Methods of Alkaline Protease Production from Bacillus Cereus Strain

Production of crude alkaline protease from Bacillus cereus strain S8 (MTCC NO 11901) was carried out by statistically optimized media supplemented with molasses, 1% (w/v); potassium nitrate, 0.75% (w/v); salt solution, 5% (v/v); {MgSO4.7H2O, 0.5% (w/v); KH2PO4, 0.5% (w/v)}; FeSO4.7H2O, 0.01% (w/v) and CaCO3, 0.5% formulated by [3].

Shrimp waste deproteinization

Shrimp waste (50%, w/v) was minced and cooked at 100В°C for 20 min to inactivate endogenous enzymes. Cooked sample was homogenized for 2 min in a Moulinex® blender. Adjust the pH of the mixture to 10.0 and then shrimp waste proteins were digested with crude protease (208 В± 0.89 U/ml). The reaction was stopped after 3 h incubation at 75В°C by heating the solution for 20 min at 100В°C. Then, the shrimp waste protein hydrolysate was centrifuged for 20 min at 5000g to separate soluble and insoluble fractions. Wash the solid phase with distilled water and dried for 1 h at 60В°C. Deproteinization (DP) was expressed as percentage and computed by the following equation as described by Rao et al., (2000).

% DP = [(PO Г— O) - (PR Г— R)] Г— 100

PO Г— O

Where PO and PR are protein concentrations (%) before and after hydrolysis; while, O and R represent the mass (g) of original sample and hydrolyzed residue in dry weight basis, respectively.

Silk degumming method

Raw silk threads were dried at 80В°C to constant weight and then treated with protease of Bacillus cereus strain S8 at 70В°C in Glycine-NaOH buffer (pH 10.0) for 1 h. Wash the treated fibres with water and dried at 80В°C. Weight difference between treated and untreated silk threads was measured. Structures of treated and untreated fibres were observed under scanning electron microscope.

Weight loss determination

Fabric weight loss was recorded as dried sample weight loss. The drying conditions were 80В°C in an air-circulated oven for 1 h. The samples were weighed, after cooling in a desiccator. The following equation was used to calculate the weight loss (wt %):

wt % =("W1-W2" /"W1" )"x100"

Where, W1 and W2 are the weights of the fabric before and after treatment, respectively (Kalantzi et al., 2008).

Dehairing studies

Dehairing of common salt preserved goat skins procured from the local market were cut into two pieces. One piece was chosen for dehairing using lime-sulphide to serve as control in the study and the other was taken for enzyme application trials. The skins were soaked with three changes of water till they are free from dirt, dung, blood and other contaminating materials and also free from sodium chloride as checked by silver nitrate solution. After soaking, the skins were piled to drain water for about an hour before the application. Control was applied on the flesh side uniformly with a paste of lime (10%) and 3% sodium sulphide. Other piece was dipped in 50 ml of Glycine-NaOH buffer (pH 10.0) supplemented with protease (206 В± 0.19 U/ml). After application, they left overnight. Next day (after about 18 h), the skins were dehaired manually on a wooden beam using a knife in tune with the commercial practice in the leather industry and the dehairing efficacy was assessed according to the depilated area of the skin at the end of the process and the quality of the dehaired skin was estimated by the naked eye after treatment.

SEM studies

Samples measuring 5Г—2 mm were cut from an identical location on control and enzyme treated leathers and mounted both vertically and horizontally on aluminum stubs and sputter coated with gold (JEOL, JSM-6610LV). The micrographs of the surface view recorded.

Destaining studies and detergent additive

Application of alkaline protease as a detergent additive was studied on white cotton cloth pieces (4x4 cm) stained with human blood and spicy food material. Effect of five different commercial detergents (Surf excel, Arial, Tide, Rin and wheel) on the enzyme activity was performed under optimal conditions at 5 mg/ml concentration. Based on the result obtained, detergent (Enzyme retained maximum activity) was selected for further study. The stained cloth pieces were taken in separate flasks. The following sets were prepared.

Set I

(a) Control: stained cloth (human blood)

(b) Stained cloth (human blood) + distilled water (100 ml)

(c) Stained cloth (human blood) + distilled water (100 ml) + 1 ml detergent (5 mg/ml)

(d) Stained cloth (human blood) + distilled water (100 ml) + 1 ml enzyme (186 U/ml)

(e) Stained cloth (human blood) + distilled water (100ml) + 1 ml detergent (5 mg/ml) + 1 ml enzyme solution (suitably diluted).

Set II

(a) Control: stained cloth (spicy food)

(b) Stained cloth (spicy food) + distilled water (100 ml)

(c) Stained cloth (spicy food) + distilled water (100 ml) + 1 ml detergent (5 mg/ml)

(d) Stained cloth (spicy food) + distilled water (100 ml) + 1 ml enzyme (186 U/ml)

(e) Stained cloth (spicy food) + distilled water (100ml) + 1 ml detergent (5 mg/ml) + 1 ml enzyme solution (suitably diluted).

The above flasks were incubated at 70°C for 15 min. Thereafter, the cloth pieces were taken out, rinsed with water and dried for visual examination. Untreated cloth pieces stained with blood and spicy food material were used as control.