Extremophiles DOI 10.1007/s00792-015-0752-3

ORIGINAL PAPER

Purification and characterization of halo‑alkali‑thermophilic protease from Halobacterium sp. strain HP25 isolated from raw salt, Lake Qarun, Fayoum, Egypt Khaled Elbanna1,2 · Ibrahim M. Ibrahim1 · Anne‑Marie Revol‑Junelles3 

Received: 15 April 2014 / Accepted: 26 April 2015 © Springer Japan 2015

Abstract  A total of 33 halophilic protease producers were isolated from different salt samples collected from Emisal salt company at Lake Qarun, Fayoum, Egypt. Of these strains, an extremely halophilic strain that grew optimally at 30 % (w/v) NaCl was characterized and identified as Halobacterium sp. strain HP25 based on 16S rRNA gene sequencing and phenotypic characterization. A halo-alkali-thermophilic protease was purified in three successive steps from the culture supernatant. The purified halophilic protease consisted of a single polypeptide chain with a molecular mass of 21 kDa and was enriched 167-fold to a specific activity of 6350 U mg−1. The purified enzyme was active over a broad pH range from 6.0 to 11.0, with maximum activity at pH 8.0, exhibited a broad temperature range from 30 to 80 °C with optimum activity at 60 °C, and was active at salt concentrations ranging from 5 to 25 % (w/v), with optimum activity at 17 % NaCl (w/v). The KM and Vmax values of the purified halophilic protease with casein as a substrate were 523 µg mL−1 and 2500 µg min−1 mL−1, respectively. In addition, this enzyme

Communicated by F. Robb. Electronic supplementary material  The online version of this article (doi:10.1007/s00792-015-0752-3) contains supplementary material, which is available to authorized users. * Khaled Elbanna [email protected] 1

Department of Agricultural Microbiology, Faculty of Agriculture, Fayoum University, Fayoum, Egypt

2

Department of Biology, Faculty of Applied Sciences, Umm Al-Qura University, Makkah, Kingdom of Saudi Arabia

3

Institut National Polytechnique de Lorraine (ENSAIA-INPL), 54505 Vandoeuvre‑lès‑Nancy Cedex, France





was stable in the tested organic solvents and laundry detergents such methanol, propanol, butanol, hexane, Persil and Ariel. The unusual properties of this enzyme allow it to be used for various applications, such as the ripening of salted fish. Furthermore, its stability and activity in the presence of organic solvents and detergents also allow the use of this enzyme for further novel applications and as an additive in detergent formulations. Keywords  Extreme halophiles · Raw salt · Halobacterium sp. strain HP25 · 16S rRNA · Halo-alkali-thermophilic protease

Introduction Halophilic archaea are salt-loving organisms that inhabit hypersaline environments such as salt ponds, soda lakes and, as dormant cells, even rock salt crystals (Woese 1993). Halophilic microorganisms play an essential role in various fermentation processes that occur in the presence of salt (Rodriguez-Valera and Lillo 1992; Ventosa and Nieto 1995; Gupta et al. 2002; Oren 2010; Delgado-García et al. 2012; Moreno et al. 2013). Their high salt tolerance allows the cultivation of extreme halophiles under conditions that naturally suppress the growth of many contaminants, thus allowing a reduction of cost (Margesin and Schiner 2001). In recent years, halophilic microorganisms have been explored for their biotechnological potential in different fields. Their unusual properties make them a potentially valuable resource in the development of novel biotechnological processes and industrial applications such as new pharmaceuticals, cosmetics, nutritional supplements, molecular probes, fine chemicals, biopolymers, carotenoids, compatible solutes, and enzymes (Rodriguez-Valera

13

Extremophiles

and Lillo 1992; Gomes and Steiner 2004; Alqueres et al. 2007). Examples of well-adapted and widely distributed extremely halophilic microorganisms include species of the archaeal genus Halobacterium, which belongs to the family Halobacteriaceae of the order Halobacteriales. Oren (2012) reported that 36 genera with a total of 129 species have been described in the family Halobacteriaceae, including Halobacterium, Haloarcula, Haloferax, Halococcus, Halorubrum, Halogeometricum, Haloterrigenia, Halobaculum, Halorhabdus, Natrialba, Natrinema, Natronobacterium, Natronococcus, Natronorubrum and Natronomonas. Halobacterium is the best-known genus of the Halobacteriaceae family, and Hbt. salinarum is the type genus of this family (Grant et al. 2002). Proteases are hydrolytic enzymes that can degrade a variety of proteins, and so find potential application in waste treatment, bioremediation, wool quality improvement. Other applications in food industries, which exploit the hydrolytic property of proteases, include hydrolysis of soy protein, casein, fish protein and meat tenderization. Proteases are also used extensively in pharmaceutical and detergent industries. However, most industrial processes are carried out under specific physicochemical conditions that may not be compatible with the conditions required for the activity of the available enzymes. Thus, it would be of great importance to identify enzymes that exhibit optimal activities under a great variety of different conditions (Sumantha et al. 2006; Rohban et al. 2009). In this sense, identifying novel enzymes showing optimal activities at various ranges of salt concentrations, temperatures and pH values is of great importance. Since halophilic proteases are adapted to extreme environments, they are unusually stable and, therefore, may be suitable candidates for industrial processes that are performed under harsh conditions. However, in spite of a growing interest in the use of halophilic enzymes for biotechnological applications, there are relatively few reports in the literature about their production and characterization (Bhatnagar et al. 2005; Alqueres et al. 2007; Oren 2010; Kumar et al. 2012; Moreno et al. 2013). Proteases can be grossly subdivided into two major groups, i.e., exopeptidases and endopeptidases, depending on their site of action. Exopeptidases cleave the peptide bond proximal to the amino or carboxy termini of the substrate, whereas endopeptidases cleave peptide bonds distant from the termini of the substrate. Based on the functional group present at the active site, proteases may also be classified into four prominent groups, i.e., serine proteases, aspartic proteases, cysteine proteases, and metalloproteases (Hartley 1960; Rao et al. 1998). Based on their structural similarities, serine proteases have been grouped into 20 families, which have been further subdivided into about six clans with common ancestors (Barett 1994). Serine

