International Journal of Environmental Health Research
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Characterization of extracellular amylase produced by haloalkalophilic strain Kocuria sp. HJ014 Marisela Y. Soto-Padilla, Pablo Gortáres-Moroyoqui, Luis A. Cira-Chávez, Anthony Levasseur, Luc Dendooven & María Isabel Estrada-Alvarado To cite this article: Marisela Y. Soto-Padilla, Pablo Gortáres-Moroyoqui, Luis A. Cira-Chávez, Anthony Levasseur, Luc Dendooven & María Isabel Estrada-Alvarado (2016): Characterization of extracellular amylase produced by haloalkalophilic strain Kocuria sp. HJ014, International Journal of Environmental Health Research, DOI: 10.1080/09603123.2015.1135310 To link to this article: http://dx.doi.org/10.1080/09603123.2015.1135310
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Date: 29 January 2016, At: 00:39
International Journal of Environmental Health Research, 2016 http://dx.doi.org/10.1080/09603123.2015.1135310
Characterization of extracellular amylase produced by haloalkalophilic strain Kocuria sp. HJ014 Marisela Y. Soto-Padillaa,b, Pablo Gortáres-Moroyoquib, Luis A. Cira-Chávezb, Anthony Levasseurc, Luc Dendoovend and María Isabel Estrada-Alvaradob Downloaded by [NUS National University of Singapore] at 00:39 29 January 2016
a
Instituto de Ingeniería y Tecnología, Universidad Autónoma de Ciudad Juárez, Chihuahua, Mexico; bBiotecnología y Ciencias Alimentarias, Instituto Tecnológico de Sonora, Obregón, Mexico; cBiotechnologie des Champignons Filamenteux, INRA, Marseille, France; dCinvestav, ABACUS, Mexico City, Mexico
ABSTRACT
The haloalkaliphilic bacterium Kocuria sp. (HJ014) has the ability to produce extracellular amylase. The aim of this study was to purify and characterize this protein. The amylase enzyme with a specific activity of 753,502 U/mg was purified 5.7– fold using Sepharose 4B and Sephacryl S-300 gel filtration columns. The molecular weight of the enzyme was 45,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amylase showed maximum activity at pH 9 and 50°C in the presence of 3.5 M NaCl. The Km was 3.0 mg/ml and Vmax 90.09 U/ml. It was found that extracellular amylase from Kocuria sp. has a high industrial potential.
ARTICLE HISTORY
Received 23 April 2015 Accepted 3 December 2015 KEYWORDS
Haloalkaliphilic; enzyme molecular weight; industrial potential; specific activity
Introduction Organisms in the soil contribute to the functioning of the ecosystem. Soil is an appropriate environment for both eukaryotic (algae, fungi, protozoa) and prokaryotic (bacteria and archaea) organisms. Viruses and bacteriophages can also be found in soil (Nogales 2005). Extreme environments are defined by an extreme pH, temperature, salinity or other soil characteristics or combinations of them (Rothschild and Mancinelli 2001). There are groups of organisms that are adapted specifically to these extreme conditions. These organisms are usually referred to as alkaliphiles, halophiles, thermophiles, and acidophiles, reflecting the particular type of extreme environment they inhabit (Ulukanli and Digrak 2002). Extremophiles produce several compounds of industrial interest and many of these are excreted in the environment. Recent research has focused on the production of extracellular enzymes from bacteria (Sastoque et al. 2007; Ninawe et al. 2008; Rohban et al. 2009). For example, it is known that hydrolytic enzymes (amylases, proteases, peptidases, esterases, and lipases) have different potential applications in biomedical science, chemical industries, and industrial processes (Ventosa et al. 1998; Margesin and Schinner 2001; Mellado et al. 2004). Amylases are one of the most important industrial enzymes that have a wide variety of applications, ranging from conversion of starch to sugar syrups (saccharification), to the production of cyclodextrins for the pharmaceutical industry. Amylases are hydrolytic enzymes widespread in nature, particularly in animals, micro-organisms, and plants (Safey and Ammar 2004). Amylases have been obtained from halophilic, alkaliphilic, thermophilic, and mesophilic micro-organisms (Chessa et al. 1999; Kiran and Chandra 2008; Nouadri et al. 2010). These enzymes account for about 30 % of the world’s enzyme
CONTACT María Isabel Estrada-Alvarado © 2016 Taylor & Francis
[email protected]
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production, as enzymatic hydrolysis is preferred over acid hydrolysis in the starch processing industry (Marc et al. 2002). Enzymatic hydrolysis has different advantages, such as specificity, stability, low energy consumption, and not neutralization step (Satyanarayana et al. 2005). Due to the increasing demand for these enzymes in various industries, there is an enormous interest in finding enzymes with suitable properties for industrial applications (Van der Maarel et al. 2002; Burhan et al. 2003). The genus Kocuria was proposed by Stackebrandt et al. (1995) and consists currently of 18 species with valid published names (http://www.bacterio.cict.fr/k/kocuria.html). The members of this genus have been isolated from different sources, such as air, fermented sea food, mammalian skin, soil, the rhizoplane, freshwater, seawater, marine sediment, and desert soil (Tang et al. 2009; Monu et al. 2012). Yamaguchi et al. (2011) reported the cloning and characterization of a halophilic–amylase obtained from the moderately halophilic bacterium K. varians. The aim of this work was to purify and characterize an amylase obtained from Kocuria sp. isolated from an extreme alkaline saline soil of the former Texcoco Lake.
