Alkaline protease from Bacillus cereus was characterized and optimized for eco-friendly degradation of feathers

doi:10.21203/rs.3.rs-1509674/v1

Purpose: To investigate the detailed analysis of alkaline protease production, media optimization, protease assay, mass production, and application in feather degradation by isolates of Bacillus cereus strain isolated from leather industry effluent, Ethiopia.

Methods: In this study, media optimization was subjected to nine different parameters like fermentation time, temperature, pH, Substrate concentration, carbon sources, nitrogen sources, metal ions, Inoculum size, and sodium chloride concentration to check the maximum alkaline protease production using Bacillus cereus. Ammonium sulfate and dialysis were used to partially purify the enzyme from Bacillus cereus. SDS-PAGE was used to test the activity and total protein content of the partially purified enzymes, and then extracellular alkaline protease from Bacillus cereus was used to examine hydrolyzed chicken feathers, and the findings were reported.

Results: The maximum enzyme activity of crude alkaline protease from Bacillus cereus was achieved at optimized conditions. The specific enzyme activity of partially purified alkaline protease after dialysis (123.35U/mg) showed high activity compared to the crude protease (24.45U/mg). Finally, the partially purified enzyme was tested for its potential to degrade feather waste. Partially purified enzyme demonstrated significant feather degradation of 76.5% on the fifth day at optimized temperature and enzyme concentration.

Conclusions: Bacillus cereus was found to cause considerable feather deterioration in this investigation. It's was also been discovered that enzyme activity gets enhanced at a specific optimized condition. Compared to the crude enzyme, partially purified and dialyzed enzymes showed considerable change in the enzyme activity, indicating that they have the potential to break down feathers.

Crude protease

feather degradation

Screening

Partially purified protease

Protease activities

Alkaline proteases isolated from a variety of microbial species have been intensively investigated to determine their industrial utility. Its qualities such as ideal pH, temperature, solubilizing potential, and inhibitory variables provide a clear understanding of its specific industrial application. They have been observed to grow in high alkaline pH and even excessive saline environments (Ellaiah et al. 2002; Chandran et al. 2014). Alkaline protease has extensive application in the detergent business and X-ray film silver recovery. They can also be potentially used as bleaching agents, hair removal agents in the leather industry, and to break down feathers into usable protein. (Kumar et al.2016; Chandran et al. 2017).

Economic and environmental pressures, as well as a growing interest in using renewable and sustainable raw materials and a desire to reduce reliance on nonrenewable petroleum resources, are forcing the industry to find better ways to deal with discarded feathers. Burning poultry manure could emit as much as or more hazardous air pollution than coal facilities. Because of its tenacious character, chicken waste, particularly feathers has become one of the most significant pollutants as a result of improper management (Manczinger et al. 2003). Landfilling, incineration, burning, or the employment of chemicals to dispose of them or convert them to protein hydrolysates are all expensive, non-eco-friendly, and dangerous to living beings. Alkaline protease is one of the promising enzymes with considerable potential in breaking down so many biological wastes. The primary objective of this study was to isolate, screen, and characterize alkaline protease-producing bacteria from leather industry effluents to control the associated harm with feather wastes. (Chandran et al.2015).

Chicken consumption is increasing every year as it is one of the cheapest and healthiest protein sources. Every year, over 3 billion pounds of chicken feathers are produced worldwide while the majority of them are discarded as garbage (Wang et al. 2016). These keratin-rich wastes and a variety of colored feather wastes are regarded as environmental contaminants frequently disposed of in landfills and other dumb sites, particularly in underdeveloped nations. Generally, the vast quantity of feathers is wasted and/or burned as garbage with only some percentage of them being utilized for product development like insulating materials (Sharma and Gupta,2016). Despite their abundance, chicken feathers are typically classified as garbage and are routinely burned or disposed of harming the environment. Using an enzyme to degrade biological waste is one of the best methods for environmental waste management and is displayed in Figure: 1 (Getachewet al. 2020).

Among all sources of organisms that could produce alkaline proteases, microorganisms are considered to be the best source of protease due to the long shelf life of produced enzymes, their fast production rate, and the large production of proteases (Razzaq et al. 2019). Also, compared to plants and animal sources, microbial protease can be obtained through the short and simple process as extracellular products from the fermentation medium.

There are several studies and efforts in Ethiopia in isolating, screening, and characterizing bacteria for producing protease enzymes from local sources for industrial applications. However, there are minimal studies to isolate and purify alkaline protease for use in local and industrial consumption to protect against environmental pollution. The study of (Haile and Gessesse, 2012) also reports the isolation of 240 alkalophilic bacteria from Chitu Soda Lake of Ethiopia and characterized proteases produced from Bacillus for keratinolytic activity on cow hair. The major aim of this study is to optimize the medium components, characterize the alkaline protease from Bacillus cereus isolated from leather industry effluent in Ethiopia. Additionally, the proteolytic activity and its potential to degrade waste chicken feathers were tested.

Modjo leather industry effluents were sampled and preserved in the microbiology lab. Using skim milk agar media, microorganisms were recovered from effluents. Finally, by gene sequencing (16S rRNA) the prospective bacteria was found to be Bacillus cereus which is one such best source of alkaline protease. Bacillus cereus was utilized in our research for the production of alkaline protease in an optimized (medium components) condition and then the protease was tested for its potency to use in feather decomposition. Finally, mass production was attempted (Chandran Masi et al. 2021).