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proteases are so named because they employ a common catalytic mechanism characterized by the involvement of a reactive serine residue that is essential for their enzymatic activity (Voet and Voet 2004). The extensive active site similarities between the various serine proteases indicate that they all have a similar catalytic mechanism. Egypt is host to various saline environments, including hypersaline lakes and salt ponds, of which the microbial diversity has not been well characterized. However, the available information about halophilic protease-producing microorganisms present in these environments is rare. Therefore, this work aims to (1) screen for the most halophilic protease-producing microorganisms from brine, multicolor solar salt, saline soil, saline mud and raw salt (Emisal salt company, Lake Qarun, Fayoum, Egypt), (2) identify the most promising strain based on phenotypic and 16S rRNA gene sequencing, (3) optimize the culture conditions for growth and protease production, and (4) purify and characterize the halophilic protease of the most promising strain.

Materials and methods Sampling and media Different salt samples such as brine (non-colored salt solution), multicolor solar salt pond water (red liquid collected from solar salt ponds), saline soil (soil from the immediate vicinity of solar salt ponds), saline mud (salt mixed with clay collected from the bottom of solar salt ponds) and raw salt (dry, untreated salt) were collected from Emisal salt, Lake Qarun, Fayoum, Egypt. Protease-producing halophilic strains were isolated using a modified S-G medium (Sehgal and Gibbons 1960), wherein skim milk was added instead of casamino acids. The medium was prepared in two parts: the first part contained 2.5 % (w/v) skim milk and 1 % (w/v) yeast extract prepared in 200 mL water and sterilized using an intermittent sterilization method (heating to 100 °C for 15 min on three successive days) to minimize changes in skim milk properties. The second part of the medium contained NaCl (250 g L−1), MgSO4 × 7H2O (20 g L−1), KCl (2 g L−1), trisodium citrate (3 g L−1), was prepared in 800 mL water, and was autoclaved at 121 °C for 15 min. After sterilization, both parts were mixed and the pH was adjusted to a value of 7.0. Enrichment and screening for protease‑producing halophilic strains For the enrichment of halophilic protease-producing strains, 90 mL of the liquid modified S-G medium was inoculated with 10 mL of liquid sample (e.g., brine) or 10 g

Extremophiles

of solid sample (e.g., soil and raw salt). The inoculated flasks were incubated at 40 °C for 1 week at 200 rpm on a rotary shaker. Serial dilutions from each enriched culture were spread onto the surface of S-G agar plates and were then incubated in sealed plastic bags at 40 °C for 2 weeks. The presence of proteases was indicated by the formation of a clear zone surrounding the colonies (Supplementary 1) where the skim milk had been degraded. Based on this protease activity assay on skim milk agar medium, strain HP25 was selected as the most halophilic protease producer for further phenotypic and genotypic characterizations. Phenotypic characterization of strain HP25 Before phenotypic and genotypic characterization of strain HP25, the purity was confirmed by streaking a single colony of strain HP25 on S-G agar medium containing 25 % (w/v) NaCl, as well as by microscopic examination. Colony morphology, cell shape, and Gram reaction were determined. The growth at different NaCl concentrations of 5–30 % (w/v), different temperatures (20–60 °C) and various pH values (5–10) was assessed according to the methods described by Reddy et al. (2007). The biochemical reactions included carbon source utilization and enzyme activities were determined using API 20 E and API 50 CH kits (Bio Mérieux, France) that were slightly modified by supplementing the media with 25 % (w/v) NaCl. Genotypic characterization of strain HP25 To determine the 16S rRNA gene sequence of strain HP25, the strain was cultivated in S-G broth medium (pH 7.0) containing 25 % (w/v) NaCl and incubated at 40 °C for 96 h. The genomic DNA was then prepared by suspending a pure colony in a safe-lock 1.5-mL Eppendorf tube containing 200 μL of sterile distilled water and incubating at 100 °C for 10 min. The tubes were immediately cooled on ice and centrifuged at 10,000×g for 10 min at 5 °C. The supernatants were subsequently kept on ice or at −20 °C. The purity and concentration of the DNA solution were determined by measuring the absorption at 260 and 280 nm (Sambrook et al. 1989). One microliter of supernatant (corresponding to approximately 50–100 ng of genomic DNA) was used as a DNA template for each 50 μL PCR (Johnsen et al. 1996). The 16S rRNA gene of strain HP25 was amplified by PCR using universal primers (Hezayen et al. 2002) and sequenced using an ABI 3730xl automated DNA sequencer (Applied Biosystems USA) through Lab Biotechnology Company, Egypt. The 16S rRNA gene sequences were initially analyzed using the program BLAST (National Center Biotechnology Information, http://www.ncbi.nml.nih.gov). Sequencing data obtained from different primers were assembled using the

