GLUCOSE OXIDASE REACTION FOR ESTIMATION OF GLUCOSE WITHOUT HORSERADISH PEROXIDASE. SOME MICROBIOLOGICAL AND FERMENTATION APPLICATIONS

An enzymic method has been developed for analysis of glucose. Glucose oxidase acts on glucose to produce hydrogen peroxide which acts to directly reduce the green Cu(II) 2–2′-bicinchoninate complex to a violet complex without horseradish peroxidase. A concentration range of 20–200 μM glucose was used but the reaction shows a linear range of 20–800 μM glucose. Interference is controlled by using a blank determination which has not been treated with glucose oxidase. The reaction has been used to estimate glucose levels in Saccharomyces cerevisiae fermentation and α-amylase, invertase and β-galactosidase reactions and coloured corn-steep fermentation media.     

The molecular mechanism of membrane proteins probed by evanescent infrared waves

The catalytic action of membrane proteins is vital to many cellular processes. Yet the molecular mechanisms remain poorly understood. We describe here the technique of evanescent infrared difference spectroscopy as a tool to decipher the structural changes associated with the enzymatic action of membrane proteins. Functional changes as minute as the protonation state of individual amino acid side chains can be observed and linked to interactions with a ligand, agonist, effector, or redox partner.     

Development of a screen-printed cholesterol biosensor: Comparing the performance of gold and platinum as the working electrode material and fabrication using a self-assembly approach

Gold (Au) and platinum (Pt) were used as the working electrode material to detect cholesterol in solution through enzymatically generated hydrogen peroxide (H₂O₂). Both gold and platinum were capable of detecting cholesterol through the electrochemical oxidation of H₂O₂, and could be used as the working electrode material. By comparison, however, Au was preferable over Pt in terms of higher response current and better sensitivity. Therefore, Au was chosen as the working electrode material for the fabrication of a thick-film screen-printed cholesterol biosensor consisting of three electrodes on an alumina substrate (working: Au, reference: Ag/AgCl, and counter: Au). The immobilization of the enzyme cholesterol oxidase (ChOx, E.C. 1.1.3.6) on the Au working electrode was achieved using a self-assembly approach. A thiol, 3-mercaptopropionic acid (MPA), was self-assembled onto the gold working electrode forming a thin organic layer that served as the anchor for the enzyme immobilization. 1-Ethyl-3(3-dimethylamino propyl)carbodiimide methiodide (EDC) was then used to immobilize the enzyme ChOx covalently on the gold working electrode through the carbodiimide coupling between the carboxyl (–COOH) groups of the self-assembled MPA layer and the amino (–NH₂) groups of the enzyme. Electrochemical measurements showed that this biosensor responded well to cholesterol, confirming that the self-assembly immobilization method was effective. The reproducibility, the interference, and the storage stability of the biosensor were studied and assessed.     

Immobilization of polyphenol oxidase on alginate–SiO2 hybrid gel: stability and preliminary applications in the removal of aqueous phenol

An organic/inorganic hybrid gel of alginate–SiO₂ (ALG–SiO₂) was used to immobilize the partially purified potato polyphenol oxidase (PPO) for the treatment of phenolic wastewater. The influences of alginate concentration, quantity of both enzyme and tetramethoxysilane (TMOS) on immobilization were investigated. The Michaelis constant for immobilized PPO was determined as 14.7 mmol L⁻¹ at 25 °C, and the highest activity of immobilized PPO was achieved at pH 7.0. The ALG–SiO₂ immobilized PPO was more stable than the free PPO or ALG(alone) immobilized PPO. This study suggests that ALG–SiO₂ immobilized PPO might be a potential tool for the removal of phenolic compounds from industrial wastewater. Copyright © 2008 Society of Chemical Industry     

Influence of ionic liquids on the direct electrochemistry of glucose oxidase entrapped in nanogold-N,N-dimethylformamide-ionic liquid composite film

Glucose oxidase (GOD) immobilized in nanogold particles (NAs)-N,N-dimethylformamide (DMF) composite film on glassy carbon (GC) electrode exhibits a pair of quasi-reversible and unstable peaks due to the redox of flavin adenine dinucleotide (FAD) of GOD. When ionic liquids (ILs) 1-butyl-3-methylimidazolium tetrafluoroborate (BMIMBF₄) or trihexyltetradecylphosphorium bis (trifluoromethylsulfony) (P_666,14 NTf₂) is introduced in the film, the peaks become small. But ILs 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF₆) and 1-octyl-3-methylimidazolium hexafluorophate (OMIMPF₆) make the peaks large and stable. In different composite films the formal potential (E⁰′) of GOD is different. UV-vis spectra show that the GOD dispersed in these films almost retains its native structure and there are weak interactions between ILs and GOD. Electrochemical impedance spectra display that NAs can promote the electron transfer between FAD and GC electrode; and ILs can affect the electron transfer through interacting with GOD. The thermal stability of GOD entrapped in NAs-DMF-ILs composite films is also influenced by ILs, and it follows such order as: in NAs-DMF-OMIMPF₆ > in NAs-DMF-BMIMPF₆ ≈ in NAs-DMF-BMIMBF₄ > in NAs-DMF. In addition, GOD immobilized in NAs-DMF-OMIMPF₆ and NAs-DMF-BMIMPF₆ films shows good catalytic activity to the oxidation of glucose. The I_max of H₂O₂ and the apparent K_m (Michaelis-Menten constant) for the enzymatic reaction are calculated.     

