Enzymatic Strategy Research Articles

Fluorescence polarization biosensor based on an aptamer enzymatic cleavage protection strategy

A novel fluorescence polarization (FP) aptasensing platform based on target-induced aptamer enzymatic cleavage protection is reported. The method relies on the FP analysis of the phosphodiesterase I mediated size variation of a dye-labeled aptamer. The tyrosinamide/antityrosinamide DNA aptamer couple was firstly tested as a model system to establish the proof-of-concept. In the absence of the target, the labeled aptamer was enzymatically cleaved into small DNA fragments, leading to a low FP signal. Upon tyrosinamide binding, the DNA substrate was partially protected against the enzymatic attack, leading to an increase in the fluorescence anisotropy response as a result of the higher average molecular volume of the weakly digested probe. The method was subsequently applied to two other systems, i.e., for the detection of ochratoxin A and adenosine. Such an approach was found to combine simplicity and general applicability features.

A review of the progress in enzymatic concentration and microencapsulation of omega-3 rich oil from fish and microbial sources

Technology continues to evolve for the concentration and stabilisation of omega-3 fatty acids for delivery into food and beverage products. The use of lipases for selective concentration of EPA and DHA, or for re-esterification reactions, is important in the production of omega-3 concentrates. Enzymatic strategies require robust enzymes that can be immobilised and multiply re-used. Novel and mild processing methods are particularly important for providing oils with good sensory properties, which are required for successful use as functional food ingredients. Although in some cases good quality oils can be used directly in some foods, such as margarine, many foods require that microencapsulated and stabilised omega-3 oils be used. This is particularly important when the oils are preconcentrated. There are a number of industrially used microencapsulation methods, but the most widely used are complex coacervates and spray dried emulsions. Fish oil is still the most widely used source of long-chain omega-3 fatty acids for addition to food, although algal oil is the primary source of DHA for infant formula use in North America. Algal oil is still significantly more expensive than fish oil for most applications, although many groups are improving both the cost and quality of omega-3 oil from algal sources. In particular, Thraustochytrid and Schizochytrid strains are a promising source of both DHA and EPA, and with further improvement could be used to provide varying ratios of these omega-3 fats. In this short review we will describe some of the current research in omega-3 fat concentration and microencapsulation, with particular emphasis on the use of lipases for concentration and complex coacervation for microencapsulation.

Unimolecular Catalytic DNA Biosensor for Amplified Detection of l-Histidine via an Enzymatic Recycling Cleavage Strategy

Fluorescence catalytic beacons have emerged as a general platform for sensing applications. However, almost all such sensing systems need covalent modification of the DNAzymes with fluorophore-quencher pairs, which may require elaborate design of the synthetic routes and many heavy and complicated synthetic steps and result in increased cost and lower synthesis yield. Here we report the construction of fluorescent cascadic catalytic beacons. With separation of the molecular recognition module from the signal reporter, this new design both avoids DNAzyme modifications and improves sensitivity through an endonuclease-based cascadic enzymatic signal amplification. This allows detection of L-histidine with high sensitivity (LOD = 200 nM) and excellent specificity. The proposed sensing system has also been used for detection of L-histidine in cellular homogenate with satisfactory results.

Ultrasensitive electrochemical immunosensor employing glucose oxidase catalyzed deposition of gold nanoparticles for signal amplification

This paper describes a novel enzymatic amplification strategy for ultrasensitive electrochemical immunosensing. This approach utilizes glucose oxidase for the enzymatic deposition of gold nanoparticles onto an indium tin oxide (ITO) electrode surface using a novel gold developer solution consisting of 20 mM of glucose, 20 mM of NaSCN, 0.5 mM of p-benzoquinone (PBQ) and 1 mM of AuCl 4 − dissolved in 0.1 M of pH 7.5 phosphate buffer solution. The amount of gold deposited was quantified electrochemically by monitoring the reduction of gold oxide in an aqueous solution of 0.5 M of H 2SO 4, which was correlated to the amount of antigens in the solution. The effectiveness of this strategy was demonstrated experimentally through the construction of an immunosensor for the detection of mouse IgG using a sandwich immunoassay in a linear dynamic range of 5 pg/ml to 50 ng/ml. A good mean apparent recovery in the range of 88–102% was obtained over the entire linear dynamic range of the sensor response in the serum samples. This suggested that the immunosensor would be useful for the testing of proteins in real clinical samples.

