Yehia AG Mahmoud¹*, Soad M Abu El Souod¹ Fatama Sombole² and Mona Ade¹

¹Botany Department, Mycology Research Lab, Faculty of Science, Tanta University, Tanta, Egypt
²Tanta University, Faculty of Pharmacy, Tanta, Egypt

*Corresponding Author: Yehia AG Mahmoud, Botany Department, Mycology Research Lab, Faculty of Science, Tanta University, Tanta, Egypt.

Received: May 17, 2018; Published: June 19, 2018

Citation: Yehia AG Mahmoud., et al. "Purification, Characterization, and Inhibition Cryptococcus neoformans Succinate Dehydrogenase". Acta Scientific Microbiology 1.7 (2018).

Abstract

Introduction: Succinate dehydrogenase (SDH) catalyzes the oxidation of succinate to fumarate in the Krebs cycle and transfers the electrons from succinate to ubiquinol. SDH, are important enzymes in the biosynthesis of ATP. The aim of this study was to purify, characterized and inhibit SDH from Cryptococcus neoformans the human pathogenic basidiomycetic yeast.

Materials and Methods: Cryptococcus neoformans was grown and its cells were harvested and subjected to breakage with glass beads. SDH was precipitated with 80% of ammonium sulfate and purified using Sephacryl S-300 gel filtration. Different natural products were tested against yeast cell growth inhibition. Furthermore, transmission electron microscopy analysis was used to examine the inhibited C. neoformans cells.

Results: SDH was 65-fold purified from Cryptococcus neoformans with an overall yield of ~ 33% and specific activity of 13.7 unit/mg. The native SDH was a multi-enzyme system with total molecular weight = 141 kDa. Analysis of the purified SDH on SDS-PAGE showed that it is composing of four subunits of molecular weights: 66, 30, 26, and 12.8 kDa. Malonate is a competitive inhibitor against succinate with Kis value of 4.3 mM, while di-methyl-malonate recorded mixed type of inhibition to succinate dehydrogenase, with Kis value of 25 mM and Kii value of 7 ml/unite. Also, diethylmalonate inhibitor exhibited competitive inhibition type, Kis value was 28 mM. SDH was inhibited by bee propolis and its effect on wild Cryptococcus neoformans capsules under electron microscope was monitored. The cell wall of the treated cells showed pores and their numbers increased when 265 mg/ml (MIC) propolis concentrations was used.

Conclusion: The obtained data indicated that Cryptococcus neoformans succinate dehydrogenase kinetic mechanism is ordered sequential enzyme mechanism. Cells treated by 256 mg/ml propolis ethanolic extract showed no capsule and the organelles inside cell appeared to be destroyed.

Keywords: Cryptococcus neoformans; Succinate Dehydrogenase; Bee Propolis; Plant Extract; Transmission Electron Microscope

Copyright: © 2018 AG Mahmoud., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.