CAP program (Contig Assembly and Genomic Expression programs). The sequence of a 1472-bp fragment of the 16S rRNA gene sequence of strain HP25 was deposited in the Genbank database under accession number KJ011554. The sequence from strain HP25 and sequences of strains belonging to the same phylogenetic group and to other representative isolates (retrieved from the NCBI database) were aligned using the computer program ClustalX (Thompson et al. 1997). The resulting trees were displayed with Tree View (Page 1996). The phylogenetic tree was calculated using the neighbor-joining method (Saitou and Nei 1987) using Methanobacterium formicicum strain DSMZ1535 (NR 025028) as an outgroup. Factors affecting the growth and protease secretion of strain HP25 To detect the best fermentation conditions for protease secretion from strain HP25, a preliminary fermentation time course experiment was conducted using S-G medium supplemented with 25 g L−1 skim milk and 250 g L−1 NaCl. Samples were withdrawn daily until the enzyme activity decreased, whereupon fermentation was ended. To evaluate the best conditions for growth and enzyme secretion of strain HP25, different salt concentrations [5, 10, 15, 20, 25 and 30 % (w/v) at pH 7.0 and 40 °C] and different pH values [5, 6, 7, 8, 9 and 10 at 25 % (w/v) NaCl and 40 °C] were tested. Different temperatures (25, 30, 35, 40, 45, 50, and 60 °C at 25 % NaCl and pH 7.0) were also tested. The inoculated flasks (50 mL in 250-mL flasks) were incubated at 40 °C on a rotary shaker at 200 rpm for 4 days to test the effect of salt concentration and pH on growth and enzyme secretion. The effect of temperature on growth was also assayed by incubation of inoculated flasks on a rotary shaker at 200 rpm for 4 days at the given temperatures (at pH 7.0 and 25 % NaCl). Cell counts during the fermentation experiments were conducted using the dilution plate method. For enzyme activity assays, the samples were centrifuged at 5000×g and the activity was measured in the supernatants. The relative activity was calculated by assigning the highest activity the value of 100 % and calculating the other values relative to it. Production and purification of halophilic protease from strain HP25 The extracellular halophilic protease from strain HP25 was purified in three successive steps: ultrafiltration, ethanol precipitation, and gel filtration using a Superdex 200 HR FPLC column. For this, a two-liter bioreactor (ADI 1030 Bio Controller Firmware Version 2.2X, French) was used. First, the modified S-G medium containing 25 % (w/v) NaCl (without casamino acids) was autoclaved, then supplemented with

13

Extremophiles

2.5 % (w/v) separately sterilized skim milk for protease induction, and the pH was adjusted to 7.0. During the fermentation process in the bioreactor, the culture was stirred at 200 rpm and aerated at 0.5 L min−1. The dissolved oxygen saturation was set to a minimum of 30 % and the temperature was kept at 40 °C. At the end of the fermentation, the culture was centrifuged at 5000×g at 4 °C for 30 min, and subsequently the cellfree supernatant was concentrated to 110 mL by ultrafiltration through an YM 10 polyethersulfone membrane filter (Sartorius Stedim Biotech, Goettingen, Germany) using an Amicon chamber (Amicon, Beverly, MA, USA). The concentrated supernatant was slowly mixed with 220 mL chilled absolute ethanol and the precipitate was allowed to settle for 2 h at 10 °C. The precipitate was recovered in 10 mL of 50 mM Tris–HCl Buffer (pH 8) containing 4.5 M NaCl and dialyzed against three changes of one liter of the same buffer at 4 °C overnight. The concentrated and dialyzed protein was tested for activity and applied onto a HiLoad 26/60 Superdex 200 HR gel filtration column (column dimension 2.6 × 60 cm) equilibrated with 50 mM Tris–HCl pH 8 buffer containing 4 M NaCl. The halophilic protease was eluted with the same buffer at a flow rate of 2 mL min−1 and a fraction size of 1 mL. The fractions containing high protease activity (supplementary 2) were tested to gel electrophoretic purity. Based on yielding only single band in SDS-PAGE gel, these fractions subsequently pooled, and concentrated using an YM 10 ultrafiltration membrane. The purified enzyme was divided into two aliquots: the first aliquot was stored at 4 °C for detailed characterization, and the second aliquot was mixed with 50 % glycerol and stored at −20 °C for further work. Total protein and halophilic protease assays Total protein was determined according to the method of Bradford (1976). The proteolytic activity of the halophilic enzyme was assayed spectrophotometrically according to the method of Folin and Ciocalteu (1929). l-Tyrosine solution with and without NaCl was used to determine standard curves. To determine the enzyme activity, 1 mL of purified enzyme solution was added to 5 mL of 0.65 % casein solution prepared in 50 mM Tris–HCl pH 7.0 containing 25 % (w/v) NaCl and incubated for 10 min at 37 °C. Protein was precipitated by adding 5 mL of 10 % (w/v) trichloroacetic acid (TCA) and incubating on ice for 30 min. The supernatant was obtained by centrifugation at 5000×g for 10 min. The assay mixture consisted of 2 mL of the supernatant, 5 mL of 500 mM NaCO3 and 1 mL of Folin and Ciocalteu’s phenol reagent (F–C). This was mixed and incubated for 30 min at 37 °C, then filtered through filter paper (0.45 µm), and subsequently the absorbance was measured at 660 nm. Blanks were prepared in which 5 mL of 10 % TCA was added before incubation. All assays were done in triplicate. One unit of protease activity was defined as

