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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.
The increasing relevance of cascade reactions in biocatalysis has sparked a great interest for enzyme co-immobilization. Enzyme co-immobilization allows access to some kinetic advantages that in some ...instances are necessary to get the desired product, avoiding side-reactions. However, the kinetic effect is very relevant mainly at the initial reaction rates, while it may be less relevant in the whole reaction course, depending on the kinetic parameters of the involved enzymes. This review not only critically discusses the advantages but also the drawbacks of enzymes co-immobilization: immobilization on the same support and surface, under similar conditions, discarding the whole biocatalyst when one of the co-immobilized enzymes is inactivated. We will discuss when co-immobilization is almost compulsory, when the advantages of co-immobilization may not be enough to compensate their problems and when it should be fully discarded. The co-immobilization of cofactors and enzymes bears special interest, as this can open up the opportunity to the building of artificial cells and extremely complex one-pot transformations. Finally, some recent strategies to overcome some the co-immobilization problems will be presented.
•Enzyme co-immobilization raises many problems in biocatalysts design.•Enzyme co-immobilization has some kinetic advantages regarding initial rates.•Enzyme co-immobilization is, in certain cases, necessary.•Co-immobilization of enzymes and cofactors opens up the possibility of building “synthetic cells”.•Some strategies have been developed to solve the problems of enzymes co-immobilization.
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.
We propose a co-immobilized chemo-enzyme cascade system to mitigate random intermediate diffusion from the mixture of individual immobilized catalysts and achieve a one-pot reaction of multi-enzyme ...and reductant. Catalyzed by lipase and lipoxygenase, unsaturated lipid hydroperoxides (HPOs) were synthesized. 13(S)-hydroperoxy-9Z, 11E-octadecadienoic acid (13-HPODE), one compound of HPOs, was subsequently reduced to 13(S)-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) by cysteine. Upon the optimized conditions, 75.28 mg of 13-HPODE and 4.01 mg of 13-HODE were produced from per milliliter of oil. The co-immobilized catalysts exhibited improved yield compared to the mixture of individually immobilized catalysts. Moreover, it demonstrated satisfactory durability and recyclability, maintaining a relative HPOs yield of 78.5% after 5 cycles. This work has achieved the co-immobilization of lipase, lipoxygenase and the reductant cysteine for the first time, successfully applying it to the conversion of soybean oil into 13-HODE. It offers a technological platform for transforming various oils into high-value products.
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•The formation of metal-amino acid frameworks was facilitated by the addition of PVP and L-cysteine.•In a one-pot cascade reaction, soybean oil was successfully converted into 13-HPODE and 13-HODE.•The in-situ co-immobilization of lipase, lipoxygenase and the reductant L-cysteine has been accomplished within MOF.
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.
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•Taguchi design-assisted Co-immobilization of Lipase.•Co-immobilization of Lipase A and B from Candida antarctica onto Chitosan (CALA-CALB-CHI).•CALA-CALB-CHI derivative evaluated in ...the kinetic resolution of halohydrins acetates.•(S)-chlorohydrin 3b produced with 98% ee, conversion of 46% and E > 200.•Molecular docking was performed to elucidate the hydrolysis interaction reaction.
In the present communication, the simultaneous co-immobilization by covalent binding of lipase A from Candida antarctica (CALA) and lipase B from Candida antarctica (CALB) in glutaraldehyde (GLU) activated chitosan (CHI) was optimized using the Taguchi method. Under optimized conditions (pH 9, 5 mM, 6:1 (protein load/g of support and 1 h), it was possible to reach 80.00 ± 0.01% for the immobilization yield (IY) and 46.01 ± 0.35 U/g for the activity of the derivative (AtD); in this case, load protein and ionic strength were the only statistically significant parameters and, therefore, those that most influenced the immobilization process. Furthermore, at pH 7, CALA-CALB-CHI had a half-life 2–6 times longer than the mixture of CALA and CALB for a temperature range of 50−80 °C. CALA-CALB showed the highest activity at pH 7, whereas CALA-CALB-CHI, except at pH 7, was more active than the soluble lipase mixture in the pH range (5–9), especially at pH 9. CHI, CHI-GLU, and CALA-CALB-CHI were characterized by X-ray powder diffraction (XRPD), Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscope (SEM), Thermogravimetry (TGA), and Energy Dispersive Spectroscopy (EDS), proving the immobilization of CALA and CALB in chitosan. CALA-CALB-CHI derivative evaluated in the kinetic resolution of halohydrins acetates rac-2-bromo-1-(2-chlorophenyl) ethyl acetate (2a) and rac-2-chloro-1-(2,4-dichlorophenyl) ethyl acetate (2b), to produce the corresponding halohydrins 3a-b, which are intermediates in the synthesis of the drugs chlorprelanine (antiarrhythmic) and luliconazol (antifungal), respectively. (S)-bromohydrin 3a was obtained with 79% enantiomeric excess (ee), whereas (S)-chlorohydrin 3b produced with 98% ee, conversion of 46% and E > 200. Additionally, molecular docking was performed to elucidate the hydrolysis interaction reaction between β-halohydrin acetates and lipases CALA-CALB.
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