Thermostabilization of Panomycocin, a novel exo-beta-1,3-glucanase isolated from Pichia anomala NCYC 434, by using excipients and computational methods


Thesis Type: Postgraduate

Institution Of The Thesis: Orta Doğu Teknik Üniversitesi, Faculty of Arts and Sciences, Department of Biology, Turkey

Approval Date: 2015

Student: MUHAMMED TİLAHUN MUHAMMED

Co-Consultant: ÇAĞDAŞ DEVRİM SON, KADRİ FATİH İZGÜ

Abstract:

As the risks for fungal infections increased, the prevalence of invasive fungal infections increased. Therefore, the demand for antifungal agents has risen. Moreover, the currently used antifungal agents have serious side effects and resistance development resulting from their mechanisms of action. Thus, novel antifungal agents with mechanisms that will not affect the host mammalian cells are in need. Yeast killer proteins which are naturally occuring toxins are good candidates for such types of agents. Panomycocin is an example for this type of killer proteins. Panomycocin is a killer toxin of Pichia anomala NCYC 434 (K5). It has exo-β-1,3-glucanase activity. It kills the sensitive cells by hydrolyzing β-1,3-glucans that are crucial in maintaining the integrity of fungal cell wall. However, its activity decreases above 37ºC. In this work various types of excipients were used to increase the thermostability of Panomycocin so that it would be active at higher temperatures. Gradient concentrations of these excipients along with the protein were tested on Saccharomyces cerevisiae NCYC 1006 at increasing temperatures. If the excipients tested increase the thermostability, this will give us the opportunity to choose suitable excipients that can be used in the formulation of Panomycocin as a novel antifungal drug. Since the effect of excipients on thermostability is limited, computational methods were also used to design a thermostable protein at much higher temperatures. Homology modeling of the protein was performed first. After the binding site of the protein was predicted, the best thermostabilizing positions in the model generated were detected utilizing various computer programs and servers. Although the excipients tested did not increase the thermostability of the protein, we found the best amino acid residues in the model whose substitutions can increase the thermostability of the protein to the desired level. In the allosteric part of the protein Leu52Arg, Phe223Arg and Gly254Arg were found to be the best thermostabilizing mutations with 6.26 K, 6.26 K and 8.27 K temperature increases respectively. In the binding site Glu186Arg was found to be the best thermostabilizer mutation with 9.58 K temperature increase. Using the results mutant thermostable protein can be obtained and this will enable the formulation of Panomycocin as a novel antifungal drug with high thermostability.