The use of chicken feather waste as a substrate for the production of keratinolytic enzyme blend has a potential to generate value-added products. In this study, Six fungal isolates were screened for this purpose. Trichoderma viride showed the highest keratinase activity (69 U ml-1) at 5-day incubation period. Three other proteolytic enzymes along with α-keratinase were produced in the medium, alkaline, neutral and acidic proteases enzymes. The alkaline protease showed the highest activity (2453 Anison unit) with weak collagenase activity. Partial purification of crude enzyme showed a purification fold of keratinase enzyme reached 5.99 times with 37% protein recovery at 80% ammonium sulphate saturation using bulk precipitation technique. By applying this fraction on hide specimen it was cleared that it was the most proper for unhairing process.
Keywords: Leather; dehairing; Keratinolytic enzymes; Trichoderma viride
Leather tanning industry considered as one of the highly polluting industries accompanied with bad effect on the environment. The worldwide production 2 of leather was about 24 billion m by 2005 using a huge amount of hazardous chemicals in their processing (about 3.5 million tons). Most of these pollutants are discharged into the effluent. The tannery processes of leather are producing not only high amount of toxic materials but also an offensive odor emanating from the decomposition of proteinous waste © material and the presence of sulphide, ammonia and other volatile organic compounds [1].
Lately, the enzyme blend was used in the tannery processes of leather. Microbial proteases are today used in soaking, unhairing, bating of hides [2]. In this respect, many enzyme preparations were developed for these purposes in the market as lederzim RV, lederzim CA, and Erhavit MC [2,3].
In fact, enzyme blends accomplished many weighty advantages, specially on quality of the final leather product and overcome all the environmental pollution by chemicals and other wastes [2,4].
The microbial isolates (five fungi and one bacterium) were screened for the production of protease blends with special reference to keratinase enzyme. The microbial isolates screened were Aspergillus flavus, Aspergillus niger, Penicillium chrysogenum, Tric hoderma viride, Trichoderma harzianum and Bacillus subtilis. These fungal and bacterial species were isolated from different soil sources of Cairo governorate and identified in Taxonomy Department, AinShams University, Cairo city, Egypt.
The fungal isolates were maintained as single spore on potato-dextrose-agar (PDA) medium, subcultured on PDA o slopes and incubated at 30 C for 7 days. The bacterial isolate B. subtilis was maintained on nutrient agar (NA) medium o and incubated at 37 C for 3 days.
The following media were used and had -1 the following composition (g L ) [5].
Potato slices, 500; dextrose, 10; agar, 20 and distilled H2O.
Peptone, 5; Yeast extract, 1; glucose, 10; MgSO .7H O, 0.5; KH PO , 0.5 and distilled 4 2 2 4 H2O.
fungi or bacteria [6] Milled white feather, 20; (NH )H PO , 1; 4 2 4 MgSO4.7H2O, 2; KCl, 0.2 and pH was adjusted to 7.5.
These were provided as dried rawhide by Radio Tannary-Misr-El Kadeema, Cairo, Egypt. This was pretreated by 25% (w/v) NaCl as preservative, then wetted just before unhairing by 3% (w/v) wetting agent, Egyptol.
This was purchased from the Chemicals Marketing Corp., Cairo, Egypt.
This was purchased from ICN Biochemicals Inc. Ohio, USA.
The fungal inoculum was prepared by cultivation of the fungal strain in growth enhancement medium and the culture was o incubated in bench-top shaker at 30 C for 48 h. 5 ml of inoculums pellets were used for fungal culture inoculation.
Five ml fungal pellets of each fungal culture were transferred to 250 ml Erlenmeyer flask containing 50 ml enzyme blend production medium. The fungal culture was incubated in bench-top o shaker at 30 C, and the culture was lasted for 4, 7 and 9 days. Thereafter, the culture was centrifuged at 6000 rpm to separate the mycelium from the culture filtrate. The clear supernatant was taken for measuring keratinase, protease (acidic, neutral and alkaline) and collagenase activities.
The protein content of the enzyme preparations was determined by the method of Lowry [7].
