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dc.contributor.authorRomero, Óscar-
dc.contributor.authorRivas, Blanca de las-
dc.contributor.authorLópez-Tejedor, David-
dc.contributor.authorPalomo, José Miguel-
dc.identifierdoi: 10.1002/cbic.201700466-
dc.identifiere-issn: 1439-7633-
dc.identifierissn: 1439-4227-
dc.identifier.citationChemBioChem 19(4): 369-378 (2018)-
dc.description.abstractTailor-made peptides were investigated for site-specific tag labeling of Geobacillus thermocatenulatus lipase (GTL). GTL was first genetically modified by introducing a unique cysteine on the lid site of the enzyme to produce two variants (GTLσ-A193C and GTLσ-S196C). Chemical modification was performed by using a small library of cysteine-containing peptides. The synthesized peptide–lipase biocatalysts were highly stable, more active, more specific, and more selective toward different substrates than unmodified GTL. Very high enzyme thermostability of GTLσ-A193C modified with peptides Ac-Cys-Phe-Gly-Phe-Gly-Phe-CONH (1) and Ac-Cys-Phe-Phe-CONH (2) (>95 % activity after 24 h at 60 °C) was observed. The incorporation of 1 and 2 in GTLσ-S196C improved its catalytic activity in the hydrolysis of p-nitrophenyl butyrate by factors of three and greater than five, respectively. The specificity for short-chain versus long-chain esters was also strongly improved. The diacylglycerol activity of GTLσ-S196C was enhanced more than tenfold by the incorporation of 1 and more than threefold by modification of this variant with Ac-Cys-(Arg)-CONH (6) in the hydrolysis of 1-stearoyl-2-arachidonoyl-sn-glycerol. The enantioselectivity of GTLσ-S196C increased for all formed bioconjugates, and the GTLσ-S196C–1 conjugate was the most active and selective in the hydrolysis of dimethylphenyl glutarate at pH 7 (72 % ee), also showing an inversion in the enzyme enantiopreference.-
dc.description.sponsorshipThis work was sponsored by the Spanish National Research Council (CSIC). We thank the Ramon Areces Foundation for financial support.-
dc.publisherJohn Wiley & Sons-
dc.titleEffect of site-specific peptide-tag labeling on the biocatalytic properties of thermoalkalophilic lipase from geobacillus thermocatenulatus-
dc.description.versionPeer Reviewed-
dc.contributor.funderFundación Ramón Areces-
dc.contributor.funderConsejo Superior de Investigaciones Científicas (España)-
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