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Purification and Characterization of Heat-Tolerant Protease Produced by Bacillus polyfermenticus SCD
Purification and Characterization of Heat-Tolerant Protease Produced by Bacillus polyfermenticus SCD
Journal of Microbiology and Biotechnology. 2013. Nov, 23(11): 1554-1559
Copyright © 2013, The Korean Society For Microbiology And Biotechnology
  • Received : July 01, 2013
  • Accepted : August 12, 2013
  • Published : November 28, 2013
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About the Authors
Gooi Hun Choi
Division of Animal Life Science, Konkuk University, Seoul 143-701, Republic of Korea
Mi Na Jo
Division of Animal Life Science, Konkuk University, Seoul 143-701, Republic of Korea
Jin-Man Kim
Lotte R&D Center, Seoul 150-104, Republic of Korea
Cheon-Jei Kim
Division of Animal Life Science, Konkuk University, Seoul 143-701, Republic of Korea
Kee-Tae Kim
Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea
Hyun-Dong Paik
Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea
hdpaik@konkuk.ac.kr

Abstract
A protease produced by Bacillus polyfermenticus SCD was purified and characterized as a new detergent material. The protease was purified from supernatant produced by B. polyfermenticus SCD, by ammonium sulfate precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, and finally gel filtration chromatography on Sephadex G-50. The molecular mass of this enzyme was 44 kDa based on SDS-PAGE. The optimum temperature and pH were 50℃ and pH 8.0. The ranges of its stability to the pH and temperature were 7.0 to 9.0 and under 40℃, respectively. The enzyme was highly stable in the presence of the surfactants like Triton X-100 (0.1%), showing a 2-fold increase in its proteolytic activity. However, the enzyme was slightly inhibited by the chelating agent EDTA (1 mM). The enzyme has a maximum activity at 50℃ and the activity can be increased by surfactants such as Triton X-100 and Tween 80.
Keywords
Introduction
Microorganisms represent an excellent source of enzymes, including proteases, because of their broad biochemical diversity. Microbial proteases represent one of the three largest groups of industrial enzymes and account for approximately 60% of the total enzyme sales in the world [30] . These enzymes have numerous applications in industrial production, including food processing, leather processing, pharmaceutical and biomediation processes, and in the textile industry to remove protein-based stains [6 , 22] .
Proteases produced by Bacillus species are the most important group of secondary metabolites that are widely exploited [19] . Proteases constitute approximately 65% of the total worldwide production of enzymes [9] and bacteria of the genus Bacillus produce most of the industrial proteases used today [5 , 7 , 32] . For application of protease to the detergent industry, it would be of great importance to have available enzymes with optimal activities at different salt concentrations, temperatures, and pH values [20 , 23 , 31] . The increasing demand for proteases with specific properties has led biotechnologists to explore newer sources of proteases.
B. polyfermenticus strains have been reported to be effective for the treatment of long-term intestinal disorders because their endospores can successfully reach the target intestine [10] . In the past, many studies have reported on the properties of B. polyfermenticus , including its industrial utility and safety [2 , 11 , 27] , capacity to inhibit carcinogeninduced DNA damage [25] , antioxidant effects [4] , anticarcinogenic and antigenotoxic effects [26] , and probiotic potential [12] . However, there are no studies to date regarding the use of any proteases from B. polyfermenticus .
The aim of this study was to purify and characterize a protease produced by B. polyfermenticus SCD as a detergent additive.
Materials and Methods
- Bacterial Strain and Culture Conditions
B. polyfermenticus SCD ( B.polyfermenticus KCCM10104) was stored at -70℃ in t ryptic soy b roth (TSB; Difco, USA) to which 20% (v/v) glycerol was added. Cultures were grown in 1,000 ml of nutrient broth (NB; Difco) as a working volume in 2,000 ml flasks. The temperature was maintained at 37℃ for 48 h and the agitation speed was 150 rpm [14] .
