Modeling cellobiose hydrolysis with integrated kinetic models

The enzyme cellobiase Novozym 188, which is used for improving hydrolysis of bagasse with cellulase, was characterized in its commercial available form and integrated kinetic models were applied to the hydrolysis of cellobiose. The specific activity of this enzyme was determined for pH values from 3.0–7.0, and temperatures from 40–75°C, with cellobiose at 2 g/L. Thermal stability was measured at pH 4.8 and temperatures from 40–70°C. Substrate inhibition was studied at the same pH, 50°C, and cellobiose concentrations from 0.4–20 g/L. Product inhibition was determined at 50°C, pH 4.8, cellobiose concentrations of 2 and 20 g/L, and initial glucose concentration nearly zero or 1.8 g/L. The enzyme has shown the greatest specific activity, 17.8 U/mg, at pH 4.5 and 65°C. Thermal activation of the enzyme followed Arrhenius equation with the Energy of Activation being equal to 11 kcal/mol for pH values 4 and 5. Thermal deactivation was adequately modeled by the exponential decay model with Energy of Deactivation giving 81.6 kcal/mol. Kinetics parameters for substrate uncompetitive inhibition were: Km=2.42 mM, V _max=16.31 U/mg, Ks=54.2 mM. Substrate inhibition was clearly observed above 10 mM cellobiose. Product inhibition at the concentration studied has usually doubled the time necessary to reach the same conversion at the lower temperature tested.     

A Microdialysis Probe Coupled with A Miniaturized Thermal Glucose Sensor for In Vivo Monitoring

Abstract

A miniaturized thermal flow injection analysis biosensor has been coupled with a microdialysis probe for continuous subcutaneous glucose monitoring. Thermal biosensors are based on the principle of measuring the heat evolved during enzyme catalysed reactions. The system presented here consists of a miniaturized thermal biosensor with a small column containing coimmibolized glucose oxidase and catalase. The analysis buffer passes through the column at a flow rate of 60μL/min via an 1μL sample loop which is connected to a microdialysis probe.

Invitro results showed constant permeability of the probe and stability of the biosensor response during 24 hours. The response time was 85 sec giving a sample rate of 42 samples/hour.

During a load experiment, the glucose profile in a healthy volunteer was followed both in the subcutaneous tissue and blood using the microdialysis set-up proposed and comparing to blood glucose analyser.     

Amperometric Determination of Picomolar Levels of Flavin Adenine Dinucleotide by Cyclic Oxidation-Reduction in Apo-Glucose Oxidase System

Abstract

Acidification of a solution of glucose oxidase in 35 percent glycerol with 10 percent sulfuric acid and a gel filtration of this acidified glucose oxidase solution yielded apo-glucose oxidase with low residual enzyme activity. Further treatment with charcoal gave apo-glucose oxidase which is devoid of enzyme activity. Using this apo-glucose oxidase, it was possible to measure flavin adenine dinucleotide amperometrically at extremely low concentrations (10_?12 M) with ease, rapidity, and convenience.     

Application of HPLC to the Study of the Chloroplast Atpase Mg2+ Dependent Mechanism

Abstract

The determination by HPLC of released ADP from ATP by chloroplast ATPase (CF₁) is described.

The enzymatic rate measured by this method is well defined, over several minutes.

In the case of ? subunit-depleted CF₁ or activated CF₁, the rate is proportional to the enzyme concentration, the steady state theory is followed and the K_m and V_m constants have been calculated.

The enzymatic activity of CF₁ is inhibited by endogenous ? subunit and the inhibition constant has been measured.

The influences of ionic strength, pH, magnesium ion, phosphate and ADP concentrations have been studied and the results obtained by this method have been compared to previously reported data based on rate determination of released phosphate.     

A Prospect for the Analysis of Species Acting as Enzyme Inhibitors as Illustrated by Heavy Metal Inhibition

Abstract

A flow system incorporating an amperometric glucose oxidase enzyme electrode has been used to study the inhibitory effects of 16 metal cations on glucose oxidase. Only copper(II), mercury(II) and silver(I) caused any significant inhibition. the enzyme electrode could be reactivated by EDTA, the reactivation being most effective for copper(II) and least so for silver(I). Other complexing agents were tried for reactivation but proved to be unsatisfactory.

