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Enzyme thermoinactivation in anhydrous organic solvents
Author(s) -
Volkin David B.,
Staubli Andrea,
Langer Robert,
Klibanov Alexander M.
Publication year - 1991
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.260370908
Subject(s) - thermostability , chemistry , anhydrous , ribonuclease , bovine pancreatic ribonuclease , aqueous solution , denaturation (fissile materials) , lysozyme , organic chemistry , chromatography , differential scanning calorimetry , size exclusion chromatography , hydrophobic effect , enzyme , biochemistry , nuclear chemistry , rna , physics , gene , thermodynamics
Abstract Three unrelated enzymes (ribonuclease, chymotrypsin, and lysozyme) display markedly enhanced thermostability in anhydrous organic solvents compared to that in aqueous solution. At 110‐145°C in nonaqueous media all three enzymes inactivate due to heat‐induced protein aggregation, as determined by gel filtration chromatography. Using bovine pancreatic ribonuclease A as a model, it has been established that enzymes are much more thermostable in hydrophobic solvents (shown to be essentially inert with respect to their interaction with the protein) than in hydrophilic ones (shown to strip water from the enzyme). The heat‐induced aggregates of ribonuclease were characterized as both physically associated and chemically crosslinked protein agglomerates, with the latter being in part due to transamidation and intermolecular disulfide interchange reactions. The thermal denaturation of ribonuclease in neat organic solvents has been examined by means of differential scanning calorimetry. In hydrophobic solvents, the enzyme exhibits greatly enhanced thermal denaturation temperatures ( T m values as high as 124°C) compared to aqueous solution. The thermostability of ribonuclease towards heat‐induced denaturation and aggregation decreases as the water content of the protein powder increases. The experimental data obtained suggest that enzymes are extremely thermostable in anhydrous organic solvents due to their conformational rigidity in the dehydrated state and their resistance to nearly all the covalent reactions causing irreversible thermoinactivation of enzymes in aqueous solution.