Enzymes lactate oxidase

The commercial availability of various lactate-specific enzymes and the high demand for lactate analysis for clinical and food processing led to the construction of numerous lactate enzyme electrodes (Tkble 8). Depending on the selected enzyme—lactate oxidase (LOD), lactate mono-oxidase (LMOD), or lactate dehydrogenase (LDH)—several mechanistic approaches may be applied ... 

Kissinger [130] carried out similar experiments following lactate changes observed in a rat subcutaneous microdialysate upon an intraperitoneal injection of lactate. The half-life of the sensor, assembled by coating the enzyme lactate oxidase (LOD) on a glassy carbon electrode with an Os-redox polymer and a Nafion overcoating, was about 24 h. 

It has been observed that enzymes are highly unstable in the solution phase and lose their activity rapidly with time. In the immobilised state, the enzyme is observed to lose its activity very slowly. The immobilisation of enzymes on conducting polymers provides increased stability but decreased electron transfer in the medium resulting in the reduced response of the biosensor. The response of the enzyme lactate oxidase (LOD) to the analyte in solution and in the immobilised state as an enzyme electrode after storing at 4-10 °C is shown in Figure 10.7. 

Figure 4. Oxidation of lactate by the enzyme lactate oxidase (LOD) to pyruvate and hydrogen peroxide.

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). 

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). 

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. 

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],... 

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 . 

Two enzymes are commonly used for amperometric biosensors, namely lactate oxidase (LOD) and lactate dehydrogenase (LDH). It should be noted that, in this instance, LOD refers to the enzyme which catalyses the reaction shown in Fig. 23.4, in which the products are pyruvate and H202. This type of enzyme was formerly assigned the E.C. number 1.1.3.2, but this was confused with lactate monooxygenase (E.C. 1.13.12.4), which is also commonly referred to as type I lactate oxidase [55] or simply lactate oxidase [56] whose products are acetate, C02 and H202. The LOD which catalyses the reaction shown in Fig. 23.4 has also been referred to as type II lactate oxidase [55] following clarification of this point in a published letter [57], current publications refer to this enzyme as E.C. 1.1.3.x. 

Torriero et al. [30] managed to estimate the concentration of lactate in untreated milk without the use of a microdialysis unit. Samples were fed into a reactor consisting of a rotating disc bearing lactate oxidase which by its motion ensured adequate mass transport of hydrogen peroxide to an enzyme electrode. The electrode consisted of horse-radish peroxidase immobilised over osmium on the surface of a glassy carbon electrode. Such an electrode can be poised at 0.0 Y so avoiding electrochemical... 

Levels of lactate in buttermilk and yoghurt (and blood) were estimated using disposable sensors formed from screen-printed graphite laminated between two polymer sheets [18]. Platinum (deposited by sputter-coating) was the transducing surface. Layers of Nation were added to reduce interference and were surmounted by lactate oxidase in a mixture of polyethyleneimine and poly (carbamoyl) sulphonate hydrogel. The samples were measured in stirred buffer. A good correlation between biosensor results and those obtained with an enzyme kit was claimed but the data had a considerable amount of scatter—if the enzyme kit is taken as the reference method then a more severe analysis of the biosensor results [33] would not have shown them in a... 

Chemically binding enzymes to nylon net is very simple and gives strong mechanically resistant membranes (135). The nylon net is first activated by methylation and then quickly treated with lysine. Finally, the enzyme is chemically bound with GA. The immobilized disks are fixed direcdy to the sensor surface or stored in a phosphate buffer. GOD, ascorbate oxidase, cholesterol oxidase, galactose oxidase, urease, alcohol oxidase (135), and lactate oxidase (142) have been immobilized by this procedure and the respective enzyme electrode performance has been established. 

The activity of the enzyme is also strongly affected by the presence of inhibitors. Fluoride ions inhibit urease (173) and oxalate ions inhibit lactate oxidase (174), but the major inhibitors are heavy-metal ions, such as Ag+, Hg +, Cu " ", organophosphates, and sulfhydryl reagents (/i-chloromercuribenzoate and phenylmercury(II) acetate), which block the free thiol groups of many enzyme active centers, especially oxidase (69). Inhibiting the enzyme electrodes makes it possible to quantify the inhibitors themselves (69), for example, H2S and HCN detection using a cytochrome oxidase immobilized electrode (176). 

Co-immobilizing a second enzyme, NADH oxidase (280) or lactate dehydrogenase (288), permits the regeneration of NAD+. Measurements may be completed in Tris-HCl or carbonate buffers, but borate and glycine buffers inhibit L-alanine dehydrogenase (289). Highly selective L-histidine electrodes are available (290, 291) to determine histidine in urine (291). 

The fundamentals on which the carbanion mechanism is founded are the early studies on the related enzj mes D-amino acid oxidase (Walsh et al., 1972 and 1971), lactate oxidase (Walsh et al., 1973) and flavocytochrome bj (Urban and Lederer, 1985 Pompon and Lederer, 1985). It is interesting that all of the key work, which established the carbanion mechanism, was done before any 3-dimensional structures were available on the respective enzymes. 

An interesting application for the oxidation of organic compounds is of electrochemical nature. Octacyano complexes have been used to monitor redox enzymes such as lactate oxidase (from Pediococcus sp.) and sarcosine oxidase (from Arthrobactersp.) in a suitable electrochemical system (114). Two equivalents of [M(CN)g] can, for example, be oxidized at the electrode surface to [M(CN)g], which in turn can oxidize the flavoproteien to its oxidized form. This in turn reacts with, for example, L-lactic acid to produce pyruvic acid. 

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 ... 

---