The ability to reactivate the enzyme on the electrode following copper(II) inhibition, and the linear response of the system to the level of this inhibitor according to I/A = -9.49 × 10^?7 log([Cu]/M) + 4.84 × 10^?8; r = 0.994 between 2.5 × 10^?4M and 5 × 10^?3M [Cu]²⁺ indicates a prospect for the use of a flow system for determining enzyme inhibitors in samples.     

Characterization of a monoclonal antibody that specifically inhibits pullulanase activity ofbacillus circulans amylase-pullulanase enzyme

A monoclonal antibody (MAb) against amylase-pullulanase enzyme fromBacillus circulons, which hydrolyzes not only theα-1,6-glycosidic linkage but also theα-1,4-glycosidic linkage to the same extent, has been produced by the fusion of BALB/c mouse spleen cells immunized with the native enzyme and P3x63Ag8U1 myeloma cells, and examined for inhibition of pullulanase activity in order to characterize the catalytic site of the pullulanase. The MAb recognizes active enzyme, but not the SDS-denatured or heat-inactivated protein, indicating that the antibody is highly conformational-dependent, specific for active enzyme. The antibody inhibited the pullulanase activity, but not amylase activity. The monoclonal antibody immunoblotted the enzyme and immunoprecipitated the enzyme. The immunoprecipitation was inhibited in the presence of substrate, pullulan, and the MAb competitively inhibited the binding of pullulan to the enzyme. The MAb, therefore, recognizes the pullulanbinding site of the enzyme. Kinetic analysis showed that the MAb inhibited pullulanase activity with inhibition constant (K _i ,) of 0.77Μg/mL, providing evidence that the antibody decreases the catalytic rate of enzyme activity and has an effect on substrate binding. These results strongly confirm the previous observations that APE may have two different active sites responsible for the expression of amylase and pullulanase activities (Kim, C. H. and Kim, Y. S.Eur. J. Biochem. 1995,227, 687–693).     

Preparation and Characterization of Pepsin Immobilized on Polymeric Supports

Abstract

Immobilization of pepsin on crosslinked resinous materials SRF (salicylic acid-resorcinol-formaldehyde), Amberlite IRA-400, and poly-(vinyl alcohol) is reported. Enzyme concentration, pH of the coupling medium, and nature and concentration of crosslinking agents were optimized for the better retention of activity of immobilized pepsin. The immobilized systems were characterized through pH, thermal, and storage stabilities. Michaelis constant (K _m) and maximum reaction velocity (V _m) for the free and immobilized enzymes were calculated from Lineweaver-Burk plots. Effect of temperature on enzyme activity was studied, and the thermoinactivation constant (K _ti) and energy of activation (E _a) for free and immobilized enzymes were also calculated. The immobilized pepsin was used in a continuous fluidized bed reactor for the study of clotting of skimmed milk. Rate of coagulation was considerably high for the treated milk sample at 50°C and pH 6–6.2.     

Design,synthesis and biological evaluation of some 2-(6-nitrobenzo[d]thiazol-2-ylthio)-N-benzyl-N-(6-nitrobenzo[d]thiazol-2-yl)acetamide derivatives as selective DprE1 inhibitors

Abstract

Tuberculosis (TB) is an infectious disease and caused by various strains of mycobacteria. In the present study, pharmacophore model was developed using single ligand by ligand-based drug discovery approach. The key features responsible for DprE1 inhibitory activity were taken into consideration for developing pharmacophore. After the virtual screening, top 1000 hits were further subjected to docking study using GLIDE module, Schrödinger. Docking studies have shown promising interaction with amino residues with better glide score. Ligand-based drug design approach yielded a series of 15, 2-(6-nitrobenzo[d]thiazol-2-ylthio)-N-benzyl-N-(6-nitrobenzo[d]thiazol-2-yl)acetamide derivatives. All synthesized derivatives were characterized using NMR, mass, CHN analysis. The synthesized compounds were screened for In vitro antitubercular activity against Mycobacterium tuberculosis (H₃₇Rv). Four compounds, 5g (MIC-1.01?μM); 5i (MIC-0.91?μM); 5k (MIC-0.82?μM); and 5o (MIC-1.04?μM) has shown promising activity compared to MIC of standard isoniazid (INH) and DprE1 enzyme inhibition was compared to BTZ043. Two halogen-substituted compounds have exhibited drastic enzyme inhibition.     

