Multi-Enzymatic Cascade Reactions via Enzyme Complex by

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Multi-Enzymatic Cascade Reactions via Enzyme Complex by Immobilization Ee Taek Hwang, and Seonbyul Lee ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b04921 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019

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Multi-Enzymatic Cascade Reactions via Enzyme Complex by Immobilization Ee Taek Hwang1*, Seonbyul Lee1

1Center

for Convergence Bioceramic Materials, Korea Institute of Ceramic Engineering &

Technology, Cheongju-si, Chungcheongbuk-do, 28160, Republic of Korea

*Corresponding author footnote Phone: + 82 43 913-1514 Fax: + 82 43 913-1597 E-mail: [email protected] 1 ACS Paragon Plus Environment

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ABSTRACT Multi-enzymatic cascade reactions are most important technology to succeed in industrial process development, such as synthesis of pharmaceutical, cosmetic, and nutritional compounds. Different strategies to construct multi-enzyme structures are widely reported. Enzymes complexes are designed by three types of routes i) fusion proteins, ii) enzyme scaffolds, or iii) immobilization. As a result, enzyme complexes can enhance cascade enzymatic activity through substrate channeling. In particular, recent advances in material science lead to various materials synthesis applicable for enzyme immobilization. This review discusses different cases for assembling multi-enzyme complexes via random coimmobilization, compartmentalization, and positional co-immobilization. The advantages of using immobilized multi-enzymes include not only improved cascade enzymatic activity via substrate channeling, but also enhanced enzyme stability and ease of recovery for reuse. In this review, we also consider the latest studies of different model enzyme reactions immobilized on various support materials, because multi-enzyme systems allow for economical product synthesis through bioprocesses.

KEYWORDS Multi-enzymatic

cascade

reactions;

Multi-enzyme

immobilization;

Random

co-

immobilization; Compartmentalization; Positional co-immobilization 2 ACS Paragon Plus Environment

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1. INTRODUCTION Biocatalysts, referring to whole cells or enzymes, are catalysts in nature. They reduce the activation energy of a reaction and dramatically increase the chemical reactions rate taking place in living organisms1. Various chemical reactions take place inside a cell to support cellular growth and survival. Enzymes have evolved to catalyze diverse chemical reactions which take place in the cells to survive in the metabolic pathways found in biological systems, creating high selectivity and specificity2. In nature, many enzyme cascade reactions can be observed in large number of metabolic pathways inside the cell, and employed to ensure the integrity of enzyme-catalyzed synthetic pathways to mimic chemical processes3. A number of enzymes carry out together in multi-reaction steps or cascade reactions, inducing two or more sequential processes without the isolation of intermediates4. The oldest examples of multienzymatic reaction was fermentation by using native microorganisms for fermentation, such as for beer brewing or vinegar production5. Compared to an in vivo fermentation process, the in vitro enzymatic synthesis hold clear benefits. The flexibility of engineering in vitro processes can overcome challenges such as cell viability, complexity, physiology, and the membranes-cell walls6. However, the classical approach used for multi-enzymatic reactions in vitro is carried out in separate stages. Such synthesis has several disadvantages, such as low yields, high operation costs, and the use of many chemicals in the separation steps. To resolve these issues, a new strategy is needed that can provide advantages such as enantioselectivity, stereoselectivity, high yield, low downstream expenses, shifting of reaction equilibria, and achieving multiple steps without product recovery. Recently, these multi-enzyme reactions are rapid growth for scientific and industrial applications, particularly biotransformation, biosensors, and biomedical engineering7, 8. Additionally, multi-enzymatic processes are regarded as an alternative pathway for the production of many pharmaceuticals, biofuel, and fine chemicals. In other words, multi3 ACS Paragon Plus Environment

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enzymes can mediate complicated chemical reactions in one-pot systems. The synthesis of chiral alcohols9, carbohydrates10, 11, and polymers12 and cellulose hydrolysis by the synergetic action of endoglucanase (EG), cellobiohydrolase, and β-glucosidase (BG)13 are typical cases of multi-enzyme reactions in one-pot. The use of multiple enzymes in one-pot has countless benefits for process design, such as fewer unit operations, shorter cycle times, smaller reactor volumes, improved space–time yields, and less waste generation14. Of course, the immobilized multiple enzyme systems cannot guarantee enhanced overall activity15. Based on arguments by the Hess group, although immobilization induces multi-enzymes to be put into proximity of

one another, the activity is seen to increase in many studies of GOD/HRP

cascade due to the microenvironment effects on the enzyme and the resulting cascade kinetics16,

17.

Additionally, decreased activity can also be observed during the co-

immobilization process, attributed to enzyme autolysis and its structural changes. Despite of substrate channeling by co-immobilization it prevents intermediates from escaping during the cascade reactions during the preparation of an industrial biocatalyst. Furthermore, by coupling several reaction together, unfavorable equilibria can be controlled and designed to achieve the formation of the desired reaction pathway18. Multi-enzyme cascade reactions recently gained significant attention due not only to their elegance, but also allowing for more complex systems to be used19. Several enzymes can work together efficiently in cascade processes. In these enzymatic pathways, each intermediate is isolated from the reaction media and provide a substrate for the next transformation. In these consecutive reactions, each enzyme is assembled into macromolecular structure that regulate reactivity by compartments formation or spatial organization2,

20.

These specific structure have a major advantage that is referred to as

metabolic channeling21. The active sites of the enzymes are close together, overcoming the diffusion limitations in the bulk phase of the cell by transferring intermediates from one 4 ACS Paragon Plus Environment

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active site to another22, maintaining high local concentrations crucial for unstable metabolite compounds. Consequently, reactions is efficiently optimized without using toxic substrate or reaction intermediates22, 23, creating facilitated metabolic pathways by regulating the activities of competing pathways. The pyruvate dehydrogenase (PDH) complex, tryptophan synthase channel, fatty acid oxidation pathway, and the citric acid cycle are examples of metabolic channeling, which use metabolic channels that mimic natural enzyme complexes in an artificial way7, 23. Substrate channeling, an example of metabolic channeling, is another advantage arising from enzyme complexes, that is, direct transfer of the product as a substrate from an enzyme to another nearby enzyme with the bulk phase without equilibration, as shown in Figure 124, 25. Such phenomena take place only when the distance between two active sites is close. In addition, overall reaction rates can be lead to more fast and efficient way26, 27. Different active sites can be closer spatially and then assembled as a complex to provide efficient reaction control in a multifunctional enzyme, multi-enzyme complex, or with separate enzymes23. Furthermore, through substrate channeling, other potential advantages such as protection of unstable intermediates and/or stabilization of unstable cofactors24, 28, prevention of substrate competition between different pathways29, 30, mitigation of toxic metabolite inhibition31, and circumvention and regulation of unfavorable equilibria32,

33

can be complemented by the

avoidance of the unfavorable energetics associated with desolvating the substrates34. Furthermore, development of cascade systems is considered to be an important future direction for sustainable synthetic biology arising from productivity and reduced operation costs, and environmental impact35, 36. Although the high cost in applications induce hurdle, various attempts have been suggested and performed by optimizing the overall reaction steps37. Basically, free enzymes are sensitive and unstable, and difficult to recover and reuse efficiently. Enzyme immobilization allows researchers to overcome these limitations by 5 ACS Paragon Plus Environment

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improving enzyme reactivity under optimal conditions (e.g., pH, organic solvents, and temperatures)38,

39

and facilitating the modification of substrate specificity (discrimination

between substrates), enantioselectivity (enzyme performance), and reactivity40,

41.

In other

words, the performance of enzymes in organic media and their pH tolerance, thermostability, and functional stability can be enhanced to facilitate important commercial applications. Immobilized enzymes have the following advantages: (i) Improved thermal and operational stability than free enzymes, (ii) improved convenience of separating reaction products from mixtures containing solvent, (iii) reduced costs owing to the ease of recycling the enzyme by removal from the reaction mixture42, 43. Therefore, immobilized multi-enzyme are gradually driven by economic and environmental considerations. For example, in many studies, coimmobilization of enzymes on a porous support is shown to have a positive impact on the observed activity that is not relevant with structural changes caused by changing enzymatic kinetics44. The kinetically controlled cascade reaction depends on consumption of the substrate and accumulation of the product the pores of the carrier by eliminating lag time45. Thus, this substrate channeling effect in conformity with the particle pores is to overcome these diffusional limitations46. Obviously, all these processes can be modulated by the enzyme structure after immobilization by allowing co-immobilized enzymes to be proximate. However, since this approach also has some challenges, the Lopez-Gallego group has recently pointed out another possibility in which the enzymes should be used under the same conditions as the co-immobilized enzymes and cofactors. They developed a self-sufficient heterogeneous multi-enzyme system by modifying cofactor and enzyme with polymer, covalently or irreversibly immobilized cofactor, and enzyme to carrier to enable efficient process47. The stabilized enzyme and self-recycled cofactor is subjected to continuous operation for obtaining optically pure amine and chiral alcohol synthesis48,

49.

In general,

enzyme complexes can be designed for simple fusion enzymes, enzyme-enzyme complexes 6 ACS Paragon Plus Environment

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linked to scaffolds, and simple co-immobilization. Multi-enzyme immobilization is an effective method for mimicking cascade enzymatic pathways, resulting in numerous advantages, such as, less operations with smaller reactor volumes, higher volumetric flow rate, and shorter reaction cycle with less waste generation50,

51.

Coupling several steps

together can lead to desired product by modifying unfavorable equilibrium52. As a result, immobilized multi-enzymes on certain supports enhanced the storage and working stability while achieving efficient enzyme cascade reactions. Additionally, simple separation and easy recycling use, while maintaining activity and selectivity, are expected to make cascade enzymatic pathway-based industrial processes possible53, 54. Latest studies on multi-enzyme immobilization methods that have been applied on various support materials such as polymer- and phospholipid-based materials, carbon- and metal-based materials, inorganic particles, glass tubes, and biomaterials have proven to be highly important in introducing relevant advantages such as enzyme recycling and substrate channeling, thereby addressing a number of several difficult in material synthesis preparation. Despite several reviews, no manuscript has been identified that focuses on how the materials have been designed, and which immobilization strategies have been applied and discussed. To understand the immobilized multi-enzyme, because the general concept of enzyme stabilization via immobilization cannot be altered. Therefore, immobilizing materials (carriers) need to be classified appropriately. In this review, we focused on multi-enzyme immobilization for enzyme complexes. First, we will introduce the multi-enzyme reaction model and the strategies to study multi-enzyme reaction complexes, such as the use of fusion proteins, protein scaffolds, and coimmobilization. Second, we will discuss detailed methods for multi-enzyme immobilization

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and its role in creating enzyme complexes that produce efficient and stable results, especially for various new functional materials.

