Glycerol-3-phosphate Oxidase Research Articles

Development of interdigitated electrode based enzyme sensor for triglyceride monitoring using engineered oxygen insensitive glycerol 3-phosphate oxidase

Triglycerides (TG) are an important biomarker of various diseases including hyper-lipidemia. Current emergent requirements for TG sensing include the development of electrochemical sensors for easy, cost-effective, disposable and single use triglyceride sensors. We propose a TG sensor using an engineered glycerol 3-phosphate oxidase (GlpO) and electrochemical detection. In this study, we aimed to develop an electrochemical TG sensor based on engineering the oxidative half reaction of GlpO to be oxygen insensitive, with the employment of an interdigitated electrode (IDE). To construct an oxygen insensitive mutant GlpO, we constructed a double mutant, Met104Phe/Asp169Glu, which revealed an engineered GlpO exhibiting repressed oxidase activity, while its dye-mediated dehydrogenase activity enhanced. The electrochemical enzyme sensor was constructed for the measurement of TG based on engineered GlpO using disposable screen-printed carbon electrode (SPCE) or disposable IDE using Ru complex as the electron mediator. By using the disposable IDE sensor, a highly sensitive electrochemical sensor for Glp was achieved, with the sensitivity of 0.4 µA/mM. The improved sensitivity was 2.4 times higher than that of SPCE (0.17 µA/mM), with LOD of 0.022 mM. Furthermore, the dry sensor strip, where the double mutant GlpO and Ru complex were dropcasted and dried on the IDE, was prepared and the Glp concentration was successfully measured in the delipidized serum solution. Therefore, by the employment of the established pretreatment method for TG to release Glp, a highly sensitive, accurate, easy and rapid electrochemical measurement method for TG will be developed using constructed M104F/D169E mutant GlpO with the combination of IDE.

Electrochemical Biosensor Based on Chitosan- and Thioctic-Acid-Modified Nanoporous Gold Co-Immobilization Enzyme for Glycerol Determination

An electrochemical biosensor based on chitosan- and thioctic-acid-modified nanoporous gold (NPG) co-immobilization glycerol kinase (GK) and glycerol-3-phosphate oxidase (GPO) was constructed for glycerol determination in wine. The NPG, with the properties of porous microstructure, large specific surface area, and high conductivity, was beneficial for protecting the enzyme from inactivation and denaturation and enhancing electron transfer in the modified electrode. The co-immobilization of the enzyme by chitosan-embedding and thioctic-acid-modified NPG covalent bonding was beneficial for improving the catalytic performance and stability of the enzyme-modified electrode. Ferrocene methanol (Fm) was used as a redox mediator to accelerate the electron transfer rate of the enzyme-modified electrode. The fabricated biosensor exhibited a wide determination range of 0.1–5 mM, low determination limit of 77.08 μM, and high sensitivity of 9.17 μA mM−1. Furthermore, it possessed good selectivity, repeatability, and stability, and could be used for the determination of glycerol in real wine samples. This work provides a simple and novel method for the construction of biosensors, which may be helpful to the application of enzymatic biosensors in different determination scenarios.

Development of Interdigitated Electrode Based Enzyme Sensor for Triglyceride Monitoring Using Engineered Oxygen Insensitive Glycerol 3-Phosphate Oxidase

Development of Interdigitated Electrode Based Enzyme Sensor for Triglyceride Monitoring Using Engineered Oxygen Insensitive Glycerol 3-Phosphate Oxidase

DYSLIPIDEMIA IN HYPOTHYROIDISM PATIENTS: A HOSPITAL BASED CASE AND CONTROL STUDY.