Synthesis,characterization, semi-empirical study,and biological activities of organotin(IV) and transition metal complexes with o-methyl carbonodithioate

Three transition metal and six organotin(IV) complexes have been synthesized by treating potassium o-methyl carbonodithioate with ZnCl₂/CdCl₂/HgCl₂ and R₂SnCl₂/R₃SnCl under stirring. The complexes were characterized by IR, ¹H, and ¹³C NMR spectroscopies. IR results show that the ligand is bidentate in 1–3 while monodentate in 4–9, which is also confirmed by semi-empirical study. NMR data reveal four-coordinate geometry in solution. HOMO–LUMO study shows that 7 and 9 are thermodynamically unstable. The enzyme inhibition study shows that 1 is a potent inhibitor of ALP, EC 3.1.3.1, resulting in very slow rate of formation and breakdown of enzyme–substrate complex. UV/visible spectroscopy was used to assess the mode of interaction and binding of the complexes with DNA which shows that 9 exhibits higher binding constant when compared to 6. In protein kinase inhibition assay, 1 was active, while antifungal activity shows that organotin(IV) complexes are more active than transition metal complexes.     

Glucose Biosensor Based on Oxygen Electrode. Part II: Long-Term Operational Stability of the Rechargeable Glucose Biosensor

Abstract

A potentially implantable glucose biosensor for measuring blood or tissue glucose levels in diabetic patients has been developed. The glucose biosensor is based on an amperometric oxygen electrode and immobilized glucose oxidase enzyme, in which the immobilized enzyme can be replaced (the sensor recharged) without surgical removal of the sensor from the patient. Recharging of the sensor is achieved by injecting fresh immobilized enzyme into the sensor using a septum. A special technique for immobilization of the enzyme on Ultra-Low Temperature Isotropic (ULTI) carbon powder held in a liquid suspension has been developed.

In vitro studies of the sensors show stable performance during several recharge cycles over a period of 3 months of continuous operation.

Diffusion membranes which ensure linear dependence of the sensor response on glucose concentration have been developed. These membranes comprise silastic latex-rubber coatings over a microporous polycarbonate membrane. Calibration curves of the amperometric signal show linearity over a wide range of glucose concentrations (up to 16 mM), covering hypoglycemic, normoglycemic and hyperglycemic conditions.

The experimental results confirm the suitability of the sensors for in vitro measurements in undiluted human sera.     

Immobilization of Cytochrome P-450 and its Application in Ehzikb Electrodes

Abstract

For application in enzyme electrodes liver microsomal cytochrome P-450 was immobilized in a membraneous form. The immobilization yielded 60% of activity and did not impair the functional stability of the enzyme. By coimmobilization of glucose oxidase with P-450 the cofactor NADPH could be replaced by H₂O₂ formed from the enzymatic glucose oxidation. Fixed to a graphite electrode the obtained preparations were employed for quantitative substrate analysis. The P-450 substrate aniline was measured by anodic oxidation of its hydroqlation product at +250mV. A linear dependence of: the current on aniline concentration up to 0.5mM was obtained.     

Urease inhibitory potential of extracts and active phytochemicals of Hypochaeris radicata (Asteraceae)

Abstract

Urease inhibition potential of compound (1), guaiane-type sesquiterpene (2), confertin (3) and scopoletin (4) was carried out with high throughout mechanism-based assay. These compounds were isolated from Hypochaeris radicata L., an Asteraceae family member. The pure compounds were screened for their urease and carbonic anhydrase inhibitory activities. The ethyl acetate fractions were subjected to column chromatography, which resulted in the isolation and purification of four compounds (1–4). On evaluation, compounds (1–4) exhibited selective activity against urease enzyme with an IC₅₀ value of 180.11 ± 2.00, 27.18 ± 0.80, 24.12 ± 0.2 and 30.12 ± 1.10 µM respectively. The compounds (1–4) were found to be inactive against carbonic anhydrase enzyme. Thiourea was used as standard inhibitor (21 ± 0.14 µM) of urease enzyme.     

