Purification, characterization and secondary structure elucidation of a detergent stable, halotolerant, thermoalkaline protease from Bacillus cereus SIU1

Process Biochemistry 47 (2012) 1479–1487

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Purification, characterization and secondary structure elucidation of a detergent stable, halotolerant, thermoalkaline protease from Bacillus cereus SIU1 Sanjay Kumar Singh, Santosh Kumar Singh, Vinayak Ram Tripathi, Satyendra Kumar Garg ∗ Center of Excellence, Department of Microbiology, Dr. Ram Manohar Lohia Avadh University, Faizabad 224001, UP, India

a r t i c l e

i n f o

Article history: Received 31 March 2012 Received in revised form 12 May 2012 Accepted 29 May 2012 Available online 8 June 2012 Keywords: Thermoalkaline protease Purification Oxidants Detergents Secondary structure CD 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 SIU1 strain using a combination of Q-Sepharose and Sephadex G-75 chromatography. The kinetic analyses revealed 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, towards a casein substrate. The protease was most active and stable at pH 9.0 and between a temperature range of 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 inhibitory with regard to enzyme activity, whereas cysteine, ␤-mercaptoethanol, calcium, magnesium, manganese and 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 and 1.0% concentrations. In the presence of 0.1 and 1.0% (w/v) detergents, the protease was fairly stable and retained 50–76% activity. Therefore, it may have a possible application in laundry formulations. An initial analysis of the circular dichroism (CD) spectrum in the ultraviolet range revealed that the protease is 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 corresponded to the altered enzyme activity. The protease combined with detergent was able to destain blood stained cloth within 30 min. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Proteases are enzymes of utmost importance and are found in all cellular forms of life. Enzymes are utilized in various industries, and proteases account for ∼66% of these industrial enzymes [1]. Among these, alkaline proteases have vast applications, principally in the food, detergent, leather and pharmaceutical industries [2]. Thermoalkaline proteases are the most commonly used of the alkaline proteases because they can function at a pH range of 7.0–12.0 and a temperature range of 35–80 ◦ C [1,3]. These properties of thermoalkaline proteases make them useful for various commercial and environmental applications. Proteases are commonly included in detergents for the removal of proteinaceous dirt, which was one of the early applications for these enzymes. Additionally, the presence of thermoalkaline proteases in detergents allows for easy washing, even at elevated temperatures [4]. Bacterial proteases are also known for their halotolerance and sustained activity in the presence of high salt concentrations

∗ Corresponding author. Tel.: +91 9454755166/5278 245330; fax: +91 5278 246330. E-mail address: sk [email protected] (S.K. Garg). 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.05.021

[5]. This property facilitates their use in saline conditions. During industrial applications, the proteases are required to function under diverse environmental conditions where surfactants, oxidants, detergents and solvents are present. Therefore, the stability of alkaline proteases in the presence of metal ions, denaturants, surfactants, oxidants, detergents and organic solvents is a highly desired characteristic for their use in industrial applications [6,7]. In general, the use of industrial proteases remains highly dependent on their stability during isolation, purification and storage, in addition to their robustness in the presence of solvents, surfactants and oxidants [8]. Therefore, it is necessary to determine the optimal physical and chemical conditions under which an enzyme is most active and stable. Additionally, there is a growing awareness that structural studies need to be performed under the conditions in which the proteins actually operate and perform their biological function [9]. Circular dichroism (CD) has become an increasingly valuable technique to elucidate insights into the structure of biological molecules. The differential absorption (absorption of leftand right-handed circularly polarized light due to the chirality centers nearby to the peptide bonds) of a peptide bond is dependent on its conformational relationship to neighboring peptide bonds. This characteristic has been exploited to obtain information

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on the number of secondary structural elements in a number of proteins. Secondary structure determination of several proteins has been successfully achieved with CD [10]. This is a beneficial technique because very little sample is used (<0.5 mg ml−1 ) and the technique is non-destructive in nature, which makes it possible to re-use the sample for other applications. In addition, relative changes in protein secondary structure due to environmental influences, such as pH, temperature, and modulators, can be monitored very accurately. Therefore, it is easy to establish the structure–function relationship of an enzyme in very little time. With this is mind, this study aimed to purify and characterize an alkaline protease. The effect of pH, surfactants, oxidants, detergents, modulators and metal ions on the secondary structure of the purified protease was also studied by CD in the far-ultraviolet (UV) range. The potential use of the protease as an enzyme for stain removal was tested on blood stained cloth. 2. Materials and methods 2.1. Microorganism Bacillus cereus SIU1 isolated in our laboratory was used in this study. The isolate was antibiotic and heavy metal resistant, halotolerant and produced a thermoalkaline protease [11]. The bacterial culture was maintained over nutrient agar slants (pH 9.0) and stored at 4 ◦ C.

