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Effect of Site‐Specific Peptide‐Tag Labeling on the Biocatalytic Properties of Thermoalkalophilic Lipase from Geobacillus thermocatenulatus
Author(s) -
Romero Oscar,
de las Rivas Blanca,
LopezTejedor David,
Palomo Jose M.
Publication year - 2018
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201700466
Subject(s) - chemistry , thermostability , lipase , hydrolysis , biocatalysis , peptide , enzyme , biochemistry , stereochemistry , catalysis , reaction mechanism
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.