Construction And Optimization Of Catalytic System For Synthesis Of ?- Hydroxy-?-amino Acid By Aldolase

The?-hydroxy-?-amino acid and the corresponding amino alcohol have important biological activities and can serve as potential chiral structural units for synthesizing biologically active molecules,such as antibiotics and immunosuppressive agents.The commercial synthesis of?-hydroxy-?-amino acids is mainly conducted through tedious chemical methods,which are insufficient in stereoselectivity,require expensive precursors or chiral auxiliary agents and result in the pollution.In contrast to chemical methods,enzymatic methods have the advantages of simple procedure,mild reaction conditions,fast reaction rate and high selectivity.As one of the most important aldolases,the study of threonine aldolases in the enzymatic synthesis of?-hydroxy-?-amino acids mainly focuses on the kinetic resolution of chemically produced dl-syn mixtures rather than aldol condensation.In aldol condensation,?-hydroxy-?-amino acid is synthesized in one step from the simple prochiral precursor glycine and aldehyde,which is simpler than kinetic resolution.Here,the study included:?1?the construction and optimization of D-threonine aldolase-catalyzed aldol condensation system,?2?the optimization of D-threonine aldolase immobilization system and?3?the development of double enzyme-catalyzed synthesis of amino alcohol.Through the screening of recombinant strains expressing aldolases,the strain E.coli BL21?DE3?/pET 28b-AcDTA was selected due to its superior catalytic performance.The strain was cultivated,expressed and then used for the preparation of lyophilized powder as a biocatalyst catalyzing aldol condensation reaction.The optimized components of reaction mixture were listed as followed:1%-2%added amount of freeze-dried powder as biocatalyst,50 mM p-sulfone benzaldehyde used as substrate,500 mM glycine used as co-substrate,100?L pyridoxal5-phosphatemonohydrate used as coenzyme,350?L MnCl₂and 10%dimethyl sulfoxide used as reaction medium.The reaction conditions were optimized to be pH9.5,30?and 800 rpm.Under optimized conditions,the yield reached 69.84%,which was twice higher than that before the optimization.In industrial operations,immobilized cells with better stability and low costs are more valuable than free cells.Therefore,the freeze-dried powder of the strain E.coli BL21?DE3?/pET 28b-AcDTA was applied for PEI/GA cross-linking immobilization.The optimized polyethyleneimine/glutaraldehyde?PEI/GA?cross-linking system was listed as followed:the freeze-dried powder?32.5 g/L?was mixed with diatomaceous earth?6 g/L?in 0.2 M sodium phosphate buffer?pH 8.0?at 45?,then 2%?v/v?of the polyethyleneimine?stocksolution concentration:5%?was added for 1 h cross-linking,and finally 0.5%?v/v?of glutaraldehyde?stock solution concentration:25%?was added for another 30 min cross-linking.The investigation of the operational stability of the immobilized cells revealed that the catalytic activity after 15 batches retained about 80%of the original catalytic activity.In addition,the cascade synthesis of amino alcohol was explored by using aldolase and decarboxylase as biocatalysts.The strains E.coli BL21?DE3?/pET28b-PsDTA and E.coli BL21?DE3?/pET 28b-DAPDC were successfully constructed to express aldolase and decarboxylase,respectively.The cascade reaction results indicated that the retention time of the product was the same as that of the standard,suggesting that the proposed approach for the synthesis of amino alcohol was feasible.