Purification, amino acid sequencing and thermostability of an extracellular low molecular weight esterase produced by Bacillus subtilis NRRL 41270 in fermentation

Abstract

Extracellular esterase activity in Bacillus subtilis NRRL 41270 fermentation broths was found to reside in a small protein with a molecular weight less than 10 kDa. Following purification, esterase activity on fluorescein dibutyrate was estimated at 12 U/min/mg proteins. Enzyme saturation was observed at 5 μM substrate concentration. The produced esterase hydrolysed tributyrin. Its specific activity was estimated to be 17.8 μmol acid released/min/mg proteins. The small protein was subjected to size exclusion chromatography, SDS-PAGE and amino acid sequencing. Analysis revealed a sequence of the following amino acid residues: eevaetysfyhitphdystshispapvqffspap, according to which the molecule has 34 amino acid residues and a calculated molecular mass of 3853, which was in accordance with the gel filtration and SDS-PAGE results. Sequence based analysis and use of bioinformatics tools showed no significant similarity with known proteins while revealed a strongly hydrophobic molecule, with a α-helical conformation in the N-terminal, the rest of the molecule being β-sheet-rich. The enzyme appeared to be thermostable with more than 85% of the original activity maintained after 120 h incubation at 60°C. The producer organism and the features of the micro enzyme, suggest the case of a biotechnologically interesting biocatalyst that should be further researched in terms of its stability and production characteristics.