Open AccessStability and activity of a thermostable malic enzyme in denaturants and water‐miscible organic solvents
Author(s) -
GUAGLIARDI Annamaria,
MANCO Giuseppe,
ROSSI Mossé,
BARTOLUCCI Simonetta
Publication year - 1989
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1989.tb14891.x
Subject(s) - chemistry , solvent , enzyme , guanidine , enzyme assay , malic enzyme , thermostability , chromatography , methanol , organic chemistry , dehydrogenase
A study was made of the effects of common protein denaturants and water‐miscible organic solvents on both the stability and activity of the malic enzyme [( S )‐malate: NADP + oxidoreductase (oxaloacetate‐decarboxylating); EC 1.1.1.40] from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus . At 25°C, the enzyme was not inactivated in 4 M urea or 0.05% SDS over 24 h, while the half‐life was 30 min in 6 M guanidine hydrochloride and 5 h in 0.075% SDS. The enzyme stability in water‐miscible organic solvents at 25°C is somewhat surprising: after a 24‐h incubation, the enzyme was completely active in 50% dimethylformamide; it lost 15% of its initial activity in 50% methanol or 15% ethanol. However, the resistance to organic solvents was greatly reduced at higher temperatures. The enzyme was able to catalyze the malate conversion even in the presence of 1.5% Triton X‐100 or sodium deoxycholate. A number of solvents were found to stimulate the malic activity independent of time. Studies with 50% methanol revealed that the activation was reversible and inversely related to the temperature; moreover, the solvent was demonstrated to exclusively affect the maximal velocity of catalysis, the K m values for both substrates being unchanged. Investigation was made to find out whether there was a correlation between enzyme stability, as well as activation, and hydrophobicity of the organic medium. The residual malic activity after incubation in the water/organic medium correlated inversly with the logarithm of the partition coefficient in octanol/H 2 O of the mixture used as a hydrophobicity index. On the other hand, the extent of activation depended directly on the logarithm of the molar concentration of the organic solvent required for maximal enzymatic activation. Because of its remarkable resistance to organic solvents and protein denaturants in general, the malic enzyme from Sulfolobus solfataricus can be considered suitable for biotechnological applications.