Feather Biodegradation For Feather Protein Lysate/ Feather Meal Mutual Formations: Optimization Using Crude YNDH Protease/Keratinase; Enzyme Partial Purication and Characterization

Incubation parameters used for the production of a protein lysate from enzymatically degraded waste feather using crude keratinase produced by laceyella sacchari YNDH was optimized using RSM, amino acids quantication was estimated as well. The optimization resulted in total soluble protein 2089.5µg/ml. The optimal conditions were, time 20.2h, feather concentration 3g% and keratinase activity 24.5U/100ml at pH, 10; and cultivation temperature 50 o C. The FPL was found enriched with essential and rare amino acids. In parallel, this YNDH enzyme group had been partially puried and some characteristics of it were studied. Crude enzymes were rst concentrated with Amicon Ultra 10k centrifugal lter, and then concentrated proteins were applied to "QFF" strong anion column chromatography. The partially puried enzyme has an estimated molecular masses ranged 6 to 10 kDa. The maximum enzyme activity was observed at 70°C and pH 10.4 when measured by both casein and keratin azure as substrates. Interestingly, keratinolytic activity of this group was not affect by EDTA, PMSF and DTT. Generally the overall characters of this group protease/keratinase nearly the same when its activity was measured with both substrates suggesting that all these 3 protein bands working together as a group of keratinases. which demonstrated, production of enzymatic feather-lysate enriched with amino acids was optimized using YNDH-keratinase enzyme. The application results indicating the great potential of produced enzymes in diverse biotechnological processes


Introduction
Keratin is predominantly found in tissues of reptiles, birds, amphibians, and mammals. He structural component of feathers, hair, nails, horns, hooves, bones, furs, claws, hides, bird beaks, skin, wool, scales, and bristle is made up of keratin Although keratin processing is important for a wide range of applications, due to the large volumes of waste generated, e cient disposal of keratinous waste is of particular concern to the poultry and leather industries.
In the poultry industry, feathers are a signi cant by-product since they account for 5-7% of the The Chicken's body Weight and constituted of 91% of keratin protein, 8% water, and 1% of lipid 1 .Approximately several million tonnes of feathers are estimated to be created annually from the global poultry industry 2 .
Feathers have elevated keratin content, and consideration should be given to using this protein source. Conventional methods of degrading feathers such as alkali hydrolysis and steam pressure cooking not only destroy the amino acids but also require a huge amount of energy 3,4 . A cost-effective alternative way may be through biodegradation of the feathers from microorganisms using keratinase. Interestingly and recently, many potential applications have been linked to keratinase and related products 5,6 ; such as feather meal production which apply as a supplement for feeding stuffs 6,7 .
The demand for industrial enzymes is estimated at USD 4.61 billion in 2016 and is expected to rise at a compound annual growth rate (CAGR) of 5.8 per cent between 2017 and 2022 and to cross USD 6.30 billion by 2022 8 . The unique characteristic that distinguishes keratinases from other pro-teases is the ability to bind to the complex and insoluble sub-strates (feathers, wool, silk, collagen, elastin, horns, stratum cor-neum, hair, azokeratin and nails) (8). Although the mechanism of enzyme adsorption is not yet well understood, it is known that the higher the adsorption capacity, the higher the degree of keratin hydrolysis 9 . In recent years, demands for keratinolytic proteases have increased in industrial applications such as feeding stuffs, fertilizers, detergents and textiles 10 .
The physical (e.g. steam pressure cooking) and chemical (e.g. alkaline hydrolysis) methods currently used for the conversion of feathers, in addition to being ecologically problematic and energy-intensive, are subject to loss of nutritionally essential amino acids [11][12][13] . Thus, feather biodegrdadation by microb /or enzyme using Keratinases formed by many microorganisms are considered to be an environmentally friendly approach to keratin waste recycling 6,7 .

