International Journal of Research in Biological Sciences

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1 Available online at International Journal of Research in Biological Sciences Universal Research Publications. All rights reserved ISSN Research Article Production and characterization of keratinolytic protease(s) from the fungus, Aspergillus parasiticus Thoomatti Sudharsan Anitha and Peramachi Palanivelu* Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai , Tamil Nadu, India. Received 09 June 2012; accepted 25 June 2012 Abstract Keratinous wastes have increasingly become a problem and accumulate in the environment mainly in the form of feathers and hairs, generated mainly from large number of poultry and leather industries. As keratins are very difficult to degrade by general proteases, they pose a major environmental problem. Therefore, microorganisms which would effectively degrade keratins are needed for recycling such wastes. Keratinolytic fungi were isolated from the soil samples collected from poultry dump areas using chicken feathers as the sole source of carbon and nitrogen. They were identified as Aspergillus parasiticus and Pectinotrichum illanense. A comparative study on keratinolytic protease production on two different substrates (chicken feathers and keratin) has been done. The keratinolytic fungi grew well utilizing the keratins as sole source of carbon and nitrogen and produced good amount of keratinolytic protease activity. The optimum ph and temperature for the keratinolytic protease activity were found to be ph 6-7 and 50 o C, respectively. The K m values were and mg/ml for feather and keratin, respectively. Studies with the protease inhibitors suggest that the keratinolytic enzymes belong to serine protease type. The keratinases produced by the fungi could be useful in decomposition of keratin-wastes and could find applications in leather, pharmaceutical and cosmetics industries as well Universal Research Publications. All rights reserved Key Words: keratin, keratinase, serine protease, Aspergillus parasiticus 1. Introduction Keratins are a family of important structural proteins found in feathers, wool, horn, hooves, nails, claws, beaks and hair. The keratin chains are tightly packed into α-helix (αkeratin) or β-sheets (β- keratin) and then further turned into supercoiled structures which give good mechanical stability for these proteins. The extensive cross-linkages and disulphide bonds in keratin make them resistant to most of the known proteases like trypsin, pepsin, papain and results in polluting the environment. Worldwide, millions of tons of feathers were released annually as a waste product mainly from poultry processing plants. The presence of β- keratin in feathers causes high degree of recalcitrance in degradation process [1]. Disposing poultry feathers and related wastes by incineration requires more energy [2, 3]. Conversion of feathers into feather meals by applying physical and chemical methods results in the loss of nutritionally essential amino acids such as methionine, lysine and tryptophan and also in the formation of non-nutritive amino acids such as lysinoalanine and lanthionine [4]. Therefore, currently the poultry feathers are converted into feather meal, a digestible dietary protein, for animal feed using keratinases. Feathers degradation through keratinolytic enzymes has been considered as an important method for efficient bioconversion, nutritional enhancement and eco-friendliness. This method is extremely attractive and could be used to produce rare amino acids like serine, cysteine and proline or for the development of glues, slow-release nitrogen fertilizers and biodegradable films [5-7]. Keratinolytic enzymes are widespread in nature and are produced by several microorganisms including bacteria such as Bacillus sp. [8-11], Fervidobacterium islandicum [12], Elizabethkingia meningoseptica KB042 [13], Pseudomonas aeruginosa KS- 1[14] and actinomycetes such as Streptomyces sp. [15, 16] 87

