Examining The Background Of Enzyme Immobilization Biology Essay

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2.1 Enzyme: The Robust Biocatalyst. Enzymes belong to ubiquitous nanoscale biocatalysts in nature, which participate into the metabolic pathways and maintain the stability of intracellular microenvironment in many organisms [1-3](change and add more reference??). Due to the high activity, specificity, and enantioselectivity [4-8], enzymes have been widely pursued for numerous applications including chemicals synthesis [9], pharmaceuticals production [10], biosensors [11] and biofuel cells fabrication [12]. By employing enzymes to traditional chemical processes, reactions can be carried out under mild condition such as ambient temperature and pressure with wastes and by-products minimization [13]. Therefore, using biocatalysts enable the manufacturing process energy-saving and environment-friendly.

2.2 Limitations of Enzyme in Industry Application

From the industry point of view to the catalyst, the large-scale manufacturing process needs not only the active but also stable catalysts to obtain more products [14]. Although enzymes have plenty of advantages over the chemical catalysts, their broad and potential applications are usually restricted by the easy deactivation [15], generally because of their three-dimensional structure change or unfolding during the catalysis [16]. Therefore, the native enzyme should be modified to fulfill the industry requirement.

Till now, plenty of studies have been launched on enzyme stability improvement and some technologies such as enzyme modification (genetic and enzymatic level) [17-20], medium engineering [21-22], and enzyme immobilization [23-24] have already been well developed.

2.3. Enzyme Immobilization: The Effective Solution

Among these enzyme stabilization methods, enzyme immobilization with supporting material has been widely investigated and developed [15, 25-27], by using strategies including adsorption [28], covalent binding [29-30], entrapment [15] and encapsulation [31]. Compared with free enzyme, immobilized enzyme can greatly improve stability with activity retention and be easily recycled from the reaction mixture. Furthermore, enzyme immobilization can also broaden the enzyme application area from aqueous environment to non-conventional condition such as in organic solvents [2, 15, 27, 32]. As a result, enzyme immobilization plays the significant role in enzyme stabilization and has been already introduced to industrial biocatalysis.

Activity retention and stability improvement are two significant aspects to estimate the quality of immobilized enzyme. In order to prepare immobilized enzyme with high quality, three parameters, enzymes, supporting materials and immobilization methods, need to be considered during the design and preparation. The introduction of enzymes used in this work is located in the corresponding chapters. This chapter focuses more on the introduction of enzyme immobilization methods as well as supporting materials.

2.4 Enzyme Immobilization Methods

Immobilization methods act as a bridge connecting supporting materials and biocatalysts and directly determine the quality of immobilized enzyme. Therefore, the development of efficient enzyme immobilization methods continues attracting the interest from scientists and engineers all over the world. Generally, these methods have been divided into two categories (nonspecific and specific) respectively according to the interaction specification between enzyme and supporting material.

2.4.1 Nonspecific Enzyme Immobilization Methods

Most of nonspecific enzyme immobilization methods have been developed from the second half of the 20th century. Some methods have already been used to prepare biocatalysts in industry. Although dazzling immobilization methods exist today, most of them are derived from four basic immobilization methods, categorized into adsorption-based immobilization, covalent enzyme immobilization, enzyme entrapment and encapsulation [33]. Adsorption-Based Enzyme Immobilization

Adsorption-based enzyme immobilization (Figure 2-1) is prepared by physical adsorption between enzymes and supporting materials. In the early 20th century, it was first developed by Nilsson and Griffin to attach invertase on charcoal by adsorption and keep its catalytic activity [34]. However, this method has not attracted the industry attention until in 1960s, amino acid acrylase had the honor to be immobilized on the Diethylaminoethyl (DEAE)-cellulose via physical adsorption [35].

Figure 2-1 Illustration of adsorption-based enzyme immobilization.