2. 1. Production of crude alkaline protease by submerged fermentation

The production of alkaline protease by submerged fermentation was conducted as described by Pant et al. 2015. The alkaline protease was a production medium containing 1% Galactose, 1% Casein, 1% Gelatin, 0.2% KH₂PO₄, 0.5% Sodium Chloride, and 0.2% MgSO₄·7H₂O, dissolved in 100ml of distilled water to maintain pH 9.0, added 2ml of 24 hours fresh inoculum in a continuous shake flask. Shaker fermentations were carried out at 37°C for 72 hours with controlled agitation at 150rpm. At the end of the fermentation period, the whole culture broth was centrifuged at 5000rpm for 30minutes to remove debris, and the supernatant was collected and used for further experiments (Adesh et al. 2002).

2. 2. Determination of alkaline protease activities of the selected isolate

2.2.1. Tyrosine and bovine serum albumin calibration curve

As a reference to protease enzyme activity, the tyrosine standard curve was generated using an appropriate amount of tyrosine diluted in water. The suitably diluted samples (0.1–1.8 mg/ml) were treated similarly to the experimental enzyme-catalyzed reaction mixture and then they were measured using a spectrophotometer at a wavelength of 660 nm. The total protein content of the samples was evaluated using Bovine Serum Albumin (BSA) as a protein standard (0.1-0.7mg/ml) as reported by Lowry et al. in 1951. The activity of proteases was measured using casein as a substrate, as reported by Saibabu and Niyongabo, 2013. 1 ml of 1% casein in 50 mM sodium phosphate buffer (pH 7) and 1 ml enzyme solution made up the reaction mixture, which had a total volume of 2 ml. After 20 minutes at 37°C, the reaction was terminated by adding 2 ml of 10% trichloroacetic acid (TCA) and incubating for another 20 minutes at 37°C (Betts and Russell, 2003).

2.2.2. Total protein estimation

The stock of the standard solution was prepared by dissolving 100mg of standard (BSA) in 100ml of distilled water, from this stock solution, the working standard solution ranged from 0 to 250µg/ml and the standard curve was constructed with the given ranges. Reagent - A contained 2% sodium carbonate mixed with 0.1N of sodium hydroxide in 1000 ml of distilled water and stirred until completely dissolved. Reagent - B containing 1ml of 1% copper sulfate plus 1ml of 2% sodium potassium tartrate well mixed. Then Lowry's solution was prepared as 50ml of reagent - A was mixed with 1ml of reagent - B and stored at 4^oC. 2ml of commercialized Folin reagent was dissolved in an equal amount of distilled water. 1.5ml of protease was mixed with 0.5ml of Lowry’s solution in test tubes and incubated at room temperature for 10minutes. After incubation time, 3ml of Folin reagent was added to test tubes well mixed by vortex and incubated for 30 minutes at room temperature. The trend of blue color change intensity shows digestion of protein. Finally, absorbance was read at 660nm using a spectrophotometer and the protein concentration was calculated from the equation of the BSA standard graph (Chandranet al. 2014)

2.3. Optimization of the Growth Conditions for Production of Alkaline Protease

Different growth conditions of the selected isolate were performed by one variable at a time method.

2.3.1. Effect of incubation time on the production of alkaline protease

To determine the time taken for maximum production of protease, the 24 hours fresh culture was inoculated into the protease production stranded medium and the condition was maintained at 37°C, pH 9 for 12 to 96 hours. 2 ml of the sample from the medium was collected every 12 hours to determine protease activity (Betts and Russell, 2003).

2.3.2. Effect of temperature on the production of alkaline protease

The optimum temperature for protease production was determined by incubating the culture at different temperatures (25, 30, 35, 37, 40, 45, and 50°C), at pH 9 in a stranded medium for an incubation period of 72 hours. At the end of the incubation period, the cell-free culture centrifuged was tested for protease activity using the method described in section 2.2.1 (Dos and Sato, 2018).

2.3.3. Effect of pH on the production of alkaline protease

The effect of pH on the production of protease was investigated by adjusting the pH of the stranded growth medium to pH 6.0, 7.0, 8.0, 9.0, and 10.0 at a constant optimized temperature and incubation time. Adjustment of pH was done using 1N NaOH and 0.1N HCl solutions. At the end of the incubation period, the protease activity was determined as mentioned in section 2.2.1 (Tork et al.2013).

2.3.4. Effect of the concentration of substrate on the production of alkaline protease

The effect of substrate concentration on the production of protease was assessed by growing the isolates in protease production media containing different concentrations of casein as a substrate (0.5%, 1.0%, 1.5%, 2.0%, and 2.5%) at optimized temperature, pH and incubation period. These fermentation media were assayed every day for protease production until the optimum incubation period (Saibabu and Niyongabo, 2013).

2.3.5. Effect of different carbon sources on the production of alkaline protease

In this study, several carbon sources were tested by using the one-factor optimization method. Different carbon sources such as glucose, fructose, maltose, sucrose, lactose, galactose, and starch were used for the better production of protease at the optimized temperature, pH, incubation period, and substrate concentration (Pant et al. 2015).

2.3.6. Effect of different nitrogen sources on the production of alkaline protease.

Different nitrogen substrates like yeast extract, peptone, gelatin, ammonium chloride, and urea were added as organic and inorganic nitrogen sources. 1% w/v of each nitrogen source was added into 100 ml of the prepared medium used for protease production and thereafter 1 ml of inoculum was inoculated into the medium and then subjected to submerged fermentation at the optimum temperature, pH, incubation period, substrate concentration, and selected carbon source (Suberu et al.2019).

