A comparative study on the stability of immobilized halophilic and non-halophilic malate dehydrogenases at various ionic strengths.

Abstract

A halophilic malate dehydrogenase was purified from Halobacterium of the Dead Sea employing general-ligand affinity chromatography on AMP-Sepharose in 4.3 M NaCl yielding an enzyme preparation with a specific activity of 49.5 U/mg. The enzyme obtained was immobilized on cyanogenbromide-activated agarose at low and high degrees of activation. When support with a high degree of activation was used, it was found that the storage stability of the resulting immobilized preparations at low salt concentration (0.15 M NaCl) was increased considerably in comparison with the soluble enzyme, which lost all of its activity in about 30 min; on the other hand preparations immobilized on gels with a low degree of activation showed little stabilization. The enzyme preparation immobilized on supports with a high degree of activation showed optimal activity at a lower salt concentration (NaCl) than the soluble enzyme; optimal activity was seen at 0.6 M NaCl instead of 1.2 M. It is suggested that immobilization prevents dissociation of the halophilic dimeric enzyme into its subunits, a process which is known to occur at low salt concentrations. Non-halophilic malate dehydrogenase was immobilized in the same manner. It was found that the enzyme immobilized on strongly activated agarose as well as the soluble enzyme showed excellent storage stability in high salt concentrations (e.g. 4.3 M NaCl). Activity assays run as a function of ionic strength revealed that strongly immobilized malate dehydrogenase retained 16% of its original activity at 4 M salt. This is in contrast to soluble enzyme and enzyme immobilized on agarose, with a low degree of activation, which were essentially inactivated at about 1 M salt concentration.