Purification, Characterization and Immobilization of a Keratinase From Aspergillus Oryzae

Enzyme and Microbial Technology 34 (2004) 85–93

Review

Purification, characterization and immobilizationof a keratinase fromAspergillus oryzae

Aida M. Faraga,∗, Maha A. Hassanba 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 purified from a feather-degrading culture ofAspergillus oryzae. Fractional precipitation of thecrude enzyme with ethanol, acetone and ammonium sulfate yielded 21 fractions. The fraction obtained at 75–85% ammonium sulfatesaturation showed the highest activity and about 3.3-fold purification. This fraction was further purified by gel filtration in Sephadex G-75followed by ion exchange chromatography on DEAE-Sephadex A-50 yielding an active major protein peak showing 11.38-fold purification.Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) indicated that the purified keratinase is a monomeric enzymewith a molecular mass of 60 kDa. The purified enzyme was able to hydrolyze different substrates showing its highest proteolytic activity onbovine serum albumin and casein followed by keratin, chicken feathers, collagen, duck feathers and sheep wool. The purified enzyme wasimmobilized on various carriers. Immobilization on sintered glass beads showed the highest activity. The optimum pH of the immobilizedenzyme shifted to a more neutral range (7.0–7.4) compared with the free enzyme (8.0). The optimum temperature of the reaction wasdetermined to be 60◦C for the immobilized enzyme and 50◦C for the free enzyme. The free keratinase enzyme was retained 42.05% ofits activity at 70◦C (60 min) while the immobilized keratinase preparation showed a higher thermal stability. The half-lives of the free andimmobilized enzyme were 45.45 and 60.00 min, respectively. The pure enzyme was activated by calcium and barium ions while EDTAand 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 hairare insoluble and resistant to degradation by common pro-teolytic enzymes such as trypsin, pepsin and papain be-cause of their high degree of cross-linking by disulphidebonds, hydrogen bonding, and hydrophobic interactions[8,16,28].

Keratinases (E.C. no. 3.4.99.11), a group of proteinaseenzymes, are important for hydrolyzing feather, hair, wool,collagen and casein to clean obstructions in the sewage sys-tem during waste water treatment[14]. These enzymes arealso used or could be applied in the food industry, textiles,medicine, cosmetics and leather and poultry processing in-dustry [6,10]. The structural protein, keratin, can be de-graded by keratinases produced by species of saprotrophic

∗ Corresponding author.

and parasitic fungi ([3,15,30,31,33]), someBacillusspecies([4]) and a few actinomycetes[7,11,21,26].

Many techniques for immobilization of enzymes on differ-ent types of supports have been developed[18,32]. The im-mobilization of proteases on solid supports has been widelyused in many investigations[9,12]. When a protease is im-mobilized, enzyme autolysis is minimized. For industrialapplications, immobilization of the enzyme in gel or solidsupports may offer several advantages such as, repeated useof the enzyme, ease of product separation and improvementof enzyme stability[9,12].

The properties of microbial keratin-degrading enzymesappear to differ according to the producing species of mi-croorganism. This study reports on the purification and char-acterization of a keratinase secreted by an osmoduric strainof Aspergillus oryzaeisolated from marine sediment. Theeffect of immobilization on the obtained enzyme was alsotaken into consideration as an important biotechnologicalaspect.

