Semi-rational Design Of Amidase From Pantoea Sp. For Efficient Biosynthesis Of 2-Chloronicotinic Acid

2-Chloronicotinic acid?2-CA?is an key intermediate for synthesis of pharmaceuticals and agrochemicals.The traditional chemical routes for synthesis of2-chloronicotinic acid suffered from expensive raw materials,by-products and drastic reaction conditions.Enzyme have been proved to be promising alternatives to trational chemical ways,due to the high acitivity,strict steroselectity and environmently friendiness.Therefore,it is of great significance to develop the biosynthesis route to produce 2-chloronicotinic acid.Amidases,as an important industrial enzyme,hydrolyze the amides to corresponded carboxylic acid.This paper focused on the effcient synthesis of 2-chloronicotinic acid.The activity and themostability had been enhanced by semi-rational design of tunnel and catalytic pocket.This paper also can provide some basis for synthesis of 2-chloronicotinic by amidase.The key residues of catalytic site and tunnel were identified by threading modeling,tunnel anylysis and molecular docking,using Pa-Ami?G175A?as template previously constructed in our laboratory.Five single mutants?A378V,A378T,V402L,L403V,L403T?with improved activitywere obtained by site-saturation mutagenesis.Then,iterated mutantion were employed to further improve enzyme activity,in which a mutant A378V/V402L/L403V was achieved with the highest specific acitivity of127.44 U·mg^-1 among 17 mutants.A378V/V402L/L403V was characterized showing the highest specific acitivity at 55?in Tris-HCl buffer?p H 8.5?.The half-time at 55?is 1.5 fold of G175A.Within 5 h,accumulation of 2-chloronicotinic acid was reached950 m M by 5 g·L^-1 recombinant E.coli whole cell using fed-batch,which was1.63-fold higher than that of G175A.The Kinetic analysis of A378V/V402L/L403V to four chloronicotinic amide were investigated.The catalytic effiency of A378V/V402L/L403V to2-chloronicotinamide was only lower than 4-chloronicotinamide among the tested substrates,with V_maxand k_cat/K_m of 117?mol·mg^-1min^-1and 124 m M^-1·min^-1,respectively.Compared with G175A,the catalytic efficiencies of A378V/V402L/L403V to 2-chloronicotinamide and 4-chloronicotinamide were increased,while 5-chloronicotinamide and 6-chloronicotinamide were decreased.The structure model of A378V/V402L/L403V was built by threading modeling method.The effect of two key domain was analyzed.The results showed that the bottleneck of the tunnel was not changed but the depth was shallowed.The binding energies of2-chloronicotinamide,4-chloronicotinamide and 6-chloronicotinamide in tunnel were decreased,while thatof 5-chloronicotinamide and were increased.The shorten distance of nucleophilic attck with0.2?and the hydrogen bond formed of Ala131 and nitrogen atom of pyridine,contributing to the hydrolysis of 2-CAamidae.Using DbD 2.0,several sites for introduction of disulfide bridge were identified and mutanted.A mutant S157C/S169C with enhancement of thermostability was achieved,and the disulfide bridge was verified by DTT.The specific activity of S157C/S169C was 33.4 U·mg^-1,with V_maxand K_m of 33.3?mol·mg^-1min^-1 and 26.19m M.The half-life at 55?is 30 min,which was 1.8-fold of G175A.Compared with G175A,the T₅₀³⁰ of S157C/S169C increased by 5?reaching 55?,while the temperature of starting unfolding improved 12?.