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

Authors : Sarah FITRIANI, Kıymet GÜVEN

Pages : 130-142

Doi:10.18036/aubtdc.322711

View : 15 | Download : 6

Publication Date : 2018-08-17

Article Type : Research Paper

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