Abstract
In the present research, a rapid, simple and efficient chemoselective method for the site-directed incorporation of tailor-made polymers into protein to create biocatalysts with excellent properties for pharmaceutical industrial purpose has been performed. First we focused on the protein engineering of the Geobacillus thermocatenulatus lipase 2 (BTL2) to replace the two cysteines (Cys65, Cys296) in the wild type enzyme (BTL-WT) by two serines. Then, by similar mode, a unique cysteine was introduced in the lid area of the protein. For the site-directed polymer incorporation, a set of different tailor-made thiol-ionic-polymers were synthesized and the protein cysteine was previously activated with 2,2-dithiodipyridine (2-PDS) to allow the disulfide exchange. The protected BTL variants were specifically modified with the different polymers in excellent yields, creating a small library of new biocatalysts. Different and important changes in the catalytic properties, possible caused by structural changes in the lid region, were observed. The different modified biocatalysts were tested in the synthesis of intermediates of antiviral and antitumor drugs, like nucleoside analogues and derivatives of phenylglutaric acid. In the hydrolysis of per-acetylated thymidine, the best biocatalyst was the BTL*-193-DextCOOH , where the activity was increased in 3-fold and the regioselectivity was improved, reaching a yield of 92% of 3’-O-acetyl-thymidine. In the case of the asymmetric hydrolysis of dimethyl phenylglutarate, the best result was found with BTL*-193-DextNH2-6000, where the enzyme activity was increased more than 5-fold and the enantiomeric excess was >99%.
Highlights
Modification of protein with polymers is a widely employed technique for applications in medicine, biotechnology and nanotechnology (Gauthier & Klok, 2010; Haag & Kratz, 2006)
Mutagenesis, purification and immobilization of BTL variants Lipase BTL has the particularity that its open configuration involves a large structural rearrangement, and the concerted movements of a tricky lid formed by two different loops
We set out to create semisynthetic BTL variants with only one cysteine located at lid zone
Summary
Modification of protein with polymers is a widely employed technique for applications in medicine, biotechnology and nanotechnology (Gauthier & Klok, 2010; Haag & Kratz, 2006). The incorporation of polymers into proteins improves their stability, solubility and biocompatibility (Fig. 1). This kind of modification can alter the catalytic properties of enzymes for the creation of novel active and selective biocatalysts (Dıaz-Rodrıguez & Davis, 2011; Romero et al, 2012). Modification of the nucleophilic thiol of a unique cysteine is a convenient and widely employed strategy to obtain a site-selective bioconjugation (Chalker et al, 2009). A unique cysteine can be introduced at virtually any position within a protein structure by site-directed mutagenesis and selectively modified using disulfide compounds, for example (Romero et al, 2012)
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