Adsorption can be generally classified into several categories via different interactions between enzymes and supporting materials: non-specific adsorption (Enzyme is absorbed by non-specific interaction such as hydrogen bond, hydrophilic interaction, and van der Waals force) [36], hydrophobic interaction (Interaction between hydrophobic domain of enzyme and corresponding materials) [37], electrostatic interaction (Charge-charge interaction) [38] and biological interaction (Interaction between ligands) [39].

Adsorption-based immobilization was continuously studied owing to its several intrinsic advantages. First, the immobilization process is simple and efficient between enzymes and supporting materials [27]. Enzyme immobilization process, therefore, can be easily carried out under mild condition. Second, in contrast with covalent method, enzymes adsorbed on the supporting materials may theoretically keep higher activity because of no chemical coupling between materials and enzyme themselves [26, 40].

Several enzymes have been successfully immobilized on different supporting materials by physical adsorption. Horseradish peroxidase (HRP) has been immobilized on single-walled carbon nanotubes (SWCNTs) to prepare SWCNT-HRP conjugate by physical adsorption and it maintained 98% activity [26], while glucose oxidase was also immobilized on SWCNTs by using a linking molecule that was bound to SWCNTs by van del Waal force [28]. Stempfer, G. and his coworkers immobilized a fusion protein (yeast α-glucosidase) to polyanionic support by polyionic interactions [41].

However, because of the relatively weak interactions, enzyme leaching from the conjugate by several desorption forces is the biggest limitation of its application [27]. For example, because of isoelectric point (pI) of enzyme, pH of the reaction environment can change the charge on the protein surface, which may weaken the electrostatic interaction. Therefore, several derivative/modified adsorption methods have been developed to solve the drawback by combining other methods together. For example, crosslinking has been introduced to post-treat the conjugates in order to prevent the enzyme leaching [42]. Recently, another potential method has been investigated via using the 12-mer polypeptides selected by phage display [43]. The bond energy between peptides and nanomaterial is higher than adsorption but lower than covalent coupling, which might alleviate the enzyme leaching. Covalent Enzyme Immobilization

Since the 1970s, covalent enzyme immobilization became one of the most important methods in enzyme immobilization [44-45]. Compared with adsorption, covalent bond provides much stronger interaction between enzymes and supporting materials [15]. Therefore, the main drawback of adsorption, enzyme leaching from supporting material, could be minimized.

Figure 2-2 Illustration of enzyme immobilization via covalent bond between enzyme and supporting material.

The covalent bonds are formed by chemical reactions between active amino acid residues on the enzyme surface and the functional groups on the supporting materials. In 20 standard amino acids, only nine of them have active sidechains that can be used to covalently immobilize enzyme on the supporting material. These amino acids and their corresponding active groups are listed in Table 2-1. In order to react with R residues on amino acids, supporting materials should be functionalized to have the corresponding active groups. The frequent used active groups on the supporting material are in the Table 2-2.

Table 2-1 Amino acid residues and their active groups

Amino acid

Active group



Lysine (Lys)

(ε)-amino group


Arginine (Arg)

guanidinyl group


Glutamic acid (Glu)

γ-carboxyl group


Aspartic acid (Asp)

β-carboxyl group


Cysteine (Cys)

sulphydryl group


Histidine (His)

imidazolyl group


Methionine (Met)

thioether moiety


Tryptophan (Trp)

indolyl group


Tyrosine (Tyr)

phenolic hydroxyl group

[47, 56]

Some essential requirements of selecting appropriate reactions have been summarized in order to achieve efficient immobilization [33]. First, the reactive groups on both enzyme and supporting material should match the certain reaction. Second, the reactive groups should have less spatial confinement in order to let them easily access to each other. Third, both active amino acid residues and functional groups need to be active prior to the immobilization.

In case of the tight binding, lots of enzymes have been covalently immobilized. Subtilisin Carlsberg (SC), chicken egg white lysozyme and soybean peroxidase (SBP) were covalently attached on SWCNTs and microchannel to generate water-soluble conjugate [25, 57-58]. In another case, the low intermolecular interaction efficiency between epoxy group and soluble enzyme was overcome by using ''tailor-made'' heterofunctional epoxy supports [59]. Pierre, S.J. and his team used carbodiimide crosslinking to immobilize lipase B from Candida antarctica (CAL-B) on solid porous material prepared by polymerization of high internal phase emulsions (polyHIPEs) [60].