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

86 A.M. Farag, M.A. Hassan / Enzyme and Microbial Technology 34 (2004) 85–93

2. Materials and methods

2.1. Organism and growth condition

A. oryzaewas isolated from marine sediment of KayetBey, Anfoshi, Alexandria, Egypt. This isolate was identifiedasAspergillusby microscopic examination and further iden-tification with the help of Microcheck Inc. Lab. (Northfield,Vermont, USA) by fatty acid analysis of the fungus. Thestrain was cultivated at 30◦C in a mineral salt medium (g/l):glucose, 3.0; K2HPO4, 2.0; KH2PO4, 1.0; MgCl2·6H2O,0.3; NaCl, 40.0; KCl, 0.5; CaCl2·2H2O, 0.2; FeSO4·7H2O,traces, pH 6.0. Chiken feather was used as carbon, nitrogenand sulphur sources at a concentration of 10 g/l. Cultivationof A. oryzae, for the production of keratinase, was carried outin 250 ml erlenmyer flasks each containing 50 ml of basalmedium of the same composition as that used for isolation.Each flask was inoculated with 2 ml of a spore suspension(2×106 spore/ml) prepared from 5-day old slants of the testorganism. The flasks were incubated at 30◦C in an incuba-tor shaker (160 rpm).

2.2. Protein determination

The amount of protein was estimated by the method ofLowry et al.[24] with bovine serum albumin as the standard.The protein content of the immobilized enzyme was calcu-lated by subtracting the amount of unbound protein from theprotein originally added.

2.3. Determination of keratinase activity

Keratinase activity was determined spectrophotometri-cally according to the method of[5], with a slight modifica-tion. The reaction mixture consisted of 0.5 diluted enzymesolution and 0.5 ml of 0.5% keratin (0.5 g keratin in 0.02 Mphosphate buffer, pH 8) was incubated for 15 min at 50◦C.The reaction was stopped with 1 ml of 10% trichloroaceticacid (TCA) for 30 min at room temperature. This mixturewas centrifuged and the released amino acids measured astyrosine by Lowry method.

One unit of keratinase activity was defined as the amountof enzyme required to liberate 1�mol of tyrosine under thespecified conditions.

2.4. Keratinase production

A. oryzaewas inoculated in 100 ml mineral salts mediumand incubated at 30◦C for 5 days (static culture). The culturemedium was filtered through glass wool to remove residualundegraded feathers, followed by centrifugation to removeany spores and other particles. The enzyme was concentratedfrom the cell-free broth by either salting out with ammoniumsulfate and precipitation with acetone or ethanol. The proteinprecipitate was dissolved in a defined volume of phosphatebuffer (0.02 M, pH 8), and used as partial purified enzyme.

2.5. Partial purification

2.5.1. Acetone or ethanolA.R acetone or absolute ethanol was cooled at 4◦C one

day before starting precipitation. Acetone or ethanol wasadded to the supernatant slowly. Several enzyme fractionswere obtained at 25, 35, 50, 65, 75, 85 and 95% concentra-tion of acetone (ethanol). These fractions were dried overanhydrous calcium chloride under reduced pressure at roomtemperature. Thereafter, each precipitate was dissolved in acertain amount of distilled water and dialyzed against dis-tilled water in a refrigerator for one day. After dialysis, theprotein content and enzyme activity of each fraction weredetermined.

2.5.2. Ammonium sulfate fractionationAmmonium sulfate was added to 100 ml of the culture

filtrate at different concentrations to obtain various fractionsat 25, 35, 50, 65, 75, 85 and 95% saturation. Each precipitatewas dissolved in a certain amount of distilled water anddialyzed against distilled water in a refrigerator overnightafter dialysis.

2.6. Purification of keratinase

2.6.1. Sephadex G-75 fractionationA glass column (2.5 cm × 45.0 cm) was packed with

Sephadex G-75 (Sigma) and equilibrated with 400 ml of0.02 M phosphate buffer at pH 8.0. A flow rate of 60 ml/hwas maintained. The precipitate resulting from ammoniumsulfate fractionation 85% saturation was dissolved in 30 mlof 0.02 M phosphate buffer and dialyzed for 24 h at 4◦Cin 2 l of 0.02 M phosphate buffer after dialysis. A portion(10 ml) was then applied to the Sephadex G-75 gel bed andprotein was eluted with 0.02 M phosphate buffer. Enzymeactivity and protein content in each fraction were measured.Fractions which showed highest protein and keratinaseactivity were collected.

