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Methods in Enzymology

C M Riccardi, C V Kumar
No abstract text is available yet for this article.
2017: Methods in Enzymology
Cameron Williams, Melissa L Dougherty, Katherine Makaroff, Jacob Stapleton, Dominik Konkolewicz, Jason A Berberich, Richard C Page
Protein-polymer conjugates are increasingly viewed as promising avenues to producing industrial enzymes with high activity capable of withstanding potentially harsh reaction conditions, or to designing novel therapeutics with triggered release, controlled masking, or increased resistance to proteolytic degradation. Common among these applications are the desire to improve the stability of protein-polymer conjugates to unfolding by exposure to chemicals or thermal stress. Thus, assays that allow researchers to robustly and easily characterize protein-polymer conjugates by obtaining thermodynamic parameters for folding stand to play an important role in the development of improved protein-polymer conjugates...
2017: Methods in Enzymology
M Rita Correro, Sabine Sykora, Philippe F-X Corvini, Patrick Shahgaldian
The availability of highly stable and reusable enzymes is one of the main challenges in bio-based industrial processes. Enzyme immobilization and encapsulation represent promising strategies to reach this goal. In this chapter, the synthetic strategy to produce hybrid organic/inorganic nanobiocatalysts (NBC) is reported. This strategy is based on the sequential immobilization of an enzyme on the surface of silica nanoparticles followed by the growth, at the surface of the nanoparticles, of a shielding layer which serves as an armor to protect the enzyme against denaturation/degradation...
2017: Methods in Enzymology
Miao Hou, Jun Ge
Enzymatic processes have great potential for applications in industrial biocatalysis, biosensing, and biomedical engineering. However, poor stability and difficulty in the reuse of enzymes still create challenges for their use in catalysis and other applications. In recent years, enzyme-incorporated metal-organic frameworks (enzyme@MOF) have emerged as a new type of armored enzymes and are thought to be an appealing solution to the above challenges. To date, the preparation of enzyme-MOF composites can be divided into three categories, including physical adsorption, covalent conjugation, and coprecipitation...
2017: Methods in Enzymology
Caterina M Riccardi, Rajeswari M Kasi, Challa V Kumar
A simple method for interlocking glucose oxidase (GOx) and horseradish peroxidase (HRP) in cellulose fibers using poly(acrylic acid) (PAA) as an armor around the enzyme, without any need for activation of the cellulose support, is reported here. The resulting enzyme paper is an inexpensive, stable, simple, wearable, and washable biosensor. PAA functions as a multifunctional tether to interlock the enzyme molecules around the paper fibers so that the enzymes are protected against thermal/chemical denaturation and not released from the paper when washed with a detergent...
2017: Methods in Enzymology
Andreas Küchler, Daniel Messmer, A Dieter Schlüter, Peter Walde
Dendronized polymer-enzyme conjugates are large, water-soluble macromolecular structures built from a linear, fully synthetic, dendronized polymer (denpol), and several copies of enzyme molecules covalently bound to the peripheral functional groups of the denpol. Since denpol chains comprise repeating units with regularly branched side chains (dendrons), denpols have a cylindrical shape and are much thicker than conventional linear polymers. Depending on the dendron generation and chemical structure, denpols may have a large number of functional groups on their surface, exposed to the aqueous medium in which they are dissolved...
2017: Methods in Enzymology
Melanie Leurs, Joerg C Tiller
The properties of enzymes can be altered significantly by modification with polymers. Numerous different methods are known to obtain such polymer-enzyme conjugates (PECs). However, there is no universal method to render enzymes into PECs that are fully soluble in organic solvents. Here, we present a method, which achieves such high degree of modification of proteins that the majority of modified enzymes will be soluble in organic solvents. This is achieved by preparing poly(2-alkyloxazoline)s (POx) with an NH2 end group and coupling this functional polymer via pyromellitic acid dianhydride onto the amino groups of the respective protein...
