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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep 6 7
Purification and characterization of novel organic solvent tolerant 98 kDa alkaline protease from isolated Stenotrophomonas maltophilia strain SK
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Q1
Shailesh R. Waghmare a, Aparna A. Gurav a, Sonal A. Mali a, Naiem H. Nadaf a, Deepak B. Jadhav a, Kailas D. Sonawane a,b,⇑ a b
Department of Microbiology, Shivaji University, Kolhapur, Maharashtra, India Department of Biochemistry, Shivaji University, Kolhapur, Maharashtra, India
a r t i c l e
i n f o
Article history: Received 12 September 2014 and in revised form 9 November 2014 Available online xxxx Keywords: Alkaline protease Organic solvent Stenotrophomonas maltophilia strain SK
a b s t r a c t Ability of microorganisms to grow at alkaline pH makes them an attractive target for several industrial applications. Thus, search for new extremozyme producing microorganisms must be a continuous exercise. Hence, we isolated a potent alkaline protease producing bacteria from slaughter house soil. The morphological, biochemical and 16S rDNA gene sequencing studies revealed that the isolated bacteria is Stenotrophomonas maltophilia strain SK. Alkaline protease from S. maltophilia strain SK was purified by using ammonium sulphate precipitation and DEAE-cellulose ion exchange column chromatography. The purified enzyme was optimally active at pH 9.0 and temperature 40 °C with broad substrate specificity. It was observed that the metal ions such as Ca++, Mg++ and Fe+++ completely repressed the enzyme activity. The enzyme was stable in presence of various water miscible solvents like ethanol, methanol, isopropanol at 25% (v/v) concentration and less stable at 37.5% (v/v) concentration. These robust properties of enzyme might be applicable for various applications in detergent and pharmaceutical industries. Ó 2014 Published by Elsevier Inc.
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Introduction
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Extremophiles from diverse and exotic environment can remain active in extreme conditions and hence they are considered as an important source of several enzymes [1]. Thus, as per earlier report, search for new extremozyme producing microorganisms must be an endless task [2]. Today, proteases account for approximately 40% of the total enzyme sales in various industrial market sectors, such as food, detergent, leather, pharmaceutical, diagnostics and waste management. This dominance of proteases in the industrial market is expected to increase further [3]. Proteases are classified into six groups: aspartate, cysteine, glutamate, metallo, serine, and threonine based on characteristic mechanistic features consistent within each member of a group [4]. Microorganisms elaborate a large array of intracellular and/or extracellular proteases. Extracellular proteases are important for the hydrolysis of proteins in cell-free environments. They enable cells to absorb
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⇑ Corresponding author at: Department of Biochemistry, Shivaji University, Kolhapur 416 004, Maharashtra (M.S.), India. Tel.: +91 9881320719, +91 231 2609153; fax: +91 231 2692333. E-mail address:
[email protected] (K.D. Sonawane).
and utilize hydrolytic products. Similarly, intracellular proteases are important for various metabolic cellular processes such as differentiation, sporulation, protein turnover, maturation of hormones, enzymes and maintenance of the cellular protein pool [5]. Diverse groups of microorganisms such as fungi, bacteria and actinomycetes have been known to produce alkaline proteases [6]. In case of bacteria Bacillus [7–11] and Streptomyces species [12–15] are predominant one. Miyaji and coworkers in 2005 described the production of extracellular alkaline serine protease from Stenotrophomonas maltophilia S-1 [16]. It has been said that alkaline protease from S. maltophilia could play an important role in the pathogenesis of several diseases [17]. Alkaline proteases are robust enzymes with considerable industrial potential in detergents [18], leather processing [19], silver recovery [20], medical purposes [21], food processing [22,23] and chemical industries [24], as well as waste treatment [25]. Earlier reports have also demonstrated the use of alkaline proteases to catalyze peptide synthesis in organic solvents [26,27]. Hence, looking at the important uses and applications of alkaline proteases in various sectors, we have isolated, purified, characterized and studied applications of alkaline protease produced by S. maltophilia strain SK.
http://dx.doi.org/10.1016/j.pep.2014.11.002 1046-5928/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: S.R. Waghmare et al., Purification and characterization of novel organic solvent tolerant 98 kDa alkaline protease from isolated Stenotrophomonas maltophilia strain SK, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.002
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Materials and methods
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Isolation and identification of alkaline protease producers
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Alkaline protease producing microorganisms were isolated from slaughter house soil samples. Serial dilutions of soil samples were prepared and spread on the Bennett’s agar containing glucose 10.0 gm, beef extract 1.0 g, yeast extract 1.0 g, casein 20.0 g, agar 20.0 g in 1000 ml D/W having different pH range such as 7, 8, 9, 10, and 12. All plates were incubated at room temperature for 24 h and then zone of hydrolysis was observed around the colonies. Three different colonies showing zone of hydrolysis were separated. After every 15 days of interval the isolated bacteria were cultured on the Bennett’s agar slant. The maximum alkaline protease producing isolate was identified on the basis of morphological; biochemical characteristics and 16S rRNA gene sequencing. The phylogenetic tree was constructed using MEGA4 software and sequence was submitted to GenBank, NCBI (Accession No. KC812737).