Materials And Methods
Enzyme production and préparation Protease/keratinase enzyme was produced in the optimized medium as reported previously by Goda 36 . After 48h fermentation time; broth was collected in a beaker and kept at room temperature in static condition for 2h. Most of the bacterial cells were settled along with feather at the bottom of beaker, supernatant was decanted and then ltered through 0.2μ lters (MDI, India) using vacuum pump. This micro ltered supernatant was concentrated and used as crude enzyme, where it was using lyophilization technique, where the lyophilized enzyme was dissolved in glycine NaOH buffer pH 10 and stored at 4°C. Concentrated enzyme was used for feather degradation and feather meal production after required dilution.

Lyophilization
Lyophilization technique was carried out (lyophilizer; acculab, USA) to concentrate feather-lysate or enzyme in a powder form.

Procurement of feather
They procured chicken feather from the local market; were washed with detergent, and some washes with tap water accompanied by distilled water added detergent. Washed feather had been dried for 8h at 50°C and used for subsequent experiments.
Optimization process by Response surface methodology (Box-Behnken Design) Box-Behnken experimental design has been implemented in the present work to maximize the nutritional content mainly soluble protein of feather-lysate during the processing of the feather meal creation. Cleaned dried feathers were used as a biological substrate for tested YNDH protease/ keratinase crude enzyme, where the waste was processed in glycine NaOH buffer pH10 and incubated with enzyme at 50°C.
The Response surface methodology was used to optimize the protein content of feather-lysate generated due to enzyme action. Two kinds of precipitates could be recognized, strong precipitate is rendered the feather meal after degradation of the waste-feather by enzyme, and the transparent supernatant constitutes the condensed soluble protein. An enhancement the protein concentration of lysate is considered the main aim of Box-Behnken Design (BBD) and reach to the end point of degradation (amino acids). Three variables (incubation time, substrate concentration and enzyme concentration were studied at three levels (high, medium and low) which were signi ed by +1, 0 and -1; respectively, as shown in Table 1 37 .
The design matrix consisting of 14 trials shown in Table (1), was created to study the interaction between  signi cant variables affecting feather degradation by the tested crude YNDH protease/keratinase enzyme; regarding soluble protein concentration as a response (μg/ml). Aliquots were collected at the stated time, centrifuged for 10min at 10,000rpm, and then the protein content of clear supernatant was measured at 750nm (μg/ml) after subtracting from the blank which was taken at zero time. All cultures were conducted in triplicate, and the ndings were used on average. This optimization method includes three key steps: conducting the statistically designed experiments, estimating the standardized mathematical model's coe cients and predicting the model's answer and testing its adequacy 38 .

Enzymes activity
Protease activity was carried out according to Anson 39 using bovine casein as substrate and standard curve of tyrosine. While, keratinolytic activity was carried out according to the method of Suntornsuk 40 using keratin azure as a substrate.

Total protein estimation
Lowery method was applied to estimate the total soluble protein concentration using standard curve of bovine serum albumin 41 .

Amino acid analysis
Amino acid contents of soluble protein lysate were determined using amino acid analyzer (chromatogaphy columns and consumables catalogue (SYKAM)

Consolidation technique
For usage of feather-lysate enriched with soluble protein as an animal supplement in adequate form, it was compressed into a dense solid using the Cold Pressing (CP) technique (Hydraulic Pressing Machine used for cold Pressing Consolidation). For cold pressing of protein lysate, a simple hydraulic press with 10 mm die is used, where protein lysate content in a powder form is put in a die, normally made of stainless steel, under a hydrostatic pressure of 5 tons (i.e. 0.6 GPa) and that pressure is sustained for 6 min.
Anion-exchange chromatography for protein puri cation The concentrated enzyme solution (10ml) was loaded into anion exchange ("QFF" strong column) coupled with a 20mM Tris base buffer pH 8.5 using an automated ÄKTA Prime Plus method for the puri cation of proteases/keratinase. The column was washed off with 5 volumes of balancing buffer columns to remove the unbound proteins. The column-bound proteins were eluted with a linear gradient (0-100M) NaCl in 20mM Tris base buffer pH 8.5 at a ow rate of 1 ml/min and fraction size of 5ml. Fractions containing keratinolytic protease were pooled, desalinated and concentrated by the Amicon-10 ultra ltration concentrator (20 kDa cut off membrane).

