Influence of different parameters on reverse micelle extraction combined with acetone precipitation to purify sn-1,3 extracellular lipase from Aspergillus niger GZUF36

Abstract

There are few reports on the feasibility of combined reverse micelle extraction and acetone precipitation to obtain electrophoretic pure enzymes. We aimed to purify a sn-1,3 extracellular lipase from a novel Aspergillus niger GZUF36 through this combination in this work. This lipase preliminarily purified by controlling the volume ratio (1:2.5) of crude enzyme solution and acetone. Then, we studied effects of different parameters on reverse micelle extraction. The suitable surfactant, pH, salt and cosolvent and extraction time for forward extraction were 125mM cetyl trimethylammonium bromide (CTAB), 9.0, 0.075M NaCl, 10% n-hexanol and 30min, respectively. Under these conditions, the forward extraction rate reached 90.3% ± 3.2%. The suitable salt, pH, extraction time and short chain alcohol for backward extraction were consecutively 1.5M KCl, 6.5, 60min and 10% ethanol. Adding 10% ethanol shows a significant advantage of improvement the extraction rate. Under these optimal conditions, the total extraction rate and purification factor of lipase reached 76.8% and 10.14, respectively. SDS-PAGE showed that molecular weight of the pure protein was 42.7kDa and TLC exhibited sn-1,3 selectivity of this lipase. LC-MS/MS analysis revealed that the lipase had 297 amino acid residues and was likely to glycosylate. Through the study of different parameters, it demonstrated that the new and simple combination of reverse micelle extraction using CTAB as surfactant and n-hexanol as cosolvent for forward extraction and adding ethanol for backward extraction and acetone precipitation is a promising method to get almost an electrophoretically pure sn-1,3 lipase.