Study On The Production Of 2-chloronicotinic Acid From 2-chloro-3-cyanopyridine By Biotransformation

Recently,biocatalysis has highly attracted attentions from industry and academia,and the synthesis of carboxylic acids and amides from nitrile compounds have become an active field in research and development.Because of mild reaction conditions,free from contamination and outstanding stereo chemical specificity,the nitrile-converting enzymes have become the most promising and first choice method in place of conventional chemical methods in industry.2-chloronicotinic acid has been widely applied as an intermediate in the synthesis of agrochemicals,pharmaceuticals and fine chemical because of its specific biological activity.The goal of this project is to construct the production process of 2-chloronicotinic acid by biotransformation using the nitrile hydratase(NHase)and amidase pathway.In this work,two genes of NHase and amidase were expressed by the recombinant E.coli BL21(DE3)/pCDFduet-1-NHα-NHβ and E.coli BL21(DE3)/pET-28b(+)-AMI,respectively,which were applied to catalyze the hydrolysis of 2-chloro-3-cyanopyridine,2-chloronicotinamide,to its corresponding 2-chloronicotinamide and 2-chloronicotinic acid.The two-strains system,E.coli BL21(DE3)/pCDFduet-1-NHα-NHβ coupled with E.coli BL21(DE3)/pET-28b(+)-AMI were studied for the preparation of 2-chloronicotinic acid.A recombinant strain of E.coli BL21(DE3)/pCDFduet-1-NHα-NHβ/pET-28b(+)-AMI was constructed to coexpress NHase and amidase,which is able to hydrolyze 2-chloro-3-cyanopyridine to 2-chloronicotinic acid.The gene of NHα and NHβ was cloned from genome of Rhodococcus erythropolis ZJB-09149 based on the known sequences encoding NHases reported on the NCBI.The 624 bp and 639 bp sequence encoding the nitrile hydratase a subunit(NHa)and β subunit(NHβ)with the predicted molecular weight of 22.9 KDa and 23.4 KDa was obtained,respectively.The amino acid sequences of NHase are in 100%homology with the reported NHase sequence of Rhodococcus erythropolis SK121.The expression vector,pCDFduet-1-NHα-NHβ,was constructed and transformed into E.coli BL21(DE3),and the recombinant E.coli BL21(DE3)/pCDFduet-1-NHα-NHβ was constructed.The single factor experiments were used to optimize the process to increase the NHase production by E.coli BL21(DE3)/pCDFduet-1-NHα-NHβ.The results indicated the optimal inducer concentration of IPTG was 0.4-0.6 mM at 1.0 of OD600.The addition of Fe2+ is one of the important factors for NHase formation.It was observed that the recombinant strain gave highest NHase production at 35℃ with 10～14 h of induction time.After optimization of inducing conditions,the specific activity of E.coli BL21(DE3)/pCDFduet-1-NHα-NHβ was 730 U/l,which was 6 times higher than before.The effects on reaction of pH,temperature,substrate and resting cell concentration were investigated.The optimal conditions were pH 7.5 and 35 ℃ with the concentration of 2-chloro-3-cyanopyridine 6 mol/l.The reaction could be inhibited by high concentration of 2-chloro-3-cy anopyridine and 2-chloronicotinamide.Under appropriate reaction conditions,the yield of 2-chloronicotinic acid reached 100%after reaction 30 min with the resting cells amount of 5 gWCW/l.In order to obtain high amidase activity,one recombinant strain E.coli BL21(DE3)/pET-28b(+)-AMI was screened to hydrolyze 2-chloronicotinamide to 2-chloronicotinic acid.Response surface technique was used to optimize the induction conditions of the recombinant strain expressing amidase.Three significant parameters including IPTG concentration,induction time and induction temperature for response were selected and utilized to optimize the process.The amidase activity was improved up to 500.3 U/l by the best combinations of the parameters at optimal levels of 0.38 mM IPTG,induction at 22.4 0C,and 8.4 h of induction time.The properties of the purified recombinant amidase were investigated.Its optimum pH and temperature were 7.5 and 45-50 ℃,respectively.The amidase exhibited excellent thermostability with half-lives of 16 and 6 h at 40 and 45 ℃,respectively,and a melting temperature(Tm)of 62.5 ℃ was corroborated by thermostability measurements by CD.The substrate specificity study showed it exhibited excellent activity towards most of the aromatic and heterocyclic amides.Homology modeling and molecular docking revealed the interactions of substrate and the active center mainly affect the catalytic activity of amidase.The optimized biotransformation conditions of 2-chloronicotinamide to 2-chloronicotinic acid were at 40 ℃,pH7.5 with the resting cells amount of 10 g/I and 60 mmol/1 of 2-chloronicotinamide.Moreover,the substrate concentration could be increased to 150 mM with batch feeding,and the product yield reached 100%.The recombinant E.coli whole cells could also be repeatedly used in the biotransformation and the residual activity was still over 50%of the initial activity after 10 batches.The two-strain coupled system of E.coli BL21(DE3)/pCDFduet-1-NHα-NHβ and E.coli BL21(DE3)/pET-28b(+)-AMI was studied in the biotransformation of 2-chloro-3-cyanopyridine to 2-chloronicotinic acid.The optimized hydrolysis conditions were:pH 7.5,40 ℃,200 rpm of rotate speed and the 1:2 in weight ratio of wet cells for E.coli BL21(DE3)/pCDFduet-1-NHα-NHβ to E.coli BL21(DE3)/pET-28b(+)-AMI.The reaction was inhibited by high substrate concentration.The yield of 2-chloronicotinic acid reached 100%when initial 2-chloro-3-cyanopyridine concentration was not higher than 60 mM.Results indicated 100%conversion was reached in one step control system after 40 min.However,more than 90 min was needed to completely hydrolysis 60 mM of substrate for other system.Further,a recombinant strain of E.coli BL21(DE3)/pCDFduet-1-NHa-NHβ/pET-28b(+)-AMI was constructed,which coexpressed NHase and amidase.After optimization of induction conditions,the enzyme specific activity was 28.1 U/gWCW.Utilizing the whole cells to transform 10 mM 2-chloro-3-cyanopyridine at 40 ℃,the conversion reached 100%after reaction 50 min.