Biochemical characterization of an extreme alkaline and surfactant-stable keratinase derived from a newly isolated actinomycete Streptomyces aureofaciens K13

Abstract

Keratinase has attracted increasing attention in the field of biocatalysis in recent years because of its critical role in keratin resource exploitation and keratin waste degradation. However, conventional studies focused on keratinases from bacterial and fungal strains, especially those of the Bacillus genus, keratinase resources from actinomycetes are far from being fully explored. In this study, a novel keratinase-producing strain was isolated with wool as the sole carbon and nitrogen source and identified as Streptomyces aureofaciens K13. The keratinase was purified to electrophoretic homogeneity with a molecular mass of 46 kDa. The purified enzyme exhibited optimum activity at 75 °C and pH of 12.0. It remained extremely stable at alkaline pH values between 6 and 12 and at a high reaction temperature of 65 °C. The keratinase displayed significant activity toward casein, keratin, BSA and wool. It could be activated in the presence of K⁺, Cu²⁺, Mn²⁺, Ca²⁺, Li⁺, and Sr²⁺. The keratinase was completely inhibited by PMSF and moderately inhibited by EDTA, indicating that this keratinase is a metallo-serine keratinase. This enzyme could remain stable and even be promoted in the presence of surfactants, including SDS, Tween, and Triton; especially, 1% of Tween 80 and Triton X-100 could substantially enhance the activity by 46% and 38%, respectively. These results indicated certain advantages over conventional keratinases. The keratinase can completely remove blood stains when combined with detergents. The improvement effect of S. aureofaciens K13 keratinase by various surfactants and the favourable washing performance might indicate its significant application potential in the detergent industry. There are rare reports on keratinase production from S. aureofaciens.

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Introduction

A large amount of feathers and wool are produced each year in the poultry industry and wool manufacturing, which mainly form keratin wastes that are not fully reused.^1,2 These keratin wastes are important resources containing valuable proteins and amino acids. However, keratin is a stable protein because of the presence of disulphide bonds, and is classified into alpha-, beta-, and gamma-keratins based on the percentage of disulfide bonds.^2,3 Thus, the hydrolysis reaction of keratin is not easily performed due to the disulfide bonds. Conventionally, chemical methods have been employed to convert these keratin wastes into keratin hydrolysates. These hydrolysates were valuable products with broad applications, including being utilized as a nitrogen-rich source for the production of biofuels and fodder additives, and improving chrome exhaustion in tanning effluents; however, chemical processing including acidolysis, alkaline hydrolysis, and oxidation treatment, may result in the degradation of certain amino acids and environmental burden due to the use of strong acid or base and various chemicals.^4,5 Alternatively, microbial keratinases (EC 3.4.21/24/99.11), which can specifically biocatalyze keratin hydrolysis, appear to be potential candidates for keratin degradation in recent years because of their environmentally friendly and economical characteristics.^6–8

On the other hand, keratinases have drawn increasing attention in the detergent industry for their critical role in clearing proteinaceous stains. They showed advantages in detergency and substrate specificity for both soluble and insoluble proteins over common proteases.⁶ These enzymes could easily clear stains, including those caused by blood, milk, and sweat, which are the most frequent and difficult stains to remove using surfactants and other washing aids.

Keratinases are generally extracellular enzymes with molecular weights between 30 and 50 kDa, optimal activity between 40 and 70 °C, and at neutral to alkaline pH values (7.5–9.0).⁹ To date, various keratinase-producing strains have been isolated from bacteria of the genera Bacillus, Brevibacillus, Chryseobacterium, Kocuria, Microbacterium, Pseudomonas, and Serratia; actinomycetes from the genus Streptomyces; and fungi from the genera Alternaria, Aspergillus, Myceliophtora, Myrothecium, Scopulariopsis, and Trichophyton.^7,9 In the literature, keratinases from bacterial and fungal strains, especially those from the Bacillus genus, were most often reported; however keratinase from actinomycetes was far from being fully explored.

