Hofmeister effects in enzymatic activity: weak and strong electrolyte influences on the activity of Candida rugosa lipase

The effects of weak and strong electrolytes on the enzymatic activity of Candida rugosa lipase are explored. Weak electrolytes, used as buffers, set the pH, while strong electrolytes regulate the ionic strength. The interplay between pH and ionic strength has been assumed to be the determinant of enzymatic activity. In experiments that probe activities by varying these parameters, there has been little attention focused on the role of specific electrolyte effects. Here we show that both buffers and the choice of background electrolyte ion strongly affect the enzymatic activity of Candida rugosa lipase. The effects here shown are dramatic at high salt concentration; indeed, a 2 M concentration of NaSCN is able to fully inactivate the lipase. By contrast, Na2SO4 acts generally as an activator, whereas NaCl shows a quasi-neutral behavior. Such specific ion effects are well-known and are classified among the "Hofmeister effects". However, there has been little awareness of them, or of their potential for optimization of activities in the enzyme community. Rather than the effects per se, the focus here is on their origin. New insights into mechanism are proposed.     

A novel fluorescent probe for NAD-consuming enzymes

A novel, fluorescent NAD derivative is processed as substrate by three different NAD-consuming enzymes. The new probe has been used to monitor enzymatic activity in a continuous format by changes in fluorescence and, in one case, to directly visualize alternative reaction pathways.     

Functionality of dielectrophoretically immobilized enzyme molecules

The enzyme horseradish peroxidase has been immobilized on nanoelectrode arrays by alternating current dielectrophoresis (DEP). Preservation of its enzymatic function after field application was demonstrated by oxidizing dihydrorhodamine 123 with hydrogen peroxide as co‐oxidant to create its fluorescent form, rhodamine 123 (Rh123). Localization of the fluorescently labeled enzyme and its product was conducted by fluorescence microscopy. Nanoelectrodes were prepared as tungsten pins arranged in square arrays. Experimental parameters for dielectrophoretic immobilization were optimized for even enzyme distribution and for enzymatic efficiency. Enzyme activity was quantified by determination of fluorescence intensities of immobilized enzyme molecules and of Rh123 produced. These results demonstrate that DEP can be applied to immobilize enzyme molecules while retaining their activity and rendering any chemical modifications unnecessary. This introduces a novel way for the preparation of bioactive surfaces for processes such as biosensing.     

Kinetic analysis of PI3K reactions with fluorescent PIP<Subscript>2</Subscript> derivatives

Phosphatidylinositol 3-kinase (PI3K) signaling plays important roles in cell differentiation, proliferation, and migration. Increased mutations and expression levels of PI3K are hallmarks for the development of certain cancers. Pharmacological targeting of PI3K activity has also been actively pursued as a novel cancer therapeutic. Consequently, measurement of PI3K activity in different cell types or patient samples holds the promise as being a novel diagnostic tool. However, the direct measurement of cellular PI3K activity has been a challenging task. We report here the characterization of two fluorescent PIP₂ derivatives as reporters for PI3K enzymatic activity. The reporters are efficiently separated from their corresponding PI3K enzymatic products through either thin layer chromatography (TLC) or capillary electrophoresis (CE), and can be detected with high sensitivity by fluorescence. The biophysical and kinetic properties of the two probes are measured, and their suitability to characterize PI3K inhibitors is explored. Both probes show similar capacity as PI3K substrates for inhibitor characterization, yet also possess distinct properties that may suggest their different applications. These characterizations have laid the groundwork to systematically measure cellular PI3K activity, and have the potential to generate molecular fingerprints for diagnostic and therapeutic applications.     

A quantitative study of the enzymatic activity of horseradish peroxidase at a planar poly(acrylic acid) brush

The enzymatic activity of horseradish peroxidase (HRP) has been quantified at a planar poly(acrylic acid) (PAA) brush. A PAA brush is known to exhibit an unusual protein binding affinity, since proteins adsorb at low ionic strength only. At elevated ionic strengths of a few 100 mM proteins desorb, and the PAA brush becomes largely protein resistant. In this study, HRP was used to catalyze the oxidation of Amplex Red to resorufin by hydrogen peroxide. The fluorescence of resorufin was recorded using a microplate reader. As compared to a bare silica surface, the enzymatic activity of HRP is higher by more than one order of magnitude at a PAA brush. This increase results from a higher degree of adsorption and a reasonable preservation of the HRP activity. Upon adsorption at a PAA brush from a 20 mU/mL (0.1 μg/mL) solution, the molecular enzymatic activity of HRP is reduced to about 11%. However, when a HRP molecule is desorbed from a PAA brush by increasing the ionic strength, a gain of the molecular enzymatic activity by only 52% can be observed. Overall, this study illustrates the potential applicability of a PAA brush as a biocompatible material coating.     