Table 2-2 Carrier-bound active groups (CAG) and their structures

CAG name

CAG chemical structure






Carboxylic acid


Carboxylic acid phenyl ester





[37, 70]



However, the main drawback of covalent enzyme immobilization is the low activity retention [27]. From the immobilization reaction, one type of covalent interaction, Schiff base[73] for example, can be nonspecific formed between supporting material and four different amino acids, lysine, histidine, cysteine, and tyrosine on one enzyme [74-75]. These nonspecific interactions could change the enzyme structure so that immobilized enzyme loses its native catalytic configuration and activity. In order to solve the problem, recently, some modified methods have been developed (Section to form specific covalent bond between enzymes and materials and the conjugate keeps better activity [76-77]. Enzyme Entrapment

Enzyme entrapment is an immobilization method that confines the enzyme molecules into the matrix. In the1950s, it was first reported that enzyme can be physically entrapped into the gel matrix with activity retention. However, no special attention was given to this method until in the 1960s when Bernfeld and Wan used acrylamide with the cross-linker N,N-bis-acrylamide to immobilize antigens and seven enzymes in the lattice by polymerization [78].

Figure 2-3 Illustration of enzyme entrapment.

In enzyme entrapment, enzymes are first dispersed in the medium solution, such as polymer precursor, and then the physical barrier with confined enzymes is fabricated by either physical or chemical methods, including cross-linking, polymerization, and physical gelation [15, 79]. No matter what method is used in entrapment, the porous matrix should be permeable to the substrate molecules.

Several porous materials have been used to entrap enzymes, especially mesoporous materials. Mesoporous silica such as SBA-15 and MCM-41 were widely used to immobilize lysozyme with high loading capacity [80-81]. Deere, J., et al immobilized cytochrome c via two different mesoporous materials, MCM-41/28 and MCM-41/45 [82]. Other materials, such as amino-functionalized mesostructured cellular foams (AF-MCFs) with large mesopores, were also introduced to immobilized glucose oxidase as the biosensor [83].

Compared with covalent enzyme immobilization, entrapment may keep three-dimensional structure better owing to no direct reaction between enzymes and supporting materials [33]. Therefore, this method can be used for the enzymes that are deactivated in covalent immobilization. However, the main drawback is that enzyme entrapment can introduce serious mass transfer limitation to the immobilized conjugates [84], which decrease the catalytic performance. Enzyme Encapsulation

Distinguished from enzyme entrapment, enzyme encapsulation is the formation of physical membrane barrier around the enzymes. Enzyme encapsulation can be categorized into four classes: solid shell formed around the liquid droplet interface, phase inversion, template leaching, and post-loading encapsulation [33]. The membrane pore size is important parameter in enzyme encapsulation design. In order to avoid enzyme leaching, the pores on the membrane must be smaller than enzyme. On the other hand, pore size should be much larger than substrate to mitigate the diffusion limitation. Compared with covalent enzyme immobilization, the condition of encapsulation is mild and usually enzymes within capsules are not modified by chemical reagents, the enzyme structure, therefore, could be held. Moreover, several enzymes can be immobilized simultaneously to prepare the multi-enzymes system for sequential reactions.

Figure 2-4 Illustration of enzyme encapsulation.

Several enzymes such as horseradish peroxidase and catalase have been immobilized by using encapsulation. Horseradish peroxidase has been encapsulated in the nanogel by the two step procedures including surface acryloylation and in situ aqueous polymerization. The similar Michaelis-Menten constant between free and immobilized enzyme indicated that the polymer shell insignificantly affected the substrate transport [85]. Controlled polymer multilayer coating was also used to encapsulate catalase, and preserved its excellent initial activity [86].

