title: Purification of different lipases from Aspergillus niger by using a highly selective adsorption on hydrophobic supports.
abstract: In this manuscript, we have purified three different lipases from crude preparations from Aspergillus niger in a simple fashion, secluding the esterases and other enzymes presented in the preparation. Firstly, the crude was offered at low ionic strength to octyl agarose. The support specifically adsorbed two lipases, with molecular weights of 43 and 65 kDa. Desorption with a gradient of Triton X-100 permitted to fully purify both lipases. The addition of octadecyl-Sepabeads support to the non-adsorbed proteins on octyl-agarose permitted to selectively adsorb a third lipase, having a molecular weight of 31 kDa. Desorption of the enzyme using Triton X-100 permitted to have also a pure sample of this enzyme. A significant percentage of esterase activity remains in the supernatant, derived from esterases or lipases unable to become adsorbed on the employed supports. Furthermore, these purified lipases were immobilized via ionic adsorption on DEAE-Sepharose and their selectivity was analyzed in the kinetic resolution of (+/-)-O-2-butyryl-2-phenylacetic acid and (+/-)-mandelic acid methyl ester. In the resolution of (+/-)-O-2-butyryl-2-phenylacetic acid, the crude extract preparation gave a low enantioselectivity value (E = 9), whereas the three immobilized preparations of purified lipases exhibited an increase in E-value from 11 (43 kDa lipase) to > 100 (31 kDa lipase). When (+/-)-mandelic acid methyl ester was used, the crude extract preparation presented low enantioselectivity hydrolyzing the S enantiomer quicker, while the purified lipase preparations preferred the R one. In this case, the 65 kDa lipase was the most selective enzyme (E = 20).
authors.affiliation: Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Gloria
authors.initials: G
authors.lastname: Fernández-Lorente
authors.forename: Claudia
authors.initials: C
authors.lastname: Ortiz
authors.forename: Rosa L
authors.initials: RL
authors.lastname: Segura
authors.forename: Roberto
authors.initials: R
authors.lastname: Fernández-Lafuente
authors.forename: Jose M
authors.initials: JM
authors.lastname: Guisán
authors.forename: Jose M
authors.initials: JM
authors.lastname: Palomo
mesh_terms: D000327:Adsorption; D001234:Aspergillus niger / Q000201:enzymology*; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized; D004952:Esters / Q000378:metabolism; D005590:Fractional Precipitation; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D007527:Isoenzymes; D008049:Lipase / Q000302:isolation & purification*; D008567:Membranes, Artificial; D013237:Stereoisomerism
chemical_list: D004800:Enzymes, Immobilized; D004952:Esters; D007527:Isoenzymes; D008567:Membranes, Artificial; D008049:Lipase