Research Journal of Microbiology

Year: 2010 | Volume: 5 | Issue: 1 | Page No.: 21-35
DOI: 10.17311/jm.2010.21.35

Keratinase Production and Biodegradation of Some Keratinous Wastes by Alternaria tenuissima and Aspergillus nidulans

W.I.A. Saber, M.M. El-Metwally and M.S. El-Hersh

Abstract: The rapid growth of poultry industry has linked with increased output of keratin containing wastes. Keratinous wastes can be readily fermented to useful products and commodity chemicals by the appropriate microbes. The present research concerning biodegradation of keratinous wastes. From 82 fungal isolates, 27 isolates have keratinolytic activity. Identification tests indicated that the potent isolates were Alternaria tenuissima K2 and Aspergillus nidulans K7. Using chicken feather powder as a sole source of carbon and nitrogen, keratinase productivity were 53.4 and 55.8 U mL^-1 by Alt. tenuissima K2 and A. nidulans K7 at the 6^th and 5^th day of incubation, respectively. Using additional carbon and nitrogen sources were not found to promote keratinase productivity, except when using starch and maltose. pH 7.5, 35°C and 7.5% inoculum ratio were the best for both keratinase production and feather solubilization by both fungi. Among different keratin containing wastes, chicken, duck and goose feathers were the most degradable keratinous wastes by Alt. tenuissima K2 and A. nidulans K7. During the course of investigation, keratinase production and degradation of keratinous wastes were positively and significantly correlated. Incubation of the produced keratinases at the optimum pH (8.5) and temperature (40°C) with different keratinous wastes led to about 70% hydrolysis of chicken, duck, goose and turkey feathers after 24 h of incubation. Goat hair, sheep wool and buffalo horn showed lower response towards keratinolytic hydrolysis. Therefore, keratinous wastes can be biologically degraded by either isolated fungi or their keratinases into useful products.

Keywords: Keratinous wastes, keratinse, biodegradation, Alternaria tenuissima and Aspergillus nidulans

INTRODUCTION

Keratinous wastes constitute a troublesome environmental contaminant that is produced in large quantities in commercial poultry processing plants and their utilization is of economic value as well as ecological significance. Feather wastes make a serious problem as environmental pollutant and recently in outbreaks of H5N1 virus. Currently, feather waste is utilized on a limited basis as a source of nitrogen for plants or as a dietary protein supplement for animal feedstuffs, prior to its use for that purpose, feathers were chemically treated to increase the digestibility and reduce the rigidity; this procedure has a disadvantage, in that, certain heat-sensitive amino acids, such as methionine, lysine and tryptophane are degraded. The degradation products may generate non-nutritive amino acids, such as lysinoalanine and lanthionine. That is why the enzymatic biodegradation may be a better alternative to improve their nutritional value and offers cheap and mild reaction conditions for the production of valuable products (El-Naghy et al., 1998; Gushterova et al., 2005; Marcondes et al., 2008). The main content of feather is keratin, which are insoluble proteins resistant to degradation by common proteolytic enzymes such as trypsin, pepsin and papain because of a high degree of cross-linking by disulphide bonds, hydrogen bonding and hydrophobic interactions (Ignatova et al., 1999; Marcondes et al., 2008). Many fungi especially that belongs to fungi imperfecti have high keratinolytic activity including the following genera: Chrysosporium, Aspergillus, Alternaria, Trichurus, Curvularia, Cladosporium, Fusarium, Geomyces, Gleomastis, Monodictys, Myrothecium Paecilomyces, Stachybotrys, Urocladium, Scopulariopsis, Sepedonium, Penicillium and Doratomyces (Santos et al., 1996; Gradisar et al., 2000; Farag and Hassan, 2004; Marcondes et al., 2008). However, the isolation of filamentous fungi that efficiently degrade keratinous wastes is very interesting.

Therefore, the utilization of keratinous wastes as a fermentation substrate (carbon and nitrogen sources) by keratin-degrading fungi offers a feasible microbial technology for obtaining keratinolytic enzymes. These enzymes, besides waste feather elimination, could find their application in the food industry, manufacturing of textiles, biodegradable films, glues and foils, cosmetics, leather industry and nitrogenous fertilizer for plants (Onifade et al., 1998; Schrooyen et al., 2001; De Toni et al., 2002; Gushterova et al., 2005).

This study is a report on isolation, screening and optimizing conditions of keratinolytic fungi for biodegradation of keratinous wastes using native isolates and their keratinases.

