Comparison of Single-Step Methods to Enrich Lipase Concentrations in Bacterial Cell Lysates

HIGHLIGHTS

Lipases were enriched from Geobacillus and Anoxybacillus lysates

For each lipase, ethanol precipitation resulted in the highest enrichment fold

Ethanol precipitation is a simple enrichment method that can result in high yields

Abstract

Lipases are currently used in food technology for the modification of fats and oils. The thermal stability of lipase is an essential characteristic for this application. This study compares four individual single-step methods (heat treatment, ethanol precipitation, ammonium sulfate precipitation, and size-exclusion chromatography) to enrich lipase concentrations from thermophilic bacterial (Geobacillius stearothermophilus and Anoxybacillus flavithermus) cell lysates. SDS-PAGE and size exclusion chromatography were used to determine the molecular weights of the lipases and the enrichment efficiencies were determined using specific enzyme activities. The molecular weight of G. stearothermophilus lipase was approximately 42 kDa, and approximately 33 kDa for A. flavithermus lipase. For each organism, ethanol precipitation resulted in the highest enrichment fold, followed by ammonium sulfate precipitation, gel filtration and heat treatment respectively. The highest yields for G. stearothermophilus lipase were obtained with ammonium sulfate precipitation, followed by get filtration, and ethanol precipitation respectively. The highest yields for A. flavithermus lipase were obtained from heat treatment followed by ammonium sulfate precipitation, gel filtration and ethanol precipitation respectively. Ethanol precipitation and heat treatment are simple methods for enzyme enrichment from cell lysates and can result in high enzyme yields with moderate enrichment folds compared to complex multi-step purification methods.

Keywords:
thermophilic lipase; enzyme enrichment; zymography