Objectives: This present study was to evaluate the incidence and abnormalities in lipid profile in patients with hypothyroidism.
 Methods: A detail history, clinical examinations and relevant investigations were performed to all subjects. Serum free T3 (fT3) and T4 (fT4) were measured by competitive enzyme-linked immunosorbent assay (ELISA) method. Serum TSH was measured by sand-witch ELISA method. Serum cholesterol was measured by cholesterol oxidase-peroxidase (CHODPAP) method. Serum triglyceride was measured by glycerol-3-phosphate oxidase (GPO) method. Serum VLDL (in mg/dl) was calculated by dividing the value of TG by 5. Serum LDL value was calculated by Fried-Wald equation. And serum HDL level was calculated by precipitation method.
 Results: Data was analysed by using software SPSS 15.0. Independent t-test was applied. Mean and standard deviations were observed. P-value was taken equal to or less than 0.05 for significant differences (p≤0.05).
 Conclusions: Hypothyroidism was commonly seen in elderly age group subjects. Females were more preponderance than males. Hypercholesterolemia, hyper triglyceridemia and increased LDL-cholestrol & VLDL-Cholestrol were extremely significant associated with hypothyroidism. Hence, hypothyroidism has an importantly associated with abnormal lipid profile. Therefore, biochemical screening for thyroid dysfunction should be perform in all dyslipidemic patients. Early diagnosis should be made for proper treatment of dyslipidemia in hypothyroidism patients.
 Keywords: hypothyroidism, dyslipidemia, age, sex.

Development of glycerol biosensor based on co-immobilization of enzyme nanoparticles onto graphene oxide nanoparticles decorated pencil graphite electrode.

An improved amperometric biosensor was fabricated by immobilizing glycerol kinase (GK) and glycerol-3-phosphate oxidase (GPO) nanoparticles (NPs) onto graphene oxide nanoparticles (GrONPs) modified pencil graphite (PG) electrode. The GKNPs, GPONPs and GrONPs were characterized by UV spectroscopy, and transmission electron microscopy (TEM). The working electrode (GKNPs/GPONPs/GrONPs/PGE) was characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques. The biosensor exhibited optimal current response at an applied potential of 0.45 V, pH 8.0, and 35 °C. The biosensor displayed a wide linear response for glycerol concentration from 0.001 to 60 mM with a detection limit of 0.002 μM. Moreover, a very high sensitivity 121.45 μA·mM-1·cm-2, rapid response time (2 s) and a good concurrence with the standard enzymic colorimetric technique with a correlation coefficient (R2 = 0.99) was offered by the present biosensor. Evidently, biosensor revealed an analytical recovery of 98.5% after addition of glycerol to the sera samples. Within and between batches studies of working electrode demonstrated coefficients of variation of 0.098% and 0.101%, respectively. The biosensor measured blood serum glycerol level in patients suffering from hyperglyceridemia. The biosensor lost 25% of its initial activity after its regular use over a period of 210 days, at 4 °C storage condition.

Fabrication of glycerol biosensor based on co-immobilization of enzyme nanoparticles onto pencil graphite electrode

Glycerol kinase (GK) and glycerol-3- phosphate oxidase (GPO) nanoparticles (NPs) were prepared, characterized and immobilized onto pencil graphite (PG) electrode to fabricate an improved amperometric glycerol biosensor (GKNPs/GPONPs/PGE). GKNPs/GPONPs/PGE worked in optimum conditions of pH 7.0, temperature 30 °C, at an applied potential of −0.3 V. The biosensor exhibited wide linear response in a concentration range of glycerol (0.01–45 mM) with detection limit 0.0001 μM. The biosensor revealed high sensitivity (7.24 μAmM−1cm−2), low response time (2.5s) and a good agreement with the standard enzymic colorimetric method with a correlation coefficient (R2 = 0.99). The evaluation study of biosensor offered a good analytical recovery of 98.73% when glycerol concentration was added to the sera sample. In addition, within and between batches study of working electrode showed coefficients of variation as 0.105% and 0.14%, respectively. The application of biosensor is performed in the serum of apparently healthy subject and patients affected by cardiogenic shock. There was a 20% loss in initial activity of biosensor after its regular use over a time period of 180 days, while being stored at 4 °C.

