Recycling system, lactate

Determination of ADP and ATP by multiple enzymes in recycling systems pyruvate kinase and hexokinase co-immobilized on aminopropyl CPG, was demonstrated by Kirstein et al. [25]. In addition, a second reactor,with L-lactate dehydrogenase, lactate oxidase, and catalase, was used to increase the sensitivity from 6x 10 5 M with no recycling, to 2 x 10 6 M in the kinase bienzyme reactor,... 

An alternative method for driving the reaction equilibrium of transaminations was developed by Hohne et al. (Figure 14.44) [65]. Alanine served as the amine donor, and pyruvate decarboxylase was used to remove the pyruvate coproduct by decarboxyl ation to acetaldehyde. An advantage of pyruvate decarboxylase over lactate dehydro genase is that it requires no cofactor recycling system, and the high volatility of the coproducts allows for the desired shift of equilibrium. 

The use of alkaline phosphatase as an enzyme label allows enhancement of the sensitivity by using phosphoenolpyruvate as substrate and the utilization of a separate detection column in the ET unit for the determination of the product (pyruvate) by substrate recycling. This is accomplished by using the substrate recycling system described above [18] comprising the coimmobilized enzymes lactate dehydrogenase (reduces pyruvate to lactate under the consumption of... 

In a sensor for lactate a bienzyme system composed of cytochrome 62 for lactate oxidation to pyruvate, and lactate dehydrogenase for conversion of pyruvate back to lactate has been used [321]. Hexacyanoferrate(III) served as electron acceptor for cytochrome b2- The reduced mediator was reoxidized at the electrode, thus giving a measuring signal depending on the analyte concentration. Attempts to determine both substrates of the recycling system have shown that, at tenfold amplification for lactate, the sensitivities for lactate and pyruvate are almost identical. The same recycling scheme has also been used in connection with Fe-EDTA as electron mediator in place of hexacyanoferrate(III) [336]. 

Ihble 14-10. Amplification factor and characteristic diffusion time in the lactate recycling system as a function of the enzyme loading. 

Figure I4-2 Schematic view of an enzyme electrode using a double recycling system for the determination of ATP or ADP. PK = pyruvate kinase HK = herokinase LOD = lactate oxidase LDH = lactate dehydrogenase PEP = phosphoenolpyruvat Reproduced from [327] with permission from Marcel Dekket, Inc.

The main problem of the co-TA-catalyzed transamination is the generally unfavorable equilibrium that lies on the substrate side, especially when alanine is used as amino donor. Several methods have been successfully established to overcome this issue, for instance, coproduct removal [e.g., with lactate dehydrogenase (LDH)] or amino donor recycling [e.g., with alanine dehydrogenase (AlaDH)] [142-145]. The nicotinamide cofactor consumed in this reductive step is regenerated by an additional recycling system. These multienzymatic systems based on co-TAs were applied to the synthesis of biologically active compounds. 

In another recycling system we co-immobolized cytochrome bz and laccase in a gelatin layer in front of an oxygen electrode. In the presence of the cytochrome bz substrate, lactate, BQ is reduced to H2Q. Therefore, this enzyme substitutes the cathode of the previous cycling system (Figure 6). However, the substrate recyling is not restricted to the phase boundary (as with electrochemical recyling) but it proceeds in the total volume of the enzyme layer. At the optimum pH of cytochrome b2 (pH 6.5) and lactate saturation a maximum amplification of 500 has been obtained. 

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],... 

Simultaneous L-lactic acid fermentation (by Rhizopus oryzae immobilized in calcium alginate beads) and separation was carried out using a three-phase fluidized-bed bioreactor as a fermenter (F), an external electrodialyzer as a separator, and a pump to recycle the fermentation broth between the bioreactor and the separator. In this way, the experimental specific lactate productivity and yield practically coincided with those obtained in the CaC03-buffered fermentation process (Xuemei et al., 1999), thus confirming the capability of the combined system to alleviate product inhibition without any addition of alkali or alkali salts. It was also shown that the adoption of ED-F for the production of inoculum reduced variability in inoculum quality, thus shortening the length of the lag phase of L-lactate production practically to zero as compared to that observed using an inoculum... 

It may also be economical to remove the inhibitory product directly from the ongoing fermentation by extraction, membranes, or sorption. The use of sorption with simultaneous fermentation and separation for succinic acid has not been investigated. Separation has been used to enhance other organic acid fermentations through in situ separation or separation from a recycled side stream. Solid sorbents have been added directly to batch fermentations (18,19). Seevarantnam et al. (20) tested a sorbent in the solvent phase to enhance recovery of lactic acid from free cell batch culture. A sorption column was also used to remove lactate from a recycled side stream in a free-cell continuously stirred tank reactor (21). Continuous sorption for in situ separation in a biparticle fermentor was successful in enhancing the production of lactic acid (16,22). Recovery in this system was tested with hot water (16). 

An enzyme electrode based on coimmobilized cytochrome b2 and laccase (Scheller et al., 1987b) allows an explanation of the principle of substrate recycling in enzyme electrodes in greater detail (Fig. 100). The advantage of this system is that the cosubstrate, oxygen, as well as the analytes, hydroquinone and benzoquinone, are electrochemically active. This permits one to study different parts of the recycling process. Recycling of the analyte in the presence of the substrate of cytochrome b2, lactate, results in an increase in the sensitivity by a factor of 500 as compared with lactate-free operation. Under conditions that are optimal for laccase the analyte is almost completely in the oxidized state, i.e. it... 

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