MATERIALS AND METHODS

Isolation and Screening of Keratinolytic Fungi on Agar Plates
Samples (10 g each) collected from chicken farm wastes containing decayed feathers were serially diluted in 90 mL sterilized tab water, shaken at 100 rpm for 10 min the resulted suspension was used as a source for fungal isolation on agar plates medium, which contain (g L^-1); agar; 15, MgSO₄.H₂O; 0.5, KH₂PO₄; 0.1, FeSO₄.7H₂O; 0.01 and ZnSO₄.7H₂O; 0.005, the pH was adjusted to 7.5. The medium was supplemented with 1% chicken feather powder as a sole source of carbon and nitrogen (Wawrzkiewicz et al., 1991). Keratinolytic activity of the fungi was detected as a clear zone around the colony after incubation up to 5 days at room temperature. The diameter of the clear zone was measured to quantify activity. By this way, 82 fungi were isolated. The most active fungi were identification according to Domsch et al. (1980).

Preparation of Chicken Feather Powder
Poultry feather was cut into small fragments washed extensively with water and detergent and dried in a ventilated oven at 40°C for 72 h. To prepare feather powder, the feathers were milled in a ball mill and passed through a small mesh grid to remove coarse particles. This feather powder was used during optimization of keratinase production, unless otherwise stated.

Inoculum Preparation
Spore suspension of the fungal isolates was prepared by adding 10 mL of sterilized water to 7 days old fungal isolates growing on plates of potato dextrose agar. The final concentration of the spore suspension was adjusted to about 2x10⁶ mL^-1.

Culture Technique and Tested Parameters
In 500 mL Erlenmeyer flasks, 100 mL of the previously mentioned broth basal medium of Wawrzkiewicz et al. (1991) was added separately to 1.5 g of poultry feather powder in each flask then autoclaved. After cooling, the flasks were inoculated by 5 mL of spore suspension and incubated under shaking (120 rpm) at 28°C. At periodic intervals, final culture pH was determined and the cultures containing the hydrolysates were centrifuged (2000 rpm) and filtered through filter paper. The filtrate was recovered to determine the keratinase activity. The residual which containing the cells and undigested feather was dried to a constant weight. The feather solubility was determined as: (The dry weight of residual feather after incubation/Initial dry weight of feather)x100. For optimization studies, the following parameters were investigated; (1) time course of keratinase production (1 to 10 days), (2) the supplementation of the production medium with additional different carbon sources at 1% (w:v) which were sterilized separately and added aseptically to the sterilized medium, (3) additional nitrogen sources at 0.5% (w:v), (4) different values of initial medium pH (5.0 to 9.0) which was adjusted before autoclaving (5) different incubation temperatures (20 to 50°C) and (6) inoculum ratio (2.5 to 20%, v/v).

Degradation of Different Keratinous Wastes by the Isolated Fungi
For studying the biodegradation of different keratinous materials, the keratinous wastes (chicken feather, duck feather, goose feather, turkey feather, sheep wool, goat hair and buffalo horn) were fragmented into pieces with about 1 cm long and added to the fermentation media as a sole source of carbon and nitrogen instead of poultry feather powder. These sources were added separately to the fermentation media at 0.5, 1.0, 1.5 and 2%, w/v. All other optimum conditions were used. Keratinase activity and the percent of keratinous waste solubilization were determined after incubation.

Determination of Keratinolytic Activity
Keratinase activity was assayed by the modified method of Yu et al. (1968). In brief, 20 mg of chicken feathers powder were suspended in 3.8 mL of 100 mM Tris-HCl buffer (pH 7.8) to which 300 μL of the culture filtrate (enzyme source) was added. The reaction mixture was incubated at 37°C for 1 h. After incubation, the assay mixture was dipped into ice-cold water for 10 min and the remaining feathers were filtered out. Then the absorbance of the clear mixture was measured at 280 nm using UV-spectrophotometer. The keratinase activity was expressed as one unit of the enzyme corresponding to an increase in the absorbance value 0.01 h^-1.

Keratinase Activity as Influenced by pH and Temperature
The effect of pH (5 to 10.5) on the keratinase activity was determined in 0.1 mol L^-1 sodium acetate (pH 5.0 and 5.5), 0.1 mol L^-1 phosphate (pH 6.0 to 7.5), 0.1 M Tris-HCl (pH 8.0 to 9.0) and 0.1 M borate (pH 9.5 to 10.5) buffers. The effect of temperature on the keratinase activity was measured at temperatures ranging from 25 to 65°C.