2.3.7. Effect of inoculum sizes on the production of alkaline protease

The effect of inoculum size on the production of protease was assessed by growing the isolates in protease production media containing different inoculum sizes of bacteria (1%, 2% 3%, 4%, and 5%) v/v at optimized temperature, pH, and incubation period. These fermentation media were assayed for protease production during the optimum incubation period (Suberu et al. 2005).

2.3.8Effect of sodium chloride (NaCl) concentration on the production of alkaline protease

In the present study, various salt concentrations from (0 to 3.0%) were used to study the effect of NaCl on the production of protease from Bacillus cereus(C2) under optimized growth conditions. These fermentation media were assayed for protease production during the optimum incubation period (Suberu et al. 2008).

2.3.9 Effect of metal ions on the production of alkaline protease

Several metal ions (CaCl₂, MgSo4, MnSo_4, NaCl₂, ZnCl₂, FeCl₂, KH₂Po₄) were added separately to culture media which was inoculated at the optimized growth conditions to find the effect of metal ions in the alkaline protease production (Asha and Palaniswamy, 2018)

2.4. Mass Production and Partial purification of alkaline protease

The alkaline protease was produced in broth contained glucose, gelatin, MgSo_4, Sodium chloride, and caseins maintained at 37^oC and pH 9.0. The crude enzyme was partially purified by using ammonium sulfate precipitation methods (Gaur et al. 2014). To prevent the denaturation of enzymes, all steps of purification processes were carried out under cold conditions using an ice bath and 4^oC temperatures. 250 ml of crude enzyme which was produced with optimized parameters (1% of casein, 60 hours incubation time, pH 9, 1% glucose, and 37^oC) was precipitated by the addition of four (30%, 50%, 70%, and 90%) saturation levels of ammonium sulfate. The crude enzyme was placed in a beaker that can hold 500ml and for the first saturation level (30%), 44g of ammonium sulfate as described in the Ammonium sulfate precipitation table was added slowly with continuous stirring in an ice bath. Then, the saturated enzyme was kept at 4^oC overnight for precipitation and centrifuged at 10,000rpm for 20minutes. The enzyme protein was collected and suspended in 4ml of 0.2M of phosphate buffer pH 7. The supernatant was subjected to the next level of ammonium sulfate saturation (50%). The volume of supernatant was measured to calculate the amount of ammonium sulfate (in grams).

The precipitation processes were continued by the addition of 56.34g ammonium sulfate to 200ml of the supernatant solution with continuous stirring in an ice bath. Then, the saturated enzyme was kept at 4^oC overnight for precipitation and centrifuged at 10,000rpm for 20minutes. The enzyme protein was collected and suspended in 4ml of 0.2M of sodium phosphate buffer pH 7. The supernatant was then subjected to the next level of ammonium sulfate precipitation (70%), 75.52g was added to160 ml of the supernatant and precipitated as described in the above steps. The enzyme was collected and suspended in 4ml of 0.2M of sodium phosphate buffer pH 7.

The supernatant was taken for the last saturation level (90%) where 66.2g of ammonium sulfate was added to the 100ml supernatant according to the procedure mentioned above. The enzyme protein was collected and suspended in a 0.2M phosphate buffer. After all the precipitation steps above, each of the precipitated enzyme proteins was finally dissolved in sodium phosphate buffer and dialyzed using a dialysis membrane for protein estimation (Gaur et al. 2014).

2.4.1. Dialysis of partially purified alkaline protease

Spectrum RC Dialysis Membrane Tubing 12,000 to 14,000 Dalton MWCO was used for dialysis and desalting unwanted debris. A dialysis tube was cut into 15cm and activated by dipping into the sterile distilled water bath for 10minutes. 20ml of the partially purified enzyme was then poured into the tube and the other end of the tube was tied with the thread.

The dialysis tube was suspended in a beaker containing 400ml of 0.2M of phosphate buffer pH 7 and kept for 24hours at 4°C. Then, the dialyzed protein enzyme product was collected. The process was repeated 4 times to get 80ml of the dialyzed protein. Finally, total protein content was estimated using the method described by (Abrar2017) and was stored at -80°C for further use.

2.4.2. Purification folds of crude and partially purified protease

The Proteolytic activities of proteases were done with both crude and partially purified extracts to determine the purification fold of the enzyme to determine any interference during the purification steps. Determination of the purification fold can help to know the purity level fold of the proteins and the specific enzyme activities fold (Abrar 2017). It was calculated by the following equation:

Purification fold = Specific activity of partially purified protease

The specific activity of crude protease

2.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

Protease samples were electrophoresed in 12% polyacrylamide gels under denaturing conditions. Gels were stained overnight with a staining solution (10% acetic acid, 25% methanol, 0.25 gL^− 1 CBB G-250) and then destained overnight in 30–10% methanol and acetic acid. Standard proteins were used as molecular mass. A total of 20µL of each sample was placed into the comb's wells independently. The cell was left to sit for 5 minutes, or until all of the samples had settled to the bottom of the well. After that, an electric field was created across the electrophoresis buffer by connecting the electrophoresis cell to a power supply. It took about 5 hours to complete the separation procedure. The gel was gently removed from the cell and the plates(Suberu et al. 2019).

