CHARACTERIZATION OF PECTATE LYASE PRODUCED BY PSEUDOMONAS MARGINALIS AND CLONING OF PECTATE LYASE GENES

摘要

Pseudomonas marginalis produced two pedate lyase isozymes in a medium containing sodium polypectate. Of the two, one had alkaline pI, 9.5, and the other had neutral pI, 7.0. Extracellular pectate lyase secretion started during mid-log phase and reached a ma,xuimum during the stationary phase of growth. Pectate lyase production was improved by the addition of 1 mM CaCl2 which also enhanced the extracellular secretion in the medium containing glucose as carbon source. Ca2+ facilitated the utilization of polyg alacluronic acid as carbon source. Pectate lyase (pel) genes were cloned in pUC18 vector. One pel clone (pME1) containing a 7.7 kb BamHI fragment coded for both alkaline and neutral isozymes. A restriction map was constructed for the plasmid pME1. pel genes were sub-cloned from plasmid pME1. Sub-cloning and restriction mapping indicated that a 2.3 kb PstI-BamH1 fragment coded for one isozyme and a 2.8 kb Pstl fragment coded for another isozyme; these two genes were separated by 26 kb. Southern hybridization analysis indicated that P. marginalis pet genes are homologous with Erwinia pel genes. Escherichia coli containing pel genes efficiently macerated potato tuber tissue.