Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida

Abstract

The highly enantioselective arylacetonitrilase of Pseudomonas putida was purified to homogeneity using a combination of (NH4)2SO4 fractionation and different chromatographic techniques. The enzyme has a molecular weight of 412 kDa and consisted of approximately nine to ten identical subunits (43 kDa). The purified enzyme exhibited a pH optimum of 7.0 and temperature optimum of 40 degrees C. The nitrilase was highly susceptible to thiol-specific reagents and metal ions and also required a reducing environment for its activity. These reflected the presence of a catalytically essential thiol group for enzyme activity which is in accordance with the proposed mechanism for nitrilase-catalyzed reaction. The enzyme was highly specific for arylacetonitriles with phenylacetonitrile and its derivatives being the most preferred substrates. Higher specificity constant (kcat/K(m)) values for phenylacetonitrile compared to mandelonitrile also revealed the same. Faster reaction rate achieved with this nitrilase for mandelonitrile hydrolysis was possibly due to the low activation energy required by the protein. Incorporation of low concentration (<5%) of organic solvent increased the enzyme activity by increasing the availability of the substrate. Higher stability of the enzyme at slightly alkaline pH and ambient temperature provides an excellent opportunity to establish a dynamic kinetic resolution process for the production of (R)-(-)-mandelic acid from readily available mandelonitrile.

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

“…2 and 3). These data agree well with other reported pseudomonad nitrilases, which have temperature optima from 40 to 55°C (27,28,(33)(34)(35). The UW4 enzyme retained Ͼ50% activity at 55°C; however, Ͻ20% activity was retained at 60°C.…”

Identification of Amino Acid Residues Responsible for the Enantioselectivity and Amide Formation Capacity of the Arylacetonitrilase fromPseudomonas fluorescensEBC191

“…The arylacetonitrilases convert substrates, such as phenylacetonitrile and ␣-substituted arylacetonitriles (e.g., 2-phenylpropionitrile , mandelonitrile [2-hydroxyphenylacetonitrile], or phenylglycinonitrile [2-aminophenylacetonitrile]). This group of nitrilases is especially interesting for applications in biotechnology because these enzymes can enantioselectively hydrolyze ␣-substituted racemic nitriles to optically active carboxylic acids and thus in principle allow the production of the enantiomers of ␣-amino-, ␣-hydroxy-, and ␣-methylcarboxylic acids (1,3,10,34). This trait has been used for the industrial production of (substituted) (R)-mandelic acid(s) from racemic (substituted) mandelonitrile(s) by dynamic kinetic resolution processes using different microorganisms (often strains of Alcaligenes faecalis) (19,34; M. Ress-Löschke, T. Friedrich, B. Hauer, and R. Mattes, 1998, DE19848129A1, German Patent Office).…”

Selection of Storage Methods for Maintenance of Different Stock Cultures

“…These can be achieved by exclusion of water which is essential for optimal functioning of life process, or by lowering the temperature to a level where normal enzymes are nonfunctioning. Exclusion of oxygen in the case of aerobic organisms also helpful in slowing down their metabolic activities (Banerjee et al, 2008).…”