2017: Methods in Enzymology
Kishore Raghupathi, Sankaran Thayumanavan
The formulation in which therapeutic proteins are administered plays a key role in retaining their biological activity. Enzyme wrapping, using synthetic polymers, is a strategy employed to provide enzymes with lower immunogenicity, longer circulation times, and better targeting capabilities. Protein-polymer complexation methods, involving covalent, noncovalent, and electrostatic interactions, that can provide means to develop formulations for retaining enzyme stability are discussed in this chapter. Amphiphilic self-cross-linkable polymer was used to encapsulate capsase-3 enzyme in the nanogel, while inverse emulsion polymerization method was used to entrap α-glucosidase enzyme in the nanogel...
2017: Methods in Enzymology
Sheiliza Carmali, Hironobu Murata, Chad Cummings, Krzysztof Matyjaszewski, Alan J Russell
Atom transfer radical polymerization (ATRP) from the surface of a protein can generate remarkably dense polymer shells that serve as armor and rationally tune protein function. Using straightforward chemistry, it is possible to covalently couple or display multiple small molecule initiators onto a protein surface. The chemistry is fine-tuned to be sequence specific (if one desires a single targeted site) at controlled density. Once the initiator is anchored on the protein surface, ATRP is used to grow polymers on protein surface, in situ...
2017: Methods in Enzymology
Anindita Das, Abhijit Chakrabarti, Puspendu K Das
Recent developments in second harmonic light scattering technique and the associated theoretical models have provided a deeper insight of molecular interactions on micro- and nanoparticle surfaces. This technique is extended to probe the thermodynamics of protein adsorption on nanoparticle surface which is crucial for understanding the fate of nanoparticle-based formulations in biomedical applications. A modified Langmuir adsorption model has been applied to extract the thermodynamic parameters from the experimental data...
2017: Methods in Enzymology
Antonella Grigoletto, Anna Mero, Katia Maso, Gianfranco Pasut
PEGylation, the covalent attachment of polyethylene glycol to bioactive molecules, is one of the leading approaches used to prolong pharmacokinetics, to improve the stability, and to reduce the immunogenicity of therapeutic proteins. PEG-conjugated products are associated with better therapy outcomes and improved patient compliance. Widely applied in clinical practice, the technology is mainly used to modify proteins, peptides, and oligonucleotides but also other drug delivery systems such as the liposomal one...
2017: Methods in Enzymology
Jose M Palomo
This chapter describes the rational design and synthesis of semisynthetic lipases by site-directed incorporation of tailor-made peptides on the lipase-lid site to improve its activity, specificity, and enantioselectivity in specific biotransformations. Cysteine was genetically introduced at a particular point of the oligopeptide lid of the enzyme, and cysteine-containing peptides, complementary to the amino acid sequence on the lid site of Geobacillus thermocatenulatus lipase (BTL), were covalently attached on the lid of two different cysteine-BTL variants based on a fast thiol-disulfide exchange ligation followed by desulfurization...
2017: Methods in Enzymology
Nisaraporn Suthiwangcharoen, Ramanathan Nagarajan
The creation of polymer nanoparticles with protein functionality is of great interest to many applications such as targeted drug or gene delivery, diagnostic imaging, cancer theranostics, delivery of protein therapeutics, sensing chemical and biomolecular analytes in complex environments, and design of protective clothing resembling a second skin. Many approaches to achieving this goal are being explored in the current literature. In this chapter, we describe a relatively simple and flexible approach of conjugating the protein to an amphiphilic block copolymer and creating polymer nanoparticles with protein functionality by taking advantage of the intrinsic self-assembly behavior of the amphiphilic block copolymer...
2017: Methods in Enzymology
Priya Katyal, Yongkun Yang, Olga Vinogradova, Yao Lin
The formation of higher-order assemblies of multiple proteins or enzymes is a general mechanism to achieve more sophisticated biological function in biological systems. For example, cellulosomes are large complexes consisting of multiple cellulolytic enzymes that rely on the concerted actions of different enzymes built onto a common protein scaffold to facilitate the breakdown of the polymeric substrate, cellulose. One strategy for mimicking these highly effective nanomachines may involve the use of synthetic scaffolds that can react to and organize multiple engineered enzymes to promote synergistic action between the enzymes on the scaffold...
