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F. Xie et al. / Bioresource Technology 101 (2010) 344–350 345 from Streptomyces sp. strain 16 could also efficiently decompose casein, collagen, and keratin azure. In this study, we describe the purification and characterization of four keratinases from Streptomyces sp. strain 16 cultured in medium containing NHFS. 2. Methods 2.1. Materials Native human foot skin waste was collected from the foot care section of the Haidian Hospital in Beijing. In preparation for NHFS experiments, the human foot skin was washed thoroughly with tap water, autoclaved, and then dried. The dried NHFS was stored under dry condition and was used for keratinase production. One part of the dried NHFS was milled to powders for the keratinase assay. 1-Ethyl- 3-(3-dimethyaminopropyl) carbodiimide (EDAC), p-chloromercuribenzoic acid (pCMB), phenylmethanesulfonyl fluoride (PMSF), DLdithiothreitol (DTT), and casein were purchased from Sigma (USA). Nanosep centrifugal device tubes (molecular size cutoff, 100 or 10 kDa) were purchased from Pall Corporation (USA), while Sephacryl S-200 resin, Superose 12 10/300 GL column, Hiprep DEAE FF column (1 ml), and Hiprep Octyl FF column (1 ml) were acquired from Amersham Biosciences (Sweden). The Folin–Ciocalteu reagent was purchased from Dingguo Company (China). All reagents used in this study were analytical grade unless otherwise indicated. 2.2. Microorganism, culture medium, and growth conditions The microorganism used in this study was Streptomyces sp. strain 16, which was isolated in our lab for keratinase production (Chao et al., 2007). The medium for keratinase production was composed of NHFS, 4 g/l; soluble starch, 5 g/l; yeast extract, 1 g/l; and K 2 HPO 4 , 0.5 g/l. The pH of the medium was 8.0. Seed cultures of Streptomyces sp. strain 16 were prepared in a 500 ml Erlenmeyer flask containing 100 ml of culture medium. After 2 days of incubation, the culture broth was transferred to a 5 L Fernbach culture flask containing 1.5 L of medium for keratinases production. After 4 days of incubation, the culture was collected for keratinase purification. All incubations were done aerobically at 30 °C on an orbital platform shaker at 200 rpm. 2.3. Zymography of the culture filtrate The zymography of the culture filtrate was based on the method of Bressollier et al. (1999).The culture broth was centrifuged (10,000 rpm, 4 °C, 10 min.), and the supernatant was mixed with 2 electrophoresis sample buffer (2 ml 4 stacking gel buffer, 1.6 ml glycerol, 3.2 ml 10% SDS, 0.8 ml 2-mercaptoethanol, and 0.4 ml 1.0% bromophenol blue) (Simpson, 2003) in a 1:1 ratio without heat denaturation. SDS–PAGE was carried out at 4 °C with a constant current of 23 mA in a 12% polyacrylamide gel. The gel was washed with 2.5% Triton X-100 (v/v) in 50 mM Tris–HCl buffer (pH 7.5) for 30 min. Then washed with 50 mM Tris–HCl buffer (pH 7.5) for 30 min. Casein (1%, w/v) dissolved in 50 mM Tris–HCl buffer (pH 7.5) was poured onto the gel. After 30 min at 40 °C, the gel was stained with Coomassie brilliant blue R-250 and then destained with a water/methanol/acetic acid (50:40:10) solvent. Peptidase bands appeared as clear zones on a blue background. 2.4. Enzyme assays 2.4.1. Assay for keratinolytic activity Keratinolytic activity was determined spectrophotometrically with a modification of the Folin–Ciocalteu method (Ledoux and Lamy, 1986; Margesin and Schinner, 1991). An appropriate amount of enzyme was incubated with 1.5 ml of 0.5% sieved (mesh size: 300 lm) NHFS, in 50 mM Tris–HCl buffer (pH 7.5) at 40 °C for 16 h. The reaction was terminated with 3 ml of 10% trichloroacetic acid (TCA) and placed at room temperature for 30 min. The reaction mixture was centrifuged, and 5 ml of 0.55 M Na 2 CO 3 was added to 1 ml of the supernatant, with subsequent addition of 1 ml Folin–Ciocalteu reagent. This mixture was incubated at 40 °C for 15 min. Keratinolytic activity was measured at 680 nm using a spectrophotometer (721 Visible Spectrometry, China). One unit (U) of keratinase activity was represented by an increase in absorbance at 680 nm of 0.01 in 1 h compared with the control reaction. The control was treated in the same way, except that TCA was added before incubation. 