Purification of an Intracellular Fibrinolytic Protease from Ganoderma Lucidum Vk12 and its Susceptibility to Different Enzyme Inhibitors

Sekar Kumaran*¹ Perumal Palani², Ramasami Nishanthi², Selvanathan Srimathi² and Venkatesan Kaviyarasan²

¹Department of Biomedical Engineering, Vel Tech Multi Tech Dr. Rangarajan and Dr. Sakunthala Engineering College, Avadi, Chennai-600062, ²Centre for Advanced Studies in Botany,  University of Madras, Guindy Campus, Chennai-600025, Tamil Nadu, India.

For correspondence: E-mail:  immunkim@jejunu.ac.kr  Tel: +82-647543427Received:

Method: The intracellular fibrinolytic protease produced by Ganoderma lucidum VK12 was isolated from the mycelia grown in MCDBF broth and was purified to homogeneity using ammonium sulfate fractionation, ion exchange chromatography and sephadex G-150 column chromatography techniques. The purity of the enzyme was verified on SDS-PAGE after silver nitrate staining. The inhibitory effect of different metal ions and commercial protease inhibitors on enzyme activity was studied. The inhibitor-treated enzyme was assayed with its substrate and the residual activity of the enzyme recorded.

Result: The fibrinolytic enzyme isolated from Ganoderma lucidum was purified to near homogeneity and it appeared as a single protein band on SDS-PAGE. Metal ions such as Ca²⁺ and Mg²⁺ inhibited the activity of the enzyme while Zn²⁺ ions enhanced the activity. . Complete inactivation occurred when the enzyme was incubated with protease inhibitors such as EDTA, 1, 10-phenanthroline, phosphoamidon while the enzyme was insensitive to protease inhibitors such as leupeptin, PMSF, TPCK and APMSF.

Conclusion: Copper sulfate completely inhibited the enzyme activity. The sensitivity of this enzyme to EDTA suggests that it might be a metalloprotease.

Keywords: Ganoderma lucidum, Fibrinolytic protease, Protease inhibitors, Copper sulfate, EDTA.