Abstract

Hydrophobins (HPs) are industrially important small surface-active proteins mostly found in fungi. In this study, new hydrophobins were detected and identified from the edible white rot mushroom Pleurotus ostreatus (GRAS strain) using a proteomic approach. HPs Hydph6, Hydph7, Hydph16, Vmh2, and Vmh3-1 were detected together after separation of the membrane proteins of P. ostreatus mycelia. Identification of Hydph6, Hydph7, and Hydph16 were reported for the 1st time in Orbitrap-HR-LC-MS/MS analysis, and the primary amino acid sequences were annotated. This study is the first report of protein-level identification and extensive in silico characterization of Hydph6, Hydph7, and Hydph16. Computational tools were used to characterize these selected HPs. ProtParam and ProtScale studies confirmed Vmh2 and Hydph6 as promising candidates for industrial applications with a high value of the grand average of hydropathicity index (0.662 and 0.996, respectively) and aliphatic index (112.11 and 122.07). Modeller 9.11 was used to obtain the most accurate three-dimensional structures of all HPs. Among all the proteins, the Z-score values and Ramachandran plot analysis of Vmh2 and Vmh3-1 suggested that the structures of these proteins were more closely aligned with existing databases of previously derived structures of HPs in comparison to other HPs. Protein– protein interaction studies showed promising interaction of Hydph6 and Vmh2 with most of the proteins involved in cell division and development, of P. ostreatus, in a way that these proteins play an important role in the growth cycle of P. ostreatus. These proteins did not show transmembrane regions and were not glycosylphosphatidylinositol (GPI)- anchored. It was the first report to evaluate the role of HPs in the hydrophobicity and interaction with the cell membrane through docking studies. Results revealed that Hydph6 had a comparable docking score of −8.1 with Chitin, similar to the chitin-interacting proteins, Beta-N-acetylglucosaminidase (−7.9 Kcal/mol), Chitinase A1 (−8.5 Kcal/mol), and Chitin-Binding Protein 21 (−8.2 Kcal/mol). Out of the five studied HPs, Hydph6, and Vmh2 had shown important characteristics such as amphipathic nature, thermostability, flexibility, and interaction with membrane molecules. Computational analysis suggests the potential of Hydph6 and Vmh2 for industrial applications in surface modification, food foams, and drug delivery systems. Vmh2 is already under evaluation for various industrial applications. In a future study, Hydph6 will be isolated, characterized, and explored for industrial applications.

Key words: Hydrophobins, P.ostreatus, Proteomics, Bioinformatics, Protein characterization