Selective Covalent Protein Immobilization: Strategies and Applications

Wong, Lu Shin; Khan, F. Nawaz; Micklefield, Jason

doi:10.1021/cr8004668

Cited by 445 publications

(375 citation statements)

References 180 publications

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“…The G3-MNP, after functionalization with glutaraldehyde, was added to a Ser127Ala GpdQ mutant stock solution and after 5 days at 4 °C the nanoparticles were washed thoroughly with buffer to remove any unbound enzyme [33]. After this time a sufficient amount of enzyme was bound to the nanoparticles via amide formation between the lysine residues of the enzyme and the pendant aldehydes on the particles.…”

Section: Immobilization Of the Ser127ala Mutant Of Gpdqmentioning

confidence: 99%

Immobilization of the enzyme GpdQ on magnetite nanoparticles for organophosphate pesticide bioremediation

Daumann

Larrabee

Ollis

et al. 2014

Journal of Inorganic Biochemistry

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Annually thousands of people die or suffer from organophosphate (pesticide) poisoning. In order to remove these toxic compounds from the environment, the use of enzymes as bioremediators has been proposed. We report here a Ser127Ala mutant based on the enzyme glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes. The mutant, with improved metal binding abilities, has been immobilized using glutaraldehyde on PAMAM dendrimer-modified magnetite nanoparticles. The immobilized system was characterized using elemental analysis as well as infrared, transmission electron and X-ray photoelectron spectroscopies. The amount of GpdQ that was immobilized with the optimized procedure was 1.488 nmol per g MNP. A kinetic assay has been designed to evaluate the activity of the system towards organophosphoester substrates. The specific activity towards BPNPP directly after immobilization was 3.55 μmol mg -1 min -1 , after one week 3.39 μmol mg -1 min -1 and after 120 days 3.36 μmol mg -1 min -1 , demonstrating that the immobilized enzyme was active for multiple cycles and could be stored on the nanoparticles for a prolonged period.3

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Section: Immobilization Of the Ser127ala Mutant Of Gpdqmentioning

confidence: 99%

Immobilization of the enzyme GpdQ on magnetite nanoparticles for organophosphate pesticide bioremediation

Daumann

Larrabee

Ollis

et al. 2014

Journal of Inorganic Biochemistry

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“…The biotin-streptavidin interaction is known to be very strong (K d ≈ 10 −15 M) and represents an example of site-specific binding (23). To this end, SPBCL was used to write a square array of approximately 10-nm AuNPs with a 2-μm pitch.…”

Section: Resultsmentioning

confidence: 99%

Single-molecule protein arrays enabled by scanning probe block copolymer lithography

Chai¹,

Wong²,

Giam

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The ability to control the placement of individual protein molecules on surfaces could enable advances in a wide range of areas, from the development of nanoscale biomolecular devices to fundamental studies in cell biology. Such control, however, remains a challenge in nanobiotechnology due to the limitations of current lithographic techniques. Herein we report an approach that combines scanning probe block copolymer lithography with site-selective immobilization strategies to create arrays of proteins down to the single-molecule level with arbitrary pattern control. Scanning probe block copolymer lithography was used to synthesize individual sub-10-nm single crystal gold nanoparticles that can act as scaffolds for the adsorption of functionalized alkylthiol monolayers, which facilitate the immobilization of specific proteins. The number of protein molecules that adsorb onto the nanoparticles is dependent upon particle size; when the particle size approaches the dimensions of a protein molecule, each particle can support a single protein. This was demonstrated with both gold nanoparticle and quantum dot labeling coupled with transmission electron microscopy imaging experiments. The immobilized proteins remain bioactive, as evidenced by enzymatic assays and antigen-antibody binding experiments. Importantly, this approach to generate single-biomolecule arrays is, in principle, applicable to many parallelized cantilever and cantilever-free scanning probe molecular printing methods.dip-pen nanolithography | scanning probe lithography | single molecule array | gold nanoparticles | protein nanoarray P rotein immobilization on solid substrates with nanoscale control has been utilized in a variety of applications, including chip-based bioassays (1), proteomics (2, 3), drug discovery (3), and cellular biology studies (4). In cellular biology research, the ability to fabricate protein nanostructures on surfaces has enabled the study of many basic cellular functions including growth, signaling, and differentiation (4-7). For combinatorial molecular biology, the miniaturization of protein nanoarrays allows for smaller and higher density chips and the need for smaller sample volumes; in certain cases, this can translate into diagnostic systems with higher sensitivity and the ability to track disease and biological processes more efficiently (2, 3). The ability to site-specifically isolate single biomolecules can also facilitate molecular level studies of such structures (8, 9). Therefore, being able to nanofabricate biomolecular features at a resolution of 10 nm or less is of significant interest because this length scale approaches the dimensions of single protein molecules and offers an opportunity to address many previously unexplored biological phenomena.The use of dip-pen nanolithography (DPN) (4, 10) for the generation of arrays of biomolecules either by a direct deposition of proteins (11-13) or indirect methods (14, 15), through DPN writing of patterns followed by capture of proteins onto the patterns, has been widel...

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“…It has also reported about 288-fold increase in the thermal stability at 65°C, after immobilization of b-glucosidase on SiO 2 nanoparticles (Singh et al 2011). This transition in stability of immobilized b-glucosidase may be attributed to the multiple covalent bonds that alter the conformational flexibility and thermal vibrations, thus preventing the immobilized protein from unfolding and denaturing (Mateo et al 2000;Wong et al 2009). …”

Section: Ph and Thermal Stability Of Free And Immobilized B-glucosidasementioning

confidence: 99%

Immobilization of β-glucosidase onto silicon oxide nanoparticles and augment of phenolics in sugarcane juice

2016

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Purified b-glucosidase was immobilized on SiO 2 nanoparticles with 52 % efficiency and 14.1 % yield. It had a temperature optima at 60°C and pH optima of 5.0. Immobilized enzyme was fairly stable at 60-70°C. After immobilization, the K m value of b-glucosidase for p-nitrophenyl-b-D-glucopyranoside (pNPG) increased from 0.9 to 1.074 mM and V max decreased from 3.5 to 1.513 U/mg. The immobilized enzyme showed improved storage stability at temperature 4 and 25°C and was reusable for up to ten cycles with 70 % residual activity in pNPG and 60 % residual activity in sugarcane juice treatment. Sugarcane juice density, viscosity; surface tension etc. changed after treatment with immobilized b-glucosidase. b-Glucosidase treated sugarcane juice showed higher phenolics than untreated sugarcane juice. Caffeic acid which was absent in juice, was detected in b-glucosidase treated juice at a concentration of about 1 mg/L.

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Selective Covalent Protein Immobilization: Strategies and Applications

Cited by 445 publications

References 180 publications

Immobilization of the enzyme GpdQ on magnetite nanoparticles for organophosphate pesticide bioremediation

Immobilization of the enzyme GpdQ on magnetite nanoparticles for organophosphate pesticide bioremediation

Single-molecule protein arrays enabled by scanning probe block copolymer lithography

Immobilization of β-glucosidase onto silicon oxide nanoparticles and augment of phenolics in sugarcane juice

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