SDS-polyacrylamide gelelectrophoresis and zymography
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to test the protein purity and to detect the protein molecular mass as de ned in 42 at 12%, 15% and 17% polyacrylamide concentrations where a protein standard was used and running under the same conditions. After running, the gel was stained with Coommassie brilliant blue R-250 at 12% SDS and silver stain at 15 and 17% SDS 43 . Zymography is a simple and sensitive electrophoretic approach that can be used by Leber 44 to image protein bands of protease with keratinolytic activity as transparent bands against the dark background, following a simple renaturation and staining procedure.

Enzyme characterization
The partially puri ed enzyme was characterized by studying the effects of temperature and pH on the activity and stability. Also, effects of some metal ions, surfactant, inhibitors and detergents were tested. The effect of substrate concentration (casein and keratin-azure) were also elucidated Effect of temperature and pH To study the effect of temperature on the protease/keratinase enzyme activity; the reaction mixture was incubated at different temperatures ranging from 40 up to 90ºC and 50 up to 90ºC in 100mM glycine-NaOH buffer, pH 10, using casein and keratinazure as substrate, respectively. The enzyme activity at different temperatures was determined and expressed in percentage relative to maximum value which constitutes 100%.
While, to study the effect of pH on activity the reaction mixture (enzyme -casein as substrate) was incubated at different pH values (7.6-11.6). The substrate was dissolved individually in 100mM of buffers: tris-HCl (pH 7.6-8.2), glycine-NaOH (pH 9.6-10), NaHCo 3 -NaOH (pH 10-10.8), Na 2 HPO 4 -NaOH (pH 11.2-11.6). The enzyme activity at different pH values was determined and expressed in percentage as relative activity, where the reaction mixture was incubated at optimum temperature for 10min. When keratin-azure was used as substrate, it was dissolved individually in 100mM buffers: glycine-NaOH (pH 9.6-10), NaHCo 3 -NaOH (pH 10-10.8). The enzyme activity at tested alkaline pH values was determined and expressed in percentage relative activity, where the reaction mixture was incubated at optimum temperature for 30min.
Thermal and pH stability Thermal stability was assayed by incubating the studied partially puri ed enzyme at temperature ranging from 50-70ºC. The enzyme was pre-incubated at temperatures 50 and 55 ºC, samples were withdrawn every hour along 24h, whereas at 60 to 75 ºC; samples were withdrawn every 10min up to 240min. The enzyme activity was measured after exposure to these conditions using casein and keratin azure as substrates and expressed in percent residual activity. To study the pH stability, the enzyme was pre-incubated for 1h at room temperature at different pHs (9.6-10) & (10-10.8) using glycine NaOH and, NaHCo 3 -NaOH, respectively. Aliquots of the mixtures were taken each 10min to measure the residual enzyme activity (%) in reference to the control (untreated enzyme), where the optimum conditions of temperature and pH were applied in the reaction mixtures. pH stability was tested for all samples using casein and keratin azure as substrates.
Effect of some (metal ions), (surfactant, solvents), (activators, inhibitors) Effect of some metal ions on enzyme activity of partially puri ed enzyme was investigated using CaCl2, MgSO4, ZnSO4, HgCl2, CuSO4, KCl, MnCl2, FeSO4, NiSO4, CoCl2, and FeSO4 at 1, 5 10 ,15 and 20 mM nal concentration. The enzyme was pre-incubated with the tested metal ion for 15min at room temperature. Where, the effects of some surfactants as Triton X-100, Tween 20, 40, 80 and SDS on enzyme activity were tested at 0.1, 0.5, 1 and 2% nal concentration and some of solvents such as ethanol, methanol and glycerol on protease activity was tested at 1 and 2% nal concentration. Also, the in uence of some inhibitors such as Phenyl Methyl Sulfonyl Fluoride (PMSF), Di-Thio-Theritol (DTT) and Ethylene Diamine Tetra Acetic acid (EDTA) on enzyme activity was investigated at 1, 5 and 10mM nal concentration. The enzyme was pre-incubated with each chemical (either solvents or inhibitors) for 15 min at room temperature; then the residual activity was determined as mentioned before using casein as substrate for all concentrations and the more promising conc. ( nal conc.) was tested using keratin-azure as substrate. The enzyme activity without any tested metal ion/or agent was taken as 100%, each under optimum conditions of temperature and pH.
Effect of different substrate concentration (casein and keratin azure) on enzyme activity Kinetic constants (V max and K m ) were determined using Lineweaver-Burk double reciprocal (1/v versus 1/S) plot 45 ,by assaying xed amount of tested enzyme with varying nal concentrations of casein substrate (0.1-2%) prepared in 100mM NaHCo3-NaOH buffer pH (10.4) incubated for 10min at 70°C. and for keratin azure as substrate by assaying xed amount of tested enzyme with varying nal concentrations of keratin-azure substrate (1mg-10mg) prepared in 100mM NaHCo3-NaOH buffer pH (10.4) incubated for 30min at 70°C.

