## 2 Mechanism of Action
### Impact of Amphiphiles on Enzyme Structure  
The presence of an interaction between the micelles or monomers of amphiphiles (surfactants and protein) and enzyme in a solution is undeniable. The primarily role of hydrophobic forces in the interaction of amphiphilic compounds (other than lipids) with \"native\" proteins from a variety of sources has been well substantiated [69]. As a result of the isothermal titration micro-calorimetry analysis of interaction between SDS and Casein, [PERSON] _et al._[70] found that when NaCl was applied to the complex, the enthalpy change corresponding to SDS binding to the casein chains was not significantly different than that in the absence of NaCl, especially at low concentrations of SDS. This observation confirmed that the interaction of molecules of surfactants and the protein (e.g., enzyme) are mainly through the hydrophobic forces rather than electrostatic (Figure 3). It was also reported that the amino acids of tyrosine (Tyr), tryptophan (Trp), glutamic acid (Glu), and aspartic acid (Asp), which are exposed to the surface of cellulase, play an important role in cellulase binding to substrate and biodegradation of substrate [72]. Therefore [PERSON] _et al._[73] hypothesized that the interaction of cellulase and PEG in solution is primarily originating from hydrophobic interactions that are formed between the aromatic rings of the so-called amino acids and the -CH\({}_{2}\)-CH\({}_{2}\) group of the PEG 4000 in solution. Regardless of whether surfactants are present as monomers at the surface of biomass [54] or as micelles in solution (at above CMC level), the enzyme structure is anticipated to be modified as a result of the contact with surfactants. Studies showed that it is the conformational changes of protein in the presence of poly-electrolitic micelles as well as monomers (obtained by disintegration with 0.7 M salts) that affected the enzyme activity (e.g., lipase) [55]. In agreement with these results, it was found that the reason behind the reduction in xylanase activity during incubation with Standapol-A surfactants was due to denaturation of enzyme that was observed as reduction in ellipticity obtained from Circular dichroism analysis [22]. Conformational changes in enzyme due to interaction with surfactants seem to occur at different sub-structures of enzymes and to different extents. This observation should originate from a variety of factors that relating to surfactant properties (CMC, charge type and districusion, hydrophilic lipophilic balance (HLB) number, _etc._), as confirmed by [PERSON] _et al._[30]. It was reported that the rheological properties of the solution is modified based on the HLB of the surfactants, which itself modifies the secondary structure of the proteins present in the solution and significantly affects the enzyme activity [73]. According to [PERSON] theory, the viscosity of the solvent is responsible for a friction against protein in solution and results in decreased motion and inhibitory impact on catalysis in motile enzyme. [PERSON] _et al._[74] found a direct correlation between viscosity (\(\eta\)) and the inhibition of the maximum rate of enzyme catalysis (\(V_{\mathrm{max}}\)). It was reported that while increasing the concentration of Brij 35 and SDS induced \(\alpha\)-helix formation in a solution of lipoxygenase (LOX\({}_{1}\)), Tween 20 increased the \(\beta\)-structure of the enzyme [19]. The extent and nature of the modifications imposed seemed to not be solely dependent to the charges of the surfactants, as two surfactants of Sodium deoxycholate (DOC) and SDS, with negative charges exhibited different effects on enzyme structure. Specifically, SDS increased both  
Figure 3: Schematic of interaction between protein molecules (e.g., enzyme) and non-ionic surfactants through hydrophobic interaction (Adopted from [PERSON], and [PERSON], [71]). \(\alpha\)-helix and aperiodic structure at higher concentrations, whereas DOC increased \(\alpha\)-helix and decreased aperiodic structure of LOX1 [19].