Study On Extractive Optimization, Structure And Characterization Of Hydrophobin From Clitocybe Maxima

In this study, we worked on a novel hydrophobin (named Cm.HYD) from aerial hyphae of edible mushroom Clitocybe maxima. Purification condition was optimized using the methods of single factor test and orthogonal design with the effect of each factor such as the ratio of sample to formic acid (FA), sample to trifluoroacetic acid (TFA) and the time of TFA exaction. By circular dichroism spectra (CD), changes of Cm.HYD in secondary structure between a monomer and self-assemble polymer were detected and the "rodlet" formed by self-assembled polymer was visualized by atom force microcopy (AFM). By testing the surface tension and water contact angle (WCA), the surface activity of Cm.HYD was comprehensively evaluated and properties of Cm.HYD, such as foarmability and foam stability, emulsibility and emulsion stability, moisture-retention property were rudimentarily discussed in the paper. The main results were as follows:1. Cm.HYD produced by Clitocybe maxima was a member of classⅠhydrophobin. Its molecular weight was approximately 5 kDa in monomer state. But it would oligomerize immediately and mainly exist in dimmer state with the approximate molecular weight of 10 kDa.2. By single factor test and orthogonal design test, we found the optimum purification parameters:the radio of sample to FA is 20:1; the ratio of sample to TFA is 100:1 and the time of TFA exaction is 6 h. The yield of hydrophobin was up to 0.5 mg/g using the above optimal purification parameters.3. The secondary structure would switch distinctly between the state of monomer and oligomer of Cm.HYD. SDS-PAGE showed that, the molecular weight of monomer was about 5 kDa and CD spectra suggested the monomer mainly containsα-helix structures (a maximum ellipticity at 190 run and a double-minimum at around 215 nm). By vigorous shaking, SDS-PAGE showed that the self-assembly Cm.HYD was mainly in tetramer form. CD spectra suggested that it mainly containedβ-sheet structures (a maximum ellipticity at 195 nm and a minimum at 217 nm). After absorbing at the interface of PTFE, SDS-PAGE showed that Cm.HYD in the form of dimmer and tetramer was detected as the bands at 10 kDa and 20 kDa respectively. CD spectra suggested that the protein contains 41.1% ofα-helix,14.4% ofβ-sheet,14.4% ofβ-turn and 25.4% of random coil. It was proposed that the so-calledβ-sheet state was an on-pathway intermediate in the process of Cm.HYD assembly.4. Hydrophobin is among the most surface-active and well-known bimolecular. And so is Cm.HYD. Dynamic surface tension test showed that the decreasing of dynamic surface tension was a time-dependent process. Finally it reached equilibrium. Detection of equilibrium surface tension showed that the ability of lowering the water surface tension was closely related to the concentration. The higher the concentration, the lower the surface tension. Two critical concentrations (c1=0.78μg/mL, c2=1.43μg/mL) were founded by the curve (γ-c). They may indicate different conditions of the interfacial self-assemble at water-air interface. Detection of water contact angle (WCA) showed the self-assemble film of Cm.HYD could reverse the wettability of the absorbed materials. Hydrophilic surface turned into hydrophobin and vice versa.5. Cm.HYD, with high surface activity can be applied as a novel surfactant and humectants. Foamability experiment showed that, the initial height of foam of Cm.HYD (100μg/mL) was 1.25 times higher than that of SDS and 1.67 times higher than that of BSA and the foam stability of Cm.HYD was 3.4 and 2.04 times higher than that of SDS and BSA respectively. Emulsifying experiment showed that, Emulsion index (EAI) of hydrophobin was 1.37 times higher and emulsion stability index (ESI) is 1.06 times higher than that of SDS. Moisture-retention experiment showed, under the RH 20%,25℃condition, the moisture retention rate displayed the following tendency:H2O> Cm.HYD(100μg/mL)> glycerin(10%)> Cm.HYD(300μg/mL)> Cm.HYD(500μg/mL). Moisture-retention property was more obvious for higher concentration.