Multienzymatic cascade reactions are a most important technology to succeed in industrial process development, such as synthesis of pharmaceutical, cosmetic, and nutritional compounds. Different ...strategies to construct multienzyme structures have been 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 materials science have led to syntheses of various materials applicable for enzyme immobilization. This review discusses different cases for assembling multienzyme complexes via random co-immobilization, compartmentalization, and positional co-immobilization. The advantages of using immobilized multienzymes 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, as multienzyme systems allow for economical product synthesis through bioprocesses.
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•Design methods of co-immobilized multi-enzymes are summarized.•There will be great demand on applications of multi-enzyme system.•The challenges and future prospective of ...multi-enzyme immobilization are discussed.
Enzyme catalysis has been attracting increasing interest in the past twenty years. Nevertheless, most reports concerning enzyme catalysis have been carried out using single enzymes. Recent years, multiple enzyme cascade reactions have a significant role for the production of many compounds at an industrial level because they permit to perform very complex reactions. Especially, the development of coimmobilized multienzymatic systems is increasingly driven by economic and environmental constraints that provide an impetus to develop alternatives to conventional multistep synthetic methods. Up to now, process optimization and novel strategies of coimmobilized multienzymatic systems hardly have been reviewed. In this review, we focus on some recent novel techniques in preparing co-immobilized multienzymatic systems and the up-to-date advances in the application of multienzymatic systems. Moreover, we also discuss the improvements that co-immobilization multienzymatic systems offer enzymes such as reusability, catalytic activity, and stability.
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•β-xylosidase and endoxylanase were co-immobilized on UiO-66-NH2 by simple mixing.•Co-immobilized β-xylosidase showed enhanced activity at higher temperature.•Co-immobilized ...β-xylosidase showed better performances in high concentration of acetone.•52% β-xylosidase and 70% endoxylanase activity were remained after five recycles.
Improving the activity of β-xylosidase at high temperature and organic solvents is important for the conversion of xylan, phytochemicals and some hydroxyl-containing substances to produce xylose and bioactive substances. In this study, a β-xylosidase R333H and an endoxylanase were simultaneously co-immobilized on the metal–organic framework UiO-66-NH2. Compared with the single R333H immobilization system, the co-immobilization enhanced the activity of R333H at high temperature and high concentration of acetone, and the relative activities at 95 °C and 50% acetone solution were >95%. The Km value of co-immobilized R333H towards p-Nitrophenyl-β-D-xylopyranoside (pNPX) shifted from 2.04 to 0.94 mM, which indicated the enhanced affinity towards pNPX. After 5 cycles, the relative activities of the co-immobilized enzymes towards pNPX and corncob xylan were 52% and 70% respectively, and the accumulated amount of reducing sugars obtained by co-immobilized enzymes degrading corncob xylan in 30% (v/v) acetone solution was 1.7 times than that with no acetone.
Surface chemistry of carriers plays a key role in enzyme loading capacity, structure rigidity, and thus catalyze activity of immobilized enzymes. In this work, the two model enzymes of horseradish ...peroxidase (HRP) and glucose oxidase (GOx) are co-immobilized on the lysozyme functionalized magnetic core-shell nanocomposites (LYZ@MCSNCs) to enhance their stability and activity. Briefly, the HRP and GOx aggregates are firstly formed under the crosslinker of trimesic acid, in which the loading amount and the rigidity of the enzyme can be further increased. Additionally, LYZ easily forms a robust anti-biofouling nanofilm on the surface of SiO2@Fe3O4 magnetic nanoparticles with abundant functional groups, which facilitate chemical crosslinking of HRP and GOx aggregates with minimized inactivation. The immobilized enzyme of HRP-GOx@LYZ@MCSNCs exhibited excellent recovery activity (95.6 %) higher than that of the free enzyme (HRP&GOx). Specifically, 85 % of relative activity was retained after seven cycles, while 73.5 % of initial activity was also remained after storage for 33 days at 4 °C. The thermal stability and pH adaptability of HRP-GOx@LYZ@MCSNCs were better than those of free enzyme of HRP&GOx. This study provides a mild and ecofriendly strategy for multienzyme co-immobilization based on LYZ functionalized magnetic nanoparticles using HRP and GOx as model enzymes.
