Screening and Strain Improvement of Protease-Producing Bacillus spp. From Various Environmental Samples with Kinetic Characterization

Abstract

Background and Objective: Proteolytic enzymes account for about 60% of total enzyme sales worldwide, making them of major industrial importance. This study aimed to isolate, screen, and enhance protease-producing Bacillus spp. from four environmental sources.                                                  

 Methods: Twenty-one proteolytic isolates were obtained in primary screening from various food sources, the maximum of which (8 isolates) was obtained from the spoiled pulses. Bacillus spp was determined as the most active isolate (casein hydrolysis zone measuring 17 mm) by gram staining, biochemical tests and Vitek 2 BCL card (more than 90 % confidence level). Strain improvement applied: UV irradiation (15 min); chemical mutagens (acridine orange, ethidium bromide (25 – 100µg/ml)). The production of enzymes was carried out at SMB at a temperature of 37°C for 72 h (150 rpm) and the amount of protein was determined by the Folin-Lowry method. The enzyme was partially purified by ammonium sulphate precipitation (80% sat.) & its kinetic parameters determined.                                                                    

   Results: UV mutagenesis produced a significantly greater increase in protease production, giving a 38 mm clearance zone compared with the chemical mutagens tested. Maximum enzyme activity after partial purification was 121.03 µM/min. The optimum temperature and pH for enzyme activity were 30°C and pH 6, respectively, indicating a mesophilic acid protease.                                                                 

   Conclusions: UV mutagenesis is an effective strategy for improving protease production in Bacillus spp., with promising applications in food processing and the dairy industry.