2.2. Inoculum preparation and protease production For inoculum preparation, the bacterial culture was grown in modified glucose yeast extract (GYE) broth containing the following (gl−1 distilled water): glucose, 8.0; peptone, 15.0; yeast extract, 4.0; CaCl2 , 0.2; and NaCl, 5.0. A loopful of culture that had been grown for 24 h was inoculated into 99.0 ml of the above medium, pH 9.0 (adjusted after autoclaving using a sterilized 1.0 M Na2 CO3 solution in distilled water) in Erlenmeyer flasks and incubated at 50 ± 1 ◦ C in a shaking incubator at 150 rpm. Briefly, 1.0 ml of the mother culture of B. cereus SIU1 isolate with an OD = 0.5 (A620 ; 1.0 cm cuvette) containing 3.4 × 107 cfu ml−1 was inoculated into 99 ml of the above modified GYE medium, pH 9.0 and incubated at 50 ± 1 ◦ C in a shaking incubator at 150 rpm. After a 20 h incubation, the culture broth was centrifuged and the cell-free supernatant was used for further studies.

2.5. Characterization of purified protease 2.5.1. Kinetic analyses The purified protease was used in kinetic studies for determination of the Km , Vmax and kcat with Hammersten casein as a substrate. The enzyme was incubated with different concentrations of casein substrate, ranging from 0.25 to 4.0 mg and the activity was assayed by standard assay method described in Section 2.8.1. The Km , Vmax and kcat were calculated using a Lineweaver–Burk double reciprocal plot of the Michaelis–Menten equation. 2.5.2. Effect of temperature and pH The 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 assessed by incubation of the enzyme for 30 min at the above temperatures. The residual protease activity was estimated under standard conditions according to the method described by Anson [14]. The effect of pH on enzyme activity was determined using casein as the substrate, which was dissolved in different buffers of pH 5.0–12.0. The enzyme stability at various pH values was determined by pre-incubating the enzyme with an equal volume of each buffer for 30 min at 55 ± 1 ◦ C. The residual protease activity was assayed under standard conditions at an optimized temperature of 55 ± 1 ◦ C. 2.5.3. Effect of NaCl The 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 cations Phenyl methyl sulfonyl fluoride (PMSF), EDTA, ␤-mercaptoethanol, ascorbic acid (vitamin C) and cysteine were used to assess their effect on protease stability at different concentrations. The solutions were prepared at concentrations ranging from 1.0 to 5.0 mM. The metal ions Ca2+ (calcium chloride), Mg2+ (magnesium sulfate), Zn2+ (zinc chloride), Fe2+ (ferrous sulfate), Ni2+ (nickel chloride), Co2+ (cobalt chloride), Cu2+ (cupric chloride), Mn2+ (manganese chloride) and Hg2+ (mercuric chloride) were also tested for their effects on protease stability. The salt solutions were used at 0.1, 1.0 and 10.0 mM. The enzyme was diluted separately in an equal volume with each solution of modulator or metal ions and incubated for 30 min at 55 ± 1 ◦ C. Following incubation, the residual protease activity was assayed under standard conditions. 2.5.5. Effect of surfactants and oxidants The surfactants and oxidants Triton X-100, Tween 80, H2 O2 (each in v/v) and sodium dodecyl sulfate, sodium perborate (each in w/v) were used to assess their effects 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 at 55 ± 1 ◦ C. The residual protease activity was assayed.

2.3. Protease purification The extracellular protease produced by B. cereus SIU1 was fractionated by graded precipitation using ammonium sulfate. Initially, a 30% saturation step was performed and the resulting precipitate was removed from the solution. Thereafter, the ammonium sulfate saturation was increased to 75%, and the resulting precipitate was collected and dissolved in a minimum volume of 50.0 mM Tris–HCl buffer, pH 9.0. This enzyme solution was dialyzed against the same buffer for 24 h at 4 ◦ C and the buffer was changed at 6 h intervals. The dialyzed protease was concentrated by lyophilization, loaded onto a Q-Sepharose column (2.0 cm × 10.0 cm) pre-equilibrated with 50.0 mM Tris–HCl buffer, pH 9.0 and eluted with a linear gradient of 0.0–0.5 M NaCl in the same buffer at a flow rate of 0.2 ml min−1 . The fractions with protease activity were collected, pooled and dialyzed as described above. The fractions were then loaded onto a Sephadex G-75 column (2.0 cm × 50.0 cm) preequilibrated with 50.0 mM Tris–HCl buffer, pH 9.0 and eluted with the same buffer at a flow rate of 0.2 ml min−1 . Fractions with activity were pooled, lyophilized and stored at −20 ◦ C.

2.4. Molecular weight determination The molecular weight of the purified protease was determined by SDS-PAGE according to the method of Laemmli [12] using a 12.5% resolving gel. Electrophoresis was performed at 200 V and the protein bands were visualized with Coomassie Brilliant Blue R-250 staining. The molecular weight of the protease was determined by comparison with standard molecular weight markers of 97.4, 66, 43, 29, 20.1 and 14.3 kDa (Bangalore Genei Pvt. Ltd., Bangalore, Karnataka, India). The molecular weight was determined by software built into the Genei-UviTech gel documentation system (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–bicarbonate buffer, pH 9.0, followed by staining as described above to visualize the zone of gelatin hydrolysis.