Results
Optimization the enzymatic degradation of waste feather for FPL production and feather meal formation using YNDH protease/keratinase crude enzyme.
In order to achieve the optimum response area for maximizing the protein contents of enzymatically degraded feather in form of concentration (µg ml -1 ), the signi cant independent variables [X1; time (h), X2; feather concentration (g), X3; keratinase activity (U/ml)] were further explored, each at three levels. Table 1 represents the design matrix of the variables in coded units with the experimental results of the protein lysate concentration. All cultures were conducted in triplicates and the observations averaged.

Multiple regression analysis and ANOVA test
The three variables with fourteen trails were analyzed using linear multiple regression analysis approach and the percentage con dence rates (percent) were determined. The p-value from the ANOVA analysis for each response was determined to analyze the relationship between the variables at 90% or higher con dence level and the analysis of variance using ANOVA test in Box-Behnken experiment was generated and summarized in Table 2 which gives p=0.0235. Since the p-value indicated in the ANOVA Table is less than 0.05, it is concluded that there is a statistically signi cant relationship among the studied variables at 95% con dence level (p=0.05). The value of the determination coe cient R 2 =0.953 for protein contents (concentration) of enzymatic feather-lysate, being a measure of t of the model, indicates that about 4.7% of the total variations are not explained by protein contents concentration. The adjusted R-squared statistic is 0.85. Presenting experimental results in the form of surface plots (Fig. 1) show that higher levels of protein contents concentration were attained with the coded values 0.52 of time, 1 of substrate concentration and 0.457 of enzyme concentration. The optimum values of the three variables analyzed as obtained from the maximum polynomial model point were calculated using the JMP programme and found to be: time, 20.2h; substrate concentration, 3g/100ml and enzyme concentration, 24.57U/100ml with prediction calculated protein contents concentration equal to 2159.85 µg /ml. Bench scale experiments showed that Y value of 2089.5 µg ml -1 was obtained, this means the calculated model accuracy was 96.7 %. In this study a statistical technique, of Box-Behnken design was shown to be e cient and reliable in selecting the statistically signi cant factors and nding the optimal concentrations of those factors. Thefore, the following conditions are considered to be near the maximum, based on the results obtained from the Box-Behnken method: time 20.2h, substrate concentration, 3g%; enzyme concentration, 24.57U%; pH 10 and cultivation temperature 50 o C, the protein contents concentration measured was 2159.85 µg/ml. Finally we can conclude that, protein contents of enzymatically degraded feather by effect of keratinase from Laceyella sacchari strain (YNDH) has been systematically improved during various experimental designs compared with basal medium.

Amino acid analysis of feather meal
In this experiment the feather lysate were analyzed for free amino acids released during feather degradation in both cell free supernatant after cultivation of laceyella sacchrii YNDH in optimized medium and feather hydrolysate produced due to enzymatic treatment as shown in Table 3 (A and B), respectively. 16 amino acids were detected in both experiments and quanti ed as shown in Table 3. Presence of essential amino acids like leucine and isoleucine indicates that feather treatment with the native isolate YNDH and YNDH crude keratinase enzyme provided feather lysate enriched with rare amino acids. The present study clearly shows that the degradation of the feather by YNDH keratinase provided by Laceyella sacchari is not only economical but also a viable method for the better use of the much disregarded feather wastes. Additionally we observed that the concentrations of all different types of amino acids produced through treatment of feather using keratinase developed by laceyella sacchari YNDH was signi cantly higher than that in feather hydrolysate produced by the native organism directly.

