Enzyme and Microbial Technology 34 (2004) 8593

Review

Purication, characterization and immobilization of a keratinase from Aspergillus oryzae

Aida M. Farag a, , Maha A. Hassan b
a

National Institute of Oceanography and Fisheries, Alexandria, Egypt b Faculty of Education, Alexandria University, Alexandria, Egypt

Received 1 August 2003; received in revised form 6 September 2003; accepted 8 September 2003

Abstract A keratinase enzyme was isolated and puried from a feather-degrading culture of Aspergillus oryzae. Fractional precipitation of the crude enzyme with ethanol, acetone and ammonium sulfate yielded 21 fractions. The fraction obtained at 7585% ammonium sulfate saturation showed the highest activity and about 3.3-fold purication. This fraction was further puried by gel ltration in Sephadex G-75 followed by ion exchange chromatography on DEAE-Sephadex A-50 yielding an active major protein peak showing 11.38-fold purication. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) indicated that the puried keratinase is a monomeric enzyme with a molecular mass of 60 kDa. The puried enzyme was able to hydrolyze different substrates showing its highest proteolytic activity on bovine serum albumin and casein followed by keratin, chicken feathers, collagen, duck feathers and sheep wool. The puried enzyme was immobilized on various carriers. Immobilization on sintered glass beads showed the highest activity. The optimum pH of the immobilized enzyme shifted to a more neutral range (7.07.4) compared with the free enzyme (8.0). The optimum temperature of the reaction was determined to be 60 C for the immobilized enzyme and 50 C for the free enzyme. The free keratinase enzyme was retained 42.05% of its activity at 70 C (60 min) while the immobilized keratinase preparation showed a higher thermal stability. The half-lives of the free and immobilized enzyme were 45.45 and 60.00 min, respectively. The pure enzyme was activated by calcium and barium ions while EDTA and Pb inhibited the activity. 2003 Elsevier Inc. All rights reserved.
Keywords: Keratinase; Proteolytic activity; Aspergillus oryzae

1. Introduction Keratinaceous materials such as feather, wool and hair are insoluble and resistant to degradation by common proteolytic enzymes such as trypsin, pepsin and papain because of their high degree of cross-linking by disulphide bonds, hydrogen bonding, and hydrophobic interactions [8, 16,28]. Keratinases (E.C. no. 3.4.99.11), a group of proteinase enzymes, are important for hydrolyzing feather, hair, wool, collagen and casein to clean obstructions in the sewage system during waste water treatment [14]. These enzymes are also used or could be applied in the food industry, textiles, medicine, cosmetics and leather and poultry processing industry [6,10]. The structural protein, keratin, can be degraded by keratinases produced by species of saprotrophic

Corresponding author.

and parasitic fungi ([3,15,30,31,33]), some Bacillus species ([4]) and a few actinomycetes [7,11,21,26]. Many techniques for immobilization of enzymes on different types of supports have been developed [18,32]. The immobilization of proteases on solid supports has been widely used in many investigations [9,12]. When a protease is immobilized, enzyme autolysis is minimized. For industrial applications, immobilization of the enzyme in gel or solid supports may offer several advantages such as, repeated use of the enzyme, ease of product separation and improvement of enzyme stability [9,12]. The properties of microbial keratin-degrading enzymes appear to differ according to the producing species of microorganism. This study reports on the purication and characterization of a keratinase secreted by an osmoduric strain of Aspergillus oryzae isolated from marine sediment. The effect of immobilization on the obtained enzyme was also taken into consideration as an important biotechnological aspect.