- Protease Assay
Protease activity was determined by a modified Anson-Hagihara’s method using casein as the substrate [8] . The enzymatic reaction mixture consisted of 1 ml of protease solution, 1 ml of 0.6% (w/v) azocasein (Sigma-Aldrich Co., USA), and 1 ml of 100 mM sodium phosphate buffer (pH7.0). The reaction was initiated by the addition of protease solution and incubated at a defined temperature for 10 min. The reaction was stopped by the addition of 2.5 ml of 5% (w/v) trichloroacetic acid (TCA) followed by a 30 min incubation at room temperature and centrifugation (20,760 × g , 20min). Enzymatically hydrolyzed casein was measured by a modified Folin-Ciocalteu method, with casein treated with inactive protease as a blank. One unit of protease activity was defined as the amount of protease that liberated 1 μg of tyrosine per minute at 37℃. Protease units were measured using tyrosine (0-100 mg) as a standard curve [36] .
- Protein Measurement
The amount of protein was determined by the method of Lowry using bovine serum albumin (BSA) (0-5 mg/ml) as the standard. In addition, during the enzyme purification with column chromatography, the protein elution profile was monitored spectrometrically at 280 nm [17] .
- Ammonium Sulfate Precipitation
Ammonium sulfate was added to the cell-free culture supernatant at up to 60% saturation, and the precipitate was allowed to form at 4℃ for 24 h. The precipitate was collected by centrifugation at 20,760 ×g for 20 min at 4℃ and resuspended in 0.1 M phosphate buffer (pH7.0) and dialyzed for overnight against the same buffer with the sample:buffer ratio of 1:1,000 (v/v) [1 , 20] .
- DEAE-Sephadex A-50 Column Chromatography
The ammonium sulfate fraction was put on a column (3 × 40 cm) of diethylaminoethyl (DEAE) Sephadex A-50 (Sigma) that was equilibrated with 50 mM phosphate buffer (pH7.0). The column was washed with the same buffer and eluted with a linear gradient of 0 to 1 M NaCl in 50 mM sodium phosphate buffer (pH7.0). The fractions with protease activity were collected and dialyzed against 10 mM sodium phosphate buffer (pH7.0) at 4℃ [1 , 18] . The enzyme solution was concentrated and stored at -20℃ until used. This dialyzed fraction was made up to a known volume and referred to as partially purified protease. The activity of purified protease was assayed as described above.
- Sephadex G-50 Chromatography
For gel filtration chromatography, the dialyzed material was loaded onto a gel column (Sephadex G-50; Sigma-Aldrich Co., USA) previously equilibrated with 10 mM sodium phosphate buffer (pH7.0). The column was then washed with 250 ml of the same buffer, and bound proteins were eluted with the same buffer at a flow rate of 0.5 mg/ml. Fractions were collected, and positive fractions were pooled together, lyophilized, and resuspended in the same buffer [24] .
- Polyacrylamide Gel Electrophoresis
PAGE was carried out to determine the molecular mass of the protease with a 10% polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) at 4℃ and 20 mA/gel, after heating the samples at 90℃ for 1 min [16] . Following SDS-PAGE, the proteins were stained with Coomassie brilliant blue R-250 (0.2%). The molecular mass of the protease was determined by comparing with the mobility of standard molecular mass marker proteins: myosin (250 kDa), phosphorylase (148 kDa), bovine albumin (98 kDa), glutamic dehydrogenase (64 kDa), alcohol dehydrogenase (50 kDa), carbonic anhydrase (36 kDa), myoglobin (22 kDa), and lysozyme (16 kDa) (Sigma-Aldrich Co., St. Louis, MO, USA).
- Influence of Temperature on Protease Activity
To investigate the effect of temperature on protease activity, the protease activity was measured in the temperature range of 0-90℃ at pH 7.0 for 10 min. Prior to the assays, the enzyme sample, substrate solution, and 0.1 M sodium phosphate buffer (pH7.0) were pre-incubated at the desired temperature for 2 min [30] .