2.6.2. Ion-exchange chromatography DEAE Sephadex A-50A column (2.5 cm×45.0 cm) was packed with a slurry of

diethylaminoethyl (DEAE) Sephadex A-50. The sephadexbed (30 cm long) was equilibrated with 200 ml of 0.02 Mphosphate buffer at pH 8.0. The fractions of highest specificactivity obtained from gel filtration on Sephadex G-75 col-umn pooled and applied to the DEAE-Sephadex A-50 col-umn. Elution was performed with 0.05 M phosphate buffer,followed by 0.05 M NaCl in 0.05 M phosphate buffer at pH8.0, at a flow rate of 60 ml/h. Fractions (5-ml) were col-lected, protein content and keratinase activity for each frac-tion were monitored. The fractions possessing highest spe-cific activity were pooled.

2.7. Polyacrylamide gel electrophoresis

The purified enzyme was analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS–PAGE) ac-

A.M. Farag, M.A. Hassan / Enzyme and Microbial Technology 34 (2004) 85–93 87

cording to Laemmli[20]. A 12.5% separating gel was used.The proteins were stained with a 0.1% solution of Coomasiiebrilliant blue R-250 (Serva, Germany).

2.8. Immobilization methods

2.8.1. Physical adsorptionCharcoal, sintered glass beads and silica gel are used as

carriers for the enzyme. The carrier was incubated with theenzyme solution (74.4 UA. oryzaekeratinase) dissolved in1 ml 0.1 mol/1 phosphate buffer (pH 8.0) at 4◦C over night.The enzyme activity and protein of unbound and immobi-lized enzyme were determined[27].

2.8.2. Ionic bindingAnion exchanger (DEAE-cellulose) equilibrated with

phosphate buffer (0.1 mol/1, pH 8.0), or cation exchanger(Dowex 50W) with Tris–HCl buffer (0.1 mol 1−1 pH 8.0),was incubated with the enzyme solution (74.4 UA. oryzaekeratinase) dissolved in the same buffer for 12 h at 4◦C. Theenzyme activity and protein of unbound and immobilizedenzyme were determined[19].

2.8.3. Covalent bindingChitosan (1 g) was dissolved in 100 ml 0.1 mol/1 HCl con-

taining 2.5% (v/v) glutaraldehyde (GA) for 2 h at 30◦C. Thesolubilized chitosan was precipitated by the addition of 1 ml1.0 mol/1 NaOH. The precipitate was separated by filtration(using a sintered glass funnel) and washed with distilled wa-ter to remove the excess GA. The wet chitosan was mixedwith 5.0 ml of the enzyme solution (74.4 UA. orzyaekerati-nase) and stirred for 1 h at 30◦C. The unbound enzyme wasremoved by washing with distilled water until no proteinor activity was detected[27]. Chitin (1 g) was shaken with10 ml 2.5% (v/v) GA. Chitin was then collected by filtra-tion (using a sintered glass funnel) and washed with distilledwater to remove the excess GA. The wet chitin was mixedwith 5.0 ml of the enzyme solution (74.4 UA. orzyaekerati-nase) for 2 h at 30◦C. The unbound enzyme was removedby washing with distilled water as described before[27].

2.8.4. EntrapmentIn Ca-alginate: 5 ml of 1% or 3% or 5% (w/v) Na-alginate

were mixed with 37.2 U ofA. orzyae keratinase. Theentrapment was carried out by dropping the mixtureinto 25 ml mol/1 CaCl2 solution. The resulting beads(1.0–1.5 mm diameter) were collected and washed withdistilled water to remove the unbound enzyme[1].

2.9. Effect of different substrates

The purified enzyme preparation was incubated in phos-phate buffer (pH 8.0) containing different substrates (feath-ers and wool were dried to a fine powder using an electricmill). Enzyme activity was determined as described beforefor keratinase (as proteolytic activity) and compared withthat 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 immobi-lized enzyme were determined with keratin as substrate. Ker-atinase activity was studied in the pH range of 3.6–10.7, us-ing the following buffers: 0.02 M acetic acid/sodium acetate,pH (3.6–5.4), 0.02 M sodium phosphate buffer (pH 5.6–8.0)and 0.02 M NaHCO3/NaOH (pH 9.2–10.7). The optimumtemperature for keratinase activity was determined by vary-ing the incubation temperature between 25 and 80◦C. Theactivation energies (Ea) of free and immobilized enzymeswere determined from the slope of logarithmic Arrheniusplots (slope=Ea/2.303R whereR (1.976 cal/mol) is the gasconstant.