2.5.6. Effect of commercial detergents Various commercially available detergents, including Rin, Surf, Ariel, Tide, Wheel, Nirma, More and Ghari were used to determine their effects on protease stability with regard to the potential use of the enzyme in industrial applications in the detergent industry. Each detergent solution was prepared at concentrations of 0.1, 1.0, 5.0 and 10.0% and the purified protease was mixed with an equal volume of each solution. After incubation for 30 min at 55 ± 1 ◦ C, the residual protease activity was 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 protease concentration of 0.1 mg ml−1 in 50.0 mM sodium carbonate–bicarbonate buffer, pH 9.0 at 25 ◦ C using a Jasco J-815 spectropolarimeter. The protease was most active and stable 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 scan rate of 50 nm min−1 . The bandwidth applied was 1.0 nm with a response time of 1 s. The ellipticity values (Â) 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 software Spectra Manager Version 1.00.00. The effects of environmental factors on the secondary structure of the protease were also studied. The effect of various pH values was determined by pre-incubating the 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 citrate buffer, pH 5.0, sodium phosphate buffer, pH 7.0 and sodium carbonate–bicarbonate buffer pH 10.0 and 11.0. To study the effects of EDTA, PMSF and ␤-mercaptoethanol on enzyme stability, the purified protease (0.2 mg ml−1 ) was pre-incubated with an equal volume of 1.0 mM solutions of the above chemicals for 30 min at 25 ± 1 ◦ C. The surfactants and oxidants Triton X-100, Tween 80, sodium dodecyl sulfate, H2 O2 and sodium perborate were also studied with regard to their effects on protease structure. The

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solutions were prepared at 1.0% concentrations and an equal volume of enzyme was incubated with each solution for 30 min at 25 ± 1 ◦ C. After the 30 min incubations under various environmental conditions, the CD spectra were recorded as described above. Blank spectra of each suspension without protease were used to correct the observed spectra. These studies were carried out at the National Institute of Pharmaceutical Education and Research (NIPER), Mohali, India. 2.7. Washing efficiency of protease To evaluate the stain removal capability of the protease, clean cotton cloth pieces (4.0 cm × 5.0 cm) were soiled with blood, dried for 30 min and then washed with water. The stained cloth pieces were incubated separately with 1.0 mg of purified alkaline protease, 0.2% (w/v) of the commercial detergent Ariel and 0.2%, w/v detergent plus 1.0 mg of purified alkaline protease for 30 min, followed by rinsing with water for 2 min. The washed cloth pieces were then dried and the extent of destaining was compared. 2.8. Analytical determinations 2.8.1. Enzyme assay The alkaline protease activity was assayed using the casein digestion method of Anson [14]. One milliliter of enzyme was used for protease assay as described earlier [11]. One unit of enzyme activity is defined as the amount of enzyme that liberates 1.0 ␮g of tyrosine min−1 . 2.8.2. Protein estimation The method of Lowry et al. [16] was followed for protein concentration estimation using BSA as the standard. 2.9. Statistical analysis The experiments were performed thrice, in triplicate each time. The standard deviation for each experimental result was calculated using Microsoft Excel. The standard deviation for each value was ≤5%.

3. Results and discussion

Fig. 1. SDS-PAGE analysis of the partially purified protease from strain SKG-1. Lane 1: crude extract; Lane 2: ammonium sulfate precipitate of protease; Lane 3: protease after Q-Sepharose chromatography; Lane 4: protease after Sephadex G-75 chromatography; Lane 5: molecular weight markers (kDa): phosphorylase b – 97.4; bovine serum albumin – 66.0; ovalbumin – 43.0; carbonic anhydrase – 29.0; soyabean trypsin inhibitor – 20.1 and lysozyme – 14.3; Lane 6: zymography of purified protease.

3.1. Protease purification and molecular weight determination The isolate, B. cereus SIU1 produced 633 U of protease ml−1 during a 20 h incubation in modified GYE broth. The protease was purified by ammonium sulfate fractionation followed by Q-Sepharose and Sephadex G-75 chromatography (Table 1). Ammonium sulfate precipitation was used to concentrate the protease, which resulted in a 2.4-fold purification with an 83% recovery rate. Q-Sepharose chromatography resulted in a 7.1-fold purification with a 60% recovery rate. After Sephadex G-75 chromatography, the purification was 9.5-fold with a final recovery of 38%. The molecular weight of this alkaline protease was determined to be 22 kDa by SDS-PAGE (Fig. 1). An activity gel analysis confirmed that the purified protease is a single monomeric protein (Fig. 1). Other researchers have also reported the isolation of proteases with low molecular weights from Bacillus isolates. Adinarayana et al. [17] purified an alkaline protease of 15 kDa from Bacillus subtilis PE-11 using ammonium sulfate precipitation and Sephadex G-200 chromatography. Gessesse et al. [18] purified an alkaline protease of 24 kDa from Bacillus pseudofirmus AL-89 to 22.6-fold purity with an 18% recovery rate. They used ammonium sulfate precipitation, DEAE-Sepharose ion exchange chromatography and

