Optimization of Protease Enzyme Production by the Halo-Tolerant Vibrio alginolyticus Isolated from Marine Sources

Abstract

A renewed interest in the study of proteolytic enzymes is chiefly attributable to its surging industrial applications apart from its pivotal role in the cellular metabolic process. We report the screening, isolation and partial purification of a low-molecular-weight halo-tolerant alkaline protease from a bacterial isolate of marine environment. Twenty four bacterial isolates were collected from marine environment, of which Vibrio alginolyticus showed relatively higher proteolytic activity on agar plate and was subjected to further characterization. Enzyme production kinetics under submerged fermentation was optimized, that included incubation time, pH and heavy metal tolerance. The bacterium produced the highest protease activity (4650 U/mL) when incubated for 48 h at pH 8.0. The organism was found to be more tolerant to iron than other metals like Cu²⁺, Mn2⁺, Zn²⁺, Co²⁺ and Hg²⁺. Natural substrates such as rice and wheat brans were evaluated for protease enzyme production under solid state fermentation. A mixture of rice and wheat bran at a ratio of 1:1 supported the higher enzyme production. Effect of NaCl, heavy metals and EDTA on the catalytic activity of the crude enzyme was assayed. The total activity of the crude enzyme was unaffected at elevated concentrations of NaCl and retained 40 % of its activity even at 4 M concentration which indicated the halo-tolerant ability of the bacterium. An increase in the catalytic activity (about 30 % or 1.2 fold) was recorded when incubated with 1 mM of Co²⁺ while considerable loss in function was recorded with other heavy metals. Similar loss of function has been recorded with EDTA indicating that it is a metal ion-dependent enzyme. The partially purified enzyme was able to digest the meat and showed high capability of removing blood stains from fabric.