2.11. pH stability and thermal stability

The free or immobilized enzyme was incubated at variouspH values at 37◦C for 2 h, and then the residual activitywas determined at the optimum pH (8.0 for the free enzymeand 7.4 for the immobilized). The thermal stability of thepurified enzyme preparation was studied at the optimum pH.Identical enzyme solutions in phosphate buffer (0.02 M, pH8.0) 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 substrateand carrying out the enzyme assay under optimum reactionconditions. The first order inactivation rate constant (ki ) wasobtained from the equation lnA = ln Ao −ki t whereAo andA are the initial activity and the activity after a timet (min).

2.12. Effect of some metal ions (activators and inhibitors)on keratinase activity

To investigate the effect of some metal ions on the enzymeactivity, the purified enzyme solution was preincubated for2 h at room temperature with the tested substance (1, 10,100 mM). The residual enzyme activity was measured byadding the substrate and carrying out the enzyme assay underthe optimum conditions. Statistical analysis (one-way anal-ysis of variance (ANOVA) test and a paired samplet-test)were carried out using SPSS 10 Software. The cut-off valuefor statistical significance wasP < 0.05.

3. Results and discussion

3.1. Purification of keratinase

The purification of the keratinase enzyme produced byA. oryzaewas effective and efficient. The crude enzymewas partially purified by fractional precipitation with am-monium sulfate, acetone or ethanol (Table 1). A total of 21fractions were obtained (seven for each of the used precip-itants) and the highest recovered protein was present in the


Table 1Fractional precipitation of keratinase fromA. oryzaecultures using different agents

Agent concentration (%) Ammonium sulfate Acetone Ethanol

PC RP KA RA PC RP KA RA PC RP KA RACrude 62.80 100 26.35 100 62.80 100 26.35 100 62.80 100 26.35 100

25 0.69 1.09 13.95 0.58 0.67 0.59 11.82 0.48 0.35 0.56 18.57 0.3935 0.96 1.53 20.83 1.21 0.85 1.37 16.74 0.87 0.62 0.99 20.16 0.7550 2.30 3.66 28.26 3.93 3.95 6.29 38.48 9.18 3.59 5.72 36.21 7.8565 3.40 5.41 28.82 5.92 6.6 10.51 48.48 19.33 7.33 11:67 47.75 21.1575 5.66 9.01 33.04 11.3 5.95 9.47 33.43 12.02 6.70 10.67 25.37 10.2785 7.38 11.75 88.07 39.27 2.99 6.35 20.90 3.78 1.87 2.98 24.06 2.7295 4.80 7.64 19.58 5.68 1.02 1.62 12.65 0.78 0.82 1.30 12.44 0.62

Total 25.19 40.09 67.89 22.03 36.20 46.44 21.28 33.89 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 to-tal of 40.09%, followed by acetone (36.20%) and ethanol(33.89%). The highest total recovered activity was also ob-tained by ammonium sulfate (67.89%) followed by ace-tone (46.44%) and ethanol (43.75%). Among all the ob-tained fractions, the 85% ammonium sulfate fraction showedthe highest keratinase activity, protein recovery and about3.3-fold purification. Similarly, active keratinase prepara-tions were obtained from thermophilicStreptomyces ther-moviolaceusculture with ammonium sulfate saturation of80%[11].