Tatineni et al. isolated an alkaline keratinase from Streptomyces sp. S7 in the slaughterhouse wastes.¹⁰ The purified Streptomyces keratinase was characterized, and showed a certain stability toward several detergents and surfactants. Another thermotolerant S. gulbargensis DAS131 keratinase was reported to display superior pH and thermostability and could effectively degrade native chicken feathers within 96 h.⁸ This DAS131 keratinase demonstrated application potential in terms of valorization of keratin-containing wastes or in the leather industry.

However, the studies on extreme alkaline and surfactant-stable keratinases from actinomycetes are rare in previous reports. Also, the activities of most available actinomycete keratinases toward keratin were still unsatisfactory in literature. Here, we isolated a potential keratinase-producing actinomycete – S. aureofaciens K13 with wool as the sole carbon and nitrogen source, since keratin was the principal component of wool.¹¹ The purified S. aureofaciens keratinase was characterized and showed special characteristics such as prominent catalytic activity as well as stability under alkaline condition and in the presence of surfactants. Furthermore, its potential in washing performance was demonstrated.

Results and discussion

Screening of wool keratin-degrading microorganisms

Screening on media with wool powder as the sole carbon and nitrogen source was a straightforward method for isolation of wool keratin-degrading microorganisms. In this study, the wool keratin-degrading microorganisms were screened from soils containing decomposed wool, which were collected by using the procedure described in the Experimental section of this paper. Several strains, including actinomycetes, bacteria, and fungi, were isolated and proven to be able to degrade keratin effectively. Strain K13 showed the highest keratinase activity and was selected for further study.

Identification of strain K13

The colonies on plate were grayish-white with dark gray spores in the center. The surface of the colonies was dry and in powder form. Strain K13 was determined to belong to actinomycetes, which was gram positive. The 16S rDNA gene sequence was determined. Based on subsequent 16S rDNA sequencing (approximately 1.5 kb) analysis, strain K13 was most closely related to S. aureofaciens, S. variabilis and S. griseoflavus compared with the sequences in the GenBank database using the BLAST program. Strain K13 displayed the highest homology of 99% with S. aureofaciens (AB326923.1). Strain K13 was also observed to be located in the same clade with S. aureofaciens (AB326923.1) from the phylogenetic tree based on 16S rDNA sequence from the 13 aligned sequences (Fig. 1). Therefore, the isolate was identified as S. aureofaciens K13 based on the morphological characteristics, 16S rDNA sequencing, and phylogenetic analysis. This strain was deposited in the China General Microbiological Culture Collection Center with accession number CGMCC 8047.

Fig. 1 Phylogenetic tree based on 16S rDNA sequence, constructed by the neighbour-joining method. Numbers in parentheses are accession numbers of published sequences in the GenBank. Bootstrap values were based on 1000 replicates. Brevibacterium antarcticum was used as the out group.

Keratinase purification

The S. aureofaciens K13 was cultured in the presence of wool powder for enzyme production and collecting the culture supernatant that harboured K13 keratinase through centrifugation and removal of cells. Keratinase purification was performed using the culture supernatant of S. aureofaciens K13. The precipitates from 40% ammonium sulfate precipitation were used for further purification. It was first applied to an anion-exchange chromatography column. The enzyme was eluted with a gradient of 0 M NaCl to 0.3 M NaCl as a single peak, coinciding with a small absorbance at 280 nm (Fig. 2). The absorption peaks displaying keratinase activity through enzyme assay were collected.

Fig. 2 Purification of the S. aureofaciens K13 keratinase by anionic exchange chromatography (Hiprep DEAE FF 16/10 column).

By using the aforementioned purification procedures, the enzyme was purified approximately 37.87 purification fold with a final total activity yield of 9.96% (see Table 1). The purified keratinase displayed a specific activity of 9734.57 U mg⁻¹. The purified enzyme showed only one protein band using SDS–PAGE. These results suggested that this enzyme was purified to homogeneity with a molecular weight of approximately 46 kDa (Fig. 3). The molecular weight of S. aureofaciens K13 keratinase was similar to that of most keratinases, which was in the range of 30 kDa to 50 kDa.

