A rationale for stabilization of oxygen-labile enzymes: application to a clostridial hydrogenase.

Abstract

ABSTRACr A general procedure for stabilization of 02-labile enzymes exploiting "salting out" of oxygen from the microenvironment in the molecular layers immediately adjacent to charged surfaces of polyionic solid adsorbents has been develope. Empirical verification of this rationale is provided. The half-life of air inactivation of the Og-labile hydrogenase (EC 1.12.7.1) from Clostridium pasteurianum is increased 20-to 2-fold simply by adsorption (noncovalent binding) in dilute Tris-HCI buffer on common anion exchange supports such as DEAE-cellulose or Dowex 1-X2. Predicted increases in degree of stabilization by using more densely charged adsorbents (such as polyethyleneimine-cellulose), as well as bulkier solvent counter-anions, are found; half-lives for air inactivation for the bound hydrogenase can be increased to 3000-fold longer than that of the free enzyme. Most of the total catalytic activity, assayed as H2 evolution from dithionite mediated by methyl viologen or ferredoxin, is retained, whereas the expected suppression of H2 uptake in the reverse reaction is observed. Stabilization of the oxygen-labile hydrogenase (1) proposed for use in various schemes for bioconversion of solar energy (2) is critical for practical applications (3-5). Efforts to achieve stabilization to date (6-10) have been empirical, involving mainly immobilization of the enzyme (6, 9, 10), but success has been limited. It is known (1, 11) that the rate of 02 inactivation in solutions of the enzyme is directly proportional to 02 concentration. We have conceived a rationale based on exploitation of the well-known "salting out" phenomenon whereby the concentration of dissolved gases in aqueous solution is reduced as the salt concentration is increased (12). This effect is thought to be caused by competition between salt and gas for solvent, the salt component progressively removing water by hydration, thereby decreasing solvent volume available to the gas. Thus, a decrease of an order of magnitude in gas solubility occurs as solvent is saturated by salt (13,14). We are able to show that relatively simple procedures based on this fact can be developed to achieve increases in hydrogenase stability in air sufficient to meet the requirements for practical applications in solar energy bioconversion.Our rationale is that if hydrogenase, or any Orlabile enzyme, is attached to a polyionic surface (it must be polycationic for an acidic enzyme such as hydrogenase to be adsorbed), the enzyme molecules will be buried in a microenvironment equivalent to that provided by an extremely concentrated salt solution. There will be little solvent available for gas solution, so that access to the adsorbed enzyme will be minimal. In all probability, the salting out of gas may be much greater than is possible with saturated salt solutions because of solubility limitations. Hence the 02 concentration in such a microenvironment, extending several molecular layers from the charged adsorbent surface, will be very small so that a relatively large increase in 02 st...

On the redox reactions and accessibility of amphiphilic flavins in artificial membrane vesicles

“…An additional, highly attractive explanation for the strong inhibition of autoxidation of vesicle-bound flavins (T< T~) is the well-known "salting out" phenomenon [17]. This effect has been originally exploited for the stabilization of oxygen-labile enzymes by charged surfaces of polyionic solid adsorbents.…”

Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance

“…The generation of artificial environments around the enzyme molecules has been proposed as a way to prevent inactivation. A hydrophilic environment may reduce the concentration of hydrophobic organic solvents or some gases near to the enzyme 108. A hydrophobic environment may greatly reduce the exposition of the enzyme to very hydrophilic deactivating reagents, like hydrogen peroxide 76,95…”

Polyethylenimine: a very useful ionic polymer in the design of immobilized enzyme biocatalysts

“…Using this strategy, it has been found that PEI can have some positive effects on enzyme stability by generating a partition of hydrophobic compounds (like organic solvents or gases like oxygen) from the enzyme environment (Figures 4 and 6). 69 In general, support preparation is not optimized to maximize the polymeric bed thickness, as just one molecule of the polymer may be enough to immobilize a protein, although this may reduce the impact of the immobilization on the enzyme properties. The fact that this support may rapidly adsorb most proteins in crude extract makes the presence of contaminants detrimental since it may reduce the final loading of the target protein.…”