Amidases are robust biocatalysts for the industrial synthesis of carboxylic acids. Amidase-catalyzed hydrolysis of 2-chloronicotinamide is regarded as a promising route for 2-chloronicotinic acid production, which is an important building block for agrochemicals and pharmaceuticals. However, the o-substituted Cl of the pyridine ring caused a strong electronic effect and steric hindrance. Engineering access tunnels is one of the most efficient strategies to improve catalytic efficiency by adjusting the transfer efficiency of ligands. Herein, we engineered a twisted access tunnel of amidase and the mutant PaAmiM1A378V+V402L+L403V was achieved, which exhibited increased catalytic efficiency toward 2-chloronicotinamide. Tunnel analysis, molecular dynamics simulations, and quantum mechanical calculations revealed that the straightened tunnel significantly improved the transport efficiency and simultaneously facilitated substrate binding and nucleophilic attack. With the best mutant as a biocatalyst, 1330 mM product was accumulated within 2 h and a high space-time-yield of 2515 gproduct L–1 d–1 was achieved, which is the highest ever reported. These results lay the foundation for industrial production of 2-chloronicotinic acid and give important guidance for engineering amidases into versatile biocatalysts.
Yameng Wan,Haixia He,Pengshuai Zhang,Fanfan Li,Xiaoqiang Gao,Yanxun Li
Solubility behavior as a significant molecular property should be known in the field of crystal form screening, dosage form along with separation and purification in the pharmaceutical industry. In this research work, solubility data of 2-chloronicotinamide (2-CNA) in ten pure solvents including methanol (MeOH), ethanol (EtOH), ethylene glycol (EG), 2-methoxyethanol (EGME), 2-ethoxyethanol (EGEE), methyl acetate (MeAc), water, 1,4-dioxane (Diox), N,N-dimethylformamide (DMF) and N,N-dimethylacetamide (DMAC) within the range of 278.15/288.15 K to 323.15 K was experimentally determined by laser monitoring method at 0.1 MPa. Solubility of 2-CNA presented a positive correlation with temperature, and increased with incremental temperature. The result was indicative of a common tendency: DMAC > DMF > EGME > EGEE > EG > MeOH > Diox > EtOH > MeAc > water. The solvation free energy, molecular electrostatic potential surface (MEPS) and Hirshfeld surface were used to study the intermolecular interactions. Moreover, six mathematical models namely “Margules, UNIQUAC, NRTL, Wilson, NRTL-SAC and Jouyban model” were utilized to correlate solubility data of 2-CNA in mono-solvents. UNIQUAC model could provide maximum regression accuracy in correlation with solubility data among six models in terms of average relative deviation (ARD) and root mean square deviation (RMSD). Finally, thermodynamic parameters obtained from UNIQUAC equation indicated that all dissolution and mixing process were always entropy-increasing, endothermic and spontaneous.
2-Chloronicotinic acid (2-CA) is an important building block for a series of agrochemicals and pharmaceuticals. Amidase-catalyzed hydrolysis of 2-chloronicotinamide is one of the most attractive approaches for 2-CA production. However, development of the bioprocess was plagued by low activity of amidase for 2-chloronicotinamide. In this work, an amidase signature (AS) family amidase from Pantoea sp. ( Pa -Ami), with superior activity for nicotinamide and its chlorinated derivatives, was exploited and characterized. Kinetic analysis and molecular docking clearly indicated that chlorine substitution in the pyridine ring of nicotinamide, especially the substitution at 2-position led to a dramatic decrease of Pa -Ami activity. The productivity of the bioprocess was significantly improved using fed-batch mode at low reaction temperature and 2-CA was produced as high as 370 mM with a substrate conversion of 94.2%. These results imply that Pa -Ami is potentially promising biocatalyst for industrial production of 2-CA.
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