An update on smart biocatalysts for industrial and biomedical applications (original) (raw)
Related papers
Enzyme immobilization on smart polymers: Catalysis on demand
Reactive and Functional Polymers, 2014
A new approach for the synthesis of hydrogel films with thermo-sensitive enzymatic activity is reported. Pepsin (PEP) was covalently immobilized on thermo-responsive hydrogels by radical polymerization in the presence of N-isopropylacrylamide and poly-(ethylene glycol) dimethacrylate 750, acting as functional monomer and crosslinking agent, respectively. Hydrogels showing lower critical solution temperatures between 32.9 and 36.1°C were synthesized by UV-irradiation of reaction batches differing in the PEP/monomers ratio. The derivatization degree of the hydrogels was expressed as mg of PEP per gram of matrix and found to be in the range of 6 to 11% as assessed by Lowry method. Scanning electron microscopy analysis and water affinity evaluation allowed to highlight the porous morphology and thermo-responsivity of hydrogels as a function of temperature. Using bovine serum albumin as a substrate, kinetics parameters were determined by Lineweaver-Burk plots and the catalyst efficiency evaluated. The influence of temperature on enzyme activity, as well as the thermal stability and reusability of devices, were also investigated.
Novel enzyme-polymer conjugates for biotechnological applications
PeerJ, 2013
In the present research, a rapid, simple and efficient chemoselective method for the site-directed incorporation of tailor-made polymers into protein to create biocatalysts with excellent properties for pharmaceutical industrial purpose has been performed. First we focused on the protein engineering of the Geobacillus thermocatenulatus lipase 2 (BTL2) to replace the two cysteines (Cys65, Cys296) in the wild type enzyme (BTL-WT) by two serines. Then, by similar mode, a unique cysteine was introduced in the lid area of the protein. For the site-directed polymer incorporation, a set of different tailor-made thiol-ionic-polymers were synthesized and the protein cysteine was previously activated with 2,2-dithiodipyridine (2-PDS) to allow the disulfide exchange. The protected BTL variants were specifically modified with the different polymers in excellent yields, creating a small library of new biocatalysts. Different and important changes in the catalytic properties, possible caused by structural changes in the lid region, were observed. The different modified biocatalysts were tested in the synthesis of intermediates of antiviral and antitumor drugs, like nucleoside analogues and derivatives of phenylglutaric acid. In the hydrolysis of per-acetylated thymidine, the best biocatalyst was the BTL*-193-DextCOOH , where the activity was increased in 3-fold and the regioselectivity was improved, reaching a yield of 92% of 3'-O-acetyl-thymidine. In the case of the asymmetric hydrolysis of dimethyl phenylglutarate, the best result was found with BTL*-193-DextNH2-6000, where the enzyme activity was increased more than 5-fold and the enantiomeric excess was >99%.
NANOTECHNOLOGIES FOR ENZYME-BASED BIOSENSORS
Inorganic and organic hybrid nanomaterials are a fascinating research area, due to the highly promising potential for versatile properties provoked by combining the merits of both sources and by the nanometer size effect, which is entirely different from that in a bulk material. Moreover, a coupling of biorecognition elements on nanostructures might allow a creation of functional hybrid systems with molecular-scale proximity between the molecular recognition and transduction element. Environment, molecular biology, medicine and other life sciences require for a new class of devices having a fast sensitive and reliable response for investigating biomolecular interactions. This need has fuelled a revolution of a new class of sensors, biosensors that are the product of the integration of nanotechnology, biology, and advanced materials. The possibility to employ new materials and innovative technologies allows realizing conceptually new devices, potentially competitive to the existing i...
