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 45oC, 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 37oC. However, between 32oC and 42oC, 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 37oC, 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 50oC 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.