Enzyme–Carbon Nanotube Conjugates in Room-temperature Ionic Liquids

Room-temperature ionic liquids (RTILs) are intriguing solvents, which are recognized as “green” alternatives to volatile organics. Although RTILs are nonvolatile and can dissolve a wide range of charged, polar, and nonpolar organic and inorganic molecules, there remain substantial challenges in their use, not the least of which is the solvents’ high viscosity that leads to potential mass transfer limitations. In the course of this work, we discovered that the simple adsorption of the bacterial protease, proteinase K, onto single-walled carbon nanotubes (SWNTs) results in intrinsically high catalytic turnover. The high surface area and the nanoscopic dimensions of SWNTs offered high enzyme loading and low mass transfer resistance. Furthermore, the enzyme–SWNT conjugates displayed enhanced thermal stability in RTILs over the native suspended enzyme counterpart and allowed facile reuse. These enzyme–SWNT conjugates may therefore provide a way to overcome key operational limitations of RTIL systems.     

Interaction between Metal in Metallo Enzyme and Small Biological Molecules

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Enzyme‐Linked Immunosorbent Assay for the Determination of Total Ginsenosides in Ginseng

Abstract

Hybridoma secreting a monoclonal antibody (MAb) against ginsenosides was produced by fusing splenocytes from a mouse immunized against a ginsenoside Re‐bovine serum albumin (G‐Re‐BSA) conjugate with myeloma cell line SP2/0‐Ag14. A MAb‐4G10 had a wide cross‐reaction with 20(S)‐protopanaxadiol and 20(S)‐protopanaxatriol type ginsenosides. An enzyme‐linked immunosorbent assay (ELISA) that had an effective measuring range of 20–400 ng/mL for total ginsenosides when G‐Re was used as a standard. Total ginsenosides concentration in ginseng were determined by ELISA and high‐performance liquid chromatography (HPLC) in a good agreement together, and the least‐squares fit had a coefficient of determination (γ²) of 0.995.     

Development of Anti-Interference Enzyme Layer for α -Amylase Measurements in Glucose Containing Samples

Abstract

A glucose oxidase-glucoamylase-bienzyme electrode has been developed and tested for determination of α-amylase activity. To eliminate interfering endogeneous glucose a glucose oxidase-catalase anti-interference layer was coupled with the bienzyme electrode. Linearity was obtained for the kinetic signal up to 1.0 I.U. α-amylase. Glucose was effectively eliminated up to 2 mM final concentration thus not influencing α-amylase determinations. A general concept for anti-interference enzyme layers is suggested.     

New Approach to Detecting Phenoxyl Free Radicals Generated in Enzyme Reaction by Stopped-flow Spectrophotometry

Quantification of free radical formation is critical for evaluation and interpretation of many radical-mediated biological processes1-4. Numerous studies have been devoted to the determination of free radicals, but the most of the reports have been focuse…     

AFB1–AP Conjugate for Enzyme Immunoassay of Aflatoxin B1 in Corn Samples

Abstract

This article describes the conjugation between aflatoxin B₁ (AFB₁), one of the major mycotoxins, and alkaline phosphatase (AP), one of the most used enzymes for immunoassays. In addition, an application of the ELISA method for aflatoxin B₁ determination in corn is presented. Three AFB₁–AP conjugates in different toxin–enzyme ratios were prepared and tested. The ELISA results, developed with the most effective conjugate obtained, showed a satisfactory working range between 2.4 and 4000 ng of toxin/g of corn. The detection limit was 2 ng/g in corn samples, and recoveries ranged from 105 to 120%.     

Development of Magnetic Particle-based Chemiluminescence Enzyme Immunoassay for the Detection of 17β-estradiol in Environmental Water

