Extracellular peptidases from Deinococcus radiodurans
Gabriel Z. L. Dalmaso, Claudia A. S. Lage, Ana Maria Mazotto, Edilma Paraguai de Souza Dias, Lucio Ayres Caldas, Davis Ferreira, et al. Extremophiles Microbial Life Under Extreme Conditions ISSN 1431-0651 Extremophiles DOI 10.1007/s00792-015-0773-y
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Author's personal copy Extremophiles DOI 10.1007/s00792-015-0773-y
ORIGINAL PAPER
Extracellular peptidases from Deinococcus radiodurans Gabriel Z. L. Dalmaso1,2 · Claudia A. S. Lage2 · Ana Maria Mazotto1 · Edilma Paraguai de Souza Dias1 · Lucio Ayres Caldas3 · Davis Ferreira4 · Alane B. Vermelho1
Received: 15 November 2014 / Accepted: 14 July 2015 © Springer Japan 2015
Abstract The extremophile Deinococcus radiodurans wild type R1 produces peptidases (metallo- and serine-) in TGY medium and in the media supplemented with human hair (HMY) and chicken feathers (FMY). Enzymatic screening on agar plates revealed peptidase activity. In TGY medium metallopeptidases were detected corresponding to a molecular mass range of 300–85 kDa (gelatinases); 280–130 (caseinases) and a 300 and a 170 kDa (keratinases); and a gelatinolytic serine peptidase (75 kDa). In HMY medium after 144 h, D. radiodurans produced keratinase (290 U/ml), gelatinase (619 U/ml) and sulfite (26 µg/ ml). TGY medium produced higher proteolytic activity: 950 U/ml of gelatinolytic (24 h); 470 U/ml of keratinolytic (24 h) and 110 U/ml of caseinolytic (72 h). In the FMY medium, we found gelatinolytic (317 U/ml), keratinolytic (43 U/ml) and caseinolytic (85 U/ml) activities. The sulfite Communicated by A. Driessen. * Alane B. Vermelho
[email protected] 1
BIOINOVAR: Unidade de Biocatálise, Bioprodutos e Bioenergia, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941‑902, Brazil
2
Laboratório de Radiações em Biologia, Departamento de Biologia Molecular, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941‑902, Brazil
3
Laboratório de Microscopia Aplicada as Ciências da Vida, Instituto Nacional de Metrologia Qualidade e Tecnologia, INMETRO, Rio de Janeiro 25250‑020, Brazil
4
Laboratório de Interação Vírus‑Célula, Departamento de Virologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941‑902, Brazil
had a maximum release at 48 h (8.1 µg/ml). Enzymography analysis revealed that the keratinases degraded keratin after 24 h of reaction. The addition of sodium sulfite (1.0 %) improved the keratin degradation. Environmental Scanning Electron microscopy revealed alterations such as damage and holes in the hair fiber cuticle after D. radiodurans growth. This work presents for the first time D. radiodurans as a new keratinolytic microorganism. Keywords Extremophiles in biotechnology · Enzymes · Keratinase · Peptidases · Deinococcus radiodurans
Introduction According to Adrio and Demain (2014), more than 500 products are produced by direct or indirect enzymatic action, and roughly 170 industrial processes are benefited from the enzymes or biocatalysis of microorganisms (Binod et al. 2013). Peptidases, including keratinases, are enzymes that catalyze the hydrolysis of peptide bonds in proteins and peptides. Through evolutionary processes, peptidases have adapted to a wide range of different conditions such as variations in pH, reducing environments and others. These enzymes use different catalytic mechanisms for substrate hydrolysis (Turk 2006). A variety of biological functions and processes depend on their activity. The extracellular peptidases are produced by bacteria with several functions including nutrition and, in some pathogens, they are considered virulence factors. In prokaryotic organisms, peptidases of the seven catalytic types are found (represented by serine, cysteine, threonine, aspartic, asparagine, glutamic and metallo catalytic types). The genera Bacillus, Pseudomonas, Clostridium and Actinobacteria are examples
