This journal is c The Royal Society of Chemistry 2012 Chem. Commun., 2012, 48, 9053–9055 9053 Cite this: Chem. Commun., 2012, 48, 9053–9055 Semisynthetic peptide–lipase conjugates for improved biotransformationsw Oscar Romero, a Marco Filice, a Blanca de las Rivas, b Cesar Carrasco-Lopez, c Javier Klett, d Antonio Morreale, d Juan A. Hermoso, c Jose M. Guisan, a Olga Abian e and Jose M. Palomo* a Received 5th July 2012, Accepted 23rd July 2012 DOI: 10.1039/c2cc34816k An efficient chemoselective method for the creation of semi- synthetic lipases by site-specific incorporation of tailor-made peptides on the lipase-lid site was developed. These new enzymes showed excellent improved specificity and regio- or enantio- selectivity in different biotransformations. Chemical modification is a fascinating approach for altering the protein function by the introduction of non-natural frag- ments into proteins. 1 The addition of unnatural moieties (such as fluorophores, sugars, peptides, etc.) to proteins 2 has also proven useful for a variety of processes and applications both in vivo and in vitro. Furthermore this specific modifica- tion on proteins has permitted the alteration of catalytic properties of enzymes for creation of novel active selective biocatalysts. 3,4 Therefore the combination of molecular biology methods and efficient synthetic approaches has made possible successful preparation of semisynthetic proteins in large amounts. 5,6 Modification of the nucleophilic thiol of a unique cysteine is a widely employed strategy for site-selective bio- conjugation. 7 A cysteine can be introduced at virtually any position within a protein structure by site-directed muta- genesis and then selectively modified using for example disulfide compounds. Furthermore, the disulfide can be effi- ciently transformed into a thioether by a desulfurization method. 8 Herein, we describe the design of new semisynthetic enzymes by the site-specific incorporation of a set of tailor-made cysteine-containing peptides on successfully engineered thermo- philic lipase from Geobacillus thermocatenulatus (BTL) with a single cysteine at different positions on the lid-site of the enzyme (Scheme 1). The chemical modifications were performed on the lipase already immobilized on CNBr-activated Sepharose. This introduces the advantages of solid-state chemistry: using an excess of peptides, quantitative transformations, or easy purifica- tion. 9 The catalytic activity of the new semisynthetic enzymes in different asymmetric biotransformations was evaluated. The incorporation of molecules on the oligopeptide lid represents an elegant strategy to control its movement and therefore the lipase catalysis. As far as we know, no example of the improvement of lipase catalytic efficiency by this metho- dology has been described. First we focused on the protein engineering of this lipase to replace the two cysteines (Cys65, Cys296) in the wild type enzyme (BTL wt) by two serines. The new-engineered enzyme (BTL C65S/C296S) was expressed in E. coli without detriment to the enzyme activity. Considering the complexity of this lipase with a tricky lid formed by two different loops, the idea was to introduce a unique cysteine at different positions of this engineered enzyme. This approach was first confirmed by a bioinformatics study using the crystallographic open confor- mation of the enzyme we have recently solved. 10 Three different positions were selected to be mutated: Ala193 (in the internal loop), Leu230 (in the external loop) and Ser196 (in the middle of both loops). These three variations represent a conservative change. The three new mutants BTL-C65S/C296S/A193C (BTL*-A193C), BTL-C65S/C296S/ L230C (BTL*-L230C) and BTL-C65S/C296S/S196C (BTL*- S196C) were obtained by site-directed mutagenesis with a good production, similar to BTL wt or BTL C65S/C296S. All BTL variants were efficiently purified by hydrophobic chromatography 10 and characterized by circular dichroism and fluorescence (see ESIw, Fig. S1 and S2). For the strategic chemical incorporation of tailor-made peptides including a cysteine on the peptide sequence, the Scheme 1 Lipase-conjugates preparation. a Departamento de Biocata ´lisis, Instituto de Cata ´lisis (CSIC), Marie Curie 2. Cantoblanco. CampusUAM, 28049 Madrid, Spain. E-mail: josempalomo@icp.csic.es; Fax: +34 915854760 b Departamento de Biotecnologı´a Microbiana, Instituto de Ciencia y Tecnologı´a de alimentos y Nutricio ´n, (ICTAN-CSIC), Madrid, Spain c Departamento de Cristalografı´a y Biologı´a Estructural, Instituto de Quı´mica-Fı´sica Rocasolano (CSIC), Madrid, Spain d Unidad de Bioinforma ´tica, Centro de Biologı´a Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain e Instituto de Biocomputacio ´n y Fı´sica de Sistemas Complejos (BIFI), Universidad de Zaragoza, Zaragoza, Spain w Electronic supplementary information (ESI) available: Experimental section, characterization data of the different BTL variants, additional tables and figures and movie files. See DOI: 10.1039/c2cc34816k ChemComm Dynamic Article Links www.rsc.org/chemcomm COMMUNICATION Published on 24 July 2012. Downloaded by Instituto de Catálisis y Petroleoquímica on 10/06/2013 14:30:49. View Article Online / Journal Homepage / Table of Contents for this issue