Sephadex G-75 gel filtration chromatography. Gupta et al. [19] purified an alkaline protease from B. pseudofirmus to 10-fold purity with an 82% yield using a single step method with a Phenyl Sepharose 6 Fast Flow column. The apparent molecular mass of this protease, based on SDS-PAGE, was estimated to be 29 kDa. A halotolerant alkaline protease of 28 kDa was purified from Bacillus clausii I52 using a combination of Diaion HPA75, phenyl-Sepharose and DEAE-Sepharose column chromatography [5]. Sareen and Mishra [7] purified a 55 kDa alkaline protease from the cell-free supernatant of Bacillus licheniformis RSP-09-37 using ammonium sulfate precipitation and affinity chromatography with ␣-casein agarose. The last step of purification resulted in a 55% yield and an 85-fold purification. Subba Rao et al. [8] purified a protease of 39.5 kDa from Bacillus circulans using ammonium sulfate precipitation and Sephadex G-100 chromatography. 3.2. Characterization of purified protease 3.2.1. Kinetic analyses Kinetic analysis of the protease using casein as a substrate revealed the Km and Vmax to be 1.09 mg ml−1 and

Table 1 Steps involved in the purification of the protease produced by B. cereus SIU1. Purification steps

Total protein (mg)

Total activity (U)

Crude extract Ammonium sulfate precipitation (30–75%) Q-Sepharose chromatography Sephadex G-75 chromatography

479 165

63 300 52 470

40.53 19.18

Specific activity (U mg−1 )

Recovery (%)

Fold purification

132 318

100 83

1 2.4

37 980

937

60

7.1

24 054

1254

38

9.5

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0.007

(A) Stability Residual protease activity (%)

-1

0.004

1/V (µg ml min )

0.005

-1

0.006

0.003 0.002

120

700

100

600 500

80

400 60 300 40

200

Stability

20

Activity 100

0.001 0

0

0 -1

-0.001

35

0

1

2

3

4

Protease units ml -1 Activity

1482

45

5

55 Temperature (oC)

65

75

-1

(B)

Fig. 2. Lineweaver–Burk plot for kinetics determinations of purified protease using Hammersten casein as the substrate.

3.2.2. Effect of temperature and pH The purified protease from strain SIU1 was active and stable throughout the temperature ranges used in this study. The temperature for maximum protease activity was in the range of 45–55 ◦ C (Fig. 3A). At both 65 and 75 ◦ C, the protease was quite active, with 90 and 59% activity, respectively. Therefore, the results reveal that the optimal temperature for SIU1 protease activity is 35–55 ◦ C. As far as protease stability is concerned, the enzyme retained 100% activity in the temperature range of 35–55 ◦ C. Even at 65 and 75 ◦ C, the protease was stable, as evidenced by residual activities of 61% and 48%, respectively (Fig. 3A). The broad, optimal temperature range of 35–55 ◦ C for maximum protease activity and stability reveals the thermostable nature of this protease from B. cereus SIU1. The activity of alkaline proteases in broad temperature ranges is a desired characteristic for their application in detergent formulations. Manachini et al. [21] have reported an alkaline protease from Bacillus thermoruber that is active in a broad temperature range of 10–80 ◦ C, with an optimum of 45 ◦ C. An alkaline protease from B. clausii I-52 was observed to be stable in the temperature range of 30–80 ◦ C, with almost 100% activity in the temperature range of 30–50 ◦ C [6]. Sareen and Mishra [7] reported a thermoalkaline protease from B. licheniformis. The purified enzyme was active at a temperature range of 30–90 ◦ C and the maximum activity of the protease was observed at 50 ◦ C. Abusham et al. [22] also reported

600

100

Stability Residual protease activity (%)

0.909 mg ml−1 min−1 , respectively (Fig. 2), as determined by a Lineweaver–Burk plot. The kcat value of 3.11 s−1 indicated both a high affinity and catalytic efficiency of this protease towards casein. Additionally, the small Km value implies strong affinity; therefore, there is little substrate requirement for the enzyme catalyzed reaction velocity of Vmax /2. Gupta et al. [19] purified an alkaline protease from B. pseudofirmus with a Km and Vmax to be 2.0 mg ml−1 and 289.8 ␮g min−1 , respectively, towards a casein substrate. Fig. 2 reveals that the Km value of the SIU1 enzyme is much less than that of the B. pseudofirmus protease, revealing a higher affinity for casein. A detergent stable, serine protease from B. circulans exhibited a Km of 0.597 mg ml−1 and Vmax of 13 825 ␮mol min−1 towards a casein substrate [8]. Deng et al. [20] cloned and expressed the AprB protease from B. subtilis WB600. This protease exhibited Km and Vmax values of 0.44 mM and 12.54 × 103 U mg−1 pure protein, respectively, towards a casein substrate. The kcat value for this enzyme was 250.86 × 103 min−1 . Therefore, it appears that the kinetic parameters vary from enzyme to enzyme and substrate to substrate.