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of proteolytic bacteria with industrial applications (Nigam 2013; Vermelho et al. 2013). The glutamic peptidase is a rare type, which has been described in the bacterium thermoacidophilic Alicyclobacillus sp. DSM 15716 (Jensen et al. 2010; Vermelho et al. 2013). Keratins are proteins that constitute the epidermal structure and their appendages such as hair, feather, horns, nails, hoof, beak and claw. The secondary molecular structure of keratins have been classified as α-keratin and β- keratin. Human hair comprises α-keratin with 40–70 kDa formed by the aggregation of intermediate keratin filaments. The fibers of α-keratin are submerged in an amorphous matrix composed of sulfur rich protein. By contrast, feathers are mainly formed by β-keratin fibers surrounded by amorphous keratin. The feather structure contains α-helix, β-sheet or disordered state keratin, in the ratio 41:38:21 (Laba and Rodziewicz 2014). Keratinases are enzymes able to hydrolyze keratins. Many bacteria and fungi have been described as keratinase producers, and special attention has been directed to the Bacillus genus (Mazotto et al. 2011). Most studies have described the chicken feather degradation, but a few others have reported on the utilization of human hair or wool as substrates for microbial growth and enzymatic production (Hassan et al. 2013). The major applications of keratinases in biotechnology and industrial processes are involved in keratin-containing waste treatment, in detergents, in the textile industry, in the leather dehairing process, in cosmetics, as a peeling agent and especially the enzymatic keratin hydrolysates could be used in nutrition and animal feed and foodstuffs as a supplement (Chojnacka et al. 2011; Vermelho et al. 2010b). Keratinases can be also used as pesticides and in biotransformations as described for the production of keratin hydrolysates and prion degradation (Gupta et al. 2013; Korniłłowicz-Kowalska and Bohacz 2011). Extremophile microorganisms are a source of enzymes with different characteristics and with activities over a broad range of temperature, pressure, salinity, pH and radiation, which gives them various applications in biocatalysis for industrial processes (Dalmaso et al. 2015). Nowadays, different groups are investigating new possible biotechnological applications for extremophiles: Bergquist et al. (2014) has discussed thermophile enzymes usage, Gabani and Singh (2013) have proposed the use of metabolites from radioresistant extremophiles in the pharmaceutical industry and Karan et al. (2012) described enzymes functionally present in non-aqueous organic solvents. The extremophile bacterium Deinococcus radiodurans is known for its radioresistance to ultraviolet and ionizing radiation (Daly 2009; Krisko and Radman 2013; PaulinoLima et al. 2010), and also has been described as a polyextremophile, tolerating different ranges of oxidative stress,
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desiccation, low temperatures and pressures (Lage et al. 2012; Slade and Radman 2011). Few studies have demonstrated the presence of extracellular peptidases, their production, quantification and classification in D. radiodurans. Zhou et al. (2005) described the variation in the expression of gelatinases and caseinases after the exposure of D. radiodurans to gamma irradiation (γ) and ultraviolet-C (UV-C), which were associated with an increase of production, due to DNA repair. Basu and Apte (2008) reported the presence of a serralysin metalloprotease, and, recently, Are et al. (2014) described a serine peptidase (APH; EC 3.4.19.1), which catalyzes the removal of an N-acylated amino acid from a blocked peptide. However, no work has been published concerning the presence of keratinases in D. radiodurans, despite the fact that Deinococcus ficus is described as a feather-degrading bacterium with the capability of enhancing the production of keratinase after UV inducible mutagenesis (Zeng et al. 2011). In this study, we describe the production of extracellular peptidases by D. radiodurans and its ability to use keratin (hair and feather). We measured the D. radiodurans keratinolytic activity in fermentation media containing human hair and the degradation of feather keratin by their extracellular enzymes.
