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Applied and Environmental Microbiology, November 2007, p. 7291-7299, Vol. 73, No. 22
0099-2240/07/$08.00+0     doi:10.1128/AEM.01176-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Enhancement of the Stability of a Prolipase from Rhizopus oryzae toward Aldehydes by Saturation Mutagenesis{triangledown}

Mirella Di Lorenzo,1,2,{dagger} Aurelio Hidalgo,1,{dagger},{ddagger} Rafael Molina,3 Juan A. Hermoso,3 Domenico Pirozzi,2 and Uwe T. Bornscheuer1*

Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, 17487 Greifswald, Germany,1 Department of Chemical Engineering, Federico II University, 80125 Napoli, Italy,2 Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química Física Rocasolano, Consejo Superior de Investigaciones Científicas, Serrano 119, 28006 Madrid, Spain3

Received 25 May 2007/ Accepted 8 September 2007

A prolipase from Rhizopus oryzae (proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, active variants were prescreened by an activity staining method on agar plates. Active mutants were expressed in Escherichia coli Origami in a 96-well microtiterplate format, and a stability test using octanal as a model deactivating agent was performed. The most stable histidine mutant (H201S) conferred a stability increase of 60%, which was further enhanced to 100% by combination with a lysine mutant (H201S/K168I). This increase in stability was also confirmed for other aldehydes. Interestingly, the mutations did not affect specific activity, as this was still similar to the wild-type enzyme.

* Corresponding author. Mailing address: Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix Hausdorff Str. 4, D-17487 Greifswald, Germany. Phone: 49 3834 86-4367. Fax: 49 3834 86-80066. E-mail: uwe.bornscheuer{at}uni-greifswald.de

{triangledown} Published ahead of print on 21 September 2007.

{dagger} M.D.L. and A.H. contributed equally to this work.

{ddagger} Present address: Centro de Biología Molecular Severo Ochoa (CSIC-UAM), 28049 Madrid, Spain.

Applied and Environmental Microbiology, November 2007, p. 7291-7299, Vol. 73, No. 22
0099-2240/07/$08.00+0     doi:10.1128/AEM.01176-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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