CHARACTERIZATION AND OVEREXPRESSION OF THE PEM GENE ENCODING PECTIN METHYLESTERASE OF ERWINIA-CHRYSANTHEMI STRAIN-3937

The pem gene encoding the pectin methylesterase (PME) of Erwinia chrys anthemi strain 3937 was subcloned and its nucleotide sequence determin ed. The gene contains an open reading frame of 1098 bp and codes for a protein of 366 amino acids (aa). The mature 37-kDa form of the protei n is 342 aa long and has a calculated isoelectric point of 9.64. A pla smid was constructed to overproduce PME: a DNA fragment carrying pem w as amplified by the polymerase chain reaction and cloned downstream fr om the p(L) promoter of the lambda phage, in a high-copy-number plasmi d. In an Escherichia coli strain transformed with this plasmid, an inc rease in PME production of more than 60-fold was obtained, compared wi th the wild-type Er. chrysanthemi strain. PME represents about 5% of t he total protein content of the cells. Comparison of this PME sequence with six PMEs from prokaryotic or eukaryotic organisms showed six hig hly conserved segments whose possible role in enzyme activity are disc ussed.