Table 1 Purification of S. aureofaciens K13 keratinase

Procedures	Total protein (mg)	Total activity (U)	Specific activity (U mg^−1)	Purification fold	Yield (%)
Culture supernatant	53.20	13675.00	257.07	1.00	100.00
Ammonium sulfate precipitation	20.13	6456.00	320.76	1.25	47.21
Hiprep DEAE FF	0.14	1362.00	9734.57	37.87	9.96

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Fig. 3 SDS–PAGE analysis of the purified keratinase. Samples were analyzed on 15% polyacrylamide gels and stained with Coomassie Brilliant Blue R-250. Lane M: standard protein marker; lane 1: keratinase-active fractions collected from Hiprep DEAE FF column.

Effects of temperature and pH on purified keratinase

S. aureofaciens K13 keratinase showed optimum temperature at 75 °C and exhibited moderate activity in the range of 60 °C to 80 °C (Fig. 4). S. aureofaciens K13 keratinase displayed several differences in optimal reaction conditions compared with other keratinases (Table 2). Its optimum reaction temperature was significantly higher than those of most keratinases from the Streptomyces genus, such as those of S. gulbargensis DAS131 (45 °C),⁸ Streptomyces strain K1-02 (40 °C to 70 °C),¹² and Streptomyces sp. (45 °C).¹⁰ The enzyme could remain stable below 65 °C but retained 37% of its activity at 70 °C after incubating for 1 h. In the literature, the keratinases from Kocuria rosea LPB-3 and S. gulbargensis DAS131 were reported to be stable between 10 and 60 °C and 30 and 45 °C, respectively.^8,13

Fig. 4 Effects of pH and temperature on keratinase activity. (A) Optimum temperature was measured under standard conditions, with the temperature ranging from 50 °C to 80 °C. Thermostability was measured under standard conditions after preincubation of the enzyme between 50 and 80 °C for 1 h. (B) The optimum pH was measured under standard conditions, with pH values varying from 6 to 12. pH stability was measured under standard conditions after preincubating the enzymes in various buffers at 40 °C for 1 h.

Table 2 Enzymatic characteristics of reported keratinase derived from Streptomyces and other genera in previous literature

Organisms	Specific activity (U mg^−1)	Molecular weight (kDa)	Optimum pH/temperature (°C)	Stability pH/temperature (°C)	Km/Vmax	Ref.
Streptomyces genus
Streptomyces aureofaciens K13	9734.57 (Keratin)	46	12/75	7–12/below 65	2.90 mg mL^−1/25.38 μg mL^−1 min (keratin)	This study
Streptomyces gulbargensis DAS131	14.3 U mL^−1 (Casein)	46	9.0/45	7–9/below 60	—	8
Streptomyces sp. S7	686.5 (Keratin azure)	44	11/45	9–12/40–50	—	10
Streptomyces albidoflavus K1-02	17600 (Keratin azure)	18	7.5/60	6–9.5/40–70	5 mM/—(Suc-(Ala)3-pNA)	12
Other genera
Aspergillus parasiticus	106232 (keratin)	36	7.0 (Phosphate buffer) or 6.0 (acetate buffer)/50	—/below 50	1.04 mg mL^−1/3463.34 U min^−1 mg (azocasein)	15
Bacillus subtilis NRC 3	5233 (keratin azure)	32	7.5/50	5–10/20–60	—	14
Bacillus cereus H10	1766.0 (azokeratin)	44.8	7.57/59	—/below 80	—	17
Kocuria rosea	22827.7 (feather keratin)	240	10/40	8–11/10–60	0.242 mM/— (feather keratin)	13
Brevibacillus brevis US575	21086 (keratin azure)	29	8/40	5–10/30–60	0.5136 mM/21150 U mg^−1 (keratin)	29