Expanding the Biocatalytic Scope of Enzyme-Loaded Polymeric Hydrogels
Gels
In recent years, polymeric hydrogels have appeared promising matrices for enzyme immobilization to design, signify and expand bio-catalysis engineering. Therefore, the development and deployment of polymeric supports in the form of hydrogels and other robust geometries are continuously growing to green the twenty-first-century bio-catalysis. Furthermore, adequately fabricated polymeric hydrogel materials offer numerous advantages that shield pristine enzymes from denaturation under harsh reaction environments. For instance, cross-linking modulation of hydrogels, distinct rheological behavior, tunable surface entities along with elasticity and mesh size, larger surface-volume area, and hydrogels’ mechanical cushioning attributes are of supreme interest makes them the ideal candidate for enzyme immobilization. Furthermore, suitable coordination of polymeric hydrogels with requisite enzyme fraction enables pronounced loading, elevated biocatalytic activity, and exceptional stability. A...
ACS Omega
The aim of this study is to develop efficient enzyme immobilization media that will enable the reuse of the biocatalysts over multiple cycles, increase their thermal stability, and attenuate their activity toward hydrophobic substrates for "green" transformations in aqueous media. For this purpose, amphiphilic AB and ABA block copolymers were synthesized and tested with laccase (a multicopper oxidase). In all cases, the hydrophilic B block consisted of poly(ethylene glycol), PEG, with molecular masses of 3, 5, 13, 20, or 13 kDa poly(ethylene oxide). The hydrophobic A blocks were made of linear poly(styrene), PS; hyperbranched poly(p-chloromethyl styrene); or dendritic poly(benzyl ether)s of generations 2, 3, and 4 (G2, G3, and G4) with molecular masses ranging from 1 to 24 kDa. A total of 23 different copolymers (self-assembling into micelles or physical networks) were evaluated. Notable activity enhancements were achieved with both micelles (up to 253%) and hydrogels (up to 408%). The highest enzymatic activity and thermal stability were observed with laccase immobilized in hydrogels consisting of the linear ABA block copolymer PS2.7k− PEG3k−PS2.7k (13 290 μkat/L, 65°C, ABTS test). This represents a 1245% improvement over native laccase at the same conditions. At 25°C, the same complex showed a 1236% higher activity than the enzyme. The highest polymerization yield for a water-insoluble monomer was achieved with laccase immobilized in hydrogels composed of linear−dendritic ABA copolymer G3−PEG5k−G3 (85.5%, 45°C, tyrosine monomer). The broad substrate specificity and reusability of the immobilized laccase were also demonstrated by the successful discoloration of bromophenol blue, methyl orange, and rhodamine B over eight repetitive cycles.
Sensors, 2012
Different classes of polymeric materials such as nanomaterials, sol-gel materials, conducting polymers, functional polymers and biomaterials have been used in the design of sensors and biosensors. Various methods have been used, for example from direct adsorption, covalent bonding, crossing-linking with glutaraldehyde on composites to mixing the enzymes or use of functionalized beads for the design of sensors and biosensors using these polymeric materials in recent years. It is widely acknowledged that analytical sensing at electrodes modified with polymeric materials results in low detection limits, high sensitivities, lower applied potential, good stability, efficient electron transfer and easier immobilization of enzymes on electrodes such that sensing and biosensing of environmental pollutants is made easier. However, there are a number of challenges to be addressed in order to fulfill the applications of polymeric based polymers such as cost and shortening the long laboratory synthetic pathways involved in sensor preparation. Furthermore, the toxicological effects on flora and fauna of some of these polymeric materials have not been well studied. Given these disadvantages, efforts are now geared towards introducing low cost biomaterials that can serve as alternatives for the development of novel electrochemical sensors and biosensors. This review highlights recent contributions in the development of the electrochemical sensors and biosensors based on
Covalently immobilized enzymes on biocompatible polymers for amperometric sensor applications
Biosensors and Bioelectronics, 1996
Glucose oxidase or choline oxidase were covalently immobilized on the surface of 2-hydroxyethyi and glycidyl methacrylate copolymer membranes. The polymerization was induced by gamma irradiation at low temperature. The enzyme modified polymers were applied on Clark-type or platinum electrodes to form amperometric sensors based on the electrochemical measurements of oxygen or hydrogen peroxide. Glucose and choline content in standard solutions were measured and linear calibration curves were determined. The sensors studied showed a response time of less than 2 min and the observed linear ranges were increased with respect to usual biosensors owing to the diffusion-limiting effects of the membranes used. The influence of the copolymer composition on the electrochemical response and on the retained enzyme activity were explored for verifying the optimum analytical performance. The immobilized enzyme membranes stored in suitable buffers were very stable and showed a decrease up to 20% in the electrode response after 3 months of constant use.