2. CLASSIFICATION OF MULTI-ENZYME CASCADE REACTIONS Multi-enzyme cascade reactions can be classified based on the types of intermediates that arise before the final product is formed8. Two or more reactions occur simultaneously in the same reactor, employing several enzymes. Biocatalytic cascade reactions can be categorized four different designs: (i) linear cascades, (ii) orthogonal cascades, (iii) parallel cascades, and (iv) cyclic cascades. In linear cascades, a single substrate is transformed into a single product through one or more intermediates in the process, as shown Figure 2a. Linear cascade processes not only help to save time and reduce waste during the synthesis, but also the unstable or toxic intermediates are transformed into final products without accumulation, leading to better yield and constant shifting of the equilibrium toward products. A typical example of a linear cascade is the deracemization process. Deracemization and stereoinversion of alcohols via stereoinversion by alcohol dehydrogenase is a recent example of this55, 56. An orthogonal cascade is the reaction by the substrate into product, coupled with additional steps, such as cofactor or cosubstrate regeneration or by-product removal (Figure 2b). Cofactor regeneration for NAD-dependent oxidoreductases with another redox enzyme is a classic case of an orthogonal enzymatic cascade reaction. As one example, the Soda group reported using d-amino acid transaminase with three additional enzymes (alanine racemase, alanine dehydrogenase and formate dehydrogenase (FDH)), which transformed keto acids into d-amino acids by supplying a cosubstrate and cofactor57. The use of a combination of alcohol dehydrogenases (ADH) and ketoreductases with a second enzyme for 8 ACS Paragon Plus Environment

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cofactor regeneration is another example of an orthogonal cascade for the asymmetric synthesis of alcohols. In parallel cascades, two substrates are converted into two products by two different enzymatic reactions with cofactors or cosubstrates (Figure 2c). In this reaction, both products are isolated as valuable compounds, while orthogonal cascades produce byproducts that are discarded. An example of parallel cascades, ADH, and Baeyer–Villiger monooxygenases were used for the incidental production of non-racemic alcohols and sulfoxides, reported by Gotor and co-workers58, 59. However, the deracemization of alcohols via stereoinversion can also classified as linear and parallel cascades involving ADH or combinations of both cascades. Last, cyclic cascades convert materials into an intermediate, which is transformed back to the starting material from a mixture of substrates lead to the accumulation of the compound (Figure 2d). Cyclic cascades have been applied to the chemoenzymatic deracemization of amino acids, hydroxy acids, and amines by an D-amino acid oxidase and a chemical reducing agent60. The combination of enzymatic and chemical synthesis offers further synergistic. However, cyclic cascades have been studied as chemoenzymatic rather than multi-enzymatic. Of course, the four classifications of enzymatic cascades are not strict; newly designed pathways can also be combined in many different ways. However, this classification system is helpful for designing new artificial multi-enzymatic pathways. Furthermore, multi-enzyme catalyzed reactions can also be designed by a combination with immobilized enzymes. Enzyme immobilization offers several advantages in terms of reuse, continuous processes, and providing stable processes without enzyme leakage39. Therefore immobilizing an enzyme should be adopted when high-cost enzymes and product separation from the reacting mixture are required. As an example, co-immobilized oxynitrilase and nitrilase as cross-linked enzyme aggregates (CLEAs) show better performance than two separate CLEAs of


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oxynitrilases and nitrilases52,

61.

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Therefore, in chapter 4, we will discuss multi-enzyme

immobilization and the strategies to achieve efficient cascade reaction systems.

3. MULTI-ENZYME COMPLEX MODELS To show substrate or metabolic channeling, much attention has already been given to efficient multi-enzyme cascade pathways via enzyme complexes. Indeed, vast experimental and computational tools are now available for developing the directed evolution of enzyme activity by the construction of synthetic pathways in biotechnological applications62. Based on numerous examples, organization of enzymes is a key aspect of design, as described below and listed in Figure 3. Enzyme complex models can be designed through (i) simple fusion of enzymes, (ii) enzyme scaffold complexes, and (iii) co-immobilization.

3-1. Multi-enzyme complex by fusion proteins A typical approach to form an artificial enzyme complex is to combine proteins using a short linker to increase enzymatic activity (Figure 3a). Mosbach and co-workers created a fusion enzyme between LacZ and dimeric galactose dehydrogenase from Pseudomonas fluorescens by sequential hydrolysis of lactose and consecutive oxidation of the galactose in vivo63. In this instance, the fusion enzyme complex exhibited kinetical advantages, such as a two-fold reduction of Km for lactose and a two- to four-fold increase in reaction rate compared to an identical native enzyme system at low substrate concentrations. To design a preferred pathway to the substrate, the construction of fusion proteins has been proposed for applications in metabolic engineering64. This suggests that the distance between enzymes enabled synergistic effect on the sequential reactions via substrate channeling, and lead to in vitro enzyme complex development. The activity of the fusion protein (choline 10 ACS Paragon Plus Environment

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dehydrogenase (CDH)/betaine aldehyde dehydrogenase (BADH)) increased more than 120%, while the free enzyme activity increased only 45%65, 66. Another enzyme fusion, glycerol-3phosphate dehydrogenase (GPDH) and glycerol-3-phosphatase (GP), was constructed with a 4-AA linker by removing a small C-terminal fragment of GPDH and displayed a seven-fold reduction in transient time, two-fold increase in production rate, and five-fold reduction in intermediate concentration67. In a similar manner, Snell and co-workers constructed Ralstonia eutropha thiolase (PhaA) and E. coli reductase (PhaB) enzymes in enzyme fusions for enhancing bacterial and plant-based biosynthesis of polyhydroxybutyrate granules68. In the PhaA/PhaB fusion protein, the length and composition of the linker sequence were randomized. When the construct was transformed into Arabidopsis, the activity was better than an E. coli system. However, compare to the free enzyme system, the fused enzymes showed poor activity due to folding and solubility. Later, Silver and co-workers compared the effects of different linker lengths (e.g., 2, 14, 24, 46, and 104 amino acids) and found the optimal linker length in the fusion of hydrogenase and ferredoxin69. Between a catalytic unit and other domain or catalytic units, N- or C-terminal linkages and type or length of linker may influence the activities of fusion proteins greatly70. This design is not currently available. For example, a fusion protein CelYZ (170 kD) composed of Clostridium stercorarium exoglucanase CelY, and EG CelZ, constructed by xylanase with an arabinofuranosidase, a xylosidase, and linker, Cellodextrin phosphorylase by addition of a family 9 CBM and the fusion of the Trichoderma reesei (EG) IV (EG4) with an additional catalytic module (EGIVCM) retained parental enzyme enhancement in mass-specific activity and exhibited synergistic effects than the sum of individual activities71-74.

3-2. Multi-enzyme complex by protein-scaffold


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As an alternative to protein fusions, recent work has explored individual linking via posttranslational assembly mechanisms involved in sequential reaction steps (Figure 3b). The one of examples is cellulosome75, a complex that efficiently degrades cellulose and plant cell walls. The engineered complexes are composed of two main parts: (1) containing a cellulose binding domain (CBD) and several cohesin binding domains and (2) the bound enzyme– dockerin fusion to the scaffolding through high-affinity dockerin–cohesin interaction76-78. By using the CBDs, cohesins, and dockerins, Fierobe et al. designed cellulosomes that were incorporated multicomponent complex through cohesin–dockerin interactions76. The Clostridium cellulolyticum cellulosomal family-5 CelA and family-48 CelF cellulases were each fused to dockerins, and assembled precisely onto a scaffoldin backbone. The cellulosome chimeras exhibited enhanced synergistic action, compared to the mixture of free cellulase. Additionally, a construction with 75 different chimeric cellulosomes was designed and identified. Activity in degrading cellulose was enhanced 1.1 to 7.2-fold compared to free enzymes and a two-fold enhancement resulted when designed enzymes were brought into close proximity on the cellulose substrate78. Within this construction, a variety of enzyme arrangements have been used successfully76, indicating the important role of in enzyme colocalization. Cellulosomes linked by non-hydrolytic scaffoldins always exhibit enhanced activities due to enzyme proximity and CBM effects79. Bayer et al. proposed cellulosomes with tailored subunit and defined spatial arrangement of enzymes80. Fierobe and his co-workers demonstrated numerous designer cellulosomes containing (non)-bacterial cellulases, fungal cellulases, and hemicellulases75-78,

81.

Later, synthetic cellulosomes containing T. fusca

cellulases (Cel48A exoglucanase and Cel5A EG) and two T. fusca xylanases (endoxylanases Xyn10B and Xyn11A) exhibited approximately two-fold enhancement of activity compared to non-complexed enzyme mixture on wheat straw82, 83. The linker length between catalytic 12 ACS Paragon Plus Environment

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modules and dockerins showed little effect on the synthetic cellulosome84. Presence of the dockerin on the C-terminal side resulted in an enhanced reactivity, indicating the potential alignment of the catalytic module and dockerin84. The cellulose–cellulosome–microbe complex without further extracellular hydrolysis exhibited a several-fold increase in cellulose hydrolysis rates as compared to free cellulosomes85. Natural cell-surfaces containing cellulosomes inspired the binding of extracellular cellulase components on the surface, such as B. subtilis86, Clostridium acetobutylicum87, 88, E. coli89, S. cerevisiae90, 91, and Lactococcus lactis92. The mini-cellulosomes on the surface of yeast exhibited that the final ethanol concentration was 2.6 times greater than that using the same amount of purified cellulases90. Zhao and his co-workers expressed scaffoldin on the surface of yeast and co-expressed three cellulose components (T. reesei EG II, T. reesei cellobiohydrolase II, and A. aculeatus BGKI). Trifunctional cellulosomes showed enhanced activity compared to the unifunctional and bifunctional minicellulosomes91. Although many studies have been made to exchange noncellulolytic microorganisms to cellulose-utilizing microorganisms79,

93, 94,

creating real

recombinant cellulolytic microorganisms is very challenging due to low expression levels without any other organic nutrients95-97.

3-3. Multi-enzyme complex by co-immobilization Co-immobilization of multi-enzymes can lead to benefits by mimicking cascade enzymatic pathways, such as lower costs of operation, separation, time, and waste treatment (Figure 2c)50, 51. Furthermore, coupling several steps together can drive the process toward the desired equilibrium reaction52. During co-immobilization, each enzyme can be randomly distributed98, 99,

assembled in specific positions100, 101, and formed into a localized compartment, as shown

in Figure 4102-105. As a result, each enzyme can be associated to regulate activity leading to 13 ACS Paragon Plus Environment

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substrate channeling. These specific assemblies of multi-enzyme complexes have a major advantage. For example, they can improve diffusion limitations by transferring of the intermediates from one active site to another106,

107.

That is, the product as a substrate is

directly transferred from an enzyme to another nearby enzyme lead to enzyme cascade throuput, merely linking enzymes can increase the efficiency due to facilitated transport. Coimmobilized enzymes on nanometer-sized carriers can cause enzymes cluster and its effects on the reaction kinetics can simply induce activity enhancement, as calculated by Idan and Hess16. Additionally, crowding effects might influence the overall enzyme activity. The intermediates prevented from escaping can create a higher local concentration. If the intermediate transport is hindered the substrate uptake and the product discharge can be also hindered. It is a compromise between its diffusion resistance and competing species in a multiple-enzyme immobilization system. This is the basic mechanism of enhanced activity in a compartment multi-enzyme system17. The catalytic activity of individual enzymes can be improved through immobilization by concentrating substrate molecules accompanied by decreased Km values obtained through electrostatic interactions, specific affinities or other interactions, if the amount of enzyme is limited. Additionally, pH-activity profile of immobilized enzymes can be shifted via attachment to charged matrices, where both enzymes can operate at optimized pH108.

