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Purification and Characterization of β-1,3-glucanase from Candida Oleophila for the Biocontrol of Penicillium expansum

Carlos Tamayo-Urbina, Victor Guerrero-Prieto, Cesar Guigon-Lopez, Francisco Vargas-Albores, David Berlanga-Reyes, Carlos Acosta-Muniz5 and Damaris Ojeda-Barrios

Candida oleophila was originally isolated from apple fruit peel in the region of Cuauhtémoc, Chihuahua, Mexico, and utilizes exo-β-1,3-glucanase production as a mode of action against Penicillium expansum in postharvest apples. β-1,3- Glucanase activity generated by C. oleophila on a minimal-salt medium using glucose, laminarin or cell wall fragments of P. expansum as a sole carbon source was determined. Highest β-1,3-glucanase activity (249 U/L) was obtained after 48 hours of incubation using only glucose. β-1,3-Glucanase was purified from a growth medium using anion exchange chromatography on DEAE Sepharose, which increased the specific activity of β-1,3-glucanase 74-fold, compared to the crude extract after ultrafiltration. SDS-PAGE demonstrated a purified β-1,3- glucanase with an estimated molecular weight of 48.3 kDa. The purified enzyme hydrolyzed laminarin to glucose as a final product and was identified on native polyacrylamide gels as a monomeric protein. Optimum activity occurred at pH 5 and at 40°C, and the enzyme was stable at 30°C for 1 hour. The purified exo-β-1,3-glucanase, when challenged against P. expansum conidia, reduced conidial germination to 51.8% and mycelial growth to 31%. Results indicate that one mode of action of C. oleophila for controlling P. expansum is by producing β-1,3-glucanase and that the purified