Antidiabetic effects of aqueous and dichloromethane/methanol stem bark extracts of Pterocarpus soyauxii Taub (Papilionaceae) on streptozotocin-induced diabetic rats

Aim of the Study:The aim is to evaluate the hypoglycemic and antidiabetic effects of aqueous and CH2Cl2/CH3OH stem bark extracts of Pterocarpus soyauxii Taub in normal and diabetic rats.Materials and Methods:Streptozotocin (STZ)-induced diabetic and normal adult Wistar rats were orally administered with aqueous and CH2Cl2/CH3OH plant extracts of P. soyauxii at various doses (38–300 mg/kg) in a single administration. In addition, STZ-induced diabetic rats received prolonged daily administration for 14 days. Glibenclamide (GB) (10 mg/kg) was used as reference treatment. In acute test, fasting blood glucose was followed for 5 h. In subacute test, body weight, food and water intakes, and blood glucose were followed weekly and serum biochemical parameters evaluated after 14 days treatment.Results:Acute administration of aqueous and CH2Cl2/CH3OH stem bark extracts moderately decreased fasting blood glucose compared to GB, significantly in normal rats (P < 0.05 to P < 0.01) but, as GB, not significantly in diabetic rats. Prolonged treatments in diabetic rats with aqueous and CH2Cl2/CH3OH extracts reduced blood glucose to an extent, respectively, superior or similar to GB. Moreover, P. soyauxii also significantly (P < 0.01) reduced weight loss, and diabetes increased serum triglycerides, total cholesterol, and transaminases (alanine aminotransferase/aspartate aminotransferase) elevations.Conclusion:P. soyauxii Taub stem bark extracts have possible value for antidiabetic oral medication.SUMMARY Aqueous and Dichloromethane/Methanol stem bark extracts of Pterocarpus soyauxii Taub have potent (compared to Glibenclamide) antidiabetic effects in STZ-diabetic rats, with specific kinetics and dose-responses.Moderate hypoglycemia effects upon acute P. soyauxii administration.Potent anti-hyperglycemic effects of sub-acute P. soyauxii administration in STZ-diabetic rats.Potent anti-hyperlipidemic effects of sub-acute P. soyauxii administration in STZ-diabetic rats.Improved hepatic and renal serum parameters after sub-acute P. soyauxii administration in STZ-diabetic rats.P. soyauxii extracts may be useful for oral treatment of diabetes and related metabolic disorders. Abbreviations Used: CH2Cl2/CH3OH: Dichloromethane/Methanol; STZ: Streptozotocin; GB: Glibenclamide; AE: Aqueous extract; OE: Organic extract; FeCl3: Iron (III) chloride; NaCl: Sodium chloride; K3Fe(CN)6: Potassium ferricyanide; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; H: Hour; BW: Body weight, W1 and W2: Weeks 1 and 2; CHOD: Cholesterol oxidase; GPO: Glycerol-3 Phosphate oxidase; PAP: Phenol + Aminophenazone

Amperometric triglyceride bionanosensor based on nanoparticles of lipase, glycerol kinase, glycerol-3-phosphate oxidase

