Highly selective purification of three lipases from Geotrichum candidum 4013 and their characterization and biotechnological applications

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• 作者：Jana Brabcov谩 ; Zuzana Demianov谩 ; Ji艡铆 Vondr谩拧ek ; Michal J谩gr ; Marie Zarev煤cka ; Jose M. Palomo
• 关键词：c ; conversion ; E ; enantiomeric ratio ; eep ; enantiomeric excess of the product ; ees ; enantiomeric excess of the substrate ; FOA ; free oleic acid ; MD ; molecular dynamics ; MOE ; modeling enviroment ; MTPA ; (R)-(+)-3 ; 3 ; 3-trifluoro-2-methoxy-2-phenylpropanoic acid ; MUF-butyrate ; methylumbelliferone butyrate ; pNPB ; p-nitrophenyl butyrate ; pNPE ; p-nitrophenyl ester ; PDB ; Protein Data Bank ; PLB ; protein loading buffer ; TAG ; triacylglycerol ; TO ; trioleoylglycerol
• 刊名：Journal of Molecular Catalysis B: Enzymatic
• 出版年：30 December, 2013
• 年：2013
• 卷：98
• 期：Complete
• 页码：62-72
• 全文大小：2409 K

文摘

Simple purification steps were used to purify two lipases (Lip1 and Lip2) and one novel lipase (Lip3) produced by Geotrichum candidum 4013. The octyl-Sepharose at low ionic strength was used to adsorbe Lip2 and novel Lip3, by addition of octadecyl-Sepabeads the Lip1 was also adsorbed from the crude mixture. Desorption of proteins with a Triton X-100 gradient or 1% (v/v) Triton X-100 permitted to fully purify Lip1, Lip2 and Lip3 lipases.

The identification of Lip1 and Lip2 was confirmed by using mass spectrometry approach and their characterization by molecular modeling approach. CD spectra and fluorescence showed differences in the secondary and tertiary structure; the apparent difference was the presence of 4 extra loops in Lip2 in contrast to Lip1 structure. The Edman degradation together with mass spectrometry revealed the presence of unique peptide AVGGGATLPEK in the novel lipase Lip3. The catalytic properties of the lipases were tested through hydrolysis of p-nitrophenyl esters, triacylglycerols and peracetylated thymidine esters. The enantioselectivity potential of purified lipases was studied for the hydrolyses of trans-2-(4-methoxybenzyl)-1-cyclohexyl acetate, where Lip3 was the only active lipase with an excellent selectivity (ee_p > 99%, E = 372). The all three purified lipases from G. candidum 4013 have shown promising catalytic properties with biotechnological application.