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Production, characterization and application of keratinase from Streptomyces gulbargensis
Bioresource Technology 100 (2009) 1868–1871
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Production, characterization and application of keratinase from Streptomyces gulbargensis Dastager G. Syed a,*, Jae Chan Lee b, Wen-Jun Li c, Chang-Jin Kim b, Dayanand Agasar d a
National Institute of Interdisciplinary Science and Technology (CSIR), Thiruvananthapuram 695019, India Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea c Yunnan Institute of Microbiology, Yunnan University, Kunming, China d Department of Studies and Research in Microbiology, Gulbarga University, Gulbarga 585 106, Karnataka, India b
a r t i c l e
i n f o
Article history: Received 19 August 2008 Received in revised form 23 September 2008 Accepted 23 September 2008 Available online 5 November 2008 Keywords: Keratinase activity Actinomycetes Streptomyces gulbargensis
a b s t r a c t A Streptomyces gulbargensis newly isolated, thermotolerant feather-degrading bacterial strain was investigated for its ability to produce keratinase enzyme. Maximum keratinolytic activity was observed at 45 °C and pH 9.0 at 120 h of incubation. Activity was completely stable (100%) between 30 and 45 °C and pH 7.0–9.0, respectively. Addition of starch to the growth medium affects the activity by means of increase in keratinase secretion. After seven days of cultivation, 10-fold increase (14.3 U ml 1) in keratinase activity was observed in the presence of 3 g starch (per liter) of the medium. The enzyme was monomeric and had a molecular mass of 46 kDa. The enzyme activity was significantly inhibited by CaCl2 and partly inhibited by EDTA, whereas, Na2SO3 enhance the enzyme activity by 2.9 times more. In addition, native chicken feather was completely degraded at 96 h of incubation. The results obtained showed that newly isolated strain S. gulbargensis could be a useful in biotechnology in terms of valorization of keratincontaining wastes or in the leather industry. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Feathers are produced in large amounts as a waste by-product of poultry processing plant (Sangali and Brandelli, 2000). A current value-added use for feathers is the conversion to feather meal, a digestible dietary protein for animal feed, using physical and chemical treatments. These methods can destroy certain amino acids and decrease protein quality and digestibility (Moritz and Latshaw, 2001; Anbu et al., 2005). Keratinolytic microorganisms and their enzymes may be used to enhance the digestibility of feather keratin. They may have important applications in processing keratin-containing wastes from poultry and leather industries through the development of non-polluting methods (Onifade et al., 1998). Keratin-containing materials (feather, hair, wool, etc.) are abundant in nature but have limited uses in practice since they are insoluble and resistant to degradation by the common proteolytic enzymes. Keratinous wastes represent a source of valuable proteins and amino acids and could find application as a fodder additive for animals or source of nitrogen for plants. Biodegradation by microorganisms possessing keratinolytic activity represents an alternative attractive method for improving the nutritional value of keratin wastes, as it offers cheap and mild reac-
* Corresponding author. Tel.: +91 471 2515276. E-mail address: [email protected] (D.G. Syed). 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.09.047
tion conditions for the production of valuable products (Kim et al., 2001). A number of keratinolytic microorganisms have been reported, including some species of Bacillus (Suh and Lee, 2001; Joo et al., 2002; Amare et al., 2003), actinomycetes (Bressollier et al., 1999) and fungi (El-Naghy et al., 1998; Gradisar et al., 2000). In the present work, we report new keratin-degrading Streptomyces strain and its ability to degrade chicken feather. 2. Methods 2.1. Feathers Feathers were obtained at a local poultry processing plant (Gulbarga, India). Raw feathers were washed with warm tap water and distilled water, and dried at 45 °C for 48 h in a circulating airdrying stove. After drying, feathers were hammer milled and autoclaved for prior to microbial treatment. 2.2. Maintenance and inoculum preparation Pure isolates were inoculated on nutrient agar slopes and incubated at 28 °C for 24 h. To each slant 10 ml sterile distilled water were added and 1 ml of bacterial suspension produced with a sterile needle used to inoculate the cultivation medium (1 ml/50 ml medium).
