One‐step selective affinity purification and immobilization of His‐tagged enzyme by recyclable magnetic nanoparticles

Zhou, Li-Jian; Li, Ruifang; Li, Xueyong; Zhang, Ye-Wang

doi:10.1002/elsc.202000093

Cited by 23 publications

(16 citation statements)

References 49 publications

(55 reference statements)

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“…Nonetheless, the immobilization enabled repeatable measurements in a flow injection system for over three days of extensive use at 60 tests/day [79]. Other methods enabling controlled quantitative enzyme immobilization, such as exploiting the nickel-histidine affinity in the case of His-tagged enzymes [84], immobilization on nanomaterials with high loading capacities [85], enzyme nanoflowers [86], CLEAs, or other recent strategies [83] could lead to better performance of immobilized F-ALDH.…”

Section: Kinetics Of the Recombinant F-aldhmentioning

confidence: 99%

Highly Stable, Cold-Active Aldehyde Dehydrogenase from the Marine Antarctic Flavobacterium sp. PL002

et al. 2021

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Stable aldehyde dehydrogenases (ALDH) from extremophilic microorganisms constitute efficient catalysts in biotechnologies. In search of active ALDHs at low temperatures and of these enzymes from cold-adapted microorganisms, we cloned and characterized a novel recombinant ALDH from the psychrotrophic Flavobacterium PL002 isolated from Antarctic seawater. The recombinant enzyme (F-ALDH) from this cold-adapted strain was obtained by cloning and expressing of the PL002 aldH gene (1506 bp) in Escherichia coli BL21(DE3). Phylogeny and structural analyses showed a high amino acid sequence identity (89%) with Flavobacterium frigidimaris ALDH and conservation of all active site residues. The purified F-ALDH by affinity chromatography was homotetrameric, preserving 80% activity at 4 °C for 18 days. F-ALDH used both NAD+ and NADP+ and a broad range of aliphatic and aromatic substrates, showing cofactor-dependent compensatory KM and kcat values and the highest catalytic efficiency (0.50 µM−1 s−1) for isovaleraldehyde. The enzyme was active in the 4–60 °C-temperature interval, with an optimal pH of 9.5, and a preference for NAD+-dependent reactions. Arrhenius plots of both NAD(P)+-dependent reactions indicated conformational changes occurring at 30 °C, with four(five)-fold lower activation energy at high temperatures. The high thermal stability and substrate-specific catalytic efficiency of this novel cold-active ALDH favoring aliphatic catalysis provided a promising catalyst for biotechnological and biosensing applications.

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Section: Kinetics Of the Recombinant F-aldhmentioning

confidence: 99%

Highly Stable, Cold-Active Aldehyde Dehydrogenase from the Marine Antarctic Flavobacterium sp. PL002

et al. 2021

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“…Lower V max values after enzyme immobilization were similarly reported before [33,81]. In another study, Zhou et al reported about a 6.7-fold decrease in the V max value after the immobilization of a His-tagged recombinant glucose dehydrogenase (GluDH) on NiFe 2 O 4 magnetic nanoparticles [74].…”

Section: Comparison Of Free and Immobilized Gdo Kinetic Parametersmentioning

confidence: 77%

“…One-step immobilization and purification methods have been applied before for a Histagged transaminase [41], a histidine-tagged green fluorescent protein [72], an extracellular His-tagged Thermomyces lanuginosus lipase [73], and even for the co-immobilization of two components of hydroxylase monooxygenase that were employed to construct a bienzymatic catalytic system [46]. Moreover, Zhou et al studied the selective purification and immobilization of a His-tagged recombinant glucose dehydrogenase onto NiFe 2 O 4 magnetic nanoparticles in a one-step procedure [74]. The abovementioned methodology allowed for the selective purification of the recombinant GDO through immobilization on the nanocarriers.…”