The nanoparticles (NPs) aggregates of lipase from porcine pancreas, glycerol kinase (GK) from Cellulomonas sp. and glycerol-3-phosphate oxidase (GPO) from Aerococcus viridanss were prepared by desolvation and glutaraldehyde crosslinking and functionalized by cysteamine. These enzyme nanoparticles (ENPs) were characterized by transmission electron microscopy (TEM) and Fourier transform infra red (FTIR) spectroscopy. The functionalzed ENPs aggregates were co-immobilized covalently onto polycrystalline Au electrode through thiolated bond. An improved amperometric triglyceride (TG) bionanosensor was constructed using this ENPs modified Au electrode as working electrode. Biosensor showed optimum current at 1.2 V within 5s, at pH 6.5 and 35 °C.A linear relationship was obtained between current (mA) and triolein concentration in lower concentration range,10–100 mg/dL and higher concentration range, 100–500 mg/dL. Limit of detection (LOD) of bionanosensor was 1.0 μg/ml. Percent analytical recovery of added trolein (50 and 100 mg/dL) in serum was 95.2 ± 0.5 and 96.0 ± 0.17. Within and between batch coefficients of variation (CV) were 2.33% and 2.15% respectively. A good correlation (R2 = 0.99) was obtained between TG values in sera measured by present biosensor and standard enzymic colorimetric method with the regression equation: y= (0.993x + 0.967). ENPs/Au electrode was used 180 times over a period of 3 months with 50% loss in its initial activity, when stored dry at 4 °C.

EFEK ANTIHIPERLIPIDEMIA EKSTRAK ETANOL BUAH STRAWBERRY (Fragraria x ananassa Duchesne) PADA TIKUS PUTIH DARI DAERAH BANDUNG

A study on triglycerides levels and total cholesterol activity in rats blood serum. This experiment use 42 rats, they were divided into 6 group . The first group was given by standart treatment of dietary and aquadest, the second group was given by dietary of high fatty and aquadest, the third group was given by simvastatin suspension dose 0.9 mg /Kg BB, the four group, the five group and the six were orally given by of ethanolic extract strawberry fruit with dose 0.21 gram/kgBB; 0,425 gram/kgBB; and 0.85 gram/kgBB. Hyperlipidemia was treated with high dietary fatty (profiltiourasil 9 mg /kgBB and egg yolk 10 ml/Kg BB). The triglycerides, total cholesterol, LDL-cholesterol and HDL-cholesterol levels content determined with GPO (Glycerol-3-Phosphate Oxidase) test. The result of the research showed that ethanolic extract strawberry fruit 2 weeks can decrease triglycerides, total cholesterol, LDL-cholesterol and increased HDL-cholesterol levels. Keywords: strawberry fruit, triglycerides, cholesterol total, LDL-cholesterol and HDL-cholesterol.

A new multienzyme-type biosensor for triglyceride determination

ABSTRACTAn amperometric multienzyme biosensor for determination of triglycerides (TGs) was constructed by mounting three gelatin membrane-bound enzymes on a glassy carbon electrode (working electrode), then connecting it to electrometer along with an Ag/AgCl reference electrode and a Pt auxiliary electrode. Characterization and optimization of the multienzyme biosensor, which is prepared with glycerol kinase (GK) (E.C.2.7.1.30), glycerol-3-phosphate oxidase (GPO) (EC 1.1.3.21), and lipase (EC 3.1.1.3), were studied. In the optimization studies for the bioactive layer components of the prepared biosensor, the optimum amounts of gelatin, bovine serum albumin (BSA), and glutaraldehyde was calculated as 1 mg/cm2, 1 mg/cm2, and 2.5%, respectively. Optimum pH and temperature of the reaction of biosensor were determined as 7.0 and 40°C, respectively. Linear range of triolein for the biosensor was found from the calibration curve between several substrate concentration and Δ Current. After optimization and characterization of the biosensor, its operationability in triglycerides was also tested.

Construction of triglyceride biosensor based on nickel oxide–chitosan/zinc oxide/zinc hexacyanoferrate film