D.G. Syed et al. / Bioresource Technology 100 (2009) 1868–1871
2.3. Organism and growth conditions Streptomyces gulbargensis DAS 131 which was isolated and identified by (Dastager et al., 2007) using polyphasic approach was used as an enzyme source. Keratinase production was carried out in the following basal medium (NH4Cl-0.5%, CaCl2-0.2%, MgSO4 7H2O-0.2%, K2HPO4-0.4%, pH 7.5) feather waste 0.1% and increased quantity of starch (0–3.5%) as a sole source of nitrogen and carbon. For growth of the organism, 1 l Erlenmeyer flask containing 250 ml of growth medium was kept at 30 °C in an orbital shaker set at 250 rev min 1 for seven days. Bacteria were removed by filtration and the filtrate was used for crude enzyme preparation (Bressollier et al., 1999). 2.4. Enzyme assay Keratinolytic activity was measured using a modified protocol of Anson (1938) with keratin azure as a substrate. The reaction mixture contains 0.2 ml of culture supernatant and 0.8 ml of 0.4% (w/v) keratin azure K-8500 (Sigma) in 10 mM Tris–HCl buffer (pH 8.5), and it was incubated at 45 °C for 1 h. The reaction was stopped with 500 ll, 0.1 M tri-chloroacetic acid (TCA) in 0.1 M Tris–HCl (pH 8.0) and then centrifuged at 15,000g for 15 min and supernatant was measured at 595 nm for amino acid liberation. The quantity was determined from standard tyrosine solution (50–500 lg ml 1) using UV-1601, Shimadzu spectrophotometer (Letourneau et al., 1998). One unit of keratinolytic activity was defined as the amount of enzyme required to liberate 1 lmol tyrosine under specific conditions. 2.5. Determination of biomass Gravimetric determination of the bacterial cell dry weight was determined by collecting cells from 50 ml cultures by centrifugation at 5000 rpm for 15 min. Cells were then washed with saline solution and dried at 60 °C to constant weight. 2.6. Protein determination The reaction mixture of 2 ml was composed of 1 ml of 1% casein in 0.2 M Tris–HCl, pH 8.0 and 1 ml of appropriately diluted enzyme. The reaction mixture was incubated for 30 min in a water bath at 40 °C. Two milliliter of 20% TCA was then added and the mixture centrifuged at 5000 rpm for 10 min. A control was processed by adding the enzyme after incubation and TCA was immediately added. The protein content of the enzyme preparations was determined in by the method of Lowry et al. (1951). 2.7. Feather-degrading activity (F-D activity) Feather-degrading activity was estimated from 24 to 96 h of incubation. One unit of FD activity was defined as the amount of enzyme that liberates one gram of amino acid equivalent to tyrosine per minute under the standard assay conditions. 2.8. Assay of protease activity Protease activity was determined using casein as a substrate according to the method reported by Gessesse et al. (2003). Unless otherwise stated, the reaction mixture in a total volume of 2 ml was composed of 1 ml of 1% casein in 0.2 M Tris–HCl, pH 8.0 and 1 ml of appropriately diluted enzyme. The reaction mixture was incubated for 30 min in a water bath at 40 °C. Two milliliter of 20% TCA was then added and the mixture centrifuged at 5000 rpm for 10 min. A control was processed by adding the enzyme after incubation and TCA was immediately added. One unit
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of enzyme activity (U) was defined as the amount of enzyme that liberates 1 mg of amino acid equivalent to tyrosine per minute under the above mentioned conditions. 2.9. Effect of pH and temperature Studies on the effect of pH and temperature on enzyme activity were performed as the standard assay using 50 mM Na2HPO4/citric acid (pH 4.0–7.5), 50 mM Tris/HCl (pH 7.5–8.5), 50 mM NaHCO3/ NaOH (pH 8.5–12.0) and a temperature range of 30–60 °C. The experiments on the effect of pH and temperature on enzyme stability were carried out by incubating the enzyme solution at pH and temperature ranges of 4–12 °C and 30–60 °C, respectively, for 30 min. The enzyme activities were then determined by the standard enzyme assay. 2.10. Effect of carbon and nitrogen sources on enzyme activity The effect of different carbon and nitrogen compounds on protease formation by S. gulbargensis was investigated in the cultivation media having carbon and nitrogen source content of 1.0% (w/v). 2.11. Effect of chemicals on enzyme activity The crude enzyme preparation was incubated in Tris (pH 9.0) containing different chemicals (EDTA, ZnCl2, MgCl2, CaCl2, Na2SO3), with and without feather. Enzyme activity was determined and compared with that of the control containing only feather at 30 °C for 30 min. 2.12. Substrate hydrolysis Soluble protein substrates (0.5%, gelatin, casein, bovine serum albumin, soluble keratin and elastin) and insoluble protein substrates (0.5%, human hair, human nail and chicken feather) were hydrolyzed by an enzyme solution, and amino acids produced by the reaction were analyzed using a free amino acid assay. One unit of protease activity was defined as the amount of enzyme that released 1 lmol of leucine after reaction with the substrate for 1 h at pH 9.0 and a temperature of 45 °C. 2.13. Ammonium sulfate precipitation The crude enzyme was first saturated up to 30% with solid (NH4)2SO4 and then centrifuged at 5000g for 15 min. The supernatant obtained was further saturated up to 70% with solid (NH4)2SO4 and again centrifuged. The pellets obtained were dissolved in minimum volume of 0.1 M Tris–HCl buffer, pH 8.0. 2.14. Gel-electrophoresis For determination of homogeneity and molecular weight, enzyme preparation and known molecular weights were subjected to electrophoresis by the method of Lundy et al. (1995) with the use of 12.5% agarose gel. After electrophoresis, gel was stained for 1 h with Coomassie Blue R-250 dye in methanol:acetic acid:water solution (4:1:5 w/v) and distained in the same solution without dye. 2.15. Statistic analysis Data were analyzed as a completely randomized design. Treatment means were separated using a significant f-test (Steel and Torrie, 1960).