Chemical changes of three native Turkish hazelnut varieties (Corylus avellana L.) during fruit development

Three native hazelnut varieties from Turkey, namely, Tombul, Palaz, and Badem, were examined for their proximate composition, minerals, and fatty acid profiles, as well as polyphenol oxidase (PPO), peroxidase (POD), and lipase activities during fruit development stages (early stage: ES, middle stage: MS, and harvest stage: HS). Proximate composition varied considerably (dry weight basis) from ES to MS. Fat was the predominant component at all stages and showed increasing trends. Six essential minerals (calcium, iron, magnesium, phosphorus, potassium, and zinc) were analysed (dry weight basis). Consuming recommended daily amount of 42.5 g hazelnut at HS supplies 23.3–25.0% of phosphorus, 11.6–18.1% of magnesium, 7.0–18.9% of iron, 4.9–8.9% of zinc, 5.1–5.7% of calcium, and 5.1–5.3% of potassium for recommended dietary allowances or adequate intake for adults. Significant (P < 0.05) decreasing trends were found in all mineral contents from early development to maturity, with some exceptions. Sixteen fatty acids were identified, among which 18:1ω9 was by far the most predominant one, followed by 18:2ω6, 16:0, and 18:0. As expected, total monounsaturated fatty acids constituted the main group of fatty acids ranging from 75.51 to 81.07% in Tombul, from 78.21 to 82.71% in Palaz, and from 73.69 to 81.65% in Badem through the maturation stages. In contrast, total polyunsaturated fatty acids showed decreasing trends from ES to HS. No significant changes (P > 0.05) were observed in total saturated fatty acids at different maturation stages. Tombul variety had the lowest PPO activity compared to those of Palaz and Badem. Badem showed highest POD activity compared to Tombul and Palaz at three stages of maturation and significant decreases (P < 0.05) in all hazelnut samples were observed in POD activity from ES to HS. No lipase activity was detected in any hazelnut samples at ES and MS, except in Badem at MS. In contrast, lipase activity was detected in all hazelnut samples at HS. These results suggest that some proximate compositions, minerals, and fatty acids gave good indications during fruit development stages, whereas enzymatic activities of PPO, POD, and lipase behaved differently among varieties and fruit development stages.     

Decaffeination of fresh green tea leaf (Camellia sinensis) by hot water treatment

Hot water treatment was used to decaffeinate fresh tea leaf in the present study. Water temperature, extraction time and ratio of leaf to water had a statistically significant effect on the decaffeination. When fresh tea leaf was decaffeinated with a ratio of tea leaf to water of 1:20 (w/v) at 100 °C for 3 min, caffeine concentration was decreased from 23.7 to 4.0 mg g⁻¹, while total tea catechins decreased from 134.5 to 127.6 mg g⁻¹; 83% of caffeine was removed and 95% of total catechins was retained in the decaffeinated leaf. It is considered that the hot water treatment is a safe and inexpensive method for decaffeinating green tea. However, a large percentage of tea catechins was lost if rolled leaf and dry tea were decaffeinated by the hot water treatment and so the process is not suitable for processing black tea.     

Analysis of methanol–ethanol mixtures from falsified beverages using a dual biosensors amperometric system based on alcohol dehydrogenase and alcohol oxidase

A portable bienzymatic analytical system was developed for the chronoamperometric analysis of methanol–ethanol mixtures. The system consists of two biosensors, one based on alcohol dehydrogenase (ADH) that responds only to the ethanol and the second one based on alcohol oxidase (AOX) that responds to both methanol and ethanol. The transducers were screen-printed electrodes (SPEs) modified with mediators: Meldola blue for ADH and Co-phthalocyanine for AOX. The calibration graph of the ADH biosensor is linear between 0.3 and 8 mmol/L ethanol. The AOX biosensor is able to quantify both analytes in mixtures that contain methanol between 3 and 70 mmol/L and ethanol ranging from 15 to 110 mmol/L. Interferences due to non-specific oxidations from common oxidizable compounds like gallic acid and ascorbic acid were smaller in the case of transducer based on Meldola blue. The analytical system was successfully tested on real samples: non-alcoholised beer (NAB) spiked with ethanol or methanol and a falsified rose wine (FRW).