Pectin methylesterase in Citrus bergamia R.: purification, biochemical characterisation and sequence of the exon related to the enzyme active site

Three forms of pectin methylesterase (PME) were purified, from bergamot fruit (Citrus bergamia R.), to homogeneity by ion-exchange and affinity chromatography. The isoforms, named PME I, PME II and PME III, according their elution order on a heparin-sepharose column, were characterized for their relative molecular mass, activity kinetic parameters and thermostability. The molecular mass was estimated to be 42 kDa for the three forms, and the apparent Km values for citrus pectin were 0.9 mg/ml for PME I and 0.5 mg/ml for PME II and PME III. The optimum pH values lie within the range 6.5-9.0, depending on salt concentration. Thermal behaviours of the three PME isoforms were studied in a temperature range from 65 °C to 80 °C with the less abundant PME I isoform showing a higher heat resistance. Moreover, the complete exon 2 sequence of PME gene was acquired (GenBank accession no. DQ458770) using a PCR based approach on well-known Citrus genomic DNA present in the NCBI database.