High constitutive peroxidase activity and constitutive thermotolerance in <i>Neurospora crassa</i>

Senczuk Anna Marie, Machwe Amrita, Kapoor Manju

doi:10.47371/mycosci.myc44129

<p>During the isolation of mutations in the heat-inducible hsp70-1 gene of Neurospora crassa by RIP (repeat-induced point mutations), several transformants were generated by electroporation of conidia with a plasmid harboring an incomplete copy of this gene. One isolate, designated E-45, containing ectopically integrated hsp70-1 DNA, exhibited a slow growth rate, low-temperature sensitivity, constitutive thermotolerance (without prior heat shock), and high constitutive peroxidase activity. The constitutive form of peroxidase (CP) was distinguishable from the heat-inducible form (HIP) by immunoinactivation employing polyclonal antiserum against the latter enzyme and by electrophoretic resolution in nondenaturing polyacrylamide gels. This enzyme was purified to near homogeneity and some of its properties examined. The relative molecular mass of native CP was in the range of 118–136kDa, as estimated by gel filtration analysis on size exclusion matrices, whereas SDS-PAGE analysis yielded a size of ~37kDa for the polypeptide. Substrate saturation kinetics studies were conducted using ABTS [2,2'-azino-bis (3-ethylbenzthiazole-6-sulfonic acid)] and H₂O₂ as substrates: K_m, V_max, and K_cat values for H₂O₂ were ~22μM, ~447nmolmg⁻¹, and 0.33s⁻¹, respectively, and those for ABTS were ~55μM, ~453nmolmg⁻¹, and 0.3s⁻¹, respectively. Guaiacol was not used as a substrate by this enzyme. CP peroxidase was shown to be a heme-containing enzyme, stable at temperatures up to 58°C.</p>

High constitutive peroxidase activity and constitutive thermotolerance in <i>Neurospora crassa</i>

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