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the amount of enzyme yielding the equivalent of 1 mmol of tyrosine per minute at 37 °C, pH 7.0 and 25 % (w/v) NaCl. Determination of the molecular weight of the halophilic protease Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (1970). After electrophoresis, proteins were visualized by staining with Coomassie Brilliant Blue. The molecular weight of the purified protein was determined according to the method described by Lorentz (1976) using the line resulting from the logarithm of the molecular weights of standard proteins plotted against the migration distance from the spacer-gel junction. Effect of salt concentration, pH and temperature on the halophilic protease activity To determine the optimum conditions for the purified halophilic protease, the enzyme activity was assayed at 5, 10, 15, 20, 25 and 30 % (w/v) NaCl, pH 7.0 and 37 °C for 10 min. The optimum temperature of this enzyme was determined at different temperatures (20–100 °C) at pH 7.0 and 25 % (w/v) NaCl for 10 min. For determining the optimal pH value, the purified enzyme was incubated at different pH values (5.0– 12.0). Different buffer systems were used according to the desired pH value: 50 mM sodium acetate (pH 4–5.5), 50 mM sodium phosphate (pH 5.0–7.5), 50 mM Tris–HCl (pH 8.0– 10.0), and 50 mM glycine/NaOH (pH 9.0–12). The protease activity was assayed at 37 °C in the presence of 25 % (w/v) NaCl. All experiments were done in triplicate. Effect of inhibitors on the halophilic protease activity Inhibitor studies on the halophilic protease of strain HP25 were performed according to the protocol described by Vidyasagar et al. (2009). The assay mixture (1 mL) was prepared by combining 500 µL of the purified halophilic protease solution (pH 8.0), followed by adding 500 µL of each inhibitor solution (Table 2). The enzyme-inhibitor mixture containing 17 % (w/v) NaCl was pre-incubated for 30 min at room temperature. The residual activity was then determined under optimal conditions (17 % NaCl, pH 8.0 and 60 °C). The enzyme activity in the absence of any solvent or detergent was defined as 100 % and the other values were calculated as relative to it. Stability of the halophilic protease in the presence of some organic solvents and detergents To determine the stability of the halophilic protease of strain HP25 in organic solvents, the purified enzyme

Extremophiles

solution was mixed with an equal volume of methanol, ethanol, propanol, butanol, or hexane. The mixtures were incubated at 30 °C with shaking at 150 rpm for 24 h. The residual activity was directly determined in the mixtures, except in the case of the hexane mixture, where it was determined in the aqueous phase. To determine the stability of the halophilic enzyme in some laundry detergents such as Persil (Henkel Egypt) and Ariel (Procter and Gamble Egypt), 1 mL of aqueous solution (1 % w/v) of each laundry detergent was prepared and boiled for 10 min to inactivate any enzymes present in the detergent, subsequently cooled on ice, then mixed with an equal volume of the enzyme solution and incubated at 30 °C for 120 min. The residual activity was assayed under optimal conditions (17 % w/v NaCl, pH 8.0 and 60 °C). The enzyme activity in the absence of any solvent or detergent was defined as 100 %. Determination of the KM value of the halophilic protease The KM value of the purified halophilic protease for protein was estimated using different concentrations of casein. The enzyme activity was determined under optimal conditions (17 % NaCl, pH 8.0 and 60 °C) and the KM value of the enzyme was calculated from a Lineweaver–Burk plot.