2.6. Test for potential application in enzymatic feather degradation

The chicken feathers were gathered from the Holeta Poultry Company and transported to the microbiological laboratory at Addis Ababa Science and Technology University. Nine sets of chicken feathers, each weighing 5.5g was washed were used in our experiments. Two different approaches such as (1) Effect of the incubation period for feather degradation and (2) Effect of varied alkaline protease concentrations on feather destruction have been experimented with. In the first test, two sets (each containing 5.5 g) of chicken feathers were used. One set (control) was placed in a flask containing 50 ml of distilled water and the second flask was filled with 50mL of alkaline protease solution (same as feather proportion) (Varela et al.2002) and incubated at room temperature for 120 hours in a shaker (121rpm). After treatments, feather fragments were analyzed every 24 hours. The extent of degradation and the effect of enzyme concentration on feather degradation was obtained.

In the second test, we used seven sets (each containing 5.5 g) of chicken feathers, the first with 50 ml of distilled water (control), the second with 10 ml of alkaline protease solution and 40 ml of distilled water, the third with 20 ml of alkaline protease solution and 30 ml of distilled water, the fourth with 30 ml of alkaline protease solution and 20 ml of distilled water, the fifth with 40 ml of alkaline protease solution and 10 ml of distilled water, sixth 50ml of alkaline protease solution and seventh 60 ml of alkaline protease solution. Finally, all of the sets were incubated at room temperature in a shaker (121rpm) for 120 hours, after which they were analyzed for degradation time, extent, and the effect of enzyme concentration on feather degradation (Suberu et al. 2019).

2.6.1. Determination of feather degradation

The percentage of feather degradation by the protease enzyme produced by the selected strain was determined by calculating the difference in residual feather dry weight between the control (Feather without the enzyme) and the treated sample. The percentage of weight loss was calculated using the following formula as described by (Nair et al. 2015).

P\(\text{e}\text{r}\text{c}\text{e}\text{n}\text{t}\text{a}\text{g}\text{e} \text{o}\text{f} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t} \text{l}\text{o}\text{s}\text{s}=\frac{\text{I}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t}-\text{f}\text{i}\text{n}\text{a}\text{l} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t} }{\text{I}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t}}\) ×100

2.6.2. The effect of protease concentration on feather degradation

The effect of enzyme concentration on feather degradation was studied by varying the enzyme concentration from 10ml to 60ml at constant featherweight.

2.6.3. The effect of incubation period on feather degradation

The effect of time on feather degradation by protease was determined by incubating it at varying times and optimizing enzyme concentration. The weight loss of feathers was measured continuously for six days.

2.6.4. Reuse of the enzyme for degradation

Reuse of enzyme for feather degradation was evaluated at 37°C, pH 9, and 150 rpm in a rotary shaker with continuous shaking until the feather was degraded. After the degradation of the feather, a new feather was incubated in the reaction mixture for up to six turns (Suntornsuk W and Suntornsuk L, 2003).

3.1 The standard calibration curve of BSA

Lowey’s method was used to determine the total protein content of the sample, which was degraded casein derived from media. The reference graph was used to calibrate the protein content. At 660nm, the sample's absorbance was measured spectrophotometrically.

3.1.1 Preparation of tyrosine standard curve

10 mg/ml Tyrosine stock solution was used to make the standard curve. Each test tube, save the blank, received the proper amount of buffer and Tyrosine. Finally, using a spectrophotometer, the optical density (OD) was measured at 660nm, and the standard curve was drawn. Based on these procedures and experimental results, the following standard curve was obtained.

3.2. Production of alkaline protease

The screened proteolytic bacterial isolates were characterized based on the formation of a large hydrolysis zone on skim milk agar plates; Isolate Bacillus cereus (C2) was subjected to fermentation and quantitative screening by measuring its protease activity. The maximum protease activity was 18.43 ± 0.36 U/ ml by Bacillus cereus (C2) after 72 hours of fermentation time at optimum pH and temperature.

3.3. Optimization of media for protease production

3.3.1. The effect of fermentation time on alkaline protease production

As it is indicated in Fig. 2, strain Bacillus cereus (C2) was evaluated at eight different incubation times to get the maximum production of protease. The protease activity started to increase from 24 hrs onwards but the maximum protease activity was attained at an optimum incubation time of 60 hrs (19.02 ± 0.18 U/ ml). Protease activity declined after the 60th hour.

3.2.2. Effect of Temperature on the Production of alkaline protease

Temperature is one of the most important factors that should be considered in protease production. It highly affects product quantity and producer growth. The effects of temperature on protease production by bacterial isolate Bacillus cereus (C2) indicated that maximum protease activity was obtained at 37^oC (Fig. 3). It is also observed that protease activity was increasing with temperature and declined after attaining the optimal temperature of 37^oC. Even though protease activity started decreasing after 37^oC surprisingly it has good activity until it reaches 45^oC. The maximum relative activity of protease was recorded as 19.62 ± 0.14U/ml of tyrosine released at 37^oC. Before reaching the optimum temperature protease activity was recorded as 4.32 ± 0.12, 8.15 ± 0.33, and 17.48 ± 0.24 at 25^oC, 30^oC, and 35^oC respectively.

3.2.3. Effect of pH on the production of alkaline protease

Different values of protease activity were recorded at different pHs. The optimum pH value for this isolate was recorded at pH 9 as 18.29 ± 0.75 which indicates that these isolates prefer alkaline pH (Fig. 4). Better protease activities were again recorded below the optimum pH as compared to that above the optimum pH. Protease activities of 8.25 ± 0.24, 12.25 ± 0.41, and 16.36 ± 0.19 were recorded at 6, 7, and 8 pH respectively. Protease activity starts reducing when the pH reaches 10 which indicates that this isolate cannot resist above PH 10.