Separation and collection of feather meal and protein lysate
After optimization of enzymatic degradation of waste feather by YNDH protease/keratinase crude enzyme by Response surface methodology (Box-Behnken Design) the feather meal was settled down, centrifuged and dried at 50°C, where, the supernatant containing protein lysate was lyophilized and consolidate the powder as shown as shown Figure 2.
Protease /keratinase puri cation The cell free supernatant developed after cultivation was collected, added to Amicon Ultra centrifugal lter (cutoff of 10kDa) to reduce the volume and remove protein impurities. This step is considered a partial puri cation step where it increased enzyme speci c activity from 166.28U/mg to 296.38U/mg and puri cation fold by 1.78 as shown in (Table 4).
For ion exchange chromatography the working pH should be at least one higher than the PI of the protein. At this pH value the protein will possess a net charge high enough to bind well to ion exchange resin. The concentrated and desalted cell free supernatant was subject to "QFF" strong anion column chromatography which was equilibrated with 20mM Tris-base buffer pH 8.5. The elution was performed with 1M NaCl in the same buffer.
The elution pro le (Figure 3) indicates that the protease with keratinolytic activity was detected in fractions 6-8.
Upon using "QFF" strong anion column, enzyme was puri ed to 11.31 fold with speci c activity and recovery of 1881.3 U/mg and 14.38%, respectively ( Table 4). The pooled fractions were loaded and run on 12, 15 and 17% SDS-PAGE to detect the purity. Fig. 4 illustrates the pattern of protein pro le before and after puri cation indicated by reduction in proteins bands to a 3 protein bands at molecular weight of (6 to 10) kDa (Fig. 4).
Characterization of the partially puri ed protease/keratinase enzyme Certain characteristics of the partially puri ed enzyme under study were evaluated using casein and keratinazure substrates. Some parameters such as optimum temperature and pH, thermal and pH stability, some enzyme inhibitors/ activators, surfactants, detergents, metal ions, and substrate concentration were investigated.

Temperature and pH optimum
The effect of temperature on the partially puri ed enzyme was studied by measuring the activity at different temperature values from 40 to 90ºC with an interval of 5ºC and 50 to 90ºC with an interval of 10ºC using casein and keratin-azure as substrate, respectively. The results presented in Figure 5 showed that the optimum temperature of the partially puri ed enzyme when measured as both protease and keratinase activity was 70ºC, so it can be classi ed as a thermo active protease with keratinolytic activity and the three isolated bands could be worked together as a group of keratinase.
While, the effect of pH on partially puri ed enzyme activity was investigated by determining of the enzyme activity, rst as protease at pH values (7.6-11.6); and second as keratinse at pH values (9.6-10.8). The pH activity pro le of the enzyme (protease/keratinase) indicates that it has a broad pH range with an optimum at pH 10.4 as shown in Fig. 6.

