Lignocellulosic biomass resources are abundant and environmentally friendly on earth. The liquid fuel produced by lignocellulose especially ethanol is an alternative energy with great development potential [1]. In recent years, lignocellulose has become a hot spot of modern biomass research. However, it is well known that enzymatic hydrolysis of lignocellulose is easily restricted due to the complex structure of lignocellulose. It is currently believed that the main reason affecting the hydrolysis of lignocellulose is the non-productive adsorption of the substrate lignin and cellulase, thereby reducing the efficiency of cellulase [2, 3].
Many studies have found that adding some additives (or synergic agents) into the lignocellulose enzymatic hydrolysis system can enhance the enzymatic hydrolysis effect by reducing the non-productive adsorption of lignin and cellulase. Lignosulfonate (LS), polyethylene glycol (PEG) and Bovine Serum Albumin (BSA) are three synergic agents under extensive studies for enzymatic hydrolysis of lignocellulose [4–8]. The chemical structures of three synergists are quite different; therefore, the promoting modes of three synergists should be different. However, it has not been fully understood how they promote the enzymatic hydrolysis efficiency. In literature, there are some controversies in the explanation of their promoting effects.
LS is a by-product of sulfite pulping and also a by-product of SBOL pretreatment[9]. Zhou et al. [10] suggested that LS can act as a surfactant to prevent the non-productive binding of substrate lignin to cellulase, thereby promoting saccharification. Wang et al. [9] proposed that LS can bind to cellulase and the formed complex increases the amount of negative charges carried by cellulase, thereby increasing the electrostatic repulsion between cellulase and residual lignin in substrate, as a consequence reducing cellulase non-productive adsorption of substrate lignin. By adding LS to the enzymatic hydrolysate of different pretreatment substrates, Wang et al. [11] found that the pretreatment method will affect the effect of LS. However, these studies were carried out in a complicated hydrolysis system, where both residual lignin and soluble lignin coexisted. As such, the action mechanism between cellulase and lignin cannot be clearly identified which part of lignin involved. In order to understand the action mode of LS, researchers studied the mechanism of LS in the enzymatic hydrolysis of lignocellulose from the perspective of substrate/substrate lignin- enzyme adsorption [12, 13]. However, at present, when studying the mechanism of LS in lignocellulose hydrolysis system, the influence of substrate lignin cannot be ruled out.
Adding PEG to the lignocellulose hydrolysate can reduce the amount of enzymes used and increases the conversion rate of cellulose [6, 8, 14]. It is currently believed that PEG as a nonionic surfactant can promote enzymatic hydrolysis of cellulose. The main role of surfactants in substrate containing lignin is that lignin with adsorbed surfactants can inhibit the interaction between enzymes and lignin, which makes the efficiency of lignin-binding enzymes low [15]. However, it was found in the research that the efficiency of PEG is not always positively correlated with the lignin content[16]. Therefore, some researchers believe that the reason why PEG can enhance the efficiency of enzymatic hydrolysis is due to that the addition of PEG can increase the activity of cellulase [17, 18].
BSA is widely used in biochemical studies, and plays an active role in the enzymatic hydrolysis of cellulose [19, 20]. BSA can be used as a synergist for lignocellulose hydrolysis by reducing the non-specific adsorption of lignin to cellulase [5, 19]. Some researchers believe that in addition to preventing the non-productive adsorption of cellulase by lignin, BSA can also reduce the inactivation of exoglycanase, thereby improving the hydrolysis of microcrystalline cellulose [21]. Jia et al. [22] compared the effects of adding BSA on the lignocellulose conversion rate of acid-pretreated poplar wood, ethanol-washing acid-pretreated poplar wood, and delignified acid-pretreated poplar wood and they found that BSA acts as a synergistic agent by coating the substrate lignin, thereby blocking the binding site of the substrate lignin.
From the literature study, it can be found that whether to form a complex between synergistic agent and cellulase is very critical in the discussion how they promote the enzymatic hydrolysis. Consequently, how synergistic agent interact with cellulase is very important to understand their role-played in enzymatic hydrolysis. Whereas in the literature, the system was too complicated, e.g., in the LS promotion cases, both residual and soluble lignin were coexisted. Therefore, it is difficult to distinguish the exact roles residual and soluble lignin played. In order to make better use of synergistic agents, it is necessary to study the mechanism of synergistic agents in the hydrolysis process. As for our best understanding, no relevant studies have investigated interactions between cellulase and synergistic agents alone. This may be attributed to both cellulase and synergistic agents are soluble in the enzymatic hydrolysis conditions.
QCM-D and SPR are two commonly used noninvasive techniques to study interface effects in recent years [23–27]. QCM is made according to the principle of piezoelectric effect of quartz crystal. When the quality on the electrode chip changes, the electrical signal Δf output by the quartz crystal system will change accordingly. The quality change of the chip can be calculated based on the data of Δf obtained. The accuracy can reach nanogram level [28]. Sauerbrey equation [29] shows that the frequency of crystal oscillation is inversely proportional to the mass of sensor coupling. However, this formula only applies to thin, uniform, rigid films, otherwise we think the quantity of chips will be underestimated [30, 31]. Recently, some literatures have reported that QCM-D can be used to monitor the formation of thin films in real time and explore the properties of each film layer-by-layer adsorption of substances on the chip [32–34]. In this case, the viscoelastic Voigt model can be established to estimate the adsorption capacity of each layer more accurately [35]. The viscous modulus, elastic modulus, and thickness of each film can be obtained by this model. SPR is a method to study intermolecular interactions by using optical properties. We usually study the information change of analyte by the Angle change of SPR obtained. Small Angle X-ray scattering (SAXS) can be used to analyze the size and shape of molecules and obtain the aggregation information of proteins in solution [36].
In this investigation, we attempted to simply the system to study the interaction between the synergistic agents and cellulase alone, ignoring the influence of morphology and chemistry (e.g., residual lignin content, chemical difference, and distribution), using QCM-D and SPR techniques to monitor the interaction between cellulase that was immobilized onto the sensors of QCM-D and SPR and synergistic agents. In our previous work, we probed the cellulase biosensor formation in situ and in real time on gold chips [31]. Based on the cellulase biosensor we monitored the interactions between cellulase and BSA/LS/PEG in situ and in real time. In addition, SAXS was used to study whether cellulase and additives form a complex and to evaluate the size of the complex. At the same time, the effect of adding these additives to the enzymatic hydrolysis system of pure cellulose (Avicel) and lignocellulose (green liquor-pretreated lignocellulose (GL) on the glucose conversion rate was investigated thoroughly.