Oxidases lactate oxidase

Flavin oxidation of carbanions has also been of much concern since active intermediates in some flavoenzyme-mediated reactions (amino acid oxidase, lactate oxidase, etc.) are carbanions (Kosman, 1977). Flavin oxidation of nitroethane carbanion (20), which had not been achieved in non-enzymatic systems, occurs with [56] bound to CTAB micelles (Shinkai etal., 1976b). This suggests that the nitroethane carbanion is also activated by the micellar environment. 

Sensors have also been constructed from some oxidases directly contacted to electrodes to give bioelectrocatalytic systems. These enzymes utilize molecular oxygen as the electron acceptor for the oxidation of their substrates. Enzymes such as catechol oxidase, amino acid oxidase, glucose oxidase, lactate oxidase, pyruvate oxidase, alcohol oxidase, xanthine oxidase and cholesterol oxidase catalyze the oxidation of their respective substrates with the concomitant reduction of O2 to H2O2 ... 

The lifetime of a biosensor array is always limited by the enzyme which loses its activity first. Compared with the stable enzymes glucose oxidase and glutamate oxidase, lactate oxidase is very brittle and unstable. However, owing to the overloading of enzyme activity in the membranes and especially by the stabilizing effect of pHEMA, even the lactate biosensor can be operated continuously in bovine serum for 1 month at 37°C. 

Following an abrupt transition of P. pentosaceum from anaerobic to aerobic metabolism (10 pM O2), the growth rate increases at first, but the formation of acetate from lactate and glucose is slowed down, propionate formation is completely repressed and pyruvate is accumulated in the medium (van Gent-Ruijters et al, 1976 Schwartz et al, 1976). A decrease in the activities of the citric acid cycle enzymes malate dehydrogenase, fumarase and NADH oxidase, lactate oxidase, NADH-dependent fiimarate reductase, lactate-dependent nitrate reductase is observed upon the transition from anaerobic to aerobic (10 pM O2) metabolism (van Gent-Ruijters, 1975). 

Glucose oxidase, lactate oxidase, L-amino acid oxidase, and alkaline oxidase are immobilized on new films based on 2,6-dihydroxynaphthalene copolymerized with 2-(4-aminophenyl)-ethylamine onto the Pt electrodes... 

L-a-glycelophosphoric acid L-lactic acid L-a-glycerolphosphate oxidase lactate oxidase 26)... 

Finally it should be mentioned that the green and orange flavo-coenzymes can serve as sensitive indicators when bound to the active site of (apo)flavoenzymes. Interaction with the protein can stabilize either of the tautomeric forms (18a, 18b) or (20a, 20b), and cause shifts of the pK s which can be readily monitored by changes in the absorption spectra. This method has been used successfully in studies of the active sites of the apoenzymes of flavodoxin, D-amino acid oxidase, lactate oxidase and Old Yellow Enzyme 47, 49). 

Glucose oxidase + galactose oxidase + lactate oxidase + peroxidase IPN PVA-Fn5-Si02 composite Glucose, galactose, lactose, lactate OBS 305... 

Multienzyme electrodes can increase sensitivity from micromolar to nanomolar detection levels (53,57). In this case the substrate is converted to a detectable product by one enzyme, then that product is recycled into the initial substrate by another enzyme resulting in an amplification of the response signal. For example, using lactate oxidase and lactate dehydrogenase immobilized in poly(vinyl chloride), an amplification of 250 was obtained for the detection oflactate (61). 

Several other biosensors have been developed usiag this oxygen-quenched fluorescence approach. Target species iaclude ethanol [64-17-5] hydrogen peroxide [7722-84-17, H2O2, lactate, and xanthine [69-89-6] C H4N402, usiag alcohol oxidase, catalase [9001-05-2] lactate oxidase, and xanthine oxidase, respectively. An additional technique for biocatalytic biosensors iavolves the firefly chemiluminescent reaction (17) ... 

Finally in this section on deracemization via cyclic oxidation/reduction methods, there has been some limited work carried out on the deracemization of secondary alcohols. Soda et al. [22] employed lactate oxidase in combination with sodium borohydride to deracemize D/i-lactate (18) via the intermediate pyruvate (19) (Figure 5.12). 

Kenausis G, Taylor C, Katakis I, Heller A. 1996. Wiring of glucose oxidase and lactate oxidase within a hydrogel made with poly(vinyl pyridine) complexed with [Os(4,4 -dimethoxy-2,2 -bipyridine)2Cl]. J Chem Soc Faraday Trans 92 4131-4136. 