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In the literature, to date, most reported keratinases from bacteria, actinomycetes and fungi were proven active under neutral to slight alkaline conditions.^8,12,14,15 The influences of reaction pH on enzyme activity and stability were evaluated in the present study (Fig. 4). Unexpectedly, the optimum reaction pH for this keratinase was determined to be an extremely alkaline pH 12. Furthermore, the enzyme displayed high activity between pH 7 and 12. It was speculated that the K13 keratinase tends to be alkali resistant. The pH stability of S. aureofaciens K13 keratinase was subsequently tested in the current study; this keratinase showed significant stability between pH 6 and 12 (Fig. 4). More than 90% of the activity could remain even at pH 12. A keratinase from Streptomyces sp. showed maximum activity at pH 11, and over 80% of the activity remained within the pH range of 9 to 12.¹⁰ S. aureofaciens K13 keratinase exhibited high stability in a wide alkaline pH range; thus, this keratinase may be suitable for extensive industrial applications such as in detergent formulation and in the leather industry.

Effects of metal ions on keratinase activity

The effects of metal ions on keratinase activities are shown in Table 3. The purified keratinase was inhibited by the presence of several metal ions, such as Fe³⁺, Sn²⁺, La³⁺, and Zn²⁺. By contrast, Cu²⁺, Mn²⁺, Ca²⁺, and Sr²⁺ increased the keratinase activity by 10%, 22%, 5%, and 10%, respectively. Other ions, including Na⁺, Mg²⁺, Rb²⁺, and Fe²⁺ caused no clear effect on the keratinase. However, the addition of Cu²⁺, which led to the inhibition of keratinases of B. subtilis NRC3,¹⁴ Bacillus sp. P7,¹⁶ and Streptomyces sp. S7,¹⁰ resulted in a slight improvement of S. aureofaciens K13 keratinase activity by 10% in this study.

Table 3 Effects of metal ions on keratinase activity^a

Chemicals	Concentration (mM)	Relative activity (%)	Chemicals	Concentration (mM)	Relative activity (%)
a NA means no activity.
Control	0	100 ± 0.57	Sr^2+	5	109.54 ± 2.61
K^+	5	103.62 ± 0.99	Sn^2+	5	NA
Na^+	5	98.68 ± 0.99	La^3+	5	45.39 ± 0.99
Cu^2+	5	109.54 ± 0.99	Rb^+	5	100.00 ± 0.57
Mn^2+	5	122.04 ± 0.57	Zn^2+	5	47.70 ± 2.05
Mg^2+	5	95.72 ± 0.99	Fe^2+	5	95.40 ± 0.57
Ba^2+	5	90.79 ± 1.97	Ni^2+	5	86.84 ± 0.99
Ca^2+	5	104.61 ± 1.97	Al^3+	5	70.07 ± 0.99
Co^2+	5	85.53 ± 0.57	Pb^2+	5	66.45 ± 2.05
Li^+	5	104.61 ± 0.99	Ag^+	5	73.03 ± 0.99
Fe^3+	5	38.82 ± 0.57

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Substrate specificity

The substrate specificity of purified keratinase was investigated (Fig. 5). The results indicate that this enzyme could hydrolyze various protein substrates, including soluble and insoluble proteins. This keratinase displayed high activity toward casein and keratin, and a wide hydrolysis ability of BSA, wool, and gelatin was observed; however, this keratinase showed only relatively low activity toward human hair and collagen. This result is similar to that of B. subtilis keratinase, which showed high specificity to casein but low activity to collagen.¹⁴ Furthermore, Chen et al. determined that B. cereus H10 keratinase displayed high activity to casein but low activity to porcine hair.¹⁷ However, K. rosea keratinase displayed high activity toward keratin of feathers and epidermis, which was approximately 1.6-fold higher than the activity toward casein and collagen.¹³ In this study, the keratinase exhibited low activity toward collagen, which suggested that S. aureofaciens K13 enzyme can be utilized in the leather and textile industries. Keratinase can also be used for collagen extraction, which was used for the degradation of heteropolymeric protein; thus, the structural integrity of collagen is observed.

Fig. 5 Substrate specificity of purified keratinase. To study the substrate specificity of keratinase, 2% substrates and enzyme were incubated at 75 °C for 1 h. The reaction was terminated with the addition of 0.4 M of TCA.