In the present work, a simple, fast, and highly sensitive chemiluminescence enzyme immunoassay for 17β-estradiol (E2) in environmental water samples was developed, using magnetic particles (MPs) labeled with secondary antibody as both the immobilization matrix and the separation tools. The specific anti-E2 polyclonal antibody (PcAb) was produced against a conjugate of estradiol–bovine serum albumin. The specificity of the anti-E2 antibody was studied. The results showed that the antibody did not cross-react with the structurally related endocrine-disrupting compounds, including estrone, ethinyl E2, estriol, E2-17-glucuronide, E2-3-sulfate-17-glucuronide, androstenedione, and dihydrotestosterone. The water samples were pretreated with solid-phase extraction using C₁₈ cartridges for the removal of matrix effects. Several physicochemical parameters including the dilution ratios of E2-6–horseradish peroxidase conjugate and anti-E2 PcAb, immunoreaction time, volume of chemiluminescent substrate and MPs, chemiluminescence reaction time, and pH of assay solution were studied and optimized. At optimal experimental conditions, it was found that the proposed method exhibited high performance with detection limit of 2.0 pg/mL, linear range of 20–1,200 pg/mL, and total assay time of 45 min. Both inter- and intra-assay coefficient of variation were less than 10%. The average recoveries of three different spiked concentration samples ranged from 86.3% to 108%. The method was successfully applied to the determination of E2 in river, waste, and tap water, and showed a good correlation with the commercially available radioimmunoassay kit.     

Enzyme–Substrate Binding Landscapes in the Process of Nitrile Biodegradation Mediated by Nitrile Hydratase and Amidase

The continuing discharge of nitriles in various industrial processes has caused serious environmental consequences of nitrile pollution. Microorganisms possess several nitrile-degrading pathways by direct interactions of nitriles with nitrile-degrading enzymes. However, these interactions are largely unknown and difficult to experimentally determine but important for interpretation of nitrile metabolisms and design of nitrile-degrading enzymes with better nitrile-converting activity. Here, we undertook a molecular modeling study of enzyme–substrate binding modes in the bi-enzyme pathway for degradation of nitrile to acid. Docking results showed that the top substrates having favorable interactions with nitrile hydratase from Rhodococcus erythropolis AJ270 (ReNHase), nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase), and amidase from Rhodococcus sp. N-771 (RhAmidase) were benzonitrile, 3-cyanopyridine, and l-methioninamide, respectively. We further analyzed the interactional profiles of these top poses with corresponding enzymes, showing that specific residues within the enzyme’s binding pockets formed diverse contacts with substrates. This information on binding landscapes and interactional profiles is of great importance for the design of nitrile-degrading enzyme mutants with better oxidation activity toward nitriles or amides in the process of pollutant treatments.     

Determination of Thyroxine with Capillary Electrophoretic Enzyme Immunoassay

Capillary electrophoretic immunoassay (CEIA) is a new analytical technique1,2. In CEIA, the detection methods were UV absorbance detection1 and laser induced fluorescence detection (LIF)2. However, The UV detection is lack of sensitivity. The LIF is difficult to use due to its high cost, and furthermore, most biological fluids are strongly luminescent when excited by the laser in the blue or green region of the spectrum. In our present work, a capillary electrophoretic enzyme immuno…     

Enzyme reactions in apolar solvents: the resolution of (±)-sulcatol with porcine pancreatic lipase.

The enantioselectivity of the transesterification of (±)-sulcatol by porcine pancreatic lipase in ether is increased ten-fold by dehydration of the enzyme and use of 2,2,2-trifluoroethyl laurate as the ester.     

Enzyme‐powered micromotors based on hierarchical porous MOFs

Bioinspired micro‐/nano‐motors are artificial micro‐/nano‐machines that can convert various forms of energy to propel their movement[1].For example,the motion of some of these micromachines can be precisely controlled by application of external physical stimuli including magnetic,electric and acoustic fields[2,3].Inspired by the study of microorganisms,researchers have been exploring also the use of available chemical energy from the local environment to trigger and sustain self‐propulsion[4].Within this research direction,Metal‐organic frameworks(MOFs)—a class of extended materials synthesized via a modular approach from inorganic(metal clusters or ions)and organic linkers[5]—offer excellent opportunities for the design and synthesis of self‐propelled micromotors.MOFs typically possess ultra‐high surface areas that allow facile access to densely populated catalytically active sites imbedded within their pore networks.Through careful design these catalytic sites can be exploited to convert chemical energy into kinetic energy resulting in self‐propulsion of the MOF crystal[6].In addition,rigidity,density,crystalline pore organization and pore size of MOFs can be optimized to carry out a swimming‐type motion[7].     

