Study of Protease Enzyme from Bacillus Species and its Application as a Contact Lens Cleanser

Aims: To study protease production from four Bacillus species. To optimize the production of extracellular protease by testing various environmental and nutritional factors and its application as a contact lens cleanser. Methodology: The assay was carried out in duplicates. Cultures of Bacillus subtilis, Bacillus licheniformis, Bacillus thermophilus, and Bacillus cereus were used for Protease production and assay. Sterile Bushnell – Hass medium containing 1% casein was used. The clear zone of casein hydrolysis indicated protease secretion. Protease assay was done using modified sigma protocol for enzymatic assay of protease. Effect of incubation time, pH, temperature and aeration on protease production was studied. Diameter of colony and zone of clearance was measured and Cx ratio was calculated. The best protease producer was selected. Extracted and crude protease enzymes were added to artificial tear solution prepared with 0.2% lysozyme. Enzyme treatment was done for 10, 30, 60 and 90 min at optimum temperatures of respective enzymes. Light transmission readings were recorded using visible range spectrophotometer at 285 nm. Results: All four species of Bacillus produced protease enzyme. Bacillus subtilis (Cx ratio=6) was the best protease producer. 37oC temperature, 48 hrs of incubation and pH 7.5 was optimum for maximum production of protease from Bacillus subtilis. Optimum temperature and pH for the crude enzyme activity was 40ºC and 8. The optimum ammonium sulfate fractionation (40% (w/v) saturation) showed 4.76 fold increase in the specific activity of the crude extract. Protein solution was degraded by purified enzyme in 30 min whereas crude enzyme required 60 min. Conclusion: Protease enzyme extracted from Bacillus subtilis showed good activity against artificial tear solution.

Author(s): Ashwini A Jadhav, Khatib Sayeed Ismail, Mahesh A Harale, Shashikant V Gadre and Manita T Williamson

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