Materials and methods Bacterial strain and growth conditions The bacterial strain used in this study was the Deinococcus radiodurans R1 wild type (ATCC-13939) and was obtained from a stock kept at the Instituto de Radioproteção e Dosimetria, Rio de Janeiro, Brazil (Paulino-Lima et al. 2011). The D. radiodurans was grown in TGY broth (0.5 % bacto tryptone, 0.3 % bacto yeast extract and 0.1 % glucose) at 30 °C under 200 rpm agitation for 40 h. Aliquots (1:100) were transferred to a fresh medium and grown for another 15 h, under the same conditions, to achieve the early stationary phase, 108 Colony Forming Unit (CFU), to be used in the following experiments. All the tests were performed in triplicates. Detection of extracellular activity on Agar plates A preliminary test on agar plates was performed to determine the extracellular hydrolase production by D. radiodurans. The substrates used were gelatin, casein and keratin to reveal the presence of gelatinase, caseinase and keratinase, respectively. All the plates were incubated at 30 °C for 3 days with 100 µl of culture. To reveal the hydrolysis zone, Amido Black was used to detect activity (Vermelho
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et al. 1996). The Enzymatic Index (I2) was measured using the ratio between the diameter of the hydrolysis zone and the diameter of the colony. A value of this ratio greater than 2 indicates a satisfactory enzyme production.
1 µg was defined as one unit of activity (1 U). The experiments were performed in triplicate.
Peptidase production on TGY medium
The production of sulfite is associated with the attack on the keratin structure, breaking the disulfide bonds. It was measured by the method described by Arikawa et al. (1967). A sodium sulfite solution (0.1 mg/ml) was used as a standard.
The peptidase production of D. radiodurans was determined during the microorganism growth on the TGY medium, for 96 h at 30 °C and 200 rpm. Aliquots of the supernatant were taken every 24 h.
Sulfite determination
Environmental scanning electron microscopy (ESEM) Peptidase production using human hair and chicken feather medium Cultures of D. radiodurans were grown in a TGY medium and then inoculated into 100 ml of TGY medium or in phosphate buffer medium supplemented with yeast extract (0.01 %) containing 2 % human hair or chicken feather (HMY and FMY, respectively). Cultures were grown for 96 h at 30 °C and 110 rpm and aliquots of supernatant were taken every 24 h. Both human hair and chicken feather were previously washed and cleaned with detergent. After that, they were delipidated in a solution of chloroform and methanol 1:1 and were cut into 3–5 cm pieces before adding to the solution (Mazotto et al. 2011). Keratinase production using human hair as substrate A propagation medium was used to induce the keratinase production of D. radiodurans. A culture of D. radiodurans was previously grown on TGY medium for 48 h and then 2 ml of this culture was transferred to 20 ml of HMY medium. After 144 h, the cells were centrifuged (8000 rpm for 5 min), washed twice in saline solution (NaCl 0.9 %) and inoculated into a fresh HMY medium. The keratinolytic and gelatinolytic activity and sulfite determination were measured after 144 h of incubation. Peptidases assay The quantification of peptidase activity was performed for gelatinases, caseinases and keratinases. The gelatinase assay was described by Jones et al. (1998) and modified by Mazotto et al. (2011). Caseinase activity was measured according to the method described by Tomarelli et al. (1949) and the keratinase assay was described by Grzywnowicz et al. (1989). The supernatants of all assays were measured by absorbance at 660 nm to determine the protein concentration through the Lowry et al. (1951) method using BSA (bovine serum albumin) as a standard. The amount of enzyme necessary to increase the protein concentration in