2 and fungi such as Chrysosporium tropicum [17], rpm. The culture filtrates from 2nd, 4th, 6th and 8th days Trichoderma atroviridae [18], Doratomyces microsporus were collected from each set of flasks by separating the [19]; Paecilomyces marquandii [20]; Scopulariopsis mycelia by filtration and stored at -20 o C. The culture filtrates brevicaulis [21]; Alternaria, Paecilomyces, Penicillium, Curvularia and several Aspergillus sp.[22]. We have isolated a new strain of keratinolytic fungus from poultry soil and identified it as A. parasiticus (MTCC 9164). This paper, describes the degradation of feathers by our isolate and a comparative account of keratinolytic enzyme production on two different substrates, viz. keratin and chicken feathers and further characterization of the keratinolytic enzymes. 2. Materials and Methods 2.1 Chemicals were used as the enzyme source. 2.5 Assay of keratinolytic protease Determination of keratinolytic activity using azocasein as substrate The assay was done according to Palanivelu [23]. The reaction mixture in a total volume of 200 μl contained 20 mm phosphate buffer, ph 7.0, 1% azocasein and 10 mm CaCl 2. The reaction was initiated by the addition of the enzyme solution. The reaction tubes were incubated at 37 o C for 30 min and the reaction was stopped by the addition of 1 ml of 5% trichloroacetic acid. After 30 min at 4 o C, the tubes Azocasein, phenylmethylsulphonyl fluoride were centrifuged for 5 min at 14,000 x g at 20 o C and the (PMSF), iodoacetamide, pepstatin-a, leupeptin, N- absorbency was measured at 340 nm. One unit of protease ethylmaleimide and bovine serum albumin (BSA) were purchased from Sigma- Aldrich Chemical Company, U.S.A. Keratin was obtained from Himedia Labs, Mumbai, India. All the other chemicals used were of analytical grade, manufactured in India. 2.2 Processing of chicken feathers The chicken feathers were washed thoroughly with tap water and were air-dried. The dried feathers were defatted by soaking in diethyl ether for 24 hrs and were washed again thoroughly with distilled water, air-dried and autoclaved. Finally, the feathers were cut into small pieces without the midrib and stored at room temperature. 2.3 Isolation and Identification of keratinolytic microorganisms The soil samples collected from poultry dump areas were plated on a selective medium containing (g/l) 0.4 Na 2 HPO 4 ; 0.3 NaH 2 PO 4 ; 5.0 NaCl; 10.0 treated chicken feathers; 15.0 agar in distilled water. The ph of the medium was adjusted to 6.0. The plates were incubated at 37 o C. Eight different bacterial colonies and two different fungal colonies were isolated from the plates. The fungal cultures were maintained in Sabouraud dextrose agar. The fungal cultures were identified as Aspergillus parasiticus and Pectinotrichum illanense by Microbial Type Culture Collection, Chandigarh, activity was defined as an increase in absorbency of 0.01 at 340 nm under the standard assay conditions Determination of keratinolytic activity using keratin as substrate Keratinolytic activity was also determined by using keratin as the substrate. The assay was done essentially according to Gradisar et al. [19]. 2.6 Determination of protein concentration Protein content in the culture supernatant fluids was determined by the method of Bradford [24] using BSA as standard. 2.7 Effects of ph and temperature on enzyme activity Effects of ph and temperature on the keratinolytic activity were determined using azocasein as substrate. Keratinolytic protease activity was studied in the ph range of 4 to 9 using the following buffers. 0.2 M sodium acetate buffer, ph (4 to 5), 0.2 M phosphate buffer (6 to 7) and 0.2 M Tris-HCl buffer (ph 8 to 9). The optimum temperature for keratinolytic protease activity was determined by performing the enzyme reaction at incubation temperatures between 30 o C and 80 o C. 2.8 Effect of protease inhibitors on enzyme activity To determine the type of the protease, the following inhibitors were tested by the azocasein assay method; India. As A. parasiticus was found to be a better producer of ethylenediaminetetraacetic acid (EDTA), keratinases, it was used for further studies. 2.4 Production of crude keratinolytic protease The primary inoculum of A. parasiticus was prepared by incubating a loop full of fungal spores in the mineral medium containing (g/l) 1.5 KH 2 PO 4 ; K 2 HPO 4 ; 0.2 MgSO 4.7H 2 O; 0.2 CaCl 2. 