Materials and methods Enzyme production The strain Kocuria sp. (HJ014) was isolated from the former Texcoco Lake (Soto-Padilla et al. 2013). It was cultivated in liquid (Bacto marine broth 2216, DIFCO) and on solid medium (Bacto marine agar 2216, DIFCO) at 37 °C for 72 h. It was grown at optimum conditions (NaCl 5 % and pH 7.5, at 37 °C and 200 rpm) in marine broth (2216, DIFCO) supplemented with 1 % soluble starch. The growth was estimated by turbidity measurement (Cintra 10e) at 600 nm (Coronado et al. 2000). Amylase of Kocuria sp. (HJ014) was produced in 500-ml Erlenmeyer flasks containing 200 ml of marine broth (DIFCO) supplemented with 1 % soluble starch and 5 % NaCl. Preinoculum was grown during 24 h until reach 0.8 absorbance (OD600), then 10 % (v/v) was used to inoculate the enzyme production medium. Incubation was done at 200 rpm and 37 °C for 48 h. The enzyme extract was obtained by centrifugation (Centrifuge max) at 9500 xg for 20 min at 4 °C. The clear supernatant was used for the amylase activity assay. Amylase activity was determined by measuring the release of reducing sugar from soluble starch by the Bernfeld method (Bernfeld 1955). The reaction mixture contained 450 μl of 1 % (w/v) starch (prepared in 100 mM phosphate buffer, pH 7.5) and 50 μl of enzyme extract. The reaction mixture was maintained at 50 °C for 10 min. The enzymatic reaction was stopped by adding dinitrosalicylic acid (DNS) solution. The amount of reducing sugar released was quantified using 3, 5-dinitrosalicylic acid with maltose as standard. One unit of amylase activity was defined as the amount of enzyme that produced 1 μmol of reducing sugars (as maltose) per minute. The specific activity was expressed as units of enzyme activity per milligram of protein. The relative amylase activity was defined as the percentage of the maximum amylase activity detected in the assay. Assays were performed in triplicate. The protein concentration was determined using the bicinchoninic acid protein assay kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer′s recommendations. Standard curve of bovine serum albumin was used as standard protein in the range of 50–250 μg/mL. Enzyme characterization The supernatant from the culture medium was concentrated by drying in an oven (Felisa) at 40 °C for 48 h. The concentrated protein extract was used for enzyme purification by chromatography into a Sepharose 4B column (GE Healthcare) previously equilibrated with buffer A (20 mM tris–HCl, pH 8) at a flow rate of 0.5 ml/min and adapted into a protein purification equipment (ÄKTA10 purifier, GE Healthcare, Fairfield, CT, USA). The column was washed with the same buffer until no absorbance at 280 nm was detected in the eluent. The absorbed proteins were eluted with a lineal gradient of NaCl concentration 0–1 M (typically linear) at a flow rate of 0.5 ml/min. Fractions that showed amylase
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activity (assayed as described before) were collected and loaded on a Sephacryl S-300 column (GE Healthcare, Fairfield, CT, USA) equilibrated with buffer A. The enzyme was eluted at a flow rate of 0.5 ml/min and collected in 2ml fractions. Fractions containing amylase activity were pooled and used for further analysis. SDS-PAGE (12 % (w/v)) was used to determine protein purity and molecular mass of the purified enzyme as described by Laemmli (1970). After electrophoresis, the protein bands were silver stained. The molecular weight of the standard proteins used were myosin (200.0 kDa), β-galactosidase (116.2 kDa), phosphorilase b (97.4 kDa), bovin serum albumin (66.2 kDa), ovalbumin (45.0 kDa), carbonic anhydrase (31.0 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa), and aprotinin (6.5 kDa). The effect of pH on the activity of the purified enzyme was investigated by preparing soluble starch substrate in various buffers (100 mM): sodium phosphate (pH 6.0–7.5), Tris–HCl (pH 8.0–9.0), and glycine–NaOH (pH 9.5–12) (Shafiei et al. 2012). The reaction was carried at 50 °C for 10 min. The temperature effect on the enzyme activity was determined in the range of 10–90 °C and the reaction was done at pH 7.5 for 10 min. The effect of NaCl concentration on enzyme activity was determined by measuring the amylase activity (described in enzyme production) in the reaction mixture containing different NaCl concentrations (0–4 M) (Shafiei et al. 2012). The amount of enzyme extract used was 50 μl at 50 °C and pH 7.5. The kinetic parameters Km and Vmax of the purified amylase were measured using soluble starch as substrate. Starch hydrolysis was colorimetrically assayed in the range of 0–4 % (w/v) in buffer Tris–HCl (pH 9) at 55 °C for 10 min (Patel et al. 1993). Km and Vmax values were obtained from the Hanes and Woolf plot (Ritchie and Prvan 1996).