The partially purified keratinase which was concentratedby precipitation with 85% saturation of ammonium sulfatewas dialyzed and subjected to gel filtration on a SephadexG-75 column. The elution profiles for keratinase and pro-tein from the Sephadex G-75 column are shown inFig. 1and indicate that there are three peaks obtained as shownin Fig. 1. The first peak contains the highest specific activ-ity (211.58 U/mg protein). The most active fractions (num-bers 8–13) from the Sephadex G-75 column were pooled

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

and further purified by DEAE-Sephadex A-50 column chro-matography (Fig. 2). An overall increase in specific activityof 11.38-fold was obtained. This second purification stepyielded a homogeneous protein as shown by SDS–PAGE(Fig. 3). A summary of the purification of keratinase fromthe culture medium ofA. oryzaeis presented inTable 2.

3.2. Characterization of pure keratinase

Some properties of the pure keratinase isolated fromA. oryzaecultures were studied, The apparent molecularmass of the purified keratinase was estimated to be 60 kDaas measured by SDS–PAGE (Fig. 3). The results showedthat the optimum protein concentration was 0.112 mg/mlreaction mixture, while higher values did not increase thereaction velocity. It was also shown that the optimum sub-strate concentration is 5 mg/ml reaction mixture. TheKmandVmax values of the pure enzyme were evaluated from aLineweaver–Burk plot and found to be 8.47± 0.74 mg/mland 71.43± 1.45 U/ml, respectively.


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

3.3. Effect of using different substrates on keratinaseactivity

The purified keratinase has a broad substrate specificityand can hydrolyze a wide variety of soluble and insoluble

Fig. 3. SDS–PAGE of purified keratinase after Sephadex G-75 andDEAE-Sephadex A-50 gel chromatographies (lane E).

protein substrates (Table 3). The soluble substrates bovineserum albumin (BSA) and casein were readily degradableshowing a high proteolytic activity, while insoluble sub-strates such as keratin, chicken feather, duck feather, col-lagen and sheep wool were less susceptible to enzyme hy-drolysis and the proteolytic activity ranged from 45 to 75%of the value obtained for hydrolyzing BSA. These resultsare close to the keratinase obtained fromBacillus licheni-formis [22]. Although the specificity toward soluble peptidesubstrates of the enzyme fromA. oryzaeis high, its abilityto hydrolyze keratins is significant showing relatively highactivity.

3.4. Immobilization of enzyme

Keratinase enzyme fromA. oryzae was immobilizedon various carriers and keratinase activity was evaluated(Table 4). The immobilized enzyme prepared by phys-ical adsorption to sintered glass beads had the highestimmobilized activity (39.2 U/g carrier) and the highest im-mobilization yield (63.64%). Thus, sintered glass beadswere selected as a carrier for further work. Lin et al.[23]used controlled-pore glass beads for the immobilization ofkeratinase isolated fromBacillus licheniformis.

3.5. Effect of pH and temperature on the enzyme activity

Maximum keratinolytic activity of free enzyme was ob-served between pHs 7 and 9 with an optimum at pH 8.0and the optimum pH of the immobilized enzyme was 7.4(Fig. 4). It was observed that the optimum pH of the stud-ied enzyme was lower than that obtained from bacterialcultures of Frevidobacterium pennavoranis[13] and Mi-crosporum canis[25]. The optimum temperature for thepurified free and immobilized enzyme preparations wasstudied. The free enzyme had an optimum temperature of


Table 2Purification of keratinase fromA. oryzae

Purification step Proteincontent

Totalactivity (U)

Keratinase activity(U/mg protein)

Recovery(%)

Purification (fold)

Culture filtrate 314 8275 26.69 100 1Ammonium sulfate (85% saturation) 36.9 3250 88.07 39.24 3.3Sephadex G-75 13.47 2850 211.58 34.44 7.93DEAE-Sephadex A-50 9.09 2760 303.63 33.35 11.38

Table 3Hydrolysis of different proteinaceous substrates by the purified keratinasepreparation

Substrate Keratinase activity(U/mgprotein)

Insoluble proteinChicken feather 274.46Duck feather 248.93Sheep wool 199.11Keratin 331.25Collagen 253.75

