Purification Of Hydrophobin From The Oyster Mushroom Pleurotus Ostreatus And Characterization Of Its Interfacial Self-assembly

Hydrophobin, designated as Po. HYD1, produced by Pleurotus ostreatus strain Pm039 was isolated from aerial hyphae and submerged culture medium. Hydrophobin production and feasibility of preparation were compared subsequentely. Culture conditions of submerged fermentation were optimized through orthogonal tests. Secondary structure changes between soluble state and self-assembled state were revealed using Fourier transform infared（FTIR） spectroscopy and circular dichroism （CD） spectroscopy. Ultrastructures of self-assembled films or particles formed by self-assembled Po. HYD1 were studied using atomic force microscopy（AFM）. Effects of solution environment on self-assembly were analyzed by determining protein concentration and SDS-PAGE analysis. Surface activity of Po. HYD1 was comprehensively evaluated by determination of surface tension, water contact angle （WCA） and emulsion stability. The main results were shown as follows:1. Hydrophobin Po. HYD1 produced by P. ostreatus strain Pm039 is a member of the classⅠhydrophobins with approximate molecular weight of 8kDa in monomer form. Following dissolved in water, Po. HYD1 monomers oligomerized immediately and mainly existed as dimmer form with approximate molecular weight of 15kDa.2. Po. HYD1 can be isolated either from aerial hyphae or submerged culture medium. Electrobubbling precipitation of submerged culture medium is a more simple and efficient method for isolating Po. HYD1 hydrophobin. The production of Po. HYD1 can be improved to around 11mg/L after optimizing by orthogonal test. The optimized culture conditions are combined medium of 40g/L glucose, 2g/L tryptone, 1g/L MgSO₄, 1g/L K₂HPO₄ and 1g/L KH₂PO₄ with 20-day-incubation and an initial pH value of 5.0.3. During the transition from the monomeric state to the assembled state, molecules of Po. HYD1 changed in secondary structural and ultrastructure. FTIR analysis showed the frequency of the arnideⅠband（C=O） assigned to the peptide bond was shift from 1642cm^-1 to 1635cm^-1. Comparison of CD spectra suggested significant conformational changes between its soluble state and self-assembled state. The spectrum of Po. HYD1 in soluble state had a maximum ellipticity at 190nm and a minimum at 209nm, which contains structural units ofα-helix,β-sheet and random coil. In contrast, the spectrum of assemblage showed the maximum at 195nm and the minimum shifted to 215nm～218nm, which forms the "stackedβ-sheet" state. AFM studies revealed distinct ultrastructures formed at different hydrophobic-hydrophilic interfaces. Monomolecular adsorption layer and partial bilayer with a higher coverage, thickness ranging from 3.2nm～3.8nm, was observed on the surface of highly oriented pyrolytic graphite（HOPG）. Highly structured rodlet layer with a lower coverage presenting uniform thickness of 4.2±0.1nm was visualized on the mica surface. Ear-like particles with similar shapes, identical orientations and divergent sizes were formed under induction of vigorous shaking.4. The self-assembly process of Po. HYD1 was remarkably affected by various solution environments. A lower incubation temperature（≤16℃） or a higher pH value（11.0） arrested Po. HYD1 molecules in soluble state and inhibited the occurrence of self-assembly. In contrast, a higher incubation temperature （37℃）, a lower pH value（3.0） or a higher ionic strength accelerated the accomplishment of self-assembly. However, a nearly neutral pH value （pH5.0～9.0） and a lower ionic strength could not bring evident influence on the process of self-assembly which is similar to samples dissolved in pure water.5. Po. HYD1 is an amphipathic molecule, with high surface activity. The decreasing of dynamic surface tension showed that is a time-dependent process. Determination of equilibrium surface tensions showed that the ability of lowering water surface tension is dependent upon the hydrophobin concentration. The maximal decreasing of water surface tension, which reached to 25.5mN/m, was achieved using 100μg/mL Po. HYD1 among the tested concentrations. Two crucial concentrations（C1≈6μg/mL and C2≈24μg/mL） were found by fitting for linear equations, likely indicating different conditions for the interfacial self-assembly at water-air interface. Moreover, Po. HYD1 molecules are able to self-assemble on various solid surfaces and reversed their wettability. By coating with Po. HYD1 films, hydrophobic surfaces turned hydrophilic and vice versa. Po. HYD1 is capable of stabilizing the emulsion consisting of water and hexane. Following homogenization, Po. HYD1 coated hexane drops perfectly dispersed in the aqueous phase for at least 24h.