The human hair fiber was analyzed by environmental scanning electron microscopy (FEI Quanta 450 FEG) after 144 h of culture with D. radiodurans in HMY medium. The fibers were cut transversely and placed on carbon conductive adhesive tapes, as described by Lopes Torres et al. (2013). Zymograms Each zymogram was performed with 50 µg of crude enzymatic extract in 8 % SDS-PAGE with 0.1 % of copolymerized substrate (gelatin and keratin feather powder) and 10 % SDS-PAGE with 0.1 % of casein substrate as described by Heussen and Dowdle (1980). The 24 h supernatant of D. radiodurans, grown in TGY medium, in HMY or FMY (for keratin substrate), was mixed with the sample buffer for zymography (125 mM Tris–HCL pH 6.8; 4 % SDS; 20 % glycerol and 0.002 % bromophenol blue) in a sample:buffer ratio 6:4, for gelatin and casein, and 8:2 for keratin. The gels were incubated overnight at 37 °C in proteolysis 50 mM glycine buffer (pH 10.0) for gelatinase and keratinase, and in 0.5 M Tris–HCl (pH 6.8) for caseinase. In order to observe the peptidase inhibitors on the proteolytic enzyme activity, the gels were incubated in the presence of the following inhibitors: 3 mmoL l−1 phenylmethylsulfonyl fluoride (PMSF) (inhibitor of serine peptidase); 10 mmoL l−1 1.10 phenanthroline (inhibitor of metallopeptidase); 10 μmoL l−1 pepstatin A (aspartic peptidase inhibitor) and 0.26 mmoL l−1 ethylenediaminetetraacetic acid (EDTA) (Mazotto et al. 2011; Vermelho et al. 2010a). Enzymography To detect the keratin hydrolysis, D. radiodurans supernatant containing 100 µg of proteins was mixed with 60 µg of feather keratin powder diluted in phosphate buffer (0.06 M Na2HPO4·7H2O/0.04 M KH2PO4, pH 7.2) and sodium sulfite at the concentrations of 0.5 and 1.0 %. The reaction mixtures were incubated for 16 and 24 h at room temperature. The reactions were stopped by freezing. The reaction
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mixtures were mixed with SDS-PAGE sample buffer as described by Mazotto et al. (2011).
Results The enzymatic screening of D. radiodurans extracellular peptidases revealed gelatinase, caseinase and keratinase activity by quantification of the Enzymatic Index of 2.78, 3.30 and 2.0 cm, respectively (Table 1). The highest activities for the D. radiodurans peptidases were obtained when the bacterium was cultured in 20 ml of TGY medium. They were 590 and 260 U/ml of gelatinase and keratinase activity, respectively, at 24 h of incubation, and 98 U/ml of caseinase activity at 96 h of incubation (Fig. 1). When D. radiodurans was cultivated in 100 ml of TGY and in the medium with human hair (HMY) or chicken feather (FMY) as a substrate for 96 h, the TGY supplemented culture was revealed to have the best peptidase production. In TGY, 950 U/ml of gelatinolytic and 470 U/ml of keratinolytic activity was observed in the first 24 h, and
Table 1 Enzymatic Index on preliminary detection of extracellular peptidases of D. radiodurans, grown for 72 h at 30 °C on agar plates containing specific substrates: gelatin, casein and keratin Substrate
Enzymatic Index
Gelatin Casein
2.78 3.30
Keratin
2.0
The plates were stained with Amido black and destained with methanol:acetic acid:water (5:1:4) for 30 min
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700.00 600.00
5
500.00
4
400.00 3 300.00 2
200.00
1
100.00
0
0.00 0
24
48
72
Time (h) Gelanase
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Keranase
Caseinase
Total Proteins
96
Total soluble proteins (mg/ml)
Enzymac acvity (U/ml)
Fig. 1 Kinetics of peptidases production of D. radiodurans grown in 20 ml of TGY medium for 96 h at 30 °C and 200 rpm. Activity of gelatinase, caseinase, keratinase and total soluble proteins were measured according to the Lowry et al. (1951) method. One unit of activity (1 U) was defined as the amount of enzyme necessary to increase the protein concentration in 1 µg in 30 min (gelatinase) or in 1 h (caseinase and keratinase)