700

120

500 80 400 60 300 40 200

Stability

20

Activity

Protease units ml -1 Activity

1/[S] (mg ml )

100

0

0 5

6

7

8

9

10

11

12

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% stability in 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 pH 9.0. 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 revealed that the protease was variably stable (18–100%) in the complete pH range 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 remarkable activity and stability over a wide pH range reveals the highly alkaline nature of this protease, which makes it suitable for applications in alkaline environments and with detergents. Several other researchers have also described alkaline proteases with broad pH activities and stabilities. Manachini et al. [21] purified an alkaline protease from B. thermoruber that is active in a broad pH range of 7.5–11.0, with maximum activity at pH 9.0. An alkaline protease from B. subtilis PE-11 was observed to be stable in the pH range of 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 in the pH range of 4.0–12.0, with maximum activity at pH ∼ 12.0. An alkaline protease from B. licheniformis RSP-09-37 was observed to be 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% residual protease activities at pH 4.0, 5.0 and 12.0, respectively [7]. 3.2.3. Effect of NaCl The protease stability was almost 100% at concentrations of 0.0–2.0% (w/v) NaCl when incubated for 30 min. Further increases in salt concentration were inhibitory for protease stability. The protease 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

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Residual protease activity (%)

120

100

80

60

40

20

0 0

0.5

1

1.5

2

2.5

3

4

5

7

9

10

11

12

NaCl concentration (% w/v) Fig. 4. Effect of NaCl on protease stability after a 30 min incubation.

chloride at even higher concentrations further reduced the protease stability, with only 12 and 3% residual activities retained at 11.0 and 12.0% salt concentrations, respectively (Fig. 4). It is well known that the presence of high salt concentrations destabilizes ion pairs and salt bridges and alters electrostatic interactions between charged amino acids, leading to enzyme denaturation [23]. The stability of the SIU1 protease in the presence of high salt concentrations revealed its moderately to highly halotolerant nature. This could be attributed to the halotolerant nature of our isolate [11]. Investigation of the protein molecular structures reveals that Na+ ions have a strong affinity for the side chain carboxylates and backbone carbonyls, thereby weakening salt bridges and secondary structure hydrogen bonds [23]. Salt tolerance of alkaline proteases makes their application for washing under saline conditions possible. Joo and Chang [5] reported an alkaline protease from halotolerant B. clausii I-52 having good activity in the range of 0.0–10.0% (w/v) NaCl. B. cereus MTCC 6840 produced an alkaline protease with activity in NaCl concentrations up to 5.0%. At an even higher NaCl concentration of 10.0% (w/v), the protease activity only decreased to 40% [24]. Protease stability at high salt concentrations is a desirable characteristic because NaCl is used as a core component in granulation of the protease prior to its addition to detergents [25]. Additionally, the ground water from different Indian geoclimatic regions is saline, which may be detrimental for the cleaning potential of detergents; therefore, the presence of a halotolerant alkaline protease in detergents will make efficient washing less difficult. 3.2.4. Effect of modulators and divalent cations PMSF, EDTA and ascorbic acid had adverse effects on protease stability and activity. PMSF was completely inhibitory for the enzyme at all concentrations tested. On the other hand, the extent of protease inhibition increased with increasing concentrations of EDTA and ascorbic acid (Fig. 5A). In contrast, 1.0–3.0 mM ␤mercaptoethanol and 1.0–5.0 mM cysteine did not have any effect on protease activity, indicating its stability in the presence of reducing agents. However, ␤-mercaptoethanol at higher concentrations of 4.0 and 5.0 mM decreased the protease activity by ∼20% (Fig. 5A). Complete inhibition of protease activity by PMSF, even at 1.0 mM, indicates that the enzyme is a serine alkaline protease with a serine residue in its active site. PMSF blocks the active site of proteases by sulfonating the essential serine residue, resulting in complete inhibition of protease activity [17]. The decrease in activity by EDTA reveals the requirement of metal ion(s) for this protease activity because EDTA removes metal ion(s) through chelation. A serine alkaline protease from B. licheniformis RSP-09-37 was inhibited completely by 10.0 mM PMSF within 20 min. Similar to our findings, this protease was also inhibited by EDTA and exhibited only 55