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D.G. Syed et al. / Bioresource Technology 100 (2009) 1868–1871
3. Results 3.1. Bacterial selection and identification A total of 108 keratinase-producing bacteria were screened for their ability to grow on feather and also for their feather degrading and protease activities. The local isolate DAS 131 was the most potent keratinase-producing organism since it could degrade chicken feathers completely within 96 h. The promising activities of this organism justified its selection for identification and characterization of its enzyme activities. It was found that the strain DAS 131 was the only thermotolerant feather-degrading strain. The isolate was confirmed as belonging to the genus Streptomyces as they possessed non-fragmented substrate mycelia, aerial hyphae and smooth spores arranged in chains. On the basis of polyphasic taxonomy it is proposed as a distinct genomic species (Dastager et al., 2007).
Fig. 1. Effect of addition of different concentrations of starch on keratinase activity by Streptomyces gulbargensis DAS 131. Each value is an average of three parallel replicates.
3.2. Keratinase production S. gulbargensis DAS 131 was able to grow and produced keratinase in medium in which feather meal served as a sole carbon and nitrogen source and acted as an enzyme inducer. Keratinase production was associated with growth at the maximum level of 1.5 U ml 1 (±0.05) in five days of cultivation. After five days of incubation, a loss of enzyme activity was observed probably because of enzymatic autolysis and end product inhibition. 3.3. Keratinase properties The enzyme had an optimum activity about 1.4 (±0.03) U ml 1 at 45 °C and was rapidly inactivated at higher temperatures. Enzyme was stable at 60 °C and was no longer active above 60 °C. It was observed that a free form of purified enzyme was less stable than an enzyme-substrate mixture under high temperature incubation. The keratinase was active in neutral and alkaline conditions with an optimum activity at pH 9.0 (1.39 U ml 1), depending on the buffer used (data not shown). It was stable over a wide range of pH values, with the highest stability at pH 7–9, and the activity was improved in basic conditions. The keratinase was significantly inhibited by CaCl2 and partly inhibited by EDTA whereas, Na2SO3 enhance the enzyme activity by 2.9 times more (Table 1). The enzyme degraded keratins from different sources. However, it hydrolyzed keratins less than soluble proteins such as casein, bovine serum albumin and gelatin. 3.4. Effect of carbon and nitrogen sources on enzyme activity The effect of different carbon and nitrogen compounds on protease formation by S. gulbargensis was investigated in the cultivation media with a carbon and nitrogen source content of 1% (w/v). Among all the carbon sources, the highest enzyme activity was achieved with starch and the least activity with sucrose (data not shown). Testing of the nitrogen source showed the highest enzyme activity with protease peptone and the lowest with yeast extract. Table 1 Effect of different chemicals on keratinolytic activity of Streptomyces gulbargensis Chemicals (0.1%) EDTA (5 mmol ZnCl2 MgCl2 CaCl2 Na2SO3
1
)
Residual activity (%)
Keratinase activity (U ml
68 44 48 00 293
0.88 ± 0.2 0.63 ± 0.1 0.67 ± 0.2 0.00 ± 0.0 4.16 ± 0.1
1
)
Fig. 2. SDS-PAGE of crude keratinase obtained from cell free broth of feather utilizing Streptomyces gulbargensis. Lane M: Molecular weight marker (19–105 kDa). Lane 1: Protein from cell free broth.
