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The effect of Hofmeister anions and protein concentration on the activity and stability of some immobilized NAD‐dependent dehydrogenases
Author(s) -
Carrea G.,
Bovara R.,
Pasta P.,
Cremonesi P.
Publication year - 1982
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260240102
Subject(s) - nad+ kinase , hofmeister series , chemistry , protein stability , chromatography , biochemistry , enzyme , organic chemistry , ion
The effect of several factors on the activity and stability of alcohol dehydrogenase, glyceraldehyde‐3‐phosphate dehydrogenase, and 20β‐hydroxysteroid dehydrogenase, both free and immobilized on CNBr‐activated Sepharose 4B, was investigated. Enzymes were im‐ mobilized under different conditions including various degrees of matrix activation, variable amounts of protein, in the presence, or in the absence of, additives (coenzymes, dithioth‐ reitol, salts). Activity recovery was in general satisfactorily high with 20β‐hydroxysteroid dehydrogenase, low with glyceraldehyde‐3‐phosphatedehydrogenase, and markedly linked to the concentration of immobilized protein with alcohol dehydrogenase. In the latter case the advantageous stabilizing effect of high enzyme concentrations was notably diminished by the parallel decrease of the effectiveness factor. The effect of high concentrations of anions of the Hofmeister series was examined. It was found that 1 M phosphate and 0.5 M sulfate dramatically stabilize both free and immobilized enzymes against inactivation by temperature and urea. K m , values of apolar substrates were considerably lowered by the two anions while K m values of polar substrates were not affected. In some cases V max values also were influenced by high concentrations of these anions. The present results appear of interest particularly in view of enzyme utilization for analytical as well as for preparative purposes.