A method is described for construction of an amperometric triglyceride (TG) biosensor based on co-immobilization of lipase, glycerol kinase (GK) and glycerol-3-phosphate oxidase (GPO) onto nickel oxide nanoparticles (NiONPs)–chitosan (CHIT) nanocomposite adsorbed onto zinc oxide/zinc hexacyanoferrate (ZnO–ZnHCF) hybrid film electrodeposited on the surface of an Au electrode. The NiONPs–CHIT/ZnO–ZnHCF hybrid film was characterized by cyclic voltammetry (CV), atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 4s at pH 6.0 and 35°C, when polarized at +0.4V against Ag/AgCl. There was a linear relationship between sensor response and triolein concentration in the range 50–700mg/dl with sensitivity of 0.05μA/mg/dl. The sensor was employed for determination of TG in serum. The detection limit of the biosensor was 10mg/dl. The biosensor was evaluated with 95–96% recovery of added triolein in sera and 2% and 3% within and between batch coefficients of variation (CVs) respectively. There was a good correlation (r=0.99) between serum TG values by standard enzymic colorimetric method and the present method. The biosensor lost 50% of its initial activity after its 100 uses over a period of 180 days, when stored at 4°C.

Optical ATP biosensor for extracellular ATP measurement

Extracellular Adenosine-5′-triphosphate (ATP) is an important multi-functional molecule which can mediate numerous physiological activities by activating purinergic P2 receptors. The objective of this study was to develop a novel optical ATP sensor for in-situ extracellular ATP measurement in biological tissues. The optical ATP sensor was made by applying two layers of sol–gel coating to the end of an optical fiber probe end. The first layer contained ruthenium complex for sensing changes in oxygen concentration which resulted from oxidation of ATP by glycerol kinase and glycerol 3-phosphate oxidase entrapped in the second layer. It was demonstrated that the optical ATP sensor was capable of detecting ATP concentration at a broad range of 10−3mM to 1.5mM.A compensation method was established to enable the optical sensor to determine ATP concentration at different oxygen levels. This study also demonstrated the capability of ATP sensor to measure extracellular ATP content in biological tissues (i.e., porcine intervertebral disc). In addition, it was shown that the optical ATP sensor was not affected by pH and derivatives of extracellular ATP. Therefore, the newly developed optical ATP sensor is a good option for in-situ extracellular ATP measurement.

Droplet-based microfluidic sensing system for rapid fish freshness determination

A microfluidic device was constructed for on-site determination of fish freshness by using adenosine-5′-triphosphate (ATP) concentration as an indicator of freshness. In the device, extracts from fish and reagent solutions were processed in the form of droplets separated by air. The volumes of the droplets of the extract solution, a solution containing enzyme substrates, and a washing buffer solution were measured in branched flow channels. To simplify the structure and procedure for mixing solutions, a wider portion was formed in one flow channel to merge the droplets. A row of droplets was ejected from each of three branched flow channels, merged in the wider portion, and transported to a sensing region located downstream. ATP was detected using two enzymatic reactions involving glycerol kinase (GK) and glycerol-3-phosphate oxidase (G3PO). A linear relationship between the generated current and ATP concentration was confirmed, and ATP detection from jack mackerel extracts was carried out. A distinct change in the current was observed with the fresh extracts as in the case of standard solutions. The obtained data correlated well with those obtained by high-performance liquid chromatography (HPLC).

Construction of a triglyceride amperometric biosensor based on chitosan–ZnO nanocomposite film

A method is described for construction of a novel amperometric triglyceride (TG) biosensor based on covalent co-immobilization of lipase, glycerol kinase (GK) and glycerol-3-phosphate oxidase (GPO) onto chitosan (CHIT) and zinc oxide nanoparticles (ZnONPs) composite film deposited on the surface of Pt electrode. The enzymes–ZnONPs–CHIT composite was characterized by X-ray diffraction (XRD), scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The sensor showed optimum response within 6s at pH 7.5 and temperature of 35°C. The sensor measures current due to electrons generated at 0.4V against Ag/AgCl from H2O2, which is produced from triolein by co-immobilized enzymes. A linear relationship was obtained between a wide triolein concentration range (50–650mg/dl) and current (mA) under optimum conditions. The biosensor showed high sensitivity, low detection limit (20mg/dl) and good storage stability (half-life of 7 months at 4°C). The biosensor was unaffected modified by a number of serum substances at their physiological concentrations. The biosensor was evaluated and employed for determination of TG in sera in apparently healthy subjects and persons suffering from hypertriglyceridemia.

