Optimization of keratinase production by keratinolytic fungus Chrysosporium tropicum and its potentiality in bidegradation of chicken feathers

The current study has been designed to investigate the possible biodegradtion of native keratin by fungi isolated from chicken feathers collected from chicken farms in Egypt. Out of 56 fungal isolates 23 species and 2 species varieties showed different degrees of keratinolytic activity, with Chrysosporium tropicum showing the highest keratinase productivity (23.15 U/ml) as well as the highest value of specific activity for keratinase (197.90 U/mg protein) indicating a great degree of purity for the enzyme. Our present study indicates that the highest production of keratinase by C. tropicum was recorded at pH 8 and a temperature of 35 °C during a period of 20 days. In addition the optimal value of ionic strength for keratinase production was 100 mM NaCl. Monitoring the influence of constituents deprivation from basal salt medium on production of keratinase enzyme showed that K+, Fe2+, Mg2+, Ca2+ and Zn2+ were necessary for keratinase production. Sulfur deprivation has no significant influence on keratinase production. Keratinase activity of C. tropicum showed that, the optimal values of temperature and ionic strength were 35–40 °C and 80 mM NaCl, individually. The optimum pH range was (7.8–9). These promising findings demonstrate a high potentiality of C. tropicum keratinase in keratin proteolysis, suggesting that keratinase producing isolates of C. tropicum may be suitable as a biodegradation agent.


Introduction
Keratinases (E.C. 3.4.99.11) are proteolytic inducible enzymes produced by different microorganisms when they growing on substrates containing keratin. Microbial keratinases attracted much attention, because their multiple industrial applications in different areas including feed, detergent, fertilizer, leather and pharmaceutical industries [1][2][3]. Mature chicken possess around 5-7% feathers from their total weights. The large quantities of the discarded chicken feathers during industrial poultryprocessing represent environmental hazard responsible for environmental pollution. Keratins as substrates are insoluble and resistant against digestion by animal, plant and different known microbial proteases, hindered the utilization of feathers as a source of valuable products [4]. In an attempt to overcome this problem, enzymatic fungal degradation of feathers may introduce a possible resolution and provide an available alternative for improving feather utilization. Some of saprophytic and parasitic fungal species possess the capability of keratin degradation [5][6][7][8][9][10]. Using natural materials such as chicken feather during new biofertilizers development has attracted more attention of research interest [11]. The application 1 3 51 of nitrogen-rich organic amendments in organic farming improves plant growth rate of and stimulates the soil microflora activity. Feather meals are rich in nitrogen (15% N), cheap and easily obtainable can be used as a potential biofertilizer [2]. Application of microorganisms to hydrolyze feathers has several advantages over than steaming process as fertilizer including, high nutrition content, simple production and economically efficient, therefore these microorganisms may considered to be a possible alternative for providing nitrogen sources in fertilizer application [11]. Microbiological decomposition of poultry biowastes during storage causes liberation of some toxic gases such as ammonia and hydrogen sulfide [12]. In addition it has been observed that the increase in pH is a good indicator of keratinolysis, as it simultaneously reflects the utilization of keratin proteins, deamination, and ammonia production during chicken feather decomposition by fungi [13]. One of the microorganisms those involved in this degradation process is the fungal genus Chrysosporium. It has been demonstrated that some Chrysosporium species isolated from soil are characterized by high mineralization potential towards native keratin [14][15][16]. Although many keratinolytic enzymes have been isolated over the years, the exact and precise mechanism of keratin biodegradation is not fully understood [17,18]. However, keratin biodegradation is thought to be composed of enzyme's adsorption to the macromolecule's surface followed by the proper catalytic action that can be further divided into reduction in disulfide bonds, (sulfitolysis) and disruption of the peptide chain (proteolysis) [19]. This study aims to isolate and characterize highly keratinase-producing fungi from chicken feathers, optimize the production of keratinase from the most powerful fungal isolate, and convert feather wastes into soluble amino acids used as animal feed additives or biofertilizers.

Isolation and maintainace of fungi
From chicken farms in Egypt, keratinolytic fungi were isolated from chicken feathers. Fungal species isolated during this current study have been cultivated in pure culture and identified macro-and micromorphologically via systematic studies [20,21].The fungi were maintained in Sabouraud Dextrose Agar medium (SDA) at 4 °C for the next work.