- Effects of pH on Protease Activity
The effect of pH on protease activity was investigated using 0.1 M acetate buffer (pH 4, 5, and 6), 0.1 M phosphate buffer (pH 6, 7, and 8), and 0.1 M glycine-NaOH buffer (pH 8, 9,and 10) at the range of pH 4-10 at 50℃, previously determined for 10 min. Substrate solution of azocasein was prepared in the respective buffers. The assay procedure was the same as described above [34] .
- Effects of Inhibitors and Surfactants
The effects of inhibitors and surfactants on protease activity was were under standard assay conditions where the assay cocktail was supplemented with EDTA (1 mM), Tween 80 (0.1%), and Triton X- 100 (0.1%). The effects were assessed relative to a control [35] .
- Statistical Analysis
Triplicate experiments were done in this study. Analysis of variance (ANOVA) for data was performed by using the SPSS software 8.0 (Statistical Package for the Social Sciences). Significance of differences was defined as p < 0.05.
Results and Discussion
- Protease Production
During growth in nutrient broth, the protease activity reached 41.8 units/ml within 48 h, when cell growth reached the late log phase or early stationary phase ( Fig. 1 ). An alkaline protease from marine yeast produced protease activity within 30 h when the cell growth reached the midlog phase [3] . The content of protease decreased because of the utilization of protein by the organism. The decline in protease activity upon prolonged incubation may be due to autolysis of the enzyme.
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Time course of protease production by Bacillus polyfermenticus SCD. △: Cell number, ▲: enzyme activity.
- Purification of Protease
The protease was purified by ammonium sulfate fractionation followed by ion-exchange and gel filtration chromatographies. A summary of the purification of the protease is shown in Table 1 . The ammonium sulfate fractionation was an effective step in the purification process, leading to 28.2-fold purification ( Table 1 ). Ion-exchange chromatography with DEAE Sephadex-A50 enhanced the specific activity to 2,973.1 units/mg protein, achieving 58.4-fold purification. During the last step of purification using gel filtration chromatography, the specific activity of the purified protease was 3,845.3 units/mg protein, with a fold purification of 75.5% and a recovery of 31.3% from the spent medium.
Purification of protease fromBacillus polyfermenticusSCD.
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Purification of protease from Bacillus polyfermenticus SCD.
- Polyacrylamide Gel Electrophoresis
Partial purification of the protease after ion-exchange chromatography with DEAE SephadexA-50 was successfully achieved, as shown by a double band corresponding to 32 and 44 kDa on SDS-PAGE.
Bacillus spp. have many kinds of extracellular protease during culturing [13 , 23] . Kim et al . [13] presented that B. cereus has two proteases with molecular masses of approximately 38 and 36 kDa. Nilegaonkar et al . [23] reported that partially purified protease from B. cereus MCM B-36 contained multiple proteases of molecular masses 45 and 36 kDa. In this study, the purified enzyme appeared as a single band on SDS-PAGE, corresponding to a molecular mass of 44 kDa after gel filtration chromatography with DEAE Sephadex G-50 ( Fig. 2 ).
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Analysis of protease purification by 10% SDS-PAGE. Lane M, Molecular mass marker; Lane 1, Crude broth; Lane 2, Fraction after 60% ammonium sulfate precipitation; Lane 3, Fraction after ion-exchange chromatography with DEAE Sephadex-A50; Lane 4, Fraction after gel filtration chromatography on Sephadex G-50.
- Effect of Temperature on Protease Activity
The effect of temperature from 0 to 90℃ on protease activity in 0.1 M sodium phosphate buffer (pH 7.0) is shown in Fig. 3 . The optimum temperature for protease activity was 50℃, beyond which there was a rapid decline. In an earlier report, Raju et al . [29] reported that the protease from Bacillus species was active in the temperature range of 20-50℃, with optimum activity at 37℃. Thus, the protease of B. polyfermenticus SCD in this study is more thermotolerant than the proteases referred to above.
