Recombinant anoxybacillus flavithermus t1 esterase/lipase: optimization of expression and recovery

Laura-Mihaela Chis1, Monica Hriscu1, Flore Chirila2, Iulia Lupan3, Monica Tosa1, Florin Dan Irimie1

1 Babes-Bolyai University, Faculty of Chemistry and Chemical Engineering, Dept. of Biochemistry and Biochemical Engineering, 11 Arany Janos St., RO-400028 Cluj-Napoca, Romania
2 Dept. of Paraclinical Education, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine,
3 -5 Calea Manastur, RO-400372 Cluj-Napoca, Romania 3Babes-Bolyai University, Institute for Interdisciplinary Experimental Research, Molecular Biology Center,

Abstract


We have previously described the molecular cloning and expression in an Escherichia coli system and the characterization of a thermostable esterase/lipase from Anoxybacillus flavithermus T1. We herein report on the optimization of the expression process. When using isopropyl-.-D-thiogalactopyranoside (IPTG) for induction, the highest protein yield was obtained at 30 C, with 0.4 mM IPTG and 1 h induction time. Similar results were obtained at 37 C, but with a higher IPTG concentration (5 mM) and after 8h of induction, which makes the former a better option in terms of cost and time-efectiveness. Better results yet were attained with lactose, a very attractive option, given its high availability, low cost and low toxicity to the host cells. Recovery of the active enzyme from the periplasmic space was highest with a lysis buffer which combines osmotic shock with a membrane destabilization effect (Tris-sucrose/EDTA 1 mM/MgSO4 5 mM). The freeze-thaw treatment yielded similar results, while treatment with 1% organic solvent (chloroform or DMSO), while effective in permeabilization of the cell membrane, exerted a certain inhibitory effect upon the enzyme.

Keywords


Anoxybacillus flavithermus T1 esterase/lipase; IPTG; lactose; membrane destabilization; periplasmic fraction

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