Characterization of the lipase and esterase multiple forms in an enzyme preparation from a Candida rugosa pilot-plant scale fed-batch fermentation

Characterization of crude extracellular enzyme preparations from Candida rugosa ATCC 14830 pilot-plant fed-batch fermentations showed the presence of multiple forms of lipases and esterases when a synthetic medium with oleic acid as a carbon source was used. Four major components were identified. An esterase of about 43 kDa, which was purified from the enzyme preparation by hydrophobic interaction and anion exchange chromatography. The N-terminal sequence for this esterase was homologous to acyl-CoA thioesterases YTE-1 and YTE-2 from C. rugosa [Diczfalusy MA, Alexson SEH. Arch Biochem Biophys 1996;334:104–112]. A 50.4-kDa protein with unknown activity whose sequence did not show any correspondence to previously described sequences reported in protein data banks. Also, two lipase isoforms were separated by means of hydrophobic interaction and ion-exchange chromatography. N-terminal sequencing of these proteins revealed that they corresponded to Lip2 and Lip3 lipases but not to Lip1, the major isoform usually present in crude lipase commercial preparations from C. rugosa (e.g. Sigma). Besides, these lipases were found to be tightly associated to a high-molecular-weight polysaccharide co-secreted by C. rugosa during the fermentation. These four proteins also have been characterized in terms of glycosylation degree.