Purification and characterization of mitomycin C-induced pectin lyase of Erwinia carotovora subsp. atroseptica strain SCRI 1043
1994
Godfrey, V. | Hyman, L.J. | McMillan, G.P. | Old, D.C. | Perombelon, M.C.M.
Erwinia carotovora supsp. atroseptica strain SCRI 1043 produces pectin lyase (PNL) which degrades highly methyl-esterified pectin by trans-elimination when induced by DNA damaging agents such as mitomycin C. Purification of the enzyme (66.5-fold) to homogeneity with 42.3% recovery was achieved by cation exchange chromatography on an S-Sepharose fast flow column equilibrated to pH 8.5 with 20 mmol l(-1) Tris buffer, followed by elution of the bound proteins with a 1 mol(-1) NaCl gradient. SDS-PAGE and IEF-activity staining analyses showed that the mol. wt and pI of the enzymes were 31 kDa and 9.4 respectively and only one isomer was present. The optimum pH and temperature were 8.0 and 35 degrees C respectively, and divalent cations, 1.37 mmol(-1) Ca2+ and 1.37 mmol(-1)Mg2+, although not essential, stimulated enzyme activity by about four and six times respectively. The endo mode of action of PNL was determined by viscometry. PNL induction by mitomycin C was determined spectrophotometrically and by ELISA, and was concomitant with bacteriocin production and bacterial cell lysis. The purified enzyme caused 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.
اظهر المزيد [+] اقل [-]الكلمات المفتاحية الخاصة بالمكنز الزراعي (أجروفوك)
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