Fully Enzymatic N→C-Directed Peptide Synthesis Using C-Terminal Peptide α-Carboxamide to Ester Interconversion

Chemoenzymatic peptide synthesis is potentially the most cost-efficient technology for the synthesis of short and medium-sized peptides with some important advantages. For instance, stoichiometric amounts of expensive coupling reagents are not required and racemisation does not occur rendering purification easier compared to chemical peptide synthesis. In this paper, a novel interconversion reaction of peptide C-terminal α-carboxamides into primary alkyl esters with alcalase was used to develop a fully enzymatic peptide synthesis strategy. For each elongation step a cost-efficient amino acid carboxamide building block was used followed by the interconversion of the elongated peptide carboxamide to the corresponding primary alkyl ester. These peptide esters are the starting materials for the next enzymatic peptide elongation step.

Systematic Review of Thiopurine Methyltransferase Genotype and Enzymatic Testing Strategies

An increased understanding of the genetic basis of disease creates a demand for personalized medicine and more accurate testing for diagnosis and treatment. Thiopurine methyltransferase (TPMT) plays an important role in the metabolism of thiopurine drugs used in pediatric leukemia, rheumatoid arthritis, and inflammatory bowel disease. The objective was to review the literature systematically to ascertain the performance characteristics of current genotype and enzymatic testing technologies for TPMT. A systematic review of the literature was conducted to describe TPMT testing technologies. Eligible studies evaluated either a TPMT genotype or TPMT phenotype technology in comparison to a reference standard and expressed results in terms of sensitivity and specificity or positive/negative predictive value. The laboratory technique was recorded, and the quality of the identified studies was assessed using a modified Critical Appraisal Skills Program tool. Seventeen studies were reviewed. The sensitivity and specificity of the genotype test ranged from 55% to 100% and from 94% to 100%, respectively. The sensitivity and specificity of the phenotype test ranged from 92% to 100% and from 86% to 98%, respectively. A variety of laboratory techniques were employed. Reviewed studies were of low methodological quality. The systematic review of TPMT test strategies found that available technologies demonstrated high values for sensitivity and specificity, however, there was a lack of a single gold standard and most studies were of poor quality. Disregard for study sample size and confounding factors such as concurrent medications and blood transfusions were the main contributors to low quality. There were also inconsistencies in the selection of a reference standard which complicated the interpretation of the findings.

Microbial Community Composition and Function in Permanently Cold Seawater and Sediments from an Arctic Fjord of Svalbard

Heterotrophic microbial communities in seawater and sediments metabolize much of the organic carbon produced in the ocean. Although carbon cycling and preservation depend critically on the capabilities of these microbial communities, their compositions and capabilities have seldom been examined simultaneously at the same site. To compare the abilities of seawater and sedimentary microbial communities to initiate organic matter degradation, we measured the extracellular enzymatic hydrolysis rates of 10 substrates (polysaccharides and algal extracts) in surface seawater and bottom water as well as in surface and anoxic sediments of an Arctic fjord. Patterns of enzyme activities differed between seawater and sediments, not just quantitatively, in accordance with higher cell numbers in sediments, but also in their more diversified enzyme spectrum. Sedimentary microbial communities hydrolyzed all of the fluorescently labeled polysaccharide and algal extracts, in most cases at higher rates in subsurface than surface sediments. In seawater, in contrast, only 5 of the 7 polysaccharides and 2 of the 3 algal extracts were hydrolyzed, and hydrolysis rates in surface and deepwater were virtually identical. To compare bacterial communities, 16S rRNA gene clone libraries were constructed from the same seawater and sediment samples; they diverged strongly in composition. Thus, the broader enzymatic capabilities of the sedimentary microbial communities may result from the compositional differences between seawater and sedimentary microbial communities, rather than from gene expression differences among compositionally similar communities. The greater number of phylum- and subphylum-level lineages and operational taxonomic units in sediments than in seawater samples may reflect the necessity of a wider range of enzymatic capabilities and strategies to access organic matter that has already been degraded during passage through the water column. When transformations of marine organic matter are considered, differences in community composition and their different abilities to access organic matter should be taken into account.