4. MULTI-ENZYME CO-IMMOBILIZATION Enzymes have been immobilized in different ways. Immobilization has been used for industrial application, to enable biocatalyst reusability, and to improve stability, substratespecificity, enantioselectivity, and reactivity. In co-immobilization, enzymes are linked to the same support in close physical proximity, mimicking the multi-enzyme complexes in cellular 14 ACS Paragon Plus Environment

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systems. Despite the significant research progress, multi-enzyme stabilization is still challenged due to a difficult material preparation and poor enzyme-complex stability.

4-1. Random co-immobilization Many researchers have attempted to create multi-enzymatic cascade reactions by immobilizing enzymes. Among them, random co-immobilization may be the simplest strategy to create an immobilization system. Generally, enzyme solutions are mixed with supports and classical immobilization methods, such as physical adsorption, covalent attachment, or cross-linking are applied. To create multi-enzymatic processes, different enzymes can be crosslinked with each other or linked to the same support in close physical proximity, mimicking multi-enzyme complex structures for reusability summarized in Table 1. A novel cross-linked enzyme aggregate (CLEA) concept called combi-CLEA has been described in the literature. A combi-CLEA can be made from heterogeneous mixtures of proteins/enzymes by the aggregation and cross-linking of two or three different enzymes, as presented by Dalal et al. Lipase, α-amylase, and phospholipase A2 activities were retained up to 100%109. The lipase in the combi-CLEA showed excellent thermal stability at 50 °C compared to free enzymes. The lipase and α-amylase activities in the combi-CLEA were preserved for up to three-repeated use. The combined aggregation of an (S)-selective oxynitrilase and a non-selective nitrilase enabled the production of enantiomerically pure (S)mandelic acid from benzaldehyde52. It was suggested that racemization of the nitrile intermediate was suppressed because of immediate hydrolysis by the nitrilase without diffusion into the buffer. Later, Pelt et al. controlled the continuous addition of low concentrations of aldehyde and pure hydrogen cyanide using the co-immobilization of the two biocatalysts in the form of a combi-CLEA110. Vafiadi and his co-workers produced a 15 ACS Paragon Plus Environment

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combi-CLEA consisting of three enzymes with feruloyl esterase111. This CLEA was considered a multifunctional biocatalyst for the esterification of different ferulic acids112. The multi-enzyme system contained three enzymes of the purine nucleotide analogue pathway and an auxiliary ATP recycling system. The aggregate showed improved stability compared to free enzyme. Recently, oxidase and catalase were used in a one-pot process for the production of 7-aminocephalosporanic acid, a key intermediate for the production of βlactam antibiotics113. As a final example of combi-CLEAs, Bhattacharya et al. generated combi-CLEAs with different protein ratios114. Hydrolysis of lime pre-treated bagasse with combi-CLEAs at specific protein ratios resulted in a 1.68-fold increase in sugar release compared to free enzymes. Similarly, hydrolysis of corn stover with combi-CLEAs resulted in a 1.58-fold increase compared to free enzymes at a certain optimized ratio. The efficiency of combi-CLEAs compared to free enzymes make them ideal candidates for cost-effective commercialization of lignocellulolytic enzymes. Combi-CLEAs can be also combined with magnetic nanoparticles for facile separation. Zhou and his co-workers fabricated glucose oxidase (GOD) and horseradish peroxidase (HRP) by co-precipitation and cross-linking onto amino-functionalized Fe3O4 particles115. The results showed that the magnetic combi-CLEAs exhibited high removal efficiency for direct black-38 (DB38) at pH 6.0 and 40 °C. Under optimal conditions, the highest removal efficiency was 92.28%, which was higher than that of free enzymes (46.82%). After 8 cycles of reuse, the DB38 removal efficiency was 55.53%, indicating that the magnetic combiCLEAs had good reusability. Multiple co-immobilized enzymes by cross-linking were reported by Moehlenbrock and colleagues116. Mitochondria from S. cerevisiae were extracted and crosslinked before the disruption of the mitochondrial membranes. The lysates were immobilized in a biofuel cell 16 ACS Paragon Plus Environment

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via a covalently linked mitochondrial enzyme and exhibited higher activity than lysed native mitochondria. This indicated that preservation of the enzyme complexes before isolation had a positive effect, and that substrate channeling took place on the bioanode. A biological recognition based method was proposed to construct cross-linked multiple glycoenzyme aggregates by Zhang et al. (Figure 5)117. With Concanavalin A (ConA) as a molecular glue, HRP and GOD were agglutinated and then cross-linked by glutaraldehyde (GA), forming a GOD-ConA-HRP complex. The affinity of ConA for glucose increased the uptake of the substrate, reducing the Km value from 51 mM to 8.8 mM. The co-localization and clustering of cascade enzymes facilitated intermediate consumption and created a 1.5fold increase in the specificity of the GOD-HRP cascade, indicating a better throughput than separately cross-linked enzyme aggregates. The simplest strategy for creating a co-immobilized complex structure is to activate or link the surface and multi-enzyme by treatment with chemical reagent. Sepharose particles118, chitin119, glyoxyl agarose113, and chitosan beads120 were used for the random immobilization of enzymes on each surface by a GA cross-linker. For example, López-Gallego et al. have developed a process for the conversion of cephalosporin C to 7-aminocephalosporanic acid (7-ACA) without the presence of hydrogen peroxide and achieved more than 80% yield (from less than 4% previously achieved). D-amino acid oxidase (DAAO) was coimmobilized with catalase (CAT), which was able to eliminate in situ hydrogen peroxide produced by the neighboring DAAO molecules118. Yang and his co-workers showed synergistic enzyme activity; they reduced the size of starch molecules via α-amylase, and the non-reducing ends of starch were further hydrolyzed by glucoamylase, producing glucose with efficient yield. It was anticipated that an optimal ratio between the two enzymes might affect the glucose production yield120. The co-immobilized enzymes (glucoamylase and α-


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amylase), with the aid of external magnetic fields with nonintrusive agitation, led to improved mass transfer in the reaction. Polymer beads or particles are also simple supports used to co-immobilize multi-enzymes on the surface, created by modifying the functional groups of their surface. Mosbach and coworkers reported example of a two biocatalyst system121. Hexokinase and glucose-6phosphate dehydrogenase (G6PDH) were covalently linked to two different matrices, such as sepharose and acrylamide–acrylic acid. After addition of a co-substrate such as glucose, ATP, and NADP+, NADPH reduction was monitored compared to the activity of the same quantity of free enzymes in solution. The diffusion of the intermediate was reduced and rapidly converted to products, which was attributed to the close proximity of the coupled enzymes. Le et al. co-immobilized rabbit muscle lactate dehydrogenase (LDH) and bovine liver glutamate dehydrogenase (GLDH) in different quantities and arrangements on Amberlite XAD-7 for NADH regeneration122. The overall efficiency of the system, based on the rates of glutamate production, suggesting that NADH formation was rate limiting under most conditions and demonstrating the importance of enzyme proximity. The immobilized bienzyme system preserved 75% of its activity after one week of recycling use. For cofactor regeneration, Wang presented FDH, formaldehyde dehydrogenase (FADH), ADH, and GLDH immobilized on polystyrene nanoparticles123. The catalysts retained over 80% activity after 11 cycles of reuse with a cumulative yield of 127%. The system was the first trial of MeOH production via co-immobilized enzyme-particle-enabled in situ cofactor regeneration. Polystyrene nanoparticles were also used to immobilize GOD and HRP, which led to a 100% increase in the conversion rate compared to the equivalent amount of free enzymes and a mixture of individual immobilized enzymes on nanoparticles124. Rocha-Martin et al. coimmobilized NADH oxidase and FDH on agarose beads activated with glyoxyl groups and immobilized peroxidase on boronate groups activated agarose beads to avoid peroxidase 18 ACS Paragon Plus Environment

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ACS Catalysis

suicide inactivation125. The surface modification of polymer beads enabled co-immobilization of a fungal nitric oxide reductase (NOR, a member of the cytochrome P450 enzyme family), and a bacterial glucose dehydrogenase (GDH) was obtained with carboxyl-functionalized microspheres, maintaining enzyme activities of 158% for NOR and 104% for GDH126. The optimal stoichiometric ratio between two enzymes enabled the occurrence of two independent chemical reactions and allowed for efficient cofactor conversion rates. Furthermore, NOR immobilization onto the microspheres enhanced enzyme stability up to 5 days at 37 °C, compared to less than 30 h for the free enzyme. The microenvironment created by the carboxyl chemistry may have favored the active site orientation, indicating conformational flexibility and substrate availability of the enzymes. The Lopez-Gallego group co-immobilized CALB and β-gal on octyl-agarose beads through simple protocols (e.g., immobilization, inactivation, desorption, PEI incubation and immobilization)127. The immobilized enzymes preserved more than 90% of their initial activity after 6 cycles. Since PEI coated on beads yielded better enzyme stability, this concept was applied to immobilize enzymes (CALB and β-gal) in bilayers using PEI as a glue to reuse the enzyme. By preventing PEI release, the immobilized enzymes were better stabilized for recycled uses128. Later, they developed co-immobilized NAD+, FADH and ADH on porous agarose microbead activated PEI to fabricate self-sufficient heterogeneous biocatalysts capable of reutilizing and retaining the cofactor by continuously shifting between the enzyme active sites without getting released from the microbeads129. Polymer gels and capsules have also been studied by many groups searching for efficient co-immobilizing supports. Jasti et al. immobilized GOD and β-galactosidase (β-Gal) on the same matrix, with no more than 10% loss of enzyme activity during immobilization. GA cross-linking after immobilization provided stable enzyme preparations130. The optimum pH was similar to those for the free enzymes, but their thermal stability was hugely improved. 19 ACS Paragon Plus Environment

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Aranaz et al. co-encapsulated D-hydantoinase and D-carboamylase in alginate–chitosan polyelectrolyte capsules131. Dubey and his co-workers immobilized pyruvate kinase (PK) and LDH on a poly(N-isopropylacrylamide)-poly(ethylenimine) (PNIPAm-PEI) microgel using GA as the cross-linker132. For cofactor dependent applications, in which the ADP monitoring ability and ATP synthesis by the conjugates were studied, a 2-fold increase of activity indicated that the distance between the enzymes is an important factor for the optimization of multi-enzyme immobilization on the support. Jiang et al. encapsulated three dehydrogenases (FDH, FADH, and ADH) into a millimeter-sized gel bead (host bead)–capsule in a bead microreactor (Figure 6)133. The capsules-in-bead scaffold significiantly improved enzymatic activity, recycling, and storage stability, thus a higher MeOH yield was achieved. The system could raise the local concentration of the intermediate products and considerably shorten the distance required for the intermediate to move between active sites, inducing substrate channeling. Polymer gelation is another immobilizing method. Bi-enzyme mediated redox initiation was used to achieve cross-linking polymerization. Co-immobilized GOD/HRP during gelation, resulting in a printable hybrid hydrogel, combines the merits to achieve higher mechanical strength and porous networks134. The hydrogel could be inverted to a nanofiber type, which has been widely utilized for enzyme immobilizing support. Liang et al. reported a facile method for multi-enzyme immobilization in metal coordinated hydrogel nanofibers135. Specifically, four enzymes, GOD, Candida rugosa lipase, α-amylase, and HRP were encapsulated in a gel nanofiber made of Zn2+ and adenosine monophosphate (AMP) via a simple mixing step. After 15 days storage, the encapsulated enzyme retained 70% activity. Compared to nanoparticles formed with AMP and lanthanide ions, the nanofiber gels allowed much higher enzyme activity. Using the gel nanofiber to co-immobilize GOD and HRP cascade system achieved a detection limit of 0.3 μM glucose with excellent selectivity. Ji et 20 ACS Paragon Plus Environment