Soluble proteinBSA 441.96Cascin 360.00

about 50◦C, whereas that of immobilized enzyme shiftedto 60◦C (Fig. 5). Arrhenius plots of temperature data ap-peared linear, activation energies were found to be 12.16and 41.86 kcal/mol for the free and immobilized keratinase,respectively. The increase in optimum temperature and ac-tivation energy may indicate some change in the physicalproperties of the enzyme molecule. Immobilization of theenzyme on sintered glass might have reduced the confor-mation flexibility, thereby resulting in a higher activationenergy for the molecule to attain the suitable conformationfor binding to substrate. This optimum temperature for ker-

Table 4Immobilization of purified keratinase obtained fromAspergillus oryzaecultures

Carrier Addedenzyme (U)

Unboundedenzyme (U)

Immobilizedenzyme (U)

Specific activity of immobilizedenzyme (U/mg protein)

Immobilizationyield (%)

Physical adsorptionCharcoal 74.4 20.6 34.2 118.33 63.57Silica gel 74.4 13.2 36.2 126.97 59.15Sintered glass 74.4 12.8 39.2 163.33 63.64

Ionic bindingDowex 74.4 21.8 28.5 89.19 54.18DEAE-cellulose 74.4 20.4 30.4 104.29 56.30

Covalent bindingChitin 74.4 14.9 35.2 111.76 59.16Chitosan 74.4 13.6 36.2 156.80 59.54

EntrapmentCa-alginate 1% 37.2 16.2 81.00 43.55

3% 37.2 17.4 96.67 46.775% 37.2 16.0 64.00 43.01

Fig. 4. Effect of pH on the activity of the purified free and immobilizedkeratinase.

atinase activity was in good agreement with other keratinasepreparations obtained fromBacillus licheniformis[22], Tri-chophyton scloenleinü[29], Streptomyces thermoviolaceus[11] andDoratomyces microsporus[15].

3.6. pH stability

The pH stability of the free and immobilizedA. oryzaekeratinase was determined in a pH range of 3.6–10.7 at room


Fig. 5. Effect of temperature on the activity of the purified free andimmobilized keratinase.

temperature for 2 h incubation periods (Fig. 6). The resultsindicated that high-pH does not decrease keratinase activityof both forms but acidic pH does, this is in good agreementwith [23].

3.7. Thermal stability

The results of the present investigation showed also thatthe free and immobilized enzyme were fairly stable to heattreatment in absence of substrate. At 60◦C the immobilizedenzyme retained most of its activity after 60 min while thefree enzyme retained 58% after the same time of exposure.The free enzyme retained 42.05% of its activity when treatedat 70◦C for 60 min. At a higher temperature the enzyme re-tained 28.03% of its original activity by heating at 80◦C for60 min (Fig. 7A). The immobilized enzyme retained 58.92%of its activity when treated at 70◦C for 60 min, while at a

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

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

higher temperature (80◦C) the enzyme retained 37.11% ofits original activity by heating for 60 min (Fig. 7B). Theseresults indicate that the enzyme may be considered as ther-mostable. The half-lives and thermal inactivation rate con-stants (ki ) of free and immobilized enzyme preparations at60, 70 and 80◦C (Table 6) suggest that the thermal stabil-ity of immobilized keratinase increased considerably as aresult of immobilization on sintered glass beads. Similar re-sults have previously been reported for other immobilizedenzymes[2,17]. The thermal stability ofA. oryzaekerati-nase is higher than the keratinase isolated fromStreptomycessp. K1-02 which showed a stability up to 60◦C [21]. Theresults are also in good agreement with purified keratinasesobtained fromS. fradiae[34], S. pactum[7] and Bacilluslicheniformis[23].