Enzymatic activity of cellulase adsorbed on cellulose and its change during hydrolysis

Hydrolysis of pure cellulose Avicel has been carried out, using Meicelase from Trichoderma viride, where the enzymatic activity of cellulase adsorbed on cellulose and its changes during the hydrolysis were investigated. A rapid drop of the hydrolysis rate during the reaction, that is always observed in enzymatic hydrolysis of cellulose, could be explained by a decline of specific activity of adsorbed enzyme, and it was implied that the decline results from a loss of synergistic action between endoglucanase and exoglucanase. An empirical equation expresses the change of hydrolysis rate during the reaction and also shows that the change of the hydrolysis rate is caused by the decline of the specific enzymatic activity of adsorbed enzyme.     

在有机溶剂中进行酶催化合成肽及肽类衍生物的研究进展

介绍了有机溶剂中含非天然组分的肽及肽类衍生物的酶催化反应,同时对研究有机溶剂中酶的活性与结构关系的谱学技术进行了评述.     

High-performance liquid chromatographic determination of D-amino acid oxidase activity.

A new procedure for the assay of D-amino acid oxidase activity has been developed. alpha-Ketoisovaleric acid, derived from D-valine, was estimated by high-performance liquid chromatography after reaction with o-phenylenediamine to give the corresponding quinoxalinol derivative. alpha-Ketovaleric acid was used as an internal standard to ensure the reproducibility of the method. As an example of application, kidney cortex homogenates were analyzed for their D-amino acid oxidase activity. The advantages of the presented procedure for the determination of the enzymatic activity in biological samples compared with previously reported procedures are discussed.     

Significance of specific activity and a possible universal unit for its definition

The growing importance of specific activity is reviewed. It concerns especially surface phenomena, toxicity, labelling of radiopharmaceuticals, isotope exchange, enzymatic and pharmacological ligand-receptor reactions. The present state of evaluating the specific activity has been analyzed. Introduction of the coefficient D_CF (deviation from true carrier free state) is proposed as a possibility for universal declaration of the specific activity.     

New Synthesis of A‐Ring Aromatic Strigolactone Analogues and Their Evaluation as Plant Hormones in Pea (Pisum sativum)

A new general access to A‐ring aromatic strigolactones, a new class of plant hormones, has been developed. The key transformations include in sequence ring‐closing metathesis, enzymatic kinetic resolution and a radical cyclization with atom transfer to install the tricyclic ABC‐ring system. The activity as plant hormones for the inhibition of shoot branching in pea of various analogues synthesized by this strategy is reported.     

Monitoring enzymatic conversions by mass spectrometry: a critical review

This review highlights recent advances in the application of electrospray ionisation and matrix-assisted laser desorption/ionisation mass spectrometry (MS) to study enzymatic reactions. Several assay schemes for different fields of application are presented. The employment of MS as a means of detection in pre-steady-state kinetic studies by rapid-mixing direct analysis and rapid-mixing quench flow techniques is discussed. Several steady-state kinetic studies of a broad range of different enzymatic systems are presented as well as enzyme inhibition studies for various target enzymes. As a promising new development multiplex assays, which monitor the conversion of several substrates simultaneously in one experiment, are described. This assay type has been used for competition studies, enzymatic activity screenings and for diagnostic purposes in clinical chemistry. Generally, it can be concluded that mass spectrometry offers an intriguing alternative as detection methodology in enzymatic bioassays. Its applicability for the monitoring the conversion of naturally occurring substrates and its overall versatility make MS an especially promising tool for the study of enzyme-catalysed processes.     

A Study on the Stability and Enzymatic Activity of Yeast Alcohol Dehydrogenase in Presence of the Self-Assembling Block Copolymer Poloxamer 407

Yeast alcohol dehydrogenase (ADH) is an enzyme widely studied for biotechnological applications due to its involvement in fermentation industry, and various attempts to improve its catalytic properties and its thermal stability have been carried out. In this paper, the influence of a block copolymer (Poloxamer 407) on ADH enzymatic activity and thermal behaviour has been studied in order to get new insights about the use of poloxamers in formulation of sustained release systems for therapeutic proteins. Poloxamer 407 has the ability to form micelles and gel due to its self-assembling and thermoresponsive properties. The effect of the copolymer towards thermal stress and pH changes, which often reduce enzymes activity it has been investigated by means of enzymatic assays and differential scanning calorimetry. Results showed that at pH?9.1 and 7.3, the Poloxamer in the form of unimeric, micellar and gel state is able to effectively preserve the enzyme from thermoinactivation. In addition by calorimetric data Poloxamer 407 has showed an effect in preserving ADH from aggregation at pH?7.3. In conclusion, Poloxamer 407 seems to be very effective in protecting ADH from stress related events, like alkaline inactivation and aggregation.     

One-step detection of T4 polynucleotide kinase activity based on single particle-confined enzyme reaction and digital particle counting

T4 polynucleotide kinase (T4 PNK) is a pivotal enzyme for DNA replication, recombination, and DNA damage repair. Herein, a robust single particle counting-based assay has been developed for the high-sensitive determination of T4 PNK activity through only a simple one-step reaction. Taking benefit of the exceptional space-confined enzymatic property of T4 PNK towards DNA substrates on a single nanoparticle, the T4 PNK activity can be precisely determined by counting the fluorescence-positive nanoparticles in a digital manner with a total internal reflection fluorescent microscope (TIRFM). Due to the featured spatial-confined enzymatic property of T4 PNK and the single particle counting-based signal readout, T4 PNK can be effectively differentiated from other interfering enzymes. This facile strategy has been also successfully applied to screen T4 PNK inhibitor and accurately determine T4 PNK activity in complex biological samples, paving a potential avenue for the digital analysis of biomarkers.     