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Alkaline protease production
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The alkaline protease production from isolated S. maltophilia strain SK was carried out in the 250 ml Erlenmeyer flask containing 100 ml Bennett’s medium. The fresh culture of S. maltophilia strain SK was inoculated into production medium containing glucose 1.0 gm, beef extract 0.1 g, yeast extract 0.1 g, 1% casein and then flasks were incubated at 37 °C. The protease activity was monitored by the method described in the enzyme assay section after every 12 h of incubation.
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Purification of alkaline protease
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Purification of enzyme was carried out at 4 °C. The fresh growth of S. maltophilia strain SK was inoculated in 100 ml medium containing casein as a protein source. After 36 h of incubation the cell density was high when broth taken for extraction of enzyme. The cell free extract was collected by centrifugation at 5000 rpm for 15 min and then precipitation was carried out at 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75% by using fractional ammonium sulphate saturation. The precipitate was collected by centrifugation at 8000 rpm for 20 min at 4 °C. The precipitate was then dissolved in 10 ml glycine-NaOH buffer (pH 9.0) and dialyzed overnight against same buffer. This was further purified by the DEAE-cellulose ion exchange column chromatography having column height 15 cm and diameter 2.5 cm. The column was eluted with the series of salt steps of 0.1–1.0 M NaCl concentration, and the fractions of 5 ml were collected at the flow rate of 0.5 ml per min. All fractions were checked for their protein content by measuring absorbance at 280 nm with a spectrophotometer. The protein content was determined by the Lowry method using standard graph of Bovine Serum Albumin [28].
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Characterization of purified alkaline protease
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Effect of temperature and pH The effect of temperature on enzyme activity was studied between 10 and 60 °C temperature range at pH 9.0. The effect of pH on enzyme activity was also studied at pH range from 4.0 to 10.0 at 40 °C, using 50 mM sodium acetate buffer (pH 4.0–5.0), sodium-phosphate buffer (pH 6.0–8.0), glycine-NaOH buffer (pH 9.0–12.0). The enzyme activity was expressed as % relative activity.
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Effect of metal ions Purified alkaline protease was pre-incubated for 1 h with 50 mM glycine-NaOH buffer pH 9.0 along with the metal ions Ca++, Na++, Mg++, Fe+++, Zn++, Co++ and Cu++ using their respective water soluble salts to attain 5 mM concentration. The activity was measured by using standard assay protocol and expressed as % residual activity.
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Substrate specificity The purified alkaline protease was assayed at 40 °C for substrate specificity by using different substrates like casein, gelatin and BSA dissolved in 50 mM glycine-NaOH buffer having pH 9.0 at concentration of 5 mg/ml.
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Effect of organic solvent Effect of various organic solvents such as ethanol, methanol, isopropanol, and acetone on protease activity was also studied. The 0.1 ml enzyme was incubated in presence of 0.1 ml of these solvents having concentration 25%, 50% and 75% at 30 °C for 1 h. After incubation, the residual protease activity was measured.
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SDS–PAGE Purity of the fractions, showing alkaline protease activity, was checked by SDS–PAGE as per the method explained by Laemmli et al. in 1970 [29]. The bands were visualized using Coomassie brilliant blue R-250 stain. The molecular weight of alkaline protease was determined by comparison with standard molecular marker proteins (phosphorylase b 98 kDa, bovine serum albumin 66 kDa, ovalbumin 43 kDa, carbonic anhydrase 29 kDa).
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Enzyme assay The reaction mixture in a total volume of 2.5 ml was composed of 1.0 ml 1% casein, 1.0 ml enzyme solution and 0.5 ml glycineNaOH buffer (pH 9.0). After 30 min incubation at 37 °C the reaction was terminated by adding 0.5 ml trichloroacetic acid. The supernatant was collected by centrifugation at 4000 rpm for 10 min. Out of that 0.5 ml supernatant was taken. Then in that supernatant 2.5 ml 0.5 M sodium carbonate buffer was added followed by 0.5 ml 1 M folin phenol reagent. The reaction mixtures were allowed to stand for 30 min at room temperature and then absorbance was measured at 660 nm. The product was determined by using standard graph of tyrosine. One unit of enzyme activity defined as the amount of enzyme required to release 1 lg tyrosine residue per minute at specified conditions.
