Lactate oxidase

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. 

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... 

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. 

Reagents i, L-lactate oxidase ii, NaBH4 iii, MeOH iv, monoamine oxidase v, proteolytic digest... 

Amplification of the sensitivity of substrate or co-en me recycling is especially efficient in thermometric analysis since all the reactions involved frequently contribute to increasing the overall temperature change. One case in point is the determination of lactate or pyruvate by substrate recycling using co-immobilized lactate oxidase and lactate dehydrogenase [160]. 

L-Iactate is oxidized by lactate oxidase to pyruvate, which is reduced back to lactate by LDH. The total enthalpy change for this system can be further increased by addition of catalase, which makes the overall enthalpy change as large as -225 kJ/mol, so signal increases greater than 1000-fold can be obtained as a result. Co-enzyme recycling was also used for the determinations of ATP/ADP [161] and NAD(H) [162],... 

The same deproteinised blood as used for lactate, pyruvate, , and ACAC assays described above is used for enzymatic methods employing spectrophotometric measurement (Fig. 1.4.3) [10, 17]. The enzymes involved are LDH for pyruvate, lactate oxidase for lactate and HBDH for ACAC and . 

Lactate oxidase, internal flavoprotein mono- Sutton 15)... 

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