An isolate of <i>Botrytis cinerea</i> produced polygalacturonase (PG) activity when grown on a modified Czapek-Dox liquid medium containing 1% citrus pectin and 1&37 glucose. Activity was detected after 3 days of growth and reached a maximum of 0.7 units after 10 days. Four polygalacturonases were purified from this medium. The purification involved liquid-phase isoelectric focusing, ion exchange chromatography and concanavalin A-affinity chromatography. The mode of cleavage of polygalacturonic acid by PGs was studied by comparison of the rate of reduction in viscosity with corresponding increase in appearance of reducing groups. Two endo-PG isozymes (endo-PGI and endo-PGII) with pI values of 8.78 and 4.9 and two exo-PGs (exo-PGI and exo-PGII) with pI values of 4.9 and 3.5 were identified. Endo-PGI and endo-PGII both had apparent M<SUB>r</SUB> values of 30 kDa; exo-PGI and exo-PGII had M<SUB>r</SUB> values of 65 and 70 kDa respectively. Each protein consisted of a single polypeptide chain. Exo-PGI and exo-PGII were shown to be high mannose-containing glycoproteins by their affinity to concanavalin A. Treatment of exo-PGI and exo-PGII with endoglycosidase-H resulted in peptides with reduced M<SUB>r</SUB>s of 46 and 60 kDa respectively. The temperature maxima for endo-PGI, endo-PGII, exo-PGI and exo-PGII were 37, 34, 48 and 50<SUP>o</SUP> respectively, and all the isozymes showed optimal pH values between 4.0 and 5.0. The inducibility of PG activity by galacturonic acid was assessed. When the fungus was grown on a modified Czapek-Dox liquid medium containing galacturonic acid or glucose, there was no consistent increase with time in the total PG activity, relative to mycelial dry weight. However, preparative isoelectric focusing identified a peak of activity between pH 3.7 and 6.3 when the fungus was grown in the presence of galacturonic acid. This activity was attributed to the induction exo-PGs (exo-PGI and/or exo-PGII).
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:592634 |
| Date | January 1993 |
| Creators | Johnston, D. |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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