2.4.2. Assay for proteolytic activity Protease activity was determined by modifying the Folin–Ciocalteu method using casein as a substrate. The appropriate amount of enzyme was incubated with 1 ml 1% casein in 50 mM Tris–HCl buffer (pH 7.5) at 40 °C for 15 min. The reaction was terminated by the addition of 3 ml of 10% TCA. Subsequent steps were the same as those described in the assay of keratinolytic activity. One unit (U) of protease activity was defined as an increase in absorbance at 680 nm of 0.01 in 1 min compared with the control reaction. The control was treated in the same way, except that TCA was added before incubation. 2.5. Protein determination Protein concentrations of the enzyme preparations were determined according to the method described by Bradford (1976), using bovine serum albumin as the standard. 2.6. Purification procedure Culture broth was centrifuged (11,366g, 4°C, 10 min) to remove cells and debris and the supernatant was collected. Solid ammonium sulfate was added to the supernatant with a saturation of 80% under gentle stirring, and then kept at 4 °C overnight. The suspension was centrifuged at 10,000g for 30 min at 4 °C. The precipitate was then dissolved in 100 ml of 50 mM Tris–HCl buffer (pH 7.5). Three milliliter of the dissolved ammonium sulfate precipitate was loaded onto a Sephacryl S-200 column (3 100 cm) equilibrated with 50 mM Tris–HCl buffer (pH 7.5) and eluted with the same buffer at a flow rate of 0.5 ml/min. Fractions of 6.0 ml in each tube were collected and assayed for keratinolytic activity. The peaks with keratinolytic activity were named KI, KII, KIII, and KIV according to the order of elution. Purity was checked via SDS– PAGE. KIV was found to be homogenous on the SDS–PAGE gel, while the other three components needed further chromatographic purification. 2.6.1. Purification of keratinases KI and KII Following Sephacryl S-200 column chromatography, the KI and KII fractions were pooled separately, and sequentially applied to a Hiprep DEAE FF column (1 ml) pre-equilibrated in 50 mM Tris–HCl buffer (pH 7.5), and a Hiprep Octyl FF column (1 ml) pre-equilibrated in 50 mM Tris–HCl buffer (pH 7.5) containing 1.5 M (NH 4 ) 2 SO 4 . The KI fraction was eluted from the Hiprep DEAE FF column with 20 ml 50 mM Tris–HCl buffer (pH 7.5) containing 0 to 1 M NaCl at a linear flow rate of 2 ml/min. The KII fraction was eluted using 0.1 M, 0.2 M, 0.5 M, and 1 M NaCl sequentially, in 50 mM Tris–HCl buffer (pH 7.5). The KI fraction was eluted from the Hiprep Octyl FF column using 20 ml of 50 mM Tris–HCl buffer (pH 7.5) containing 0 to 1.5 M (NH 4 ) 2 SO 4 at a linear flow rate of
346 F. Xie et al. / Bioresource Technology 101 (2010) 344–350 2 ml/min. The KII fraction was eluted from the Hiprep Octyl FF column using a reverse step gradient of 50 mM Tris–HCl buffer (pH 7.5) containing 1.0, 0.75, 0.5, 0.3, and 0 M (NH 4 ) 2 SO 4 , sequentially, at a flow rate of 2 ml/min. 2.6.2. Purification of keratinase KIII After Sephacryl S-200 column chromatography, fractions of peak KIII were pooled and adjusted to 1.5 M (NH 4 ) 2 SO 4 . KIII was eluted by applying 20 ml of 50 mM Tris–HCl buffer (pH 7.5) containing 0 to 1.5 M (NH 4 ) 2 SO 4 at a linear flow rate of 2 ml/min. 3. Results 3.1. Keratinases production Streptomyces sp. strain 16 was able to grow and produce keratinase in a medium that contained 4 g/L NHFS. To expedite growth and increase the biomass of the strain, limited nitrogen sourceyeast extract was added to the medium in this study. NHFS began to decrease in the second day of cultivation, and completely disappeared in the fourth day of cultivation. Keratinase production reached maximal activity of 74.9 U/ml at four days of cultivation. 