ISOLATION, SCREENING, PARTIAL PURIFICATION AND CHARACTERIZATION OF PROTEASE FROM HALOPHILIC BACTERIA ISOLATED FROM INDONESIAN FERMENTED FOOD

Abstract

The protease producing bacteria were screened from Indonesian traditional fermented food, tauco and terasi, and 4 halophilicprotease producers were isolated. Among these isolates, halophilic bacterial isolate TANN 4 was recorded as the best proteaseproducer. Extracellular protease from isolate TANN 4 was partially purified using ammonium sulfate precipitation. Theprotease was partially purified with final yield of 72.87 % and 25.41 fold purity. This moderate thermoactive and alkaliphilicprotease showed a pH optimum of 8.0 and temperature optimum was 50 °C. The enzyme was also active at salt concentrationsranging from 1 to 15 % (w/v), with optimum activity at 1 % NaCl (w/v). Ethylenediaminetetraacetic acid (EDTA) completelyinhibited the enzyme activity suggesting that it was a metalloprotease. Among metal ions, the Ca2+, K+and Mg2+ ions enhancedthe activity of enzyme. The KM and Vmax values exhibited by partially purified protease were 0.0649 mM and 216.45 U mg−1using casein as substrate. The molecular weight was estimated to be 19.8 kDa on SDS PAGE. The enzyme was also fairlystable in Triton X-100, SDS, 1 % commercial detergents (OMO and Ariel) and 25 % methanol and it was capable ofhydrolyzing casein, hemoglobin and bovine serum albumin (BSA). These characteristics make this halophilic bacterialextracellular metalloprotease seem to be potentially useful for biotechnological and industrial applications. Automatedribotyping analysis revealed that 3 isolates (TANN 4, TR 2 and TR 4) resembled Halobacillus trueperi that exhibited 71, 68and 69 % similarity respectively, and isolate (TR 1) resembled Virgibacillus pantothenticus with 64 % similarity.