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

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 (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.

Ultra-Small Pd(0) Nanoparticles into a Designed Semisynthetic Lipase: An Efficient and Recyclable Heterogeneous Biohybrid Catalyst for the Heck Reaction under Mild Conditions

“…First, a tailor-made enzyme was created following the protocol previously described [ 16 , 17 ]. Lipase from Geobacillus thermocatenulatus (GTL) was genetically modified by site-directed mutagenesis, changing Ala193 to Cys ( Figure 2 ), without alteration in the properties of the enzyme.…”

“…Then the peptide was conjugated to the Cys193 by a disulfide bond exchange. This modification generated a new semisynthetic GTL lipase (GTLσ-A193Cp) with improved properties and specifically increased the hydrophobicity of the final protein structure [ 16 ].…”

Biocatalysis at Extreme Temperatures: Enantioselective Synthesis of both Enantiomers of Mandelic Acid by Transesterification Catalyzed by a Thermophilic Lipase in Ionic Liquids at 120 °C

“…The stereoselectivity of BTL2 towards 29 chiral substrates was initially tested by Liu et al [44], reporting only good results for the acylation of 1-phenylethanol and 1-phenylpropanol with vinyl acetate (as well as for the hydrolysis of the corresponding esters). Later, this enzyme-immobilized on different supports via diverse methodologies-has been extensively tested on different substrates, some of them chiral [45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64], generally with moderate results. Additionally, chemical [55,58,[65][66][67] and genetic [56,[68][69][70][71] modifications of BTL2 lipase for improving its catalytic behavior (typically, to reduce the steric hindrance around the active site) have been also reported.…”

Disulfide Engineered Lipase to Enhance the Catalytic Activity: A Structure-Based Approach on BTL2

“…This double lid consists in a main lid (residues 169 to 200) that changes its secondary structure during the process of interfacial activation revealing the active site groove (open conformation), and a secondary lid (residues 217 to 234) that plays the role of other common lipase lids, and that in BTL2 structurally supports the main lid and helps stabilizing the enzymatically active state [5]. Several strategies have been applied to enhance its catalytic properties, including chemical modifications with small molecules, polymers, peptides, and/or through molecular biology tools [19,20,21,22,23]. Thus, its enzymatic activity is substantially impacted when the double lid of BTL2 is modified [13,19,23,24].…”

“…Several strategies have been applied to enhance its catalytic properties, including chemical modifications with small molecules, polymers, peptides, and/or through molecular biology tools [19,20,21,22,23]. Thus, its enzymatic activity is substantially impacted when the double lid of BTL2 is modified [13,19,23,24].…”