2.4.2 Specific Enzyme Immobilization Methods

Traditionally adsorption-based enzyme immobilization, covalent enzyme immobilization, enzyme entrapment and encapsulation belong to non-specific enzyme immobilization. For example, in covalent enzyme immobilization, one type of covalent bond can be formed between different amino acid residues and supporting materials. The non-specific interactions sometimes alter the enzyme configuration and differentiate the enzyme orientation on materials so that the enzyme activity decreases. Furthermore, enzymes need to be purified before immobilization, which could increase the cost of products obtained by biocatalysis. Therefore, in order to improve activity retention and cut the costly purification step, specific enzyme immobilization methods have been developed. These methods can be assorted into covalent and non-covalent categories. Specific Covalent Immobilization

Although most of covalent enzyme immobilization methods belong to the non-specific immobilization, the novel covalent strategies, appending covalent adducts to enzyme, have already been developed. These fusion proteins can specifically form the covalent bond between the adducts and supporting materials.

Azido, a chemical inert group, has been exploited to enzyme immobilization by using either azide-alkyne cycloaddition (Figure 2-5a) [77, 87] or Staudinger ligation (Figure 2-5b) [76, 88-89]. Both reactions are carried out between azido on enzymes and alkyne or phosphate on supporting materials. Ronald T. Raines and collaborates appended azido group to the C-terminus of bovine pancreatic ribonuclease (RNase A) and reported the specific interaction between enzyme and its supporting material [90]. Duckworth, B.P., et al. also used the similar strategy via introducing azido group to the C-terminus of protein farnesyltransferase (PFTase) to immobilize enzyme on agarose bead [91].

Figure 2-5 Reaction of specific enzyme immobilization by (a) azide-alkyne cycloaddition [92] and (b) Staudinger ligation [88]:

Besides the above two methods, phosphopantetheinylation can also be used to specifically immobilize enzyme. With phosphopantetheinyl transferase (Sfp), enzyme can specifically react with coenzyme A (CoA) modified supporting material to form the covalent bond (Figure 2-6). Jason Micklefield with his collaborators immobilized luciferase (Luc) and glutathione-S-transferase (GST) ybbR-fusion proteins on CoA-derivatized PEGA resin and retain high level enzyme activity [75].

Figure 2-6 Reaction of specific enzyme immobilization by phosphopantetheinylation site catalyzed by Sfp [75]. Specific Non-covalent Immobilization

Non-covalent specific enzyme immobilization usually utilizes the biological interactions between different receptors and ligands such as antibody-antigen [93-96]. These interactions are specific between enzymes and supporting materials and provide enzymes the favorable orientation on the supporting materials [74]. Furthermore, this immobilization may be reversible, which means the supporting materials can be recycled when enzymes totally lost their activity. Biotin-Avidin System

Biotin is a water-soluble B-complex vitamin, which is vital for cell growth [97], fatty acid synthesis [98], and amino acid catabolism [99]. From its structure, biotin is fused ring structure containing an ureido (tetrahydroimidizalone) ring with a tetrahydrothiophene ring attached by valeric acid. Avidin, a homotetramer protein with MW 69 kDa, is produced from the white of the bird's egg [100]. In Figure 2-7, each avidin subunit can bind one molecule of biotin with a tight and specific non-covalent bond (KD ≈ 10-15) [101].

Figure 2-7 Reaction of biotin and avidin (PDB code: 1RAV) to the biotin-avidin complex (PDB code: 1LDQ).