Section: Immobilization Of Recombinant Gentisate 12-dioxygenasementioning

confidence: 99%

Characterization of Gentisate 1,2-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and Its Stabilization by Immobilization on Nickel-Functionalized Magnetic Nanoparticles

Asimakoula

Giannakopoulou

Lappa

et al. 2022

Applied Microbiology

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The aim of this study was the biochemical and kinetic characterization of the gentisate 1,2-dioxygenase (GDO) from Pseudarthrobacter phenanthrenivorans Sphe3 and the development of a nanobiocatalyst by its immobilization on Ni2+-functionalized Fe3O4-polydopamine magnetic nanoparticles (Ni2+-PDA-MNPs). This is the first GDO to be immobilized. The gene encoding the GDO was cloned with an N-terminal His-tag and overexpressed in E. coli. The nanoparticles showed a high purification efficiency of GDO from crude cell lysates with a maximum activity recovery of 97%. The immobilized enzyme was characterized by Fourier transform infrared spectroscopy (FTIR). The reaction product was identified by 1H NMR. Both free and immobilized GDO exhibited Michaelis–Menten kinetics with Km values of 25.9 ± 4.4 and 82.5 ± 14.2 μM and Vmax values of 1.2 ± 0.1 and 0.03 ± 0.002 mM*s−1, respectively. The thermal stability of the immobilized GDO was enhanced at 30 °C, 40 °C, and 50 °C, compared to the free GDO. Stored at −20 °C, immobilized GDO retained more than 60% of its initial activity after 30 d, while the free enzyme completely lost its activity after 10 d. Furthermore, the immobilized nanoparticle–enzyme conjugate retained more than 50% enzyme activity up to the fifth cycle.

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“…Adsorption and ionic binding are quite simple and low-cost methods, but non-specific. Affinity binding is highly selective, enables one pot purification and immobilization and allows oriented enzyme immobilization, yet it is costly [44,[65][66][67][68][69]. Chemical attachment to the carrier involves covalent binding through amide, carbamate, ether or thio-ether bonds established between groups of suitable residues on the enzyme surface and on the carrier (Figure 1d 1 ) [36,67].…”

Section: Classification Of Immobilization Methods and Their Key Featuresmentioning

confidence: 99%

Enzyme Immobilization and Co-Immobilization: Main Framework, Advances and Some Applications

et al. 2022

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Enzymes are outstanding (bio)catalysts, not solely on account of their ability to increase reaction rates by up to several orders of magnitude but also for the high degree of substrate specificity, regiospecificity and stereospecificity. The use and development of enzymes as robust biocatalysts is one of the main challenges in biotechnology. However, despite the high specificities and turnover of enzymes, there are also drawbacks. At the industrial level, these drawbacks are typically overcome by resorting to immobilized enzymes to enhance stability. Immobilization of biocatalysts allows their reuse, increases stability, facilitates process control, eases product recovery, and enhances product yield and quality. This is especially important for expensive enzymes, for those obtained in low fermentation yield and with relatively low activity. This review provides an integrated perspective on (multi)enzyme immobilization that abridges a critical evaluation of immobilization methods and carriers, biocatalyst metrics, impact of key carrier features on biocatalyst performance, trends towards miniaturization and detailed illustrative examples that are representative of biocatalytic applications promoting sustainability.

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One‐step selective affinity purification and immobilization of His‐tagged enzyme by recyclable magnetic nanoparticles

Cited by 23 publications

References 49 publications

Highly Stable, Cold-Active Aldehyde Dehydrogenase from the Marine Antarctic Flavobacterium sp. PL002

Highly Stable, Cold-Active Aldehyde Dehydrogenase from the Marine Antarctic Flavobacterium sp. PL002

Characterization of Gentisate 1,2-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and Its Stabilization by Immobilization on Nickel-Functionalized Magnetic Nanoparticles

Enzyme Immobilization and Co-Immobilization: Main Framework, Advances and Some Applications

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