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Results
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Identification of alkaline protease producer
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The soil sample was spread over the Bennett’s agar medium. After the incubation period, morphologically distinct colonies were observed on the agar plates. Some of the colonies were showing zone of hydrolysis indicating production of extracellular protease. It was found that as pH increases the number of colonies decreases. Therefore, only few microorganisms which possess alkaline protease activity at pH 11 were isolated and labeled as Iso-A, Iso-B, and Iso-C. Among these Iso-B showed maximum zone of hydrolysis. The Iso-B was identified by standard morphological, physiological and biochemical tests according to Bergey’s Manual of Systematic Bacteriology. The organism was a Gram negative rod shaped bacteria capable of growing at alkaline pH. The organism was further confirmed by 16S rRNA gene sequencing having length of 1446 bp nucleotides which confirmed that the isolated strain is S. maltophilia strain SK. This sequence was
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deposited in GenBank with accession number KC812737. The phylogenetic relationship of S. maltophilia strain SK is shown in Fig. 1.
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Production of alkaline protease
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Bennett’s medium containing casein as a protein source having pH 10.0 was used for the production of alkaline protease from S. maltophilia strain SK. The enzyme was produced after 12 h and reached maximum up to 36 h. After 36 h enzyme production started decreasing rapidly as can be seen in Fig. 2. The S. maltophilia strain SK produces alkaline protease during log phase indicating that the hydrolyzed protein was required for the growth of organism.
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Purification of alkaline protease
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Extracellular alkaline protease produced by S. maltophilia strain SK was precipitated at 70% ammonium sulphate saturation which was then further purified by the DEAE-cellulose ion exchange column chromatography as shown in Fig. 3A. Other proteins were eluted in between 0.1 and 0.5 M NaCl concentrations, but our protein of interest i.e. alkaline protease was eluted at 0.2 M NaCl concentration. No other eluted peaks showed alkaline protease activity which confirm that the S. maltophilia strain SK produces only single protease. The purity of enzyme increased up to 5.19-fold with 24% yield when enzyme was purified by ion exchange chromatography (Table 1).
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Characterization of purified alkaline protease
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Molecular weight determination SDS–PAGE analysis of purified alkaline protease revealed the enzyme was completely purified by the ion exchange chromatography. The molecular mass of purified alkaline protease was found approximately 98 kDa as shown in Fig. 3B. The single band on the SDS–PAGE indicates the alkaline protease secreted by the
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Fig. 2. Alkaline protease production by using S. maltophilia strain SK at different time intervals. The fresh growth of S. maltophilia strain SK was inoculated into flask containing 100 ml Bennett’s medium and enzyme activity was monitored after every 12 h of interval.
S. maltophilia strain SK is made up of by the single polypeptide chain.
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Effect of temperature and pH The alkaline protease activity of purified enzyme was studied at different pH and temperature range. Enzyme found more active at alkaline pH in the range of pH 7.0–10.0, but was optimally active at pH 9.0 (Fig. 4). The activity of purified alkaline protease was observed in between temperature range 15–45 °C with optimally active at 40 °C (Fig. 5). Thus, it confirms that alkaline protease produced by S. maltophilia strain SK is active in mesophilic temperature range.
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Effect of metal ions Effect of metal ions on purified protease shows complete inhibition of enzyme activity in presence of CaCl2, MgSO4 and FeCl3 (Fig. 6). Nearly, 80% inhibition was observed in presence of NaCl, ZnSO4, and CuSO4 and 50% inhibition by CoCl2 as can be seen in Fig. 6.
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Fig. 1. Shows phylogenetic tree of isolated S. maltophilia strain SK. The isolated strain was identified using 16S rDNA gene sequencing technique and submitted to NCBI’s GenBank database (Accession Number KC812737). Blastn programme was used to search homologous sequences. Sequences showing more homology were downloaded and the tree was constructed by neighbor-joining method using MEGA4 software.
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Fig. 3. (A) Purification profile of alkaline protease by DEAE-cellulose ion exchange column chromatography. The extracellular alkaline protease produced by S. maltophilia strain SK was precipitated by 70% ammonium sulphate and loaded on DEAE-cellulose column. The proteins from column was eluted at 0.1–1.0 M NaCl and protein content (N) was checked by taking an absorbance at 280 nm, where as alkaline protease activity (j) was determined by standard assay method. (B) SDS–PAGE analysis of purified alkaline protease. The enzyme extracted at different steps such as crude enzyme (C) and purified enzyme (P) analyzed by SDS–PAGE and molecular weight was determined by comparing with standard molecular eight marker proteins (M).