Results Screening and identification of selected halophilic protease‑producing isolates A total of 33 halophilic protease producers were isolated from brine, multicolor solar salt, saline soil, saline mud and raw salt (Emisal Salt Company, Lake Qarun, Fayoum, Egypt). All strains were retested for purity and efficiency of enzyme production on skim milk S-G agar containing the same salt concentration. Among these isolates, Gramnegative halophilic strain HP25 was selected for further identification and characterization of its superior halophilic protease. The data in Table 1 showed that strain HP25 can grow optimally at 25 % NaCl and not at 15 % (w/v). In addition, it can utilize a narrow range of organic substrates. Phylogenetic tree based on 16S rRNA gene sequences (Fig.  1) showed that strain HP25 represented a member of the genus Halobacterium with similarities to Halobacterium salinarum strain R1 DSM 671 (gi 444303782) (97 % identity) and Halobacterium piscisalsi HPC1-2 (gi 343200866) (95 % identity). Strain HP25 was deposited in the culture collection of the Agricultural Microbiology Department, Faculty of Agriculture, Fayoum University, Fayoum, Egypt.

Factors affecting the growth and the secretion of halophilic protease of strain HP25 To find the best conditions for growth and secretion of halophilic protease by strain HP25, experiments varying parameters such as fermentation duration, salt concentration, pH and temperature were conducted using S-G medium supplemented with 2.5 % (w/v) skim milk and 250 g L−1 NaCl. In these studies, growth and secretion of the halophilic protease were monitored by colony counts on dilution plates and by measuring the protease activity of the cell-free supernatant. To determine the best fermentation period, growth and enzyme activity of strain HP25 were monitored during 5 days. The minimal enzyme activity was detected after 1 day and reached its maximal activity after 4 days (at 25 % w/v NaCl, pH 7 and 40 °C), then drastically decreased after the 5th day (Fig.  2a). So, the fermentation period was fixed for all other experiments. Regarding the effect of different salt concentrations on the growth and protease activity, it was noticed that no growth was detected at 10 % (w/v) salt. At 15 %, the growth of strain HP25 was flourished and increased as the salt increased and the maximum growth was detected at 30 % (w/v) salt, pH 7 and 40 °C. Meanwhile, the minimal protease activity of strain HP25 was detected at 15 % (w/v) salt and reached its maximal value at 25 % salt, then slightly decreased with 30 % salt (Fig. 2b). The data illustrated in Fig. 2c show the effect of different pH values (5, 6, 7, 8, 9 and 10) on the growth and protease secretion of strain HP25. The minimal growth and protease activity were detected at pH 5 and the optimal value was at pH 7.0 at 25 % (w/v) salt and 40 °C, then the growth and protease secretion decreased gradually with increasing the pH value up to pH 10. The effect of different temperature (20–60 °C) on the growth and protease secretion of strain HP25 is presented in Fig. 2d. The data clearly indicate that strain HP25 exhibited its maximal growth and protease secretion at 40 °C, pH 7.0 and 25 % (w/v) salt, but drastically decreased at 55 °C. Purification of halophilic protease from strain HP25 The extracellular halophilic protease of strain HP25 was purified in three successive steps: ultrafiltration, ethanol precipitation, and subsequent gel filtration using a Superdex 200 HR FPLC column (Table 2). The enzyme activity of halophilic protease was detected in Amicon concentrated supernatant, indicating that the molecular weight of this enzyme was exceeding 10 kDa. As presented in the purification table (Table 2), the specific activity of the concentrated enzyme increased from 380 to 563 U mg−1

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Extremophiles Table 1  Differential characteristics of strain HP25 and other related strains

Characteristic

HP25T (This study)

Ref (1)

Ref (2)

Ref (3)

Ref (4)

Ref (5)

Cell morphology Pigmentation pH optimum Temperature range (°C) Optimum NaCl conc. (%) Acid production from glycerol Utilization of carbon sources  Citrate  Inulin  Lactose  d-Mannose  Melezitose  Maltose  d-Xylose Hydrolysis of  Casein  Gelatin Enzyme assay  Esterase  Lipase  Oxidase  Lysine decarboxylase

Short rods Pink 7.0 20–55 20–30 +

Short rods Red 7.0 20–60 20–25 +

Rods nr 7.0 20–55 20 −

Rods nr 7.0 20–55 24–30 nr

Rods Red 7.5 20–60 20–25 +

Rods Red 5–8 20–60 20–25 −

+ − − + − + +

+ + − − − + +

− nr − − nr nr −

nr nr nr nr nr nr nr

+ − + − + − −

− + − + − − −

+ +

+ +

nr −

nr +

+ +

+ +

− − + −

− − + −

nr nr + +

nr nr + +

+ + + −

+ − + +

 Ornithine decarboxylase

nr

−

+

+

nr

nr

Biochemical and physiological characteristics were determined using an API kit (BioMerieux, Lyon, France) that was slightly modified by supplementing the media with 25 % NaCl. Other tests were applied according to Reddy et al. (2007). Data of the reference strains: Ref (1) Hbt. Salinarum strain R1 (DSM3754), Ref (2) Hbt. salinarum (DSM 671), Ref (3) Hbtsalinarum (published type strain), Ref (4) Hbt. Piscisalsi HPC1-2 and Ref (5) Hbt. jilantaiense were obtained from Ventosa and Oren (1996), Mormile et al. (2003), Oren (2002), Yachai et al. (2008) and Yang et al. (2006), respectively + growth; − no growth; nr not determined

