Biosynthesis in an on-line immobilized-enzyme reactor containing phenylethanolamine N-methyltransferase in single-enzyme and coupled-enzyme formats

An immobilized-enzyme reactor (IMER) based upon phenylethanolamnie N-methyltransferase (PNMT) has been developed. The activity of the PNMT-IMER and its applicability for on-line N-methylation of normetanephrine was investigated. The reactor was connected through a switching valve to a cyano (CN) and ODS stationary phase connected in series. The substrate was injected onto the PNMT-IMER and the unreacted substrate and product were eluted and transported via a switching valve onto the analytical columns. The results from the PNMT-IMER/CN–ODS chromatographic system demonstrate that the enzyme retained its catalytic activity. Known substrates and inhibitors for PNMT were examined and the chromatographic system was utilized to carry out both quantitative and qualitative determinations. The PNMT-IMER/CN–ODS system proves to be useful in basic biochemical studies, an ideal for the high throughput screening of substances for PNMT substrate–inhibitor properties. The PNMT-IMER was then coupled in series using switching-valve technology with a previously developed dopamine β-hydroxylase immobilized-enzyme reactor and used to carry out the on-line two-step synthesis of epinephrine from dopamine.