2.7. Molecular weight of the purified keratinases The purity and subunit number of each peptidase was estimated by SDS–PAGE (12% gel) according to Laemmli (1970). After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 dissolved in a water/methanol/acetic acid (50:40:10) solvent for 1 h, then destained in the same solvent. The molecular weight of the native peptidases was determined using a Superose 12 10/300 GL column, using thyroglobulin (669,000 Da), ferritin (440,000 Da), catalase (232,000 Da), lactate dehydrogenase (140,000 Da), and bovine serum albumin (67,000 Da) (Amersham Biosciences, Sweden) as standards. 2.8. Effect of pH and temperature on enzyme activity and stability The optimal pH for enzyme activities was determined at 40 °C at various pH levels (pH 6.0–12.0). Sodium phosphate was used for pH 6.0–7.0, Tris–HCl was used for pH 7.5–9.0, and sodium carbonate–bicarbonate was used for pH 10.0–12.0. For pH stability, residual activity was measured at pH 7.5 after 1 h of incubation at 40 °C in the pH buffers being tested. Enzyme activities were tested at temperatures ranging from 30 °C to80°C in 50 mM Tris–HCl buffer (pH 7.5). Each of the purified enzymes was incubated for 1 h at the tested temperature, and residual activity was measured at 40 °C in 50 mM Tris–HCl buffer (pH 7.5). 3.2. Purification of the keratinases produced by the Streptomyces sp. strain 16 The zymogram of culture supernatant shown in Fig. 1 indicated the presence of five bands that showed peptidase activity in the culture supernatant. The purification procedures of the four enzymes is outlined in Fig. 2. When the precipitated enzyme was loaded onto Sephacryl S-200 column, four major peaks eluted that showed keratinase activity. The main keratinase in each peak was designated KI, KII, KIII, and KIV based on their order of elution. However, the fifth band appeared on the zymogram gel was not detected from the elution. After one to two further steps of chromatography, all four keratinases from Streptomyces sp. strain 16 were ultimately purified to homogeneity, which was confirmed by SDS–PAGE (Fig. 3). Molecular weights of subunits of the four enzymes were calculated as 25 kDa (KI), 50 kDa (KII), 34 kDa (KIII) and 19 kDa (KIV). The determination of the molecular weight of each of the four keratinases was performed by gel filtration chromatography on a 2.9. Effects of protease inhibitors, reducing agents, and metal ions on keratinase activity To investigate the effects of different inhibitors, reducing agents, and metal ions on keratinase activity, the residual activity after allowing the enzyme to stand with the reagent for 30 min at 30 °C was measured. Phenylmethyl sulphonyl fluoride (PMSF), para-chloro mercuric benzoate (pCMB), EDAC, and ethylene diamine tetra acetic acid (EDTA) were used at a concentration of 1 mM as protease inhibitors. Dithiothreitol (DTT) and ascorbic acid were used at a concentration of 1 mM, and Na 2 SO 3 ,Ca 2+ , and Mg 2+ were used at a concentration of 10 mM. 2.10. N-terminal amino acid sequencing of the purified keratinases After SDS–PAGE, each purified ketatinase band was transferred onto a polyvinylidene fluoride (PVDF) membrane (Pall Corporation, USA). The keratinase band on the PVDF membrane was cut out and dissolved in 20% (v/v) acetonitrile/water containing 0.001% trifluoroacetic acid (TFA). The N-terminal sequences of the purified keratinases were determined by automated Edman degradation using a pulsed-liquid sequence analyzer (ABI PROCISE TM 492cLC protein sequencing system, Applied Biosystems, USA). Fig. 1. Zymogram analysis of keratinases secreted by Streptomyces sp. strain 16. Arrows indicate the keratinase bands.
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