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ACS Catalysis

al. co-encapsulated 3a-hydroxysteroid dehydrogenase (3a-HSD), diaphorase (DP), and NADH in an electrospun, hollow nanofiber by a facile co-axial electrospinning process136. Retained activities as high as 76% and 82% were observed for the encapsulated 3a-HSD and DP, respectively, as shown in Figure 7. In addition, the nanofiber provided a confined multienzyme system inside the hollow fibers with a unique stabilizing mechanism. More than a 170-fold increase in half-life at 25 °C was obtained for the 3a-HSD/DP cascade reaction. Greatly stabilizing the enzymes, the half-lives of the 3a-HSD and DP were 183 and 174 times greater than free enzyme before encapsulation. Paper fibers with covalently interlocked GOD and HRP in the voids of paper were crosslinked with poly(acrylic acid) via carbodiimide chemistry and formed enzyme–polymer spider webs, shown by Riccardi and coworkers137. These interlocked 3-dimensional networks prevented enzyme activity loss and enhanced the stability. Polymer modified materials and phospholipid surfaces have been also utilized as multienzyme immobilization supports. Polymer nanoparticles were prepared by Watanabe group by combining a phospholipid polymer shell with a polystyrene core138. The ester groups for conjugation and phospholipid polar groups were incorporated into the phospholipid polymer backbone. Acetylcholinesterase, choline oxidase, and HRP-labeled IgG were immobilized onto the nanoparticles for sequential enzymatic reactions. When incubated at 4°C, over 80% of the residual peroxidase activity was observed, even when stored for 2 weeks. Glycerol dehydrogenase (GlyDH) catalyzed the production of 1,3-dihydroxyacetone (DHA) from glycerol, while xylose reductase (XR) enabled the reduction of xylose to xylitol from released protons from glycerol. Both enzymes were co-immobilized with P(MMA–EDMA–MAA) nanoparticles139. Interestingly, the immobilized multi-enzyme system showed greatly improved yield and stability compared to native enzymes. The total turnover number (TTN) reached 82 for cofactor regeneration while the yield reached 160 g/g-immobilized GlyDH for 21 ACS Paragon Plus Environment

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DHA production. Jia et al. designed a micelle to co-localize multiple enzymes (GOD and HRP) by controlling local position140. The immobilized enzymes on the same micelles exhibited an enhanced product conversion yield of 100%. Carbon material electrode surfaces have been widely used in electrochemical cascade enzymatic reactions by immobilization. An electrochemiluminescence (ECL) immunosensor based on the peroxydisulfate was first proposed by coupling GOD and HRP with palladium nanoparticles (PdNPs) coated carbon nanotubes to detect α-1-fetoprotein (AFP). The system exhibited a wide linear range of 1×10−5-100 ng mL−1, with a low detection limit of 3.3 fg mL−1, presented by Niu and his co-workers141. A facile enzymatic polymerization protocol to prepare enzyme-poly(thiophene-3-boronic acid) polymeric biocomposites for highperformance GOD/HRP amperometric biosensing with a sensitivity of 75.1 μA mM-1 cm-2 and a LOD of 1 mM was reported by Huang and his co-workers142. Fang and his co-workers co-immobilized salicylate hydroxylase and tyrosinase on carbon nanotube modified electrodes. The system exhibited a sensitivity 30.6±2.7 µA cm−2 µM−1 and the limit of detection and quantification were 13 nM (1.80 ppb) and 39 nM (5.39 ppb), respectively during 10 repeatability tests143. Zor and his co-workers implemented a system consisting of immobilized hydrogenase, NAD+ reductase, alcohol dehydrogenase and L-alanine reductase on carbon nanotube-line column for H2-driven NADH recycling. About 40% conversion was obtained at higher pyruvate concentration (12.5 mM) equating to a TTN of 454,000 products per NAD+ reductase, indicating stabilized enzymes in the CNC144.

The carbon felt-based

bioanode was fabricated by Komaba group. They immobilized three enzymes (maltase, mutarotase and GOD) by crosslinking for stabilized high power system145. After one week the system still delivered about 50% of the initial current output. Furthermore, at least onethird of the initial chronoamperometric current response was maintained during the 10 h operation. Mathesh et al. presented immobilization of HRP followed by GOD on a 2D 22 ACS Paragon Plus Environment

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ACS Catalysis

graphene-oxide-based scaffold that exhibited 2.2 times greater cascade activity (Figure 8). The immobilization of the enzymes led to good stability for two months with 15–17% activity loss and good reusability with 30% activity loss, attributed to efficient substrate channeling compared to the free enzymes146. Metal based nanomaterials were also used to assemble multi-enzyme systems. Qui et al. co-immobilized GOD with lignin peroxidase (LiP) on 40-50 nm sized nanoporous gold surfaces147. By co-immobilizing with GOD, in situ H2O2 release, high LiP activity was achieved. In addition to greater reusability, the co-immobilized enzyme system also showed higher dye conversion when compared to an external one-batch H2O2 supplying strategy. After 7 cycles, the residual activity of the immobilized LiP was still ca. 85% of its original value. Keighron and his co-workers used AuNPs (30 nm) to immobilize malate dehydrogenase (MDH) and citrate synthase (CS)148. The system had 3-fold increase in sequential activity (per-particle) for conversion of malate to citrate. Zhuo et al. modified a gold electrode with composite magnetic particles to design an electrochemical immunosensor. The system exhibited high selectivity and good reproducibility by coupling bienzyme GOD and HRP149. The metal–organic framework (MOF) ZIF-8 can provide a biomolecule-friendly environment. Wu et al. co-encapsulated GOD and HRP in the MOF, randomly150. The GOD & HRP in ZIF-8 retained 80% of the initial overall activity for 7 days. The structural rigidity and confinement of the MOF scaffolds led to greatly enhanced thermal stability by shielding the embedded enzymes from proteolysis and chelating. Inorganic materials are the most promising support for enzyme immobilization. Silica materials have been widely used because of their mesoporous structure with high loading capacity. Sol-gel silica matrices have been synthesized and used as enzyme immobilizing supports because of their easy preparation. Obert et al. co-immobilized FDH, FADH, and 23 ACS Paragon Plus Environment

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ADH for the reduction of CO2 to MeOH151. In this process, the reduced NADH acts as an electron donor for each dehydrogenase-catalyzed reduction. For 1 mol of MeOH produced, 3 mol of NADH are consumed. As such, for 100% yield, the number of moles of MeOH produced should be 1/3 of the NADH added. Silica sol–gel obtained from a water-in-oil microemulsion was used to encapsulate polyethyleneimine (PEI)-grafted superoxide dismutase (PEI-SOD) and CAT (PEI-CAT) and exhibited excellent cascade transformation of superoxide to water152. Porous silica glass supports were utilized by El-Zahab and his coworkers for the covalent immobilization of LDH, GDH, and cofactor NADH98. Effective shuttling of the covalently bound NADH between LDH and GDH inside of two silica supports, which had 30 and 100 nm diameters, provided better enzyme-cofactor integration. The effect of the spacer size was also examined. The use of longer spacers increased the reaction rates 18 times compared to the rates achieved using GA linkages by optimizing the flexibility of spacers that manipulate the interactions between the immobilized components. Betancor et al. co-immobilized nitrobenzene nitroreductase and G6PDH in silica particles, which enabled continuous conversion of nitrobenzene to hydroxylaminobenzene with NADPH recycling50. The system described significantly enhanced product formation (up to a 125-fold increase compared to the non-coupled system) with a total turnover number for NADPH of 62 under the tested conditions. Liu and his co-workers reported that covalently attached GLDH, LDH, and NAD(H) on silica nanoparticle produced α-ketoglutarate and lactate with the cofactor153. Interactions between the catalytic components achieved dynamic shuttling of the particle-supported cofactor between the two enzymes to continue the reaction cycles. Total turnover numbers (TTNs) as high as 20,000 h−1 were observed for the cofactor. Mukai et al. co-immobilized enzymes on Ni-NTA functionalized silica nanoparticles (500 nm)154. The efficacy of the 10-step pathway, measured by conversion of glucose to lactate, was significantly higher when tethered, enabling a three-fold increase in the complexity of 24 ACS Paragon Plus Environment

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ACS Catalysis

the sequential tethered reactions, inducing an elegant, high-throughput glycolytic system. A macroporous silica foam (MSF) also was reported by Cao and his co-workers155. Random coimmobilization of enzymes in MSF was successfully synthesized and assembled to create nanoreactors for co-confining GOD and HRP. Mutti group co-immobilized ADH with an amine dehydrogenase (AmDH) based on its FeIII ion-affinity for specific binding with Histag on porous glass beads to enhance the efficiency of hydrogen-borrowing biocatalytic amination. The product formed by the amination of (S)-phenylpropan-2-ol substrate resulted in 90% conversion and 80% yield in 24 h and was able to perform up to 5 recycle uses156. Hybrid silica particles can be synthesized and applied to the immobilization system by a random co-immobilization method. Xu et al. encapsulated three dehydrogenases in an alginate-silica hybrid gel for MeOH production157. The immobilized enzymes retained activity as high as 76.2% after 60 days storage and as high as 78.5% after recycling 10 times. The MeOH yield reached a value of 98.8%, and a yield of 78.5% was observed after 10 cycles. Au-doped magnetic silica nanoparticles (25~27 nm) were created for the coimmobilization of three cystein-tagged cellulases by Cho and his co-workers158. The hydrolytic degradation of cellulose of these co-immobilized enzymes was compared to those of mixtures. The yields remained at almost 85% after up to seven catalytic cycles, and the yields of cellobiose and glucose increased by 158% and 179%, respectively. An alginatesilica based hybrid matrix was reported by Dibenedetto group. “One-pot” conversion of CO2 into MeOH by co-encapsulated FDH, FADH, and ADH enzymes with NAD+ reduction to NADH was successfully studied159. Other inorganic materials, such iron oxide160, 161, titanium dioxide162-164, aluminum oxide165, copper phosphate166, cadmium167, and magnesium and zinc based materials168 have been reported for multi-enzyme immobilizing supports. Especially, magnetic properties allow facile separation for recycling. A bacterial cytochrome P450 monooxygenase was 25 ACS Paragon Plus Environment