Enzymatic Determination of Diglyceride Using an Iridium Nano-Particle Based Single Use, Disposable Biosensor

A single use, disposable iridium-nano particle contained biosensor had been developed for the determination of diglyceride (DG). In this study hydrogen peroxide, formed through the enzymatic breakdown of DG via lipase, glycerol kinase and glycerol 3-phosphate oxidase, was electrochemically oxidized at an applied potential of +0.5 V versus the Ag/AgCl reference electrode. The oxidation current was then used to quantify the diglyceride concentration. Optimum enzyme concentrations and the surfactant loading used were established for successful sensor response. Good linear performance was observed over a DG concentration range of 0 to 25 μM in phosphate buffer and bovine serum media.

Lab-on-a-chip for analysis of triglycerides based on a replaceable enzyme carrier using magnetic beads

In this paper an enzyme-carrier-based microfluidic chip coupled with a gold nanoband microelectrode as electrochemical detector for Triglyceride (TG) determination was developed by co-immobilized lipase, Glycerokinase (GK) and glycerol-3-phosphate oxidase (GPOx) on chitosan/Fe(3)O(4) composite nanoparticles with a shell-core structure, which combined the advantageous features of microfluidic chips technology with magnetic beads. This procedure enabled the easy renewal of the microchip enzyme carrier after each determination in a highly reproducible manner. Several operational parameters such as working potential, buffer pH, adenosine triphosphate concentrations (ATP, mM), separation voltage and temperature were evaluated and optimized. The performance of enzyme-carrier-based microfluidic chip for TG determination was modulated by changing the length of enzyme carrier from 1.0 to 3.0 cm, and the linear ranges were changed from 0-4.0 mM to 0-10.0 mM with the detection limits from 15 μM to 6.0 μM. The enzyme carrier remained its 70% activity after 40 days storage. This system was successfully employed for on-line detection of TG in serums. The experimental results demonstrated that this enzyme carrier using magnetic beads based microfluidic chip provided a relatively simple, sensitive, miniature, and replaceable means for the accurate determination of TG in serum.

Fabrication of an Amperometric Triglyceride Biosensor based on PVC Membrane

An amperometric enzyme sensor for determination of triglycerides (TGs) was constructed by mounting polyvinyl chloride (PVC) membrane bound lipase, glycerol kinase (GK), glycerol-3-phosphate oxidase (GPO), and horseradish peroxidase (HRP) on a platinum electrode (working electrode), then connecting it to electrometer along with Ag/AgCl reference electrode and Cu auxiliary electrode. The biosensor measures the electrons generated from H2O2 at 0.4 V, which is formed from triolein/triglycerides by co-immobilized lipase, GK, GPO, and HRP. The concentration of triolein/TG was directly proportional to the current measured. The enzyme electrode showed optimum response when operated at 25°C in 0.1 M sodium phosphate buffer, pH 7.5, for 30 s. A linear relationship was obtained between triolein concentration ranging from 0.56 to 2.25 mM and amount of current (mA). The minimum detection limit of the method was 0.11 mM. The levels of TG in serum of apparently healthy persons and persons suffering from cardiovascular disease and pancreatitis, as measured by this sensor, were in the range 50–216 mg/dl and 200–499 mg/dl respectively. The analytical recovery of added triolein was 85.2%. Within-batch and between-batch coefficients of variation were <2.53% and <3.21% respectively. A good correlation (r = 0.91) was found between serum TG values obtained by commercial enzymic colorimetric and the present method. Among the various serum substances tested, only cholesterol and bilirubin caused inhibition, 15% and 8% respectively. The enzyme electrode was used 100 times over 40 days without any considerable loss of activity when stored at 4 °C.

