Function Of Surfactants In Immobilization of Cellulase And Multiphase Hydrolysis: A Review

Surfactants, especially non-ionic surfactants, play an important role in the preparation of nanocarriers and can also promote the enzymatic hydrolysis of lignocellulose. A broad overview of the current status of surfactants on the immobilization of cellulase is provided in this review. In addition, the restricting factors in cellulase immobilization in the complex multiphase hydrolysis system are discussed, including the carrier structure characteristics, solid-solid contact obstacles, external diffusion resistance, limited recycling frequency, and invalid combination of enzyme active centers. Furthermore, promising prospects of cellulase-oriented immobilization are proposed, including the hydrophilic-hydrophobic interaction of surfactants and cellulase in the oil-water reaction system, the reversed micelle system of surfactants, and the possible oriented immobilization mechanism.


Introduction
Bioethanol, as a renewable, economically affordable, and environmentally safe energy material, will gradually become a substitute for fossil fuels. It has far-reaching research signi cance and application value for the development of a sustainable energy strategy (Adewuyi 2020;Thatoi et al. 2016;Zhao et al. 2017). Due to competition with food supply in the rst generation of bioethanol production, lignocellulose, a non-starch material, has become an important raw material for bioethanol production importantly, the non-speci c binding of free cellulase on lignocellulosic substrates may account for the low rate of hydrolysis at the action mechanism level during enzymatic hydrolysis. Some enzymes remain free after the enzymatic hydrolysis of lignocellulosic substrates, whilenon-speci c binding to the residual substrates also prevents the e cient recycling of cellulase (Kellock et al. 2017; Kuhad et al. 2011; Rahikainen et al. 2011). Moreover, the utility of cellulases has been limited due to their low operational stability, high costs, and poor reutilization when used in the native form ). To overcome these barriers, immobilization is usually used to improve enzyme stability and even activity or selectivity when properly designed, which can also facilitate the reuse of enzymes and effective cost of catalytic processes (Li et  . However, enzymes often display drastically lower activity in organic solvents than in water, and the water layer on the molecular surface of enzymes determines their activity in organic media (Zhang et al. 2012). Therefore, among several approaches to resolve the challenges, one of the most effective methods is immobilization of the enzymes within an aqueous microenvironment in the organic solvents. Microemulsions formed by amphiphilic surfactants have been widely reviewed as effective media for the immobilization of enzymes in hydrophobic solvents (Itabaiana et al. 2014;Pavlidis et al. 2010;Uskokovi and Drofenik 2007). The MBGS method based on microemulsions has been used to form matrices for enzyme immobilization to achieve enzymatic catalysis in nonconventional medium as they appear to be rigid and stable for a long time, even within the reaction solution (Zhang et al. 2012). Therefore, the MBGS method has unique advantages of improving the chemical stability of immobilized enzymes and maintaining high catalytic activity (Itabaiana et

Effects Of Nanocarriers On Immobilization Of Cellulase
The structure and properties of carrier materials have great in uence on the properties of immobilized cellulase, such as internal geometry (e.g., at surfaces or thin bers), speci c surface area, super cial activation degree, mechanical resistance, and pore diameter (Begum et  ; 4) the mechanical properties of the carriers need to be controlled considering the nal con guration of the reactor. If the reactor is a xed-bed reactor, it should possess very high rigidity to withstand high pressures without pressure problems, but the situation is different if a stirred-tank reactor is used (Cristina et al. 2011;Santos et al. 2015); 5) after the cellulase penetrates the carriers, the internal morphology of carriers will determine the possibility of obtaining a very intense or very limited enzymecarrier interaction (Santos et al. 2015). When the diameter of the carriers is smaller than that of the enzyme, it is di cult to obtain an intense enzyme-carrier interaction (Cristina et al. 2011), but if the carriers have su ciently large internal surfaces, it is possible to get an intense interaction with a similar at surface (e.g., agarose beads, porous glass, or silicates) (Malar et al. 2018).
In particular, the special superparamagnetism of magnetic nanocarriers has attracted increasing interest as they allow easy recycling and separation of catalysts and biomolecules from high-viscosity liqueurs and high-solid-content broths. This unique characteristic has been well-applied to immobilization of cellulase, and a better hydrolysis e ciency and recycling feasibility have been observed ( Fig. 3. In this process, the CAGs directly participate in binding with enzyme molecules, but the carrier-bound inert groups are not directly involved. This interaction inevitably disturbs the maintenance of the natural conformation of the enzyme, leading to structural and functional changes in the enzyme molecules. No obvious stability change has been observed when the newly formed conformation is similar to that of the natural enzyme. The covalent binding between carriers and active sites of the enzyme not only causes pore plugging of the surface, but also leads to the drag increment of in-diffusion. Although an initial high dosage of cellulase is added, the inhomogeneous distribution of the carrier surface structure results in the uncontrollable immobilization site, and ineffective immobilization may lead to a signi cant loss of enzymatic activity and reduce the accessibility of the substrate to the functional site. Moreover, the partition and mass transport limitations of nanocarriers may cause spatial variation in local reaction rates and further affect enzymatic hydrolysis (Du et al. 2017). The chitosan molecules are mostly used because of the large number of -OH and amino groups (-NH 3