(1.48-fold enrichment) at 87 % yield. The specific activity after ethanol precipitation and dialysis increased from 563 to 714 U mg−1 (18.78-fold enrichment) at 64 % yield. When the concentrated enzyme from the precipitation and dialysis step was applied to a gel filtration column, the enzyme activity of halophilic protease was detected in the fractions between 223 and 238 mL (Supplementary 2). The purification of the halophilic protease by gel filtration resulted in a specific activity of 6350 U mg−1 (167-fold enrichment) and a yield of 31 % (Table 2). Effect of the salt concentration on the purified halophilic protease The purified halophilic protease of strain H25 showed an optimum activity at 17 % (w/v) salt, 60 °C and pH 8.0 (Fig.  3a). Furthermore, the enzyme retained about

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88 and 45 % of its activity at 20 and 25 % (w/v) NaCl, respectively. Effect of the pH on the purified halophilic protease The halophilic protease was active over a broad pH range (6.0–11.0), and exhibited maximum (100 %) activity at pH 8.0, 60 °C and 17 % salt (Fig. 3b). It retained more than 70 % of its activity up to pH 9.0 and 10.0, which indicated that it is an alkalophilic enzyme. Effect of temperature on the purified halophilic protease The purified halophilic protease from strain HP25 showed activity over a wide temperature range with an optimum activity (100 %) at 60 °C, 17 % (w/v) salt and pH 8.0 (Fig. 3c). In addition, it retained more than 94 % of

Extremophiles Halobacterium halobium (gb|M11583)

Fig. 1  Neighbor-joining tree showing the estimated phylogenetic relationships of HP25 (accession number KJ011554) and the closest members of the halophiles. Accession numbers are given in parentheses. Bootstrap values are shown as percentages of 1000 replicates. Bar 0.1 % sequence divergence

410

Halobacterium salinarum DSM 3754 (gi|21953240) 989 361

Halobacterium salinarum strain R1 DSM 671 (gi|444303782)

Halobacterium piscisalsi HPC1-2 (gi|343200866) 975

HP25 (KJ011554)

940

Halobacterium jilantaiense JCM 13558 (gi|631252227)

Methanobacterium formicicum DSMZ1535 (gi|219857440) 0.1

its activity at 70 °C, indicating that it is a thermophilic protease.

The Michealis–Menten kinetics of the purified halophilic protease is shown in Fig. 3d. The casein hydrolysis rate was estimated using a concentration of 10 µg mL−1 of purified enzyme. The Km value and Vmax of the purified halophilic protease for casein as a substrate were 523 µg mL−1 and 2500 µg min−1 mL−1, respectively.

The reductant β-mercaptoethanol also caused a moderate inhibition (22 %) of protease activity, whereas KCl and CaCl2 had no inhibitory effect on the enzyme activity. In contrast, Zn2+ and Cu2+ ions inhibited the enzyme activity of the purified protease by 44 and 17 %, respectively. As shown in Table 3, the halophilic protease from strain HP25 exhibited extreme stability in the presence of tested organic solvents and laundry detergents. Ethanol had no inhibitory effect on this enzyme. In addition, a slight inhibition of protease activity was detected with methanol, propanol, butanol, hexane, Persil and Ariel which were 1, 3, 4, 5, 7 and 9 %, respectively.

Effect of inhibitors, organic solvents and some laundry detergents on enzyme activity

Molecular mass of the halophilic protease of strain HP25

The effects of various enzyme inhibitors on the activity of purified halophilic protease of strain HP25 are summarized in Table 3. In the presence of anionic surfactants SDS (0.1 %), practically all activity was lost. Also, noticeable inhibition of the enzyme was observed by PMSF, urea and EDTA which were 87, 69 and 66 %, respectively.

The effectiveness of the purification steps was evaluated by SDS-PAGE (Fig. 4). Protein bands were visualized by the Coomassie Brilliant Blue staining method, and only a single polypeptide band was detected after the final purification step. The apparent molecular mass of the purified halophilic protease of strain HP25 was 21 kDa.

Effect of casein concentration on the purified halophilic protease of strain HP25

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Extremophiles

250

107

200

106

150

105

100

104

50

1

2

3

4

5

6

300

109

250

108

200

107

150

106

100

105

50

0

5

10

15

Fermentation period (day)

300

109

250

108

200

107

150

106

100

105

50 5

6

7

8

30

0

35

9

10

400

10 11

350

1010

4

25

(d)

400

Living-cell count (cells/ml) Specific activity (U/mg)

Living cell count (cells/ml)

Living cell count (cells/ml)

1011

20

NaCl concentration (%)

Specific activity (U/mg)

(c)

350

1010

Specific activity (U/mg)

108

400

Living-cell count (cells/ml) Specific activity (U/mg)

