Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida.
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.
Banerjee
Anirban
A
Department of Pharmaceutical Technology, National Institute of Pharmaceutical Education and Research, Sector-67, 160 062, SAS Nagar, Punjab, India.
Kaul
Praveen
P
Banerjee
U C
UC
D000097:Acetonitriles / Q000378:metabolism; D000619:Aminohydrolases / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016958:Pseudomonas putida / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D000097:Acetonitriles; C045516:mandelonitrile; D000619:Aminohydrolases; C510080:arylacetonitrile aminohydrolase, Pseudomonas putida