2024-03-29T14:54:22Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/1709172020-12-13T09:21:56Zcom_10261_70com_10261_2com_10261_101com_10261_5col_10261_323col_10261_354
Effect of site-specific peptide-tag labeling on the biocatalytic properties of thermoalkalophilic lipase from geobacillus thermocatenulatus
Romero, Óscar
Rivas, Blanca de las
López-Tejedor, David
Palomo, José Miguel
Fundación Ramón Areces
Consejo Superior de Investigaciones Científicas (España)
Tailor-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.
2018-10-10T12:32:08Z
2018-10-10T12:32:08Z
2018
2018-10-10T12:32:09Z
artículo
ChemBioChem 19(4): 369-378 (2018)
http://hdl.handle.net/10261/170917
10.1002/cbic.201700466
http://dx.doi.org/10.13039/100008054
http://dx.doi.org/10.13039/501100003339
29193524
eng
Sí
closedAccess
John Wiley & Sons