Results The maximum cell concentration was achieved after 72 h (Figure 1). Amylase activity was 8900 U/ml after 1 h and reached a maximum of 133,772 U/ml after 39 h. The results of the purification process are summarized in Table 1. In the first purification step, a specific activity of 203,681 U/mg was obtained. In the second purification step, the fractions containing amylase activity were applied to the Sephacryl S-300 column and had a specific activity of 753,502 U/mg and purified 5.7–fold. A single protein band was observed after electrophoresis of the fraction obtained from the column purification Sephacryl S-300 in 12 % SDS-PAGE (Figure 2). The molecular weight of the purified amylase was 45 kDa. Figure 3 shows the single and symmetrical absorption
Figure 1. Kinetic growth of HJ014 and amylase activity on Marine Broth supplemented with 1 % soluble starch and 5 % NaCl.
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Table 1. Purification protein profile at different steps of the amylase from Kocuria sp. strain HJ014.
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Fraction Raw extract [Raw extract] Sepharose 4B Sephacryl S-300
Activity (U/ml) 252000 362533.3 285600 97955.3
Total activity (U) 20160000 15588933.3 9139200 1959106.4
Protein (mg/ml) 152.56 139.75 44.87 2.6
Specific activity (U/mg) 132144.72 111548.71 203681.74 753502.46
Purification (×times) 1 0.8 1.5 5.7
peak parallel to the peak of the amylase activity in the fraction obtained from the Sephacryl S-300 gel filtration column. The enzymatic characteristics of the purified amylase were determined using soluble starch as substrate and the maximum specific activity of the enzyme was 51,701 U/mg at pH 9 (Figure 4). An increase in alkalinity from pH 8–11 decreased the relative activity with 20 %. Figure 5 indicate that the maximum enzyme activity expressed as relative activity was obtained at 40–50 °C with a maximum specific activity of 41,236 U/mg protein.
Figure 2. Separation of protein by SDS-PAGE.
Figure 3. Purification profile of amylase produced by Kocuria sp. strain HJ014.
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Figure 4. Effect of pH on the amylase activity of the purified enzyme produced from Kocuria sp. strain HJ014.
Figure 5. Effect of temperature on the amylase activity of the purified enzyme produced from Kocuria sp. strain HJ014.
The amylase activity was determined at various NaCl concentrations. The amylase showed maximum activity in 3.5 M NaCl (40,969 U/mg protein) (Figure 6). Relative activity was highest at 2 M NaCl. The effect of different concentrations of starch on the activity of amylase was studied and plotted ([S]/Vo vs. [S]) to determine the maximum velocity and Michaelis–Menten constant Km (Figure 7). The Km and Vmax values were 3.0 mg/ml and 90.09 U/ml, respectively.
Discussion Recently, extremophiles micro-organisms and their extremozymes have become important due to their potential application in industry (Madigan and Marrs 1997; Rothschild and Mancinelli 2001; Satyanarayana et al. 2005; Podar and Reysenbach 2006; Ferrer et al. 2007). As far as we know, this study is the first report about characteristics of an extracellular amylase derived from Kocuria sp. Pandey et al. (2000) reported that strains of Aspergillus sp. and Bacillus sp. were a source of amylase enzymes. Most enzymes from these bacteria might be purified by chromatographic techniques after crude isolation by precipitation and membrane separations. The demand for large-scale effective
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Figure 6. Effect of NaCl on the amylase activity of the purified enzyme produced from Kocuria sp. strain HJ014.