Biotin-avidin system has several advantages over direct coupling biomarker with antibody. First of all, biotin-avidin conjugate can be rapidly and stably formed owing to low dissociation constant [102]. Second, biotin-avidin can be used in fluorescence application such as fluorescence activated cell sorting (FACS) because of the high fluorochrome/protein ratio [103]. Third, biotin-avidin system can mitigate the background fluorescence noises encountered when using of heavily fluorescein-labeled antibody [104]. Due to these advantages, several research groups have used this method to prepare protein-material conjugate. Marassa, A.M., et al prepared the enzyme-linked immunosorbent assay (ELISA) using this method to detect mosquito 28 blood-fed Culex quinquefasciatus [105]. Horseradish peroxide-labelled avidin (HRP-avidin) coupled with biotinylated anti-E2 antibody have been used to determine estradiol (E2) [106]. Polyhistidine-Co2+/Ni2+ System

The chelation between polyhistidine-tag (6 - His tag) and bivalent ion (Typically Co2+, Ni2+, Cu2+, Fe2+ and Zn2+) is one of the most popular non-covalent specific interactions. The imidazole ring on the histidine sidechain, can be coordinated with metal ions to form the stable conjugate with low KD value [107-108]. Because of easy-to-use, high specificity and affinity, this interaction has been widely industrialized for protein purification in the form of immobilized metal affinity chromatography (IMAC).

Owning to the high affinity and specificity, His-tagged enzyme could be selectively immobilized from cell lysate and may not be affected during the process so that its 3D structure and activity should be maintained. Furthermore, another advantage of using His-tag compared to the covalent binding is that the conjugate could be regenerated when enzymes on the supporting materials totally lost their activity [74, 109-111]. This feature enables to save the supporting material with high cost and complex modification. Therefore, His-tag becomes the potential ligand choice for enzyme immobilization (Figure 2-8).

Figure 2-8 Scheme of using His-tag to immobilize enzyme on functionalized SWCNTs.

His-tag immobilization has been widely used to prepare protein-nanomaterial conjugates for biosensor fabrication by immobilizing green fluorescent protein (GFP) or its derivatives, such as yellow fluorescent protein (YFP) and cyan fluorescent protein (CYP), on either nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) modified nanomaterials. It was reported that NTA-Ni2+ modified Fe/Pt and Co/Fe2O3 core/shell magnetic nanoparticles had high specificity with His-tagged GFP and the conjugates showed outstanding fluorescence [112-113]. Besides fluorescent proteins, NTA modified nanoparticles can also be used as the probe to directly detect certain proteins. Li, Y.C., et al synthesized superparamagnetic Fe3O4 nanoparticle coated with NTA to detect streptopain [114]. Kim, S.H, et al synthesized NTA-Ni2+ modified silica nanoparticles and it showed high specific binding with estrogen receptor α ligand binding domain [115].

Till now, most of the targets using His-tag to immobilize include fluorescent proteins and some polypeptides (Table 2-3). Although many recombinant enzymes with His-tag are purified by affinity chromatography, only a few of them have been immobilized on materials by His-tag immobilization and the characteristics of these conjugates have not been thoroughly investigated yet.

Table 2-3 Various types of His-tag immobilized proteins


Supporting Material



Horseradish peroxidase

Au nanoparticle

Nitrilotriacetic-Co (II)


Green fluorescence protein

Au/FePt/Fe2O3 nanoparticle

Nitrilotriacetic-Ni (II)


Green fluorescence protein

SmCo5.2/Fe2O3 nanoparticle

Nitrilotriacetic-Ni (II)


D-Amino acid oxidase

Magnetic bead

Nitrilotriacetic-Ni (II)


Photsynthetic reaction center


Nitrilotriacetic-Ni (II)


MS2 coat protein

SiO2/Fe3O4 core/shell nanoparticle

Nitrilotriacetic-Co (II)



Fe3O4 nanoparticle

Nitrilotriacetic-Ni (II)


Green fluorescence protein


Nitrilotriacetic-Ni (II)


Green fluorescence protein

Au nanoparticle

Nitrilotriacetic-Ni (II)


Peptide (AB-G5HG2)

CdSe/ZnS core/shell quantum dot

Nitrilotriacetic-Ni (II)


T4 DNA ligase

ϒ-Fe2O3 nanoparticle

Iminodiacetic-Cu (II)


Maltose binding protein

Multivalent chelator head

Tri-Nitrilotriacetic -Ni (II)


Green fluorescence protein

Agarose bead

Nitrilotriacetic-Ni (II)


Human estrogen receptor alpha

SiO2-TMR-NTA nanoparticle

Nitrilotriacetic-Ni (II)


2.5 Supporting Materials

Supporting materials are tightly correlated with the performance of immobilized enzyme because enzyme kinetics and stability are affected by material properties like surface morphology and charge. In this thesis, supporting materials have been divided into two categories, macroscale and nanoscale.

