Effect of His.sub.6-tag Position on the Expression and Properties of Phenylacetone Monooxygenase from Thermobifida fusca

Phenylacetone monooxygenase (EC 1.14.13.92, PAMO) catalyzes oxidation of ketones with molecular oxygen and NADPH with the formation of esters. PAMO is a promising enzyme for biotechnological processes. In this work, we generated genetic constructs coding for PAMO from Thermobifida fusca, containing N- or C-terminal His.sub.6-tags (PAMO N and PAMO C, respectively), as well as PAMO L with the His.sub.6-tag attached to the enzyme C-terminus via a 19-a.a. spacer. All PAMO variants were expressed as catalytically active proteins in Escherichia coli BL21(DE3) cells; however, the expression level of PAMO N was 3 to 5 times higher than for the other two enzymes. The catalytic constants (k.sub.cat) of PAMO C and PAMO L were similar to that published for PAMO L produced in a different expression system; the catalytic constant for PAMO N was slightly lower (by 15%). The values of Michaelis constants with NADPH for all PAMO variants were in agreement within the published data for PAMO L (within the experimental error); however, the K.sub.M for benzylacetone was several times higher. Thermal inactivation studies and differential scanning calorimetry demonstrated that the thermal stability of PAMO N was 3 to 4 times higher compared to that of the enzymes with the C-terminal His.sub.6-tag.