0141-0229/$ see front matter 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2003.09.002

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2. Materials and methods 2.1. Organism and growth condition A. oryzae was isolated from marine sediment of Kayet Bey, Anfoshi, Alexandria, Egypt. This isolate was identied as Aspergillus by microscopic examination and further identication with the help of Microcheck Inc. Lab. (Northeld, Vermont, USA) by fatty acid analysis of the fungus. The strain was cultivated at 30 C in a mineral salt medium (g/l): glucose, 3.0; K2 HPO4 , 2.0; KH2 PO4 , 1.0; MgCl2 6H2 O, 0.3; NaCl, 40.0; KCl, 0.5; CaCl2 2H2 O, 0.2; FeSO4 7H2 O, traces, pH 6.0. Chiken feather was used as carbon, nitrogen and sulphur sources at a concentration of 10 g/l. Cultivation of A. oryzae, for the production of keratinase, was carried out in 250 ml erlenmyer asks each containing 50 ml of basal medium of the same composition as that used for isolation. Each ask was inoculated with 2 ml of a spore suspension (2 106 spore/ml) prepared from 5-day old slants of the test organism. The asks were incubated at 30 C in an incubator shaker (160 rpm). 2.2. Protein determination The amount of protein was estimated by the method of Lowry et al. [24] with bovine serum albumin as the standard. The protein content of the immobilized enzyme was calculated by subtracting the amount of unbound protein from the protein originally added. 2.3. Determination of keratinase activity Keratinase activity was determined spectrophotometrically according to the method of [5], with a slight modication. The reaction mixture consisted of 0.5 diluted enzyme solution and 0.5 ml of 0.5% keratin (0.5 g keratin in 0.02 M phosphate buffer, pH 8) was incubated for 15 min at 50 C. The reaction was stopped with 1 ml of 10% trichloroacetic acid (TCA) for 30 min at room temperature. This mixture was centrifuged and the released amino acids measured as tyrosine by Lowry method. One unit of keratinase activity was dened as the amount of enzyme required to liberate 1 mol of tyrosine under the specied conditions. 2.4. Keratinase production A. oryzae was inoculated in 100 ml mineral salts medium and incubated at 30 C for 5 days (static culture). The culture medium was ltered through glass wool to remove residual undegraded feathers, followed by centrifugation to remove any spores and other particles. The enzyme was concentrated from the cell-free broth by either salting out with ammonium sulfate and precipitation with acetone or ethanol. The protein precipitate was dissolved in a dened volume of phosphate buffer (0.02 M, pH 8), and used as partial puried enzyme.

2.5. Partial purication 2.5.1. Acetone or ethanol A.R acetone or absolute ethanol was cooled at 4 C one day before starting precipitation. Acetone or ethanol was added to the supernatant slowly. Several enzyme fractions were obtained at 25, 35, 50, 65, 75, 85 and 95% concentration of acetone (ethanol). These fractions were dried over anhydrous calcium chloride under reduced pressure at room temperature. Thereafter, each precipitate was dissolved in a certain amount of distilled water and dialyzed against distilled water in a refrigerator for one day. After dialysis, the protein content and enzyme activity of each fraction were determined. 2.5.2. Ammonium sulfate fractionation Ammonium sulfate was added to 100 ml of the culture ltrate at different concentrations to obtain various fractions at 25, 35, 50, 65, 75, 85 and 95% saturation. Each precipitate was dissolved in a certain amount of distilled water and dialyzed against distilled water in a refrigerator overnight after dialysis. 2.6. Purication of keratinase 2.6.1. Sephadex G-75 fractionation A glass column (2.5 cm 45.0 cm) was packed with Sephadex G-75 (Sigma) and equilibrated with 400 ml of 0.02 M phosphate buffer at pH 8.0. A ow rate of 60 ml/h was maintained. The precipitate resulting from ammonium sulfate fractionation 85% saturation was dissolved in 30 ml of 0.02 M phosphate buffer and dialyzed for 24 h at 4 C in 2 l of 0.02 M phosphate buffer after dialysis. A portion (10 ml) was then applied to the Sephadex G-75 gel bed and protein was eluted with 0.02 M phosphate buffer. Enzyme activity and protein content in each fraction were measured. Fractions which showed highest protein and keratinase activity were collected. 2.6.2. Ion-exchange chromatography DEAE Sephadex A-50 A column (2.5 cm 45.0 cm) was packed with a slurry of diethylaminoethyl (DEAE) Sephadex A-50. The sephadex bed (30 cm long) was equilibrated with 200 ml of 0.02 M phosphate buffer at pH 8.0. The fractions of highest specic activity obtained from gel ltration on Sephadex G-75 column pooled and applied to the DEAE-Sephadex A-50 column. Elution was performed with 0.05 M phosphate buffer, followed by 0.05 M NaCl in 0.05 M phosphate buffer at pH 8.0, at a ow rate of 60 ml/h. Fractions (5-ml) were collected, protein content and keratinase activity for each fraction were monitored. The fractions possessing highest specic activity were pooled. 2.7. Polyacrylamide gel electrophoresis The puried enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) ac-