Living-cell count (cells/ml) Specific activity (U/mg)

10 10

300

10 9

250

10 8

200

10 7

150

10 6

100

10 5

50

0

20

25

30

35

40

45

50

55

60

0

Temperature ( oC)

pH

Fig. 2  Effect of fermentation period (a) salt concentration (b), pH (c) and temperature (d) on the growth of strain HP25 and halophilic protease secretion. Bacterial growth and halophilic protease secretion

350

Specific activity (U/mg)

Living cell count (cells/ml)

300

109

0

1011

350

1010

103

(b)

400

Living-cell count (cells/ml) Specific activity (U/mg)

Specific activity (U/mg)

1011

Living cell count (cells/ml)

(a)

of strain HP25 were monitored by counting the viable cells and by measuring the enzyme activity of cell-free supernatant

Table 2  Purification table of the halophilic protease from strain HP25 Purification step

Volume (mL) Volume activity (U/mL)

Total activity (U) Total protein (mg/mL)

Supernatant without treatment

1000

113

113666

299

380

1.4

100

Ultrafiltration by Amicon (10 kDa cut off)

110

899

98889

175

563

14

87

Dialysis after ethanol precipitation

50

1455

72746

101

714

18

64

Gel filtration (Superdex 200 HR)

15

2349

35236

5

6350

167

31

Discussion Halophiles are excellent sources of enzymes that are not only salt stable but can also withstand and carry out reactions efficiently under extreme conditions (Kumar et al. 2012). Among the 33 halophilic protease producers isolated

13

Specific activity (U/mg)

Purification (fold) Yield (%)

from different salt samples, the obligate halophilic strain HP25 isolated from raw salt was the best protease producer. This strain grew optimally at 25 % (w/v) NaCl and failed to grow at NaCl concentrations of 15 % or lower, indicating that this strain is an obligate halophile. This result is in agreement with Oren et al. (1995) and Kanlayakrit et al.

Extremophiles

(a)

(b) 100

100

90

90

80

Relative activity (%)

80 Relative activity (%)

70 60 50 40 30

70 60 50 40 30

20

20

10

10

0

0

0

5

10

15

20

25

30

35

1

NaCl concentration (%)

2

3

4

5

6

7

8

9

10

11

12

13

pH

(d)

(c)

100 90

Relative activity (%)

80 70 60 50 40 30 20 10 0 0

10

20

30

40

50

60

70

80

90

100

Temperature ( o C)

Fig. 3  Effect of salt concentration on the purified halophilic protease (a), effect of the pH value on the purified halophilic protease (b), effect of temperature on the purified halophilic protease (c), and Km

value Lineweaver–Burk (µg min−1mL−1) (d). All assays were done in triplicate. The maximum activity in each case was considered as 100 % and the other data were calculated as relative to it

(2004), who found that the extremely halophilic archaea are the dominant heterotrophic organisms in hypersaline environments in which salt concentrations exceed 25–30 % (w/v). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain HP25 revealed identities to its nearest phylogenetic neighbors of Halobacterium salinarum and Halobacterium piscisalsi. However, the phylogenetic assignment of strain HP25 by analysis of the 16S ribosomal RNA gene suggests that this strain is distinct from Halobacterium salinarum and Halobacterium piscisalsi and in a separate subcluster. In this context, Yachai et al. (2008) argued that the name Hbt. piscisalsi is problematic and that this name should now be considered as a junior synonym of Hbt. salinarum. Also, Oren (2012) reported that Hbt. piscisalsi JCM 14661T shares a high 16S rRNA gene sequence similarity and a high DNA–DNA

relatedness with Hbt. salinarum; this was confirmed by 16S rRNA gene sequencing of the type strain obtained from different culture collections. The distinctness of strain HP25 from related strains is supported by physiological characteristics, such as gelatin hydrolysis, lysine decarboxylase activity, ornithine decarboxylase activity, and their ability to utilize citrate, inulin and d-mannose (Table 1). In addition, they differed with regard to pigmentation, whereas HP25 formed pink colonies, and Hbt. salinarum formed red colonies as reported by Grant et al. (2002). These results indicate that strain HP25 could be classified as a new species in the genus of Halobacterium. Gibbons (1974) and Oren (2012) proposed the family Halobacteriaceae to include rods and cocci that require more than 12 % (w/v) of NaCl to grow, and included two genera, Halobacterium and Halococcus. Within each genus, very few species were recognized, and very little phenotypic and genotypic variety

13

Extremophiles Table 3  Effect of inhibitors, metal ions, organic solvents and detergents on the purified halophilic protease activity Enzyme inhibitors

Residual activity (%)

Standard deviation

Control 100 ± 0.12 EDTA (10 mM) 33 ± 0.58 β-Mercaptoethanol (0.1 %) 77 ± 0.23 PMSF (1 mM) 12 ± 0.29 Urea (8 M) 30 ± 0.14 SDS (0.1 %) 5 ± 0.23 83 ± 0.23 CuSO4 (2 mM) 97 ± 0.12 CaCl2 (2 mM) 95 ± 0.32 MgCl2 (2 mM) 56 ± 0.61 ZnSO4 (2 mM) KCl (2 mM) 99 ± 0.03 Methanol 99 ± 0.12 Ethanol 100 ± 0.12 Propanol 97 ± 0.12 Butanol 96 ± 0.12 Hexane 95 ± 0.12 Ariel 93 ± 0.46

