Bacteria (Tang et al., 2008). The incubation was

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
Casein degradation reaction was done as protease activity assay which illustrate by Kembhavi and Kulkami with slight modification (Kembhavi et al., 1993). Briefly, 50µl of partial purified enzyme was added to a microtube containing 200µl of 2% (w/v) casein (immerse in 20 mM Tris–HCl buffer, pH 8.0) and 150µl 20 mM Tris–HCl buffer, pH 8.0. After incubation for 15 min in 45°C, 300µl of 10% trichloroacetic acid (TCA) was added to disrupt casein degradation reaction and was incubated at 4 °C for 30 min. Tubes were centrifuged at 13000 rpm for 15 min and absorbance of supernatant was measured using UV-visible spectroscopy. One unit of enzyme activity refers to 1µg product liberated by the amount of enzyme per minute.
2.5. Synthesis and modification of magnetite nanoparticle (Fe3O4)
Magnetic Fe3O4 nanoparticles were prepared and its surface modified with amine group by method of REZA et al (Reza et al., 2010). For this 1.351 g of Ferric chloride (FeCl3-6H2O) were mixed with 0.6852 g of Ferrous sulphate (FeSO4-7H2O) and 25 mL deionized water, then precipitate was formed by adding ammonium hydroxide in room temperature. Product purification was done by centrifuge the solvent. Sediment has been washed with deionized water for several time until a pH value of 7.0 was obtained. Pellet was dried at 100 ?C for 2 h. In order to obtain amino functionalized groups on magnetic particle, surface salinization reaction with 3-aminopropyl triethoxysilane (APTES) was done. The procedure consisted of mixture of deionized water (25µl), APTES (100µl), magnetite nanoparticles (0.02 g) dissolve in methanol (2.5ml) and sonicate for 30 min. Then, the mixture was heated at 90?C for 6 h with mechanical agitation. For prevent of evaporation, glycerol (1.5ml) was added to solution. Sediment obtained was washed with water and methanol three times.
2.6. Enzyme modification and coupling reaction
Enzyme modification was carried out with following method. 2 ml of solution containing enzyme and 5ml of saturated ammonium sulphate solution was added to Erlenmeyer flasks containing magnetic stirrer bar. For enzyme precipitation keep stirring for 30 min at 4 °C, then glutaraldehyde at the final concentration of 40 mM was added, and agitated with a stirrer bar at 30 °C for 3 h. Finally, it centrifuged for 10 min in 10000 rpm and precipitate was washed three times and stored in 20 mM Tris-HCl buffer at 4°C. Lastly, linkage reaction and crosslinking between enzymes was established by glutaraldehyde. So, magnetic macromolecule nanocomposite were prepared as following. 1 ml of modified enzyme was mixed with 5 mg of amino functionalized magnetite nanoparticles and agitated for 15 min at 30 °C in a shaker flask, then 5 ml of saturated ammonium sulphate solution was used in mixture with mechanical agitation at 4 °C for 30 min. After precipitation reaction glutaraldehyde added to a final concentration of 40 mM to mixture and stirred for 3 h at 30 °C. Finally, magnetic nanocomposite ware separated using magnet and washed three times with 20 mM Tris-HCl buffer and stored in this buffer at 4 °C.

2.7. Immobilization yield and activity recovery
Activity recovery of immobilized enzyme was determined as following:
Activity recovery (%) = (total activity of immobilized enzyme (U)×100)/(total activity of enzyme used for immobilization reaction)
Bradford method and UV-visible spectroscopy were also applied for determining the immobilization capacity that was calculated by following formula:
That Ci and Cw are initial protein concentration and the protein found in washing solution after immobilization, respectively. And Cb is total protein in unbound after immobilization.
2.8. Physicochemical characterization of magnetic CLEA-nanocomposite
Some features of enzyme coupled nanoparticle and free form of nanoparticle were analyzed by Fourier-transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). FT-IR spectroscopy studies of lyophilized form of samples were measured by the FT-IR spectra were scanned in a range from 4000 to 400 cm-1. The surface morphology investigation by SEM under acceleration voltage of 26 kV was transparent.
2.9. Biochemical characterization of the enzyme
2.9.1. Effect of temperature and pH on the enzyme performance
Optimum activity condition for free and immobilized enzyme was determined by protease assay at different pH ranging from 4.0-12.0 (temperature 45 °C). Best temperature for enzyme activity was investigated in various temperatures from 30-90 °C in Tris-HCl buffer (20 mM, pH 8.0). Enzyme stability in temperature 50 °C was also investigated at different time range of 15-240 min.
2.9.2. Effect of metallic salts and inhibitors on free enzyme activity
All metallic salt was dissolved in Tris-HCl buffer (20 mM, pH 8.0), with final concentration of 1 mM. Enzyme was added and pre-incubated for 1 h after that the residual activity (%) was determined under standard conditions by protease assay. With the same strategy the effect of some inhibitors and detergents such as SDS, Triton X-100, Tween 20, EDTA, HgCl2, B-Mercaptoethanol, and H2O2 were also studied.
2.9.3. Stability in organic solvents and Kinetic Parameters
In order to study the effect of organic solvent on enzyme stability, 200µl of protease was mixed with different organic solvents in two final concentrations of 20 and 40% (v/v). Mixture was incubated at 30 °C and agitated on a rotary shaker at 180 rpm for 5 days. Remaining protease activity was calculated by protease assay as described previously.
Km and Vmax values of casein degradation activity of the free and immobilized enzyme were compared by Lineweaver–Burk plot. In this method different concentration of casein as a substrate and by plotting the values of 1/v as a function of 1/S.
2.9.4. Substrate specificity and reusability of immobilized enzyme
For finding specific substrate for extracted enzyme, we apply 1% concentration of different protein including albumin, casein, and gelatin as substrate. 20µl of enzyme was mixed with 200µl substrate solution and 80µl of 20mM Tris- HCl buffer pH 8.0. Then, the reaction stopped with TCA (5 %) and read absorbance in 280 nm as explained previously.
Stability of immobilized enzyme was done via casein hydrolysis reaction which followed in 10 cycles. At the end of every cycle, protease assay was performed. Then enzyme coupled nanoparticle collected by a magnet, wash them by Tris-HCl buffer (20 mM, pH 8.0) to separate enzyme from substrate and reuse them in the next cycles.


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