Fig. 5. Effects of modulators (A) and divalent cations (B) on protease stability after a 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 requirement of metal ions for the activity of this enzyme [7]. In our study, the decreased activity of the SIU1 protease in the presence of ascorbic acid may be due to the acidic environment. The presence of ascorbic acid 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 mM cysteine, protease activity remained unaffected, indicating that SH groups are not essential for catalytic activity, but are necessary for the maintenance of the three-dimensional structure of the enzyme [3,27]. The stability of the SIU1 protease in the presence of reducing agents is also demonstrated by SDS-PAGE analysis, where it was observed to be a monomeric polypeptide. Furthermore, zymography analysis confirmed the monomeric structure of this protease (Fig. 1). Correa et al. [3] also reported the activity of a keratinase produced by Bacillus sp. P7 that was only slightly inhibited by the reducing agent, ␤-mercaptoethanol. The residual protease 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 increased the protease activity up to 112, 105 and 102%, respectively, at a concentration of 0.1 mM, while magnesium and zinc had no effect. Mercury, cobalt, iron and nickel reduced the protease activity to 68, 81, 94 and 98%, respectively (Fig. 5B). Furthermore, at a concentration 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 concentration of 1.0 mM, while other metal ions inhibited the protease activity to a variable extent (Fig. 5B). At the highest concentration

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Fig. 6. Effects of oxidants and surfactants (0.1–10.0%) on protease stability after a 30 min incubation.

of 10.0 mM, each metal ion exerted inhibitory effects except calcium. In the presence of 10.0 mM calcium, the protease activity was slightly increased to 105%. Mercury drastically reduced the protease activity and only 5% of the residual activity remained after a 30 min incubation with 10.0 mM mercury. The protease retained 30–98% activity at a 10.0 mM concentration of other metal ions tested (Fig. 5B). Metal ions are well known to induce both enzyme activity and thermostability. Calcium, magnesium, copper and manganese exhibited positive effects on protease activity, indicating that such metal ion(s) are required for increased activity of this protease. Several other researchers have also demonstrated increased protease activity in the presence of different metal ion(s) [17]. Joshi et al. [24] reported the positive effects of iron and cobalt on the protease activity of B. cereus MTCC 6840. Sareen and Mishra [7] reported a metal-dependent serine alkaline protease from B. licheniformis RSP-09-37. The protease activity increased in the presence of Ca2+ , Mg2+ and Mn2+ metal ions, while Zn2+ and Cu2+ had inhibitory effects. This protease exhibited only 55% activity in presence of 1.0 mM EDTA, indicating that removal of metal ions through chelation by EDTA was responsible for the decreased protease activity. 3.2.5. Effect of oxidants and surfactants The protease demonstrated significant stability in the presence of surfactants, detergents and oxidants. Sodium dodecyl sulfate (SDS), Triton X-100, Tween 80, H2 O2 and sodium perborate at concentration of 0.1 and 1.0% (w/v) increased the protease activity to a maximum of 129%. Furthermore, higher concentrations of Triton X-100, Tween 80, H2 O2 and sodium perborate did not affect the protease activity significantly and the residual protease activity was in the range of 65–100%. In contrast, higher concentrations of SDS were inhibitory to protease stability and the protease activity decreased to 58 and 16% at 5.0 and 10.0% (w/v) concentrations, respectively (Fig. 6). The action of the detergents on the protein can be correlated to their hydrophilic/lipophilic balance (HLB), which is defined as how a detergent distributes between polar and nonpolar phases. Triton X-100, with a HLB of 13.5, has a less detrimental effect compared to SDS, with a HLB of 40 at a 0.5% concentration [7]. It is interesting to observe that the protease activity significantly increased, up to 129%, in the presence of surfactants, detergents and oxidants at concentrations of 0.1 and 1.0% (w/v), making it suitable as a detergent additive. Commercially available proteases, such as Subtilisin Carlsberg, Subtilisin BPN’, Alcalase, Esparase and Savinase, exhibit great stability in the presence of detergents; however, most are unstable in the presence of oxidants and bleaches [2]. Therefore, the stability of the B. cereus SIU1 protease suggests that it is a better candidate for a detergent additive. Proteases from Bacillus 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 of strain 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 gammaProteobacteria was completely stable in the presence of the laboratory detergents Tween 80 and Triton X-100, oxidizing agents, reducing agents, commercial detergents and bleaches, including hydrogen peroxide and sodium perborate. Beena et al. [29] reported an alkaline protease from a B. cereus isolate. The protease activity increased in the presence of 10.0 mM SDS and Tween 80 after a 30 min incubation. Even in the presence of 10.0 mM H2 O2 , the activity was almost completely (99%) retained. 3.2.6. Effect of commercial detergents The protease from B. cereus SIU1 was remarkably stable (50–93%) in the presence of 0.1 and 1.0% (w/v) commercial detergents. Furthermore, at a 5.0% concentration of commercial detergent, the protease retained residual activity in the range of 15–28% (Fig. 7). Even at a concentration of 10.0%, the residual protease activity was 7, 10 and 5% in Rin, Ariel and Wheel detergents, respectively. Such a remarkable stability of our protease in the presence of higher detergent concentrations reveals its usefulness as a detergent additive. Detergent stability of a protease is an important trait for its industrial application. Generally, detergent powders are used at <1.0% (w/v) concentrations for washing clothes. Because our protease 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 of different detergents have been studied by several other researchers [5,8,28]. Adinarayana et al. [17] reported an alkaline protease from B. subtilis PE-11 with 87–96% residual activity when incubated with 0.7% (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 was used as a reference structure. An initial analysis of the far-UV CD spectrum (Fig. 8A) revealed that the native protein at pH 9.0 is an ␣-, ␤-polypeptide with a majority of ␤-structure. Although negative ellipticity was present, no clear negative peak, characteristic of ␣-helical structure, was evident at 222 nm. These results strongly suggested that the protease from B. cereus SIU1 is predominantly a ␤-rich protein (Fig. 8A). The detailed structural composition of the protease indicated a large fraction (∼50%) of ␤-sheets (Table 2). CDbased conformational evaluation of the protease after incubation at different pH values and with modulators, metal ions and detergents, demonstrated that deviation in the ␤-content was directly