110 U/ml of caseinolytic activity after 72 h. The release of sulfite increased through to 96 h (9.6 µg/ml) (Fig. 2a). In the HMY medium, we found 425 U/ml of gelatinolytic activity (48 h), 85 U/ml of keratinolytic activity (72 h) and 37 U/ml of caseinolytic activity in 24 h. The sulfite had a maximum release at 48 h (8.6 µg/ml) (Fig. 2b). In the FMY medium, we found 317 U/ml of gelatinolytic and 43 U/ml of keratinolytic activity, in the first 24 h, and 85 U/ml of caseinolytic activity in 96 h. The sulfite had a maximum release at 48 h (8.1 µg/ml) (Fig. 2c). Keratinase and gelatinase production in HMY fermentation medium increased by 18 % (290 U/ml) and 15 fold (619 U/ml), respectively, compared to the propagation medium (Fig. 3). The sulfite determination showed a constant increase in sulfite released by the bacteria into the supernatant, during the 96 h of cultivation in the 20 ml TGY medium, reaching values of 17 µg/ml. Table 2 shows the values for sulfite release for HMY propagation (6.5 µg/ ml) and fermentation (26 µg/ml) medium. The environmental scanning electronic microscopy revealed significant alterations in the cuticle of hair fiber, after the 144 h of D. radiodurans interaction. An eroded cuticle and the presence of holes were observed for hair samples that had been cultured with D. radiodurans (Fig. 4). Four different extracellular enzymes with molecular masses of approximately 75, 85, 200, and 300 kDa were detected on the zymogram co-polymerized with a gelatin substrate. The enzymatic class for the 85, 200 and 300 kDa proteins was defined as metallopeptidases due to their inhibition by phenanthroline and EDTA and as a serine peptidase for the 75 kDa protein due to its inhibition by PMSF (Fig. 5). The zymogram with casein
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A
10
800
8
600
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400
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2
0
Sulfite concentra on (mg/ml)
12 1000 Enzyma c ac vity (U/ml)
Fig. 2 Kinetics of peptidase production and sulfite release of D. radiodurans grown in 100 ml of TGY (a), HMY (b) and FMY (c) media for 96 h at 30 °C and 110 rpm. Activity of gelatinase, caseinase and keratinase were measured according to the Lowry et al. (1951) method. One unit of activity (1 U) was defined as the amount of enzyme necessary to increase the protein concentration in 1 µg in 30 min (gelatinase) or in 1 h (caseinase and keratinase)
0 0
24 Gela nase
48 Time (h) Kera nase
72
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Caseinase
Sulfite
Enzyma c ac vity (U/ml)
B
10
800
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0
Sulfite concentra on (mg/ml)
12 1000
0 0
24 Gela nase
48 Time (h) Kera nase
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Caseinase
Sulfite
Enzyma c ac vity (U/ml)
C
10
800
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600
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400
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24 Gela nase
as a substrate showed the presence of three enzymes, with molecular masses of approximately 280, 150 and 130 kDa. We suggest that the 280 kDa enzyme is a serine peptidase, due to its inhibition by PMSF, and the 130 kDa peptide is a metallopeptidase, due to its inhibition by phenanthroline. The 150 kDa peptide was not inhibited by any of the inhibitors tested in this study (Fig. 5). On the other hand, we reported the presence of two metallopeptidases on the zymogram containing keratin as a substrate, with molecular masses of approximately 300
48 Time (h) Kera nase
72
96
Caseinase
Sulfite
and 170 kDa (Fig. 5a). A major expression of a 170 kDa keratinase was also observed after 144 h in the HMY medium (Fig. 5b). However, only a metallopeptidase of approximately 300 kDa in the FMY medium was observed (Fig. 5c). The enzymography analysis demonstrated that keratin was hydrolyzed by keratinases from D. radiodurans. Keratin migrated at 10 kDa (Fig. 6, line A). The degradation was progressive with time (16 and 24 h) and sulfite addition (0.5 and 1.0 %) as showed in Fig. 6 (lines D–I).