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ffects of t emperature ( A) and pH (B) on protease activity. The protease assay was carried out at pH 7.0 and at different temperatures in the range of 10-80℃. Activity at 50℃ was referred to as 100% and the relative activity was calculated for other temperatures. pH optima were determined by incubating the enzyme with the substrate at different pH values at 50℃: acetate buffer (pH 4.0-6.0); phosphate buffer (pH 6.0-8.0), and glycine-NaOH buffer (pH 8.0-10.0).
- Effect of pH on Protease Activity
The activity of the protease at the range of pH 4.0-10.0 was determined after the enzyme was incubated at 50℃ for 10 min. The protease showed high activity at pH 7.0-9.0, and the highest activity was obtained at pH 8.0 ( Fig. 3 ). The effect of pH on the protease activity shows that the protease is a type of alkaline protease active in a broad range of pH values. Preliminary studies on the extracellular de-hairing protease secreted by Bacillus sp. showed that it has dual pH maxima at 7.5 and 9.0 [29] . These findings are in accordance with several earlier reports showing pH optima of 8.0-9.5 for fungal proteases such as those from Aspergillus fumigatus, A. parasiticus , and A. clavatus CCT2759 [21 , 33 , 34] .
- Effects of Inhibitors and Surfactants on Protease Activity
The effects of inhibitors and surfactants on the purified protease is detailed in Fig. 4 . The protease was highly stable in the presence of the surfactants such as Triton X-100 (0.1%), showing a 2-fold increase in proteolytic activity. The enzyme was slightly inhibited by the chelating agent EDTA (1 mM). The high activity of the protease in the presence of EDTA is very important for its potential application as a detergent additive because most detergents contain such chelating agents. This was similar to results obtained by Kim et al . [15] and Rahman et al . [28] .
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Effects of inhibitors and surfactants on protease activity. The standard protease assay was supplemented with EDTA (1 mM), Triton X-100 (0.1%), and Tween 80 (0.1%). The effects of these additives were assessed relative to the control. Percent residual activity was calculated by considering the activity of control to be 100%.
Despite the fact that many different proteases have been identified and some of them have been used in biotechnological and industrial applications, the presently available proteases are not sufficient to meet most industrial demands. Industrial processes are carried out under specific physical and chemical conditions, which cannot always be adjusted to the optimal values required for the activity and stability of the available enzymes. From this study, it appeared that protease produced from B. polyfermenticus SCD has a single molecular mass unlike other Bacillus species, so it would be more economical and convenient to apply it to industrial production for the detergent industry than those produced from other strains. The stability of the protease in detergent appears to be an attractive feature for its use in industrial applications. In general, the optimum temperature of most proteases in nature is at the range of 30-40℃, but enzymatic activity at higher temperature such as 50℃ can increase the yield of proteolysis industrially. In addition, detergents and EDTA as major detergent ingredients tested in this study have no significant inhibitory effects on the protease produced from B. polyfermenticus . Therefore, these results suggest that protease produced from B. polyfermenticus SCD is a potential material as a detergent additive for laundry and may be useful in other industries such as textile or leather processing.
Acknowledgements
This research was supported by the Priority Research Centers Program (Grant #2012-0006686) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology.