Figure 7. Hanes & Woolf plot of purified enzyme from strain HJ014 to calculate the Vmax and Km values.
purification of proteins has resulted in the evolution of techniques that provide fast, efficient, and economical protocols requiring few separation steps (Takeuchi et al. 2006). The α-amylase obtained in this study was purified using a chromatographic method applying Sepharose 4B and Sephacryl S-300 gel filtration columns, which is fast, efficient, and a low-cost technique. Purified amylolytic enzymes have been reported with molecular weights ranging from 42 to 75 kDa (Young et al. 1995; Safey and Ammar 2004; Kumar et al. 2005; Nouadri et al. 2010). The enzyme reported in this study had an apparent molecular weight of 45 kDa. Different studies have reported the purification and characterization of amylases obtained from other micro-organisms, but not all of them report their activities (Young et al. 1995; Safey and Ammar 2004). Takeuchi et al. (2006) reported 40,700 U of amylase activity for Pichia burtonii, which, compared to our results, 1,959,106 U, is lower than our activity obtained. The maximum activity in this study was reached within the first 48 h, whereas amylase from Penicillium camemberti had a maximum enzyme activity after 168 h (Nouadri et al. 2010). Amylase is usually produced from strains of the genus Bacillus as well as from some thermophilic strains. Production and purification of proteins from Kocuria sp. isolated from a haloalkaliphilic soil was done to obtain halophilic α-amylase with the capacity of an
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enzymatic catalysis in high NaCl concentrations and pH. Bacillus sp. is widely used for thermostable α-amylase production to meet industrial needs. Bacillus subtilis, B. stearothermophilus, B. licheniformis, and B. amyloliquefaciens are excellent producers of α-amylase and these bacteria have been widely used for commercial production (Sivaramakrishnan et al. 2006). The properties of α-amylases, such as thermostability and pH profile, should match their industrial application. Hence, the range of possible applications creates the need to search for new α-amylases with novel and improved properties for specific conditions. The rate of amylolytic hydrolysis depends on process conditions, such as temperature, pH, starch substrate (type and concentration), enzyme (catalytic properties and concentration), and the presence of NaCl. Kocuria sp. showed amylase activity in a pH range from 8.0 to 11.0 with maximum at pH 9.0, indicating its alkalitolerance. Kobayashi et al. (1986) reported an amylase obtained from the archaebacterium Natronococcus sp. with maximum activity at pH 8.7 (i.e. an alkalitolerant enzyme) similar to the alkalitolerant amylase of Bacillus sp. strain TSCVKK (Kiran and Chandra 2008) and Halobacillus sp. strain MA-2 (Amoozegar et al. 2003). The pH is one of the most important factors that determine the growth and morphology of micro-organisms as they are sensitive to hydrogen ion concentrations in the medium. Bacterial cultures, such as B. subtilis, B. licheniformis, and B. amyloliquefaciens required an initial pH of 7 (Syu and Chen 1997; Haq et al. 2005; Tanyildizi et al. 2005). Rhodothermus marinus was reported to have an amylase with an optimum pH range of 7.5–8 (Gomes et al. 2003). The effect of temperature on amylase production is related to the growth of the organism. Bacterial amylases are produced in a wide range of temperatures. Bacillus amyloliquefaciens, B. subtilis, B. licheniformis, and B. stearothermophilus are among the most commonly used Bacillus sp. that produces α-amylase at temperatures between 37 and 60 °C (Mendu et al. 2005; Mishra et al. 2005). The enzyme obtained in this study showed a maximum amylase activity at 50 °C similar to amylase from Halobacillus sp. strain MA-2 (Amoozegar et al. 2003) and Nesterenkonia sp. strain F with α-amylase activity between 45–50 °C (Sivaramakrishnan et al. 2006). Halophilic enzymes are therefore uniquely adapted to function in conditions with low water potential, this property is important for a biotechnological view. Consequently, these organisms can be exploited for industrial uses. These amylases are active in low water activity and showed interesting applications in the treatment of saline waters or waste solutions with starch residues and high salt content (Coronado et al. 2000; Amoozegar et al. 2003). The amylase produced by strain Kocuria sp. HJ014 showed maximum activity in 3.5 M NaCl. The amylase was active over a range of salt concentrations as it showed > 60 % activity in the 0–4 M NaCl range processes. The Km of fungal and yeast amylases have been reported to be between 0.13 and 5 mg/ml (Pandey et al. 2000; Gupta et al. 2003; Kubilay et al. 2010; Irshad et al. 2012). In bacteria, Km values for amylases of 1.8–8.3 mg/ml have been reported (Demirkan 2010; Ikram et al. 2010; Yandri and Sutopo 2010; Saeeda et al. 2011). The amylase used in this study had a higher catalytic activity compared to values reported in literature that range from 1.9 to 464,000 U/mg (Amoozegar et al. 2003; Sivaramakrishnan et al. 2006; Takeuchi et al. 2006).
Conclusions HJ014 bacterium is capable of producing extracellular amylase. The enzyme had haloalkaliphilic enzyme characteristics with maximum activity at pH 9.0 and 3.5 M NaCl. Kinetic values exhibited by the purified enzyme haloalkaliphilic, Km was 3.0 mg/ml and Vmax 90.09 U/ml. Since lower Km values allows faster and easier industrial processes, the low Km values of Kocuria sp. extracellular amylase potentiates its use in industrial applications.
Disclosure statement No potential conflict of interest was reported by the authors.
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Funding This work was supported by the Conacyt (Mexico).