Hydrolysis of Keratin Containing Wastes by the Crude Enzyme
Keratinous wastes were cut into small fragments, washed extensively with water and detergent and dried in a ventilated oven at 40°C for 72 h, milled in a ball mill and sieved to remove coarse particles. 500 unites of enzyme was added to 0.5 g of each of the milled keratinous materials in 45 mL of 0.1 M Tris-HCL buffer, pH 8.5 and incubated at 40°C in a shaking water bath at 130 rpm for up to 24 h. Controls of keratinous materials were done in the same conditions, using culture filtrate previously boiled for 5 min. The dry weight of the remaining keratinous materials was determined on membrane filters (pore size, 0.2 mL) after drying at 105°C for 12 h (Moreira et al., 2007).

RESULT AND DISCUSSION

Isolation and Identification of Keratinolytic Fungi
Concerning the isolation trials, 82 fungi belonging to more than six genera were isolated from chicken farm wastes containing decayed feathers on agar plates containing feather keratin (Table 1). The primary screening showed that most of the isolated fungi were able to grow in the given environmental conditions. Some of them showed weak growth but did not clarify the keratin agar, presumably due to the lack of extracellular keratinase. Meanwhile, the others (27 isolates) exhibited growth and clear zone on agar plates. It appeared that Alternaria, Aspergillus, Fusarium, Penicillium and Trichoderma were the most common active keratinolytic genera, representing more than 88% of the total positive keratinolytic fungi. The results are in accordance with those obtained by Friedrich et al. (1999).

As described by Onifade et al. (1998) and Farag and Hassan (2004), most of keratinolytic fungi are belonging to fungi imperfecti. The identification and characterization of new fungal species able to degrade keratinous wastes may help to understand the role of fungi in the degradation of complex keratinous substrates in nature, In addition, the ability of such fungi to grow and to produce appreciable levels of keratinase using keratinous wastes as substrates could be potentially useful for the development of biotechnological methods aiming to obtain useful hydrolysis products (Gioppo et al., 2009).

Screening of the Most Active Keratinolytic Fungi
Based on the previous results, the most active isolates on keratin plates, were identified as Aspergillus nidulans K7, Fusarium culmorum K23, Alternaria tenuissima K2, Aspergillus niveus K11, Aspergillus flavus K6, Trichoderma viride K63, Alternaria alternate K1 and Penicillium sp. K40, the corresponding clear zone diameter on feather keratin agar plates were 43, 42, 41, 41, 35, 26, 16 and 14 mm, respectively. These isolates were also quantitatively tested in submerged culture for keratinolytic activity using poultry feather powder as the only source of carbon and nitrogen (Fig. 1). It was observed that the highest activity was reached with A. nidulans K7, Alt. tenuissima K2, A. niveus K11 and F. culmorum K23, which have the highest extracellular keratinase production as they recorded 45.2, 41.7, 39.8 and 38.3 U mL^-1, respectively.

In this respect, Marcondes et al. (2008) found that among 106 filamentous fungi isolated from poultry farm waste, 13 species belonging to seven genera (Aspergillus, Acremonium, Alternaria, Beauvaria, Curvularia, Paecilomyces and Penicillium) were able to grow and produce keratinase in stationary cultures using poultry feather powder as the only substrate.

Friedrich et al. (1999) screened 300 common fungi for synthesis of extracellular keratinase, about 54% of the fungi grew on agar plates with soluble keratin and excreted the enzyme. Some representatives of Fusarium, Acremonium and Geotrichum were the most active and others (A. flavus, Alt. radicina, Trichurus spiralis and Stachybotrys atra) proved to be powerful producer of extracellular keratinases when cultivated in submerged conditions in a medium with porcine nail as the sole source of carbon and nitrogen. Some species of fungi are already known to colonize feathers or wool and some, such as Alt. alternata and Alt. tenuissima, A. flavus, Stachybotrys chartarum and Trichurus spiralis, are known as protease producers (Domsch et al., 1980). In the present research, quite important differences were detected among isolated fungi, hereby; A. nidulans K7, Alt. tenuissima K2, A. niveus K11 and F. culmorum K 23 were selected according to their keratinolysis.