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Bhaskar N , Sudeepa ES , Rashmi HN , Tamil SA 2007 Partial purification and characterization of protease of Bacillus proteolyticus CFR3001 isolated from fish processing waste and its antibacterial activities Bioresour. Technol 98 2758 - 2764    DOI : 10.1016/j.biortech.2006.09.033
Chang KH , Park JS , Choi JH , Kim CJ , Paik HD 2007 Cytotoxic effects of partially purified substances from Bacillus polyfermenticus SCD supernatant toward a variety of tumor cell lines. Food Sci. Biotechnol 16 163 - 166
Chi Z , Ma C , Wang P , Li HF 2007 Optimization of medium and cultivation conditions for alkaline protease production by the marine yeast Aureobasidium pullulans. Bioresour. Technol 98 534 - 538    DOI : 10.1016/j.biortech.2006.02.006
Choi GH , Lee JH , Jo MN , Yoon YC , Paik HD 2008 Growth and antioxidant production of Bacillus polyfermenticus SCD in whey protein concentrate (WPC)-based medium. Korean J. Food Sci. Anim. Resour 28 105 - 108    DOI : 10.5851/kosfa.2008.28.1.105
Genckal H , Tari C 2006 Alkaline protease production from alkalophilic Bacillus sp. isolated from natural habitats. Enzyme Microb. Technol 39 703 - 710    DOI : 10.1016/j.enzmictec.2005.12.004
Gupta R , Beg QK , Lorenz P 2002 Bacterial alkaline proteases; molecular approaches and industrial applications. Appl. Microbiol. Biotechnol 59 15 - 32    DOI : 10.1007/s00253-002-0975-y
Gupta RK , Prasad D , Sathesh J , Naidu RB , Kamini NR , Palanivel S 2012 Scale-up of an alkaline protease from Bacillus pumilus MTCC 7514 utilizing fish meal as a sole source of nutrients. J. Microbiol. Biotechnol 22 1230 - 1236    DOI : 10.4014/jmb.1203.03021
Hagihara B 1958 The Enzymes. Vol. 4. Academic Press Inc. NY
Johnvesly B , Naik GR 2001 Studies on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp. JB-99 in a chemically defined medium. Process Biochem 37 139 - 144    DOI : 10.1016/S0032-9592(01)00191-1
Jun KD , Lee KH , Kim WS , Paik HD 2000 Microbiological identification of medical probiotic Bispan strain. Kor. J. Microbiol. Biotechnol 28 124 - 127
Jun KD , Kim HJ , Lee KH , Paik HD , Kang JS 2002 Characterization of Bacillus polyfermenticus SCD as a probiotic. Kor. J. Appl. Microbiol. Biotechnol 30 359 - 366
Jung JH , Lee MY , Chang HC 2012 Evaluation of the probiotic potential of Bacillus polyfermenticus CJ6 isolated from meju, a Korean soybean fermentation starter. J Microbiol. Biotechnol 22 1510 - 1517    DOI : 10.4014/jmb.1205.05049
Kim SS , Kim Y , Rhee IK 2001 Purification and characterization of a novel extracellular protease from Bacillus cereus KCTC 3674. Arch. Microbiol 175 458 - 461    DOI : 10.1007/s002030100282
Kim TH , Lee NK , Chang KH , Park EJ , Choi SY , Paik HD 2006 Antioxidant activity of partially purified extracts isolated from Bacillus polyfermenticus SCD culture. Food Sci. Biotechnol 15 482 - 484
Kim Y , Bae JH , Oh BK , Lee WH , Choi JW 2002 Enhancement of proteolytic enzyme activity excreted from Bacillus stearothermophilus for a thermophilic aerobic digestion process. Bioresour. Technol 82 157 - 164    DOI : 10.1016/S0960-8524(01)00177-8
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 680 - 685    DOI : 10.1038/227680a0
Lowry OH , Rosebrough NJ , Farr AL , Randall RJ 1951 Protein measurement with the folin-phenol reagents. J. Biol. Chem 193 265 - 275
Lu F , Lu M , Lu Z , Bie X , Zhao H , Wang Y 2008 Purification and characterization of xylanase from Aspergillus ficuum AF-98. Bioresour. Technol 99 5938 - 5941    DOI : 10.1016/j.biortech.2007.10.051