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immobilized on magnetic beads using a standard amine coupling method by Tan and his coworkers160. Yang group developed a multienzyme system (GOD and HRP) by using dopamine derivatives with functionalized magnetic nanoparticles through DNA-directed immobilization. The Km and kcat/Km of the multienzyme system were approximately twice compared to free GOD & HRP. In addition to this, the system preserved more than 63% of its initial activity after 13 cycles and 81.2% of the initial activity was preserved after incubation for 30 days161. The co-immobilized enzymes catalyzed monooxygenation and retained their electron-transport function with reusability. Sun et al. encapsulated three dehydrogenase (FDH, FADH, and ADH) within titania particles through a mild biomimetic mineralization process162. Compared to the free enzyme, the immobilized enzymes showed improved yields for production of MeOH, due to the more favorable molecular interactions among enzymes. Under identical conditions, the yields of MeOH ranged from 35% to 60% in the coimmobilization system, while there was low yield in the aqueous solution (5-10%). More than 50% of the initial activity was retained after eight cycles. Wu et al. co-immobilized GlyDH, 1,3-propanediol-oxidoreductase (PDOR), and glycerol-dehydratase (GDHt) on microfloccules of TiO2 nanoparticles, which were prepared by adsorption-flocculation with polyacrylamide163. The stabilities of GDH against pH and temperature was significantly higher than that those of free GDH, and simultaneous NAD(H) regeneration was feasible in the glycerol redox system, promoting the formation of 1, 3-propanediol (1, 3-PD) with yields up to 11.62 g/L (46.48%). Shi et al. co-immobilized GOD and HRP in titania based organic– inorganic hybrid microcapsules164. This route might be a facile, and efficient way to prepare permeable hybrid materials for various applications. Crestini et al. co-immobilized laccase and HRP on alumina (3 mm) and then applied a layer-by-layer (LbL) coating via crosslinking with polyelectrolyte for the development of an oxidative cascade reaction with lignin165. Randomly co-encapsulated GOD and HRP in Cu3(PO4)2·3H2O nanoflowers, 26 ACS Paragon Plus Environment

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ACS Catalysis

presented by Sun et al., showed improved thermal stability at temperatures as high as 65°C, whereas free GOD lost a large amount of its reactivity at temperatures higher than 45 °C166. CdSe-ZnS quantum dots have also been used with enzyme immobilizing supports167. By constituting multienzyme−QD assemblies in the menaquinone synthetic pathway, the catalytic

efficiency

of

multienzyme−QD

was

predominantly

determined

by

the

enzyme−enzyme distance, and an appropriate stoichiometry of the enzyme assembly was discussed. Mahdi and his co-workers reported inorganic Mg2Al, Zn2Al, and MgZnAl LDH matrices for four enzymes (dihydroxyacetone kinase, pyruvate kinase, triosephosphate isomerase, and fructose-6-phosphate aldolase) on the surface of biohybrid nanoreactors by exhibiting one-por cascade reaction168. Concerning the stability of each enzyme in the bionanoreactor, it was observed that the immobilized enzyme nanoreactor was preserved in Tris buffer (15 mm), MgCl2 (5 mm), and KCl (5 mm); the activities of the immobilized enzymes were unchanged after 1 month. A functionalized microfluidic glass chip is a good candidate for an immobilization support for cascade enzymatic reactions. Invertase, GOD, and peroxidase were co-immobilized for poly(p-cresol) production by Lee and his co-workers169. Co-immobilized GOD and HRP were used for glucose monitoring by Costantini et al.170 and was also reported by Lin et al.171. Biomolecule mediated materials such as DNA directed assembly nanocomplexes (invertase, GOD, and HRP)172, bacteriophage P22 virus-like particles (tetrameric BG), monomeric ATPdependent galactokinase, and dimeric ADP dependent glucokinase173, have been reported. For example, a 24-fold increase in turnover rate was observed in nanocomplexes (invertase – GOD-HRP), and over 70% of their activity was retained after incubation at 65° C for 60 min, with significant enhanced stability. The co-located enzymes, in both studies, increased the


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catalytic efficiency of the cascade reaction, probably by reducing the diffusion limitation associated with the transfer of intermediates in each multi-enzymatic process. As indicated by the majority of the examples described above, it has been demonstrated that co-immobilization had a favorable effect on overall catalytic activity and stability. This positive effect can be attributed to substrate channeling via the formation of enzyme complexes, regardless of their randomized structure.

4-2. Compartmentalization As discussed above, multi-enzyme immobilization via random co-immobilization may be the simplest strategy for improving overall catalytic activity and stability. However, to control the immobilization pattern, the ratio of the immobilized enzymes or the distribution of functional groups for enzyme attachment at the surface could present a challenge. Recently, cellular environments have been found to have highly sophisticated structures that allow multi-enzymatic reactions to occur with efficiency and specificity. However, these microenvironments are delicate. To mimic natural enzyme organization, immobilization by compartmentalization has been used. For example, several materials, such as polymer capsules, phospholipid liposome vesicles, polymersomes, and nano/micro sized-particle, were synthesized and integrated with encapsulation and surface attachments via a LbL strategy as described in Table 2. Polymer capsules have been the simplest materials used to form compartment structures. Kreft et al. presented a simple method for the preparation of micrometer-sized shell-in-shell polyelectrolyte capsules. The fabrication of spherical ball-in-ball particles consisting of calcium carbonate allowed sequential deposition of polyelectrolyte multilayers on the surface, forming capsule-in-capsule compartment systems, as shown in Figure 9174. The ball-in-ball particles had a size ranging from 8–10 μm, with an inner core diameter of 3–4 μm. Bäumler 28 ACS Paragon Plus Environment

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ACS Catalysis

and his co-workers also presented a similar concept by preparing spherical biopolymer particles through co-precipitation of calcium carbonate, followed by cross-linking and dissolution of the calcium carbonate. BG, GOD, and HRP have been incorporated stepwise in such particles and formed separate compartments in a desired sequence at defined positions175. Thus, the substrates and products of the reaction were distributed and equilibrated between the two compartments and then were visualized using confocal laser scanning microscopy within few seconds. Shi et al. slightly modified the previous sacrificial CaCO3 based method176. A robust multicompartment system was constructed through the LbL selfassembly and biomimetic mineralization. Protamine-soaked enzyme (FDH)-containing CaCO3 microspheres were synthesized, and the assembly process was repeated several times for a specific formation of silica layer. Another kind of enzyme (FADH) in the silicate aqueous solution was entrapped in the silica layer. Next, protamine was adsorbed onto the surface of the silica layer to form a titania layer from a titanium precursor. A CO2 substrate transferred through three compartments (outer membrane, intermembrane space, inner membrane) into the lumen to be converted to formic acid. The formic acid had to pass the compartments to escape, and to convert into formaldehyde. The amount of formaldehyde was significantly higher compared to free enzymes after equilibrium (50% to 95%), as shown in Figure 10, indicating that the equilibrium was shifted toward the products due to a reduced distance between the active sites of the enzymes. Another example of the application of three enzymes were reported by the same group176. To explore the application potential of the hybrid polydopamine (PDA) microcapsules consisting of PDA, a multienzyme system for converting starch to isomaltooligosaccharide (IMOs) was used. The α-amylase, β-amylase, and BG were respectively immobilized. After eight repeated uses, the overall catalytic activity of the system was only reduced by 15%, showing almost no catalytic activity loss. The excellent recycling stability arose from the appropriate choice of microenvironment, 29 ACS Paragon Plus Environment

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including the biocompatibility and mechanical and structural stability of these hybrid microcapsules. Additionally, the wall thickness of the microcapsules was controlled by the dopamine concentration, and the three enzymes were respectively immobilized through physical encapsulation in the lumen, in situ entrapment within the wall, and chemical attachment on the out surface under mild conditions, as shown in Figure 11177. Compared to free enzyme (maximum yield 39% (w/w)), the maximum yield of IMOs was 53% (w/w) after 30 min by the immobilized enzymes, due to the substrate channeling (Figure 12). The GelCSi hybrid microcapsules were prepared through the deposition of catechol-modified gelatin (GelC) followed by growth of silica nanoparticles on the GelC layer by same group178. Three enzymes, FDH, FADH, and ADH, were immobilized through physical entrapment in the capsule lumen and covalent attachment. The microcapsules enabled the ideal localization of substrates/intermediates of the enzymatic cascades within short distances, which allowed for rapid mass transfer. The yield and selectivity of the system were 71.6% and 86.7%, respectively, remarkably higher than those of the free enzyme system (35.5%, 47.3%). Furthermore, a methanol yield of 71.6% was achieved much higher than that of the free system (35.5%), attributed the direct transfer of intermediates between three enzymes without equilibration. After nine cycles, the methanol yield decreased from 71.0% to 52.6%, indicating significant enzyme stability during storage. Hwang et al. achieved CO2 conversion using a multi-enzymatic microbead composed of immobilized carbonic anhydrase (CA) and phosphoenolpyruvate carboxylase (PEPCase)179. The CA and PEPCase in the microbeads preserved their activity, even after 20 reuses, and the CA/PEPCase retained about 75% of the oxaloacetate (OAA) production rate of free CA/PEPCase at room temperature, with facile magnetic separability, as shown in Figure 13. By forming multi-enzyme complex structures, the system might offer shorter enzyme−enzyme distances, reducing the mass transfer distance, and allowing an enhanced reaction rate. 30 ACS Paragon Plus Environment

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ACS Catalysis

Phospholipid liposome vesicles have been used to make ideal compartments by mimicking cell membrane structures. Huang et al. co-assembled and integrated three enzymes (glucoamylase, GOD, and HRP) into the cross-linked proteinosome membrane103. The location within the membrane increased the catalytic efficiency, by reducing the diffusional limitation associated with the transfer of intermediates in the cascade process. Elani et al. constructed lipid vesicle-based artificial cells as chemical microreactors180. They achieved this by constructing multi-compartment vesicles within which an engineered multi-step enzymatic pathway is performed. The individual reactions are isolated in each compartments, and their products are moved into neighboring compartments with the aid of transmembrane protein pores. Polymersomes, which have a bilayer structure identical to liposomes, have been synthesized and used in multi-enzyme compartment system development. The van Hest group described a procedure to create a PS–PIAT(diblock copolymer polystyrene-b-polyisocyanoalanine(2thiophene-3-yl-ethyl)amide) polymersome nanoreactor as a proof of concept (Figure 14)181. Three enzymes: Candida antarctica lipase B (CALB), HRP, and GOD were selected. The diameters of the enzyme encapsulated polymersomes ranged from 50 to 1100 nm with an average 517 nm. The polymersome nanoreactors, composed of HRP–polystyrene (HRP–PS) and HRP–poly(methyl methacrylate) (HRP–PMMA) giant amphiphiles, contained GOD inside the self-assembled structures182. These all-enzyme assemblies were able to catalyze cascade reaction. Prepared porous polymersomes based on block copolymers of isocyanopeptides and styrene have been used to anchor enzymes at three different locations, namely, in their lumen (GOD), in their bilayer membrane (CALB), and on their surface (HRP)183. The enzyme-decorated polymersome nanoreactors converted glucose acetate to glucose by CALB, which was oxidized by GOD to gluconolactone in a second step. The hydrogen peroxide produced by HRP oxidized 2,2′-azinobis (3-ethylbenzothiazoline-631 ACS Paragon Plus Environment

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sulfonic acid) (ABTS) to ABTS+. PS–PIAT polymersome nanoreactors was applied for selected self-sufficient Baeyer–Villiger monooxygenase (PAMO) and G6PDH for encapsulation as shown in Figure 15184. Once NADPH entered, it was oxidized and reduced in the same confined space. Encapsulation of the NADP+ regenerated G6PDH, while the PAMO also exhibited good performance. Later, they slightly modified the method to construct a different compartment system, which exhibited a combination of the structural cell mimicry. Enzyme-filled (PS-b-PIAT) nanoreactors were encapsulated together with free enzymes and substrates in a larger polybutadiene-b-poly(ethylene oxide) (PB-b-PEO) polymersome, forming a multi-compartmentalized structure, which showed a structural similarity to organelle mimics into a single system185. A cofactor-dependent three-enzyme cascade reaction (FDH, FADH, and ADH) was performed, using either compatible or incompatible enzymes, in the polymersome-in-polymersome structure, and the functional aspects of the enzymatic reactions took place across multiple compartments (Figure 16). Both cascade systems showed small decreases in the overall reaction rate of 17% and 14%, respectively, relative to that of the same reaction by the free enzymes. It was found that the reaction intermediates easily traveled across multiple compartments to react with the different enzymes in confined cavities. The small decrease in the reaction rate was due to the encapsulation procedure, and the ADH catalyzed reaction caused decreased diffusion of the polar cofactor into the nanoreactor. The Appelhans group constructed polymersomes by mimicking different cell membrane characteristics. The system possessed pH-switchable organelle mimics and temperature- and pH-responsive biomimetic cell membranes with a polyethylene glycol (PEG) surface obtained by combining with enzymatic cascade reactions using GOD, myoglobin (Myo) and CAT for metabolism mimicry. Highly versatile and effective multi-compartmentalized systems were presented that showed flux balance