3.5. Stimulation of enzyme activity by starch The keratinase activity was enhanced in presence of starch as carbon source. It was added in increasing amount from 0% to 3.5%, to stimulate the keratinase activity. After seven days of incubation in submerged broth there is remarkable increase in the enzyme activity was recorded. It was observed that, 10-(14.3 U ml 1, ±0.02) fold higher activities in presence of 3% starch were observed (Fig. 1). Since starch is a main component in the media for isolation of actinomycetes, it was added in order to stimulate the keratinase secretion. 3.6. Gel-electrophoresis SDS–PAGE analysis revealed that the crude enzyme preparations were quite homogeneous and there was only a single protein band which had proteolytic activity. The molecular weight of band was found to be about 46 kDa (Fig. 2). The usefulness of this preparation in its crude form for industrial applications could be exploited. 4. Discussion Many Bacillus species have been reported to produce keratinolytic protease (Lal et al., 1999; Wang and Shih, 1999). Strain DAS
D.G. Syed et al. / Bioresource Technology 100 (2009) 1868–1871
131 isolated from Gulbarga soil grew and degrade feather at elevated temperatures. The keratinase production from S. gulbargensis DAS 131 reached a maximum in the late logarithmic growth phase similar to those reported by Kim et al. (2001). The molecular weight of keratinase from S. gulbargensis DAS 131 (46 kDa) was similar to that of B. licheniformis PWD-1, S. pactum DSM 40530 and Doratomyces microsporus (30–33 kDa) (Bressollier et al., 1999; Chitte et al., 1999; Gradisar et al., 2000). The optimum temperature for keratinase from S. gulbargensis DAS 131 (45 °C) was slightly lower than that of other keratinolytic proteases (50–55 °C) (Brouta et al., 2001). The enzyme was stable at 60 °C temperatures partly because its catalytic site was well protected by a substrate. Additionally, its optimum pH was similar to that of previous reports (Rozs et al., 2001). Stimulation of keratinase activity in S. gulbargensis DAS 131 was in presence of increasing amounts of starch. Since starch is a main component in the media for isolation of actinomycetes. After seven days of cultivation, an increase of 10-fold higher activity in presence of 3 g starch/l was observed. The purified keratinase from S. gulbargensis DAS 131 easily digested soluble proteins and a few insoluble proteins such as human hair, nail and chicken feather in accordance with other observations (Gradisar et al., 2000). The enzyme showed complete digestion on chicken feather since this substrates contained disulphide linkages which are a crucial structural feature of their molecules. Additionally, high protein content and amino acids were also accumulated in fermented broth as results of feather degraded by bacterial activities. In conclusion, an optimal substrate concentration and optimal cultivation conditions (including pH of the medium, temperature and rotation speed) improve feather degradation using S. gulbargensis DAS 131. In this study, the optimum conditions for keratinase synthesis by the S. gulbargensis DAS 131 were determined, when compared to enzymes in Bacillus (Johnvesly and Naik, 2001), Streptomyces have less thermostability, but keratinase from S. gulbargensis DAS 131 stable up to 60 °C this report was quite similar with Streptomyces S.K1-02 reported by Letourneau et al., (1998), were in maximum keratinolytic activity of DAS 131 was observed at pH 9.0, this result was in good agreement with other known enzymes of keratinolytic Streptomyces such as Streptomyces sp. S.K1-02 (Letourneau et al.,1998), S. albidoflavus (Bressollier et al., 1999), S. thermonitrificans (Mohamedin, 1999) and Streptomyces strain BA7 (Korkmaz et al., 2003). In conclusion results obtained from the present study reveals that, the bacterium and its enzyme could be used to improve nutritional values of animal feeds containing feather or keratin, or poultry processing wastes in a tropical country. The usefulness of crude form can be exploited in future for processing of keratin-containing wastes as well as in leather industry under suitable conditions. Acknowledgement The authors would like to thank CSIR Task force network programme on exploration of India’s Rich Microbial Diversity (NWP 0006) for providing the financial support.
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References Amare, G., Rajni, H.K., Berhanu, A., Gashe, B.M., 2003. Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather. Enzyme Microb. Technol. 32, 519–524. Anbu, P., Gopinatha, S.C.B., Hildaa, A., Lakshmi priyab, T., Annaduraic, G., 2005. Purification of keratinase from poultry farm isolate – Scopulariopsis brevicaulis and statistical optimization of enzyme activity. Enzyme Microb. Technol. 36, 639–647. Anson, M.L., 1938. Plant proteolytic enzymes. J. Gen. Physiol. 22, 79–84. Bressollier, P., Letourneau, F., Urdaci, M., Verneuil, B., 1999. Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus. Appl. Environ. Microbiol. 65 (6), 2570–2576. Brouta, F., Descamps, F., Fett, T., Losson, B., Gerday, Ch., Mignon, B., 2001. Purification and characterization a 43.5 kDa keratinolytic metalloprotease from Microsporum canis. Med. Mycol. 39 (3), 269–275. Chitte, R.R., Nalawade, V.K., Dey, S., 1999. Keratinolytic activity from the broth of a feather-degrading thermophilic Streptomyces thermoviolaceus strain SD8. Lett. Appl. Microbiol. 28, 131–136. Dastager, S.G., Wen-Jun, Li, Dayanand, A., Sulochana, M.B., Shu-Kun, Tang, Xin-Peng, Tian, Xiao-Yang, Zhi, 2007. Streptomyces gulbargensis sp. nov., isolated from soil in Karnataka, India. Antonie Van Leeuwenhoek 9 (2), 99–104. El-Naghy, M.A., El-Ktatny, M.S., Fadl-Allah, E.M., Nazeer, W.W., 1998. Degradation of chicken feathers by Chrysosporium georgiae. Mycopathology 143, 77–84. Gessesse, A., Hatti-Kaul, R., Gashe, B.A., Mattiasson, B., 2003. Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather. Enzyme Microb. Technol. 32, 519–524. Gradisar, H., Kern, S., Friedrich, J., 2000. Keratinase of Doratomyces microsporus. Appl. Microbiol. Biotechnol. 53, 196–200. Johnvesly, B., Naik, G.R., 2001. Studies on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp. JB-99 in a chemically defined medium. Process Biochem. 37, 139–144. Joo, H.S., Kumar, C.G., Park, G.C., Kim, K.T., Paik, S.R., Chang, C.S., 2002. Optimization of the production of an extracellular alkaline protease from Bacillus hirikoshii. Process Biochem. 38, 155–159. Kim, J.M., Lim, W.J., Suh, H.J., 2001. Feather-degrading Bacillus species from poultry waste. Process Biochem. 37, 287–291. Korkmaz, H., Aldi, M.N., Aslan, B., Coral, G., Arikan, B., Din, S., Lak, R., 2003. Keratinolytic activity of Streptomyces strain BA7, a new isolate from Turkey. Ann. Microbiol. 53, 85–93. Lal, S., Rajak, R., Hasijia, C., 1999. In vitro degradation of keratin by two species of Bacillus. J. Gen. Appl. Microbiol. 45, 283–287. Letourneau, F., Soussotte, V., Bressollier, P., Branlan, P., Verneuil, B., 1998. Keratinolytic activity of Streptomyces sp. S.K1-02; a new isolated strain. Lett. Appl. Microbiol. 26, 77–80. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Ranall, R.T., 1951. Protein measurement with 338 the folin phenol reagent. J. Biol. Chem. 193, 265–273. Lundy, F.T., Magee, A.C., Blair, L.S., McDowell, D.A., 1995. A new method for the detection of proteolytic activity in Pseudomonas lundensis after sodium dodecyl sulfate polyacrylamide gel electrophoresis. Electrophoresis 16, 43–45. Mohamedin, A.H., 1999. Isolation, identification and some cultural condition of a protease-producing thermophilic Streptomyces strain grown on chicken feather as a substrate. Int. Biodeter. Biodegr. 43, 13–21. Moritz, J.S., Latshaw, J.D., 2001. Indicators of nutritional value of hydrolyzed feather meal. Poultry Sci. 80, 79–86. Onifade, A.A., Al-Sane, N.A., Al-Musallan, A.A., Al-Zarban, S., 1998. Potentials for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resources. Bioresour. Technol. 66, 1–11. Rozs, M., Manczinger, L., Vagvolgyi, C., Kevei, F., 2001. Secretion of a trypsin-like thiol protease by a new keratinolytic strain of Bacillus licheniformis. FEMS Microbiol. Lett. 205, 221–224. Sangali, S., Brandelli, A., 2000. Isolation and characterization of a novel featherdegrading bacterial strain. Appl. Biochem. Biotechnol. 87, 17–24. Steel, R.G.D., Torrie, J.H., 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc., New York. p. 907. Suh, H.J., Lee, H.K., 2001. Characterization of a keratinolytic serine protease from Bacillus subtilis KS.l. J. Protein Chem. 20, 165–169. Wang, J.-J., Shih, J.C.H., 1999. Fermentation production of keratinase from Bacillus licheniformis PWD-1 and B. subtilis FDB-29. J. Ind. Microbiol. Biotechnol. 22, 608–616.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Production, characterization and application of keratinase from Streptomyces gulbargensis Dastager G. Syed a,*, Jae Chan Lee b, Wen-Jun Li c, Chang-Jin Kim b, Dayanand Agasar d a
National Institute of Interdisciplinary Science and Technology (CSIR), Thiruvananthapuram 695019, India Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea c Yunnan Institute of Microbiology, Yunnan University, Kunming, China d Department of Studies and Research in Microbiology, Gulbarga University, Gulbarga 585 106, Karnataka, India b
a r t i c l e
i n f o
Article history: Received 19 August 2008 Received in revised form 23 September 2008 Accepted 23 September 2008 Available online 5 November 2008 Keywords: Keratinase activity Actinomycetes Streptomyces gulbargensis
a b s t r a c t A Streptomyces gulbargensis newly isolated, thermotolerant feather-degrading bacterial strain was investigated for its ability to produce keratinase enzyme. Maximum keratinolytic activity was observed at 45 °C and pH 9.0 at 120 h of incubation. Activity was completely stable (100%) between 30 and 45 °C and pH 7.0–9.0, respectively. Addition of starch to the growth medium affects the activity by means of increase in keratinase secretion. After seven days of cultivation, 10-fold increase (14.3 U ml 1) in keratinase activity was observed in the presence of 3 g starch (per liter) of the medium. The enzyme was monomeric and had a molecular mass of 46 kDa. The enzyme activity was significantly inhibited by CaCl2 and partly inhibited by EDTA, whereas, Na2SO3 enhance the enzyme activity by 2.9 times more. In addition, native chicken feather was completely degraded at 96 h of incubation. The results obtained showed that newly isolated strain S. gulbargensis could be a useful in biotechnology in terms of valorization of keratincontaining wastes or in the leather industry. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Feathers are produced in large amounts as a waste by-product of poultry processing plant (Sangali and Brandelli, 2000). A current value-added use for feathers is the conversion to feather meal, a digestible dietary protein for animal feed, using physical and chemical treatments. These methods can destroy certain amino acids and decrease protein quality and digestibility (Moritz and Latshaw, 2001; Anbu et al., 2005). Keratinolytic microorganisms and their enzymes may be used to enhance the digestibility of feather keratin. They may have important applications in processing keratin-containing wastes from poultry and leather industries through the development of non-polluting methods (Onifade et al., 1998). Keratin-containing materials (feather, hair, wool, etc.) are abundant in nature but have limited uses in practice since they are insoluble and resistant to degradation by the common proteolytic enzymes. Keratinous wastes represent a source of valuable proteins and amino acids and could find application as a fodder additive for animals or source of nitrogen for plants. Biodegradation by microorganisms possessing keratinolytic activity represents an alternative attractive method for improving the nutritional value of keratin wastes, as it offers cheap and mild reac-
