Hydrophobins: Exceptional proteins for many applications in brewery environment and other bio-industries

Abstract

Hydrophobins are exceptional proteins produced by fungi. Research over the last decade has led to a better understanding of their role in spontaneous self-assembly at hydrophobic/hydrophilic interfaces. This has resulted in many proposals for using hydrophobins in many important scientific and technological applications. Hydrophobins may become attractive as special biosurfactants, as foaming agents and for protein immobilization in the food industries and in biosensors. Moreover, they can be interesting as stabilizers for flavors, and as, encapsulating agents of trace ingredients in beverages. The use of hydrophobins in pharmaceutical formulations and in medicine is another interesting application as they cause an increased stabilization of drugs. The study of hydrophobins must also lead to a better understanding of the gushing phenomenon in beverages like beers, wines and ciders, which causes great economic losses in those fields. To recognize the positive and the negative aspects of hydrophobins these proteins should be commercially available at large scale which however is not the case. An overview of existing possibilities for applications may help to understand their behavior in different environmental conditions and to stimulate finding improved methods for isolation and purification, and possibly other unexpected applications.

Introduction

Hydrophobins are a large family of small cysteine rich proteins (about 100 amino acids) (Chaplin and Kennedy, 1994) with a molecular mass of around 10 kDa (Cooper and Kennedy, 2010) (Fig. 1). The name hydrophobin was originally used due to their high content of hydrophobic amino acids (Armenante, 2008). The proteins are secreted by fungi into liquid media or remain at the surface of aerial mycelia (Stubner et al., 2010). Recent evidence demonstrates that a broad spectrum of functions in fungal growth and development is mediated by hydrophobins (Armenante, 2008). They have been detected in fungal plant pathogens (Talbot et al., 1996, Bidochka et al., 2001), insect pathogens (St Leger et al., 1992), and human pathogens (Parta et al., 1994) and saprophytes (Bell-Pedersen et al., 1996). More than one type of hydrophobin has been found in most moulds. For example, in Schizophyllum commune, at least four hydrophobin-encoding genes (Sc1–Sc4) with dissimilar functions have been isolated (Wessels, 1993). Due to the distribution of the cysteines and the clustering of hydrophobic and hydrophilic residues, hydrophobins are divided into two classes: class I such as SC3 and EAS, and class II like HFBI and HFBII. The aggregates formed by both classes are distinguished on the basis of their solubility and morphology (Linder, 2009a, Linder, 2009b).

Hydrophobins can assemble at a hydrophilic–hydrophobic interface forming an amphipathic film (Wessels, 2000) (Fig. 2). This film makes hydrophobic surfaces of liquid or solid materials wettable, while hydrophilic surfaces are becoming hydrophobic. Thanks to their extraordinary properties, hydrophobins were consequently first suggested for a number of applications involving the modification of surfaces properties (Janssen et al., 2002a, Janssen et al., 2002b) leading to improving biocompatibility, reducing friction, or providing specific sites for protein immobilization (Kisko, 2008) (Table 1). The self-assembly of hydrophobins (Wösten and Wessels, 1997) is interesting in the field of surfactants, emulsifiers, and surface coating. Using the hydrophobin in future nanotechnology applications (e.g. patterning molecules at a surface with nanometer accuracy) (Scholtmeijer et al., 2001, Wang et al., 2004, Thomas, 1995), may lead to improving biosensors (Bilewicz et al., 2001), and applications in tissue engineering (Lumsdon et al., 2005). Remarkable is the possibility to separate and isolate nutritional proteins of interest by fusing them to the hydrophobins. This could result in a generic, rapid and efficient protein purification system (Linder et al., 2004). As hydrophobins are adsorbed easily to both hydrophilic and hydrophobic surfaces, such as glass, mica, parafilm, and Teflon, resulting in modification of their properties (Wösten et al., 1994) and the ability of hydrophobins to assemble into strong monolayers and to modify the hydrophobicity of a surface these proteins may have a wide range of potential usages as biotechnological tools (Mackay et al., 2001). Increasing demands for hydrophobins leads to a challenge in terms of the production and purification of this product (Subkowski et al., 2007, Winterburn et al., 2011a, Winterburn et al., 2011b).

For the stabilization of oil vesicles, or oil emulsions, in pharmaceutical applications hydrophobins might be used instead of traditional surfactants (Scholtmeijer et al., 2001). Hydrophobins with suitable properties are potential candidates for native biosurfactants to treat repellency. On the other hand, understanding their involvement in modification of hydrophobicity could open up new applications, for instance in using fungal field inoculants (Rillig, 2005). The amphiphilic nature of hydrophobins can also be exploited using its surfactant-like behavior in solutions, since they are quite soluble in water (Lahtinen et al., 2008). Recently, the ability of hydrophobins to increase the dispersion of hydrophobic particles in water has been described (Wang et al., 2010a, Wang et al., 2010b, Wang et al., 2010c).

Although various applications are expected for native hydrophobins, the modification of hydrophobin structures might offer more possibilities as it may lead to changing the biophysical properties of the hydrophobin surface. By changing the surface hydrophobicity, the binding of various molecules and cells can be manipulated (Hektor and Scholtmeijer, 2005). The self-assembly at hydrophobic/hydrophilic interfaces as well as the nontoxic nature of hydrophobins allow them to be introduced into a number of products (Kershaw and Talbot, 1998). A negative aspect of hydrophobins is that they are produced by fungi when growing on grains (e.g. barley) and during grain treatment. Barley is a very important vegetal food with high economic value as it is used in the production of beer and other drinks (Ehrenbergerova et al., 2006, Jood and Singh, 2001, Sehgal and Kawatra, 1998). The presence of hydrophobins leads to gushing, which has many economic drawbacks for brewing industries. Consequently, improvement in rapid and early detection methods for hydrophobins is required (Shokribousjein et al., 2011).

The aim of this report is to emphasize the different applications of hydrophobins and to stimulate more studies for finding methods to detect, isolate and produce these valuable proteins.