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Fig. 8. Far-UV CD spectra of (A) purified protease (0.1 mg ml−1 ) in 50.0 mM sodium carbonate–bicarbonate buffer, pH 9.0; (B) protease incubated at pH 5.0 for 30 min; (C) protease incubated at pH 11.0 for 30 min; and (D) protease incubated with H2 O2 for 30 min.

correlated with the altered (increased/decreased) protease activity (Table 2). At an acidic pH of 5.0, the protease lost its secondary structure. The ␤-structures were greatly reduced and the percentage of unordered structures were dominant (Fig. 8B). There was a concomitant reduction in protease activity to only 28%. Similarly, at the highly alkaline pH of 11.0, the percentage of ␤-structures were reduced, while the percentage of unordered structure increased

(Fig. 8C), compared to the native protein at pH 9.0. The protease activity also corresponded in a similar manner. Therefore, it was clear that with a reduction in ␤-content, the protease activity was significantly 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 and the protease activity also decreased. Although PMSF had no significant effect on the secondary structure of the protein, its presence

Table 2 CD secondary structure and residual activity of purified protease (0.1 mg ml−1 ) at pH 9.0 and all other environmental exposures after a 30 min incubation. Environmental condition

pH 9.0 (reference) pH 5.0 pH 7.0 pH 10.0 pH 11.0 Protease + Ca2+ (1.0 mM) Protease + EDTA (1.0 mM) Protease + PMSF (1.0 mM) Protease + ␤-mercaptoethanol (1.0 mM) Protease + SDS (1.0%) Protease + Triton X-100 (1.0%) Protease + Tween 80 (1.0%) Protease + H2 O2 (1.0%) Protease + Sodium perborate (1.0%)

CD secondary structure content (%)

Protease activity (%)

␣-Helix

␤-Sheets

Turns

Unordered

13.1 4.1 8.0 12.7 15.2 12.6 18.6 13.9 16.1 11.5 12.1 10.4 13.5 12.7

49.8 15.1 40.0 44.1 33.8 52.4 39.2 48.2 51.6 53.0 52.6 52.2 53.4 54.0

17.4 27.6 21.2 20.4 19.8 18.5 12.4 16.6 13.3 18.7 17.3 18.1 10.7 15.9

19.7 53.2 30.8 22.8 31.2 16.5 29.8 21.3 19.0 16.8 18.0 19.3 22.4 17.4

100 28 91 92 70 120 91 0 106 126 105 118 124 105

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Fig. 9. Effect of protease on blood stain removal: (A) untreated; (B) treated with detergent alone; (C) treated with protease alone; and (D) treated with detergent plus protease.

completely inhibited the protease activity. In the presence of Ca2+ , ␤-mercaptoethanol, surfactants, oxidants and detergents, the percentage of ␤-structures increased. Accordingly, these treatments increased the protease activity to variable extents (Table 2). Therefore, the findings indicated that the purified protease from B. cereus SIU1 is predominantly a ␤-protein. In the presence of H2 O2 , the percentage of ␤- and unordered structures increased more than in the native protease at pH 9.0 (Fig. 8D and Table 2). However, the protease activity increased to 124%, indicating that the activity is affected more by the ␤-structure, and once again confirming that this protease is predominantly a ␤-protein. Tornvall et al. [30] reported that hydrogen peroxide has been shown to oxidize the methionine, cysteine, tyrosine and tryptophan residues in various proteins. However, the number of oxidized residues, and the effect thereof, differs widely from protein to protein, depending on the position of the sensitive amino acid and its role in the stability or activity of the enzyme. The SIU1 protease is a ␤-rich protein and the change in protease activity 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 and Subtilisin BPN’ from Bacillus amyloliquefaciens because both of these proteins are ␣/␤ proteins as demonstrated by their secondary and tertiary structures [10]. At an acidic pH of 5.0, the loss of ␤-structures and increase in unordered structures occurred, leading to a dramatic loss of protease activity. Other environmental factors did not exert such a profound effect on the ␤-content of the protease. The factors under which the ␤-structure of the protease increased also exerted a positive effect on the alkaline protease activity (Table 2). Our findings

