Gene Cloning, Expression, and Characterization of a Nitrilase from Alcaligenes faecalis ZJUTB10

Abstract

Nitrilases are important industrial enzymes that convert nitriles directly into the corresponding carboxylic acids. In the current work, the fragment with a length of 1068 bp that encodes the A. faecalis ZJUTB10 nitrilase was obtained. Moreover, a catalytic triad was proposed and verified by site-directed mutagenesis, and the detailed mechanism of this nitrilase was clarified. The substrate specificity study demonstrated that the A. faecalis ZJUTB10 nitrilase belongs to the family of arylacetonitrilases. The optimum pH and temperature for the purified nitrilase was 7-8 and 40 °C, respectively. Mg(2+) stimulated hydrolytic activity, whereas Cu(2+), Co(2+), Ni(2+), Ag(+), and Hg(2+) showed a strong inhibitory effect. The K(m) and v(max) for mandelonitrile were 4.74 mM and 15.85 μmol min(-1) mg(-1) protein, respectively. After 30 min reaction using the nitrilase, mandelonitrile at the concentration of 20 mM was completely hydrolyzed and the enantiomeric excess against (R)-(-)-mandelic acid was >99%. Characteristics investigation indicates that this nitrilase is promising in catalysis applications.

Efficient production of methionine from 2-amino-4-methylthiobutanenitrile by recombinant Escherichia coli harboring nitrilase

“…For aromatic and heterocyclic nitriles such as phenylacetonitrile, mandelonitrile and 2-chloro-3-cyanopyridine, no hydrolysis was found, which is similar to the report of liu [14]. these results further confirmed that this nitrilase belongs to the family of aliphatic nitrilases [12,14,23].…”

“…Fe 3+ , al 3+ , li 2+ , cu 2+ , Fe 2+ and co 2+ could partially inhibit the catalytic activity. the metal-chelating agent EDta also did not inhibit the catalytic activity, indicating that this enzyme has no metal ion requirement, which is similar to the reported nitrilase from A. facilis ZJUtB10 [12].…”

“…there are several factors that influence the function of the recombinant enzyme. From this study, we found that this nitrilase was more stable to heat compared to other nitrilases, and pH 7.5 favored its activity [12]. Just like the reported nitrilase from A. facilis ZJUtB10 [12], this nitrilase had no metal ion requirements.…”

“…at 40 and 50 °c, the half-lives decreased to 5.52 and 0.58 h, respectively. It demonstrated that the nitrilase obtained in this study was more thermostable than that of nitrilases from A. facilis ZJUtB10 with half-lives of 57 min at 40 °c and 22 min at 50 °c, and P. putida with half-lives of 76 min at 40 °c and 9 min at 50 °c, respectively [12]. .…”

Biosynthesis of Iminodiacetic Acid from Iminodiacetonitrile by Immobilized Recombinant <i>Escherichia coli</i> Harboring Nitrilase

“…Traditionally, the manufacturing process of IDA employs a strong base catalyst, such as NaOH, resulting in the byproduct of salts due to the neutralization of the base used in the final product separation, which causes serious environment problems [Duan et al, 2007]. Compared to the chemical synthesis of IDA, nitrile-converting biocatalysts have attracted more attention because of their high activity, mild reaction conditions and environmental compatibility [Chiao and Sun, 2007;Liu et al, 2011a;Shanmugam and Ingram, 2008]. In our lab, we firstly explored a newly catalytic route by biotransformation of iminodiacetonitrile (IDAN) to produce IDA, which paved the foundation of IDA production by biological method [Liu et al, 2011b;Zhang et al, 2012].…”