Gene Mining And Semi-rational Modification Of ?-transaminase For Preparation Of Heterocyclic Amines

Chiral amines are important active pharmaceutical ingredients.At present,almost half of the active phararmaceutical ingredients contain at least one chiral amine unit,making chiral amines a really valuable structural unit in the pharmaceutical industry.Heterocyclic amines,such as optically pure 3-aminopiperidine and 3-aminopyrrole,have strong biological activity and can be used to develop a variety of drugs,for example,anti-bacterial,anti-virus,anti-tumor and anti tuberculosis drugs.The industrial production of amines mainly depends on the metal-catalyzed hydrogenation of enamides.However,the cost of this method is high,the production process is dangerous and seriously polluted,and most of the amines are achiral.Enzymatic synthesis of chiral amines has gradually attracted attention due to its low cost,green synthetic routes,mild reaction conditions and high preparation efficiency.Among the method of enzymatic synthesis,the asymmetric synthesis of chiral amines from corresponding precursor ketones by transaminase has become the most potential alternative for industrial production of chiral amines due to its excellent enantioselectivity.In order to prepare chiral heterocyclic amines efficiently,we developed the method of gene mining to explore novel co-transaminases,and conducted activity-based molecular modification of the target ?-transaminase through semi-rational design,which has laid a good foundation for the enzymatic industrialization of chiral heterocyclic amines.The main research contents are listed as follows:(1)Mining and screening of ?-transaminase.Based on the NCBI database,40 novel ?-transaminases were excavated from 9 strains including Paenarthrobacter aurescens TC1?Alcaligenes faecalis ATCC8750 and Aeropyrum pernix K1 and so on,and successfully expressed in E.coli BL21(DE 3).Combined with the existing ?-transaminases in the laboratory,a transaminase library which contain 300 ?-transaminases was constructed.Using N-Boc-3-piperidone and N-Boc-3-pyrrolidone as substrates,the enzyme library was screened.Finally,Cc-TA showed high catalytic activity for N-Boc-3-piperidone and N-Boc-3-pyrrolidone.When isopropylamine was used as amino donor,the conversion of N-Boc-3-piperidone and N-Boc-3-pyrrolidone reached 100%.Although N-Boc-3-piperidone and N-Boc-3-pyrrolidone with concentration up to 1 M can be consumed efficiently by Cc-TA,but a large amount of dry cell need to be invested in the reaction process,and the reaction time is long.In order to improve its industrial application potential,it is necessary to further increase the catalytic activity of Cc-TA to realize the economic efficiency of the catalytic process.(2)Based on semi-rational design to improve the catalytic activity of enzyme.Considering the hydrophobic properties of N-Boc-3-piperidone,the hot spots Q25 and R424 were chosen by sequence alignment and molecular docking in order to create a hydrophobic cavity and substrate channel.First,the saturation mutagenesis of Q25 was performed,and the best mutant Q25F in the first round was screened,which showed 8.85-fold activity improvement for N-Boc-3-piperidone.On the basis of the first round of mutations,combinatorial mutagenesis was performed to change the polar charged R424 to other hydrophobic amino acid residues,and finally a double-point mutant Cc-TA-Q25F/R424L was obtained.There is a 15.82-fold higher specific activity of Cc-TA-Q25F/R424L for N-Boc-3-piperidone and a 35.27-fold higher value of kcat/Km than that of wild type.The molecular mechanism of Q25F and R424L,which affects the catalytic activity of the enzyme was analyzed by the methods of bioinformatics.The reason for the improvement of the catalytic activity of the mutants was explained from the aspects of molecular structure and interaction.(3)The establishment and optimization of the synthesis process of chiral heterocyclic amines by transaminase.The effects of different dry cell dosages on the preparation of(S)-(+)-1-Boc-3-aminopiperidine and(S)-(-)-1-Boc-3-aminopyrrole in 50 mL scale were investigated while the concentration of substrates was kept at 1 M.For N-Boc-3-piperidone,when the enzyme loading was 30 g/L,the double-point mutant Cc-TA-Q25F/R424L reached the reaction equilibrium after 6 h(ee>99.9%),and the conversion reached 96%.However,the reaction equilibrium of wild type was reached after 30 h,and the conversion was 93%.When the enzyme loading was 15 g/L,the double-point mutant Cc-TA-Q25F/R424L reached the reaction equilibrium after 12 h,and the conversion was 93%.Compared with the wild type,the time is shortened by 18 hours than the wild type when the enzyme loading was 30 g/L.For N-Boc-3-pyrrolidone,the double-point mutant Cc-TA-Q25F/R424L(ee>99.7%)and the wild type reached equilibrium at 24 h at different enzyme loading,and the conversion of the mutant increased slightly.This research successfully achieved the goal of efficiently preparing high-concentration chiral heterocyclic amines by the ?-transaminase,laying a good foundation for its futher industrial application.