* Corresponding author. Tel.: +91 471 2515276. E-mail address: [email protected] (D.G. Syed). 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.09.047
tion conditions for the production of valuable products (Kim et al., 2001). A number of keratinolytic microorganisms have been reported, including some species of Bacillus (Suh and Lee, 2001; Joo et al., 2002; Amare et al., 2003), actinomycetes (Bressollier et al., 1999) and fungi (El-Naghy et al., 1998; Gradisar et al., 2000). In the present work, we report new keratin-degrading Streptomyces strain and its ability to degrade chicken feather. 2. Methods 2.1. Feathers Feathers were obtained at a local poultry processing plant (Gulbarga, India). Raw feathers were washed with warm tap water and distilled water, and dried at 45 °C for 48 h in a circulating airdrying stove. After drying, feathers were hammer milled and autoclaved for prior to microbial treatment. 2.2. Maintenance and inoculum preparation Pure isolates were inoculated on nutrient agar slopes and incubated at 28 °C for 24 h. To each slant 10 ml sterile distilled water were added and 1 ml of bacterial suspension produced with a sterile needle used to inoculate the cultivation medium (1 ml/50 ml medium).
D.G. Syed et al. / Bioresource Technology 100 (2009) 1868–1871
2.3. Organism and growth conditions Streptomyces gulbargensis DAS 131 which was isolated and identified by (Dastager et al., 2007) using polyphasic approach was used as an enzyme source. Keratinase production was carried out in the following basal medium (NH4Cl-0.5%, CaCl2-0.2%, MgSO4 7H2O-0.2%, K2HPO4-0.4%, pH 7.5) feather waste 0.1% and increased quantity of starch (0–3.5%) as a sole source of nitrogen and carbon. For growth of the organism, 1 l Erlenmeyer flask containing 250 ml of growth medium was kept at 30 °C in an orbital shaker set at 250 rev min 1 for seven days. Bacteria were removed by filtration and the filtrate was used for crude enzyme preparation (Bressollier et al., 1999). 2.4. Enzyme assay Keratinolytic activity was measured using a modified protocol of Anson (1938) with keratin azure as a substrate. The reaction mixture contains 0.2 ml of culture supernatant and 0.8 ml of 0.4% (w/v) keratin azure K-8500 (Sigma) in 10 mM Tris–HCl buffer (pH 8.5), and it was incubated at 45 °C for 1 h. The reaction was stopped with 500 ll, 0.1 M tri-chloroacetic acid (TCA) in 0.1 M Tris–HCl (pH 8.0) and then centrifuged at 15,000g for 15 min and supernatant was measured at 595 nm for amino acid liberation. The quantity was determined from standard tyrosine solution (50–500 lg ml 1) using UV-1601, Shimadzu spectrophotometer (Letourneau et al., 1998). One unit of keratinolytic activity was defined as the amount of enzyme required to liberate 1 lmol tyrosine under specific conditions. 2.5. Determination of biomass Gravimetric determination of the bacterial cell dry weight was determined by collecting cells from 50 ml cultures by centrifugation at 5000 rpm for 15 min. Cells were then washed with saline solution and dried at 60 °C to constant weight. 2.6. Protein determination The reaction mixture of 2 ml was composed of 1 ml of 1% casein in 0.2 M Tris–HCl, pH 8.0 and 1 ml of appropriately diluted enzyme. The reaction mixture was incubated for 30 min in a water bath at 40 °C. Two milliliter of 20% TCA was then added and the mixture centrifuged at 5000 rpm for 10 min. A control was processed by adding the enzyme after incubation and TCA was immediately added. The protein content of the enzyme preparations was determined in by the method of Lowry et al. (1951). 2.7. Feather-degrading activity (F-D activity) Feather-degrading activity was estimated from 24 to 96 h of incubation. One unit of FD activity was defined as the amount of enzyme that liberates one gram of amino acid equivalent to tyrosine per minute under the standard assay conditions. 2.8. Assay of protease activity Protease activity was determined using casein as a substrate according to the method reported by Gessesse et al. (2003). Unless otherwise stated, the reaction mixture in a total volume of 2 ml was composed of 1 ml of 1% casein in 0.2 M Tris–HCl, pH 8.0 and 1 ml of appropriately diluted enzyme. The reaction mixture was incubated for 30 min in a water bath at 40 °C. Two milliliter of 20% TCA was then added and the mixture centrifuged at 5000 rpm for 10 min. A control was processed by adding the enzyme after incubation and TCA was immediately added. One unit
