EXPRESSION, PURIFICATION AND CHARACTERIZATION OF RECOMBINANT PHOSPHOMANNOMUTASE AND GDP-ALPHA-D-MANNOSE PYROPHOSPHORYLASE FROM SALMONELLA-ENTERICA, GROUP-B, FOR THE SYNTHESIS OF GDP-ALPHA-D-MANNOSE FROM D-MANNOSE

The genes rfbK and rfbM from the rfb cluster (O-antigen biosynthesis) of Salmonella enterica, group B, encoding for the enzymes phosphomanno mutase (EC 5.4.2.8) and GDP-alpha-D-mannose pyrophosphorylase (EC 2.7. 7.13) were overexpressed in E. coli BL21 (DE3) with specific activitie s of 0.1 U/mg and 0.3-0.6 U/mg, respectively, Both enzymes were partia lly purified to give specific activities of 0.26 U/mg and 2.75 U/mg, r espectively, Kinetic characterization of the homodimeric (108 kDa) GDP -alpha-D-mannose pyrophosphorylase revealed a K-m for GTP and mannose- 1-P of 0.2 mM and 0.01 mM with substrate surplus inhibition constants (K-iS) of 10.9 mM and 0.7 mM, respectively. The product GDP-alpha-D-ma nnose gave a competitive inhibition with respect to GTP (K-i 14.7 mu M ) and an uncompetitive inhibition with respect to mannose-1-P (K-i 115 mu M). Both recombinant enzymes were used for repetitive batch synthe sis of GDP-cw-D-mannose starting from D-mannose and GTP, In three subs equent batches 581 mg (960 mu mol) GDP-alpha-D-mannose was synthesized with 80% average yield, The overall yield after product isolation was 22.9% (329 mu mol, 199 mg).