM, Showell MS, Venegal GM, Barnett BL, Wertz WC. Laundry per-formance of Subtilisin protease. In: Bott R, Betzel C, editors. Subtilisin

enzymes: practical protein engineering. New York: Plenum Press; 1996.p. 113–20.

36] Oberoi R, Beg QK, Puri S, Saxena RK, Gupta R. Characterization and wash per-formance analysis of an SDS-stable alkaline protease from a Bacillus sp. WorldJ Microbiol Biotechnol 2001;17:493–7.