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of enzyme activity (U) was defined as the amount of enzyme that liberates 1 mg of amino acid equivalent to tyrosine per minute under the above mentioned conditions. 2.9. Effect of pH and temperature Studies on the effect of pH and temperature on enzyme activity were performed as the standard assay using 50 mM Na2HPO4/citric acid (pH 4.0–7.5), 50 mM Tris/HCl (pH 7.5–8.5), 50 mM NaHCO3/ NaOH (pH 8.5–12.0) and a temperature range of 30–60 °C. The experiments on the effect of pH and temperature on enzyme stability were carried out by incubating the enzyme solution at pH and temperature ranges of 4–12 °C and 30–60 °C, respectively, for 30 min. The enzyme activities were then determined by the standard enzyme assay. 2.10. Effect of carbon and nitrogen sources on enzyme activity The effect of different carbon and nitrogen compounds on protease formation by S. gulbargensis was investigated in the cultivation media having carbon and nitrogen source content of 1.0% (w/v). 2.11. Effect of chemicals on enzyme activity The crude enzyme preparation was incubated in Tris (pH 9.0) containing different chemicals (EDTA, ZnCl2, MgCl2, CaCl2, Na2SO3), with and without feather. Enzyme activity was determined and compared with that of the control containing only feather at 30 °C for 30 min. 2.12. Substrate hydrolysis Soluble protein substrates (0.5%, gelatin, casein, bovine serum albumin, soluble keratin and elastin) and insoluble protein substrates (0.5%, human hair, human nail and chicken feather) were hydrolyzed by an enzyme solution, and amino acids produced by the reaction were analyzed using a free amino acid assay. One unit of protease activity was defined as the amount of enzyme that released 1 lmol of leucine after reaction with the substrate for 1 h at pH 9.0 and a temperature of 45 °C. 2.13. Ammonium sulfate precipitation The crude enzyme was first saturated up to 30% with solid (NH4)2SO4 and then centrifuged at 5000g for 15 min. The supernatant obtained was further saturated up to 70% with solid (NH4)2SO4 and again centrifuged. The pellets obtained were dissolved in minimum volume of 0.1 M Tris–HCl buffer, pH 8.0. 2.14. Gel-electrophoresis For determination of homogeneity and molecular weight, enzyme preparation and known molecular weights were subjected to electrophoresis by the method of Lundy et al. (1995) with the use of 12.5% agarose gel. After electrophoresis, gel was stained for 1 h with Coomassie Blue R-250 dye in methanol:acetic acid:water solution (4:1:5 w/v) and distained in the same solution without dye. 2.15. Statistic analysis Data were analyzed as a completely randomized design. Treatment means were separated using a significant f-test (Steel and Torrie, 1960).
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3. Results 3.1. Bacterial selection and identification A total of 108 keratinase-producing bacteria were screened for their ability to grow on feather and also for their feather degrading and protease activities. The local isolate DAS 131 was the most potent keratinase-producing organism since it could degrade chicken feathers completely within 96 h. The promising activities of this organism justified its selection for identification and characterization of its enzyme activities. It was found that the strain DAS 131 was the only thermotolerant feather-degrading strain. The isolate was confirmed as belonging to the genus Streptomyces as they possessed non-fragmented substrate mycelia, aerial hyphae and smooth spores arranged in chains. On the basis of polyphasic taxonomy it is proposed as a distinct genomic species (Dastager et al., 2007).
Fig. 1. Effect of addition of different concentrations of starch on keratinase activity by Streptomyces gulbargensis DAS 131. Each value is an average of three parallel replicates.
3.2. Keratinase production S. gulbargensis DAS 131 was able to grow and produced keratinase in medium in which feather meal served as a sole carbon and nitrogen source and acted as an enzyme inducer. Keratinase production was associated with growth at the maximum level of 1.5 U ml 1 (±0.05) in five days of cultivation. After five days of incubation, a loss of enzyme activity was observed probably because of enzymatic autolysis and end product inhibition. 3.3. Keratinase properties The enzyme had an optimum activity about 1.4 (±0.03) U ml 1 at 45 °C and was rapidly inactivated at higher temperatures. Enzyme was stable at 60 °C and was no longer active above 60 °C. It was observed that a free form of purified enzyme was less stable than an enzyme-substrate mixture under high temperature incubation. The keratinase was active in neutral and alkaline conditions with an optimum activity at pH 9.0 (1.39 U ml 1), depending on the buffer used (data not shown). It was stable over a wide range of pH values, with the highest stability at pH 7–9, and the activity was improved in basic conditions. The keratinase was significantly inhibited by CaCl2 and partly inhibited by EDTA whereas, Na2SO3 enhance the enzyme activity by 2.9 times more (Table 1). The enzyme degraded keratins from different sources. However, it hydrolyzed keratins less than soluble proteins such as casein, bovine serum albumin and gelatin. 3.4. Effect of carbon and nitrogen sources on enzyme activity The effect of different carbon and nitrogen compounds on protease formation by S. gulbargensis was investigated in the cultivation media with a carbon and nitrogen source content of 1% (w/v). Among all the carbon sources, the highest enzyme activity was achieved with starch and the least activity with sucrose (data not shown). Testing of the nitrogen source showed the highest enzyme activity with protease peptone and the lowest with yeast extract. Table 1 Effect of different chemicals on keratinolytic activity of Streptomyces gulbargensis Chemicals (0.1%) EDTA (5 mmol ZnCl2 MgCl2 CaCl2 Na2SO3
1
)
Residual activity (%)
Keratinase activity (U ml
68 44 48 00 293
0.88 ± 0.2 0.63 ± 0.1 0.67 ± 0.2 0.00 ± 0.0 4.16 ± 0.1
1
)
Fig. 2. SDS-PAGE of crude keratinase obtained from cell free broth of feather utilizing Streptomyces gulbargensis. Lane M: Molecular weight marker (19–105 kDa). Lane 1: Protein from cell free broth.
