EFFICIENT LACTAMIDE SYNTHESIS BY FED-BATCH METHOD USING NITRILE HYDRATASE OF RHODOCOCCUS PYRIDINIVORANS NIT-36

Abstract

Lactamide is used in the synthesis of lactic acid esters and as a solvent in many ecofriendly formulations. Till date lactamide synthesis was accomplished by employing chemical methods involving harsh reaction conditions while the enzyme-based approach ensures high purity lactamide synthesis under ambient environment without the intervention of acid byproducts using lactonitrile as substrate. Nitrile hydratase of Rhodococcus pyridinivorans NIT-36 was expressed by supplementing the growth medium with inducer i.e. lactamide. A 1.7-fold increase in enzyme activity was recorded by double feeding of the inducer. A further two-fold enhancement in enzymatic activity was achieved by the statistical optimization of process parameters for lactamide production by Response Surface Methodology. The substrate, lactonitrile inhibited the enzyme at higher concentrations, hence a fed-batch approach was adopted. The enzyme substrate ratio was standardized and the enzyme completely converted 15 mM of lactonitrile to lactamide in 30 minutes using 2 mg/ml resting cells. The fed-batch was performed at 1 L scale and 100% biotransformation of 360 mM of lactonitrile to lactamide was achieved at 45°C in 720 minutes. The catalytic productivity of 14.5 g/g dcw/h for lactamide formation was finally recorded in the 1 L fed-batch reaction.