Enzymatic measurement of phosphatidic acid in cultured cells

In this work, we developed a novel enzymatic method for measuring phosphatidic acid (PA) in cultured cells. The enzymatic reaction sequence of the method involves hydrolysis of PA to produce glycerol-3-phosphate (G3P), which is then oxidized by G3P oxidase to generate hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacted with Amplex Red to produce highly fluorescent resorufin. We found that lipase from Pseudomonas sp. can completely hydrolyze PA to G3P and FAs. The calibration curve for PA measurement was linear between 20 and 250 microM, and the detection limit was 5 microM (50 pmol in the reaction mixture). We also modified the method for the enzymatic measurement of lysophosphatidic acid. By this new method, we determined the PA content in the lipid extract from HEK293 cells. The cellular content of PA was decreased with increasing cell density but not correlated with the proliferation rate. The diacylglycerol kinase inhibitor R59949 markedly reduced the cellular PA content, suggesting the diacylglycerol kinase activity was involved in a large part of the PA production in HEK293 cells. This novel method for PA quantification is simple, rapid, specific, sensitive, and high-throughput and will help to study the biological functions of PA and its related enzymes.

Glycerol Metabolism Is Important for Cytotoxicity of Mycoplasma pneumoniae

Glycerol is one of the few carbon sources that can be utilized by Mycoplasma pneumoniae. Glycerol metabolism involves uptake by facilitated diffusion, phosphorylation, and the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate, a glycolytic intermediate. We have analyzed the expression of the genes involved in glycerol metabolism and observed constitutive expression irrespective of the presence of glycerol or preferred carbon sources. Similarly, the enzymatic activity of glycerol kinase is not modulated by HPr-dependent phosphorylation. This lack of regulation is unique among the bacteria for which glycerol metabolism has been studied so far. Two types of enzymes catalyze the oxidation of glycerol 3-phosphate: oxidases and dehydrogenases. Here, we demonstrate that the enzyme encoded by the M. pneumoniae glpD gene is a glycerol 3-phosphate oxidase that forms hydrogen peroxide rather than NADH(2). The formation of hydrogen peroxide by GlpD is crucial for cytotoxic effects of M. pneumoniae. A glpD mutant exhibited a significantly reduced formation of hydrogen peroxide and a severely reduced cytotoxicity. Attempts to isolate mutants affected in the genes of glycerol metabolism revealed that only the glpD gene, encoding the glycerol 3-phosphate oxidase, is dispensable. In contrast, the glpF and glpK genes, encoding the glycerol facilitator and the glycerol kinase, respectively, are essential in M. pneumoniae. Thus, the enzymes of glycerol metabolism are crucial for the pathogenicity of M. pneumoniae but also for other essential, yet-to-be-identified functions in the M. pneumoniae cell.

Construction of an amperometric enzymic sensor for triglyceride determination

Abstract An amperometric enzyme sensor for determination of triglycerides (TGs) was constructed by mounting cellulose acetate (CA) membrane-bound commercial lipase (E.C.3.1.1.3), glycerol kinase (GK) (E.C.2.7.1.30), and glycerol-3-phosphate oxidase (GPO) (E.C.1.1.3.21), on a platinum electrode (working electrode), then connecting it to potentiostat along with Ag/AgCl reference electrode and Ag auxillary electrode. The biosensor measures the electrons generated from H2O2 under a potential of 0.4 V which in turn formed from triolein/triglycerides by co-immobilized lipase, GK and GPO. The concentration of triolein/TG was directly proportional to the current measured. The enzyme electrode showed optimum response when operated at 25 °C in 0.1 M sodium phosphate buffer, pH 6.5 for 40 s. A linear relationship was obtained between triolein concentration ranging from 0.2 to 3.5 mM and amount of current (mA). The minimum detection limit of the method was 0.2 mM. The level of TG in serum of healthy and persons suffering from cardiovascular disease and pancreatitis was in the range 53.2–174.5 and 189–990 mg/dl, respectively. The analytical recovery of added triolein was 89.0%. Within batch and between batches CV (coefficient of variation) were