3.8. Effect of some chemicals on enzyme activity

An ANOVA test showed that the different concentrationsof each of the tested ions significantly affected the activityof the purified keratinase fromA. oryzae. The enzyme wasactivated by Ca2+, Ba2+, Cu2+, Na+, K+ and Mg2+ ions,with Ca2+ showing the highest rank (170.1%± 4.62). Onthe other hand, the enzyme was inhibited by Hg2+, Cd2+,


Table 5Effect of some metal ions on the enzyme activity

Substance Relative keratinase activity (%) at differentmetal ions concentrations of

l mM 10 mM 100 mM

None 100.00 100.00 100.00CaCl2 170.11 120.30 116.98BaCl2 136.16 121.77 92.62CuSO4 125.83 104.40 90.78NaCl 122.14 110.70 100.00KCl 117.34 97.05 87.09MgSO4·7H2O 114.02 96.68 81.55MnCl2 100.00 91.88 70.85ZnCl2 93.36 85.61 63.10HgCl2 71.20 44.28 29.10CdCO3 61.99 34.69 19.89PbCl2 32.84 18.45 8.85EDTA 24.72 8.49 2.00

Pb2+ and EDTA, with EDTA showing the highest inhibitionat 100 mM concentration (Table 5). A paired samplet-testbetween the three ions leading to highest activation (Ca2+,Ba2+ and Cu2+) and the three ions leading to highest in-hibition (Cd2+, Pb2+ and EDTA) showed that their was asignificant difference between the three ions activating orinhibiting the enzyme (P < 0.0001) except Ca2+ and Ba2+at 10 mM concentration (P = 0.160). The results are in par-tial agreement with those obtained by Letourneau et al.[21]and Ignatova et al.[16] who observed a partial inhibition byEDTA of keratinase enzyme fromThermoactinomyces can-didus, and noticed a high activation by Ca2+. The increasedactivity in the presence of Ca2+ implies that the cation playsan important role in the regulation of enzyme active confor-mation and in this way increases keratinolytic activity.

3.9. Amino acid analysis

The amino acid of the purified preparation obtained fromA. oryzae(Table 7) showed that it contained a high pro-

Table 6Comparison of thermal properties of both free and immobilized keratinaseenzyme preparations

Property Free enzyme Immobilizedenzyme

Optimum temperature (◦C) 50 60Activation energy (kcal/mol) 12.16 41.86

Half-life (mm)60◦C 77.30 545.1170◦C 45.45 60.0080◦C 30.00 39.00

Thermal inactivation rate constant (min−1)60◦C 3.88× 10−3 0.55 × 10−3

70◦C 6.60× 10−3 5.00 × 10−3

80◦C 10.00× 10−3 7.70 × 10−3

Table 7Amino acid composition of the purified keratinase obtained fromA. oryzaeculture

Amino acid Relative concentration (%)

Aspartic add 7.21Threonine 5.20Serine 9.44Glutamic acid 10.29Proline 1.71Glycine 21.70Alanine 5.90Cystine 0.64Valine 3.30Methionine 1.28Isoleucine 2.10Leucine 4.92Tyrosine 2.33Phenylalanine 3.34Histidine 7.14Lysine 6.53Arginine 6.97

portion of glycine (21.70%). Glutamic acid (10.29%) andserine (9.44%). The amino acid composition of the studiedenzyme is partially comparable to other keratinase enzymesobtained by[22,23].

4. Conclusion

This work may add some new information on the pro-duction of keratinase from Aspergilli. The usefulness ofthis enzyme preparation in its pure form could be exploitedfor waste treatment, leather technology, and also as animalfeed supplement. The pH tolerance and thermal stability ofthe immobilized keratinase could be exploited in some in-dustrial applications. For example, a bioreactor with immo-bilized keratinase can convert ground feathers to peptidesand amino acids which can be separated by filtration andion-exchange chromatography. The enzyme could be use-ful for industrial processing as it showed a significant activ-ity against many keratinaceous substrates. Purification andcharacterization of the enzyme have provided the basis todevelop further production in large scale and possible usesof the enzyme preparation.

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Purification, Characterization and Immobilization of a Keratinase From Aspergillus Oryzae