87 The different protease (acidic, neutral or alkaline) activities were assayed in each culture filtrate as follows; one ml of clear enzyme was mixed with 1 ml of 1.5% (w/v) casein solution in 0.03 M phosphate buffer for acidic (pH 5.7) and neutral protease and of 0.1 M carbonatebicarbonate buffer for alkaline protease (pH 10). The mixture was incubated at 37 o C for 60 min then the reaction was stopped by adding 3 ml of 15% TCA and left for 30 min. at room temperature. The mixture was centrifuged at 4000 rpm for 5 min and the supernatant was used for measurement of released soluble proteins and amino acids released according to Lowry [7]. Control was prepared by adding TCA solution at zero time. One unit of any protease was defined as the amount of enzyme required to liberate 1 microgram of tyrosine during one minute of reaction (Anson unit, AU).
Keratinase activity was determined according to the method of Nickerson et al. [8] as follows, one ml enzyme solution was added to 5 ml borate buffer ( pH 8.5), 50 mg α-keratin, 2.5 ml distilled water and 0.5 ml of 1 mM-MgCl2. The mixture was o incubated for 2 h at 37 C, then stopped by 1 ml of 10% (w/v) trichloroacetic acid and left for 30 min at room temperature, then centrifuged and the released amino acids in the clear supernatant were measured as tyrosine [7]. One unit of keratinase activity was defined as the amount of enzyme required to liberate one microgram tyrosine under the specified conditions.
This was detected by Gover and Ramanthan [9] method using Scanning electron microscope for detection. The surface holes and the opening up of fiber bundles of grain surface which composed of collagen indicates the activity of collagenase enzyme.
The skin specimen was examined using SEM (JEOL/ 1230, Services Central Lab, National Research Centre, Cairo, Egypt) to notice the quality of the skin grain and estimate the effect of collagenase enzyme.
A fresh fleshed cow-hide skin was soaked in sodium chloride solution for one hour then washed twice with tab water. The specimen was treated with a commercial detergent solution (Egyptol) and cut into 2 5 cm pieces then washed twice with sterile water. The hide pieces was sterile with alcohol and left to dry at sterile conditions. A set of conical flasks with different pHs (7, 8.5, 8.9, 10) buffers (50 ml) was prepared and inoculated with 2 ml of crude enzyme. The hide pieces were transferred to these flasks and shaked for o two hours at 37 C in incubated-shaker. The skin pieces were gently scraped with fingers to remove loose hairs. Lime and Na2S were used instead of the protease blend for comparison. At the end of the process, the skin fragments were fixed as described by Lopes et al.[10] and the unhaired leather was evaluated by scanning electron microscope (SEM) analysis to detect the quality of enzyme treated skin.
The enzyme was first precipitated from the cell free supernatant by the addition of ammonium sulphate at different saturation levels ranging from 20-80% (v/v) then stirred for 1 h at 4°C. The o mixture was stored overnight at 4 C and the precipitated protein was collected by 88
centrifugation at 12,000 Xg for 20 min and 4°C then re-suspended in a minimal volume of 0.1M phosphate buffer, pH 8.0 to obtain the concentrated enzyme suspension. The enzyme suspension was finally dialyzed overnight in the same buffer, then dried and stored at -20°C.
The main goal of this study is to produce a potent enzyme blend used in dehairing process of leather to overcome the pollution problems accompanied with the traditional chemical methods without deformation of the grain layer of leather.
Six fungal isolates were screened for study of their abilities to produce keratinase enzyme using the production medium recommended by Cantera [6] using dry ground feather as a sole carbon source and o incubation periods 5,10,17 days at 28 C by static culture method.
Table 1 showed that all fungal strains had the ability to produce keratinase enzyme with different activities. Trichoderma viride showed the highest keratinase activity (69 -1 Uml ) at 5-day incubation period.
The final pHs of all culture was in alkaline range (7.5-8.6) during all incubation periods and were not a significant parameter for keratinase enzyme production.
The protein content of the culture filtrate was increased exponentially with the age of the culture and reached the highest values at 17 days incubation period. There was no clear relation between the protein content and keratinase activity.
The effect of different feather concentrations on keratinase production is displayed in Figure 1. The fungal growth and keratinase production increased with the increase of feather concentration. The optimal concentration was 2.0% at which -1 the highest keratinase activity (69 U ml ) was reached.
