Characterization and Application of Protease Enzyme from Oceanobacillus Iheyensis KB7
Molecular identification of bacteria
The 16S rRNA gene of the Oceanobacillus iheyensis strain was amplified using Polymerase Chain Reaction (PCR) with the help of 16S rRNA Universal primers. The 16S rRNA gene sequences were submitted to the BANK it and the ID number is (O. iheyensis _ MF192763).
The Phylogenetic relationship was obtained using neighbor joining by pair wise comparison among the 16S rRNA gene sequence of selected isolate with species. The dendrogram was constructed for their Phylogenetic relationship and it revealed that the isolate was distinctly placed under separate clusters (Fig. 1).
Fig.1. Phylogenetic tree
Effect of agricultural residues on protease production
The maximum amount of enzyme production in whitegram husk (1.013 IU/ml) and minimum amount of protease production in rice bran (0.512 IU/ml) showed in fig. 2.
Fig. 2. Effect of agricultural residues on protease production
pH stability for protease activity
The pH stability of enzyme activity was found to be Glycine NaOH buffer (0.666 IU/ml) at 72 hours showed in fig. 3. The protease stability was increased at higher pH like 10. This gives the credit that as pH decreases much lower than inactivation of enzyme would be occurred.
Fig. 3. pH stability for protease activity
Temperature stability for protease activity
The effect of temperature on protease stability was maximum at 40?—¦C (0.813 IU/ml), the minimum stability at 60?—¦C (0.196 IU/ml) showed in fig. 4. At higher temperature the protein and nucleic acids would be denatured. So at higher temperature the inactivation of protein would be carried out.
Fig. 4. Temperature stability for protease activity
Effect of metal ions on activity and stability of protease
The effect of metal ions for protease activity was maximum stability of protease enzyme at 72 hours Calcium chloride (0.724 IU/ml) showed in fig.5. The protease enzyme stability was improved by supplementing Calcium carbonate and Potassium chloride. The enzyme activity was superior by adding K+, Ca+2 salts to the medium for better protease stability and production. By adding sodium sulfate would be decreased the production and stability of enzyme.
Fig. 5. Effect of metal ions on activity and stability of protease
Effect of surfactants on activity and stability of protease
The maximum protease stability is Tween 20 (0.812 IU/ml) and minimum protease stability is Triton X-100 (0.499 IU/ml) showed in fig. 6.
Fig. 6. Effect of surfactants on activity and stability of protease
Effect of inhibitor (HgCl2) and activator (CaCl2) on protease activity
The inhibitor (HgCl2) is high amount of protease production is (0.556 IU/ml) at 72 hours. The Activator (CaCl2) is high amount of protease production is (0.654 IU/ml) at 72 hours showed in fig. 7.
Fig.7. Effect of inhibitor (HgCl2) and activator (CaCl2) on protease activity
Wash performance for removal of various stains
A clean piece of cloths was soaked in soy sauce, juice, tomato, green leaves, greeze, tea, chocolate, coffee and blood to dry overnight. The dried stained cloths were incubated with the protease solution 40 IU/ml.
Fig.8a. various stains applied to clean cloth
Fig.8b. After washing with protease enzyme
One g of X-ray film cut into 2 x 2 cm pieces was then incubated with 10 ml of crude protease (such that the film is completely immersed in the solution) at 40?°C, (pH 10) in a water bath with continuous shaking. Turbidity of the reaction mixture (hydrolysate) increased with time and no further increase in turbidity was observed when hydrolysis was complete. Hence, turbidity was monitored by measuring the absorbance at 660 nm. So, Oceanobacillus iheyensis KB7 recovered the silver from X-ray film. The turbidity was measured at 660 nm, maximum amount of turbidity recorded at (1.552 ?± 0.003), minimum amount of turbidity was recorded at (0.223 ?± 0.004) showed in fig. 9a and 9b.
Fig.9a. Before silver recovery of x-ray film