Time Course of Keratinase Production by Selected Fungi
After the previous screening, the selected fungi were cultivated in submerged shaken cultures. As shown in Fig. 2, A. nidulans K7 and Alt. tenuissima K2 started with relatively high activity in first day (13.8 and 10.2 U mL^-1, respectively), with gradual increase to reach their maximum activities at the 5^th day for A. nidulans K7 (55.8 U mL^-1) and 6^th day for Alt. tenuissima K2 (53.4 U mL^-1), with slight decrease in activity to reach 45.2 and 39.8 U mL^-1 at the 10^th day for A. nidulans K7 and Alt. tenuissima K2, respectively. On the other hand, A niveus K11 and F. culmorum K23 started with low level of activity i.e., 3.3 and 6.6 U mL^-1 and reached to the maximum activity after 6 and 7 days of incubation, recording 44.5 and 45.6 U mL^-1, respectively. Noronha et al. (2002) found the maximum yield of keratinase after 3 days by A. fumigates, meanwhile, Gradisar et al. (2000) and Gioppo et al. (2009) reported 4 days as the optimum incubation period and they considered this period as a very short time, whereas, 5 days was found to be the optimum for Penicillium sp. keratinase production (El-Gendy, 2009).

It is apparent (Fig. 2) that there was a change in the final culture pH of the medium towards alkalinity. The four fungal isolates showed significant positive correlation coefficient (r) between pH and production of keratinase, the values of r ranged from 0.88 to 0.98 at p = 0.01. Several studies (Malviya et al., 1992; Kaul and Sumbali, 2000; Muhsin and Hadi, 2001) documented that saprophytic fungi grown on keratinous substrates have the ability to extract nutrients from such substrates; apparently, there is a change in the pH of the media towards alkalinity after keratinase production and protein releasing by the tested fungi. The ability of such fungi to alkalinize culture media, can be explained by accumulation of ammonium (the products of deamination) and other keratin degradation products (Marcondes et al., 2008; Gioppo et al., 2009) this variation in pH towards alkalinity also suggest keratinolytic activity.

From the previous results and according to keratinase production, A. niveus K11 and F. culmorum K23 were excluded from this investigation.

Additional Carbon and Nitrogen Sources
Most of keratinolytic fungi can utilize feather as the sole source of carbon and nitrogen. To improve the enzyme production, many additional carbon sources were examined. In Table 2, the highest keratinolytic activity and feather solubilization were recorded with starch and maltose. It is worthy to note that, with the exception of starch and maltose, the extra addition of carbon sources to the fermentation medium led to remarkable reduction in feather solubilization (Fig. 3), in spite the obvious improvement in keratinase production. On the other hand, CMC and pectin negatively modulated keratinase production and feather solubilization in both fungal isolates. This means that this enzyme is inducible. In general, enzyme production is not totally repressed by the tested carbon sources and the major regulatory mechanism is induction of keratinase production by the substrate. These results are in accordance with the findings of Ignatova et al. (1999) and Ramnani and Gupta (2004). Simple sugars have been reported to suppress the synthesis of keratinase (Moreira et al.,2007).

Mabrouk (2008) reported that starch and glucose highly inhibited keratinase production by Streptomyces sp. MS-2. However, in some organisms the opposite was found, since increasing in keratinase production with the extra addition of carbon sources was previously reported (El-Naghy et al., 1998; Anbu et al., 2008; Son et al., 2008). The absence of carbon source drive the fungus to assimilate the keratin as carbon source which increase the percentage of solubilization as the keratinolytic organisms are capable of using keratin as the sole source of carbon and nitrogen (Szabo et al., 2000; Gousterova et al., 2005). Malviya et al. (1992) on Chrysosporium queenslandicum and El-Naghy et al. (1998) on Chrysosporium georgiae noted that the keratinase enzyme was inducible by keratin and its production was stimulated by glucose and inhibited by ammonia.

Concerning the use of nitrogen sources different from keratin, the extra addition of nitrogen sources have no or depressive effect on keratinase production as well as solubilization of faether keratin (Table 3 and Fig. 4).

