Genetic and biochemical characterization of an exopolygalacturonase and a pectate lyase fromYersinia enterocolitica

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Abstract
Yersinia enterocolitica, an invasive foodborne human pathogen, degrades polypectate by producing two depolymerizing enzymes, pectate lyase (PL) and polygalacturonase (PG). The gene encoding the PG activity, designated pehY, was located in a 3-kb genomic fragment of Y. enterocolitica ATCC 49397. The complete nucleotide sequence of this 3-kb fragment was determined and an open reading frame consisting of 1803 bp was predicted to encode a PG protein with an estimated Mrof 66 kDa and pI of 6.3. The amino acid sequence of prePG showed 59 and 43% identity to that of the exopolygalacturonase (exoPG) of Erwinia chrysanthemi and Ralstonia solanacearum, respectively. The Y. enterocolitica PG overproduced in Escherichia coli was purified to near homogeneity using perfusion cation exchange chromatography. Analysis of the PG depolymerization products by high performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD) revealed the exolytic nature of this enzyme. The Y. enterocolitica PL overproduced in E. coli was also partially purified and the Mrand pI were estimated to be 55 kDa and 5.2, respectively. HPAEC-PAD analysis of the PL depolymerization products indicated the endolytic nature of this enzyme. Southern hybridization analyses revealed that pehY and pel genes of Y. enterocolitica are possibly encoded in the chromosome rather than in the plasmid. Purified exopolygalacturonase (over 10 activity units) was unable to macerate plant tissues.Key words: pectinase activities, human pathogen, HPLC analysis, pehY gene.