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Characterization of extracellular amylase produced by haloalkalophilic strain Kocuria sp. HJ014 Marisela Y. Soto-Padilla, Pablo Gortáres-Moroyoqui, Luis A. Cira-Chávez, Anthony Levasseur, Luc Dendooven & María Isabel Estrada-Alvarado To cite this article: Marisela Y. Soto-Padilla, Pablo Gortáres-Moroyoqui, Luis A. Cira-Chávez, Anthony Levasseur, Luc Dendooven & María Isabel Estrada-Alvarado (2016): Characterization of extracellular amylase produced by haloalkalophilic strain Kocuria sp. HJ014, International Journal of Environmental Health Research, DOI: 10.1080/09603123.2015.1135310 To link to this article: http://dx.doi.org/10.1080/09603123.2015.1135310
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Date: 29 January 2016, At: 00:39
International Journal of Environmental Health Research, 2016 http://dx.doi.org/10.1080/09603123.2015.1135310
Characterization of extracellular amylase produced by haloalkalophilic strain Kocuria sp. HJ014 Marisela Y. Soto-Padillaa,b, Pablo Gortáres-Moroyoquib, Luis A. Cira-Chávezb, Anthony Levasseurc, Luc Dendoovend and María Isabel Estrada-Alvaradob Downloaded by [NUS National University of Singapore] at 00:39 29 January 2016
a
Instituto de Ingeniería y Tecnología, Universidad Autónoma de Ciudad Juárez, Chihuahua, Mexico; bBiotecnología y Ciencias Alimentarias, Instituto Tecnológico de Sonora, Obregón, Mexico; cBiotechnologie des Champignons Filamenteux, INRA, Marseille, France; dCinvestav, ABACUS, Mexico City, Mexico
ABSTRACT
The haloalkaliphilic bacterium Kocuria sp. (HJ014) has the ability to produce extracellular amylase. The aim of this study was to purify and characterize this protein. The amylase enzyme with a specific activity of 753,502 U/mg was purified 5.7– fold using Sepharose 4B and Sephacryl S-300 gel filtration columns. The molecular weight of the enzyme was 45,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amylase showed maximum activity at pH 9 and 50°C in the presence of 3.5 M NaCl. The Km was 3.0 mg/ml and Vmax 90.09 U/ml. It was found that extracellular amylase from Kocuria sp. has a high industrial potential.
ARTICLE HISTORY
Received 23 April 2015 Accepted 3 December 2015 KEYWORDS
Haloalkaliphilic; enzyme molecular weight; industrial potential; specific activity
Introduction Organisms in the soil contribute to the functioning of the ecosystem. Soil is an appropriate environment for both eukaryotic (algae, fungi, protozoa) and prokaryotic (bacteria and archaea) organisms. Viruses and bacteriophages can also be found in soil (Nogales 2005). Extreme environments are defined by an extreme pH, temperature, salinity or other soil characteristics or combinations of them (Rothschild and Mancinelli 2001). There are groups of organisms that are adapted specifically to these extreme conditions. These organisms are usually referred to as alkaliphiles, halophiles, thermophiles, and acidophiles, reflecting the particular type of extreme environment they inhabit (Ulukanli and Digrak 2002). Extremophiles produce several compounds of industrial interest and many of these are excreted in the environment. Recent research has focused on the production of extracellular enzymes from bacteria (Sastoque et al. 2007; Ninawe et al. 2008; Rohban et al. 2009). For example, it is known that hydrolytic enzymes (amylases, proteases, peptidases, esterases, and lipases) have different potential applications in biomedical science, chemical industries, and industrial processes (Ventosa et al. 1998; Margesin and Schinner 2001; Mellado et al. 2004). Amylases are one of the most important industrial enzymes that have a wide variety of applications, ranging from conversion of starch to sugar syrups (saccharification), to the production of cyclodextrins for the pharmaceutical industry. Amylases are hydrolytic enzymes widespread in nature, particularly in animals, micro-organisms, and plants (Safey and Ammar 2004). Amylases have been obtained from halophilic, alkaliphilic, thermophilic, and mesophilic micro-organisms (Chessa et al. 1999; Kiran and Chandra 2008; Nouadri et al. 2010). These enzymes account for about 30 % of the world’s enzyme
CONTACT María Isabel Estrada-Alvarado © 2016 Taylor & Francis
[email protected]
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production, as enzymatic hydrolysis is preferred over acid hydrolysis in the starch processing industry (Marc et al. 2002). Enzymatic hydrolysis has different advantages, such as specificity, stability, low energy consumption, and not neutralization step (Satyanarayana et al. 2005). Due to the increasing demand for these enzymes in various industries, there is an enormous interest in finding enzymes with suitable properties for industrial applications (Van der Maarel et al. 2002; Burhan et al. 2003). The genus Kocuria was proposed by Stackebrandt et al. (1995) and consists currently of 18 species with valid published names (http://www.bacterio.cict.fr/k/kocuria.html). The members of this genus have been isolated from different sources, such as air, fermented sea food, mammalian skin, soil, the rhizoplane, freshwater, seawater, marine sediment, and desert soil (Tang et al. 2009; Monu et al. 2012). Yamaguchi et al. (2011) reported the cloning and characterization of a halophilic–amylase obtained from the moderately halophilic bacterium K. varians. The aim of this work was to purify and characterize an amylase obtained from Kocuria sp. isolated from an extreme alkaline saline soil of the former Texcoco Lake.