•LYZ functionalized MCSNCs exhibited rich functional groups for enzyme immobilization.•Trimesic acid was firstly used as a crosslinker for enzyme aggregates formation.•Through cross-linking, the structure rigidity and loading capacity of the enzyme were enhanced.•The multienzyme co-immobilization technique improved the stability and activity of the enzyme.
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•Recent works addressing the topic of co-immobilization of organic acids and bases are summarized.•The rational design and engineering of these catalysts for cooperative and tandem ...catalysis are reviewed.•The structure–activity relationships of these catalysts are discussed.•Interdisciplinary efforts are required for the development of ideal supports and procedures.
Acid–base bifunctional catalysts have been extensively used for a series of CC bond formation reactions. Among these catalysts, organic acid–base catalysts have attracted wide attention due to their structural variety, conformational dynamics, and enantioselectivity. To prevent mutual deactivation, a simple, effective, and versatile strategy is the attachment of both acid and base onto the surfaces of supports. In particular, the co-immobilization of organic acids and bases not only enhances synergistic effects in cooperative catalysis but also improves yields to desired products by controlling the diffusion of intermediates in tandem catalysis, leading to significant improvements in energy and atom efficiency. In this review, we highlight recent works addressing the broad topic of the co-immobilization of organic acids and bases for cooperative and tandem catalysis. We mainly focus on the synthetic strategies for silica-supported organic acid–base catalysts and polymer-supported organic acid–base catalysts. Furthermore, we summarize and discuss the structure–activity relationships of these catalysts. Last, the remaining issues and prospects will be discussed to advance the rational design and engineering of co-immobilized acid–base bifunctional catalysts.
The adsorption kinetics of cellulase and xylanase immobilized on magnetically separable, hierarchically ordered mesocellular mesoporous silica (M‐HMMS) was investigated. The adsorption of cellulase ...on M‐HMMS followed pseudo‐second‐order kinetics while the xylanase adsorption followed the Avrami model. Intraparticle and film diffusion also affected this process. These results enable in‐depth knowledge of the cellulase and xylanase adsorption mechanisms on M‐HMMS for better future applications by improving the stability of the immobilization.
Cellulase and xylanase were co‐immobilized in magnetically separable hierarchically ordered mesocellular mesoporous magnetic silica (M‐HMMS) through adsorption. The adsorption mechanism was investigated. Cellulase and xylanase adsorption took place on the external surface and inside the pores of M‐HMMS.
C‐glycosyltransferase (CGT) and sucrose synthase (SuSy), each fused to the cationic binding module Zbasic2, were co‐immobilized on anionic carrier (ReliSorb SP400) and assessed for continuous ...production of the natural C‐glycoside nothofagin. The overall reaction was 3ʹ‐C‐β‐glycosylation of the polyphenol phloretin from uridine 5ʹ‐diphosphate (UDP)‐glucose that was released in situ from sucrose and UDP. Using solid catalyst optimized for total (∼28 mg/g) as well as relative protein loading (CGT/SuSy = ∼1) and assembled into a packed bed (1 ml), we demonstrate flow synthesis of nothofagin (up to 52 mg/ml; 120 mM) from phloretin (≥95% conversion) solubilized by inclusion complexation in hydroxypropyl β‐cyclodextrin. About 1.8 g nothofagin (90 ml; 12–26 mg/ml) were produced continuously over 90 reactor cycles (2.3 h/cycle) with a space‐time yield of approximately 11 mg/(ml h) and a total enzyme turnover number of up to 2.9 × 103 mg/mg (=3.8 × 105 mol/mol). The co‐immobilized enzymes exhibited useful effectiveness (∼40% of the enzymes in solution), with limitations on the conversion rate arising partly from external liquid–solid mass transfer of UDP under packed‐bed flow conditions. The operational half‐life of the catalyst (∼200 h; 30°C) was governed by the binding stability of the glycosyltransferases (≤35% loss of activity) on the solid carrier. Collectively, the current study shows integrated process technology for flow synthesis with co‐immobilized sugar nucleotide‐dependent glycosyltransferases, using efficient glycosylation from sucrose via the internally recycled UDP‐glucose. This provides a basis from engineering science to promote glycosyltransferase applications for natural product glycosides and oligosaccharides.