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(substrate channeling effect) and imbalance, as well as the exclusion of proteins from the cell mimics186. Several inorganic nanoparticles, such as titanium oxide187, copper phosphate188, and silica189 nanoparticles have been used to create multi-enzyme immobilization by forming compartmental structure materials. Shi et al. proposed to construct a multi-enzyme system for conversion of CO2 to formaldehyde. Specifically, FDH was entrapped during the formation of titania nanoparticles (NPs) through bio-inspired titanification187. After in situ surface functionalization of the NPs with oligodopa, the FADH was immobilized on the surface of the NPs. The system exhibited significantly enhanced formaldehyde yield, and after being stored for 20 days at 4°C, it retained as high as 70% of its initial activity. Once particle size increased, the formaldehyde yield, selectivity, and initial specific activity decreased. Li and his co-workers presented a GOD-HRP compartment system, which had GOD on the outer shell and HRP in the core of the complex in Cu3(PO4)2·3H2O nanoparticles188. The system gave the highest overall activity, about 310% of the activity of the free enzyme system and retained 90% of its initial activity after incubating in a water solution at 25°C for 7 days. Begum et al. attempted to mimic nature’s celluar organization by controlling the positioning of enzymes through immobilization in ordered compartments189. This work utilized peptidemediated LbL mineralization as a facile and generic method in nanoscale silica layers. The 30–50 nm thick silica coating was mechanically stabilized. Finally, sequential assembly of the multilayer enabled positioning and incorporation of specified amounts of the cascade enzymes. To ensure a high overall cascade reaction rate, the first reaction step needs to occur close to the surface. Also, the likelihood of efficient enzyme–substrate interactions of a reactant leaving a silica layer might be higher when enzymes are positioned in neighboring layers, indicating that the proximity of the enzymes reduces the diffusion path lengths of the intermediate reactants. 33 ACS Paragon Plus Environment

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Microparticles can also be used to assemble compartment structure. Caruso et al. presented colloidal particle, comprising PS carrier coated with enzyme multilayers via the LbL selfassembly method190. GOD, HRP, or preformed enzyme-polyelectrolyte complexes were assembled in alternating layers with oppositely charged polyelectrolytes onto PS particles. Pescador et al. assembled multilayer films of GOD and HRP with polyelectrolyte layers on the surface of silica microparticles191. Pre-complexation of the enzymes with a polyvinylpyridine-based polyamine allowed stable adsorption of enzyme layers, and different polyelectrolyte combinations on the immobilization and functionality of the enzymes were tested for several multilayer arrangement. Zhou group presented MOF to assemble the multienzyme system (GOD and HRP). They developed a cage-containing MOF that acted as an efficient molecular trap between the framework and enzymes, leading to high loading and excellent catalytic performance. Moreover, the system provided protection from enzyme leaching and trypsin digestion, indicating a highly stabilized nanoreactor192. Niemeyer group established enzyme cascades combined with (R)-selective ADH, (S)selective methylglyoxal reductase and GDH by specific and directional immobilization by genetic fusion with streptavidin binding peptide, Spy and Halo-based tags on magnetic microbeads coated with coresponding receptor in microfluidic packed-bed reactors. The system operated up to 14 days by exhibiting up to 95% conversion and excellent stereoselectivity (d.r. > 99:1) in a continuous flow process indicating a stable and robust enzyme reactor193. Of course, based on reaction rate or pathway, enzyme loading and their ideal ratio have been controlled and optimized. This method allows the formation of enzyme complexes that can induce substrate channeling by mimicking natural enzyme organization.


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4-3. Positional co-immobilization Positional co-immobilization is a good strategy for assembling a sequential order of enzymes, depending on the reaction rate or pathway and enzyme loading. Therefore, this method results in making enzyme complexes in a different manner than compartmentalization (Table 3). A typical example of positional co-immobilization is the application of enzymes to microfluidic channel. Logan and his coworkers developed photopatterning porous polymer monoliths within microfluidic devices and used them to perform spatially separated multienzymatic reactions194. To reduce nonspecific adsorption, the pore surface was modified by grafting PEG, followed by surface photoactivation and enzyme immobilization in the presence of a nonionic surfactant. A three-enzyme cascade reaction was performed using invertase, GOD, and HRP. Different immobilization sequences were tested, but significant product formation was only observed when the enzymes were in the correct sequential order. Luckarift et al. assembled silica-immobilized enzymes within microfluidic devices195. Metallic zinc, silica-immobilized hydroxylaminobenzene mutase, and silica-immobilized soybean peroxidase were connected in series inside of individual microfluidic chips to perform cascade enzymatic synthesis. Ono et al. designed and constructed a microfluidic chip with three reaction chambers to examine the three sequential glycosyltransfer reactions196. The synthesis of tetrasaccharide in a microfluidic system allowed more complex compounds to be synthesized biologically and chemically. Furthermore, a 3-D matrix created inside the microchannel was found to overcome the difficulty associated with introducing beads into the reaction chamber for analysis platform. The Walde group immobilized biotinylated enzymes in three steps197. Microchannels were modified with dendronized polymer (de-PG2) to form a soft organic layer and avidin was adsorbed to the de-PG2. Finally, binding of either Gal, GOD, or HRP to the bound avidin was performed. Simple filling of the microchannel with 35 ACS Paragon Plus Environment

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aqueous solutions of the molecules was used and local immobilization of enzymes in the microchip was achieved by separating a microchannel into three compartments, realized by the integration of valves as shown in Figure 17. Glass tubes have been good candidate for cascade enzymatic reactions by coimmobilizing enzymes at specified positions. Vong et al. realized a three-enzyme cascade reaction in a continuous flow microreactor (Figure 18)198. The first (lipase B) and third (HRP) enzymes were immobilized in a mild non-contact method via an ssDNA-ssDNA interaction in separate zones on the capillary wall, whereas the second enzyme (GOD) was kept in the mobile phase. By using photolithography in a closed fused-silica microchannel, the noncovalent immobilization method enabled a facile reuse of the capillary microreactor. The productivity was increased as the spatial distance between the first and last enzyme was enlarged. The three-enzyme cascade reaction system was used to obtain more insight about the kinetics of the complicated reaction sequence in this study. The Walde group presented immobilized GOD and HRP prepared using a simple procedure inside micropipette glass tubes through avidin–biotin interactions using biotinylated polycationic de-PG2199. The dePG2 strongly adhered to the SiO2 surfaces as organic layer for non-covalently gluing the biotinylated enzyme–avidin interaction to the solid glass surface as described above. The system remained highly active for several weeks. Furthermore, a simple flow reactor to produce D-glucose in an aqueous solution via a cascade reaction was been used. Slight modifications of this method was also later reported. They synthesized dendronized polymer (denpol de-PG2)-bis-aryl hydrazine-enzyme conjugates on unmodified silicate glass surfaces via simple adsorption in one step200. The adsorbed conjugates strongly adhered to the glass surface due to multiple interactions. The conjugate adsorption formed a homogenous thin layer, confirmed by a transmission interferometric adsorption sensor and AFM imaging. Additionally, a simple fabrication of enzymatic flow reactors was determined and the 36 ACS Paragon Plus Environment

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conjugates were used for both enzymes, either through a sequential immobilization of the two enzymes, or through a co-immobilization. Later, these concepts were demonstrated by using a hybrid structure of a synthetic de-PG2201. They prepared a de-PG2−enzyme hybrid and two enzymes, HRP and SOD, were covalently bound to the same polymer chain, with controlled positions. This was the first study on the preparation of a polymer−enzyme conjugate containing two enzymes on the same polymer chain. Several biomolecules, such as DNA origami202-205, cellulose-binding domains206, and protein scaffolds207 were used for the positional co-immobilization of enzymes. A modular DNA origami-based enzyme cascade nanoreactor was reported by Linko and his co-workers by co-immobilizing enzymes (GOD-HRP) inside of each DNA origami unit202. The nanoreactor was assembled with the DNA origami building blocks. Two different structural units were fabricated by annealing an M13mp18 scaffold strand with the set of either 187 (GOD-origami) or 183 staple strands (HRP-origami). Each of the units contained 3 strands with biotin exposed surface of the tubular structure. Later, the Morri group utilized these DNA origami structures as a scaffold to co-assemble for cofactor regeneration203. Yang’s group developed substrate channeling system using DNA origami using a photoresponsive molecule. G6PDH and LDH were immobilized for NADH regeneration and lactate production to continue the cascade reaction. By fixing the location of G6PDH on the origami, the location of LDH was changed by the swing arm. With decreasing inter-enzyme distance, enzyme

cascade

activity

improved

significantly,

exhibiting

substrate

channeling.

Construction of DNA origami raft-based real-time imaging at the single-molecule level was designed by Fan and his co-workers by adhering catalase on cholesterol-labeled doublestranded (ds) DNA and GOD on cholesterol-labeled origami rafts in supported lipid bilayers (SLB) via the cholesterol−lipid interaction205. Xylose reductase (XR) and xylitol dehydrogenase (XDH) were located in the space of the DNA scaffold at predesigned 37 ACS Paragon Plus Environment

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positions in a distance-dependent strategy. The system showed that the close proximity of the two localized enzymes facilitated the transport of reaction intermediates in the cascade reaction, causing significantly higher product yields and in situ recycling of cofactors. The xylose metabolism through the simultaneous transport of xylitol and NAD+ for recycling of NADH were systematically evaluated. The efficiency of the cascade reaction was significantly dependent on the inter-enzyme distance between XR and XDH. You et al. suggested a one-step purification of a multi-enzyme complex based on a three cellulosebinding module (CBM3)-containing scaffolding through high-affinity adsorption206. The three-enzymes, triosephosphate isomerase, aldolase, and fructose 1,6-biphosphatase were self-assembled through a high-affinity interaction between dockerin-cohesins scaffold. The immobilized enzymes exhibited 48 times larger initial reaction rates at the same enzyme loading. Such reaction rate enhancements were induced by substrate channeling, due to ideal spatial organization of the cascade enzymes. Protein scaffolds have been used stoichiometrically to control immobilization of multiple enzymes by the Yoshino group207. The immobilization of cellulases on magnetic nanoparticles (MNPs), called magnetosomes, were prepared by mimicking natural multiple cellulase complexes (cellulosomes). The EG and BG were immobilized on MNPs to form EG/BG-MNPs, and then used for rapid hydrolysis of carboxymethyl cellulose. The fusion of the cellulose-binding domain to EG/BG-MNPs encouraged improved hydrolysis activity against the insoluble cellulose by mimicking natural cellulosome organization on MNPs as shown in Figure 19. Hybrid nanomaterials (gold–mesoporous silica Janus nanoparticles) were used for immobilization by Villalonga group208. Two different faces (Au and silica nanoparticle faces) with different chemical compositions allowed selective GOD-HRP immobilization via different specific ligands (thiol and silane derivatives) and linking strategies. The nanoparticles were deposited on glassy carbon electrodes coated with single-walled carbon 38 ACS Paragon Plus Environment

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nanotubes and used for glucose detection. The enzymatic electrode retained about 98% of its initial response after 15 days of storage, and even 64% of the initial activity after 1 month. Positional co-immobilization is very effective for modifying surfaces, channels, and tubes for cascade enzymatic reactions. By changing the enzyme immobilization sequence, the cascade enzymatic reaction rate and its process stability can be also controlled and improved.

