Production, optimization and characterization of extracellular amylase from halophilic Bacillus lichineformis AH214

Abstract

Twenty one moderately halophilic bacterial strains were isolated from seawater and sediment in Alexandria Eastern Harbour, Egypt. The isolates were screened for the production of four extracellular degradative enzymes. The majority of isolates (57.1%) possessed significant enzyme activities, 43% of them have potentiality to produce amylase enzyme. The most active isolate for the production of amylase enzyme was identified by using a 16S rRNA sequence analysis as Bacillus lichineformis AH214. Optimization of the fermentation medium components and environmental factors using One Variable at a Time Approach and Plackett-Burman design was applied to enhance the amylase production by Bacillus lichineformis AH214. The maximum microbial amylase production could be achieved using an optimized medium of the following composition (g/l): 1.0 g yeast extract, 0.05 g K2HPO4, 0.25 g FeCl3, 15.0 g starch, 30.0 g NaCl, 0.75 g MgSO4.7H2O and inoculums size of 1.5 ml/50 ml and incubated at optimum conditions of pH 7, agitation speed 160 rpm, time 30 h and temperature 40°C. On applying optimized medium in the fermentation process, an enzyme productivity of 13.44 U/mg protein was achieved with two fold increase compared to the basal one. The crude amylase produced by Bacillus lichineformis was stable up to 40°C, pH 7.5 and 1.5 M NaCl.

Key words: Halophiles, amylase, Bacillus lichineformis, Plackett-Burman, optimization.