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cording to Laemmli [20]. A 12.5% separating gel was used. The proteins were stained with a 0.1% solution of Coomasiie brilliant blue R-250 (Serva, Germany). 2.8. Immobilization methods 2.8.1. Physical adsorption Charcoal, sintered glass beads and silica gel are used as carriers for the enzyme. The carrier was incubated with the enzyme solution (74.4 U A. oryzae keratinase) dissolved in 1 ml 0.1 mol/1 phosphate buffer (pH 8.0) at 4 C over night. The enzyme activity and protein of unbound and immobilized enzyme were determined [27]. 2.8.2. Ionic binding Anion exchanger (DEAE-cellulose) equilibrated with phosphate buffer (0.1 mol/1, pH 8.0), or cation exchanger (Dowex 50W) with TrisHCl buffer (0.1 mol 11 pH 8.0), was incubated with the enzyme solution (74.4 U A. oryzae keratinase) dissolved in the same buffer for 12 h at 4 C. The enzyme activity and protein of unbound and immobilized enzyme were determined [19]. 2.8.3. Covalent binding Chitosan (1 g) was dissolved in 100 ml 0.1 mol/1 HCl containing 2.5% (v/v) glutaraldehyde (GA) for 2 h at 30 C. The solubilized chitosan was precipitated by the addition of 1 ml 1.0 mol/1 NaOH. The precipitate was separated by ltration (using a sintered glass funnel) and washed with distilled water to remove the excess GA. The wet chitosan was mixed with 5.0 ml of the enzyme solution (74.4 U A. orzyae keratinase) and stirred for 1 h at 30 C. The unbound enzyme was removed by washing with distilled water until no protein or activity was detected [27]. Chitin (1 g) was shaken with 10 ml 2.5% (v/v) GA. Chitin was then collected by ltration (using a sintered glass funnel) and washed with distilled water to remove the excess GA. The wet chitin was mixed with 5.0 ml of the enzyme solution (74.4 U A. orzyae keratinase) for 2 h at 30 C. The unbound enzyme was removed by washing with distilled water as described before [27]. 2.8.4. Entrapment In Ca-alginate: 5 ml of 1% or 3% or 5% (w/v) Na-alginate were mixed with 37.2 U of A. orzyae keratinase. The entrapment was carried out by dropping the mixture into 25 ml mol/1 CaCl2 solution. The resulting beads (1.01.5 mm diameter) were collected and washed with distilled water to remove the unbound enzyme [1]. 2.9. Effect of different substrates The puried enzyme preparation was incubated in phosphate buffer (pH 8.0) containing different substrates (feathers and wool were dried to a ne powder using an electric mill). Enzyme activity was determined as described before for keratinase (as proteolytic activity) and compared with that of the control containing keratin.

