Sephadex G-200, bovine serum albumin (BSA), ethylene diamine tetra acetic acid (EDTA), casein, β-mercaptoethanol, phenyl methyl sulphonyl fluoride (PMSF) and media components were products of Sigma-Aldrich, St Louis, MO, USA. All other chemicals used were of analytical grade and obtained from Fisher Scientific (Waltham, MA, USA). Commercial laundry detergents used were Omo and Sunlight from Unilever (Rotterdam, Netherlands); Ariel was a product of Procter and Gamble (Cincinnati, OH, USA).
Isolation and identification
Sub-soil samples were collected aseptically from organic waste sites which included beds of effluent treatment plants of selected brewery, dairy and food industries, drainage from abattoir, poultry litter site and locust bean processing farm in the south western part of Nigeria for the preparation of initial culture. These were sub-cultured to obtain pure isolates of Bacillus species using the method of Aslim et al. . The Bacillus species were identified based on methods described in Bergey's Manual of Systematic Bacteriology ,.
Screening for proteolytic activity of Bacillus species
The identified bacterial isolates were plated onto casein milk agar plates and were incubated at 37°C for 24 h. 15% HgCl2 in 20% HCl was added to the plates and examined for clearing zone around the bacterial growth. The diameter of the zone was measured in millimeters. A clear zone of casein hydrolysis gave an indication of protease-producing organisms. Depending on the zone of clearance, B. brevis MWB-01 was selected for further studies.
Production of protease
Production of protease from Bacillus brevis MWB-01 was carried out in a culture medium containing the following: 0.5% glucose, 0.75% peptone, and 5% salt solution made up of 0.5% MgSO4.7H2O and 0.1% NaCl, maintained at 37°C for 72 h in a shaking incubator (180 rpm). At the end of each cultivation period, the broth was centrifuged at 10,000 rpm at 4°C for 15 min. The cell-free supernatant was collected as crude enzyme preparation and subjected to purification procedures.
Assay of protease activity
Extracellular protease activity was determined using a modified procedure of Fujiwara et al.  with 1.0% casein in 50 mM Tris-HCl buffer pH 8.0 as substrate. The assay mixture consisted of 0.4 ml of substrate and 0.1 ml of enzyme solution in 50 mM Tris-HCl buffer pH 8.0. The assay mixture was incubated at 40°C for 30 min and reaction was terminated by the addition of 2.5 ml of 10% (w/v) trichloroacetic acid (TCA). The mixture was allowed to stand for 15 min and then centrifuged at 10,000 rpm for 10 min at 4°C to remove the resulting precipitate. Protease activity was determined by estimating the amount of tyrosine in the supernatant which was done by measuring the absorbance at 280 nm. One unit of protease activity was defined as the amount of enzyme required to release 1 μg of tyrosine per milliliter per minute under the specified assay conditions.
Purification of protease from B. brevis MWB-01
The cell-free supernatant was fractionated by precipitation with ammonium sulphate of 80% saturation. The precipitated protein collected by centrifugation was dissolved in 50 mM Tris-HCl buffer pH 7.5, and dialyzed against the same buffer at 4°C with three buffer changes, each for 12 h using Spectra/Por dialysis membrane (MWCO 3,500; Serva, Heidelberg, Germany). The resulting dialysate was centrifuged at 10,000 rpm, 4°C for 15 min, and the supernatant was applied on Sephadex G-200 (1.5 × 24 cm) column (Sigma-Aldrich, St Louis, MO, USA) equilibrated with 50 mM Tris-HCl buffer, pH 7.5. The column was eluted at a flow rate of 0.5 ml/min. Protease activity was assayed in all eluted fractions. The fractions (25 to 27) with high protease activity corresponding to the highest peak on the chromatogram were pooled, and subsequently used for characterization studies. The concentration of protein during purification studies was determined by Bradford method .
Partial characterization of purified protease
Effect of temperature on the activity and stability of protease
The optimum temperature of purified protease was determined by measuring enzyme activity at varied temperatures (30°C to 70°C). The reaction mixture was incubated at respective temperatures for 30 min before determining protease activity according to the standard assay procedure earlier described. Thermal stability was determined by measuring the residual protease activity after 30 min and 60 min of pre-incubation of purified enzyme at temperatures ranging from 30°C to 70°C in 50 mM Tris-HCl buffer pH 8.0.
Effect of pH on activity and stability of protease
Effect of pH on activity of protease was determined by assaying for enzyme activity at different pH values ranging from 4.0 to 12.0. The pH was adjusted using 50 mM of the following buffer solutions: sodium acetate (pH 4.0 to 5.0), sodium citrate (pH 6.0), Tris-HCl (pH 7.0 to 8.0) and glycine-NaOH (pH 9.0 to 12.0). Reaction mixtures were incubated at 40°C for 30 min and the activity of the protease was measured. To determine the effect of pH on stability of protease, the purified protease was incubated in relevant buffers of varying pH (4.0 to 12.0) without substrate for 60 min at 40°C. The residual protease activity was determined as previously described.
Effect of inhibitors on protease activity
Effect of inhibitors (phenylmethylsulphonyl fluoride [PMSF], β-mercaptoethanol [β-ME] and ethylene diamine tetra acetic acid [EDTA]) at 5 mM on protease activity was determined by pre-incubating the purified enzyme solution with inhibitor for 30 min at 40°C before the addition of substrate following the standard assay procedure.
Effect of metal ions on protease activity
The effects of metal ions on enzyme activity (Ca2+, Mg2+, Al3+, Mn2+, Zn2+, and Hg2+) at 5 mM was investigated by pre-incubating the purified protease with each of the metallic chlorides without substrate for 30 min at 40°C. The residual protease activity was measured as previously described.
Substrate specificity of purified protease
Substrate specificity of purified protease from B. brevis MWB-01 was studied by examining proteolytic activity on protein substrates. The substrates studied were casein, bovine serum albumin (BSA), egg albumin, and gelatin. Purified protease (0.5 ml) was added to 2.0 ml of 50 mM Tris-HCl buffer pH 8.0 containing 1% substrate. After incubation at 40°C for 30 min, the reaction was stopped by adding 2.5 ml of 10% TCA. Protease activity was determined by following the standard assay procedure.
Detergent compatibility of purified protease
The compatibility of B. brevis protease with commercial laundry detergents, Sunlight (Unilever), Ariel (Procter and Gamble), and Omo (Unilever), was studied. The effect of 5 mg/ml of each detergent on the stability of purified protease was determined. The diluted detergent solution was heated to 60°C for 1 h to denature the enzymes present in the detergent and left to cool at room temperature for 1 h. The purified protease was incubated with the diluted detergent for 1 h at 40°C and the residual activity was determined. The enzyme activity of the control was taken as 100%.