Solid support based nanocomposite open ample opportunities for large scale application in industrial biocatalyst market. In this study, we isolated a high potent protease producing Bacillus cereus. Results showed that, it was an organic solvent tolerant, thermophile protease which showed 20 % increase in protease stability in the presence of n-hexane. It was active in a wide range of pHs and temperatures. A magnetic CLEA-protease nanocomposite (mCLEA-P-NC) was constructed successfully which confirmed by SEM and FTIR analysis. Results showed that mCLEA-P-NC displayed Km value 4.6 folds lower than free enzyme. Biotechnological applications showed nearly 44 % of wwly proteins were degraded by mCLEA-P-NC. In addition, it showed high potential in silver recovery and de-haring process. A simple mechanic system was manifested to use mCLEA-P-NC in washing performance. These results indicated high potential of this protease nanocomposite in biotechnological application especially in recycling of waste proteins and washing performance.
Key words: protease, Bacillus cereus, immobilization, nanocomposite
Solid support based nanocomposite open ample opportunities for large scale application in industrial biocatalyst market. Among such attractive nanomaterials, magnetic nanoparticles provide a great positive point such as biocompatibility, catalytic activity, efficient brownian motion in solution, and enhanced surface to volume ratio due to small size (Gubin et al., 2005). These features made them an efficient solid surface for macromolecules coupling like enzymes. To date, some enzyme such as laccase (Das et al., 2017), cellulose (Grewal et al., 2017), keratinase (Konwarh et al., 2009), lipase (Huang et al., 2003) and ?-amylase (Namdeo & Bajpai, 2009) have been immobilized on the magnetic nanoparticles. Proteases are a dominant component of industrial enzymes. They share approximately 60% of the total sales in the world (Banik & Prakash, 2004; Sangeetha & Abraham, 2006). So, there is an unmet need for them to be easy to recycle and stability under harsh condition (Chang & Tang, 2014; Luckarift et al., 2004; Park et al., 2013). One of the effective existing strategies is immobilization of enzymes on magnetic nanocomposite surface which made the hydrolysis process more controllable.
In recent years, biocatalyst processes by proteases opened their place in different commercial purposes like pharmaceuticals, leather processing, food production, and detergent industry and they have attracted substantial interest as a promising candidate among industrial enzymes. Diversity and potential growing condition of bacteria made extracellular microbial enzymes to popular tools in biotechnology application scale. Also applying this technology based on enzymes lead to pollutants abatement in environment (Huang et al., 2005; Mao et al., 2010; Wang et al., 2012). So, we screen an organic solvent tolerant, thermophilic protease with robust signs of efficient hydrolysis rate of substrates. This enzyme was secreted from bacteria belonged to the genus Bacillus which isolated from slaughterhouse wastes and deposited in Gene Bank databases (accession code: MG009251.1).
Development of immobilization protease technology has a lot of application in biotechnology. For example, high efficient recovery of waste materials as one of the major environmental problem can be achieved by some immobilized enzymes. On the other side, natural resources of silver are decreasing and high applications of this metal in photography, biomedical, medicines, radiography, and electronics cannot be ignore (Liang et al., 2007; Tao et al., 2012). In this respect, bio-treatment of waste X-ray films as a recyclable silver source was done with protease as eco-friendly methods which result in efficient re-use of the other cheap source of silver in nanoscale (Nakibo?lu et al., 2003; Shankar et al., 2010). In addition, protease immobilization on nano-surface has efficient application in protein hydrolysates which has wide usage in biotechnology and food industry (Heidemann et al., 2000; Onuh et al., 2014; Pasupuleti et al., 2008; Shobharani & Agrawal, 2009). Low-cost waste materials as enzyme substrates has the great advantage to enable reuse of them during a cycle. Wastewater of local yogurt is a large waste resource. High protein content of this waste material paved the way for spectacular changes in protein hydrolysates industry. Furthermore, extreme application of proteases in detergent additive industry showed severe need to a desirable tool in detergent to control wash approaches. Using of magnetic immobilized protease under a variable electromagnetic field is one of the key procedure for control wash approaches was also considered in this study.
2. Material and Method
2.1. Screening organic solvent resistant protease secreting bacteria
Bacteria isolated from the slaughterhouse wastes samples were screened based on tolerant level against toluene and cyclohexane enrichment in modified Luria-Bertani medium (MLB) containing tryptone (10.0 g/L); NaCl (10 g/L); yeast extract (5.0 g/L); and MgSO4 (0.5 g/L), as previously described (Badoei-Dalfard et al., 2010; Ogino et al., 1995). After autoclaving, both toluene and cyclohexane were added to the medium at final concentration at 10 and 20%, respectively. To prevent toluene and cyclohexane evaporation, the cultivation flask with content 20 ml of medium in 250 ml shake flasks was plugged with a chloroprene–rubber stopper (Tang et al., 2008). The incubation was carried out in shaker incubator at 30 °C with agitation at 200 rpm for two days. Repeated transfer in the same culture conditions with the same strategy should be done for cultures acclimate, then samples were diluted and spread on MLB-plates. Colonies able to grow on the selective plates were purified by repeated streaking.
2.2. Isolation of high efficient colony for secretion of protease
The colony with high ratios of clear zone diameter to colony diameter were elected after 24-48 h of incubation in skim milk agar plate (SMA) which containing yeast extract, 3.0(g/l), tryptone, 5.0(g/l); skimmed milk powder, 25(g/l); and bacteriological agar 12(g/l). For following experiments, the selected colony stored at –80 °C in LB supplemented with 25% (v/v) glycerol.
2.3. Production and partial purification of protease
Extraction of protease was conducted in two step. At first, selected colony was inoculate in pre-culture medium containing nutrient broth (8.0 g/L), NaCl (5.0 g/L), starch (10 g/L), and yeast extract (10 g/L). After incubation at 37 °C with agitation 160 rpm for 16 h, 10% (v/v) of this medium content was inoculate with production of protease culture containing sucrose (5.0 g/L), citric acid (5.0 g/L), yeast extract (10 g/L), K2HPO4 (1.0 g/L), MgSO4·7H2O (0.1 g/L) and of CaCl2·2H2O (0.1 g/L) (Moradian et al., 2009). pH were adjusted to 7.0 and incubation were taken for grow and protease secreted for 48 h with agitation 200 rpm. Then, for remove cell and additional large particle centrifuge with 6000 rpm for 5 min was done. Subsequently crude protease precipitation was conduct with adding 0-100% of ((NH4)2SO4). Precipitates were collected by centrifuged at 10000 rpm for 30 min in 4 °C and were dissolved in a small amount of 20 mM Tris–HCl buffer (pH 8.0). Finally, dialyzed protein were obtained overnight in the cold condition against 100 volumes of 20 mM Tris–HCl buffer to release ammonium sulphate salt.
2.4. Protease assay