3

3.7.2. Silver recovery
Treatment of X-ray films with protease lead to degradation of gelatin bounded to silver. So, this reaction was done by 20 µl of both forms of protease. We prepared humidify condition for prevention of evaporation. After two hours a clear zone showed gelatin degradation reaction and release of silver from it. Results in Fig. 6 shown that more gelatin hydrolysis was occurred by immobilized form compared to free protease.

3.7.3. De-haring process
Using some chemical materials for leather processing cause environmental pollution and low leather quality whereas enzymatic de-hairing mechanism give certain desired characteristics to the processed leather. Enzymatic dehairing by protease SO24 showed a big clear region derived from immobilized protease activity which demonstrated higher efficiency of immobilized protease activity compare to free enzyme in leather industry.
3.7.4. Applying as additive to detergent
Magnetic properties of nanoparticle help us to design a simple machine for moving this nanoparticle through cloth fibers (Fig. 1). We use a piece of cloth which soaked to blood for finding out clear efficiency of immobilized enzyme as additive in detergent industrial. Nanoparticles movement under electromechanical force were done in 20 second and results demonstrated all the blood stains were removed as shown in Fig. 6.

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4. Discussion
Bacillus SO24 which produced a high potent protease thrive in the alkaline environment among slaughterhouse wastes. It was immobilized on the functionalized Fe3O4 magnetic nanoparticles. Optimal temperature for protease activity was 50 °C for free protease whereas it was maximum in the range of 50-70 °C in the case of immobilized enzyme. It showed that the activity of immobilized enzyme was increased in higher temperatures in comparison with free enzyme. It showed an appreciated activity in the high temperatures in which its activity was retained about 90 % of the initial activity at 80 °C (Fig. 4a). Protease SO24 has the same optimum temperature for protease activity of B. subtilis PE-11. Also it is more thermostable than the Bacillus protease reported in some previous studies (Mukherjee et al., 2008; Sen et al., 2011). Moreover, its stability significantly increased after immobilization reaction (Jin et al., 2010). Results demonstrated that the immobilized enzyme retained 40% of its initial activity after 4 h in 50 °C whereas free form showed about 10 % activity during the same condition (Fig. 4c).
As in Fig. 4b) shown, both forms of protease were active over a wide range of pH (4.0-11.0). The maximum activity of free protease was observed in pH 8.0 which it was agreement with previous study on Bacillus cereus BG1 (Horikoshi, 1990). Many reports demonstrated Bacillus protease with alkaline stability in comparison with this study (Mala & Srividya, 2010; Sareen & Mishra, 2008; Sen et al., 2011). Optimum pH for immobilized form was pH 9.0, it showed immobilization process cause increase enzyme tolerant toward alkaline in comparison with free form. Furthermore, the reusability as a key factor in industry was also tested. Results showed residual activity after an 8th cycle was unchanged but a sharp decline of the activity was observed in 10th (Fig. 4d). Although, Hu et al. 2015, reported that protease immobilized onto amino-functionalized Fe3O4 nanoparticles showed 75% of initial activity retained after 6th cycle (Sinha & Khare, 2015). Furthermore, Atacan et al. 2016 and Hu et al. 2015 reported 79 and 50% remaining activity after 5th and 10th, respectively (Atacan et al., 2016). Among different substrates (albumin, casein, and gelatin) which used as substrate specificity of protease, gelatin was an attractive substrate for protease SO24 in comparison with other protein substrates. It can improve significantly of protease performance in silver recovery. Protease recovery and immobilization capacity were about 84 and 60%, respectively. It showed high efficiency of immobilization reaction and almost, in all case of hydrolysis reaction, immobilized form of enzyme was more efficient than its free form.
In this study we also evaluated application of protease SO24 in protease hydrolysis, leather process, silver recovery and using as additive in detergent industrial. Whey contains high value of soluble proteins which obtains from byproduct of local and industrial dairy waste (about 20 % of the total milk proteins). In addition, it contains high amount of lactose, vitamin B, and essential amino acids (e Silva & Silveira, 2013). Some proteolytic enzyme such as subtilisin, trypsin, and corolase have been used for enzymatic hydrolysis of whey (Silvestre et al., 2014; Wang et al., 2010). It is mentioned that, degree of hydrolysis for Whey was reported in the range of 5 to 23 %. Sinha et al, reported that DH was about 35 % for immobilized halophilic Bacillus sp. EMB9 protease after 30 min of incubation (Sinha & Khare, 2015). Our results showed that degree of hydrolysis for Whey was 44 % for immobilized thermophilic protease after 20 min of incubation at 60 % whereas it was 25 % for free enzyme at the same condition. Maximum DH (44 %) after 20 min of incubation for the immobilized protease indicating the improved process efficiency. It has been previously reported that, protein hydrolysates of whey proteins display some valuable properties such as antihypertensive, antimicrobial, lipid-lowering and antioxidant (Conesa & FitzGerald, 2013).
Merging biological and magnetic science in nanoscale level made a high number of notable advancement in the clinical and sensor technology (Haes et al., 2005; Ivkov, 2013). A simple mechanic system was manifested to use magnetic property of protease coupled nanoparticle. Appling this system for different forms of wash-up methods including protease coupled nanoparticle+ detergent, detergent alone, protease coupled nanoparticle alone and detergent alone. Results showed high potential level of immobilized protease SO24 coupled detergents as additive in detergent industry. Protease recovery showed compatibility of this enzyme in combination with detergent which its performance is also shown in Fig. 6.
5. Conclusion
A facile method to immobilize CLEAs-protease onto amino-coated magnetite nanoparticles was established to attain enhanced activity and stability. Hydrolysis degree for Whey was 44 % for immobilized thermophilic protease after 20 min of incubation at 60 %. In addition, a simple and efficient mechanic system was manifested to use of mCLEA-P-NC in washing performance. mCLEA-P-NC showed appreciated potential for treatment of waste protein of local wwly. Taken to gather, unique properties of this magnetic-CLEAs protease open an attractive way towards commercialize the production of high value product from waste protein and in washing system.