Enzymatic kinetics

The kinetic parameters K_m and V_max of the purified S. aureofaciens K13 keratinase were determined with keratin as substrate according to the Michaelis–Menten equation. K_m and V_max were calculated to be 2.90 mg mL⁻¹ and 25.38 μg mL⁻¹ min, respectively. Whereas the kinetic parameters of Aspergillus parasiticus keratinase were determined to be 1.04 mg mL⁻¹ and 3463.34 U min⁻¹ mg⁻¹ with azocasein as the substrate.¹⁵

Effects of enzyme inhibitors and surfactants on keratinase activity

The effects of enzyme inhibitors and surfactants on keratinase activity are shown in Table 4. S. aureofaciens K13 keratinase was completely inhibited by 5 mM of PMSF, characteristic of serine proteases. By contrast, a severe inhibition effect of 5 mM of EDTA on enzyme activity, combined with the activation effect of 5 mM of Ca²⁺ on the keratinase activity as stated above, may denote that this keratinase belongs to a metalloprotease. Similar results were reported for metallo-serine keratinases from Streptomyces species,¹⁰ Bacillus sp. P7,¹⁶ and B. cereus H10.¹⁷ Furthermore, the reducing agent DTT could completely inhibit the enzyme activity, whereas another reducing agent β-mercaptoethanol could retain 79% of the enzyme activity. The inhibition effects of DTT and β-mercaptoethanol on S. aureofaciens keratinase were different from those of most reported keratinases, such as those from B. pumilus KS12,¹⁸ Streptomyces sp. S7,¹⁰ and Aspergillus parasiticus,¹⁵ which could be substantially improved in the presence of DTT and β-mercaptoethanol. For example, the A. parasiticus keratinase activity was increased by approximately 0.5- and 2-fold with the addition of 10 mM of DTT and β-mercaptoethanol, respectively.

Table 4 Effects of inhibitors and surfactants on keratinase activity^a

Chemicals	Concentration (mM or %)	Relative activity (%)	Chemicals	Concentration (mM or %)	Relative activity (%)
a NA means no activity.
Control	0	100 ± 0.57	Tween 20	1%	120.07 ± 3.74
PMSF	5	NA	Tween 40	1%	128.62 ± 7.27
EDTA	5	19.74 ± 0.99	Tween 80	1%	146.71 ± 1.51
DTT	5	NA	Triton X-100	1%	138.16 ± 0.99
β-Mercaptoethanol	1%	78.95 ± 3.42	Triton X-114	1%	99.67 ± 0.99
SDS	1%	107.57 ± 0.99

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Surfactants are among the major components in commercially available detergents; therefore, the keratinase activity in the presence of surfactants was determined in this study. Table 4 shows that 1% SDS could improve the keratinase activity by approximately 8%. In most literature, SDS generally resulted in the inhibition of keratinases, which may be caused by the denaturation effect of SDS on keratinase protein. The activities of Keratinibaculum paraultunense gen. nov., sp. nov.,¹⁹ Chryseobacterium L99 sp. nov.,²⁰ A. parasiticus,¹⁵ and Streptomyces sp. S7 (ref. 10) were reduced by 47% (11.6 mM of SDS), 54% (0.2%), 50% (0.5%), and 62% (0.1%), respectively. Moreover, Tween and Triton increased the enzyme activity to varying degrees; especially Tween 80 and Triton X-100 (1%), which could substantially enhance the activity by 47% and 38%, respectively. Recently, two keratinases from Stenotrophomonas maltophilia BBE11-1 were reported to be improved by 3% and 2% of activity, respectively, in the presence of 0.5% Triton X-100.²¹ S. albidoflavus keratinase activity was increased by 37% and 4% using 0.2% and 0.5% of Triton X-100, respectively.¹² A. parasiticus¹⁵ and K. rosea¹³ keratinase could also remain stable in the presence of detergents, such as Triton X-100 or Tween-80. However, Streptomyces sp. keratinase activity was decreased by 6% and 14% with 0.1% and 0.5% Triton X-100, respectively.¹⁰ The stability of S. aureofaciens K13 keratinase in the presence of surfactants is an important characteristic of this biocatalyst, which may lay the foundation for its future application in detergent formulations.