Langmuir, 2003
A new organized biomimetic nanostructure embedding a monoclonal antibody in a lipidic matrix has been designed to sequester a hydrophilic enzyme in an oriented position and allowed to preserve the enzyme activity over a few months. The nanostructure was constituted of a glycolipid and a noninhibitory monoclonal IgG directed against the soluble form of acetylcholinesterase (AChE). A mixed monolayer (IgG-glycolipid) was obtained by spreading mixed IgG-glycolipid vesicles at the air/buffer interface. Several measurements (π-A isotherms, surface potential measurements, and compression-decompression cycles) allowed us to demonstrate the presence of IgG in the monolayer, as well as a reorientation of IgG molecules during the compression. After transfer on solid supports by the Langmuir-Blodgett technique, the presence of IgG in the mixed monolayer was characterized by ATR FTIR spectroscopy. Linking of the AChE on the IgG-glycolipid matrix was realized by immunoaffinity, and the enzyme was shown to retain its activity. The opportunity to detect a strong enzymatic activity, even after transfer at high surface pressures, suggested a preferential orientation of the antibody, favorable to retain the enzyme active at the surface of the nanostructure. The homogeneity of the transferred monolayer before and after immunoassociation, observed by Nomarski microscopy, did not display any structural modification. The enzyme kinetics was typical of the biocatalytic behavior of an immobilized enzyme, with a decrease of reaction rates due to the lower accessibility to the substrate at higher enzyme content. With the advantages of stability and favorable orientation of IgG, this new active matrix induces, in turn, a favorable orientation of the enzyme bound by immunoaffinity. The typical enzymatic behavior of the ternary nanostructure (glycolipid-IgG-AChE) demonstrates the usefulness of such a functional molecular assembly for biocatalysis study in a biomimetic situation.
Biosensing Devices: Conjugated Polymer Based Scaffolds
Encyclopedia of Polymer Applications, 2018
Conjugated polymer (CP)-based biosensors are recognized to be a next generation building architecture for highly sensitive and fast biosensing systems. This entry highlights an overview on the recent expansion of research in the field of CP-based biosensors. We start by first introducing some of the important knowledge about the CPs and biosensors. This is followed by emphasizing the importance of CPs in biosensor construction. Lastly, an overall survey of CP-based biosensor studies reported between 2010 and 2015 is discussed. Several reviews in literature mostly either refer to a specific enzyme or focus on the techniques of biosensor construction together with general information on conducting polymers. Hence, we tend to emphasize on a number of enzymes studied for the past 5 years.
Recent advances in nanostructured biocatalysts
Biochemical Engineering Journal, 2009
Recent years have witnessed a renaissance in the field of chemically re-engineering of enzymes to obtain highly selective and efficient biocatalysts for catalyzing processes under various conditions. The incorporation of enzyme into nanostructured materials is particularly noteworthy from a structural perspective since there are unprecedented opportunities in such systems to establish suitable microenvironments for chosen enzymes. This review summarizes recent developments in the nanostructured biocatalyst with emphasis on those formed with polymers. Based on the synthetic procedures employed, the established methods are grouped into three major categories-"grafting onto", "grafting from", and "self-assembly". The merits of the methods in enhancing enzyme stability at adverse conditions and their potential for large-scale preparation and the use of the nanostructured biocatalysts are discussed. The molecular fundamentals underlying each method are highlighted, and the use of molecular simulation as a tool for the design and application of nanostructured biocatalysts, although at a nascent stage, is presented. Finally, the problems encountered with nanostructured biocatalysts are discussed together with the future prospects of such systems.