Purification and characterization of a salt, solvent, detergent and bleach tolerant protease from a new gamma-Proteobacterium isolated from the marine environment of the Sundarbans

Abstract

In this communication we report purification and characterization of a single salt, solvent, detergent and bleach tolerant alkaline serine protease produced by a truly marine bacterium. Based on the 16S rRNA gene sequence, absence of polyhydroxybutyrate accumulation, growth with very high NaCl levels as well as with hydrocarbons as sole carbon source, the isolate was classified as a new bacterium belonging to the gamma-Proteobacteria family. A 69-fold purification (specific activity 791.7 U/mg protein, unit expressed as μmole of tyrosine liberated per minute) was achieved by a three-step purification procedure. The enzyme is active over a broad range of pH (6.0–11.0), the optimum being at 9.0. This protease enzyme shows activity from 30 to 70 °C and Ca 2+ increase the thermostability. This enzyme exhibits appreciable activity in presence of up to 30% NaCl and is highly stable even after 18 h pre-incubation with 35% (w/v) NaCl. While Ba 2+ and Ca 2+ enhance the enzyme activity, heavy metals like Co 2+, Zn 2+, Hg 2+ inactivate the enzyme. The enzyme is completely stable in presence of laboratory detergents (Tween 80 and Triton X-100), oxidizing agents, reducing agents, commercial detergents and bleaches (hydrogen peroxide and sodium perborate) after 1 h of pre-incubation. Water miscible and immiscible organic solvents like ethylene glycol, ethanol, butanol, acetone, DMSO, xylene and perchloroethylene enhance as well as stabilize the enzyme activity. Reflectance measurements during wash performance studies show that our enzyme is capable of almost complete removal of recalcitrant blood and egg stains in both wet and dry wash operations. Enzymatic activity against a wide variety of substrates indicates that our enzyme can be investigated for a range of commercial applications especially for soy protein and gelatin hydrolysis in the food processing industry as well as for the dehairing process in the leather industry in addition to catalyzing hydrolysis reactions at high salt concentrations.