Thermal and pH stability
In the present experiment, The effect of temperature on the thermal stability of partially puri ed enzyme was studied by exposing it to a range of temperatures (50-55ºC) for 24h and (60,70 and75 ºC) for 4h then the residual activity was calculated where (100%) is considered the activity of enzyme kept at room temperature.
The enzyme activity was measured as described before and expressed in percent residual activity using casein/keratin-azure substrates (Data not shown).
In summary, the results showed that the enzyme was highly activated (238.46% as protease and 185.4% as keratinase) at temperature 50ºC, moreover it still steady at 55ºC up to 24h. However, the enzyme possessed its stability until reached 90min at 60ºC however, it lost about half of its activity at temperature 70ºC (the optimum T for enzyme activity) after 60min. Moreover it was less stable when exposed to higher temperatures (75ºC).
The pH stability of the partially puri ed enzyme was investigated as protease and keratinase by measuring the residual activities after incubating the enzyme at pH ranging from 9.6 to 10.8 at room temperature for 60min with time interval of 10min and using casein and keratin azure as substrates. The obtained results indicated that the enzyme was highly stable in all tested pHs (Data not shown).
Effect of some metal ions,detergent, surfactant, solvents, and activators/ inhibitors The effect of some metal ions on the partial puri ed protease enzyme activity was examined by measuring the residual activity in the presence of (1mM, 5mM, 10mM, 15mM and 20mM) of each metal ion. All metal ions at concentrations (1, 5 and 10mM) signi cantly activated protease activity while Hg +2 and Cu +2 inhibited the enzyme to 3.3% and 18.3%, respectively when measured as protease. Moreover, the most signi cant metals activated the proteolytic activity are Mg +2 and Fe +2 where, Mg +2 signi cantly increased protease activity to 274.477 and 232.11 at 15mM and 20mM, respectively. While Fe +2 signi cantly activated protease activity to 411.11% and 1577%, at 15mM and 20mM, respectively as shown in Table 5. The response of studied enzyme as keratinase was investigated towards the previously mentioned; metal ions; detergent, surfactant, solvents; activators/inhibitors, at the most signi cant concentration (20mM, 2%& 10Mm), respectively. As shown in Table   6; keratinolytic activity of the enzyme signi cantly activated (410.1% &179.33%) by, Fe +2 and Mg +2 , respectively.
On the other hand, the enzyme activity was investigated as protease and keratinase activity in the presence of some surfactants, detergents and solvents. The results represented in Table 5 indicated that a slight increase in the activity of partially puri ed protease/keratiinase enzyme when treated with Tween-20 and Tween-80 at different concentrations, where Tween-40 highly activated the proteolytic activity and keratiolytic activities to 149.3% and 205.4%, respectively. A sharp increase in proteolytic activity with 219.6% was resulted by incubating the enzyme with TritonX-100 at (1%), but it did not show any effect on keratinolytic activity. However, sharp decrease in the proteolytic/keratinilytic activities was resulted by incubating the enzyme with SDS. Moreover, ethanol, methanol and glycerol had no effect on protease/keratinase enzyme activity while glycerol activated keratinase activity.
Also, the enzyme activity was investigated in the presence of some activators and inhibitors such as EDTA (inhibitor of metallo-type proteases), DTT (reducing agent) and PMSF (a serine protease inhibitor), the results represented in Table 5& 6 showed that both EDTA and DTT caused a slight increase in activity of the partially puri ed protease/keratinase when measured by both casein and keratin azure. While, PMSF a serine protease inhibitor signi cantly increased enzyme activity as both protease and keratinase at concentration of 10mM to 120.13% and 176.07%, respectively.

Effect of substrate concentrations
The kinetic parameters (Km and Vmax) of the extracellular YNDH protease/keratinase enzyme for hydrolysis of casein and keratin-azure at 70°C and pH10.4 were determined by double reciprocal Lineweaver-Burk plot. Fig. 7A and these are 7mg/ml and 384.6 U/mg, respectively. While, the hydrolysis e ciency represented by Km and Vmax for keratin azure is shown in Fig. 7B, these are 7.2mg/ml and 103U/mg, respectively. The estimated Km value indicated the a nity of enzyme towards the substrate, while Vmax is an indication of the catalytic activity of an enzyme which is usually desired to be as high as possible; the high Vmax estimated indicates the high e ciency of tested enzyme towards the substrate.