Manni L , Jellouli K , Agrebi R , Bayoudh A , Nasri M 2008 Biochemical and molecular characterization of a novel calciumdependent metalloprotease from Bacillus cereus SV1. Process Biochem 43 522 - 530    DOI : 10.1016/j.procbio.2008.01.016
Mohamed H , Safia K , Moncef N , Neji G 2007 Purification and characterization of an alkaline serine-protease produced by a new isolated Aspergillus clavatus ES1. Process Biochem 42 791 - 797    DOI : 10.1016/j.procbio.2007.01.011
Monod M , Togni G , Rahalison L , Frenk E 1991 Isolation and characterization of an extracellular alkaline protease of Aspergillus fumigatus. J. Med. Microbiol 35 23 - 28    DOI : 10.1099/00222615-35-1-23
Najafi MF , Deobagkar D 2005 Potential application of protease isolated from Pseudomonas aeruginosa PD100. Electron. J. Biotechnol 8 198 - 207    DOI : 10.2225/vol8-issue2-fulltext-5
Nilegaonkar SS , Zambare VP , Kanekar PP , Dhakephalkar PK , Sarnaik SS 2007 Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326. Bioresour. Technol 98 1238 - 1245    DOI : 10.1016/j.biortech.2006.05.003
Nonaka H , Ishikawa Y , Otsuk Y , Toda Y , Sato M , Nakamura R 1983 Purification and some properties of neuraminidase isolated from the culture medium of oral bacterium Streptococcus mitis ATCC 9811. J. Dent. Res 62 792 - 797    DOI : 10.1177/00220345830620070301
Park E , Park JS , Paik HD 2004 Effect of Bacillus polyfermenticus SCD and its bacteriocin on MNNG induced DNA damage. Food Sci. Biotechnol 13 684 - 688
Park E , Kim KT , Kim CJ , Kim CH , Paik HD 2004 Anticarcinogenic and antigenotoxic effects of Bacillus polyfermenticus. J. Microbiol. Biotechnol. 14 852 - 858
Paik HD , Jung MY , Jung HY , Kim WS , Kim KT 2002 Characterization of Bacillus polyfermenticus SCD for oral bacteriotherapy of gastrointestinal disorders. Korean J. Food Sci. Technol 34 73 - 78
Rahman R , Razak C , Ampon K , Basri M , Yunus W , Salleh A 1994 Purification and characterization of a heat stable protease from Bacillus stearothermophilus F1. Appl. Microbiol. Biotechnol 40 822 - 827    DOI : 10.1007/BF00173982
Raju AA , Chandrababu NK , Samivelu N , Rose C , Rao NM 1996 Eco-friendly enzymatic dehairing using extracellular protease from Bacillus species isolate. J. Am. Leath. Chem. Assoc. 91 115 - 119
Rao MB , Tanksale AM , Ghatge MS , Deshpande VV 1998 Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev 62 597 - 635
Sana B , Ghosh D , Saha M , Mukherjee J 2006 Purification and characterization of a salt, solvent, detergent and bleach tolerant protease from a new gamma-Proteobacterium isolated from the marine environment of the Sundarbans. Process Biochem 41 208 - 215    DOI : 10.1016/j.procbio.2005.09.010
Shikha Adhyayan S , Nandan SD 2007 Improved production of alkaline protease from a mutant of alkalophilic Bacillus pantotheneticus using molasses as a substrate. Bioresour. Technol. 98 881 - 885    DOI : 10.1016/j.biortech.2006.03.023
Studdert CA , Seitz MKH , Gilv MIP , Sanchez JJ , De Castro RE 2001 Purification and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease. J. Basic Microbiol. 6 375 - 683
Tremacoldi CR , Monti R , Selistre-De-Araujo HS , Carmona EC 2007 Purification and properties of an alkaline protease of Aspergillus clavatus. World J. Microbiol. Biotechnol. 23 295 - 299    DOI : 10.1007/s11274-006-9211-8
Tunga R , Shrivastava B , Banerjee R. 2003 Purification and characterization of a protease from solid state cultures of Aspergillus parasiticus. Process Biochem. 38 1553 - 1558    DOI : 10.1016/S0032-9592(03)00048-7
Whooley MA , O’Callaghan JA , McLoughlin AJ 1983 Effect of substrate on the regulation of exoprotease production by Pseudomonas aeruginosa ATCC 10145. J. Gen. Microbiol. 129 981 - 988