Among nine different nitrogen sources, NH₄H₂PO₄, ammonium citrate ((NH₄)₂C₆H₆O₇), NH₄Cl and KNO₃ highly inhibited keratinase production and feather solubilization by Alt. tenuissima K2 and A. nidulans K7. These data confirm the inducible nature of keratinase. Malviya et al. (1992) and El-Naghy et al. (1998) noted that the keratinase enzyme was inhibited by ammonia. Santos et al. (1996) reported that additional nitrate supported the mycelial growth, but it repressed keratinase production in A. fumigates. However, most reports describe a partial or complete repressive effect of the supplementation of cultures with small nitrogen molecules (Malviya et al., 1992; El-Naghy et al., 1998; Son et al., 2008). However, the drastic reduction in the keratinase production is due, or at least overwhelmingly due to catabolite repression by the nitrogen sources (Gioppo et al., 2009). Contrary, when Veselal and Friedrich (2009) used microorganisms directly for the biodegradation of different keratin containing wastes, less solubilization of keratinous wastes could be observed than with the use of additional carbon and nitrogen sources since the microorganism itself consumes the released products. An inductive effect of soybean meal on keratinase production has been also reported to occur (Gradisar et al., 2000).

Effect of pH on Keratinase Production
Regarding to the keratinase production vis pH, Fig. 5 shows that Alt. tenuissima K2 and A. nidulans K7 yielded the maximum amount of keratinase as well as feather solubilization at pH 7.5 and declined, thereafter, with the initial medium alkalinity. Keratinase production and feather solubilization was positively and significantly correlated (r = 0.99 and 0.98, at p = 0.01, respectively). Several reports are available on keratinolytic microorganisms and their biotechnological potential with respect to keratinases production at pH 6.0 to 9.0 (Gupta and Ramnani, 2006; El-Gendy, 2009) with high activity at 8.0 for Trichophyton sp. HA-2 (Anbu et al., 2008). However, very few microorganisms were reported to be active above pH 11.0 (Gessesse et al., 2003).

Keratinase Production in Relation to Incubation Temperature
As depicted in Fig. 6, the keratinolytic activity was detectable between 20 and 50°C, but showing a maximum activity at 35°C for both Alt. tenuissima K2 (80.8 U mL^-1) and A. nidulans K7 (79.7 U mL^-1). At the same manner, feather solubilization (%) increased with temperature and recorded 44.8 and 46.5%, respectively, at 35°C. Both isolates still show a positive significant correlation between keratinase production and feather solubilization. These results are similar to that reported for keratinases of other microorganisms, such as Trichophyton sp. (Anbu et al., 2008), Streptomyces pactum (Bockle and Muller, 1997) and Bacillus licheniformis (Lin et al., 1992), the above-mentioned studies reported optimum temperatures near 40°C. Whereas, El-Gendy (2009) reported that 26°C was the optimum for Penicillium sp.

Keratinase Production at Different Inoculum Ratios
Inoculum ratio is one of important factor affecting keratinase production and feather solubilization. The pattern of keratinase production with respect to inoculum ratio indicated that, with the gradual increase in inoculum ratio, Alt. tenuissima K2 and A. nidulans K7 showed positive improvements in their activities and reached the maximum keratinase productivity (83.2 and 80.9 U mL^-1, respectively) and feather solubilization (56.3 and 53.4%, respectively) at 7.5% inoculum ratio (Fig. 7), both of the tested criteria, slightly, decreased when inoculum ratios were out of this point. Larger inoculum ratio has been shown to affect adversely the yield of keratinase by Beauveria bassiana (Suresh and Chandrasekaran, 1999) and Penicillium sp. (El-Gendy, 2009). Data (Fig. 7) of the analysis of correlation of coefficient continue providing evidence for the significant positive correlation between keratinase production and feather solubilization for both isolates. Conversely, Singh (1997) did not find any relation between the rate of enzyme production and keratin degradation.

Biodegradation of Keratinous Wastes by Alt. tenuissima K2 and A. nidulans K7
Different keratinous wastes were used as a sole source of carbon and nitrogen in the growth medium of the tested fungi. The fungi under investigation were able to grow normally, using all keratin-containing wastes as their sole source of carbon and nitrogen (Tables 4 and 5). Generally, A. nidulans K7 possessed better keratinase production and solubilization percent than Alt. tenuissima K2 on different keratinous wastes. Chicken feather recorded high ability to be degraded by both fungi compared to the other keratin sources. Additionally 1.5% of different keratinous wastes was found to be optimum for the highest keratinase production and waste solubilization percent. Moreover, the mathematical relation between enzyme production and biodegradation of keratinous wastes at 1.5% waste concentration is depicted in Fig. 8. It is clear the positive correlation coefficient which shown on the linear regression; as the enzyme production increased, remarkable degradation of keratinous wastes was induced by both fungi. Alt. tenuissima K2 recorded positive significant correlation while, A. nidulans K7 recorded positive non-significant correlation. Marcondes et al. (2008) found similar results.