Washing performance

As mentioned above, the S. aureofaciens keratinase displayed moderate thermal and alkaline pH stability, as well as prominent tolerance towards several surfactants, which, together with the enzymes, were the major components of commercial detergents. As it is expected that the keratinase would improve the washing performance of detergents, the washing performance of S. aureofaciens K13 keratinase was tested in this study. As shown in Fig. 6, the removal of blood stain from cotton cloth was obviously improved with the addition of S. aureofaciens K13 keratinase (1000 units) in the washing system, compared with the addition of commercial detergent (WhiteCat, Shanghai, China). Especially, the washing effect could be further enhanced, when the S. aureofaciens K13 keratinase was combined with 1% commercial detergent. B. laterosporus-AK1 (ref. 22) and S. fungicidicus MML1614 (ref. 23) proteases could also enhance the washing performances and removal of blood stains to a great degree at 3000 units and 3200 units of protease activity (with specific activity of 796.0 and 315.66 U mg⁻¹ using casein as the substrate), respectively.

Fig. 6 Washing performance of the keratinase. Washing effect of S. aureofaciens K13 keratinase was studied on white cotton cloth pieces pre-stained with blood. They were kept immersed in (A) stained cloth without treatment; (B) stained cloth + 100 mL distilled water; (C) stained cloth + 100 mL distilled water + 1 mL of detergent solution at 10 mg mL⁻¹; (D) stained cloth + 100 mL distilled water + 2 mL enzyme solution; (E) stained cloth + 100 mL distilled water + 1 mL of detergent solution at 10 mg mL⁻¹ + 2 mL enzyme solution.

Conclusion

S. aureofaciens K13 was isolated with wool as the sole carbon and nitrogen source and showed excellent stability at alkaline pH values between 6 and 12. This enzyme could also remain stable in the presence of several surfactants. To date, limited information reported about keratinases, which can remain stable in extreme alkaline pH and in the presence of SDS, has been available. This study suggests the actinomycetic keratinase has a potential of scientific and commercial applications in detergents industry due to its moderate operational stability and superior washing performance.

Experimental

Materials

Wool powder was obtained from a livestock farm (Chifeng City, Neimenggu Province, China) and prepared through shattering in our lab. The keratin for enzyme assay was purchased from J&K Chemical Ltd. (Shanghai, China). The PCR reagents were purchased from Takara (Japan). All other chemicals were of analytical grade and obtained from commercial sources.

Strain screening

Six soil samples for screening of keratinase-producing strains were collected from a livestock farm (Wuxi City, China). These samples contained decomposed wool that lost from sheep and stored for long-term in the ground. About 1 g of soil sample was serially diluted by 0.9% saline solution, plated on wool powder agar medium (10 g L⁻¹ wool powder, 0.5 g L⁻¹ NaCl, 0.4 g L⁻¹ K₂HPO₄, and 20 g L⁻¹ agar), and incubated at 30 °C for 72 h. Single colonies were subcultured twice on wool powder agar plates to obtain microbial monoculture. The purified isolates obtained in this step were subcultured in liquid media (10 g L⁻¹ wool powder, 0.5 g L⁻¹ NaCl, and 0.4 g L⁻¹ K₂HPO₄) at 30 °C on a rotary shaker. After incubation for 2 d, the culture broth was centrifuged to remove biomass. The supernatants were collected to determine the keratinase activity. Strains displaying high keratinase activity were selected for further study.