2H 2 O; 0.2 NaCl; ZnSO 4.7H 2 O; Yeast extract, 6.0 Glucose, 2.0 ml Glycerol, ph ~ 6 for about 3 days. For enzyme production, 3% of the inoculum was transferred to the production medium which was the minimal medium containing 10 g/l glucose and either treated chicken feathers (10 g/l) or keratin (5 g/l) and the flasks were incubated up to 8 days at room temperature (30 o C ± 3 o C) on a gyratory shaker with constant shaking at 140 phenylmethylsulphonyl fluoride, iodoacetamide, pepstatin A, leupeptin and N-ethylmaleimide. Each inhibitor was added to a final concentration of mm and mm and the reactions were carried out for 30 min at 37 o C. A control reaction was run without any inhibitor. 2.9 Determination of kinetic constants The kinetic constant for the keratinolytic protease activity was determined by performing the azocasein assay at different substrate concentrations ( to 3.0 mg). The hydrolysis was followed continuously and the initial velocities were determined. The kinetic constants K m and V max were calculated using Lineweaver Bürk plot using Ez- Fit software. 88

3 Activity (U/ml) Relative Activity (%) Activity (U/ml) Feather Keratin ph Figure 1 Effect of ph on hydrolysis of chicken feather (- -) and keratin (- -) as substrates by the keratinolytic protease from Aspergillus parasiticus. Figure 3 Lineweaver-Bürk plot for the keratinolytic protease from A. parasiticus. (A Enzyme obtained from feather spent medium, B Enzyme obtained from keratin spent medium) Temperature ( o C) Feather Keratin Figure 2 Effect of temperature on hydrolysis of chicken feather (- -) and keratin (- -) as substrates by the keratinolytic protease from Aspergillus parasiticus Effect of metal ions on enzyme activity Effect of metal ions on the enzyme activity was determined by incubating the crude enzyme with the following metal ions (Ca 2+, Cu 2+, Mn 2+, Mg 2+, Na +, Cd 2+, Zn 2+ and Pb 2+ ) at a concentration of 5 mm. The activity was measured by the azocasein assay as described earlier. A control reaction was run without any metal ion SDS-PAGE Protein profiles of the culture filtrates, obtained from keratin and feather media, were determined by SDS-PAGE according to the method of Laemmli [25]. Before loading, the samples were concentrated by equal volume of cold (-20 o C) acetone. The gels were stained by silver staining method [23]. The relative molecular masses of the proteins were determined by comparing with standard molecular weight markers Zymogram analysis Zymogram analysis was done according to the method of Palanivelu [23] incorporating casein in the separating gel. 3. Results Keratinolytic microorganisms were isolated from soil samples using selective media as described elsewhere. Among the isolated cultures, the two fungal strains digested the chicken feathers better than the bacterial strains and were Control Sodium Chloride Calcium Chloride Copper Sulphate Manganese Chloride Magnesium Sulphate Metal Ion ( 5 mm) Zinc Sulphate Cadmium Chloride Lead Nitrate Feather medium Keratin medium Figure 4 Effect of metal ions on the keratinolytic protease activity from A. parasiticus. used for further studies (data not shown). The feather digesting fungi were morphologically different, that is, one is sporulating type (MKU-S) and the other is non-sporulating (MKU-NS) type. These two fungi were identified by Microbial Type Culture Collection at Chandigarh as Aspergillus parasiticus (MKU-S, MTCC 9164) and Pectinotrichum illanense (MKU-NS, MTCC 9168). Among the two fungi, A. parasiticus grew faster in the medium containing chicken feather (data not shown) and therefore, it was selected for further studies. The culture filtrates from A. parasiticus, grown on keratin and feather media were tested for their protease/keratinase activities. It was found that the spent medium containing feather showed higher specific activity for protease and keratinase as compared to the spent keratin medium (Table 1). Highest specific activities were found on the fourth and second day of growth in spent media with feather and keratin, respectively. The ratio of keratinase/caseinase activity was found to be 0.5 for the culture filtrates from feather media whereas in the case of keratin media, the ratio was found be less than 0.5. However, treatment of the keratin with 5 mm of DTT and β -mercaptoethanol increased the ratio (data not shown). The optimum ph of the enzyme from feather containing spent medium was found to be 6.0. The activity was decreasing under acidic conditions and stable at basic 89