The effect of different feather concentrations on keratinase production is displayed in Figure 1. The fungal growth and keratinase production increased with the increase of feather concentration. The optimal concentration was 2.0% at which -1 the highest keratinase activity (69 Uml ) was reached.
These results are matching with those reported with Hamdy & Mohamed et al.[11, 12]. Suntornsuk and Suntornsuk reported that higher concentrations (3 and 5%) might cause substrate inhibition or repression of keratinase production [13]. The effect of different percentages of keratinolytic wastes (feather and cow hair) on the productivity of keratinolytic enzymes is presented in Table 2.
The effect of different percentages of keratinolytic wastes (feather and cow hair) on the productivity of keratinolytic enzymes showed that the cow hair is not suitable for keratinolytic enzymes production and this may be related to the highly crystalline, α- helical structure of the α-keratin of hair containing a high percent of disulphide bonds. In comparison, the chicken feather has β-pleated sheet structure or β-keratin with low percent of disulphide bonds and weak crystalline structure. This vision is matched with Williams & Ferdinand [14, 15].
There were three other proteolytic enzymes along with α-keratinase were produced in the medium, alkaline, neutral and acidic proteases enzymes. The alkaline protease showed the highest activity (2453 Anison unit). Cantera & Feigel pointed to the synergistic action of the alkaline and neutral proteases with α-keratinase in leather unhairing process [16, 17].
To optimize the enzyme blend properties ammonium sulphate precipitation technique was applied for partially purification of the crude enzyme. From the results summarized in Table 3, an extracellular keratinolytic enzymes were precipitated from the culture filtrate of T. viride using different saturation levels of ammonium sulphate ranging from 25-100% saturation. The results showed that the fractional precipitation by 60% ammonium sulfate saturation was the best, showing the highest activity (615.14 U/ml) and specific activity 95.67 U/mg with recovered protein reached 22.97%.
While, the highest activity was achieved at 80% ammonium sulphate saturation using bulk precipitation technique (Table 4); where, the purification fold of keratinase enzyme reached 5.99 times with 37% protein recovery.
Figures 2&3 showed the effect of partial purification by ammonium sulphate on the activity of the other proteases in culture filtrate of T. viride. The highest activities for alkaline, neutral and acidic proteases were found in the precipitate resulted from 80% ammonium sulfate saturation using bulk precipitation technique. On the other hand, the collagenase enzyme showed weak activity (+1) in 60% and 80% saturations, while this activity was slightly increased (+2) in 100% saturation. These results assured that the 80% ammonium sulphate saturation part was the most proper for unhairing process. The purification fold of keratinase enzyme reached 5.99 times more than crude one at 80% amm. Sulphate saturation.
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These results are matched with Abdallah et al.[18] who found that the highest
recovered activity was achieved with 50% saturation ammonium sulphate which exhibited 13.18 fold purification of extracellular alkaline protease produced by S. rochei NRC 24. Also, proteases from S. albidoflavus and S. alboniger are precipitated by 45% and 40% ammonium sulphate saturation, respectively [19, 20]. On the other hand, Al-Askar et al. reported that the maximum precipitation of protease content from the culture filtrate of S. griseorubens E44G was reached at saturation of 70% of ammonium sulphate, giving the highest proteolytic activity and specific activities [21]. Salting out with ammonium sulphate was also reported by many investigators [22, 23].
By applying the crude enzyme produced from T. viride strain on cow leather the unhairing process was completely accomplished in only 24 hour at pH 10 without any deformations in the grain layer of leather using BLC technique (Figure 4) and this assured that there was no collagenase activity. The visual observation showed that the leather specimen kept with its natural features without any deformations in the grain layer and this was assured with SEM examination (Figure 5). These results refer to the success of this enzyme blend in leather industry with good 92 leather quality. In comparison with the crude enzyme the total time of unhairing was lowered by about five hours in 80% ammonium sulphate saturation using bulk
Finally, we can conclude that the partially purified enzyme blend produced from the fungal strain T. viride can be employed in leather industry with low-cost production and high leather quality.
Funding: This study was not funded by any organization.