They observed abrupt changes in the slope of Arrhenius plots for reactions catalyzed by NADH oxidase and p-lactate oxidase that correlate well with phase transitions detected by the ESR spectra of the nitroxide spin labels bound covalently to the enzymes (Table 5.4). 

NADH oxidase p-Lactate oxidase Phase trans. temp (°C)... 

The work in the biosensor industry permitted the testing and proved of stability and reproducibility of enzymes, within the conditions employed in that area. Enzymes with demonstrated stability include lactate oxidase, malate dehydrogenase, alcohol oxidase, and glutamate oxidase. 

It should be pointed out that the addition of substances, which could improve the biocompatibility of sol-gel processing and the functional characteristics of the silica matrix, is practiced rather widely. Polyethylene glycol) is one of such additives [110— 113]. Enzyme stabilization was favored by formation of polyelectrolyte complexes with polymers. For example, an increase in the lactate oxidase and glycolate oxidase activity and lifetime took place when they were combined with poly(N-vinylimida-zole) and poly(ethyleneimine), respectively, prior to their immobilization [87,114]. To improve the functional efficiency of entrapped horseradish peroxidase, a graft copolymer of polyvinylimidazole and polyvinylpyridine was added [115,116]. As shown in Refs. [117,118], the denaturation of calcium-binding proteins, cod III parvalbumin and oncomodulin, in the course of sol-gel processing could be decreased by complexation with calcium cations. 

These examples demonstrate that additives can have a beneficial effect on the entrapped biopolymers. Unfortunately, they are generally not universal. The additives need to be found for individual immobilized biopolymers and that is not so easy to do. For instance, lactate oxidase retained its activity in a silica matrix if the enzyme was taken as a complex with poly(N-vinylimidazole) prior to the immobilization, but the polymer did not stabilize glycolate oxidase [87,114], Its stabilization was observed after an exchange of poly(N-vinylimidazole) for poly(ethyleneimine). This is a decisive disadvantage of the approaches because they do not offer a general solution that might be extended to any immobilized biopolymer. 

With the specific suitable oxidases, lactate, choline and glucose could be assayed. Concentration measurements of these metabolites could be performed over at least two decades wit a detection limit of 10 pM for lactate and choline and 20 pM for glucose. 

Berger A., Blum L.J., Enhancement of the response of a lactate oxidase/peroxidase-based fiberoptic sensor by compartimentalization of the enzyme layer, Enzyme Microb. Technol., 1994 16 979-984. 

Nicotinamide Adenine Dinucleotide (NAD+) Alcohol dehydrogenase, Lactate oxidase... 

Several enzymes have been immobilized in sol-gel matrices effectively and employed in diverse applications. Urease, catalase, and adenylic acid deaminase were first encapsulated in sol-gel matrices [72], The encapsulated urease and catalase retained partial activity but adenylic acid deaminase completely lost its activity. After three decades considerable attention has been paid again towards the bioencapsulation using sol-gel glasses. Braun et al. [73] successfully encapsulated alkaline phosphatase in silica gel, which retained its activity up to 2 months (30% of initial) with improved thermal stability. Further Shtelzer et al. [58] sequestered trypsin within a binary sol-gel-derived composite using TEOS and PEG. Ellerby et al. [74] entrapped other proteins such as cytochrome c and Mb in TEOS sol-gel. Later several proteins such as Mb [8], hemoglobin (Hb) [56], cyt c [55, 75], bacteriorhodopsin (bR) [76], lactate oxidase [77], alkaline phosphatase (AP) [78], GOD [51], HRP [79], urease [80], superoxide dismutase [8], tyrosinase [81], acetylcholinesterase [82], etc. have been immobilized into different sol-gel matrices. Hitherto some reports have described the various aspects of sol-gel entrapped biomolecules such as conformation [50, 60], dynamics [12, 83], accessibility [46], reaction kinetics [50, 54], activity [7, 84], and stability [1, 80],... 

B. Lillis, C. Grogan, H. Bemey, and W.A. Lane, Investigation into immobilisation of lactate oxidase to improve stability. Sens. Actual. B 68, 109—114 (2000). 

F. Ghamouss, S. Ledru, N. Ruille, F. Lantier, and M. Boujtita, Bulk-modified modified screen-printing carbon electrodes with both lactate oxidase (LOD) and horseradish peroxide (HRP) for the determination of 1-lactate in flow injection analysis mode. Anal. Chim. Acta 570, 158-164 (2006). 

Lactic acid Immobilized lactate oxidase, luminol-H202 CL 5 mg/L 104... 

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