2.10. Effect of pH and temperature on the enzyme activity The effect of pH and temperature on the free and immobilized enzyme were determined with keratin as substrate. Keratinase activity was studied in the pH range of 3.610.7, using the following buffers: 0.02 M acetic acid/sodium acetate, pH (3.65.4), 0.02 M sodium phosphate buffer (pH 5.68.0) and 0.02 M NaHCO3 /NaOH (pH 9.210.7). The optimum temperature for keratinase activity was determined by varying the incubation temperature between 25 and 80 C. The activation energies (Ea ) of free and immobilized enzymes were determined from the slope of logarithmic Arrhenius plots (slope =Ea /2.303R where R (1.976 cal/mol) is the gas constant. 2.11. pH stability and thermal stability The free or immobilized enzyme was incubated at various pH values at 37 C for 2 h, and then the residual activity was determined at the optimum pH (8.0 for the free enzyme and 7.4 for the immobilized). The thermal stability of the puried enzyme preparation was studied at the optimum pH. Identical enzyme solutions in phosphate buffer (0.02 M, pH 8.0) were preheated separately at different temperatures (50, 60, 70 and 80 C) for various time periods (15, 30, 60 min). The residual activity was determined by adding the substrate and carrying out the enzyme assay under optimum reaction conditions. The rst order inactivation rate constant (ki ) was obtained from the equation ln A = ln Ao ki t where Ao and A are the initial activity and the activity after a time t (min). 2.12. Effect of some metal ions (activators and inhibitors) on keratinase activity To investigate the effect of some metal ions on the enzyme activity, the puried enzyme solution was preincubated for 2 h at room temperature with the tested substance (1, 10, 100 mM). The residual enzyme activity was measured by adding the substrate and carrying out the enzyme assay under the optimum conditions. Statistical analysis (one-way analysis of variance (ANOVA) test and a paired sample t-test) were carried out using SPSS 10 Software. The cut-off value for statistical signicance was P < 0.05.

3. Results and discussion 3.1. Purication of keratinase The purication of the keratinase enzyme produced by A. oryzae was effective and efcient. The crude enzyme was partially puried by fractional precipitation with ammonium sulfate, acetone or ethanol (Table 1). A total of 21 fractions were obtained (seven for each of the used precipitants) and the highest recovered protein was present in the

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Table 1 Fractional precipitation of keratinase from A. oryzae cultures using different agents Agent concentration (%) Crude 25 35 50 65 75 85 95 Total Ammonium sulfate PC 62.80 0.69 0.96 2.30 3.40 5.66 7.38 4.80 25.19 RP 100 1.09 1.53 3.66 5.41 9.01 11.75 7.64 40.09 KA 26.35 13.95 20.83 28.26 28.82 33.04 88.07 19.58 RA 100 0.58 1.21 3.93 5.92 11.3 39.27 5.68 67.89 Acetone PC 62.80 0.67 0.85 3.95 6.6 5.95 2.99 1.02 22.03 RP 100 0.59 1.37 6.29 10.51 9.47 6.35 1.62 36.20 KA 26.35 11.82 16.74 38.48 48.48 33.43 20.90 12.65 RA 100 0.48 0.87 9.18 19.33 12.02 3.78 0.78 46.44 Ethanol PC 62.80 0.35 0.62 3.59 7.33 6.70 1.87 0.82 21.28 RP 100 0.56 0.99 5.72 11:67 10.67 2.98 1.30 33.89 KA 26.35 18.57 20.16 36.21 47.75 25.37 24.06 12.44 RA 100 0.39 0.75 7.85 21.15 10.27 2.72 0.62 43.75

PC: protein content (mg), RP: relative protein (%), KA: keratinase activity (U/mg protein), RA: relative activity (%).

fractions precipitated with ammonium sulfate yielding a total of 40.09%, followed by acetone (36.20%) and ethanol (33.89%). The highest total recovered activity was also obtained by ammonium sulfate (67.89%) followed by acetone (46.44%) and ethanol (43.75%). Among all the obtained fractions, the 85% ammonium sulfate fraction showed the highest keratinase activity, protein recovery and about 3.3-fold purication. Similarly, active keratinase preparations were obtained from thermophilic Streptomyces thermoviolaceus culture with ammonium sulfate saturation of 80% [11]. The partially puried keratinase which was concentrated by precipitation with 85% saturation of ammonium sulfate was dialyzed and subjected to gel ltration on a Sephadex G-75 column. The elution proles for keratinase and protein from the Sephadex G-75 column are shown in Fig. 1 and indicate that there are three peaks obtained as shown in Fig. 1. The rst peak contains the highest specic activity (211.58 U/mg protein). The most active fractions (numbers 813) from the Sephadex G-75 column were pooled