Identification

The target strain was identified through 16S rDNA sequencing after PCR amplification with primers designed based on the highly homologous region of 16S rDNA. The 16S rDNA gene of the isolate was amplified using the upstream primer P₀: 5′-GAGAGTTTGATCCTGGCTCAG-3′ and the downstream primer P₆: 5′-CTACGGCTACCTTGTTACGA-3′. PCR conditions were as follows: 1 cycle of 95 °C for 4 min; 30 cycles of 95 °C for 45 s, 56 °C for 45 s, and 72 °C for 1.5 min; and a final cycle of 72 °C for 10 min. The 16S rDNA sequence obtained was compared with sequences in the GenBank database using the BLAST program.

Keratinase-producing strain cultivation

Single colonies of S. aureofaciens K13 in the plates were picked and inoculated into the LB medium (10 mL medium in a 50 mL flask) for 48 h in a shaker of 220 rpm at 30 °C. The culture liquid was transferred into fresh fermentation medium. This cultivation was performed in the 250 mL shake flask containing 50 mL medium in a shaker of 220 rpm. The conditions for keratinase production contained the following parameters: 30 °C of cultivation temperatures; 10% of inoculum size; and the medium consisting of 10 g L⁻¹ wool powder, 0.5 g L⁻¹ NaCl, and 0.4 g L⁻¹ K₂HPO₄, pH7.0.

After incubation for 48 h, the culture broth was centrifuged at 4 °C and 12 000 × g for 20 min. The culture supernatant was used for further experiment.

Keratinase purification

All the purification procedures were carried out at 4 °C. The culture broth harbouring S. aureofaciens keratinase were harvested by centrifugation and the cells were removed. Ammonium sulfate after grinding was gradually added into the supernatants with a final concentration of 40%. The mixture was kept in a 4 °C refrigerator for over 12 h. The enzyme precipitates were collected by centrifugation and dissolved in a minimal amount of 10 mM of Tris–HCl buffer (pH 8.0, buffer A), and dialyzed over 12 h against the same buffer at 4 °C to remove the ammonium sulfate salts. Following centrifugation of the culture, the supernatant was filtered through a 0.45 μm-size membrane. Keratinase purification was performed using a AKTA purifier (GE Healthcare, USA).

The proper dialyzed solution was applied to anion-exchange chromatography with a Hiprep DEAE FF 16/10 column (GE Healthcare, USA), equilibrated with the Tris–HCl buffer. And this procedure was followed by step elution with 1 M NaCl in the same buffer. Subsequently, the column was washed using the same buffer and eluted with a gradient of 0 M of NaCl to 0.5 M NaCl in 10 mM of Tris–HCl buffer (pH 8.0). 5 mL fractions were collected at a flow rate of 2 mL min⁻¹. Fractions were collected and tested for keratinase activity and protein concentration. The protein compositions were estimated by SDS–PAGE. Active fractions that eluted with the NaCl gradient were pooled, dialyzed, and combined for subsequent characterization.

Protein determination

Protein concentrations were determined by the Bradford method with bovine serum albumin as the standard.²⁴ Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed based on the method of Laemmli,²⁵ using 15% (w/v) polyacrylamide resolving gels and 5% (w/v) polyacrylamide stacking gels, and traced against standard molecular weight markers (14.4 kDa to 116 kDa). The protein bands were stained with a solution of Coomassie Brilliant Blue R-250.

Keratinase assay

Keratinase activity was determined by the modified method of Yamamura et al., which employed the Folin Ciocalteau's reagent for enzyme assay.²⁶ 1 mL of diluted enzyme solution was incubated with 1 mL of 1% keratin in 50 mM of Tris–HCl buffer (pH 8.0) at 55 °C for 15 min. The reaction was terminated with 2 mL of 0.4 M trichloroacetic acid (TCA), and then allowed to stand for 5 min. After centrifugation at 12 000 × g for 10 min, the supernatant (1 mL) of the reaction mixture was mixed with 4 mL of 0.4 M Na₂CO₃ and 1 mL of Folin Ciocalteau's reagent, and incubated at 40 °C for 20 min. The absorbance was measured at 680 nm using a spectrophotometer (Phenix UV-1700PC, Shanghai, China). One unit of keratinase activity (U) is defined as an increased absorbance of 0.01 at 680 nm in 15 min.^15,27

Effects of temperature and pH on enzyme activity and stability

The optimum reaction temperature for keratinase was investigated by incubating the purified enzyme with 1% keratin at temperatures ranging from 50 °C to 80 °C under the standard assay conditions. Thermal stability was determined by pre-incubating the enzyme without substrate in Tris–HCl buffer (pH 8.0) for 1 h at temperatures from 50 °C to 80 °C. The residual activity was measured under standard conditions.