4 Figure 5 SDS-PAGE analysis of proteins from the culture filtrates of feather (A) and keratin (B) media from A. parasiticus. M Protein molecular weight markers ( kda), lane 1 (A & B) - 2nd day culture filtrates, lane 2 (A & B) 4th day culture filtrates, lane 3 (A & B) 6th day culture filtrates, lane 4 (A & B) 8th day culture filtrates. Two major bands of similar molecular weights were observed in both the cases (indicated by arrows). Figure 6 Zymogram analysis of keratinase. 1, 2, 3 & 4 2 nd, 4 th, 6 th and 8 th day of spent feather medium. 5, 6, 7 & 8-2 nd, 4 th, 6 th and 8 th day of spent keratin medium. The two proteolytic bands were clearly visible (Fig 4B). phs in which more than 90% activity was retained. In the culture medium with keratin, the optimum ph for the crude keratinase enzyme was found to be 7 (Fig. 1). The optimum temperature was found to be 50 o C for the enzymes from both the sources (Fig. 2). To determine the type of protease, the protease activity was measured in the presence of different protease inhibitors. The results are summarized in Table 2. PMSF at 1mM inhibited 97% of keratinolytic protease activity obtained from both the sources. At mm, it inhibited 85% of the activity in the culture filtrate of spent feather medium and 94% in the culture filtrate of spent keratin medium. The general protease inhibitor leupeptin had partial inhibitory effect on the enzyme activity. The residual activity was ~ 38% - 43% and ~ 77% - 79% at mm and mm, respectively. Iodoacetamide and EDTA, did not show any significant inhibition. Using the Lineweaver Bürk plot, K m and V max were found to be mg/ml and units/min/mg protein (feather extract) and mg/ml and units/min/mg protein, respectively (Fig. 3). The effect of metal ions (Ca 2+, Cu 2+, Mg 2+, Mn 2+, Na +, Zn 2+, Cd 2+ and Pb 2+ ) on the keratinolytic activity was investigated by adding metal ions to the enzymes extract at a concentration of 5 mm. These metal ions had similar effect with the enzymes obtained from both the sources. Ca 2+, Mg 2+, Mn 2+, Zn 2+, Cd 2+ and Pb 2+ did not show any significant effect on enzyme activity and more than 75% activity was found in all cases. Addition of CuSO 4 inhibited the enzyme activity by about 50% whereas NaCl slightly enhanced the activity (103%, Fig. 4). In order to find out the protein profiles in the culture filtrates obtained from two different keratin sources, SDS- PAGE was performed. The protein profiles of the culture filtrates from feather and keratin media were analyzed. Increased numbers of proteins bands were observed in spent medium containing feathers as compared to the spent medium with keratin (Figs. 5A & B). Two prominent bands with approximate molecular masses of 37 kda and 25 kda were seen in both the substrates. Culture filtrates of A. parasiticus from feather and keratin media showed more than one type of proteases as observed by zymogram analysis. Two proteolytic bands were observed in the culture filtrates obtained from the spent feather meal and keratin media (Fig. 6). Discussion Large amount of feathers are produced as waste at poultry processing plants, reaching millions of tons per year with potential environmental impact. Feathers are mainly made of the structural protein, keratin which is an insoluble protein and can be only digested by microbial keratinases. In this work, two feather-degrading fungi were isolated from soil using selective media and identified as A. parasiticus and P. illanense. A comparative study of keratinase production by A. parasiticus on two different substrates (chicken feather and keratin) has been carried out. Spent medium with feather elaborated more protease and keratinase activities as compared to the spent medium that contained keratin. There are several limitations of using feather keratin for growth of poultry because of poor digestibility (due to structural rigidity) and nutritional limitations (due to low content of essential amino acids) [1]. However, the keratinolytic fungi isolated in this work overcame these limitations and utilized feathers and produced good amount of the enzyme. The keratinase produced from the two different culture media were found to be active with a broad ph range, with the optimum ph of 6.0 (feather medium) and ph 7.0 (keratin medium). This result is in good agreement with keratinases obtained from other fungal sources like Trichophyton granulosum [26] and Trichophyton rubrum [27]. However, the optimum ph of some of the keratinolytic enzymes from other fungal sources were towards alkaline 90