Electrochemical Determination of Cortisol by Capillary Electrophoretic Enzyme Immunoassay

In recent years, capillary electrophoretic immunoassay (CEIA) has become a primary analytical tool in clinical diagnostics. The primary detection methods in CEIA are UV absorbance detection and laser induced fluorescence detection (LIF)1. The major disadvantages of the UV detector are the lack of sensitivity and not available for many antigens which are small molecules without strong UV absorbance. LIF is a more general approach to improve sensitivity. However, it is difficult to u…     

The Amperometric Glucose Enzyme Electrode by Immobilized Glucose Oxidase in Glucose Oxidase/N-ethyl-Poly (4-vinyl)pyridine Multilayer

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Nucleic Acid-based Enzyme Cascades—Current Trends and Future Perspectives

The natural micro- and nanoscale organization of biomacromolecules is a remarkable principle within living cells, allowing for the control of cellular functions by compartmentalization, dimensional diffusion and substrate channeling. In order to explore these biological mechanisms and harness their potential for applications such as sensing and catalysis, molecular scaffolding has emerged as a promising approach. In the case of synthetic enzyme cascades, developments in DNA nanotechnology have produced particularly powerful scaffolds whose addressability can be programmed with nanometer precision. In this minireview, we summarize recent developments in the field of biomimetic multicatalytic cascade reactions organized on DNA nanostructures. We emphasize the impact of the underlying design principles like DNA origami, efficient strategies for enzyme immobilization, as well as the importance of experimental design parameters and theoretical modeling. We show how DNA nanostructures have enabled a better understanding of diffusion and compartmentalization effects at the nanometer length scale, and discuss the challenges and future potential for commercial applications.     

Detection of Liposome Lysis Utilizing an Enzyme–Substrate System

A novel optical reporter system was developed to verify encapsulation and subsequent release of a foreign molecule in liposomes. The protocol utilizes a single enzyme and substrate. We encapsulate o-nitrophenyl-β,d-galactopyranoside (ONPG) and measure its release by detecting the levels of o-nitrophenol created when the encapsulated ONPG is released and hydrolyzed by β-galactosidase. Using this method, liposome formation and subsequent lysis with Triton X-100 were verified. This new protocol eliminates the complications of multiple reaction enzyme detection methods, along with the chance for false negatives and unreliable data seen when using fluorescent particles as reporters.     

Calix[6] arene—bismetalloporphyrins as Enzyme Models for P450 V.Catalytic Performance in Epoxidation of Styrene

In this paper the catalytic performance of the calix[6]arene-bismetalloporphyrins in the epoxidation of styrene was studied.The influence of phase transfer catalyst,pH value,buffer agent,substrate concentration,substituent on the benzene ring of porphyrin,and the central metal ion,etc.,on the reaction rates were investigated meticulously.The results showed that the calix[6] arene-bismetalloporphyrins had a much higher catalytic activity than that of the corresponding metalloporphyrins.     

Study on the Model for Regulation of the Allosteric Enzyme Activity

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Synthesis and Enzyme Inhibitory Activities of Highly Functionalized Pyridylmethyl-C-β-D-Glycosides

Several pyridylmethyl-C-β-D-glycosides (3a–3l, 6a, and 6h) were synthesized by refluxing 3-(β-D-glucopyranosyl)/(β-D-cellobiosyl)-propanones and dicyanobenzylidenes with ammonium acetate in anhydrous toluene in moderate to good yields. The reaction involves a C?C Michael addition of enamine, formed from glycosyl ketone and ammonium acetate, to the dicayanobenzylidene derivative; subsequent dehydrative cyclization; and oxidative aromatization. Two of these prototypes, compounds 3e and 3k, were deacetylated to the respective glucopyranosyl methyl pyridines 4e and 4k with NaOMe/MeOH. The synthesized compounds were screened for their in vitro α-glucosidase inhibitory activities and one of the compounds showed 20% inhibition as compared to standard drug acarbose displaying 39% inhibition.     

Improving the Enzyme Catalytic Efficiency Using Ionic Liquids with Kosmotropic Anions

The kosmotropicity of cations and anions in ionic liquids has a strong influence on the enzyme catalytic efficiency in aqueous environments. The kosmotropic anion CF3COO^- seemed to activate the protease, and the chaotropic anions tended to destabilize the enzyme.     

Chromatography of A Polydisperse Enzyme,Yeast β-Fructofurancsidase,On Deae-Cellulose

Abstract

When yeast β-fructofuranosidase is chromatographed on DEAE-callulose with a linear gradient, subfractions of the emergent peak can be individually rechromatographed on new columns as essentially separate entities. This behavior is related to the polydispersity in polysaccharide to protein ratio which is encountered in this glycoprotein enzyme.