2.5.1 Macroscale Supporting Materials

At the outset, scientists and engineers used macroscale materials, such as glass bead and different membranes, as the supporting materials to immobilize enzymes. These materials are convenient to obtain and easy to functionalize. Uncountable of immobilized enzymes have been made, which accelerated the process of biocatalyst application. Transparent porous silicate glass helped to encapsulate copper-zinc superoxide dismutase (CuZnSOD) and the conjugate has the similar properties as the free enzyme [121]. β-glucosidase was also immobilized on calcium alginate-silicate bead and demonstrated excellent operational stability of the sol-gel immobilization system [122]. However, due to relative low surface/volume ratio and mobility in reaction mixture, enzyme immobilization on macroscale material usually causes low loading capacity and enzyme functions have been confined.

2.5.2 Nanoscale Supporting Materials

Bionanotechnology involves the combination of the physics, chemistry and biology together in the research and application. With the development of physics and chemistry, new nanomaterials were synthesized, characterized and functionalized, which could be applied in fabrication of different kinds of devices in electronics [123-124] and biomedical diagnosis [125-126]. Because nanoscale materials have significantly different properties from the macroscales due to their dimensions, biomolecules including nucleic acids, peptides, and proteins may tailor their structures and functions, and even gain the novel characteristics when shared with nanotechnology [127-129]. Novel hybrids, therefore, can also be generated combining nanotechnology and biotechnology with new physicochemical properties and better biocompatibility to solve the problems in industry.

Nanomaterials (carbon nanotubes [130-132], nanoparticles [11, 108, 115], fullerenes [133], nanofibers [16, 134-135], and mesoporous/nanoporous materials [3, 136-137]) used as solid support could provide the platform for enzymes to show and improve their functions [27]. The advantage of using nanoscale materials as support is that nanomaterials could provide upper limits on enzyme-efficiency-determining factors such as high surface area/volume ratios and enzyme loading capacity [1-2, 23, 138]. Moreover, nanomaterials expand the field of enzyme application from bioengineering to chemical and even electronic engineering, and let the enzyme function in some special conditions. Various types of enzymes have been used to make enzyme-nanomaterial conjugate. α-Chymotrypsin (α-CT) [139], alkaline phosphatase (AP) [140-141], glucose oxidase (GOx) [142-144] and horseradish peroxidase (HRP) [94], for instance, have been immobilized on different nanoparticles as the biocatalyst or biosensor. With the progress of bionanotechnology, more and more enzyme-nanomaterial conjugates have been made, which are applied in biosensor [28, 145], drug delivery [146-147], cell therapeutics [125, 148], and biocatalysis [2, 149].

2.6 Scope of This Thesis

My thesis will focus on obtaining immobilized oxidoreductases with high activity retention and stability improvement. As discussed in section 2.3, besides enzyme itself, two different factors can possibly lead to this goal: (1) selection of suitable enzyme immobilization methods and (2) choice of nanomaterials as the supporting materials. This thesis fulfills two requirements based on the following considerations: (1) The immobilization method should be efficient and specific to have high loading capacity and avoid unpredictable enzyme configuration changes. Furthermore, the specific immobilization could also avoid the costly enzyme purification step. (2) The series of nanomaterials used in the study should be easily functionalized and separated. Moreover, nanomaterials should have certain similarities as well as differences, which help to investigate the relationship between the performances of immobilized enzymes and properties of different supporting materials. (3) The immobilization system should be verified by different enzymes to demonstrate the robustness of the method. In addition, this thesis also includes the application of these novel nanoscale biocatalysts via building cofactor regeneration system to improve pharmaceutical intermediate productivity.