Lactate dehydrogenase stabilizing

Figure 9 Effects of polyethylene glycols and sucrose on lactate dehydrogenase stability during freeze-thawing. (Data taken from Refs. 59 and 70.)...

Examples of other recombinant enzymes in which an alteration using site-directed mutagenesis resulted in altered substrate binding efficiencies, rates of catalysis, or stability include carbonic anhydrase (Alexander, Nair Christianson, 1991), lactate dehydrogenase (Feeney, Clarke Holbrook, 1990), and several industrially important proteases (Wells etal., 1987 Siezenera/., 1991 Teplyakovcra/., 1992 Aehle et al., 1993 Rheinnecker et al., 1994). 

A key structural and mechanistic feature of lactate and malate dehydrogenases is the active site loop, residues 98-110 of the lactate enzyme, which was seen in the crystal structure to close over the reagents in the ternary complex.49,50 The loop has two functions it carries Arg-109, which helps to stabilize the transition state during hydride transfer and contacts around 101-103 are the main determinants of specificity. Tryptophan residues were placed in various parts of lactate dehydrogenase to monitor conformational changes during catalysis.54,59,60 Loop closure is the slowest of the motions. 

Miller et al. [3.83] freeze-dried lactate dehydrogenase (LDH) in the presence of trehalose and trehalose plus sodium tetraborate (TST) to stabilize LDH for storage at high humidity (100%) or warm temperature (45 °C). The freeze-dried LDH with TST had a considerably higher Tg than with trehalose alone and was more stable for several weeks under the conditions given above. 

Enzymes in the cross-linked crystal form are essentially impervious to degradation by exogenous proteases and from autolysis, in the case of CLCs of proteases themselves [5], This stability makes the enzyme-catalyzed preparation of peptides and peptide mimics truly practical [6], Examples will be discussed in more detail in Sec. IV. Further, one could conceive of using multiple enzymes in one-pot reaction systems mimicking natural biosynthetic cascades. Indeed, the application of this concept has been reported for a mixture of lipoamide dehydrogenase and lactate dehydrogenase [19],... 

Trimethylamine oxide stabilizes teleost and mammalian lactate dehydrogenases against inactivation by hydrostatic pressure and trypsinolysis. J. Exp. Biol. 202 3597-3603. 

Holland, L.Z., M. McFall-Ngai, and G.N. Somero (1997). Evolution of lactate dehydrogenase-A homologs of barracuda fishes (genus Sphyraena) from different thermal environments Differences in kinetic properties and thermal stability are due to amino acid substitutions outside the active site. Biochemistry 36 3207-3215. 

S., Increased stabilizing effects of amphiphilic excipients on freeze-drying of lactate dehydrogenase (LDH) by dispersion into sugar matrices, Pharm. Res. 12, 838-843, 1995 Jennings, T.A., Lyophilization Introduction and Basic Principles, Interpharm Press, Denver, CO, 1999 Royall, P.G., Huang, C.Y., Tang, S.W. et al., The development of DMA for the detection... 

K. Izutsu, S. Yoshioka, and T. Terao, Stabilizing effect of amphiphilic excipients on the freeze-thawing and freeze-drying of lactate dehydrogenase. Biotechnol. Bio-engneer. 43.1102-1107 (1994). 

An electrode for measuring urea has been described (Gll), consisting of a thin film of urease, immobilized in acrylamide gel, on the surface of a glass electrode responsive to NH. Conditions are carefully selected to ensure stability of the enzyme, and the potential developed is proportional to the logarithm of the urea concentration. Blood glucose and lactate have been determined with a membrane electrode in which the enzyme (glucose oxidase or lactate dehydrogenase) is trapped in a porous or jellied layer at the membrane surface (W20). 

Sum) or hollow spherical particles (such as sodium chloride, mannitol, or tobramycin sulfate) are formed depending on the compound. Protein powders such as lysozyme or lactate dehydrogenase can also be produced by this process and can be stabilized through the use of sugars, buffers, and surfactant additives in the formulations. Depending on the solute and conditions of drying, the particles are crystalline in some cases and amorphous in others. ° ... 

Miller, D.P. Anderson, R.E. de Pablo, J.J. Stabilization of lactate dehydrogenase following freeze-thawing and vacuum-drying in the presence of trehalose and borate. Pharm. Res. 1998,15 (8), 1215-1221. 

Niven, R.W. Ip, A.Y. Mittelman, S.D. Farrar, C. Arakawa, T. Prestrelski, S.J. Protein nebulization I. Stability of lactate dehydrogenase and recombinant granulo- 56. cyte-colony stimulating factor to air-jet nebulization. Int. 

Gorton et al. reported carbon paste electrodes based on Toluidine Blue O (TBO)-methacrylate co-polymers or ethylenediamine polymer derivative and NAD" " with yeast alcohol dehydrogenase for the analysis of ethanol [152,153] and with D-lactate dehydrogenase for the analysis of D-lactic acid [154]. Use of electrodes prepared with dye-modified polymeric electron transfer systems and NAD+/NADH to detect vitamin K and pyruvic acid has also been reported by Okamoto et al. [153]. Although these sensors showed acceptable performances, insensitivity to ambient oxygen concentration, sensor stability and lifetime still need to be improved to obtain optimal dehydrogenase based enzyme biosensors. 

Fry has used a similar system for the enzymatic reduction of pyruvate to L-lactate. In this case, the one-electron transfer redox catalyst, methyl viologen, and the lipoamide dehydrogenase are coimmobilized within a Nafion cation-exchange layer on the surface of a reticulated vitreous carbon electrode. As production enzyme, L-lactate dehydrogenase (LDH) was employed [48], which was later stabilized considerably in the form of cross-... 

Mi YI, Wood J. The application and mechanisms of polyethylene glycol 8000 on stabilizing lactate dehydrogenase during lyophilization. PDA ] Pharm Sci Technol 2004 58(4) 192-202. 

Adler M, Lee G. Stability and surface activity of lactate dehydrogenase in spray-dried trehalose. J Pharm Sci 1999 88 199-208. 

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