SITE-DIRECTED MUTAGENESIS OF THE ACTIVE-SITE OF PECTIN METHYLESTERASEFROM ASPERGILLUS-NIGER RH5344

The role of two histidine residues of pectin methylesterase (PME) were analysed by site-directed mutagenesis. Mutant and wild-type pmeA-cDNA were expressed in A. niger strain NRRL3. Both mutant enzymes exhibite d the same mobility on SDS-polyacrylamide gel electrophoresis and gave similar circular dichroism spectra to that of the wild-type enzyme. S ubstitution of His-137 to alanine caused a loss of PME activity. In co ntrast, replacement of His-188 had no effect on the PME activity. Thes e results revealed that the histidine residue at position 137 is essen tial for enzyme activity and probably located in the active site of PM E.