and further puried by DEAE-Sephadex A-50 column chromatography (Fig. 2). An overall increase in specic activity of 11.38-fold was obtained. This second purication step yielded a homogeneous protein as shown by SDSPAGE (Fig. 3). A summary of the purication of keratinase from the culture medium of A. oryzae is presented in Table 2. 3.2. Characterization of pure keratinase Some properties of the pure keratinase isolated from A. oryzae cultures were studied, The apparent molecular mass of the puried keratinase was estimated to be 60 kDa as measured by SDSPAGE (Fig. 3). The results showed that the optimum protein concentration was 0.112 mg/ml reaction mixture, while higher values did not increase the reaction velocity. It was also shown that the optimum substrate concentration is 5 mg/ml reaction mixture. The Km and Vmax values of the pure enzyme were evaluated from a LineweaverBurk plot and found to be 8.47 0.74 mg/ml and 71.43 1.45 U/ml, respectively.

Fig. 1. Gel ltration in Sephadex G-100 of the partially puried keratinase preparation (85% ammonium sulfate fraction).

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Fig. 2. Ion-exchange chromatography on DEAE-Sephadex A-50 of the major activity keratinase peak obtained by gel ltration.

3.3. Effect of using different substrates on keratinase activity The puried keratinase has a broad substrate specicity and can hydrolyze a wide variety of soluble and insoluble

protein substrates (Table 3). The soluble substrates bovine serum albumin (BSA) and casein were readily degradable showing a high proteolytic activity, while insoluble substrates such as keratin, chicken feather, duck feather, collagen and sheep wool were less susceptible to enzyme hydrolysis and the proteolytic activity ranged from 45 to 75% of the value obtained for hydrolyzing BSA. These results are close to the keratinase obtained from Bacillus licheniformis [22]. Although the specicity toward soluble peptide substrates of the enzyme from A. oryzae is high, its ability to hydrolyze keratins is signicant showing relatively high activity. 3.4. Immobilization of enzyme Keratinase enzyme from A. oryzae was immobilized on various carriers and keratinase activity was evaluated (Table 4). The immobilized enzyme prepared by physical adsorption to sintered glass beads had the highest immobilized activity (39.2 U/g carrier) and the highest immobilization yield (63.64%). Thus, sintered glass beads were selected as a carrier for further work. Lin et al. [23] used controlled-pore glass beads for the immobilization of keratinase isolated from Bacillus licheniformis. 3.5. Effect of pH and temperature on the enzyme activity Maximum keratinolytic activity of free enzyme was observed between pHs 7 and 9 with an optimum at pH 8.0 and the optimum pH of the immobilized enzyme was 7.4 (Fig. 4). It was observed that the optimum pH of the studied enzyme was lower than that obtained from bacterial cultures of Frevidobacterium pennavoranis [13] and Microsporum canis [25]. The optimum temperature for the puried free and immobilized enzyme preparations was studied. The free enzyme had an optimum temperature of

Fig. 3. SDSPAGE of puried keratinase after Sephadex G-75 and DEAE-Sephadex A-50 gel chromatographies (lane E).

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Table 2 Purication of keratinase from A. oryzae Purication step Culture ltrate Ammonium sulfate (85% saturation) Sephadex G-75 DEAE-Sephadex A-50 Protein content 314 36.9 13.47 9.09 Total activity (U) 8275 3250 2850 2760 Keratinase activity (U/mg protein) 26.69 88.07 211.58 303.63 Recovery (%) 100 39.24 34.44 33.35 Purication (fold) 1 3.3 7.93 11.38

Table 3 Hydrolysis of different proteinaceous substrates by the puried keratinase preparation Substrate Keratinase activity (U/mg protein) 274.46 248.93 199.11 331.25 253.75 441.96 360.00 Fig. 4. Effect of pH on the activity of the puried free and immobilized keratinase.

