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Purification and biochemical characterization of an extracellular endoglucanase from the necrotrophic oomycete, Pythium myriotylum Dreschler
Author(s) -
Geethu C.,
Nair R. Aswati
Publication year - 2014
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.201400323
Subject(s) - hydrolysis , extracellular , chemistry , cellulase , sodium dodecyl sulfate , cellulose , enzyme , chromatography , nuclear chemistry , gel electrophoresis , polyacrylamide gel electrophoresis , biochemistry
An extracellular endoglucanase (EG) that catalyzes the hydrolysis of carboxy‐methyl cellulose (CMC) as substrate was purified to homogeneity from the soft‐rot causing oomycete P. myriotylum with maximum EG production observed in presence of 1% (w/v) sucrose. The enzyme designated Pm EG was observed to be monomeric with a molecular weight of 78 kDa as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Optimal activity of Pm EG was determined at pH 5.0 and 25 °C with stability observed at pH extending over acidic to alkaline ranges viz ., 3.0–10.0 and thermal stability upto 75 °C for 1 h. Optimal Pm EG activity was obtained by addition of metal ions viz ., Ca 2+ , Fe 3+ , Zn 2+ , Cu 2+ , Al 3+ , and also in presence of DTT and β‐mercaptoethanol while it was inhibited by Cr 2+ . Various organic solvents, surfactants, and the oxidant, H 2 O 2 had little/no effect on Pm EG activity reflecting its robustness and potential commercial significance. Kinetic constants of Pm EG, K m and V max were determined as 1.1 mM and 407 µmol min −1 mg −1 protein, respectively. Glucose was observed to cause mixed non‐competitive inhibition of Pm EG.