Multi-enzyme Cascade Biocatalysis For Deracemization Of 2-hydroxy Acids

Enantiopure 2-hydroxyacids are key intermediates for the synthesis of pharmaceuticals and fine chemicals.We present an enantioselective cascade biocatalysis for deracemization of racemic 2-hydroxyacids that allows for efficient production of enantiopure 2-hydroxyacids.The method was realized by a genetic engineered E.coli strain coexpressing three enzymes:(S)-2-hydroxyacids dehydrogenase((S)-2-HADH),(R)-2-ketoacid reductase((R)-2-KAR)and glucose dehydrogenase(GDH).One enantiomer((S)-2-hydroxyacid)is firstly oxidized to the ketoacid with(S)-2-HADH,while the other enantiomer((R)-2-hydroxyacid)remains unchanged.The ketoacid obtained is then reduced to the opposite enantiomer with(R)-2-KAR plus cofactor regeneration enzyme GDH subsequently.First,the(S)-2-HADH gene from pseudomonas aeruginosa CCTCC M 2011394(KU612124)was cloned and expressed in E.coli BL21(DE3).The result showed that the activity was lower.Genome mining strategy conducted by using(S)-2-HADH from P.aeruginosa CCTCCM 2011394 as the template in the NCBI database.the relatively higher activity(S)-2-HADH from P.aeruginosa NSUT was selected for futher studies.In the previous work,we had presented an engineering of E.coli BL21(DE3)/pCDFDuet-KAR-GDH.The pET28b-HADH and pCDFDuetKAR-GDH with different antibiotic selection were introduced to E.coli BL21(DE3).A three-enzyme coexpression strain E.coli BL21(DE3)/ pET28b-HADH/pCDFDuet-KAR-GDH was screened.Then,E.coli BL21(DE3)/pET28b-HADH and E.coli BL21(DE3)/ pCDFDuet-KAR-GDH were used to deracemization of 2-hydroxyacids.The result showed that the process was efficient.To our delight,most of those substrates(1a-1m)were obtained in high yields(>90%)with > 99% ee.Thus,the method by three enzymes system from two cells was successfully used to deracemization of 2-hydroxyacids.Then,The recombinant E.coli BL21(DE3)/pET28b-HADH/ pCDFDuet-KAR-GDH was cultured to achieve the resting cells.The cascade oxidation-reduction reaction catalyzed by the resting cells of the recombinant E.coli was performed at 35°C,pH 7.5,cell concentration 8 g DCW/L and substrate concentration 20 mM.The results showed that most of those substrates(1a-1c,1e-1m)can be obtained in high yield(>90%)with >99% e.e in a shorter reaction time as compared to the mixtures of two recombinant E.coli.The use of constructed E.coli BL21(DE3)/pET28b-HADH/pCDFDuet-KAR-GDH as biocatalyst can not only simplify cultivation of bacteria,but also reduce reaction time.We speculate that multienzyme in one engineering strain avoided the transfer of substrates in different cells.Finally,the E.coli BL21(DE3)/pET28b-HADH/pCDFDuet-KARGDH was used to deracemization of racemic 2-chloromandelic acid.The cascade oxidation-reduction reaction catalyzed by the resting cells of the recombinant E.coli was performed at 35°C,pH 7.5,cell concentration 8 g DCW/L and glucose: substrate = 1.5:1(c/c).The result showed the whole racemic 2-chloromandelic acid was converted to(R)-2-chloromandelic acid by the three enzymes system when the concentration of substrate was 20-100 mM.