The complete mechanism of keratin degradation is not fully understood. Basically, microbial keratinolysis is a proteolytic, protein-degrading process for the simple reason that keratin is a protein (Gupta and Ramnani, 2006). The high mechanical stability of keratin and its resistance to proteolytic degradation is due to the tight packing of the protein chains through intensive interlinkage by cystine bridges (Bockle and Muller, 1997). The capability of filamentous fungi to degrade keratin may be the result of a combination of extracellular keratinase, mechanical keratinolysis (mycelial pressure and/or penetration of the keratinous substrate), sulphitolysis (reduction of disulphide bonds by sulphite excreted by mycelia) and proteolysis (Bockle and Muller, 1997; Onifade et al., 1998; Gupta and Ramnani, 2006). Enzymatic or chemical reducing agents in form of disulfide reductases, sulfite, thiosulfate or cellular membrane potential may play a significant role in the degradation of this insoluble protein, additionally, the initial attack by keratinases and disulfide reductases may allow other less specific proteases to act, resulting in an extensive keratin hydrolysis (Gioppo et al., 2009).

Effect of pH and Temperature on Enzymatic Reaction
The keratinolytic activities of the culture fltrates of both fungi were detectable over a wide range of pH and temperature. Keratinase activity was found to be better in the alkaline conditions ranging from pH 8.0 to 9.0 for Alt. tenuissima K2 and from pH 7.5 to 9.0 for A. nidulans K7, with an optimum at 8.5 for both enzymes (Fig. 9). Over a wide range of temperatures (from 25 to 65°C), keratinase showed maximum activity at 40°C (Fig. 10). Keratinase from A. nidulans K7 was found to be more active at the higher temperatures than keratinase of Alt. tenuissima K2.

Keratinases from most bacteria, actinomycetes and fungi have pH optima in neutral to alkaline range (Riffel et al., 2003; Farag and Hassan, 2004; Thys et al., 2004; Anbu et al., 2005). Other keratinolytic enzymes have been also proved to be active at alkaline pH, as those secreted by Streptomyces sp. (Letourneau et al., 1982) and Streptomyces albidoflavus (Bressolier et al., 1999). However, few keratinases possess extreme alkalophilic optima of pH>12 (Gupta and Ramnani, 2006). Moreover, the optimum temperature of keratinases ranges from 30 to 80°C (Gupta and Ramnani, 2006) but, the enzyme from Chrysosporium keratinophilum (Dozie et al., 1994) and thermophile Fervidobacterium islandicum (Nam et al., 2002) showed exceptionally high temperature optima of 90 and 100°C, respectively.

Degradation of Keratinous Wastes by the Crude Keratinase
The crude culture filtrates of both fungi were able to catalyze the biodegradation of native keratin of different keratinous wastes. At pH 8.5 and 40°C, both enzymes were able to hydrolyze efficiently keratin of all tested feather (Table 6). The degradation of different keratinous wastes by the two tested enzymes increased with the prolongation of time up to 24 h. More than 70% hydrolysis of chicken, duck, goose and turkey feathers was observed, after 24 h of enzyme-waste incubation. Goat hair, sheep wool and buffalo horn poorly hydrolyzed and showed lower response towards keratinolytic hydrolysis. The keratinolytic activity of crude enzyme was confirmed because the action of enzyme into poultry feather meal reduces its weight (Moreira et al., 2007). The keratinolytic activity of microorganisms is normally associated with the production of serine proteases or metalloproteases, irrespective of the microorganism (Gupta and Ramnani, 2006), with the exception of yeasts, which produce keratinases belonging to the aspartic proteases (Monod et al., 2002). The lower level of degradation of buffalo horns and goat hair at the expense of higher release of enzyme might be due to the restricted substrate specificity of the enzyme or removal of some accessory proteins capable of splitting the disulphide bonds present in the keratin molecules during the hydrolysis process (Singh, 1997).

Finally, in Egypt like other many countries with high population and limited resources, recycling of wastes like keratin containing wastes is very important, in this study A. nidulans and Alt. tenuissima showed good result in this trend. The association of cheap and readily available keratinous wastes could result in a substantial reduction in the costs of enzyme production. Additionally, the hydrolysis of such wastes provide beneficial product that could find their application in several industries e.g., biodegradable films, glues and leather as well as in agriculture as nitrogenous fertilizer for plants.

REFERENCES