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Enzymac acvity (U/ml)
700 600 500 400 300 200 100 0
D. radiodurans PM Gelanase
D. radiodurans FM Keranase
Fig. 3 Inducible production of keratinase by D. radiodurans using human hair as substrate (HMY medium). Gelatinase and keratinase activity were measured after 144 h growth at 30 °C and 110 rpm in propagation medium (PM) and in fermentation medium (FM). A higher turbidity was observed in the fermentation medium compared to the propagation medium (pictures above the graph) Table 2 Sulfite determination measured from the supernatant culture of D. radiodurans, produced over a 96 h period at 30 °C and 200 rpm in TGY medium incubation as a constitutive production and after 144 h of incubation in propagation medium (PM) and fermentation medium (FM) at 30 °C and 110 rpm Medium
Time (h)
Sulfite concentration (µg/ml)
TGY
Propagation Medium
0 24 48 72 96 144
5.26 ± 0.16 11.57 ± 1.21 14.90 ± 2.37 17.13 ± 0.96 17.78 ± 1.05 6.56 ± 0.73
Fermentation Medium
144
26.11 ± 0.84
Discussion Deinococcus sp. is a chemoorganotrophic bacterium that has been isolated from several environmental habitats including air, arid soil, water and activated sludge, alpine environments, rhizosphere, aquifer, marine fish, radioactive sites, Antarctica soil and hot springs (Chaturvedi and Archana 2012; Murray 1992; Slade and Radman 2011). Some examples include Deinococcus radiophilus isolated from Oyster Extract (Chan and O’Toole 1999) and Deinococcus geothermalis in deep-ocean subsurface environments (Kimura et al. 2003; Liedert et al. 2012). In this work, we demonstrated the production of extracellular peptidases with a focus on keratinases by the polyextremophile
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bacteria D. radiodurans. This is the first report of keratinolytic activity in D. radiodurans. Only a few studies have explored the D. radiodurans extracellular peptidase activity, specifically, the presence of a functional serralysin metalloprotease and the peptidase expression subsequently to UV and gamma irradiation (Basu and Apte 2008; Zhou et al. 2005). Peptidases have been described in the Deinococcus genus, such as a serine peptidase, with a molecular mass of 24 kDa, described in D. geothermalis by Pietrow et al. (2013). Database such as Merops have described the presence of 103 (known and putative) peptidases in D. radiodurans (Rawlings et al. 2014). We attempted to investigate the degradation of keratin and the results showed that D. radiodurans was a good producer of extracellular peptidases. According to Korniłłowicz-Kowalska (1997), the most reliable indicator of microbial keratinolytic abilities is the mass loss of the keratin substrate. The keratinase activity, and the release of peptides, amino groups, amino acids, ammonia, sulfhydryl groups and sulfate are also other indicators of keratinolytic microorganisms skills (Korniłłowicz-Kowalska and Bohacz 2011). Several bacteria have been cited as keratinolytic, most of them from the Bacillus and Streptomyces genera. Peptidases from extremophiles such as Fervidobacterium and Thermoanaerobacter genera have been described (Brandelli 2008; Cedrola et al. 2012; Friedrich and Antranikian 1996; Riessen and Antranikian 2001; Suzuki et al. 2006). Most keratinolytic microorganisms degrade chicken feather but dermatophyte fungi are frequently been associated to with human hair degradation (Brandelli et al. 2010; Gopinath et al. 2015; Grumbt et al. 2013; Gupta and Ramnani 2006; Mazotto et al. 2011). Keratinolytic fungi are usually found colonizing natural environments inhabited by humans, birds and mammals, where a supply of keratin are regular (KorniłłowiczKowalska et al. 2011). They can be divided in two types: true keratinolytic organisms degrade hard keratin and completely solubilize keratin structures; and potentially keratinolytic microorganisms with strong proteolytic properties, which plays an important role converting non-keratin proteins associated to keratin structures (KorniłłowiczKowalska and Bohacz 2011). Microorganisms such as the fungi genera Trichophyton sp. and Chrysosporium sp. are specialized in degrading hair keratin (Korniłłowicz-Kowalska and Bohacz 2011). Micromorphological, biochemical and cytochemical investigations, shows that the process of hair degradation is associated with enzymatic and mechanical destruction (Kunert 2000; Marchisio 2000). Mazotto et al. (2010) described the partial degradation of fiber hair by Bacillus subtilis, but only the cuticle was hydrolyzed, due to the β-conformation of this structure.
