(247d) The Protein Surface Hydrophilic Lipophilic Balance As a New Approach for Characterizing Large Biosurfactants

Surfactants are molecules with the capacity to decrease the interfacial tension between two immiscible phases. Within the non-ethoxylated surfactants, it is possible to find an emerging group of biomolecules derived from microorganisms; these are called biosurfactants or bioemulsifiers. As a case study, there is a growing tendency in the food and cosmetic products industry to replace surfactants derived from petroleum or chemical synthesis, with “green” alternatives such as biosurfactants. The latter are formed from amino acids, fatty acids, carboxylic acids or sugars with low molecular weight (e.g. glycolipids), or high molecular weight (e.g. proteins, lipoproteins, heteropolysaccharides and lipopolysaccharides). Some of them are proteins with a large amphiphilic structure that have more than one hydrophobic zone. Depending on their molecular weight, biosurfactant have the capacity to emulsify and lead to interfacial tension reduction. [1].

Over the years, the hydrophilic – lipophilic balance (HLB) metric has been employed to characterize their behavior, and technological capacity to form stable emulsions with a determinate lipophilic phase. This approach was originally designed for ethoxylated surfactants, which vary widely from other types of surfactants from the molecular point of view. Polyethoxilated surfactants exhibit a non-conventional interaction pattern with water molecules; for example, there is a significant conformational change associated with the temperature during the emulsification process. For this reason, some researchers have suggested that it is not possible to extrapolate the HLB parameter to cationic and ionic surfactants. For their part, Yamashita and Sakamoto [2], [3] have argued that the HLB is not a good descriptor for non-POE surfactants. In spite of all those findings, researchers and practitioners still use the HLB to describe surfactant emulsification capacity. In this context, the HLB value continues to be a universal tool for comparing surfactants [4], [5]. To address the inadequacy of the HLB metric, over the years, several HLB-like concepts have been proposed [2], [6], [7]. Despite the fact a lot of research has been done to understand monomeric surfactants behavior, the characterization of polymeric biosurfactants, especially proteins, are conspicuously absent.

Unfortunately most of the few proposed models for biosurfactants have been limited to monomeric biosurfactants (e.g. rhamnolipids [8]–[10]). As a result, little is known about how proteins can decrease the interfacial tension between immiscible mixtures [11], [12], and even less is known about how to formulate products using it. Since the relevant surfactants are smaller molecules than proteins, their three-dimensional structure does not have a significant effect; thus, proteins do not factor into the assessment. We note that due to the big structure of a protein, it makes sense that those amino-acids located deep inside the protein molecule well away from the surface, will not interact with water or oil molecules. Instead, the amino-acids located on the protein surface have the most effect on the protein emulsification capacity [13].

In recent years, membrane proteins (such as OmpA, a membrane protein of mainly Gram-negative bacteria) have become an active area of research, not only for their natural role in biological processes but also for their potential impact on industrial applications. Most of the interest centers on their amphiphilic nature; specifically with regard to OmpA, the capacity to stabilize emulsions of dodecane in water[11] has exciting and widespread implications. Those proteins have a large amphiphilic structure and more than one hydrophobic zone.

We have focused on the surface activity of two membrane proteins of Escherichia coli, (OmpA and OmpN) in order to compare their emulsifier capacity within a media composed of water and triglycerides. Using the Kawakami equation [14], the HLB of a surfactant can be determined through the relation between the molecular weight of its hydrophilic groups and its lipophilic groups. Following up this idea, and considering Ala, Cys, Phe, Ile, Leu, Met, and Val, as the hydrophobic residues of a protein, we applied the Kawakami equation to the entire protein sequence which results in an HLB of 11.2 and 11.6 for OmpA and OmpN respectively. Although, experimental results showed that OmpA and OmpN have different behaviors when they are used as emulsifier agents in o/w emulsions made with vegetable oils. To resolve this, we performed molecular dynamic simulation of OmpA and OmpN in a water – triglycerides media. Changes in solvent accessible surface area (SASA) for both proteins were detected, and they were considered an indicator of protein flexibility. Additionally, we found when we calculate the HLB value taking into account only the residues located at the outer layer (7 Å) surface of each protein (during the final stage of the production of the MD simulation), we obtained a HLB value of 9 to 10 for OmpA, while of 6 to 7 for OmpN. We call this new HLB value at the surface approach the “protein surface hydrophilic lipophilic balance (PS – HLB)”. This calculation allows us to consider only the surface residues as responsible of the interaction with water and hydrophobic molecules at the interface. The PS-HLB values are more accurate than the traditional HLB for describing the experimental behavior of those two proteins when emulsifying vegetable oils.

References

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