Site-specific Immobilization Of Haloacid Dehalogenase ST2570

Formylglycine-generating enzymes can selectively recognise and oxidise cysteine residues within the sulphatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine(FGly) residues; these residues can form covalent bonds with amino functionalised fluoresceins or supports through the Schiff base reaction. In this experiment, aldehyde-tagged proteins were prepared using a previously designed set of the p ET28 a plasmid system. The C-terminal aldehyde-tagged protein(ST2570CQ) exhibits significant enzymological properties, such as generation of new free aldehyde groups, high level of protein expression and improved enzyme activity. The immobilization of ST2570 CQ on SBA-15 modified with 2% APTES ethanol solution(V/V) was performed under the following optimal conditions: 2 mg of ST2570 CQ per 10 mg of SBA15-NH2 was incubated in 50 m M PB buffer(p H 6.5) at 10°C for more than 3 h. The immobilized ST2570 CQ shows 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The immobilized ST2570 CQ retains 60% of its original activity after seven cycles of batch operation and 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. These results provide a basis for industrial-scale production of immobilized ST2570.