Effect of Site-Specific Peptide-Tag Labeling on the Biocatalytic Properties of Thermoalkalophilic Lipase from Geobacillus thermocatenulatus

Oscar Romero, Blanca de las Rivas, David Lopez-Tejedor, Jose M. Palomo

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5 Scopus citations

Abstract

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 2 (1) and Ac-Cys-Phe-Phe-CONH 2 (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) 7 -CONH 2 (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.

Original languageEnglish
Pages (from-to)369-378
Number of pages10
JournalChemBioChem
Volume19
Issue number4
DOIs
StatePublished - 16 Feb 2018
Externally publishedYes

Keywords

  • biocatalysis
  • enantioselectivity
  • lipases
  • peptides
  • site-specific modification

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