Fast Amperometric Determination of Enzymatic Activity of Glutaminase

ABSTRACT

The activity of the enzyme glutaminase has been measured using a glutamate electrochemical biosensor based on H₂O₂ detection. Calibration curves for glutamate detection and for glutaminase activity using standard glutaminase from Escherichia coli demonstrated the high sensitivity and the rapid analysis time of this novel amperometric procedure, which was 100 times more sensitive than that reported in liternature.

Porcine liver and kidney tissue and human kidney tissue samples have been tested for glutaminase activity, demonstrating the possibility to perform measurement directly on whole tissues, with no need of sample extraction and purification.     

Neue Methode zur viskosimetrischen Bestimmung der Hemmkonstante KI für Enzymreaktionen,die in der Art einer reversiblen,kompetitiven Hemmung verlaufen

Zusammenfassung Es wird eine Theorie entwickelt, die es ermöglicht, viskosimetrisch die Kinetik von Enzymreaktionen zu verfolgen, an denen hochmolekulare Substrate teilnehmen und die in der Art einer reversiblen, kompetitiven Hemmung verlaufen. Die abgeleiteten Relationen geben die Möglichkeit, die Hemmkonstante (K _I) der verlaufenden Enzymreaktion bei dieser art Hemmung zu bestimmen.

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A new method for the viscosimetric determination of the inhibition constant K _I for enzymatic reactions of the reversible competitive inhibition type

A theory has been developed making it possible to viscosimetrically trace enzyme reactions in which high molecular substrates are involved and which belong to the reversible competitive-inhibition type. The equations derived permit of a determination of the inhibition constantK _I.

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Mit 1 Abbildung     

Calixarenes functionalised water-soluble iron oxide magnetite nanoparticles for enzyme immobilisation

ABSTRACT

In this study, we first used water-soluble iron oxide nanoparticles for Candida rugosa lipase immobilisation. Moreover, two new complexation phenomena of the prepared water-soluble Fe₃O₄ nanoparticles with an enzyme might address interesting results in terms of enzyme activity and stability in typical enzymatic reactions. The catalytic activity on pH and temperature dependence, and reusability of the immobilised lipases ( p-SCX4-NP-E and p-SCX8-NP-E) were also investigated with the hydrolysis reaction of p-nitrophenyl palmitate (p-NPP). The results show that the highest catalytic affinities for p-SCX4-NP-E and p-SCX8-NP-E were obtained at pH 5.0 and pH 7.0, respectively.     

New copper(II) complexes including pyridine-2,5-dicarboxylic acid: synthesis,spectroscopic, thermal properties,crystal structure and how these complexes interact with purified PON 1 enzyme

We report the synthesis of two square-pyramidal copper(II) complexes, [Cu(2,5-pydc)(2-aepy)(H₂O)]·H₂O, 1, and [Cu(2,5-pydc)(2-ampy)(H₂O)]·H₂O, 2 (2-aepy = 2-(aminoethyl)pyridine, 2-ampy = 2-(aminomethyl)pyridine, 2,5-pydc = pyridine-2,5-dicarboxylic acid or isocinchomeronic acid). The synthesized complexes have been characterized by X-ray diffraction, FT-IR, elemental, and thermal analysis techniques. The crystal structure of 1 was established by X-ray analysis. Powder X-ray diffraction analysis showed that the complexes are pure. The inhibition of human serum paraoxonase 1 (PON 1, EC 3.1.8.1) enzyme with these complexes were investigated. We used diethyl 4-nitrophenyl phosphate as a substrate to measure the paraoxonase activity of PON 1 enzyme spectrophotometrically. Complexes 1 and 2 decreased the in vitro PON 1 activity with different inhibition mechanisms. Complexes 1 and 2 inhibited paraoxonase activity of this enzyme as competitively and noncompetitively, respectively.     