Partial Characterization of Inulinases Obtained by Submerged and Solid-State Fermentation Using Agroindustrial Residues as Substrates: A Comparative Study

Inulinase belongs to an important class of enzymes as it can be used to produce high-fructose syrups by enzymatic hydrolysis of inulin and fructooligosaccharides, which has been used as functional food. This work aimed to carry out a partial characterization of the crude enzymatic extract of two different inulinases, obtained by solid-state fermentation (SSF) and submerged fermentation (SmF), using agroindustrial residues as substrates. The crude enzymatic extract obtained by SmF showed an optimal pH and temperature for hydrolytic activity of 4.5 and 55?°C, respectively; and that obtained by SSF conducted to optimal pH and temperature of 5.0 and 55?°C, respectively. Both enzymes presented high thermostability, with a D value of 230.4 h and 123.1 h for SmF and SSF, respectively. The inulinase produced by SmF showed highest stability at pH?4.4, while inulinase obtained by SSF was more stable at pH?4.8. The results showed that inulinase obtained by SmF is less susceptible to pH effect and the inulinase obtained by SSF is more resistant to higher temperatures.     

The catalytic mechanism of indole-3-glycerol phosphate synthase (IGPS) investigated by electrospray ionization (tandem) mass spectrometry

An enzymatic reaction has been monitored by on-line direct infusion electrospray ionization (tandem) mass spectrometry. Using this fast and sensitive technique, a key and transient intermediate of Mycobacterium tuberculosis indole-3-glycerol phosphate synthase (IGPS)-catalyzed reaction has been trapped. The reaction catalyzed by indole-3-glycerol phosphate synthase is part of the tryptophan biosynthetic pathway, and is not present in mammals, including humans. This peculiarity renders this enzyme a potential target for the development of biospecific agents with potential anti-TB activity. The present results indicate the presence of two intermediates in the mechanism of this enzymatic reaction.     

Immobilized hepatic microsomes for the synthesis of glucuronides

A method has been developed for the direct immobilization of hepatic microsomes. The technique is simple and efficient. The immobilized microsomes retain biological activity and can be used to perform enzymatic reactions.     

The catalatic and peroxidatic activity of irradiated dilute aqueous solutions of catalase

The influence of -irradiation on both catalatic and peroxidatic activity of bovine liver catalase in dilute aqueous solutions has been studied. An attempt to find correlations between radiation-induced conformational changes of the enzyme molecule and the loss of enzymatic activity has been undertaken. The role of radicals of water radiolysis on the radiosensitivity of the enzyme and on post-radiation recovery of enzyme activity has been also investigated.     

Time-dependent nanogel aggregation for naked-eye assays of α-amylase activity

This work designs an enzyme-stimulated nanogel aggregation system for the naked-eye assays of α-amylase activity. The visible aggregation of the starch-stabilized CdTe nanogels may be accelerated by α-amylase through its efficient cleavage of glycosidic bonds in the starch network, which has been verified by the evidences from transmission electron microscopy and dynamic light scattering spectra. The required aggregation time, as validated by both the theoretical deduction and the experimental results, is inversely proportional to the enzymatic activity. Therefore a facile method has been proposed for the detection of enzyme activity, with an excellent linear range and a low detection limit. This nanogel-based protocol can be successfully applied in the fast and accurate assays of α-amylase activity in saliva samples with a satisfactory correlation with the standard protocol, suggesting its promising applications in the biomedical and clinical fields, especially in point-of-care testing.     

Kinetic evaluation of phenolase activity of tyrosinase using simplified catalytic reaction system

A very simple tyrosinase reaction system has been developed using borate anion as a trapping agent of catechols and hydroxylamine as an external reductant to evaluate the phenolase activity without the interference of catecholase activity. Reactivities of variously para-substituted phenols in this system were compared directly to those of the phenols in the model reactions, demonstrating that the enzymatic oxygenation reaction of phenols proceeds via the same mechanism as the model reaction, that is, electrophilic aromatic substitution mechanism.     

Determination of the urease activity and the relative inhibition in the presence of some metal ions: a microcalorimetric study

Batch microcalorimetry has been employed to obtain a calibration curve for the enzymatic activity of urease in solution.This method is simplier, more reliable and easier to handle than the more common techniques (spectrophotometry and potentiometry), because it is based on direct investigation of the enzymatic reaction.By comparison with calorimetric studies employing the thermistor combined with the immobilized, enzyme, this method also allows the catalytic activity to be measured.Variations in the urease activity in the presence of nine metal ions [Hg(II), Ag(I), Cu(II),Zn(II), Cd(II), Ni(II), Co(II), Mn(II) and Mg(II)] are also described.A graphic method has been devised for immediate identification of the minimum inhibitor concentration,determining the start, 50% and complete inhibition of ureasic activity.