Constitutive Expression of Acidovorax facilis Nitrilase in E.coli and Its Application in the Hydrolysis of Nicotinonitrile in High Concentration
The constitutive expression of nitrilase derived from Acidovorax facilis in E.coli was studied,and the constitutively expressed nitrilase recombinant plasmid pET3b-NIT was constructed,and the optimal host strain for its expression was determined to be E.coli BL21Gold(DE3).The enzyme activity and specific activity of recombinant E.coli were 4 392.39 U·L-1 and 953.25 U·g-1,which were 2.19 and 1.94 times higher than those of the original inducible expression strain,respectively.On this basis,the process conditions for the hydrolysis of high concentration of nicotinonitrile for the synthesis of nicotinic acid by resting cells were systematically investigated.It was found that 2 mol·L-1 nicotinonitrile could be completely converted to nicotinic acid by 12 g·L-1 cells for 2 h at 35℃,and the space-time yield was up to 246.22 g·g-1·d-1.Preparation experiments on the gram scale of nicotinic acid were also performed,and 2.23 g of nicotinic acid was obtained in 90.65%yield and over 99%HPLC purity,achieving efficient preparation of nicotinic acid at high substrate concentration.
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