Table 1 Purification of protease from S. maltophilia strain SK. Purification steps
Total activity (U)
Total protein (mg)
Specific activity (U/mg protein)
Yield (%)
Purification fold
Crude enzyme Ammonium sulphate precipitation DEAE-cellulose ion exchange column chromatography
250.0 ± 0.05 160.0 ± 0.07 60.0 ± 0.03
52.3 ± 0.08 13.6 ± 0.04 2.7 ± 0.04
4.78 ± 0.06 11.76 ± 0.05 22.22 ± 0.03
100 64 24
1 2.74 5.19
Each value represents the mean ± standard error values. The total volume of fermentation medium was 100 ml.
100 90 Relative activity (%)
80 70 60 50 40 30 20 10 0
Fig. 4. Effect of pH on alkaline protease activity. The enzyme activity was studied at different pH range between pH 4 and pH 10 and activity expressed as % relative activity.
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Substrate specificity Substrate specificity study of purified alkaline protease of S. maltophilia strain SK was performed with various substrates such as casein, gelatin, and bovine serum albumin (BSA)1. It was found that the enzyme has more activity towards gelatin than casein and BSA, which confirms the broad substrate specificity of alkaline protease extracted from S. maltophilia strain SK (Fig. 7).
Effect of organic solvents Table 2 shows the stability of alkaline protease in presence of various solvents at different concentrations such as 12.5%, 25% 1
Abbreviations used: BSA, bovine serum albumin.
10
20
30
40
50
60
Temperature ˚C Fig. 5. Effect of temperature on alkaline protease activity. Enzyme activity was checked at different temperature range from 10 to 60 °C and activity expressed as % relative activity.
and 37.5%. The protease was found stable particularly at 12.5% and 25% (v/v) concentration of ethanol, acetone, methanol, isopropanol but at 37.5% (v/v) showed less stability. The enzyme was found more stable at 25% (v/v) acetone concentration.
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Discussion
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In this study, we isolated and identified alkaline protease producer S. maltophilia strain SK from slaughter house soil. Earlier, Miyaji and coworkers had isolated alkaline serine protease from
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The purified alkaline protease of S. maltophilia strain SK was found more sensitive to metal ions such as Ca+2, Mg+2, and Fe+3, whereas alkaline protease from Bacillus sp. showed stimulatory effects as per earlier report [31]. It has been known that the natural proteases which remain stable in organic solvents are more useful for various applications employing organic solvents as a reaction media because they can be used without any modification to stabilize the enzymes [32]. Our results depict that the purified alkaline protease from S. maltophilia strain SK is more stable in various water miscible organic solvents such as ethanol, methanol, isopropanol and acetone similar to protease from Bacillus sp. JER02 [33]. SDS–PAGE analysis of purified alkaline protease of S. maltophilia strain SK revealed a single band of 98 kDa molecular weight which is comparatively larger than the 75 and 48 kDa molecular weight alkaline proteases produced from S. maltophilia MTCC 7528 [30] and S. maltophilia [17] respectively. The characteristics of alkaline protease produced from S. maltophilia strain SK such as broad substrate specificity and stability in presence of various organic solvents might be more applicable for peptide production in non aqueous environment.
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Conclusion
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The S. maltophilia strain SK isolated from slaughter house soil has capability to produce 98 kDa alkaline protease. This enzyme has ability to work in harsh conditions such as alkaline pH, elevated temperature and in presence of organic solvents. These properties of enzyme explore applicability in leather industry, detergent industry, silver recovery, feed industry and management of proteinaceous waste. Similarly, this enzyme could also be used to produce peptides in non-aqueous phase which might minimize the production cost.
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Fig. 7. Substrate specificity of purified alkaline protease. The enzyme substrate specificity was studied using gelatin, bovine serum albumin and casein as substrate.
Acknowledgments
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Q2 This work is supported by Department of Biotechnology (DBT) Q3 and Department of Science and Technology (DST), Government of India, New Delhi under the ‘‘DBT-IPLS’’ and DST-PURSE Schemes sanctioned to Shivaji University, Kolhapur, Maharashtra, India.
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Table 2 Effect of organic solvents on protease activity.
References
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Fig. 6. Effect of different metal ions on alkaline protease activity. The enzyme was pre-incubated with different metal ions at 5 mM concentration for 1 h at 40 °C and enzyme activity was expressed as % residual activity.
Organic solvents
255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271
5
Residual activity in % at different concentration 12.5%
25%
37.5%
Ethanol Methanol Isopropanol Acetone
106 ± 1.2 103 ± 1.1 109 ± 1.4 100 ± 1.1
100 ± 0.8 100 ± 0.9 102 ± 0.8 116 ± 1.2
86 ± 1.3 93 ± 1.1 78 ± 1.1 68 ± 0.9
Control
100 ± 0.8
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Please cite this article in press as: S.R. Waghmare et al., Purification and characterization of novel organic solvent tolerant 98 kDa alkaline protease from isolated Stenotrophomonas maltophilia strain SK, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.11.002
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