Materials and methods Enzyme production The strain Kocuria sp. (HJ014) was isolated from the former Texcoco Lake (Soto-Padilla et al. 2013). It was cultivated in liquid (Bacto marine broth 2216, DIFCO) and on solid medium (Bacto marine agar 2216, DIFCO) at 37 °C for 72 h. It was grown at optimum conditions (NaCl 5 % and pH 7.5, at 37 °C and 200 rpm) in marine broth (2216, DIFCO) supplemented with 1 % soluble starch. The growth was estimated by turbidity measurement (Cintra 10e) at 600 nm (Coronado et al. 2000). Amylase of Kocuria sp. (HJ014) was produced in 500-ml Erlenmeyer flasks containing 200 ml of marine broth (DIFCO) supplemented with 1 % soluble starch and 5 % NaCl. Preinoculum was grown during 24 h until reach 0.8 absorbance (OD600), then 10 % (v/v) was used to inoculate the enzyme production medium. Incubation was done at 200 rpm and 37 °C for 48 h. The enzyme extract was obtained by centrifugation (Centrifuge max) at 9500 xg for 20 min at 4 °C. The clear supernatant was used for the amylase activity assay. Amylase activity was determined by measuring the release of reducing sugar from soluble starch by the Bernfeld method (Bernfeld 1955). The reaction mixture contained 450 μl of 1 % (w/v) starch (prepared in 100 mM phosphate buffer, pH 7.5) and 50 μl of enzyme extract. The reaction mixture was maintained at 50 °C for 10 min. The enzymatic reaction was stopped by adding dinitrosalicylic acid (DNS) solution. The amount of reducing sugar released was quantified using 3, 5-dinitrosalicylic acid with maltose as standard. One unit of amylase activity was defined as the amount of enzyme that produced 1 μmol of reducing sugars (as maltose) per minute. The specific activity was expressed as units of enzyme activity per milligram of protein. The relative amylase activity was defined as the percentage of the maximum amylase activity detected in the assay. Assays were performed in triplicate. The protein concentration was determined using the bicinchoninic acid protein assay kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer′s recommendations. Standard curve of bovine serum albumin was used as standard protein in the range of 50–250 μg/mL. Enzyme characterization The supernatant from the culture medium was concentrated by drying in an oven (Felisa) at 40 °C for 48 h. The concentrated protein extract was used for enzyme purification by chromatography into a Sepharose 4B column (GE Healthcare) previously equilibrated with buffer A (20 mM tris–HCl, pH 8) at a flow rate of 0.5 ml/min and adapted into a protein purification equipment (ÄKTA10 purifier, GE Healthcare, Fairfield, CT, USA). The column was washed with the same buffer until no absorbance at 280 nm was detected in the eluent. The absorbed proteins were eluted with a lineal gradient of NaCl concentration 0–1 M (typically linear) at a flow rate of 0.5 ml/min. Fractions that showed amylase
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activity (assayed as described before) were collected and loaded on a Sephacryl S-300 column (GE Healthcare, Fairfield, CT, USA) equilibrated with buffer A. The enzyme was eluted at a flow rate of 0.5 ml/min and collected in 2ml fractions. Fractions containing amylase activity were pooled and used for further analysis. SDS-PAGE (12 % (w/v)) was used to determine protein purity and molecular mass of the purified enzyme as described by Laemmli (1970). After electrophoresis, the protein bands were silver stained. The molecular weight of the standard proteins used were myosin (200.0 kDa), β-galactosidase (116.2 kDa), phosphorilase b (97.4 kDa), bovin serum albumin (66.2 kDa), ovalbumin (45.0 kDa), carbonic anhydrase (31.0 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa), and aprotinin (6.5 kDa). The effect of pH on the activity of the purified enzyme was investigated by preparing soluble starch substrate in various buffers (100 mM): sodium phosphate (pH 6.0–7.5), Tris–HCl (pH 8.0–9.0), and glycine–NaOH (pH 9.5–12) (Shafiei et al. 2012). The reaction was carried at 50 °C for 10 min. The temperature effect on the enzyme activity was determined in the range of 10–90 °C and the reaction was done at pH 7.5 for 10 min. The effect of NaCl concentration on enzyme activity was determined by measuring the amylase activity (described in enzyme production) in the reaction mixture containing different NaCl concentrations (0–4 M) (Shafiei et al. 2012). The amount of enzyme extract used was 50 μl at 50 °C and pH 7.5. The kinetic parameters Km and Vmax of the purified amylase were measured using soluble starch as substrate. Starch hydrolysis was colorimetrically assayed in the range of 0–4 % (w/v) in buffer Tris–HCl (pH 9) at 55 °C for 10 min (Patel et al. 1993). Km and Vmax values were obtained from the Hanes and Woolf plot (Ritchie and Prvan 1996).