Discussion
Keratinases are very interesting from a biotechnological point of view; especially those of microbial origin compared with plant or animal sources, especially because they possess almost all the features desired for biotechnological applications. Among these biocatalysts high alkaline proteases, which alone account for about 40% of global enzyme sales worldwide 14 , were especially suitable for industrial use. It is primarily because of their high stability and performance in tough conditions.
Feather is degraded to feather meal used as animal feed, organic fertilizers, feed supplements as it consists of > 90% protein and is rich in hydrophobic amino acids and major amino acids such as cystine, arginine, threonine 5,15 . Hydrothermal processes are the most common method for making feeder meal where the feather is cooked at high temperature at high pressure. However, hydrothermal treatment results in degradation of essential amino acids such as methionine, lysine, tyrosine and tryptophan. Also, it has poor digestibility and low nutritional bene ts 3,16 . In this respect, the microbial degradation of feather into feather meal has become more signi cant and new microbes are being considered for the e cient degradation of feather, up to now no more than 10% of feather degradation is recorded in the presence of keratinases 17 . Interestingly, the studied keratinase of laceyella sacchari YNDH strain was observed to completely degrade the feather into feather meal and protein lysate, in order to achieve this particular process, the parameters that regulated the enzymatic degradation of the feather to protein lysate were studied in an experimental design by Box-Behnken. A further analysis of variables (time, feather conc, and enzyme conc) were performed at three leveles, each of -1.0 and+1, to address the optimum response region of the protein concentration in the feather lysate directly via action by the crude protease with keratinolytic activity. The R value for protein concentration (μg/ml) was 0.97 in this experiment.
This value refers to the high correlation of the experimental value to the predicted values. As the optimal conditions realized from the optimization experiment were veri ed experimentally and compared to the predicted optimum of the model. The estimated protein conc. was 2089.5µg/ml, where, the predicted value from the polynomial model was 2159.8µg/ml. This high degree of accuracy (96.7%) is an evidence of the model validation under the following optimal conditions: time, 20.2h, feather conc., 3g% and keratinase activity 24.5U/100ml at pH, 10; and cultivation temperature 50 o C.
The current process of bioconversion of feathers into feather protein lysate and feather meal is an enzymatic process in which no additional redox reactions have been observed. The process is not only simple and timesaving, but also economically viable as it does not require any bioreactor for feather degradation. Thus, bulk feathers can be easily recycled into amino acid and feather meal using this predicted new found keratinase produced by laceyella sacchari strain YNDH within 20h.
Worldwide, commercial poultry processing generates more than a million tons of feathers per year 18 , which are currently converted to feather meal by steam pressure and chemical treatments, these methods therefore degrade amino acids and require signi cant energy input. Consequently, keratin can be transformed to usable biomass, protein concentrate or amino acids using keratinolytic microorganism-derived proteases 7 . The nutritional improvement of the meal and the use of microbial feather protein lysate in feed studies have shown that keratinase therapy may signi cantly increase the digestibility of amino acids in feather keratin 19, 20 .
In the present study, analysis of the feather lysate resulted from effect of crude keratinolytic protease of laceyella sacchari YNDH showed presence of 16 different amino acids. The feather hydrolysate contained good amount of essential amino acids (phenylalanine, valine, leucine and isoleucine) and rare amino acids as serine and proline, along with the other amino acids more importantly the sulphur containing amino acids like methionine, these ndings are in agreement with 21 . They reported that the feather lysates developed by a thermotolerant Streptomyces graminofaciens contained 20 amino acids. Additionally in this study it was observed that the concentrations of all different types of amino acids in feather protein lysate (feather treated by enzyme) was signi cantly higher than that in feather hydrolysate produced by the native organism directly, these ndings are in agreement with Tiwary 4 who reported that during fermentation more than 0.5-2% w/v of feather can be used and also essential amino acids are utilized by micro-organism which decreases the nutritional value of feather meal.
