Authors: Romero, O; de las Rivas, B; Lopez-Tejedor, D; Palomo, JM

ChemBioChem. vol: 19. page: 1439-4227.
Date: feb-16. 2018.
Doi: 10.1002/cbic.201700466.

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 (GTL sigma-A193C and GTL sigma-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 GTL sigma-A193C modified with peptides Ac-Cys-Phe-Gly-Phe-Gly-Phe-CONH2 (1) and Ac-Cys-Phe-Phe-CONH2 (2) (>95% activity after 24h at 60 degrees C) was observed. The incorporation of 1 and 2 in GTL sigma-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 GTL sigma-S196C was enhanced more than tenfold by the incorporation of 1 and more than threefold by modification of this variant with Ac-Cys-(Arg)(7)-CONH2 (6) in the hydrolysis of 1-stearoyl-2-arachidonoyl-sn-glycerol. The enantioselectivity of GTL sigma-S196C increased for all formed bioconjugates, and the GTL sigma-S196C-1 conjugate was the most active and selective in the hydrolysis of dimethylphenyl glutarate at pH7 (72%ee), also showing an inversion in the enzyme enantiopreference..