Identification and Characterization of a Novel-type Ferric Siderophore Reductase from a Gram-positive Extremophile

Iron limitation is one major constraint of microbial life, and a plethora of microbes use siderophores for high affinity iron acquisition. Because specific enzymes for reductive iron release in gram-positives are not known, we searched Firmicute genomes and found a novel association pattern of putative ferric siderophore reductases and uptake genes. The reductase from the schizokinen-producing alkaliphile Bacillus halodurans was found to cluster with a ferric citrate-hydroxamate uptake system and to catalyze iron release efficiently from Fe[III]-dicitrate, Fe[III]-schizokinen, Fe[III]-aerobactin, and ferrichrome. The gene was hence named fchR for ferric citrate and hydroxamate reductase. The tightly bound [2Fe-2S] cofactor of FchR was identified by UV-visible, EPR, CD spectroscopy, and mass spectrometry. Iron release kinetics were determined with several substrates by using ferredoxin as electron donor. Catalytic efficiencies were strongly enhanced in the presence of an iron-sulfur scaffold protein scavenging the released ferrous iron. Competitive inhibition of FchR was observed with Ga(III)-charged siderophores with K(i) values in the micromolar range. The principal catalytic mechanism was found to couple increasing K(m) and K(D) values of substrate binding with increasing k(cat) values, resulting in high catalytic efficiencies over a wide redox range. Physiologically, a chromosomal fchR deletion led to strongly impaired growth during iron limitation even in the presence of ferric siderophores. Inductively coupled plasma-MS analysis of ΔfchR revealed intracellular iron accumulation, indicating that the ferric substrates were not efficiently metabolized. We further show that FchR can be efficiently inhibited by redox-inert siderophore mimics in vivo, suggesting that substrate-specific ferric siderophore reductases may present future targets for microbial pathogen control.

An ultrasensitive peroxidase DNAzyme-associated aptasensor that utilizes a target-triggered enzymatic signal amplification strategy

By rationally employing enzymes for signal amplification, an ultrasensitive aptasensor was developed and successfully tested in a system designed to detect lysozyme with a detection limit of 0.1 fM. This detection limit is nearly three orders of magnitude lower than those of any previously reported DNAzyme-based aptasensors.

Enzymes for Antifouling Strategies

During the past decades, much effort has been made to find efficient alternative solutions to prevent and/or disrupt the adhesion of fouling organisms to surfaces. The use of enzymes emerges among the investigated approaches as one of the favorite candidate antifouling technologies due to enzymes' biodegradability and affordable prices. An overview of the different enzymatic antifouling strategies is presented, highlighting the most promising groups of enzymes, and their utilization upon surface-confinement to control biofouling. While the main strategies to control marine biofouling include the degradation of secreted adhesives and the production of antifouling compounds, the main concepts to control pathogenic biofilms are based on cell lysis and on the degradation of extracellular matrix polymers. Although immobilization can improve enzyme stability, activity and antifouling performance, up to date relatively few scientific articles concerning the use of immobilized enzymes to control biofouling have been published. The successful incorporation of enzymes into coatings yielding surfaces with broad antifouling spectrum and long-term efficacy remains a challenge.

Enzymatic diastereo- and enantioselective synthesis of α-alkyl-α,β-dihydroxyketones

An enzymatic strategy for the preparation of optically pure α-alkyl-α,β-dihydroxyketones is reported. Homo- and cross-coupling reactions of α-diketones catalyzed by acetylacetoin synthase (AAS) produce a set of α-alkyl-α-hydroxy-β-diketones (30-60%, ee 67-90%), which in turn are reduced regio-, diastereo-, and enantioselectively to the corresponding chiral α-alkyl-α,β-dihydroxyketones (60-70%, ee >95%) using acetylacetoin reductase (AAR) as catalyst. Both enzymes are obtained from Bacillus licheniformis and used in a crude form. The relative syn stereochemistry of the enantiopure α,β-dihydroxy products is assigned by NOE experiments, whereas their absolute configuration is determined by conversion of the selected 3,4-dihydroxy-3-methyl-pentan-2-one to the natural product (+)-citreodiol.

Chemistry in the Center for Catalytic Hydrocarbon Functionalization: An Energy Frontier Research Center

Selective catalysts that activate small molecules such as hydrocarbons, dioxygen, water, carbon dioxide and dihydrogen are central to new technologies for the use of alternative energy sources. For example, controlled hydrocarbon functionalization can lead to high impact technologies, but such catalysts require a level of molecular control beyond current means. The Center for Catalytic Hydrocarbon Functionalization facilitates collaborations among research groups in catalysis, materials, electrochemistry, bioinorganic chemistry and quantum mechanics to develop, validate and optimize new methods to rearrange the bonds of hydrocarbons, activate and transform water and carbon dioxide, implement enzymatic strategies into synthetic systems and design optimal environments for catalysis.