Preparation and properties of glucose isomerase immobilized on Indion 48-R

Partially purified glucose isomerase fromStreptomyces thermonitrificans when coupled to glutaraldehyde-activated Indion 48-R, retained 30–40% activity of the soluble enzyme. However, an approximately twofold increase in the activity could be achieved by binding the enzyme in the presence of glucose. Binding the enzyme to matrices presaturated with either glucose or fructose and influence of lysine modification on the activity of the soluble enzyme revealed that the comparatively low activity observed in case of the enzyme bound in the absence of substrate is the result of the nonspecific binding of either substrate or product to the matrix. Immobilization did not affect the pH and temperature optima of the enzyme, but it lowered the temperature stability. Immobilization resulted in a marginal increase in theK _m and a threefold decrease in theV _max . Substrate concentrations as high as 36% glucose could be converted to 18.5% fructose in 5 h, at pH 7.0 and 70‡C. The bound enzyme, however, showed inferior stability to repeated use and lost approx 40% of its initial activity after five cycles of use. Indion 48-R bound glucose isomerase could be stored, in wet state, for 30 d without any apparent loss in its initial activity.     

The Application of Engineered Glucose Dehydrogenase to a Direct Electron–Transfer‐Type Continuous Glucose Monitoring System and a Compartmentless Biofuel Cell

Abstract

Continuous glucose monitoring (CGM) is expected to become an ideal way to monitor glycemic levels in diabetic patients. On the other hand, biofuel cells can be used as an alternative energy source in future implantable devices, such as implantable glucose sensors in the artificial pancreas. Glucose dehydrogenase from Acinetobacter calcoaceticus, which harbors pyrroloquinoline quinone as the prosthetic group (PQQGDH), is one of the enzymes most attractive as a glucose sensor constituent and as the anode enzyme in biofuel cells, due to its high catalytic activity and insensitivity to oxygen. However, the application of PQQGDH for these purposes is inherently limited because an electron mediator is required for the electron transfer to the electrode.

We have recently reported on the development of an engineered enzyme, quinohemoprotein glucose dehydrogenase (QH‐GDH), in which the cytochrome c domain of the quinohemoprotein ethanol dehydrogenase (QH‐EDH) was fused with PQQGDH, to enable electron transfer to the electrode in the absence of an artificial mediator. In this study, we constructed a direct electron‐transfer‐type CGM system employing QH‐GDH. This CGM system showed sufficient current response and high operational stability. Furthermore, we successfully constructed a compartmentless biofuel cell employing QH‐GDH.     

A Direct OnFlow Assay to Monitor the Activity of Purine Nucleoside Phosphorylase from Mycobacterium tuberculosis

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides to the corresponding purine bases and (deoxy)ribosyl-1-phosphate. PNP plays a central role in purine recycling and salvage pathway and has been considered an attractive chemotherapeutic target for several diseases. In this work, PNP from Mycobacterium tuberculosis (MtPNP) was covalently immobilized into fused-silica capillaries. The activity of the produced immobilized enzyme reactor (MtPNP-IMER) was monitored onflow in a multidimensional liquid chromatography system containing the MtPNP-IMER in the first dimension. A C18 analytical column inserted in the second dimension furnished the rapid chromatographic separation of the substrate (inosine) and product (hypoxanthine) from the MtPNP catalyzed reaction, allowing monitoring MtPNP-IMER activity through the direct quantification of hypoxanthine formed. Kinetic studies showed that the K_M values (59.19 μmol L^?1) for substrates using the immobilized enzyme were consistent with those reported for the free enzyme in solution (40 μmol L^?1). To validate the use of the proposed methodology for screening purposes, a fourth-generation immucillin derivative (DI4G), known as a PNP inhibitor, was used as a standard inhibitor. The studies revealed that the immobilized enzyme retained the ability to recognize enzyme inhibitors and the onflow assay allowed the characterization of the inhibitor by determining the IC₅₀ (29.85?±?1.02 nmol L^?1), inhibition mechanism and constant (K_i?=?34.8?±?0.2 nmol L^?1). The results revealed that the immobilized MtPNP retained its catalytic activity (up to 60%) and the ability to recognize ligands with high stability for up to 10 days. The proposed model has as advantages the automation and the possibility of reusing the same amount of enzyme in several assays.