SCREENING, ISOLATION AND CHARACTERISATION OF PROTEASE PRODUCING MODERATELY HALOPHILIC MICROORGANISMSANNAPURNA S.A., AMARNATH SINGH, SHASHANK GARG , ANUPAM KUMAR AND HARSH KUMARAbstract The present study is focused on isolation of moderately halophilic bacteria and fungi that are capable of producing protease from the samples collected from Sambhar lake of Rajasthan and Mumbai seashore. Extremely and moderately halophilic bacteria dominate in saline environments (0.5% to saturated NaCl). Moderately halophilic microorganisms include a broad variety of bacteria and fungi that are able to grow in media containing a wide range of elevated NaCl concentrations (3- 15% NaCl). Considerable attention has been focused on enzymes of moderately halophilic bacteria, since they have substantial biotechnological potential. While several proteases from extreme halophiles, members of the haloarchaea have been characterized, fewer proteases from moderately halophilic bacteria have been purified and studied in depth. These microorganisms use different strategies for preserving their cell structure and function in highly saline conditions. They may produce compounds of industrial interest, such as extracellular hydrolytic enzymes with diverse potential applications in the industries. Therefore, an attempt is done in the present study for isolation of moderately halophilic bacteria and fungi able to produce protease which is of industrial importance. The samples collected from Sambhar lake of Rajasthan and Mumbai seashore were processed for the isolation and characterization of the bacteria and fungi able to produce the protease enzyme which further was shown to have the proteolytic activity demonstrated by using blood stained surgical instruments, hair, and casein. The recovery of silver from the X ray photographic film was also attempted. The molecular sequencing of the fungus and bacteria with the phylogenetic tree formulated revealed the presence of Aspergillus flavus in the sample from Mumbai sea shore and Bacillus subtilis from Sambhar lake of Rajasthan. In the subsequent phase effect of temperature, pH, activator and inhibitor was also studied on the activity of protease enzyme by using casein and hair as substrates. The enzyme further was shown to have the ability to remove the blood stain from the surgical instruments as well as to recover silver from the X ray film. The isolate Bacillus subtilis optimally grown at pH-8 and temperature 37°C and 10% NaCl with protease activity on casein and human hair. Protease enzyme obtained in the present study by using Bacillus subtilis and Aspergillus flavus is found to be stable and active so could be of significant use for detergent and leather processing technology as well as for dehairing and silver recovery.
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