Biocatalyzed mineralization in an aqueous two-phase system: effect of background polymers and enzyme partitioning

Cacace, David N.; Keating, Christine D.

doi:10.1039/c3tb00550j

Cited by 31 publications

(44 citation statements)

References 89 publications

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“…We previously reported on this mineralization system in an unstabilized PEG/dextran ATPS with less extreme volume ratios (1:1 to 1:9), where we compared reaction rates in the different phases to buffer alone and found both that the reaction was nearly quantitatively contained to the dextran-rich phase and that the resulting CaCO 3 material was not greatly impacted by the presence of the PEG and dextran polymers. 34 The PEG and dextran polymers used to generate the ATPS are nonionic and do not bind 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 Based on their binding affinities for Ca 2+ , GLDA, EDTA and EDDS were expected to prevent LUV aggregation by Ca 2+ . We tested this prediction by imaging LUV-stabilized droplet samples that 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 3...…”

Section: Resultsmentioning

confidence: 97%

Aqueous Emulsion Droplets Stabilized by Lipid Vesicles as Microcompartments for Biomimetic Mineralization

et al. 2015

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Mineral deposition within living cells relies on control over the distribution and availability of precursors as well as the location and rates of nucleation and growth. This control is provided in large part by biomolecular chelators, which bind precursors and regulate their availability, and compartmentalization within specialized mineralizing vesicles. Biomimetic mineralization in self-assembled lipid vesicles is an attractive means of studying the mineralization process, but has proven challenging due to vesicle heterogeneity in lamellarity, contents, and size across a population, difficulties encapsulating high and uniform precursor concentrations, and the need to transport reagents across an intact lipid bilayer membrane. Here, we report the use of liposome-stabilized all-aqueous emulsion droplets as simple artificial mineralizing vesicles (AMVs). These biomimetic microreactors allow the entry of precursors while retaining a protein catalyst by equilibrium partitioning between internal and external polymer-rich phases. Small molecule chelators with intermediate binding affinity were employed to control Ca(2+) availability during CaCO3 mineralization, providing protection against liposome aggregation while allowing CaCO3 formation. Mineral deposition was limited to the AMV interior, due to localized production of CO3(2-) by compartmentalized urease. Particle formation was uniform across the entire population of AMVs, with multiple submicrometer amorphous CaCO3 particles produced in each one. The all-aqueous emulsion-based approach to biomimetic giant mineral deposition vesicles introduced here should be adaptable for enzyme-catalyzed synthesis of a wide variety of materials, by varying the metal ion, enzyme, and/or chelator.

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Section: Resultsmentioning

confidence: 97%

Aqueous Emulsion Droplets Stabilized by Lipid Vesicles as Microcompartments for Biomimetic Mineralization

et al. 2015

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“…Aqueous two-phase systems (ATPS), such as the well-known PEG/dextran system 12 , are attractive as biomimetic media because they offer coexisting macromolecularly crowded aqueous compartments with different chemical and physical properties separated by a low-energy boundary, across which solutes such as proteins or nucleic acids can equilibrate without denaturation 10,13,14 . This is similar to biological fluids such as the cytoplasm and nucleoplasm, which contain 17-40 wt% biomacromolecules 15,16 .…”

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confidence: 99%

Bioreactor droplets from liposome-stabilized all-aqueous emulsions

et al. 2014

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Artificial bioreactors are desirable for in vitro biochemical studies and as protocells. A key challenge is maintaining a favourable internal environment while allowing substrate entry and product departure. We show that semipermeable, size-controlled bioreactors with aqueous, macromolecularly crowded interiors can be assembled by liposome stabilization of an all-aqueous emulsion. Dextran-rich aqueous droplets are dispersed in a continuous polyethylene glycol (PEG)-rich aqueous phase, with coalescence inhibited by adsorbed B130-nm diameter liposomes. Fluorescence recovery after photobleaching and dynamic light scattering data indicate that the liposomes, which are PEGylated and negatively charged, remain intact at the interface for extended time. Inter-droplet repulsion provides electrostatic stabilization of the emulsion, with droplet coalescence prevented even for submonolayer interfacial coatings. RNA and DNA can enter and exit aqueous droplets by diffusion, with final concentrations dictated by partitioning. The capacity to serve as microscale bioreactors is established by demonstrating a ribozyme cleavage reaction within the liposome-coated droplets.

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“…For example, compared to a polymer free solution, RNA catalysis by the hammerhead ribozyme was increased approximately 70-fold when partitioned preferentially into the dextran-rich phase of a polyethylene glycol/ dextran phase-separated system [60 ]. Similarly, urease activity was significantly increased by preferential sequestration into the dextran phase, and could be further regulated by tuning the relative volumes of the polyethylene glycol/dextran phases [70]. In other studies, enhanced levels of gene expression were reported in a cell lysate that was subjected to phase separation in polyethylene glycol-rich droplets by in situ dehydration of the aqueous phase within a microfluidic device [71 ].…”

Section: Membrane-free Protocell Modelsmentioning

confidence: 91%

Synthetic cellularity based on non-lipid micro-compartments and protocell models

Huang

Tang

et al. 2014

Current Opinion in Chemical Biology

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Biocatalyzed mineralization in an aqueous two-phase system: effect of background polymers and enzyme partitioning

Cited by 31 publications

References 89 publications

Aqueous Emulsion Droplets Stabilized by Lipid Vesicles as Microcompartments for Biomimetic Mineralization

Aqueous Emulsion Droplets Stabilized by Lipid Vesicles as Microcompartments for Biomimetic Mineralization

Bioreactor droplets from liposome-stabilized all-aqueous emulsions

Synthetic cellularity based on non-lipid micro-compartments and protocell models

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