Sugar nucleotide‐dependent (Leloir) glycosyltransferases are important enzymes of glycoside synthesis, but their development for flow chemistry applications based on heterogeneous bio‐catalysis is lacking. Here, the authors show continuous production of the natural C‐glycoside nothofagin using co‐immobilized C‐glycosyltransferase (Z‐OsCGT) and sucrose synthase (Z‐GmSuSy) in a packed‐bed flow reactor. Cascade reaction to glycosylate phloretin from glucose via intermediary UDP‐glucose is demonstrated at excellent yield, product concentration, space time yield (STY) and total turnover number of enzyme (TTN).
Glucose oxidase (GOX) and cholesterol oxidase (COX) are enzymes with numerous practical applications in medicine and industry. Although various methods for improving of enzyme stability have been ...proposed, we present novel approach for enzyme compartmentalization using polyelectrolytes layers deposited on electrospun fibers made of poly(D,L-lactide-co-glycolide) and commercial membrane UFX5. Compartmentalization has a series of advantages over simple co-immobilization offering providing of suitable microenvironment for the immobilized enzymes, ensuring proper substrate/product channeling and improving stability and reusability of biomolecules. The biosystems produced via compartmentalization, resulted in higher conversion efficiency of model reaction at 45 °C and 65 °C compared to free enzymes as well as negative effect of hydrogen peroxide was minimalized after immobilization. Moreover, after 5 catalytic cycles, biosystems based on UFX5 membrane and HRP or GOX or COX immobilized by compartmentalization reached efficiencies of 89% and 34%, respectively, with less than 10% enzyme elution from the support. Enzymes compartmentalized using ultrafiltration membrane showed also significant improvement of their biocatalytic productivity over repeated use, resulting in possible costs reduction of the desired process. Use of biosystems with electrospun fibers and co-immobilized enzymes resulted in enzyme elution of over 90% and very limited conversion efficiency.
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•Novel approach for simultaneous compartmentalization of three enzymes on membranes.•Compartmentalization yield of over 90% was noticed.•Compartmentalized enzymes showed over 90% of conversion efficiency.•Over 40% improvement of enzyme stability against thermal and H2O2 inactivation.•After 5 catalytic cycles UFX5/HRP/LbL/GOX retained 89% conversion efficiency.
The synergistic combination of current biotechnological and nanotechnological research has turned to multienzyme co-immobilization as a promising concept to design biocatalysis engineering. It has ...also intensified the development and deployment of multipurpose biocatalysts, for instance, multienzyme co-immobilized constructs, via biocatalysis/protein engineering to scale-up and fulfil the ever-increasing industrial demands. Considering the characteristic features of both the loaded multienzymes and nanostructure carriers, i.e., selectivity, specificity, stability, resistivity, induce activity, reaction efficacy, multi-usability, high catalytic turnover, optimal yield, ease in recovery, and cost-effectiveness, multienzyme-based green biocatalysts have become a powerful norm in biocatalysis/protein engineering sectors. In this context, the current state-of-the-art in enzyme engineering with a synergistic combination of nanotechnology, at large, and nanomaterials, in particular, are significantly contributing and providing robust tools to engineer and/or tailor enzymes to fulfil the growing catalytic and contemporary industrial needs. Considering the above critics and unique structural, physicochemical, and functional attributes, herein, we spotlight important aspects spanning across prospective nano-carriers for multienzyme co-immobilization. Further, this work comprehensively discuss the current advances in deploying multienzyme-based cascade reactions in numerous sectors, including environmental remediation and protection, drug delivery systems (DDS), biofuel cells development and energy production, bio-electroanalytical devices (biosensors), therapeutical, nutraceutical, cosmeceutical, and pharmaceutical oriented applications. In conclusion, the continuous developments in nano-assembling the multienzyme loaded co-immobilized nanostructure carriers would be a unique way that could act as a core of modern biotechnological research.
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•Immobilized multienzymatic systems represent a versatile platform for biocatalysis.•Prospective nano-carriers as host for immobilized multienzymatic systems are reviewed.•Biotechnological applications of immobilized multienzymatic systems are outlined.•Improvement of stability and reusability of the co-immobilized enzymes is emphasized.•Future trends for widespread applications of multienzymatic systems are highlighted.
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