Insoluble protein Chicken feather Duck feather Sheep wool Keratin Collagen Soluble protein BSA Cascin

about 50 C, whereas that of immobilized enzyme shifted to 60 C (Fig. 5). Arrhenius plots of temperature data appeared linear, activation energies were found to be 12.16 and 41.86 kcal/mol for the free and immobilized keratinase, respectively. The increase in optimum temperature and activation energy may indicate some change in the physical properties of the enzyme molecule. Immobilization of the enzyme on sintered glass might have reduced the conformation exibility, thereby resulting in a higher activation energy for the molecule to attain the suitable conformation for binding to substrate. This optimum temperature for kerTable 4 Immobilization of puried keratinase obtained from Aspergillus oryzae cultures Carrier Physical adsorption Charcoal Silica gel Sintered glass Ionic binding Dowex DEAE-cellulose Covalent binding Chitin Chitosan Entrapment Ca-alginate Added enzyme (U) 74.4 74.4 74.4 74.4 74.4 74.4 74.4 1% 3% 5% Unbounded enzyme (U) 20.6 13.2 12.8 21.8 20.4 14.9 13.6 37.2 37.2 37.2

atinase activity was in good agreement with other keratinase preparations obtained from Bacillus licheniformis [22], Trichophyton scloenlein [29], Streptomyces thermoviolaceus [11] and Doratomyces microsporus [15]. 3.6. pH stability The pH stability of the free and immobilized A. oryzae keratinase was determined in a pH range of 3.610.7 at room

Immobilized enzyme (U) 34.2 36.2 39.2 28.5 30.4 35.2 36.2 16.2 17.4 16.0

Specic activity of immobilized enzyme (U/mg protein) 118.33 126.97 163.33 89.19 104.29 111.76 156.80 81.00 96.67 64.00

Immobilization yield (%) 63.57 59.15 63.64 54.18 56.30 59.16 59.54 43.55 46.77 43.01

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Fig. 5. Effect of temperature on the activity of the puried free and immobilized keratinase.

temperature for 2 h incubation periods (Fig. 6). The results indicated that high-pH does not decrease keratinase activity of both forms but acidic pH does, this is in good agreement with [23]. 3.7. Thermal stability The results of the present investigation showed also that the free and immobilized enzyme were fairly stable to heat treatment in absence of substrate. At 60 C the immobilized enzyme retained most of its activity after 60 min while the free enzyme retained 58% after the same time of exposure. The free enzyme retained 42.05% of its activity when treated at 70 C for 60 min. At a higher temperature the enzyme retained 28.03% of its original activity by heating at 80 C for 60 min (Fig. 7A). The immobilized enzyme retained 58.92% of its activity when treated at 70 C for 60 min, while at a

Fig. 7. Thermal stability of free (A) and immobilized (B) keratinase from A. oryzae.

higher temperature (80 C) the enzyme retained 37.11% of its original activity by heating for 60 min (Fig. 7B). These results indicate that the enzyme may be considered as thermostable. The half-lives and thermal inactivation rate constants (ki ) of free and immobilized enzyme preparations at 60, 70 and 80 C (Table 6) suggest that the thermal stability of immobilized keratinase increased considerably as a result of immobilization on sintered glass beads. Similar results have previously been reported for other immobilized enzymes [2,17]. The thermal stability of A. oryzae keratinase is higher than the keratinase isolated from Streptomyces sp. K1-02 which showed a stability up to 60 C [21]. The results are also in good agreement with puried keratinases obtained from S. fradiae [34], S. pactum [7] and Bacillus licheniformis [23]. 3.8. Effect of some chemicals on enzyme activity An ANOVA test showed that the different concentrations of each of the tested ions signicantly affected the activity of the puried keratinase from A. oryzae. The enzyme was activated by Ca2+ , Ba2+ , Cu2+ , Na+ , K+ and Mg2+ ions, with Ca2+ showing the highest rank (170.1% 4.62). On the other hand, the enzyme was inhibited by Hg2+ , Cd2+ ,

Fig. 6. pH stability of the free and immobilized keratinase enzyme.