3.5. Stimulation of enzyme activity by starch The keratinase activity was enhanced in presence of starch as carbon source. It was added in increasing amount from 0% to 3.5%, to stimulate the keratinase activity. After seven days of incubation in submerged broth there is remarkable increase in the enzyme activity was recorded. It was observed that, 10-(14.3 U ml 1, ±0.02) fold higher activities in presence of 3% starch were observed (Fig. 1). Since starch is a main component in the media for isolation of actinomycetes, it was added in order to stimulate the keratinase secretion. 3.6. Gel-electrophoresis SDS–PAGE analysis revealed that the crude enzyme preparations were quite homogeneous and there was only a single protein band which had proteolytic activity. The molecular weight of band was found to be about 46 kDa (Fig. 2). The usefulness of this preparation in its crude form for industrial applications could be exploited. 4. Discussion Many Bacillus species have been reported to produce keratinolytic protease (Lal et al., 1999; Wang and Shih, 1999). Strain DAS
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131 isolated from Gulbarga soil grew and degrade feather at elevated temperatures. The keratinase production from S. gulbargensis DAS 131 reached a maximum in the late logarithmic growth phase similar to those reported by Kim et al. (2001). The molecular weight of keratinase from S. gulbargensis DAS 131 (46 kDa) was similar to that of B. licheniformis PWD-1, S. pactum DSM 40530 and Doratomyces microsporus (30–33 kDa) (Bressollier et al., 1999; Chitte et al., 1999; Gradisar et al., 2000). The optimum temperature for keratinase from S. gulbargensis DAS 131 (45 °C) was slightly lower than that of other keratinolytic proteases (50–55 °C) (Brouta et al., 2001). The enzyme was stable at 60 °C temperatures partly because its catalytic site was well protected by a substrate. Additionally, its optimum pH was similar to that of previous reports (Rozs et al., 2001). Stimulation of keratinase activity in S. gulbargensis DAS 131 was in presence of increasing amounts of starch. Since starch is a main component in the media for isolation of actinomycetes. After seven days of cultivation, an increase of 10-fold higher activity in presence of 3 g starch/l was observed. The purified keratinase from S. gulbargensis DAS 131 easily digested soluble proteins and a few insoluble proteins such as human hair, nail and chicken feather in accordance with other observations (Gradisar et al., 2000). The enzyme showed complete digestion on chicken feather since this substrates contained disulphide linkages which are a crucial structural feature of their molecules. Additionally, high protein content and amino acids were also accumulated in fermented broth as results of feather degraded by bacterial activities. In conclusion, an optimal substrate concentration and optimal cultivation conditions (including pH of the medium, temperature and rotation speed) improve feather degradation using S. gulbargensis DAS 131. In this study, the optimum conditions for keratinase synthesis by the S. gulbargensis DAS 131 were determined, when compared to enzymes in Bacillus (Johnvesly and Naik, 2001), Streptomyces have less thermostability, but keratinase from S. gulbargensis DAS 131 stable up to 60 °C this report was quite similar with Streptomyces S.K1-02 reported by Letourneau et al., (1998), were in maximum keratinolytic activity of DAS 131 was observed at pH 9.0, this result was in good agreement with other known enzymes of keratinolytic Streptomyces such as Streptomyces sp. S.K1-02 (Letourneau et al.,1998), S. albidoflavus (Bressollier et al., 1999), S. thermonitrificans (Mohamedin, 1999) and Streptomyces strain BA7 (Korkmaz et al., 2003). In conclusion results obtained from the present study reveals that, the bacterium and its enzyme could be used to improve nutritional values of animal feeds containing feather or keratin, or poultry processing wastes in a tropical country. The usefulness of crude form can be exploited in future for processing of keratin-containing wastes as well as in leather industry under suitable conditions. Acknowledgement The authors would like to thank CSIR Task force network programme on exploration of India’s Rich Microbial Diversity (NWP 0006) for providing the financial support.
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