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Fig. 4 Scanning electronic microscopy showing the degradation of a hair fiber by D. radiodurans after 144 h of incubation in HMY medium. Control, (a–c), and after 144 h of cultivation (d–f). Bars 10 µm
Our analysis with ESEM demonstrated that the D. radiodurans produced holes at the surface of hair fiber, suggesting that the degradation is initiated at 144 h. In HMY culture, a keratinase of approximately 170 kDa was more expressed, suggesting that this enzyme takes part in the mechanism to degrade the α-keratin presented in human hair. This can be supported by the fact that the 170 kDa keratinase was not observed in the feather medium, where the major keratin found in feathers is the β-keratin. For keratin degradation, two mechanisms were described. The first one was the sulfitolysis, when the sulfite is responsible for the breakdown of sulfide bridges followed by keratin hydrolysis and peptidases (Kunert 1972). The second mechanism was the synergic action of two enzymes, peptidase and disulfide reductase (Cedrola et al. 2012; Yamamura et al. 2002). It is interesting to note that sulfite was produced by D. radiodurans in all media tested, indicating sulfitolysis and peptidases action together as a possible mechanism of keratin degradation. Sulfitolysis
mechanism was reported to other bacteria, such as in Bacillus genus (Cedrola et al. 2012; Gupta and Ramnani 2006; Laba and Szczekala 2013). Proteolytic keratinase activity is usually associated with serine peptidases and less frequently with metallopeptidases (Brandelli et al. 2010; Gupta and Ramnani 2006). It is possible that the 300 kDa keratinase with metallopeptidases activity is the same enzyme that was detected in the gelatinase zymogram, suggesting a peptidase acting in both gelatin and keratin substrates. Gelatinases (300, 200, 75, 85 kDa) and caseinases (130– 280 kDa) were detected with zymograms. Zhou et al. (2005) also described gelatinases and caseinases in D. radiodurans with high molecular mass, approximately of 140 and 120 kDa, respectively. The presence of peptidases with high molecular masses is unusual. This could happen due to glycosylation process or protein aggregation. The periodic acid-Schiff staining is a method used to detect polysaccharides and indicates if extracellular proteins might be glycosylated. Regarding this, Saarimaa et al. (2006) reported the presence of glyconjugates and serine peptidases with conservative motifs in Deinococcus geothermalis. A purified serine peptidase, described in D. radiodurans as an acyl peptide hydrolase (APH; EC 3.4.19.1), showed a molecular mass of 265 kDa (Are et al. 2014). This suggests that this uncommon characteristic is found in Deinococcus genus. Peptidases with high molecular weight have been reported in other microorganisms. Keratinases from Kocuria rosea (240 kDa), Fervidobacterium islandicum (200 kDa) and Bacillus cereus 1268 (200 kDa) have been found (Bernal et al. 2006; Mazotto et al. 2011; Nam et al. 2002). A peptidase from the pathogenic bacterium Campylobacter jejuni was described with high molecular mass (170 kDa) (Karlyshev et al. 2014). Recently, Farci et al. (2014) detected two peptidases associated to the cell wall of D. radiodurans that had been predicted by the genes DR_2310 and DR_A0283. According to White et al. (1999), an uncharacterized peptidase from the catalytic domain of metallopeptidases (DR_2310) was described with a molecular mass of 84.2 kDa and a probable subtilase-type serine protease (DR_A0283) was described with a molecular mass of 76.6 kDa. These putative enzymes match the same mass and enzymatic class of that found in our gelatin zymograms, a 85 kDa metallopeptidase and a 75 kDa serine peptidase. However, more tests need to be done to address these enzymes within their genes. The production of gelatinase and collagenase is particularly interesting for industrial processes, as they can be applied in the food industry as a meat tenderizer, in drink industry, in healthcare products, drugs and cosmetics (Watanabe 2004). As for the caseinases, their industrial