3.2.4. Effect of the concentration of substrate (Casein) on alkaline protease production

The effect of substrate concentration on the production of protease was assessed by growing the isolates in media containing different concentrations of casein as a substrate. Protease activities were found to vary with the casein concentration. The maximum protease activity was obtained at 1% casein substrate which is about 20.47 ± 0.33 U/ml (Fig. 5). About 5.74 ± 0.14 U/ml of protease activity was obtained when the casein concentration is 0.5% and this was recorded as the minimum relative activity of the protease. Beyond optimum casein concentration, the protease activity starts decreasing and is recorded as 15.97 ± 0.28 U/ml, 11.29 ± 97U/ml, and 7.34 ± 0.26U/ml at 1.5%, 2%, and 2.5% respectively.

3.2.5. Effect of carbon source on alkaline protease production

Among the several carbon sources, glucose had the greatest impact on enzyme synthesis with a protease activity of 22.27 ± 0.22 U/mL, whereas all other carbon sources yielded lower yields when compared to glucose (Fig. 6). Carbon sources had a significant impact on protease synthesis and various bacteria used different carbon sources for growth and metabolism. When maltose was employed as a carbon source, the minimum protease activity was 12.44 ± 0.29 U/mL.

3.2.6. The effect of different nitrogen sources on alkaline protease Production

By adding different nitrogen sources such as Yeast extract, Urea, Peptone, Gelatin, and Ammonium chloride different protease activities were recorded. In this study, the maximum protease activity recorded was 21.47 ± 0.78 when 1% (w/v) of gelatin was used as a nitrogen source (Fig. 7). Yeast extract produced a low enzyme yield. Also, it was found that, though inorganic nitrogen sources were not as effective as organic sources, urea was found to be better among them for the production of protease.

3.2.7. Effect of inoculum sizes on alkaline protease production

The highest production was achieved when 2% v/v of inoculum was used and it was found to be the optimum inoculum size for protease production followed by 3% and 1% inoculum size (Fig. 8). 19.34 ± 0.23 protease activity was recorded when 2% inoculum size was used for fermentation. 9.45 ± 0.55 protease was recorded while using 1% inoculum size.

3.2.8. Effect of sodium chloride (NaCl) concentration on protease production

In the present study, various salt concentrations from (0–3%) were used to study the effect of NaCl on the production of protease from Bacillus cereus C2 under optimized growth conditions (Fig. 9). These fermentation media were assayed for protease activity by the addition of different concentrations of sodium chloride. 1% sodium chloride concentration was found to be optimum for the production of alkaline protease with about 19.25 ± 0.36 U/ml protease activity. Minimum protease activity was observed by the addition of 3% sodium chloride concentration which shows a high salt concentration effect on the alkaline protease producer bacteria. Only about 6.85 ± 0.23 protease activity was recorded by the addition of 3% sodium chloride as the medium component.

3.2.9. Effect of metal ions on alkaline protease production

Mn was found to be the most important metal ion for the growth of alkaline protease-producing bacteria with protease activity of 17.65 ± 0.66 U/ml (Fig. 10). Fe was the second important metal ion that activates the growth of alkaline protease producer bacteria with 12.15 ± 0.43U/ml. Protease activity observed Na and Ca was found to reduce the alkaline protease production in our research.

3.3. Purification of alkaline protease by Ammonium Sulfate precipitation

Alkaline protease that was extracted from isolate Bacillus cereus (C2) was subjected to different saturation levels ranging from 30 to 90% of ammonium sulfate (Table 1). The protein content of the partially purified protease showed decreasing from crude Protease to partially purified one with ammonium salt followed by dialysis, which might be due to precipitation of the enzyme proteins particularly, protease by ammonium as ammonium changes the solubility of the protein in water and the proteins precipitate. The specific activities of protease increased from crude to partially purified and from partially purified to dialyzed protease.

Table 1

Purification trend of protease from Isolate *Bacillus cereus (*C2)
Purification steps	Total volume(ml)	Total activity(U)	Total protein (mg)	Specific activity/mg)	Partial Purification fold
Crude Protease	250	4890	200	24.45	1.00
Ammonium sulfate (30%)	250	5055	175	28.89	1.18
Ammonium sulfate (50%)	200	4246	120	35.38	1.22
Ammonium sulfate (70%)	160	3592	60	59.87	1.32
Ammonium sulfate (90%)	100	2365	30	78.83	1.40
After dialysis	80	1973.6	16	123.35	1.56

3.4. SDS-PAGE analysis of partially purified alkaline protease

Purified alkaline protease bands with a relative molecular mass of approximately 76 kDa were found on SDS-PAGE with a protein marker. SDS-PAGE analysis of the purified enzyme revealed a single band, indicating that the preparation was homogeneous. Protease recovery and specific activity were computed using molecular weight data (Fig. 11).

3.5. Application of alkaline protease in feather degradation

Alkaline protease has wide application in feather degradation. The protease produced showed a promising result in feather degradation. About 76.5% of degradation was recorded by this alkaline protease after 120hours (5 days) (Fig. 12& Table 2).

Table 2

Effect of incubation period on feather degradation
Treatment steps	Weight of feather (g)	Percentage of weight loss	Total Protein concentration (mg/ml)	Total Protease activity (U/ml)
Before treatment	5.5 + 50 ml of Protease enzyme	-	-	-
After 1 day	4.44	19.2	34.1	98.3.
After 2 days	3.77	31.5	38.5	104.9
After 3 days	2.58	53.1	43.5	109.8
After 4 days	1.92	65.1	46.2	113.7
After 5 days	1.29	76.5	47.1	114.8
Control	5.5 + 50 ml of Distilled water	2.8	3.8	-

Table 3 explains the effect of different concentrations of alkaline protease on feather degradation. After 120 hours (5 days), the weight of the feather, percentage of weight loss, protein concentration, and specific protease activity were calculated. 76% of weight loss was achieved with 50ml of alkaline protease and it was only 75.6% with 60 ml. Thus it is inferred that there is much effect in degradation for increasing the alkaline protease from 50 to 60 ml.

