Cloning and biochemical properties of a highly thermostable and enantioselective nitrilase from Alcaligenes sp. ECU0401 and its potential for (R)-(−)-mandelic acid production

Abstract

A nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli BL21 (DE3) in a soluble form. The encoded protein with a His₆-tag was purified to nearly homogeneity as revealed by SDS-PAGE with a molecular weight of approximately 38.5 kDa, and the holoenzyme was estimated to be composed of 10 subunits of identical size by size exclusion chromatography. The V(max) and K(m) parameters were determined to be 27.9 μmol min⁻¹ mg⁻¹ protein and 21.8 mM, respectively, with mandelonitrile as the substrate. The purified enzyme was highly thermostable with a half life of 155 h at 30 °C and 94 h at 40 °C. Racemic mandelonitrile (50 mM) could be enantioselectively hydrolyzed to (R)-(-)-mandelic acid by the purified nitrilase with an enantiomeric excess of 97%. The extreme stability, high activity and enantioselectivity of this nitrilase provide a solid base for its practical application in the production of (R)-(-)-mandelic acid.

Efficient production of (R)-o-chloromandelic acid by deracemization of o-chloromandelonitrile with a new nitrilase mined from Labrenzia aggregata

“…Aqueous-organic biphasic system has been demonstrated in our previous study on mandelonitrile to be a useful strategy in relieving substrate inhibition of nitrilase (Zhang et al 2011b). Therefore, enzymatic hydrolysis of o-chloromandelonitrile was also attempted in toluene-water biphasic system.…”

“…ECU0401 (Zhang et al 2011b) but better performance in deracemization of o-chloromandelonitrile. It was then overexpressed in Escherichia coli, purified to homogeneity and characterized with respect to its biochemical properties.…”

“…Although the nitrilase that we cloned from Alcaligenes sp. ECU0401 (Zhang et al , 2011b has been utilized in biphasic system for the hydrolysis of o-chloromandelonitrile, the maximum substrate concentration that the enzyme could tolerate was 200 mM (apparent concentration, based on the total volume of two phases), yielding 153 mM (R)-o-chloromandelic acid with merely 90.4% ee (Zhang et al 2011a). These results and further experiments indicated that the performance of this nitrilase was not sufficiently satisfactory for the production of (R)-o-chloromandelic acid.…”

Characterization and optimization of heterologous expression in Escherichia coli of the chitinase encoded by the chiA gene of Bacillus halodurans C-125

“…The effect of the heterologous expression system used (the combination of strain/vector) on solubility and even other properties of the enzymes were also described for other proteins. Thus, for a recombinant nitrilase the expression system based on the pUC10 resulted in a more soluble product and than that one based on the pET11 and pET28 plasmids [35].…”

The influence of hydroxypropyl-β-cyclodextrin on the enantioselective hydrolysis of 2-amino phenylpropionitrile catalyzed by recombinant nitrilase

“…Some important chiral pharmaceutical intermediates and bulk products, such as acrylic acid [4], (R)-(−)-mandelic acid [5], 3-hydroxyvaleric acid [6], and nicotinic acid [7], are produced by nitrile hydrolysis. For example, (R)-(−)-mandelic acid, widely used for the production of semisynthetic cephalosporins and anti-obesity agents, is produced by hydrolyzing mandelonitrile [5,8]. In addition, other studies focused on strategic optimizations for increasing the reaction rate, reducing substrate inhibition, and improving enzyme enantioselectivity [9][10][11].…”