Keratinase activity assay
The enzyme activity of Keratinase was detected according to Yu et al. [22] with some changes. Reaction mixture containing 50 mM Tris-HCl buffer (pH 7.8), 20 mg powder of keratin and 1 ml of fungal filtrate containing crude extracellular keratinase in a total volume of 4 ml. This reaction mixture was left for 4 h at temperature 37 °C under shaking and thereafter the liberated amino acids during proteolysis of keratin were detected by the ninhydrin method as described by Muting and Kaiser [23]. Unit of enzyme activity was identified as equal to the amount of enzyme that catalyzes the liberate of 1 μg amino acids per hour. The specific activity of keratinase was defined as the number of keratinase units per mg extracellular protein.
Total amount of extracellular protein produced by the keratinolytic fungi was measured in extracellular crude enzyme according to Bradford [24].

Keratinase production optimization
After screening of fungal species for keratinase production and specific activity of enzyme, Chrysosporium tropicum has been chosen for consequent investigations (temperature, incubation period and initial pH) for production of keratinase and keratinase activity.

Metal ions dependency and ionic strength
The dependency of keratinase production on ionic strength has been monitored by using several concentrations of salt varying from 0.0 to 2 M NaCl in basal salt medium (BSM). The fungus C. tropicum was grown in BSM medium as a control and the same medium deprived of K + , Ca 2+ , Mg 2+ , Zn 2+ or Fe 2+ at both the optimum temperature and initial pH. After 2 weeks of incubation period the yield of keratinase produced was measured.

Screening of fungal keratinase production in liquid medium
Screening of fungal species for keratinase production in this current study has been designed of the of fungi in submerged culture and obtained results have been summarized in Table 1. Twenty three and 2 species varieties among 105 fungal isolates showed different levels of keratinolytic activities when cultured on chicken feather medium and 80 fungal isolates exhibited no keratinolytic activity (data not shown). Five fungal isolates among 25 keratinolytic isolates were considered to be highly keratinase producers. Within the first group, the highest activity was recorded by C. tropicum followed by Chrysosporium keratinophilum, Scopulariopsis brevicaulis, Aspergillus fumigatus and A. terreus. While the second group includes producers with moderate activity for keratinases production such as Absida corymbifera, Acremonium strictum, Aspergillus flavus, A. flavus var. columinaris, Botryotrichum piluliferum, Fusarium oxysporum, F. solani, Penicillium funiculosum, Scopulariopsis brumptii and Trichoderma harzianum. The rest of fungal isolates show low keratinase production, as presented in Table 1. The fungus C. tropicum showed the highest productivity value for keratinase (23.15 U/ml) and also the highest keratinase specific activity (197.90 U/mg protein). This indicates a greater purity of the C. tropicum extracellular keratinase, so it has been selected for further optimization of keratinase production and activity experiments. Among common fungi the keratinolytic activity is widespread and many fungal species release enzymes into the surrounding environment and can be used as producers of keratinolytic enzymes. The non-dermatophytes kerationlytic fungi attract much of research interest due to safety approach in fungal handling and manipulation.
In this current investigation, five efficient keratinase producing non-dermatophytic fungal species have been isolated. Among the non-dermatophytic fungi, keratinases possessing attractive biochemical characteristics were reported to be produced by Aspergillus [10,25,26], Trichoderma [27], Doratomyces [28], Myrothecium [29], Paecilomyces [30], Scopulariopsis [31], also Acremonium, Alternaria, Beauveria, Curvularia, and Penicillium [32]. Chrysosporium tropicum strain, showed outstanding activity in comparison with other fungi which showed the highest keratinase productivity (23.15 U/ml) and also the highest specific activity for keratinase was (197.90 U/mg protein), this indicates a higher purity of the C. tropicum extracellular keratinase. El-Naghy et al. [33] reported that the degradation of chicken feathers by 28 different fungal species indicated that fungi belonging to the genus Chrysosporium were the most active in degrading feathers, and C. georgiae was the most active one. More recently the keratinolytic activity of C. tropicum has been assessed [34]  The keratinase production was measured after 2 weeks of inoculation of fungi H high production of keratinase (more than 10 U/ml), M moderate production of keratinase (6-10 U/ml), L low production of keratinase (less than 6 U/ml)

