PURIFICATION AND CHARACTERIZATION OF MITOMYCIN C-INDUCED PECTIN LYASEOF ERWINIA-CAROTOVORA SUBSP ATROSEPTICA STRAIN SCRI-1043

Erwinia carotovora supsp. atroseptica strain SCRI 1043 produces pectin lyase (PNL) which degrades highly methyl-esterified pectin by trans-e limination when induced by DNA damaging agents such as mitomycin C. Pu rification of the enzyme (66.5-fold) to homogeneity with 42.3% recover y was achieved by cation exchange chromatography on an S-Sepharose fas t flow column equilibrated to pH 8.5 with 20 mmol l(-1) Tris buffer, f ollowed by elution of the bound proteins with a l mol(-1) NaCl gradien t. SDS-PAGE and IEF-activity staining analyses showed that the mel. wt and pI of the enzymes were 31 kDa and 9.4 respectively and only one i somer was present. The optimum pH and temperature were 8.0 and 35 degr ees C respectively, and divalent cations, 1.37 mmol l(-1) Ca2+ and 1.3 7 mmol l(-1) Mg2+, although not essential, stimulated enzyme activity by about four and six times respectively. The endo mode of action of P NL was determined by viscometry. PNL induction by mitomycin C was dete rmined spectrophotometrically and by ELISA, and was concomitant with b acteriocin production and bacterial cell lysis. The purified enzyme ca used rapid maceration of potato tuber discs and IEF-activity staining of PNL in extracts of rotting tuber discs inoculated with strain SCRI 1043 showed that two isoenzymes were present, one corresponding to the enzyme produced in vitro and the other with a slightly higher pI.