Results The maximum cell concentration was achieved after 72 h (Figure 1). Amylase activity was 8900 U/ml after 1 h and reached a maximum of 133,772 U/ml after 39 h. The results of the purification process are summarized in Table 1. In the first purification step, a specific activity of 203,681 U/mg was obtained. In the second purification step, the fractions containing amylase activity were applied to the Sephacryl S-300 column and had a specific activity of 753,502 U/mg and purified 5.7–fold. A single protein band was observed after electrophoresis of the fraction obtained from the column purification Sephacryl S-300 in 12 % SDS-PAGE (Figure 2). The molecular weight of the purified amylase was 45 kDa. Figure 3 shows the single and symmetrical absorption
Figure 1. Kinetic growth of HJ014 and amylase activity on Marine Broth supplemented with 1 % soluble starch and 5 % NaCl.
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Table 1. Purification protein profile at different steps of the amylase from Kocuria sp. strain HJ014.
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Fraction Raw extract [Raw extract] Sepharose 4B Sephacryl S-300
Activity (U/ml) 252000 362533.3 285600 97955.3
Total activity (U) 20160000 15588933.3 9139200 1959106.4
Protein (mg/ml) 152.56 139.75 44.87 2.6
Specific activity (U/mg) 132144.72 111548.71 203681.74 753502.46
Purification (×times) 1 0.8 1.5 5.7
peak parallel to the peak of the amylase activity in the fraction obtained from the Sephacryl S-300 gel filtration column. The enzymatic characteristics of the purified amylase were determined using soluble starch as substrate and the maximum specific activity of the enzyme was 51,701 U/mg at pH 9 (Figure 4). An increase in alkalinity from pH 8–11 decreased the relative activity with 20 %. Figure 5 indicate that the maximum enzyme activity expressed as relative activity was obtained at 40–50 °C with a maximum specific activity of 41,236 U/mg protein.
Figure 2. Separation of protein by SDS-PAGE.
Figure 3. Purification profile of amylase produced by Kocuria sp. strain HJ014.
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Figure 4. Effect of pH on the amylase activity of the purified enzyme produced from Kocuria sp. strain HJ014.
Figure 5. Effect of temperature on the amylase activity of the purified enzyme produced from Kocuria sp. strain HJ014.
The amylase activity was determined at various NaCl concentrations. The amylase showed maximum activity in 3.5 M NaCl (40,969 U/mg protein) (Figure 6). Relative activity was highest at 2 M NaCl. The effect of different concentrations of starch on the activity of amylase was studied and plotted ([S]/Vo vs. [S]) to determine the maximum velocity and Michaelis–Menten constant Km (Figure 7). The Km and Vmax values were 3.0 mg/ml and 90.09 U/ml, respectively.
Discussion Recently, extremophiles micro-organisms and their extremozymes have become important due to their potential application in industry (Madigan and Marrs 1997; Rothschild and Mancinelli 2001; Satyanarayana et al. 2005; Podar and Reysenbach 2006; Ferrer et al. 2007). As far as we know, this study is the first report about characteristics of an extracellular amylase derived from Kocuria sp. Pandey et al. (2000) reported that strains of Aspergillus sp. and Bacillus sp. were a source of amylase enzymes. Most enzymes from these bacteria might be purified by chromatographic techniques after crude isolation by precipitation and membrane separations. The demand for large-scale effective
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Figure 6. Effect of NaCl on the amylase activity of the purified enzyme produced from Kocuria sp. strain HJ014.
