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

Year 2018, Volume: 7 Issue: 2, 130 - 142, 17.08.2018

Sarah Fıtrıanı Kıymet Güven

https://doi.org/10.18036/aubtdc.322711

Cited By: 1

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

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