2017: Methods in Enzymology
Gayan Premaratne, Leslie Coats, Sadagopan Krishnan
Polymer-armored enzymes loaded onto magnetic nanoparticles, as efficient nanobioreactors with enhanced properties, are described in this chapter. Polymers are useful macromolecules carrying a large number of surface charges and repeating units of desired chemical functional groups for linking enzymes onto them. Magnetic micro/nanoparticles have been widely used as enzyme carriers with the incorporation of suitable polymer layers. Synthesized iron oxide magnetic nanoparticles have been used to immobilize a peroxide-catalyzing enzyme-like heme protein: myoglobin using covalent and noncovalent strategies...
2017: Methods in Enzymology
Matthew Paeth, Jacob Stapleton, Melissa L Dougherty, Henry Fischesser, Jerry Shepherd, Matthew McCauley, Rebecca Falatach, Richard C Page, Jason A Berberich, Dominik Konkolewicz
A series of methods are outlined for attaching functional polymers to proteins. Polymers with good control over structure, functionality, and composition can be created using reversible addition-fragmentation chain transfer (RAFT) polymerization. These polymers can be covalently linked to enzymes and proteins using either the "grafting-to" approach, where a preformed polymer is attached to the protein surface, or the "grafting-from" approach, where the polymer is grown from the protein surface. Methods for grafting-to, or attaching the RAFT chain transfer agent to the protein surface outlined include the commonly used carbodiimide/activated ester (EDC/NHS) coupling...
2017: Methods in Enzymology
Omkar V Zore, Rajeswari M Kasi, Challa V Kumar
Synthesis and characterization of highly stable and functional bienzyme-polymer triads assembled on layered graphene oxide (GO) are described here. Glucose oxidase (GOx) and horseradish peroxidase (HRP) were used as model enzymes and polyacrylic acid (PAA) as model polymer to armor the enzymes. PAA-armored GOx and HRP covalent conjugates were further protected from denaturation by adsorption onto GO nanosheets. Structure and morphology of this enzyme-polymer-nanosheet hybrid biocatalyst (GOx-HRP-PAA/GO) were confirmed by agarose gel electrophoresis, zeta potential, circular dichroism, and transmission electron microscopy...
2017: Methods in Enzymology
Konda R Kunduru, S N Raju Kutcherlapati, Dhamodaran Arunbabu, Tushar Jana
Jack bean urease is an important enzyme not only because of its numerous uses in medical and other fields but also because of its historical significance-the first enzyme to be crystallized and also the first nickel metalloenzyme. This enzyme hydrolyzes urea into ammonia and carbon dioxide; however, the stability of this enzyme at ambient temperature is a bottleneck for its applicability. To improve urease stability, it was immobilized on different substrates, particularly on polymeric hydrogels. In this study, the enzyme was coupled covalently with poly(acrylamide) hydrogel with an yield of 18μmol/cm(3)...
2017: Methods in Enzymology
Jingjing Xu, Paulina X Medina-Rangel, Karsten Haupt, Bernadette Tse Sum Bui
Molecularly imprinted polymers (MIPs) are synthetic antibody mimics possessing specific cavities designed for a target molecule. Nowadays, molecular imprinting of proteins still remains a challenge as the generation of selective imprinted cavities is extremely difficult, due to their flexible structure and the presence of a multitude of functional sites. To overcome this difficulty, we propose a solid-phase synthesis strategy to prepare MIPs specific for any protein that can be immobilized in an oriented way on a solid support...
2017: Methods in Enzymology
Yi Liu, Aoneng Cao
Encapsulation of proteins in nanoparticles (NPs) can greatly improve the properties of proteins such as their stability against denaturation and degradation by proteases, and branches out the applications of natural proteins from their intrinsic localizations and functions in living organisms for biomedical and industrial applications. We recently developed several methods to armor proteins in NPs with sizes from nanometers up to >100nm, batch by batch or one by one, covalently or noncovalently, for a wide range of applications from biocatalysis to bioimaging and drug delivery...
2017: Methods in Enzymology
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