The Oriented Immobilization Of Cellulase In The Srm System
Construction of the SRM system The SRM system has been widely used in the preparation of immobilized enzymes (Dong et al. 2010;Marhuendaegea et al. 2015). The special structure of surfactant molecules caused a water-oil amphipathy with a hydrophobic nonpolar hydrocarbon chain (alkyl) and a hydrophilic polar group (such as -OH, -COOH, -NH 2 , and -SO 3 H) distributed at different ends. In the water-oil (W/O) system, the surfactants are dissolved in the nonpolar organic solvent when a trace of water is provided, and the reversed micelles are formed when the concentration exceeds the CMC (Takagi et al. 2019;Xiaodong et al. 2018). In reversed micelles, the nonpolar groups of the surfactants are exposed to the nonpolar organic solvents, while the polar groups are arranged inside. Therefore, a polar core with the ability to dissolve polar substances in the microreactors is formed. The SRMs are nanoscale aggregates that are formed spontaneously, and the W/O microemulsion with low water content provides a stable thermodynamic system (Tao et al. 2013). According to the hydrophilic-hydrophobic interaction of surfactants and cellulase in the oil-water reaction system, the large number of oil-water interfaces in the system provides a good environment for the catalytic reaction of enzyme molecules (Brady and Jordaan 2009 Mechanism of oriented-immobilized cellulase in the SRM system Multipoint covalent attachment is likely the most effective strategy for immobilization, but it is di cult to allow the immobilization of enzymes at a well-de ned position since the proteins are usually attached to the solid surface by uncontrolled chemical bonds Hernandez and Fernandez-Lafuente 2011;Li et al. 2016). The uncontrolled conformational changes were caused by random immobilization, which may lead to a signi cant loss of enzyme activity, and the disordered enzyme orientation may also reduce the accessibility of the substrate to functional sites (Orellana et al. 2018;Steen Redeker et al. 2013;Yu et al. 2012). However, the hydrophilic cellulase will be dissolved in the SRM system due to the existence of surfactants, which can maintain the activity of the enzyme and prevent the toxic effects of organic solvents (Tao et al. 2013). The active centers of cellulase molecules are usually cracks, which provide a different microenvironment  because the structures of cellulase active centers are mainly composed of eight kinds of amino acids (tryptophan, tyrosine, histidine, phenylalanine, aspartic acid, glutamic acid, and arginine), most of which are hydrophobic . Hydrophobic active centers are conducive to the combination of catalyzed groups of cellulase and substrates. When the speci c substrate is close to the active centers, a change in the conformation of the cellulase molecule can be induced so that the reaction groups of the enzyme active centers and substrate are aligned correctly. Meanwhile, the molecular orbitals between the reaction groups of the active centers are strictly located in the right direction for easier enzymatic reactions. Therefore, cellulase is distributed in the W/O interface, and the catalytic active center is toward the organic solvent and the other side toward the "pool". Moreover, the addition of surfactants can enhance the aggregation effects of cellulase on the W/O interface, and the existence of a crosslinking agent promotes the covalent crosslinking of enzyme molecules (Hyemin et al. 2012). The catalytic activity centers of the cross-linked microspheres are distributed uniformly and toward the outside, which solves the challenge of the uncontrollable attachment sites of the cellulase molecules in the immobilization process Steen Redeker et al. 2013;Yu et al. 2012). In the SRM system, the hydrophobic active molecules are exposed to the outside, which is bene cial for the further combination of immobilized cellulase and lignocellulosic substrates. However, the immobilized sites of cellulase molecules remain stochastic and heterogeneous, which may lead to covalent binding between the carriers and the active center of the enzyme, which can cause ineffective immobilization and enzymatic reactions . Therefore, to achieve oriented immobilization and improve the recycling times of cellulase, C-MNPs can be used as carriers as shown in Fig. 4. This method can effectively prevent the ineffectiveness of cellulase immobilization.In this process, glutaraldehyde is used as the crosslinking agent, and EDC and N-hydroxysuccinimide are the coupling agents (Fig. 5). In the W/O system, the free carboxyl group (-COOH) in the adsorption zone of the cellulase molecules can realize covalent binding with a large number of amino terminal catalytic residues of chitosan molecules ). The process cannot destroy the catalytic center of cellulase, and the exposed active sites increase the effective attachment of immobilized cellulase to solid substrates, which further promotes enzymatic hydrolysis. Therefore, the oriented immobilization of enzymes was obtained in the SRM system, which can prevent invalid combinations effectively and further promote enzymatic hydrolysis.

Conclusion
Cellulase plays an important role in the production of fuel ethanol by the enzymatic hydrolysis of lignocellulose, and the immobilization of cellulase on the nanocarriers is an effective way to improve hydrolysis e ciency. However, the nanocarrier structure characteristics, solid-solid contact obstacles, external diffusion resistance, limited recycling frequency of nanocarriers, and invalid combination of enzyme active centers restricted the further improvement of hydrolysis e ciency in the complex multiphase system. Surfactants can promote the enzymatic hydrolysis of lignocellulose and play an important role in the preparation of nanocarriers. The special SRM system caused by the amphiphilicity in the oil-water reaction system can provide effective protection to obtain the immobilization of singlelayer cellulase, which can effectively prevent the immobilization of cellulase and increase the effective attachment of immobilized cellulase and solid substrates, which further promotes enzymatic hydrolysis.

Declarations Acknowledgments
The authors acknowledge that this work was supported by the National Natural Science Foundation of China (22078194) and National Key Research and Development Program (2017YFE0127100).

Funding
Funding provided by the National Natural Science Foundation of China (22078194) and National Key Research and Development Program (2017YFE0127100).

Con icts of interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper      The oriented immobilization diagrammatic sketch of single-layer cellulase in the surfactant reversed micelles system Figure 5 The oriented immobilization of cellulase on magnetic nanoparticles