The optimum pH of keratinase was measured in the pH range of 6.0–12.0, using different buffers with a concentration of 50 mM (pH 6.0 sodium phosphate buffer, 7.0–9.0 Tris–HCl, 10.0 glycine–NaOH, and 11.0–12.0 KCl–NaOH)^13,28 under the standard assay conditions. Here, Tris–HCl softly improved the enzyme activity. pH stability was determined by pre-incubating the enzyme at various pH conditions without substrates in the aforementioned buffer systems at 40 °C for 1 h and then subjected to enzyme assay under standard conditions. All assays were performed in triplicate.

Effects of various chemicals on keratinase activity

The effects of various metal ions on enzyme activity were investigated with a final concentration of 5 mM in the reaction mixture. Keratinase activity was determined under the standard assay conditions. Relative activity was expressed at the percentage of the activity without addition of metal ions. The effects of enzyme inhibitors were studied using 1% (final concentration) of phenylmethyl sulfonylfluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), and β-mercaptoethanol. The effects of surfactants on activity were studied using 1% (final concentration) of sodium dodecyl sulfate (SDS), Tween 20, Tween 40, Tween 80, Triton X-100 and Triton X-114. Moreover, the effects of surfactants and inhibitors on ultraviolet absorption values were excluded.

Substrate specificity

Substrate specificity of S. aureofaciens K13 keratinase was investigated by comparing the proteolysis of both soluble (including BSA, type I collagen, gelatin, casein, and keratin) and insoluble protein substrates (including feather powder, wool powder, and human hair powder). 0.5 mL enzyme solution was incubated in 0.5 mL Tris–HCl buffer (pH 8.0) containing different substrates (2%) at 75 °C for up to 1 h. The reaction mixture was terminated with 1 mL of 0.4 M trichloroacetic acid (TCA), centrifuged at 12 000 × g for 10 min. The supernatant (1 mL) of the reaction mixture was mixed with 4 mL of 0.4 M Na₂CO₃ and 1 mL of Bradford reagent, incubated at 40 °C for 20 min. The keratinase activity was measured at 680 nm with a spectrophotometer. The activity measured with the substrate of casein was set at 100%.

Determination of kinetic parameters

The kinetic parameters K_m and V_max of the purified enzyme were calculated based on the Michaelis–Menten equation generated from the activity with increasing substrate concentrations (from 0.125 mg mL⁻¹ keratin to 10 mg mL⁻¹ keratin).

Washing performance of the keratinase

Washing effect of S. aureofaciens K13 keratinase (500 U mL⁻¹) was studied on white cotton cloth pieces (6 × 6 cm) pre-stained with 100 μL blood at 60 °C for 30 min. The specific activity of keratinase was 9734.57 U mg⁻¹ with keratin as the substrate. The cloth pieces with blood stains were kept immersed in (A) stained cloth without treatment; (B) stained cloth + 100 mL distilled water; (C) stained cloth + 100 mL distilled water + 1 mL of detergent solution at 10 mg mL⁻¹; (D) stained cloth + 100 mL distilled water + 2 mL enzyme solution; (E) stained cloth + 100 mL distilled water + 1 mL of detergent solution at 10 mg mL⁻¹ + 2 mL enzyme solution. Then the samples were incubated at 60 °C for 30 min and rinsed with distilled water and dried. Visual examination of various pieces was made on the effect of enzyme in stain removal.

Acknowledgements

This work was financially supported by the National High Technology Research and Development Program of China (no. 2012AA022204C) and the Natural Science Foundation of Jiangsu Province (no. BK20140133).

Notes and references

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Footnote

† These authors contributed equally to this work.

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