An anchor-chain molecular system was constructed for controlled orientation
and high activity in enzyme immobilization. A streptavidin recognition pep
tide (streptag) coding sequence was fused to the 3' end of the phoA gene, w
hich codes for E. coli alkaline phosphatase (EAP). Both the wild-type (WT)
and the Asp-101 --> Ser (D1O1S) mutant were modified with the streptag sequ
ence with or without the insertion of a flexible linker peptide [-(Gly-Ser)
(5)-] coding sequence. The fused genes were cloned into the vector pASK75 a
nd expressed in the periplasm of the host cell Escherichia coli SM547. The
proteins were released by osmotic shack and purified by ion-exchange chroma
tography. Enzyme activities of all proteins were measured spectrophotometri
cally with p-nitrophenyl phosphate as the substrate. Specific activities of
D1O1S-streptag and D1O1S-linker-streptag enzymes were increased 25- or 34-
foId over the WT, respectively. These fusion proteins were then immobilized
on microtiter plates through streptag-streptavidin binding reaction. After
immobilization, the D1O1S-linker-streptag enzyme displayed the highest res
idual activity and the ratio of enzyme activities of the linker to nonlinke
r enzymes was 8.4. These results show that the addition of a linker peptide
provides a spacer so as to minimize steric hindrance between the enzyme an
d streptavidin. The method provides a solution for controlled enzyme immobi
lization with high recover activity, which is especially important in const
ruction of biosensors, biochips, or other biodevices.