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Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Published on: April 19, 2014
A novel nitrilase from Ralstonia eutropha H16 and its application to nicotinic acid production
Haiyang Fan1, Lifeng Chen1, Huihui Sun2
1State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
Abstract:
A novel aliphatic nitrilase, REH16, was found in Ralstonia eutropha H16 and overexpressed in Escherichia coli BL21(DE3), and its enzymatic properties were studied. The temperature and pH optima were 37 °C and 6.6, respectively, and the best thermostability of the nitrilase was observed at 25 °C, which preserved 95% of activity after 120 h of incubation. REH16 has a broad hydrolytic activity toward aliphatic and heterocyclic nitriles and showed high tolerance of 3-cyanopyridine; this enzyme could hydrolyze as high as 100 mM 3-cyanopyridine completely. To improve the 3-cyanopyridine conversion efficiency in an aqueous reaction system, water-miscible organic solvents were tested, and ethanol (10% v/v) was chosen as the optimal co-solvent. Finally, under optimized conditions, using the fed-batch reaction mode, total of 1050 mM 3-cyanopyridine was hydrolyzed completely in 20.8 h with eight substrate feedings, yielding 129.2 g/L production of nicotinic acid and thus showing a potential for industrial application.
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