Biotransformation of p-methoxyphenylacetonitrile into p-methoxyphenylacetic acid by resting cells of Bacillus subtilis.

Resting cells of Bacillus subtilis ZJB-063 were used for the direct transformation of MOPAN (p-methoxyphenylacetonitrile) to MOPAA (p-methoxyphenylacetic acid), which is an important pharmaceutical intermediate. The B. subtilis ZJB-063 culture conditions for the production of nitrilase and the reaction conditions for this nitrilase-mediated conversion were optimized. The maximum production of nitrilase was achieved when glucose and a combination of ammonium sulfate and yeast powder were added as carbon and nitrogen sources respectively. Previously reported inducers were found to be unnecessary for the production of nitrilase from B. subtilis ZJB-063, which indicated that this nitrilase appeared to be constitutive. However, when epsilon-caprolactam (6-hexanolactam) was added as the inducer, B. subtilis ZJB-063 exhibited nitrile hydratase and amidase activity. The maximum conversion of MOPAN into MOPAA (specific activity 17.03 units.g(-1)(DCW); DCW is dry cell weight) was observed in a solution containing 50 mM phosphate buffer (pH 7.0), 10 mM MOPAN, 2.7 mg DCW.ml(-1) wet resting cells and 5% (v/v) DMSO for 4 h at 32 degrees C. MOPAN (10 mM) was completely converted into MOPAA (9.65 mM) in 5 h in shake flasks without the formation of p-methoxyphenylacetamide. The small deviation of MOPAA (9.65 mM) from the theoretical amount (10 mM) may be due to partial consumption of the products by B. subtilis ZJB-063. Both MOPAN and MOPAA inhibited the hydrolysis at concentrations above 15 mM. Scale up of the reaction to 200 ml in a bubble bioreactor shortened the reaction time compared with the reactions performed in shake flasks.