Novel glycosaminoglycan biosynthetic inhibitors affect tumor-associated angiogenesis

Heparan sulfate proteoglycans (HSPGs) are essential players in several steps of tumor-associated angiogenesis. As co-receptors for several pro-angiogenic factors such as VEGF and FGF, HSPGs regulate receptor–ligand interactions and play a vital role in signal transduction. Previously, we have employed an enzymatic strategy to show the importance of cell surface HSPGs in endothelial tube formation in vitro. We have recently found several fluoro-xylosides that can selectively inhibit proteoglycan synthesis in endothelial cells. The current study demonstrates that these fluoro-xylosides are effective inhibitors of endothelial tube formation in vitro using a matrigel based assay to simulate tumor-associated angiogenesis. These first generation scaffolds offer a promising stepping-stone to the discovery of more potent fluoro-xylosides that can effectively neutralize tumor growth.

Gold coated ferric oxide nanoparticles based disposable magnetic genosensors for the detection of DNA hybridization processes

In this article, a disposable magnetic DNA sensor using an enzymatic amplification strategy for the detection of specific hybridization processes, based on the coupling of streptavidin-peroxidase to biotinylated target sequences, has been developed. A thiolated 19-mer capture probe was attached to gold coated ferric oxide nanoparticles and hybridization with the biotinylated target was allowed to proceed. Then, a streptavidin-peroxide was attached to the biotinylated target and the resulting modified gold coated ferric oxide nanoparticles were captured by a magnetic field on the surface of a home-made carbon screen printed electrode (SPE). Using hydroquinone as a mediator, a square wave voltammetric procedure was chosen to detect the hybridization process after the addition of hydrogen peroxide. Different aspects concerning the assay protocol and nanoparticles fabrication were optimized in order to improve the sensitivity of the developed methodology. A low detection limit (31 pM) with good stability (RSD = 7.04%, n = 10) was obtained without the need of polymerase chain reaction (PCR) amplification.

The genetic and molecular basis for sunscreen biosynthesis in cyanobacteria.

Ultraviolet UV-A and UV-B radiation is harmful to living systems, causing damage to biological macromolecules. An important strategy for dealing with UV exposure is the biosynthesis of small-molecule sunscreens. Among such metabolites, the mycosporine and mycosporine-like amino acids (MAAs) are remarkable for their wide phylogenetic distribution and their unique chemical structures. Here, we report the identification of a MAA biosynthetic gene cluster in a cyanobacterium and the discovery of analogous pathways in other sequenced organisms. We have expressed the cluster in a heterologous bacterial host and characterized all four biosynthetic enzymes in vitro. In addition to clarifying the origin of the MAAs, these efforts have revealed two unprecedented enzymatic strategies for imine formation.

Regioselective synthesis of amphiphilic metoprolol–saccharide conjugates by enzymatic strategy in organic media

An efficient protocol to prepare metoprolol–saccharide conjugates by a selective enzymatic synthesis method was developed. Firstly, the transesterification of metoprolol with three divinyl dicarboxylates (divinyl succinate, divinyl adipate and divinyl sebacate) was performed. The influences of organic solvents, sources of enzymes and acylating reagents on the synthesis of N-(vinyloxycarbonyl)metoprolol were investigated. A series of lipophilic metoprolol derivatives with vinyl group were obtained by using a lipase from porcine pancreas (PPL) in anhydrous tetrachloromethane at 50 °C. Subsequently, alkaline protease from Bacillus subtilis catalyzed highly regioselective acylation of three monosaccharides (glucose, mannose and galactose) and two disaccharides (maltose and sucrose) with N-(5-vinyloxycarbonylpentanoyl)metoprolol in anhydrous pyridine at 50 °C to give metoprolol–saccharide conjugates in good yields. The partition coefficients of the products were investigated. The results indicated that the aqueous solubility of metoprolol–monosaccharide and metoprolol–disaccharide conjugates was improved markedly compared with the parent drug of metoprolol, and the aqueous solubility of metoprolol–disaccharide conjugates was much better than that of metoprolol–monosaccharide conjugates.