5. CONCLUSION Many industrial processes, such as synthesis of pharmaceutical, cosmetic, and nutrition, are catalyzed by two or more enzymes simultaneously or sequentially. These biocatalysts have been arranged with short distances between each active enzyme site to mimic the arrangement in cellular organisms. By assembling enzyme complexes through fusion proteins and protein scaffolds, multi-enzymatic structures have been successfully designed and used. The co-localized enzymes contribute to the improvement of activity via substrate channeling. In particular, it may be very useful to co-immobilize enzymes in an efficient support to overcome diffusion limitations. Of particular interest, these multi-enzyme assemblies may be able to mimic the multi-enzymatic reactions which take place in nature, as described above. The development of new multi-enzyme complex structures by immobilization has received attention for several years, and new strategies for stabilizing enzymes with improved enzymatic cascade activity and operational stabilities have been achieved by classical immobilization methods with specific strategies, such as random co-immobilization, compartmentalization, and positional co-immobilization. Many chemical biologists have tried to increase multi-enzyme performance, whereas materials scientists have proposed new supports to achieve their objectives. This review has demonstrated multi-enzyme immobilization with different types of materials, including polymers, organic/inorganic 39 ACS Paragon Plus Environment

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materials, biomaterials, nano/micro-sized materials, mesoporous materials, and microfluidic channels. These immobilization technologies have led to progress in various areas involving several multi-enzyme processes. As these approaches are applied to design multiple enzymemediated processes, they will result in practical applications in many fields, including medicine, cosmetic, the food industry, biodiesel production, and bioremediation.

SYMBOLS Endoglucanase = EG β-glucosidase = BG Pyruvate dehydrogenase = PDH Alcohol dehydrogenase = ADH Formate dehydrogenase = FDH Cross-linked enzyme aggregates = CLEAs Choline dehydrogenase = CDH Betaine aldehyde dehydrogenase = BADH Glycerol-3-phophatate dehydrogenase = GPDH Glycerol-3-phosphatase = GP Cellulose binding domain = CBD Glucose oxidase = GOD Horseradish peroxidase = HRP Concananvalin A = ConA Glutaraldehyde = GA 7-aminocephalosporanic acid = 7-ACA D-amino acid oxidase = DAAO Catalase = CAT Glucose-6-phosphate dehydrogenase = G6PDH 40 ACS Paragon Plus Environment

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Lactate dehydrogenase = LDH Glutamate dehydrogenase = GLDH Formaldehyde dehydrogenase = FADH Nitric oxide reductase = NOR Glucose dehydrogenase = GDH β-galactosidase = β-Gal Pyruvate kinase = PK Adenosine monophosphate = AMP 3a-hydroxysteroid dehydrogenase = 3a-HSD diaphorase = DP Glycerol dehydrogenase = GlyDH 1,3-dihydroxyacetone = DHA Xylose reductase = XR Electrochemiluminescence = ECL Lignin peroxidase = LiP Malate dehydrogenase = MDH Citrate synthase = CS Metal–organic frameworks = MOF Polyethyleneimine = PEI Superoxide dismutase = SOD Macroporous silica foam = MSF Amine dehydrogenase = AmDH 1,3-propanediol-oxidoreductase = PDOR Glycerol-dehydratase = GDHt Layer-by-layer = LbL Polydopamine = PDA isomaltooligosaccharide = IMOs Catechol-modified gelatin = GelC 41 ACS Paragon Plus Environment

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Carbonic anhydrase = CA Phosphoenolpyruvate carboxylase = PEPCase Oxaloactate = OAA PS–PIAT= diblock ethyl)amide)

copolymer

polystyrene-b-polyisocyanoalanine(2-thiophene-3-yl-

Candida antarctica lipase B = CALB 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) = ABTS Baeyer–Villiger monooxygenase = PAMO Polybutadiene-b-poly(ethylene oxide) = PB-b-PEO Polyethylene glycol = PEG Dendronized polymer = de-PG2 Supported lipid bilayer = SLB Xylitol dehydrogenase = XDH 3 cellulose-binding module = CBM3 Magnetic nanoparticles = MNPs

ACKNOWLEDGMENTS This research was supported by the Ministry of Trade, Industry and Energy(MOTIE), Korea Institute for Advancement of Technology(KIAT) through the Encouragement Program for The Industries of Economic Cooperation Region (P0006145). This work was also supported by a grant from Fundamental R&D program and funded by the Korea Institute of Ceramic Engineering & Technology (KICET) and Ministry of Trade, Industry and Energy(MOTIE), Republic of Korea.


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Table 1. Examples of immobilization of multi-enzyme systems using various materials by random co-immobilization

Materials

　

Model enzyme

　

Immobilization method

Sepharose particles

　

DAAO was co-immobilized with CAT

　

Crosslinking

　

118

Glyoxyl agarose

　

α-amylase, glucoamylase

　

Crosslinking

　

120

Sepharose and acrylamide–acrylic acid

　

Hexokinase and G6PDH

　

H

　

121

Amberlite XAD-7

　

LDH and GDH

　

Physical adsorption

　

122

　

FDH, FADH, ADH

　

　

123

　

124

　

125

　

126

Polystyrene nanoparticle

Covalent attachment 　

GOD and HRP

　

　

NADH oxidase and FDH, peroxidase

　

Agarose beads

Octyl-agarose beads

　 Reference

Covalent attachment 　

NOR and GDH

　

　

CALB and β-gal

　

Covalent attachment

　 127, 128


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ACS Catalysis

　

FADH and ADH

　

　

129

Alginate–chitosan polyelectrolyte capsule

　

D-hydantoinase and D-carboamylase

　

Encapsulation

　

131

(PNIPAm-PEI) microgel

　

PK and LDH

　

Crosslinking

　

132

Polymer Capsule in bead microreactor

　

FDH, FADH, and ADH

　

Encapsulation

　

133

Hybrid hydrogel

　

GOD and HRP

　 Cross-linking and polymerization

　

134

Hydrogel nanofibers

　

GOD, Candida rugosa lipase, α-amylase, and HRP

　

Encapsulation

　

135

Electrospun hollow nanofiber

　

3a-HSD and DP

　

Encapsulation

　

136

Paper fiber

　

GOD and HRP

　

Crosslinking

　

137

Polystyrene nanoparticle

　

Acetylcholinesterase, choline oxidase

　

Covalent attachment

　

138

P(MMA–EDMA–MAA) nanoparticles

　

GlyDH and XR

　

Covalent attachment

　

139

Copolymer-quantum dot micelle

　

GOD and HRP

　

Adsorption

　

140

Palladium nanoparticles (PdNPs) coated carbon nanotubes

　

GOD and HRP

　

Adsorption

　

141

　

Hydroxylase and tyrosinase

　

Dropcasting

　

142

　

hydrogenase, NAD+ reductase, ADH, and Lalanine reductase

　

Adsorption

　

143

　

Maltase, mutarotase, and GOD

　

Crosslinking

Graphene-oxide

　

GOD and HRP

　

Adsorption

　

145

Nanoporous gold

　

GOD and LiP

　

Adsorption

　

146

AuNPs

　

MDH and CS

　

Adsorption

　

147

ZIF-8

　

GOD and HRP

　

Encapsulation

　

140

　

FDH, FADH, and ADH

　

Encapsulation

　

141

　

SOD and CAT

　

Encapsulation

　

142

　

LDH and GDH

　

Covalent attachment

　

98

　

Nitrobenzene nitroreductase and G6PDH

　

Entrapment

　

153

　

10 enzymes (glucose to lactate)

　

Ni-NTA-Histag

　

154

Macroporous silica foam (MSF)

　

GOD and HRP

　

Entrapment

　

155

Glass bead

　

ADH and mine dehydrogenase (AmDH)

　

FeIII ion-affinity for the His-tag

　

156

Alginate-silica (ALGSiO2) hybrid gel

　

FDH, FADH, and ADH

　

Encapsulation

Au-doped magnetic silica nanoparticles

　

Endo-glucanase, exo-glucanase, and βglucosidase

　

Covalent attachment

Carbon nanotube modified electrodes

144

Sol-gel silica

Silica particles

Silica nanoparticles

　 157, 158 　

158


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GOD and HRP

　

Crosslinking

　

115

　

P450cam, PdX, and PdR

　

Covalent attachment

　

160

　

GOD and HRP

DNA-directed immobilization 　 (Watson-Crick base pairing (A−T, G−C))

　

161

Titania particles

　

FDH, FADH, and ADH

　

Encapsulation

　

162

TiO2 nanoparticle

　

GlyDH, PDOR, and GDHt

　

Adsorption

　

163

Titania based organic– inorganic hybrid microcapsules

　

GOD and CAT

　

Encapsulation and adsorption

　

164

Almunia

　

Laccase and HRP

　

Covalent attachment

　

165

Copper phosphate

　

GOD and HRP

　

Encapsulation

　

166

CdSe-ZnS quantum dots

　

GOD and HRP

　

Site-specific protein–nanoparticle conjugation

　

167

Mg2Al, Zn2Al, and MgZnAl LDH

　

Dihydroxyacetone kinase, PK, triosephosphate isomerase, and fructose-6-phosphate aldolase

　

Encapsulation

　

168

　

Invertase, GOD, and peroxidase

　

Covalent attachment

　

169

　

GOD and HRP

　

Covalent attachment

　 170, 171

DNA directed assembly nanocomplexes

　

Invertase, GOD, and HRP

　

Encapsulation

　

172

Bacteriophage P22 VirusLike Particle

　

Monomeric ATP-dependent galactokinase, and dimeric ADP dependent glucokinase

　

Encapsulation

　

173

Fe3O4 nanoparticles

Microfluidic glass chip

　　

　　

　　

Table 2. Examples of immobilization of multi-enzyme systems using various materials by compartmentalization

Materials and approach

　


Phenylacetone 　 Monooxygenase,CALB, and ADH

　

Encapsulation

　

174

　

BG, GOD, and HRP

　

Encapsulation and then cross-linking

　

175

　

FDH, FADH, and ADH

　

Encapsulation, entrapment, and covalent attachment

　

176

　 α-amylase, β-amylase, and BG

　


　 177, 178

　

CA and PEPCase

　


　

179

Proteinosome membrane by self assemlby

　

Glucoamylase, GOD, and HRP

　


　

103

Lipid vesicle assembly

　

Lactase, GOD, and HRP

　

Encapsulation

　

180

Polymer capsule with sacrificial core removal

Polymer capsule and layer modification

　

Model enzyme

　 Reference


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ACS Catalysis

　