Table 3

Effect of Different alkaline protease concentration on feather degradation
Protease concentration	Weight of feather in (g)	Percentage of weight loss	Protein concentration (mg/ml)	Specific Protease activity (U/ml)
Control (50 ml of Distilled water)	5.50	-	0.47	-
Treatment with 10ml protease + 40 ml of Distilled water	4.29	22	0.62	18.93
Treatment with 20ml protease + 30 ml of Distilled water	3.57	35	0.79	19.96
Treatment with 30ml protease + 20 ml of Distilled water	2.51	54	0.83	21.28
Treatment with 40ml protease + 10 ml of Distilled water	1.82	67	0.89	21.43
Treatment with 50ml protease	1.32	76	0.95	21.85
Treatment with 60ml protease	1.34	75.6	0.91	20.45

3.5.1 Reuse of protease in feather degradation

Figures 13 indicate the reuse of protease for feather degradation. The partially purified protease was used six-cycle to test the reuse of enzyme for feather degradation. In the first cycle, 75.56% weight loss of feathers was recorded. Removing this and replacing it with a new feather (5.5g), about 68.75% weight loss was recorded on the second cycle (Figure.13). The weight loss started decreasing and a minimum result was observed on the sixth cycle. The reason for the decreasing power of enzymes in feather degradation is the formation of high debris by degraded protein in the enzyme. Even though enzymes do not participate or change during a reaction, they can be affected by the protein content of the sample and prolonged usage.

The optimum fermentation time for protease production in this study was shown at sixty hours (60 hours) of incubation with the production of 18.98 ± 0.21 U/ml of tyrosine indicating the highest protease activity by using casein as a substrate. The amount of tyrosine released after 12, 24, 36, 48, 72, and 84 hours of fermentation time was 7.70 ± 0.15 U/ml, 8.59 ± 0.32 U/ml, 11.18 ± 0.18 U/ml, 12.62 ± 0.25 U/ml, 15.22 ± 0.46 U/ml, and 7.35 ± 0.74 U/ml respectively. In the present study, the optimum time for protease production for C2 isolates was found to be 60 hours with protease activities of 18.98 U/ml. The decline in protease activity thereafter might be due to the decrease in microbial growth associated with the depletion of available nutrients, production of toxic metabolites, and autolysis caused by the protease produced (Sumantha et al.2006). This protease which was produced within 84hours was found to be closer to the previous studies (Hadash et al. 2017). In their studies, It was reported as 4.2 U/ml by Bacillus species for the same incubation time. The protease activity obtained at 60 hours was lower than the previous report (Asha and Palaniswamy, 2018) and about 140 U/ml of after seventy-two hours of fermentation time using Bacillus cereus FT 1 was reported. SDS-PAGE analysis showed purified protease bands with a relative molecular mass of approximately 76 kDa.

In another investigation reported by (Shine et al.2016), the highest protease activity obtained was 250 U/ml after 60 hours of fermentation time using bacillus strain CEMB 10370 (Sangeetha2012). Also, Bacillus pumilus SG 2 produces about 40U/ml at 60 hours of incubation. The variation in the protease activity was dependent on bacterial species even if the production of protease is carried out in the same incubation time, some bacterial species might have the capacity to degrade casein in the short period of incubation time and others might have the ability to degrade casein in a long incubation time. In addition to this, the activity might also depend on the environment from which the bacterium is isolated. Hadush et al. 2017 reported that, the optimum temperature for the production of proteases by Bacillus spp. Ew-9 and Sw-11 were found to be 37°C, which resulted in protease activities of 10.1 U/ml and 9.0 U/ml, respectively. The protease activity obtained at 37°C is about two-fold higher than the previous research report and the reason might be due to the environmental condition (high-temperature area) from where the species was isolated from.

Protease production by Bacillus sp. KW2 increased with increasing incubation temperature, peaking at 30°C (246 16 U/ml). At 40°C the enzyme production was 50.6 U/ml (Kshetriand Ningombam, 2016). Bacillus cereus demonstrated a progressive increase in protease production up to a temperature of 35°C and then a gradual drop thereafter (Asha and Palaniswamy, 2018). Bacillus cereus FT 1 produced enzymes between 25 and 45^oC, with a maximum enzyme activity of 168 U/mL when incubated at 35°C. According to Sangeetha, 2012, the optimum activity temperature for Bacillus pumilus SG 2 protease production was found to be 37^oC. However, between 32^oC and 42^oC, significant enzyme production was observed. Khusro, 2016 has found that Bacillus licheniformis had the highest enzyme activity of 60.552 U/mL at 35°C.In our research, the optimal activity was 40 U/ml at 37^oC, which is consistent with the findings of other research investigations.

