Biotransformation of 4-hydroxyphenylacetonitrile to 4-hydroxyphenylacetic acid using whole cell arylacetonitrilase of Alcaligenes faecalis MTCC 12629

Abstract

Abstract The whole cell arylacetonitrilase of Alcaligenes faecalis MTCC 12629 was employed to develop a bioprocess for the synthesis of 4-hydroxyphenylacetic acid (4-HPAA) from 4-hydroxyphenylacetonitrile (4-HPAN). Isobutyronitrile emerged as the most appropriate inducer with a 1.2-fold enhanced nitrilase production in inducer feeding approach. A. faecalis MTCC 12629 nitrilase preferably hydrolyzed 4-HPAN, 4-aminoyphenylacetonitrile, mandelonitrile and phenylacetonitrile to their corresponding acids. Thermostability profile revealed the stability of nitrilase at 40, 45 and 50°C with the half-life of 45 h, 36 h and 13 h, respectively. Michaelis constant (Km) and reaction velocity (Vmax) of nitrilase were calculated as 20 mM and 2 U/mg dcw, respectively. This nitrilase exhibited complete conversion of 4-HPAN to 4-HPAA at 45°C in 50 mM K2HPO4/KH2PO4 buffer (pH 7.0) in 30 min. A fed batch process designed at 500 ml with five feedings of substrate resulted in 150 mM product formation using 18 U/ml whole cells nitrilase (16 mg/ml dcw) at 45°C. This is the first report on the production of 4-HPAA through nitrilase mediated pathway with the volumetric and catalytic productivity of 23 g/l and 4.13 g/g dcw/h, respectively.