For further understanding the applied YNDH enzyme which characterize by dual functions protease/keratina; puri cation and intensive biochemical characterization were followed. Through puri cation steps from crude extract to the nal step of separation using anion exchange ("QFF" strong anion column); the purity have been gradually improved which revealed 3 distinctive bands with low molecular weights (<=10 kDa) indicating a partial puri cation. Generally, bacterial keratinases molecular weights are varied between bacterial species. But, the reported results in this study disagreement with others 22,23 ; they observed keratinase with a relatively low molecular mass (18 kDa) in Streptomyces albido avus and the higher molecular weight was observed in Kocuria rosea (240 kDa), respectively, and agree with Lange 24 which reported that a combination of endoprotease, exoproteases, and oligopeptidase is required to bring about keratin degradation. Hence, the result of this work may be showed a novel group of keratinases with very low molecular weight equal and/or below 10 kDa.
The effect of temperature on the activity of the partial puri ed protease with keratinolytic activity showed that the isolated enzyme and/or isozymes was capable of hydrolyzing casein and keratin azure substrates at a wide range of temperature from 40ºC to 75ºC, but maximal at 70ºC by using both casein and keratin azure. Therefore, this protein(s) could be classi ed as a thermoactive protease/keratinase enzyme and the three isolated bands could be worked together as a group of keratinase. Similarly, Habbeche 25 have reported the optimum temperature of keratinase was 70ºC.While, pH activity pro le of this enzyme indicates a broad pH range (7.6-11.6) of activity, with a maximum at pH 10.4 when used casein/keratin-azure substrate. In summary the optimum protease/keratinase activity was observed at pH 10.4, but by increasing the pH beyond 10.4 a rapid decline in protease activity was noticed. These ndings are in agreement with several earlier reports showing pH optima of alkaline protease being close to 10.0 [26][27][28][29] . The thermal stability of the partial puri ed protease/keratinase showed that it was activated at 50ºC for 24h. Also it's possessed its stability until reached 90min at 60ºC. On the other hand the investigated enzyme was completely inactivated at 75°C after 20min. The increased proteolysis rate of proteases at elevated temperatures has been reported to be one of the factors responsible for rapid thermal inactivation of these enzymes. As described by 30 . This is in agreement with alkaline protease obtained from Bacillus sp. 26,31 , and from Streptomyces clavligerus 32 . Where, the enzyme was stable at pH 9.6 to 10.8 after 1h incubation at each tested pH. The effect of different metal ions and chemical compounds on the activity of partially puri ed protease/keratinase enzyme indicate that the enzyme activity was completely inhibited by 1mM HgCl 2 and CuSO 4, which are in agreement with Patel and Sana 28,33 . While, the non-ionic surfactant like Tween-20, Tween-40 Tween-80 and Triton-X-100 demonstrate slight enhancement of enzyme activity with increase of its concentration from 0.1% to 2% and 0.1% to 1%, respectively which are in agreement with Joo and Sana 31,33 . They reported an enhancement in alkaline protease activity by Triton-X-100 and Tween-80. Also, PMSF is known to sulphonate the essential serine residue in the active site of the protease, resulting in a total loss of enzyme activity 34 . Unlikely, it caused activation for studied partially puri ed protease/keratinase enzyme at 10mM concentration.
For the kinetic parameters (K m and V max ) of the protease with keratinolytic activity from Laceyella sacchari YNDH, the present work showed that our group of keratinase enzymes have the same a nity toward keratin azure and casein because K m for keratin azure was nearly equal that for casein. So that, we can concluded that our group of keratinase enzymes working together as protease with keratinolytic activity enzymes. Moreover the K m is independent of enzyme concentration and it is a true characteristic of the enzyme under de ned condition of temperature, pH, 35 . Higher V max and lower K m indicate the e ciency of protease with keratinolytic activity from laceyella sacchari YNDH. The present study is the rst one reported the enzymatic degradation of waste feather for feather protein lysate production and feather meal formation using a new found crude protease/keratinase from a local isolate laceyella sacchari YNDH. In addition the characteristic features of the partially puri ed enzyme concerning unusual low molecular weight make it may be classify as a new or novel protein.

Conclusion
The extracellular protease with keratinolytic activity enzyme produced from Laceyella sacchari YNDH, was puri ed using FPLC to almost 11.31 puri cation fold with speci c activity of 1881.3 U/mg protein. The molecular weight of the partially puri ed Laceyella sacchari YNDH group (three active bands) of protease with keratinolytic activity was found to be ranged below 10KDa, using sodium dodecyl sulphate polyacrylamide gel electrophoresis technique, representing three subunits and this enzyme possess promising characteristics.
The present study showed that the isolation of a novel strain of laceyella sacchari for the production of protease with keratinolytic activity it may produce a novel group of keratinases which demonstrated, production of enzymatic feather-lysate enriched with amino acids was optimized using YNDH-keratinase enzyme. The application results indicating the great potential of produced enzymes in diverse biotechnological processes Declarations Tables   Table 1. BBD for selected 3 variables with coded and