0.20 1.00 0.40 0.50 0.25 0.40 0.40 0.20 0.20 1.05 0.05 0.20 0.20 0.20 0.20 0.20 0.80

Persil

1.00

91 ± 0.58

Fig. 4  SDS-PAGE of halophilic protease from strain HP25 at different purification levels. SDS-denatured samples of 20–40 µL were separated on a 12 % SDS-polyacrylamide gel and stained using Coomassie Brilliant Blue R-250. Lane 1 molecular mass standard; lane 2 (8 µg) protein of the purified enzyme and lane 3 (15 µg) of concentrated culture supernatant

13

was considered to exist among them. In this context, Colwell et al. (1995) reported that “a bacterial species is generally considered to be a collection of strains that show a high degree of overall similarity and differ considerably from related strain groups with respect to many independent characteristics”. Also, it should be noted that phylogenetic studies of the family Halobacteriaceae based on 16S rRNA gene sequences are complicated by the fact that some species contain more than one copy of the 16S rRNA gene, and these copies can be very different (Oren 2012). Therefore, when discriminating between closely related species of the same genus, DNA–DNA hybridization as well as housekeeping gene sequences should be the methods of choice, in accordance with the proposed molecular definition of species (Murray et al. 1990). The purified halophilic protease of strain HP25 exhibited its maximal activity at 17 % NaCl (w/v), pH 8.0 and 60 °C, indicating that this enzyme is a halo-alkalithermophilic protease. The Km value of the purified halophilic protease towards casein was 523 µg mL−1. This result is comparable to the results of Juhasz and Skarka (1990), Takii et al. (1990) and Mesbah and Wiegel (2014), who reported that proteases with low Km values (400 and 1300 µg mL−1) towards casein substrate were produced by Bacillus alkalophilus, Brevibacterium linens and Alkalibacillus sp. NM-Fa4, respectively. Higher Km values (7400 and 7500 µg mL−1) have been reported for the proteases of Halomonas sp. ES 10 (Kim et al. 1992) and Virgibacillus sp. EMB 13 (Sinha and Khare 2012), respectively. In addition, the purified enzyme exhibited a considerable stability in the presence of the tested organic solvents and laundry detergents. The unusual properties of this enzyme may allow it to be used in various applications such as the ripening of salted fish to avoid the toxicity risk associated with the traditional manufacturing methods. Furthermore, its stability in the presence of the tested organic solvents and laundry detergents may allow this enzyme to be used for further novel applications and as an additive in laundry detergent formulations. However, it is important to highlight that the use of enzymes from halophiles in industrial applications is not limited to high salt conditions, as these extremozymes are usually also tolerant of high temperatures and are stable in the presence of organic solvents (Oren 2010). Similar results were recently reported by Mesbah and Wiegel (2014) who purified and characterized an alkaline and thermostable protease from Alkalibacillus sp. isolated from alkaline, hypersaline lakes of Wadi An Natrun, Egypt. The inhibition of the purified halophilic protease of strain HP25 by EDTA indicated that this enzyme may require metal ions as cofactor for its activity. Also, the drastic inactivation of the halophilic protease of strain HP25 by

Extremophiles

PMSF indicated that this enzyme is likely to belong to the class of serine proteases. Beynon and Bond (2001) reported that serine proteases are irreversibly inhibited by PMSF, and PMSF inhibited the activity of the purified protease by up to 80 %. PMSF causes a sulfonation of the serine residue at the active site of serine proteases, resulting in the complete loss of enzyme activity. Serine proteases follow a two-step reaction mechanism for proteolysis in which a covalently linked enzyme–peptide intermediate is formed, with the loss of an amino acid or peptide fragment from the substrate. This acylation step is followed by a deacylation reaction that occurs by a nucleophilic attack on the enzyme–peptide intermediate by water, resulting in hydrolysis and liberation of the amino acyl moiety (Fastrez and Fersht 1973; Voet and Voet 2004). The apparent molecular mass of the purified halophilic protease of strain HP25 is considerably lower than of those purified from Halobacterium halobium (Izotova et al. 1983), Halophilic archaebacterium (Kamekura and Seno 1990), Natronolimnobius innermongolicus WN18 (Selim et al. 2014), Natrialba magdii (Gimenez et al. 2000), Halobacterium halobium, and Natronococcus occultus (Studdert et al. 2001). This result indicates that the halophilic protease of strain HP25 is a novel halophilic protease. However, further studies, such as the determination of the N-terminal amino acid sequence and the cloning and expression of the halophilic protease of strain HP25, are in progress. Acknowledgments  The authors would like to thank Dr. Martin Krehenbrink for helpful revision of the manuscript.

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