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A.M. Farag, M.A. Hassan / Enzyme and Microbial Technology 34 (2004) 8593 Table 7 Amino acid composition of the puried keratinase obtained from A. oryzae culture Amino acid Aspartic add Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Relative concentration (%) 7.21 5.20 9.44 10.29 1.71 21.70 5.90 0.64 3.30 1.28 2.10 4.92 2.33 3.34 7.14 6.53 6.97

Table 5 Effect of some metal ions on the enzyme activity Substance Relative keratinase activity (%) at different metal ions concentrations of l mM None CaCl2 BaCl2 CuSO4 NaCl KCl MgSO4 7H2 O MnCl2 ZnCl2 HgCl2 CdCO3 PbCl2 EDTA 100.00 170.11 136.16 125.83 122.14 117.34 114.02 100.00 93.36 71.20 61.99 32.84 24.72 10 mM 100.00 120.30 121.77 104.40 110.70 97.05 96.68 91.88 85.61 44.28 34.69 18.45 8.49 100 mM 100.00 116.98 92.62 90.78 100.00 87.09 81.55 70.85 63.10 29.10 19.89 8.85 2.00

Pb2+ and EDTA, with EDTA showing the highest inhibition at 100 mM concentration (Table 5). A paired sample t-test between the three ions leading to highest activation (Ca2+ , Ba2+ and Cu2+ ) and the three ions leading to highest inhibition (Cd2+ , Pb2+ and EDTA) showed that their was a signicant difference between the three ions activating or inhibiting the enzyme (P < 0.0001) except Ca2+ and Ba2+ at 10 mM concentration (P = 0.160). The results are in partial agreement with those obtained by Letourneau et al. [21] and Ignatova et al. [16] who observed a partial inhibition by EDTA of keratinase enzyme from Thermoactinomyces candidus, and noticed a high activation by Ca2+ . The increased activity in the presence of Ca2+ implies that the cation plays an important role in the regulation of enzyme active conformation and in this way increases keratinolytic activity. 3.9. Amino acid analysis The amino acid of the puried preparation obtained from A. oryzae (Table 7) showed that it contained a high pro-

portion of glycine (21.70%). Glutamic acid (10.29%) and serine (9.44%). The amino acid composition of the studied enzyme is partially comparable to other keratinase enzymes obtained by [22,23].

4. Conclusion This work may add some new information on the production of keratinase from Aspergilli. The usefulness of this enzyme preparation in its pure form could be exploited for waste treatment, leather technology, and also as animal feed supplement. The pH tolerance and thermal stability of the immobilized keratinase could be exploited in some industrial applications. For example, a bioreactor with immobilized keratinase can convert ground feathers to peptides and amino acids which can be separated by ltration and ion-exchange chromatography. The enzyme could be useful for industrial processing as it showed a signicant activity against many keratinaceous substrates. Purication and characterization of the enzyme have provided the basis to develop further production in large scale and possible uses of the enzyme preparation. References
[1] Abdel-Naby MA. Immobilization of Aspergillus niger NRC 107 xylanase and B-xylosidase and properties of the immobilized enzymes. Appl Biochem Biotechnol 1993;38:6981. [2] Arica MY, Alaeddinoglu NG, Hasirci V. Immobilization of glucoamylase onto activated pHEMA/EGDMA microspheres: properties and application to a packed-bed reactor. Enz Microbial Tech 1998;22:1527. [3] Asahi MR, Lindquist K, Fukuyama G, Apodaca WL, Epstein, Mckerrow JH. Purication and charaterization of major extracellular proteinases from Trichophyton rubrum. Biochem J 1985;232:13944.

Table 6 Comparison of thermal properties of both free and immobilized keratinase enzyme preparations Property Optimum temperature ( C) Activation energy (kcal/mol) Half-life (mm) 60 C 70 C 80 C Free enzyme 50 12.16 77.30 45.45 30.00 Immobilized enzyme 60 41.86 545.11 60.00 39.00 0.55 103 5.00 103 7.70 103

Thermal inactivation rate constant (min1 ) 60 C 3.88 103 70 C 6.60 103 C 80 10.00 103

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