Covalent Immobilization Of Recombinant Dehalogenase ST2570

Dehalogenase, which belongs to hydrolase, can catalyze the cleavage ofcarbon-halogen bonds in of halogen-containing compounds. So far, it plays animportant role in the fine chemical industry，biodegradation and the remediationof environmental pollution. The ST2570dehalogenase from thermophilicarchaeon Sulfolobus tokodaii is substrate-stereospecific, belongs to theL-2-haloacid dehalogenase. Catalyzing the L-2-haloacid convert toD-2-hydroxyalkanoates and the halogen substituent is replaced by hydrogen.Accordingly, racemic2-chloropropionic acid and recombinant ST2570dehalogenase will be used as substrate and catalyzing enzyme, respectively, toproduct D-lactic acid.Compared to the free enzyme, immobilized enzyme has the advantage ofstability, reusable and easily separation, therefore, we attempt to immobilizeST2570dehalogenase. In the conventional covalent immobilization progress,the amino acid residue in enzyme active center are always randomly linked tothe functional groups of carrier, giving rise to the decrease or loss of enzymeactivity.We introduce a new method of selectively immobilization, it inject aconsensus sequence (contains a cysteine) at ST2570N terminal, then exploits thecoexpression formylglycine(FGE), which can identify the consensus sequence, tocatalyze cysteine convert to formylglycine (contains an aldehyde group). So thatwe add a aldehyde label to N-terminus, and obtain the recombinant ST2570.According to the Schiff-base generation reaction, we apply the amino-resinas carrier to covalently immobilize recombinant ST2570NQ, and then reduce thedouble bond with the reducing agent sodium cyanoborohydride. Ultimately, werealize the specific covalent immobilization of ST2570NQ to carrier, reducingthe possibility of damage to the enzyme spatial structure.Finally, the enzymatic properties of the immobilized ST2570NQ aremeasured, including optimum temperature and pH, thermal stability, pH stability,and storage stability. After immobilization, the stability, pH and temperature tolerance range are improved. We expect it to provide ideas for the applicationof ST2570dehalogenase in industry to product optically pure D-lactic acid.