Optimization of keratinase production
During keratinase production process C. tropicum showed an optimum pH ranging from 6 to 9 (Fig. 1A), this finding indicate that the fungus productivity is not influenced by the increase in pH value of the medium due to ammonium release. The incubation temperature influence on the production of keratinase by C. tropicum was presented in (Fig. 1B), maximum keratinase enzyme production was recorded at 35 °C. After 2 days of incubation period, Keratinase production was measured. The maximum productivity has been detected after 20 days (Fig. 2A). Study of the dependency of keratinase production on the ionic strength was conducted; maximum keratinase production was observed at (100 mM NaCl) and declined as NaCl concentration was increased in the culture medium (Fig. 2B). By depriving the medium of each constituent individually, we studied the influence of the constituent BSM on keratinase production. Keratinase production was not significantly influenced by sulfur deprivation (Fig. 2C). Deprivation of K + or Zn 2+ strongly decrease enzyme production indicating that these elements are necessary for keratinase enzyme production. While deprivation of Ca 2+ , Mg 2+ or Fe 2+ moderately affected the keratinase production. In this current study, during the incubation period of 20 days, the highest production of keratinase enzyme(s) was observed from C. tropicum.. Within the incubation time range of 2-20 days, the production of keratinase increased proportionally. Thereafter, production of keratinase has declined. The optimum production of keratinase enzyme(s) was measured within an incubation temperature of (35 °C). The optimum pH value for keratinase production ranges from 6 to 9, pointing out that the fungus productivity is not affected by the increase in pH value of the medium due to ammonium release as suggested by Gupta and Ramnani [2]. El-Naghy et al. [33] found that, optimum degradation of feather keratin by Chrysosporium georgiae was obtained after 21 days at pH 6 or pH 8 and an optimal incubation temperature of 30 °C. The optimal concentration values of NaCl for keratinase production by C. tropicum is 100 mM NaCl, and then it decreased by increasing NaCl concentration in the culture medium. Sulfur deprivation did not show significant effect on the keratinase productivity by C. tropicum, this might be attributed to the release of amino acids containing sulfur and sulfahydral groups from keratin degradation which might provide the fungus with its sulfur requirements for growth and keratinase production, as reported by Gupta and Ramnani [2]. In spite of strong effect of Zn 2+ or k + on keratinase production, deprivation of Ca 2+ , Mg 2+ or Fe 2+ had moderate effected. It has been reported that Ca 2+ and Mg 2+ cause a threefold increase in the enzymatic activity [35]. These metal ions possibly engaged with the stability of the tertiary structure conformation of metalloproteases and protect these enzymes against autoproteolysis [36,37].

Kinetics of extracellular keratinase activity of C. tropicum
Optimum pH value for keratinase activity was 7.8, however keratinase activity slightly decreased at higher pH values. In contrast, the keratinase activity decreased sharply at low pH values (Fig. 3A). The optimum temperature for C. tropicum keratinase was 35-40 °C (Fig. 3B). The optimal ionic strength for C. tropicum keratinase activity was 80 mM NaCl. The keratinase activity sharply decreased at ionic strength higher than 100 Mm NaCl (Fig. 3C). As the enzyme under investigation showed an optimum pH range of 7.8-9, it can be classified as alkaline keratinase.
In agreement with our results, keratinase isolated from some other fungi such as Scopulariopsis brevicaulis, Microsporum canis, Doratomyces microsporus and Trichophyton marquandii were also reported as alkaline enzymes [28,30,38,39]. It has been reported that keratinase activity from Trichophyton vanbruseghemii was optimum at pH 8 and remained high in the range of pH 7-11 [40]. The optimum temperature for C. tropicum keratinase was 35-40 °C. Keratinases from Scopulariopsis brevicaulis and Trichophyton sp. HA-2 showed similarity in optimum temperature 35-40 °C [38, 41]. The optimal ionic strength for keratinase activity was 80 mM NaCl. By increasing NaCl concentrations the keratinase activity sharply reduced, perhaps by inhibiting its interaction with keratin substrates. Moreover, NaCl concentration of more than 100 mM sharply reduced both growth and keratinase production by C. tropicum. The decrease in keratinase activity at high ionic strength might indicate an electrostatic interaction between the enzyme and its keratin substrate which would be adversely affected by high ionic strength. The activity of the major keratinolytic serine Proteinase of Streptomyces albidoflavus significantly decreased at NaCl concentration of more than 0.5 M which was attributed to the adverse effect of 55 high ionic strength on the electrostatic interaction of the enzyme with its keratin substrate [42]. Our results were in agreement of an earlier report on keratin degradation [43,44]. According to the current results, the feather hydrolyzate containing various amino acids could be utilized as feed additives or fertilizers. Also it has been found that amino acids play key roles in living organisms, there has been an increased demand for amino acids to be used in many areas such as pharmaceutical drug manufacturing, feedstuffs and feed additives. It is known that feather keratin can be converted to feedstuffs, fertilizers, and edible films or used for the production of the rare amino acids serine, cyteine and proline [4,45,46].

Conclusion
In conclusion, C. tropicum might be considered to be a potent keratinase producing fungus. The keratinase production optimization and characterization analyses revealed potential characteristics for its probable applicability in commercial keratin degradation process and conversion of keratin wastes to feedstock additives and effective soil fertilizer.
Funding The authors declare that no funding was received during the preparation of this manuscript.
Data availability Our manuscript has no associated data.

Conflict of interest
The authors have no relevant financial or nonfinancial interests to disclose.
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