Mining And Transform Of Aminotransferase And Its Application In Enzymatic Synthesis Of Chiral Amines

Niraparib has excellent curative effect on recurrent ovarian cancer.The key chiral intermediate(S)-5-(4-bromophenyl)piperidin-2-one of niraparib was prepared by biological enzymatic process.It has the advantages of high chiral purity and green environmental protection.The recombinant aminotransferase excavated and mutated in this paper can effectively catalyze the preparation of key chiral intermediates of niraparib,which has laid a solid theoretical foundation for the synthesis and preparation of niraparib.In this paper,the transaminase At AT(Aspergillus terreus)preserved in the laboratory was used as the template.Through the method of gene mining and virtual screening,4 transaminase strains with possible catalytic activity were mined in the NCBI database.Through gene synthesis,p ET-28a(+)plasmid vector,and introduced into Escherichia coli BL21(DE3)for induced expression;all four transaminases were successfully expressed in soluble,used to catalyze the chiral intermediate precursor ketone(4-(4-bromophenyl)-5-oxobutyric acid isopropyl ester);the enzyme activity assay results show that the transaminase number ATArt has catalytic activity to the substrate precursor ketone,the conversion rate is 41.0%,ee>99%,In order to further improve the catalytic activity of the enzyme to the niraparib chiral intermediate precursor ketone,this paper used the screened ATArt as the starting template,and after two rounds of error-prone PCR mutation,a total of 480 mutant strains were screened.Among them,the transformation rate of mutants of Y60 F,S124I,G136 W and A284G(numbered ATA301)was significantly improved,reaching 64.0%,and ee>99.0%.The conversion rate is 56.1% higher than that of ATArt.In order to continue to improve the catalytic activity of the enzyme for niraparib intermediates,site-directed saturation mutation was carried out on the site F122 near the catalytic pocket of transaminase,and a total of 19 mutants were obtained.The rate reached 81%,which was 26.5% higher than that before site-directed mutagenesis,and ee>99.0%,indicating that Y60,S124,G136 and A284 may be the key amino acids that affect the activity of transaminase.Based on the molecular docking strategy,the mutant enzymes and substrates were analyzed.The interaction force of 4-(4-bromophenyl)-5-oxobutyric acid isopropyl ester shows that the mutant enzyme has stronger substrate binding ability and better steric conformation,which may be responsible for the increased conversion rate s reason.Subsequently,the enzymatic properties of ATA301-F122 Itransaminase were studied,the effects of p H and temperature on enzyme activity,the storage stability of ATA301 and the determination of enzyme kinetic parameters were investigated.The results showed that the most suitable p H of ATA301-F122 Iwas 9.5 and the most suitable temperature At 50 ℃,the stability of the enzyme showed that the mutant strain ATA301-F122 I had good stability.The enzyme kinetic parameter Km value was 6.632 mmol/L,and the maximum initial reaction velocity Vmax= 1.235mmol/L/min.In this paper,the transaminase activity of the mutant enzyme on other ketones was investigated,and the substrate spectrum of ATA301-F122 I was further expanded.It was found that the catalytic activity of 4-methoxyacetophenone was 15.7%,and its catalytic activity was 15.7% compared to the original enzyme preserved in the laboratory.The improvement is obvious,which further enriches the substrate spectrum of the mutant enzyme.