Tuning the Properties of Natural Promiscuous Enzymes by Engineering Their Nano-environment

Abstract

Owing to their outstanding catalytic properties, enzymes represent powerful tools for carrying out a wide range of (bio)­chemical transformations with high proficiency. In this context, enzymes with high biocatalytic promiscuity are somewhat neglected. Here, we demonstrate that a meticulous modification of a synthetic shell that surrounds an immobilized enzyme possessing broad substrate specificity allows the resulting nanobiocatalyst to be endowed with enantio­selective properties while maintaining a high level of substrate promiscuity. Our results show that control of the enzyme nano-environment enables tuning of both substrate specificity and enantio­selectivity. Further, we demonstrate that our strategy of enzyme supramolecular engineering allows the enzyme to be endowed with markedly enhanced stability in an organic solvent (i.e., acetonitrile). The versatility of the method was assessed with two additional substrate-promiscuous and structurally different enzymes, for which improvements in enantio­selectivity and stability were confirmed. We expect this method to promote the use of supra­molecularly engineered promiscuous enzymes in industrially relevant biocatalytic processes.

Metagenomic Mining for Esterases in the Microbial Community of Los Rueldos Acid Mine Drainage Formation

“…The primers used for amplification are listed in Supplementary Material . All proteins studied here were N-terminally His 6 -tagged, and the soluble His-tagged proteins were produced and purified at room temperature after binding to a nickel–nitrilotriacetic acid (Ni–NTA) His-Bind resin (from Merck Life Science S.L.U., Madrid, Spain) as described previously ( Giunta et al, 2020 ), with slight modifications (the expression culture was scaled up to 1 L using 50 ml pre-inoculum). The purity was assessed as >98% using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE; Supplementary Figure 1 ) in a Bio-Rad Mini Protein system ( Laemmli, 1970 ).…”

“…The acid produced after ester bond cleavage by the hydrolytic enzyme induced a color change in the pH indicator that was measured spectrophotometrically at 550 nm. The experimental conditions were as detailed previously ( Giunta et al, 2020 ), with the absence of activity defined as at least a twofold background signal. For V max determination, (protein): 270 μg/ml; (ester): 20 mM; reaction volume: 44 μl; T: 30°C; and pH: 8.0.…”

The Komagataeibacter europaeus GqqA is the prototype of a novel bifunctional N-Acyl-homoserine lactone acylase with prephenate dehydratase activity

“…Conversion rate (V max ) ) and affinity (K m ) for 3-oxo-hexanoyl-, 3-oxo-octanoyl-, 3-oxo-decanoyl-, and 3-oxo-dodecanoyl- homoserine lactones, all from Merck Life Science S.L.U. (Madrid, SPAIN), was calculated using a pH indicator assay in 96-well plates, at 30 °C and pH 8.0 in a Synergy HT Multi-Mode Microplate Reader in continuous mode at 550 nm over 20 min, as described 53 . For enzyme assays, conditions were as detailed previously: [protein]: 10 μg ml-1; [3-oxo acyl homoserine lactone]: 0–65 mM; reaction volume: 200 μl; buffer: 5 mM EPPS buffer pH 8.0 containing 0.5 mM Phenol Red; T: 30 °C; and pH: 8.0.…”

“…The absorbance readings in all wells were measured immediately after the plate was gently shaken for 3 s. The rate of hydrolysis in each triplicate assay was recorded at 15 s intervals over 20 min. The absorbance readings for the reaction steady state (within the first minute of the assay) were used to generate a progress curve (arbitrary absorbance units vs. time) from which a line slope value was determined, and enzyme units calculated as described 53 . All values, in triplicate, were corrected for non-enzymatic transformation, with absence of activity defined as at least a two-fold background signal as described 53 .…”

Crystal structure of a family VIII β‐lactamase fold hydrolase reveals the molecular mechanism for its broad substrate scope

“…nr. KY483644) [4], which was produced and purified at 4 °C after binding to a Ni-NTA His-Bind resin (from Merck Life Science S.L.U., Madrid, Spain) as described previously [33].…”