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Fig. 5 Zymograms of D. radiodurans grown for 144 h at 30 °C in TGY medium (a), in HMY medium (b) and FMY medium (c) for keratin substrate and zymograms of D. radiodurans grown for 24 h in TGY medium for gelatin and casein substrate. Gel strips containing culture supernatants were incubated overnight at 37 °C in proteolysis 50 mM glycine buffer (pH 10.0) for gelatinase and keratinase,
and in 0.5 M Tris–HCl (pH 6.8) for caseinase. The zymogram shows the effects of proteolytic inhibitors on each extracellular peptidase, in absence (control) or in the presence of different proteolytic inhibitors: phenylmethylsulfonyl fluoride (PMSF), phenanthroline (Phen), pepstatin A (Peps) and ethylenediaminetetraacetic acid (EDTA)
Fig. 6 Enzymography of keratinolytic hydrolysis by extracellular enzymes of D. radiodurans. The reaction mixtures containing culture supernatant and feather keratin were incubated for 16 and 24 h at room temperature in the absence or presence of sodium sulfite (0.5 or 1.0 %). The enzymography reveals that the enzymes of D. radiodurans shows keratinolytic activity and the hydrolysis was increased by adding sulfite. Keratin and supernatant were placed separately as
a control. a Keratin (control); b supernatant (control); c keratin and supernatant (0 h); d keratin and supernatant (16 h); e keratin and supernatant supplemented sulfite 0.5 % (16 h); f keratin and supernatant supplemented sulfite 1.0 % (16 h); g keratin and supernatant (24 h); h keratin and supernatant supplemented sulfite 0.5 % (24 h); i keratin and supernatant supplemented sulfite 1.0 % (24 h)
applications lie mainly in cheese production and in reducing allergenic properties of milk. Moreover, their hydrolysates are used in energetic drinks, dietetic food and healthcare (Claverie-Martín and Vega-Hernández 2007; Urista et al. 2011).
The industrial and household enzymes market has been growing continuously over growing in the last decades (Sarrouh et al. 2012) and the search for novel, more stable and active enzymes and their respective producer microorganisms is of great interest. In this context, extremophiles
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are a source of special enzymes (Elleuche et al. 2014; Morozkina et al. 2010). The production of keratinase by a polyextremophile is an interesting characteristic for an industrial microorganism.
Conclusion This study describes the radioresistant bacterium Deinococcus radiodurans as a source of new enzymes. We demonstrated that D. radiodurans expresses proteolytic enzymes using gelatin, casein, and keratin. The peptidases produced belong to the metallopeptidases or serine-peptidases classes. We demonstrated that D. radiodurans is able to grow with feather and hair as a substrate. We also demonstrated that D. radiodurans is capable of interacting with α-keratin (hair), eroding and creating holes in the hair cuticle, and increasing the expression of gelatinases, keratinase and sulfite release in a fermentation medium. Finally, we proposed that the D. radiodurans’ ability to degrade keratin is associated with the sulfitolysis mechanism. The world market is under constant pressure for ecofriendly processes and products, and this microorganism has a great potential and applicability in different sectors. Acknowledgments We would like to thank Dr. Veronica Cardoso, Samyra Tibúrcio, João Barreto and Luiz Eduardo Ferreira for their assistance and give our thanks to Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem (INBEB). This study was supported by grants from Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (MCTi/CNPq) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.
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