Some researchers also tried to study the production of protease from thermophilic bacterial species and their findings deviate to some extent from our present study. The reason is the sampling area and the impact on the growth condition of bacteria. (Sarhan and Alamrri, 2014) studied the capability of the thermophilic bacteria Brevibacterium linens and Bacillus subtilis for the production of proteolytic enzymes. The optimum conditions for the production of those enzymes were achieved at 50^oC for both strains. Various researchers have tried to optimize the pH for different bacterial isolates. Sangeetha, 2012 stated that the protease activity was maximum when the pH was 8.0 and the production decreased significantly above and below this value for Bacillus pumilus SG 2. Hadash et al.2017 stated that the protease activities for bacillus spp, D-9 were recorded as 12.5 U/ml at pH 7 which strongly agrees with our research findings. However, in our study, higher protease activity was reported at pH 9It also correlates well with protease produced from Bacillus sp. THZ14. (Abrar2017). The optimum pH may differ much based on the sampling site and also the ethnicity of the protease-producing bacterial species. The present study agrees with the research previously conducted by (Lakshmi et al. 2014) who suggested that the optimum casein concentration for protease production was 1% by Bacillus licheniformis. On the other hand, recent research (Asha and Palaniswamy2018) suggested that 1.25% of casein concentration produces high protease activity by Bacillus cereus FT1. Some bacteria have the potential of degrading a high concentration of casein and release tyrosine quickly and some others may not survive in the high concentration of casein.

As reported by various researchers, the ideal carbon source for the production of protease differs for different protease-producing bacteria. Accordingly, lactose was found to have a strong influence on enzyme production with a protease activity of 151 U/mL, whereas all other carbon sources tested yielded only 50% enzyme yield when compared to maltose by Bacillus cereus FT 1. Bacillus odyssey, a halophilic bacterium, was found to use lactose as a carbon source for the highest protease synthesis when compared to fructose, maltose, or starch (Sneha et al. 2014). Lactose was shown to be the best carbon source for maximal protease synthesis in a Vibrio GA CAS2 strain, according to Azhar et al. 2014. Previous research has identified glucose as another major carbon source for protease synthesis (Suzuki et al. 2006). According to these researchers, glucose is the best carbon source for bacillus species to produce protease at a concentration of 1%. The latest research findings also support the fact that glucose might be the best carbon source for this isolate.

The maximum protease activity was 21.47 ± 0.78 with 1% (w/v) of gelatin as a primary nitrogen source. When the media was supplemented with gelatin as the nitrogen source, protease activity of 151 U/mL was observed. According to (Sangeetha2012), gelatin had a significant effect on the yield of protease by Bacillus pumilus SG 2 among the organic nitrogen sources studied. Also, the enzyme output was slightly reduced by beef extract and yeast extract. Although inorganic nitrogen sources were not as effective as organic nitrogen sources, urea was shown to be the best among them for the generation of protease, according to this researcher. In our studies also it was found that the activity was only 12.750.71U/ml, with urea as a nitrogen source. Hadash et al. 2017 discovered that employing casein, peptone, and yeast extract as organic nitrogen sources result in the highest protease production. The presence of high nutritional amino acids in these organic nitrogen sources is the reason why bacillus isolates produce so much protease. Using ammonium chloride as the nitrogen source, on the other hand, resulted in the least amount of protease synthesis. The conclusions were mostly consistent with those of the prior research. The inability of the bacterial isolates to utilize these nitrogen sources or the inhibitory effect of the inorganic nitrogen sources results in low-level protease synthesis. Berg et al. 2002 have suggested that Mg is the most important metal ion for bacterium which was isolated from tannery wastewater in Tunisia.

With 16mg of protein concentration after dialyzing, the maximal activity achieved was 123.35U/mg, showing that the protein molecules separated by ammonium primarily included protease enzyme and that the proportion of protein other than protease was higher in the crude form of the enzyme (Frey and Hegeman2007). Purification processes have resulted in the removal of interfering components seen in the crude cell-free extract, allowing for improved enzyme activity. This could be due to protease's larger molecular weight and poorer solubility in ammonium compared to other proteins in the crude enzyme, which aided protease separation (Agarwal et al.2012).

Protease activity started falling after using 3% inoculum size. The current results are in line with the work (Lakshmi et al.2014) reported 2% inoculum size as optimum for protease production by Bacillus licheniformis isolated out of leather effluents from IMTECH, Chandigarh, India. On the other hand, (Sarhan and Alamrri2014) suggested that the inoculum concentration of 4% v/v gave maximum alkaline protease activity by Bacillus licheniformis isolated from leather industry effluents. The effect of temperature on the stability of proteases was also measured by pre incubating them at the optimum pH of 12 hrs. According to reports of instability of enzymes, the protease activity was relatively stable at temperatures ranging from 60–65°C and 85.2% of the activity was retained after incubation at 70°C (Kumar et al.2016).

Based on the purification fold for the protease extraction, casein substrates increase with the folding of 1.56 when it is dialyzed. Berg et al. 2002 in their studies on the proteases have suggested that the purification fold would increase after every step of purification, including ammonium sulfate precipitation and dialysis. The present study results agree with the previous research and the purification fold of this enzyme increases from crude to dialyzed one. El-Betagy et al. 2004 reported an increased purification fold of 4.16 by ammonium sulfate precipitation to 4.33 by dialysis by a protease from the viscera of bolti fish (Tilapia nilotica).

After five days of feather degradation by Bacillus sp. FK 46 under ideal conditions, degraded feather (residue), and untreated feather were evaluated for crude proteins, in vitro pepsin digestibility, and amino acids, according to Suntornsuk W and Suntornsuk L (2003). The findings are comparable to those of Elmayergi and Smith (1971), who discovered that feather meal fermented by S. fradiae had higher levels of methionine, lysine, tyrosine, and histidine than the unfermented meal (Verela et al. 2002). Partially purified enzyme demonstrated significant feather degradation of 76.5% on the fifth day at optimized temperature and enzyme concentration. Because this enzyme candidate was found to have a promising potential in feather degradation it should be fully characterized.