Identification of the Unpaired Cysteine Status and Complete Mapping of the 17 Disulfides of Recombinant Tissue Plasminogen Activator Using LC−MS with Electron Transfer Dissociation/Collision Induced Dissociation

Recombinant tissue plasminogen (rt-PA) with 35 cysteine residues has been completely assigned by mapping the 17 disulfide linkages and the unpaired cysteine. The result is consistent with the prediction from homology except for the unassigned cysteine, which was identified at Cys83. This cysteine was found to be blocked and paired with either a glutathione or cysteine residue in an approximately 60:40 ratio, respectively. The analysis was conducted using a multifragmentation approach consisting of electron transfer dissociation (ETD) and collision induced dissociation (CID), in combination with a multienzyme digestion strategy (Lys-C, trypsin, and Glu-C). The disulfide-linked peptides, even those containing N- or O-linked glycosylation, could be assigned since the disulfide bonds were still preferably cleaved over the glycosidic cleavages under ETD fragmentation. The use of a multiple and sequential enzymatic digestion strategy was important in producing fragment sizes suitable for analysis. For the analysis of complex intertwined disulfides, the use of CID-MS(3) to target partially disulfide-dissociated peptides from the ETD fragmentation was necessary for linkage assignment. The ability to identify the exact location and status of the unpaired cysteine (free or blocked with a glutathione or cysteine) could shed light on the activation of rt-PA, upon stimulation by either oxidative or ischemic stress.

Identification, Quantification, and Functional Aspects of Skeletal Muscle Protein-Carbonylation in Vivo during Acute Oxidative Stress

Reactive oxidative species (ROS) play important roles in cellular signaling but can also modify and often functionally inactivate other biomolecules. Thus, cells have developed effective enzymatic and nonenzymatic strategies to scavenge ROS. However, under oxidative stress, ROS production is able to overwhelm the scavenging systems, increasing the levels of functionally impaired proteins. A major class of irreversible oxidative modifications is carbonylation, which refers to reactive carbonyl-groups. In this investigation, we have studied the production and clearance rates for skeletal muscle proteins in a rat model of acute oxidative stress over a time period of 24 h using a gel-based proteomics approach. Optimized ELISA and Western blots with 10-fold improved sensitivities showed that the carbonylation level was stable at 4 nmol per mg protein 3 h following ROS induction. The carbonylation level then increased 3-fold over 6 h and then remained stable. In total, the oxidative stress changed the steady state levels of 20 proteins and resulted in the carbonylation of 38 skeletal muscle proteins. Carbonylation of these proteins followed diverse kinetics with some proteins being highly carbonylated very quickly, whereas others peaked in the 9 h sample or continued to increase up to 24 h after oxidative stress was induced.

A new enzymatic strategy for the preparation of (2 R,3 S)-3-phenylisoserine: a key intermediate for the Taxol side chain

Burkholderia cepacia lipase PS-IM catalysed the hydrolysis of racemic ethyl 3-amino-3-phenyl-2-hydroxypropionate with excellent enantioselectivity ( E >200), when the reaction was performed with added H 2O as a nucleophile, in iPr 2O, at 50 °C. The hydrolysis of the less reactive enantiomeric ethyl 3-amino-3-phenyl-2-hydroxypropionate with 18% HCl afforded the corresponding enantiomerically pure (2 R,3 S)-3-amino-3-phenyl-2-hydroxypropionic acid hydrochloride, a key intermediate for the Taxol side chain.

Pharmacodynamic Monitoring of Calcineurin Inhibition Therapy: Principles, Performance, and Perspectives

The calcineurin inhibitors (CNIs) cyclosporin A and tacrolimus are immunosuppressive drugs used extensively in allograft recipients. These drugs show large interindividual pharmacokinetic variation and are associated with severe adverse affects, including nephrotoxicity and cardiovascular disease. In current practice, CNIs are combined with other immunosuppressive drugs such as steroids and mycophenolate mofetil. Dosage is titrated based on blood concentration measurement. For further optimization of calcineurin (CN) inhibition therapy, new monitoring strategies are required. Pharmacodynamic-monitoring strategies constitute novel approaches for optimization of CNIs therapy. This review focuses on the general aspects of immunosuppressive drug pharmacodynamic monitoring and describes the methodologies used for monitoring CN inhibition therapy. Two different types of pharmacodynamic-monitoring strategies can be distinguished: (1) enzymatic strategies, which monitor inhibition of drug-target enzyme activity, and (2) immunologic strategies, which measure cellular responsiveness after in vitro simulated immunologic responses. Enzymatic tests are drug type-specific markers in which CN activity is directly determined. Immunologic strategies measure immune responsiveness at several levels, such as mRNA transcripts (intracellular) concentrations/excretion of cytokines, expression of surface activation markers, and cell proliferation. This review also discusses analytical issues and clinical experience with these techniques. The call for new methodologies to evaluate immunosuppressive therapy has led to the development of a large variety of pharmacodynamic-monitoring strategies. The first reports of their clinical relevance are available, but further understanding of the analytical and clinical variables involved are required for the development of accurate, reproducible, and clinically relevant markers.