Organic co‐solvents affect activity, stability and enantioselectivity of haloalkane dehalogenases

Abstract

Haloalkane dehalogenases are microbial enzymes with a wide range of biotechnological applications, including biocatalysis. The use of organic co-solvents to solubilize their hydrophobic substrates is often necessary. In order to choose the most compatible co-solvent, the effects of 14 co-solvents on activity, stability and enantioselectivity of three model enzymes, DbjA, DhaA, and LinB, were evaluated. All co-solvents caused at high concentration loss of activity and conformational changes. The highest inactivation was induced by tetrahydrofuran, while more hydrophilic co-solvents, such as ethylene glycol and dimethyl sulfoxide, were better tolerated. The effects of co-solvents at low concentration were different for each enzyme-solvent pair. An increase in DbjA activity was induced by the majority of organic co-solvents tested, while activities of DhaA and LinB decreased at comparable concentrations of the same co-solvent. Moreover, a high increase of DbjA enantioselectivity was observed. Ethylene glycol and 1,4-dioxane were shown to have the most positive impact on the enantioselectivity. The favorable influence of these co-solvents on both activity and enantioselectivity makes DbjA suitable for biocatalytic applications. This study represents the first investigation of the effects of organic co-solvents on the biocatalytic performance of haloalkane dehalogenases and will pave the way for their broader use in industrial processes.

Catalytic activity and structural stability of three different Bacillus enzymes in water/organic co-solvent mixtures

“…Among the employed organic co-solvents, the worst effect on the thermal stability of BsAPII and BlALDH went to acetonitrile and DMF. The negative impact of watermiscible organic co-solvents on the thermal stability of Bacillus enzymes are closely consistent with other previous findings [36,44,45]. One explanation for this observation is that the hydrophilic tendency of water-miscible organic co-solvents strips off water from the protein surface and competes with the non-covalent interactions of enzymes, especially at the elevated temperatures [46].…”

Development of Fluorescent Assay for Monitoring of Dehalogenase Activity

“…Since reactions of haloalkane dehalogenases result in a drop of pH, use of a proper pH indicator is the simplest way to assay dehalogenase activity. The selected indicator has to fulfill several criteria: 1) possess proper excitation and emission spectra — the use of common microtiter plate reader for evaluation of the assay is desirable, ratiometric measurement can be advantageous; 2) exhibit pH sensitivity overlapping the typical pH optimum of haloalkane dehalogenases — these enzymes usually possess pH optimum in weakly alkaline or alkaline pH ; 3) be soluble in aqueous buffer or buffer with low content of organic co‐solvent — haloalkane dehalogenases are prone to damage by higher concentrations of organic solvents, however, their substrates are more soluble in organic solvents than in water . Potential pH indicators were identified by the literature search and their applicability for the assay was verified experimentally.…”

Bridges to Stability: Engineering Disulfide Bonds Towards Enhanced Lipase Biodiesel Synthesis

“…Nonetheless, limitations include their relatively low stability in the non‐natural environment. Polar organic solvents (such as alcohols) may act as denaturing agents, stripping off essential water molecules and penetrating the hydrophobic core . Alcohol at high concentrations may induce partial protein unfolding followed by irreversible denaturation and aggregation .…”

“…As for protein engineering, three main approaches can be applied; (i) random mutagenesis, (ii) rational design, and (iii) semi‐rational design. Rational design is based on the protein 3D structure (solved with either X‐ray crystallography, NMR or other methods) and utilizes precise amino acid substitutions to obtain altered performance …”

“…Polar organic solvents (such as alcohols) may act as denaturing agents, stripping off essential water molecules and penetrating the hydrophobic core. [5] Alcohol at high concentrations may induce partial protein unfolding followed by irreversible denaturation and aggregation. [3d] Developments in protein engineering techniques, computational design and enzyme immobilization have promoted the generation of many stable biocatalysts engaging various reactions in organic synthesis.…”