Partially purification and characterization of protease from Clostridium botulinum Type A

In this study, partially purification and characterization of protease from Clostridium botulinum Type A ATCC 7948 was investigated. Protease production was grown in %1 tripton, %1 peptone, %0,5 yeast extract , %0,1 soluble starch, %0,3 CH3COONa, % 0,05 L-cysteine, %0,03 sodium thioglycolate, %0,5 NaCl and % 1 glucose at 37 0C and 20 hour. Enzyme was purified by %85 ammonium sulfate precipitation and DEAE cellulose anion exchange chromatography with %6,12 yield and 3,67 fold. Specific activity was 650 U/mg. The optimum pH and temperature of protease was 8 and 50 0C, respectively. Protease activity was maintained %100 after 3 hours at 40 0C. Enzyme deactivated at 60 and 70 0C for 15 minute. While enzyme activity increased in the presence of Zn2+, Ca2+, Co2+, Mg2+ and Ni2+, Cd2+ and Sn2+ inhibited enzyme. Al3+, Fe3+, Na+ and Sr2+ showed no effect on protease activity. Protease showed highest activity towards casein among other native proteins such as gelatin, BSA, soy bean. Enzyme maintained activity in the presence of ethanol, methanol, DMSO, 2-propanol and acetone. But enzyme deactivated %39 and %23 in the presence of n-butanol and hexane, respectively. While Triton X-100 and Tween 80 increased enzyme activity, enzyme maintained activity towards SDS. It was observed that enzyme activity increased in the presence of %2 and %5 H2O2. Enzyme was inhibited by metalloprotease inhibitor EDTA. Also 2 and 5 mM PMSF inhibited enzyme at the rate of %18 and %30, respectively. This inhibition profile showed that enzyme was metalloprotease and there were serine residues near the active site.Science Code : 201.1.020Key Words : Metalloprotease, Clostridium botulinum, purification, characterizationPage Number : 81Adviser : Prof. Dr. Elif LOĞOĞLU?