ALB, HRP, and GOD

　


　

181, 182, 183

　

G6PDH and PAMO

　

Encapsulation and azide-Alkyne Cycloaddition

　

184

　

FDH, FADH, and ADH

　


　

185

　 GOD, myoglobin, and CAT

　

Encapsulation

　

186

Titanium oxide nanoparticle in situ surface modification

　

FDH and FADH

　 Entrapment and

　

187

Copper phosphate nanoparticle with outer shell

　

GOD and HRP

　

Entrapment and

adsoprtion

　

188

Silica nanoparticle with peptidemediated LbL modification

　

Cellulase, glucokinase, and G6PDH

　

Entrapment and

adsoprtion

　

189

Multi-layered polymer microparticle via LbL self-assembly

　

GOD and HRP

　

Adsoprtion

　

190

Silica microparticle and layer modification

　

GOD and HRP

　

Adsoprtion

　

191

Metal-organic framework (MOF)

　

GOD and HRP

　

Step-wise encapsulation

　

192

Microfluidic packed-bed reactors

(R)-selective ADH, (S)　 selective methylglyoxal reductase, and GDH

　 Specific and directional immobilization

　

193

　　

　　

　　

Polymersome with surface modification

　

covalent attachment

Table 3. Examples of immobilization of multi-enzyme systems using several systems by positional co-immobilization

Immobilization system

Microfluidic channel

Glass tube

　

Model enzyme

　


　 Reference

　

Invertase, GOD, and HRP

　

Covalent attachemnt

　

194

　

Hydroxylaminobenzene mutase and soybean peroxidase

　

Silica-immobilized enzyme and then packed into the channel

　

195

β1,4 -galactosyltransferase I (GalT-I), β1,3-galactosyltransferase II (GalT　 II), and β1,3-glucuronic acid transferase (GlcAT-I)

Co-immobilization of enzyme/3-D nano-structured 　 protein hydrogel in a microchanne

　

196

　

Gal, GOD, and HRP

　

Avidin–biotin interaction

　

198

　

Lipase, GOD, and HRP

　

Reversible immobilization via the DDI technique

　

198

　

β-GAL, GOD, and HRP

　

Avidin–biotin interaction

　

199

　

GOD and HRP

　

Adsorption

　

200


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SOD and HRP

　

Covalent attachemnt

　

201

　

GOD and HRP

　

Enatrapment

　

202

　

XR and XDH

　

Bound to the specific sites on the DNA scaffold

　

203

　

G6PDH and LDH

　

Bound to the specific sites on the DNA scaffold

　

204

　

GOD and CAT

　

DNA-enzyme conjugation

　

205

Cellulose-binding domain

　

XR and XDH

　

Interaction between the cohesin and dockerin domains

　

206

Protein scaffold

　

EG and BG

　

High-affinity adsorption

　

207

Gold–mesoporous silica Janus nanoparticles

　

GOD and HRP

　

Specific ligands (thiol and silane derivatives) linking

　

208

DNA origami

　

　　

　　

　　

(A) Free floating two enzymes

(B) Proximate two enzymes for substrate channeling


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ACS Catalysis

Figure 1. Schematic illustration of the substrate channeling effect between (A) Free floating two enzymes (E1 and E2) and (B) proximate two enzyme (E1/E2), diffusion of substrate (S1 and S2) and enzyme (E1 and E2) are assumed to be directional. E and S indicate enzyme and substrate, respectively.

(A)

(B)


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(C)

(D)

Figure 2. Classification of four different multi-enzyme cascade processes: (A) linear cascade, (B) orthogonal cascade, (C) parallel cascade, and (D) cyclic cascade

(A)

(B)


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ACS Catalysis

(C)

Figure 3. Schematic illustration of multi-enzyme complex models by co-localizing enzymes to be proximate: (A) multi-enzyme immobilization, (B) fusion protein, and (C) enzyme complex through scaffold unit

(A)

(B) 71 ACS Paragon Plus Environment

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(C)

Figure 4. Schematic illustration of multi-enzyme immobilization strategies: (A) random coimmobilization, (B) compartmentalization, and (C) positional co-immobilization


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ACS Catalysis

Figure 5. Schematic illustration of protein aggregates using ConA as agglutinant and glutaraldehyde as cross-linker. Reprinted with permission from ref. 117. Copyright 2016, American Chemical Society.


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Figure 6. Schematic illustration of Construction of capsules-in-bead structured microreactor. Reprinted with permission from ref. 133. Copyright 2009, Royal Society of Chemistry.


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ACS Catalysis

Figure 7. Relative activities of 3a-HSD and DP in 50 mM Tris-HClbuffer (pH 8.0), in the core phase solution, and in the lumen of the PU hollow nanofiber produced at different core– shell phase solution flow rates. Activities of enzymes in 50 mM Tris-HClbuffer (pH 8.0) are normalized as 100%. Reprinted with permission from ref. 136. Copyright 2014, Royal Society of Chemistry.


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Figure 8. Product-conversion rate test for studying the effect of different types of immobilization and sequence of enzyme loading. Sequential immobilization showed 2.2 times more activity than random immobilization. GOD@HRP showed double the activity of HRP@GOD. Reprinted with permission from ref. 146. Copyright 2017, Wiley-VCH.


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ACS Catalysis

Figure 9. General route for the synthesis of shell-in-shell microcapsules. A=initial core; B=core–shell particle; C=ball-in-ball particle (type I); D=ball-in-ball particle (type II); E=shell-in-shell microcapsule. Reprinted with permission from ref. 174. Copyright 2007, Wiley-VCH.


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Figure 10. Plot of (a) NADH consumption and formaldehyde product as a function of time; (b) reaction rate of the three types of multienzyme systems as a function of reaction time, and (c) formaldehyde selectivity for different multienzyme systems at equilibrium; (d) proposed mechanism for conversion of CO2 to formaldehyde in the (i) SCMCs and (ii) HDMMCs. Reprinted with permission from ref. 176. Copyright 2011, American Chemical Society.


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ACS Catalysis

(A)

(B)

Figure 11. (A) Schematic for the construction process of multienzyme system, and (B) Starch conversion with reaction time (50 ◦C, pH 6.0, starch concentration 10% (w/v)). Reprinted with permission from ref. 177. Copyright 2011, Royal Society of Chemistry.


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Figure 12. Plots of (a) NADH conversion ratio, (b) methanol yield, and (c) specific activity as a function of reaction time for converting CO2 to methanol of the free and GelCSi-based enzyme cascade systems. (d) Recycling stability and (e) storage stability (the reaction was carried out for 6 h at 37 °C and pH 7.0). (f) Proposed reaction process of CO2 to methanol catalyzed by the GelCSi-based system.Reprinted with permission from ref. 178. Copyright 2014, American Chemical Society.


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ACS Catalysis

Figure 13. Overall activities of the multi-enzyme incorporated compartment. Comparison of the OAA production rates of the CA/PEPCase microbead compartment and the free CA/PEPCase. The overall enzymatic activities of CA/PEPCase were measured using 1 mM PEP as the co-substrate in a constant CO2 pressure controlled chamber (5% CO2, 30 °C). Reprinted with permission from ref. 179. Copyright 2016, Royal Society of Chemistry.


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Figure 14. Schematic representation of the multistep reaction taking place in the threeenzyme–polymersome system. Reprinted with permission from ref. 181. Copyright 2007, Wiley-VCH.


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ACS Catalysis

Figure 15. Diagram depicting the regeneration of cofactor NADP+ by G6PDH inside PS-bPIAT polymersomes to sustain the conversion of phenylacetone catalyzed by PAMO. Two systems with encapsulated G6PDH were separately examined: PAMO in solution and PAMO covalently immobilized on the surface. Reprinted with permission from ref. 184. Copyright 2011, Royal Society of Chemistry.


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Figure 16. (A) The concept of the cell mimic, which shows the initial encapsulation of different enzymes in polystyrene-b-poly(3-(isocyano-lalanyl-amino-ethyl)-thiophene) (PS-bPIAT) nanoreactors (1), followed by mixing of the organelle mimics, cytosolic enzymes, and reagents (2), before encapsulation of the reaction mixture in polybutadiene-bpoly(ethylene oxide) (PB-b-PEO) vesicles (3) to create the functional cell mimic (4), inside which enzymatic multicompartment catalysis takes place. (B) Detailed cascade reaction scheme. Profluorescent substrate 1 undergoes a Baeyer–Villiger reaction catalyzed by phenylacetone monooxygenase (PAMO), with one unit of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) being consumed, to yield ester 2, which is subsequently hydrolyzed by Candida antarctica lipase B (CalB) or alcalase to provide primary alcohol 3. Alcohol dehydrogenase (ADH) oxidizes the alcohol, by using the cofactor nicotinamide adenine dinucleotide (NAD+), to give aldehyde 4, which then undergoes spontaneous betaelimination to yield resorufin (5) as the final fluorescent product. Reprinted with permission from ref. 185. Copyright 2014, Wiley-VCH.


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ACS Catalysis

Figure 17. Outline of the enzymatic cascade reaction system used for the quantification of lactose with the enzymes b-Gal, GOD, and HRP immobilized in the three compartments of the chip. In compartment A, b-Gal catalyzes the hydrolysis of lactose (1) into d-glucose (2) and d-galactose (3). In compartment B, the b-form of d-glucose is oxidized by GOD to glucono-d-lactone (4) and H2O2. Hydrogen peroxide activates HRP which then oxidizesAmplex Red (5) to fluorescent resorufin (6) in compartment C. For the sake of clarity only the most relevant b-form of the sugars is given in the scheme. The microchip comprises three individual bioreactor compartments (A, B, and C) which are separated during enzyme loading (pressure P1 activated). After immobilization, a mixture of lactose and Amplex Red is flushed through the whole channel (pressure P2 activated) resulting in a fluorescent signal.. Reprinted with permission from ref. 197. Copyright 2012, Wiley-VCH.


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Figure 18. (A) Reaction scheme of the 3-enzyme cascade reaction. Glucose mono-acetate (1O-acetyl-b-D-glucopyranose) 1 is hydrolyzed by CalB to produce glucose 2, which is subsequently oxidized by glucose oxidase (GOx) to gluconolactone 3 producing H2O2 as a side product. HRP uses H2O2 to convert ABTS (4) into ABTS+ (5). (B) Schematic representation of the microfluidic set-up used for performing a cascade reaction. The patches represent the immobilized enzymes CalB and HRP (GOx is carried along by the mobile phase; see text) and the block represent the zero dead-volume PEEK connectors. Reprinted with permission from ref. 198. Copyright 2011, Royal Society of Chemistry. .


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ACS Catalysis

Figure 19. (A) CMC hydrolysis using EG/BG-MNPs (top) and EG-MNPs + BG-MNPs (bottom). (B) Variation in reducing sugar contents in the supernatant with time in the presence of various MNPs using 0.5% CMC. EG/BG: 50 μg of EG/BG-MNPs. EG + BG: 50 μg of EG-MNPs + 50 μg of BG-MNPs. WT: 50 μg of MNPs from wild type. (C) Reusability of MNPs (50 μg), determined by repetition of the hydrolysis assay. Error bars represent the standard deviation measured for three independent experiments. Reprinted with permission from ref. 207. Copyright 2015, American Chemical Society.


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Table of Contents


Multi-Enzymatic Cascade Reactions via Enzyme Complex by

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