Enhanced catalytic stability and reusability of nitrilase encapsulated in ethyleneamine-mediated biosilica for regioselective hydrolysis of 1-cyanocycloalkaneacetonitrile

Xu, Zhe; Huang, Jiuzuo; Xia, Chao-Jie; Zou, Shu-Ping; Xue, Ya‐Ping; Zheng, Yu‐Guo

doi:10.1016/j.ijbiomac.2019.02.131

Cited by 20 publications

(6 citation statements)

References 58 publications

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Section: Introductionmentioning

confidence: 99%

“…28 Here, we demonstrate the encapsulation of the active integral membrane protein DGK in biomimetic silica with the aim to extend the use of functionally encapsulated IMPs to applications as catalysts in biotechnology or as sensors in biomedical applications, similar to the scope of applications for encapsulated soluble enzymes. 29,30 ■ RESULTS AND DISCUSSION R5-MSP-R5 Fusion Construct Enables the Formation of Silica-Encapsulated Nanodiscs. The silica-precipitating R5 peptide was genetically fused to both the N-and the Cterminus of the membrane scaffold protein (MSP, variant MSP1E3D1), 31 which stabilizes the lipid-bilayer nanodiscs in solution.…”

Section: ■ Introductionmentioning

confidence: 99%

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Biomimetic Silica Encapsulation of Lipid Nanodiscs and β-Sheet-Stabilized Diacylglycerol Kinase

Bialas

Becker

2021

Bioconjugate Chem.

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Integral membrane proteins (IMPs) comprise highly important classes of proteins such as transporters, sensors, and channels, but their investigation and biotechnological application are complicated by the difficulty to stabilize them in solution. We set out to develop a biomimetic procedure to encapsulate functional integral membrane proteins in silica to facilitate their handling under otherwise detrimental conditions and thereby extend their applicability. To this end, we designed and expressed new fusion constructs of the membrane scaffold protein MSP with silica-precipitating peptides based on the R5 sequence from the diatom Cylindrotheca fusiformis . Transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed that membrane lipid nanodiscs surrounded by our MSP variants fused to an R5 peptide, so-called nanodiscs, were formed. Exposing them to silicic acid led to silica-encapsulated nanodiscs, a new material for stabilizing membrane structures and a first step toward incorporating membrane proteins in such structures. In an alternative approach, four fusion constructs based on the amphiphilic β-sheet peptide BP-1 and the R5 peptide were generated and successfully employed toward silica encapsulation of functional diacylglycerol kinase (DGK). Silica-encapsulated DGK was significantly more stable against protease exposure and incubation with simulated gastric fluid (SGF) and intestinal fluid (SIF).

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Section: Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Biomimetic Silica Encapsulation of Lipid Nanodiscs and β-Sheet-Stabilized Diacylglycerol Kinase

Bialas

Becker

2021

Bioconjugate Chem.

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“…We sought to explore whether the substrate binding and chemistry functions of an enzymatic reaction could be partially separated by colocalization of a substrate-binding DNA aptamer and a substrate-converting enzyme inside the confines of a stable virus-like particle (VLP) shell. Encapsulation of enzymes in a wide variety of nanocompartments, including liposomes, polymersomes, nucleic acid cages, carbohydrates, hydrogels, mineral capsules, and VLPs, has been described, mostly to aid in enzyme stabilization. , In many of these cases, no or only modest decreases in enzyme activity (often defined as k cat / K M ) are observed while stability is enhanced. ,− However, few examples of rate improvement upon encapsulation have been reported, including the use of DNA nanostructures , and alginate- or carboxymethylcellulose-coated silica …”

Section: Introductionmentioning

confidence: 99%

Modular Catalysis: Aptamer Enhancement of Enzyme Kinetics in a Nanoparticle Reactor

et al. 2023

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Enzyme activity requires sequential binding and chemical transformation of substrates. While directed evolution and random mutagenesis are common methods for improving catalytic activity, these methods do not allow for independent control of K M and k cat. To achieve such control, we envisioned that the colocalization of aptamers and enzymes that act on the same molecule could increase catalytic efficiency through preconcentration of substrate. We explored this concept with cocaine esterase and anticocaine aptamers having varying K D values, both encapsulated in MS2 virus-like particles. Rate enhancements were observed with magnitudes dependent on both aptamer:enzyme stoichiometry and aptamer K D, peaking when aptamer K D and enzyme K M were roughly equivalent. This beneficial effect was lost when either aptamer binding was too tight or the aptamers were not constrained to be close to the catalyst. This work demonstrates a modular way to enhance catalysis by independently controlling substrate capture and release to the processing enzyme.

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“…[3][4][5][6] To satisfy the requirements of industrial application, robust nitrilases with excellent thermostability and activity are crucial to resist harsh reaction conditions. 7 However, except for few nitrilases from thermophilic and hyperthermophilic organisms, the majority of nitrilases suffer from rapid inactivation under practical operation conditions and thereby hinder their extensive industrial applications. 1 Although great efforts have been devoted to improving nitrilase thermostability, including directed evolution, identification of novel enzymes from hyperthermophile and enzyme immobilization, [7][8][9] deficiency of precise structure-function information plagued the development of efficient strategies for the evolution of thermostable nitrilases.…”

Section: Introductionmentioning

confidence: 99%

Engineering residues on C interface to improve thermostability of nitrilase for biosynthesis of Pregabalin precursor

Tang

et al. 2023

AIChE Journal

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Nitrilase‐mediated bioprocesses exhibited great potential in the production of value‐added carboxylic acids. However, poor thermostability of nitrilases usually restricts their industrial applications. Herein, the thermostability of nitrilase BaNITM0 was significantly improved by engineering the amino acid residues on the intersection of two dimers (C interface). Except for simultaneous enhancement of enantioselectivity and activity, the best variant V82L/M127I/L159M/F166Q/C237S/Q260H (BaNITM4) showed a 10.8‐fold increase in half‐life at 30°C compared with BaNITM0. Structural analysis demonstrated that additional hydrogen bonds were formed between the residues on the C interface, which strengthened the interactions of two symmetrical regions and spirals. Subsequently, the engineered nitrilase was immobilized onto epoxy resins LXTE‐603 and the immobilized nitrilase exhibited excellent stability over 12 repeated cycles, which indicated a great industrial potential for biosynthesis of Pregabalin precursor.

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Enhanced catalytic stability and reusability of nitrilase encapsulated in ethyleneamine-mediated biosilica for regioselective hydrolysis of 1-cyanocycloalkaneacetonitrile

Cited by 20 publications

References 58 publications

Biomimetic Silica Encapsulation of Lipid Nanodiscs and β-Sheet-Stabilized Diacylglycerol Kinase

Biomimetic Silica Encapsulation of Lipid Nanodiscs and β-Sheet-Stabilized Diacylglycerol Kinase

Modular Catalysis: Aptamer Enhancement of Enzyme Kinetics in a Nanoparticle Reactor

Engineering residues on C interface to improve thermostability of nitrilase for biosynthesis of Pregabalin precursor

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