Figure 7. Hanes & Woolf plot of purified enzyme from strain HJ014 to calculate the Vmax and Km values.
purification of proteins has resulted in the evolution of techniques that provide fast, efficient, and economical protocols requiring few separation steps (Takeuchi et al. 2006). The α-amylase obtained in this study was purified using a chromatographic method applying Sepharose 4B and Sephacryl S-300 gel filtration columns, which is fast, efficient, and a low-cost technique. Purified amylolytic enzymes have been reported with molecular weights ranging from 42 to 75 kDa (Young et al. 1995; Safey and Ammar 2004; Kumar et al. 2005; Nouadri et al. 2010). The enzyme reported in this study had an apparent molecular weight of 45 kDa. Different studies have reported the purification and characterization of amylases obtained from other micro-organisms, but not all of them report their activities (Young et al. 1995; Safey and Ammar 2004). Takeuchi et al. (2006) reported 40,700 U of amylase activity for Pichia burtonii, which, compared to our results, 1,959,106 U, is lower than our activity obtained. The maximum activity in this study was reached within the first 48 h, whereas amylase from Penicillium camemberti had a maximum enzyme activity after 168 h (Nouadri et al. 2010). Amylase is usually produced from strains of the genus Bacillus as well as from some thermophilic strains. Production and purification of proteins from Kocuria sp. isolated from a haloalkaliphilic soil was done to obtain halophilic α-amylase with the capacity of an
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enzymatic catalysis in high NaCl concentrations and pH. Bacillus sp. is widely used for thermostable α-amylase production to meet industrial needs. Bacillus subtilis, B. stearothermophilus, B. licheniformis, and B. amyloliquefaciens are excellent producers of α-amylase and these bacteria have been widely used for commercial production (Sivaramakrishnan et al. 2006). The properties of α-amylases, such as thermostability and pH profile, should match their industrial application. Hence, the range of possible applications creates the need to search for new α-amylases with novel and improved properties for specific conditions. The rate of amylolytic hydrolysis depends on process conditions, such as temperature, pH, starch substrate (type and concentration), enzyme (catalytic properties and concentration), and the presence of NaCl. Kocuria sp. showed amylase activity in a pH range from 8.0 to 11.0 with maximum at pH 9.0, indicating its alkalitolerance. Kobayashi et al. (1986) reported an amylase obtained from the archaebacterium Natronococcus sp. with maximum activity at pH 8.7 (i.e. an alkalitolerant enzyme) similar to the alkalitolerant amylase of Bacillus sp. strain TSCVKK (Kiran and Chandra 2008) and Halobacillus sp. strain MA-2 (Amoozegar et al. 2003). The pH is one of the most important factors that determine the growth and morphology of micro-organisms as they are sensitive to hydrogen ion concentrations in the medium. Bacterial cultures, such as B. subtilis, B. licheniformis, and B. amyloliquefaciens required an initial pH of 7 (Syu and Chen 1997; Haq et al. 2005; Tanyildizi et al. 2005). Rhodothermus marinus was reported to have an amylase with an optimum pH range of 7.5–8 (Gomes et al. 2003). The effect of temperature on amylase production is related to the growth of the organism. Bacterial amylases are produced in a wide range of temperatures. Bacillus amyloliquefaciens, B. subtilis, B. licheniformis, and B. stearothermophilus are among the most commonly used Bacillus sp. that produces α-amylase at temperatures between 37 and 60 °C (Mendu et al. 2005; Mishra et al. 2005). The enzyme obtained in this study showed a maximum amylase activity at 50 °C similar to amylase from Halobacillus sp. strain MA-2 (Amoozegar et al. 2003) and Nesterenkonia sp. strain F with α-amylase activity between 45–50 °C (Sivaramakrishnan et al. 2006). Halophilic enzymes are therefore uniquely adapted to function in conditions with low water potential, this property is important for a biotechnological view. Consequently, these organisms can be exploited for industrial uses. These amylases are active in low water activity and showed interesting applications in the treatment of saline waters or waste solutions with starch residues and high salt content (Coronado et al. 2000; Amoozegar et al. 2003). The amylase produced by strain Kocuria sp. HJ014 showed maximum activity in 3.5 M NaCl. The amylase was active over a range of salt concentrations as it showed > 60 % activity in the 0–4 M NaCl range processes. The Km of fungal and yeast amylases have been reported to be between 0.13 and 5 mg/ml (Pandey et al. 2000; Gupta et al. 2003; Kubilay et al. 2010; Irshad et al. 2012). In bacteria, Km values for amylases of 1.8–8.3 mg/ml have been reported (Demirkan 2010; Ikram et al. 2010; Yandri and Sutopo 2010; Saeeda et al. 2011). The amylase used in this study had a higher catalytic activity compared to values reported in literature that range from 1.9 to 464,000 U/mg (Amoozegar et al. 2003; Sivaramakrishnan et al. 2006; Takeuchi et al. 2006).
Conclusions HJ014 bacterium is capable of producing extracellular amylase. The enzyme had haloalkaliphilic enzyme characteristics with maximum activity at pH 9.0 and 3.5 M NaCl. Kinetic values exhibited by the purified enzyme haloalkaliphilic, Km was 3.0 mg/ml and Vmax 90.09 U/ml. Since lower Km values allows faster and easier industrial processes, the low Km values of Kocuria sp. extracellular amylase potentiates its use in industrial applications.
Disclosure statement No potential conflict of interest was reported by the authors.
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Funding This work was supported by the Conacyt (Mexico).
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