5 side, e.g., ph 8.0 for Paecilomyces marquandii and Doratomyces microsporus keratinases [20], ph 9.0 for Table 1. Specific activities of Protease and Keratinase produced from A. parasiticus in chicken feather and keratin media. GROWN ON Specific activity of enzyme Chicken Feather Medium (Keratin Medium*) 2 nd Day 4 th Day 6 th Day 8 th Day Protease Activity using azocasein (1757.6) (1531.5) (1453.3) (1348.3) Keratinase activity using keratin (301.2) (257.1) 21 (201.6) (195.4) *Activities from keratin spent medium are shown in brackets. Table 2. Effect of protease inhibitors on keratinolytic protease activity from A. parasiticus. Inhibitor Concentration (mm) From feather From keratin medium Medium Control EDTA PMSF Iodoacetamide Pepstatin A Leupeptin N-Ethylmaleimide bacterial (Streptomyces sp.) keratinases were also inhibited to a similar extent [18, 35]. Addition of Zn 2+ did not show any Aspergillus fumigatus keratinase [28]. The enzyme obtained from both the media was found to be stable at wide range of phs from 5 to 9. In the effect of temperature study, the optimum temperature was found to be 50 o C for the enzymes from both the sources. Keratinases from other fungi like Trichoderma atroviridae [18] D. microsporus [19] and Aspergillus oryzae, [29] also exhibited similar optimum temperatures. In general, most of the keratinolytic proteases have the optimum temperatures between 50 o C and 55 o C [30]. The crude keratinase was strongly inhibited by PMSF which suggests that it belong to serine protease type. Among actinomycetes, several keratinases described from Streptomyces sp. are also found to be serine proteases [31, 32]. Other bacterial keratinolytic proteases from Chryseobacterium sp. [33], Bacillus sp. [1] and fungal keratinases from D. microsporus [19], and S. brevicaulis [21] are also reported to be serine protease type. The K m values obtained in this study were found to be lower than the K m value of a purified keratinase from Bacillus strains, expressed as recombinant protein in Pichia [34] and also from P. marquandii [20]. The addition of various metal ions (Ca 2+, Cu 2+, Mg 2+, Mn 2+, Na +, Zn 2+, Cd 2+ and Pb 2+ ) to crude enzymes preparations did not have any significant effect. Only Cu 2+ inhibited the enzyme activity by about 50% obtained from both the substrates (Fig. 4). Fungal (T. atroviride) and inhibitory effect on the activity of the enzyme. This is similar to the fungal keratinase from S. brevicaulis [21], but in contrast to the characteristics of a bacterial keratinase from Bacillus [10] which was inhibited by Zn 2+. Conclusion Keratinase production by the feather degrading fungus A. parasiticus in two different keratin sources was investigated and the keratinolytic activities were further characterized. Efficient feather hydrolysis by the fungus revealed production of good amount of keratinase activity with broad optimum ph and higher temperature optima which could find application in converting the large amount of waste generated from poultry into digestible animal feed. Since, this fungus uses feathers as the sole sources of carbon and nitrogen, the production of enzyme will be cost effective. Acknowledgements The authors wish to thank University Grants Commission, New Delhi, India for the financial assistance under the University with Potential for Excellence programme to Madurai Kamaraj University, Madurai, India. The authors also wish to thank Prof. P. Gunasekaran, School of Biological Sciences and Prof. R. Usha, School of Biotechnology for useful suggestions on the manuscript. References 91

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