are in accordance with Bhattacharyya and Babu [32], who reported a trypsin protease inhibitor to be a ␤-protein (∼40% ␤-sheets). Similar to our findings, they also observed that under extreme heat and acid-alkali conditions, deviation occurred between the ␣helix and ␤-sheet content. This deviation was in accordance with the protease inhibitory activity. The increase in ␤-sheet content correlated with the increase in the protease inhibitory activity. Similarly, the far-UV (200–260 nm) CD spectra of Wrightin, a serine protease from the plant Wrightia tinctoria, revealed information regarding the secondary structure of the protein. The native spectra at pH 7.0 showed a negative peak at 215 nm, suggesting that the secondary structure was predominantly ␤-sheets [33]. The secondary structure of an alkaline protease, AL-20, from Nesterenkonia sp. was studied by Bakhtiar et al. [15]. The far-UV CD spectrum of the AL20 protease at pH 10.0 (10.0 mM glycine–NaOH buffer) revealed its secondary structure to be a combination of ␣-helix and ␤-sheet structures. The secondary structure was found unaffected even after a prolonged storage of 24 h at 50 ◦ C. Zhang et al. [34] studied the secondary structure of the Ca2+ -binding domain (RTX) of a Pseudomonas 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, consistent with a ␤-rich structure. At and above 50.0 mM Ca2+ , the absorbance shifted to a single minimum at 217 nm, revealing the complete 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

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

the protease with blood stained cotton cloth destained the cloth within 30 min without application of any detergent (Fig. 9C). However, enhanced removal of the blood stain by the alkaline protease was observed when supplemented with commercially available detergent (Fig. 9D). The purified protease from B. cereus SIU1 in combination with detergent removed the blood stain rapidly and efficiently within a 30 min incubation. Our findings are in accordance with the results of Wolff et al. [35]. They studied the effects of Subtilisin on laundry performance and demonstrated that the protease acted synergistically with the detergent to efficiently remove the stain by hydrolyzing large insoluble protein fragments strongly adhered to the fabric. Similar results were observed with the proteases from B. subtilis PE-11 (2003). An alkaline protease (100 U) from Bacillus sp. was found effective for removal of blood stains when the stained cloth was incubated with the enzyme plus detergent (1.0%, w/v) for 30 min at 45 ◦ C [36]. Subba Rao et al. [8] purified an alkaline protease from B. circulans that was capable of blood stain removal both alone and with detergents during a 30 min incubation. Our findings revealed the suitability of the B. cereus SIU1 protease for use in industrial applications, especially in laundry formulations. 4. Conclusions The molecular weight of the purified protease was 22 kDa based on SDS-PAGE and zymography analysis. The protease was active and stable in a broad range of pH values and temperatures, with an optimum pH of 9.0 and temperature range of 35–55 ◦ C. It was remarkably stable in the presence of NaCl, surfactants, oxidants and detergents. These properties make this protease suitable for applications in the detergent, food, pharmaceutical, leather and agriculture industries. Determination of the secondary structure of the protease by CD analysis in the far-UV range revealed that ␤structures were predominant and were responsible for the activity of the enzyme. Efficient removal of a blood stain within 30 min proved the potential use of this protease as a laundry additive. Acknowledgements The senior author, Sanjay Kumar Singh, is thankful to the University Grants Commission for providing a research fellowship under the scheme “Research Fellowships in Science for Meritorious Students”. The financial assistance provided by the Government of Uttar Pradesh and Department of Science and Technology, Government of India, under the schemes of Center of Excellence and DST-FIST, respectively, are duly acknowledged. The assistance with CD analysis by Dr. Vikas Grover, NIPER, Mohali, India, and Dr. Suman K. Jha, NIT, Rourkela, Odisha, India is gratefully acknowledged. References [1] Rao MB, Tanksale AM, Ghatge MS, Deshpande VV. Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 1998;62:597–635. [2] Gupta R, Beg QK, Lorenz P. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 2002;59:15–32. [3] Correa APF, Daroit DJ, Brandelli A. Characterization of a keratinase produced by Bacillus sp. P7 isolated from an Amazonian environment. Int Biodeter Biodegr 2010;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-tolerant Bacillus clausii I-52: enhanced production and simple purification. J Appl Microbiol 2005;98:491–7. [6] Joo HS, Kumar CG, Park GC, Paik SR, Chang CS. Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: production and some properties. J Appl Microbiol 2003;95:267–72. [7] Sareen R, Mishra P. Purification and characterization of organic solvent stable protease from Bacillus licheniformis RSP-09-37. Appl Microbiol Biotechnol 2008;79:399–405.

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