This bacterial strain (Bacillus subtilis-C2) was isolated from Modji leather industry effluents, Addis Ababa. Nine different major parameters such as fermentation time, temperature, pH, casein concentration, metal ions sodium chloride concentration, inoculum sizes, carbon sources, and nitrogen sources were optimized during the production of alkaline protease and its utilization in feather degradation. The best optimum protease activity exhibited was at 60 hours, pH 9, 37^oC, 1% of casein concentration, glucose, gelatin, and sodium chloride, 2% inoculum size, and Mn as metal source. Based on the optimization studies, mass production and particle purification experiments were performed. Characterization by SDS-PAGE analysis showed purified alkaline protease bands with a relative molecular mass of approximately 76 kDa. Many more characteristic features to be revealed in the future through various analytic techniques and the characterization studies will pave a feasible way forward to potentially utilize alkaline protease extracted from Bacillus cereus (C2) in waste management applications. The maximum degradation of the feather was recorded as 76.5% under optimized reaction conditions. From this study, it was observed that alkaline protease extracted from Bacillus cereus has a promising potential in feather degradation and proteinaceous waste removal options especially, portentous wastes that are considered recalcitrant. Strain improvement of the isolates could also be considered for efficient degradation of the feather. Furthermore, the protease-degraded feathers contain a high protein quantity and they could potentially be used as animal feed additives. Based on this report the alkaline protease was found to be stable under a nitrogen environment (liquid nitrogen at -196^oC). More investigations on its ability to degrade other recalcitrant materials may widen its potential application.

BSA - Bovine serum albumin

OD - Optical Density

DNS - Dinitrosalicylic acid

EC - Enzyme Commission

SDS-PAGE - Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TCA - Trichloroacetic Acid

NaCl - Sodium Chloride

kDa - kilo Delta

mM - milli molar

Acknowledgments

We are grateful for the help of the College Dean, Head of Department, and Lab Coordinator from the Department of Biotechnology at the College of Biological and Chemical Engineering with this project.

Funding

This research work was supported by the Directorate of Research and Technology Transfer, Addis Ababa Science and Technology University in funding an internal Research grant (Ref No: IBC 06/2011).

Availability of Data and Materials -

On reasonable request, the corresponding author will provide the datasets used and/or analyzed during the current work.

Ethics Approval and Consent to Participate -

Not applicable

Competing Interests -

The authors declare that they have no competing interests.

Consent for publication -

Not applicable

Authors’ Contributions

A sample of the Modjo leather industry wastewater was collected by all of the writers. CM&GG was in charge of bacterial isolation and media optimization. The microorganisms were identified by MT&CM, and the application phase was completed. The final confirmation of possible alkaline protease-producing bacteria and paper amendments were done by all authors. The final version of the paper has been approved by all authors, and they agree to be held liable for its content.

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No competing interests reported.

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Alkaline protease from Bacillus cereus was characterized and optimized for eco-friendly degradation of feathers

Status:

Version 1

Abstract

Figures

1. Introduction

2. Material And Methods

2. 1. Production of crude alkaline protease by submerged fermentation

2. 2. Determination of alkaline protease activities of the selected isolate

2.2.1. Tyrosine and bovine serum albumin calibration curve

2.2.2. Total protein estimation

2.3. Optimization of the Growth Conditions for Production of Alkaline Protease

2.3.1. Effect of incubation time on the production of alkaline protease

2.3.2. Effect of temperature on the production of alkaline protease

2.3.3. Effect of pH on the production of alkaline protease

2.3.4. Effect of the concentration of substrate on the production of alkaline protease

2.3.5. Effect of different carbon sources on the production of alkaline protease

2.3.6. Effect of different nitrogen sources on the production of alkaline protease.

2.3.7. Effect of inoculum sizes on the production of alkaline protease

2.3.9 Effect of metal ions on the production of alkaline protease

2.4. Mass Production and Partial purification of alkaline protease

2.4.1. Dialysis of partially purified alkaline protease

2.4.2. Purification folds of crude and partially purified protease

2.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

2.6. Test for potential application in enzymatic feather degradation

2.6.1. Determination of feather degradation

2.6.2. The effect of protease concentration on feather degradation

2.6.3. The effect of incubation period on feather degradation

2.6.4. Reuse of the enzyme for degradation

3. Results

3.1 The standard calibration curve of BSA

3.1.1 Preparation of tyrosine standard curve

3.2. Production of alkaline protease

3.3. Optimization of media for protease production

3.3.1. The effect of fermentation time on alkaline protease production

3.2.2. Effect of Temperature on the Production of alkaline protease

3.2.3. Effect of pH on the production of alkaline protease

3.2.4. Effect of the concentration of substrate (Casein) on alkaline protease production

3.2.5. Effect of carbon source on alkaline protease production

3.2.6. The effect of different nitrogen sources on alkaline protease Production

3.2.7. Effect of inoculum sizes on alkaline protease production

3.2.8. Effect of sodium chloride (NaCl) concentration on protease production

3.2.9. Effect of metal ions on alkaline protease production

3.3. Purification of alkaline protease by Ammonium Sulfate precipitation

3.4. SDS-PAGE analysis of partially purified alkaline protease

3.5. Application of alkaline protease in feather degradation

3.5.1 Reuse of protease in feather degradation

4. Discussions

5. Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1