Predicting enzyme behavior in nonconventional media: correlating nitrilase function with solvent properties

Abstract

The insolubility of nitrile substrates in aqueous reaction mixture decreases the enzymatic reaction rate. We studied the interaction of fourteen water miscible organic solvents with immobilized nitrile hydrolyzing biocatalyst. Correlation of nitrilase function with physico-chemical properties of the solvents has allowed us to predict the enzyme behavior in such non-conventional media. Addition of organic solvent up to a critical concentration leads to an enhancement in reaction rate, however, any further increase beyond the critical concentration in the latter leads to the decrease in catalytic efficiency of the enzyme, probably due to protein denaturation. The solvent dielectric constant (epsilon) showed a linear correlation with the critical concentration of the solvent used and the extent of nitrile hydrolysis. Unlike alcohols, the reaction rate in case of aprotic solvents could be linearly correlated to solvent log P. Further, kinetic analysis confirmed that the affinity of the enzyme for its substrate (K (m)) was highly dependent upon the aprotic solvent used. Finally, the prospect of solvent engineering also permitted the control of enzyme enantioselectivity by regulating enantiomer traffic at the active site.

Stereo-selective conversion of mandelonitrile to (R)-(−)-mandelic acid using immobilized cells of recombinant Escherichia coli

“…The final concentration of immobilized cells was 20 mg/mL. Mandelonitrile is not soluble in aqueous solvent (sparingly soluble); hence 10 % (v/v) methanol was used as a co-solvent to make it soluble (Kaul and Banerjee 2008). It was added to an initial concentration of 30 mM in reaction mixture.…”

Characterization of an atypical, thermostable, organic solvent- and acid-tolerant 2′-deoxyribosyltransferase from Chroococcidiopsis thermalis

“…We assayed CtNDT activity in the presence of 20% (v/v) mixtures of water with different water-miscible polar and apolar organic co-solvents, namely glycerol (log P = − 3.03), ethylene glycol (log P = − 1.80), DMSO (log P = − 1.3), N,Ndimethylformamide (DMF) (log P = − 1.0), methanol (log P = − 0.76), propylene glycol (log P = − 0.64), acetonitrile (log P = − 0.3), ethanol (log P = − 0.24), acetone (log P = − 0.21), isopropanol (log P = 0.05), ethyl acetate (log P = 0.73), and chloroform (log P = 1.83) (Arroyo et al 2000;Kaul and Banerjee 2008). In most cases (Fig.…”

Process Optimisation Studies and Aminonitrile Substrate Evaluation of Rhodococcus erythropolis SET1, A Nitrile Hydrolyzing Bacterium

“…Several solvent based approaches have been reported in the literature including monophasic systems with solvents such as DMSO and biphasic systems such as toluene/H 2 O and hexane/H 2 O and some systems can operate in both buffer‐organic solvent monophasic or biphasic mixtures . In several cases, enhancement of the enzyme activity and enantioselectivity of hydrolysis has also been observed in organic solvent systems . However it has been noted that the mechanism of solvent interaction with nitrile hydrolysing reactions is much less studied than in the case of other hydrolytic enzymatic systems …”