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Immobilization‐stabilization of α‐chymotrypsin by covalent attachment to aldehyde‐agarose gels
Author(s) -
Guisán José M.,
Bastida Agatha,
Cuesta Carmen,
FernandezLufuente Roberto,
Rosell Cristina 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.260381005
Subject(s) - chemistry , covalent bond , chymotrypsin , immobilized enzyme , agarose , aldehyde , autolysis (biology) , enzyme , trypsin , catalysis , glutaraldehyde , combinatorial chemistry , organic chemistry , chromatography
We have developed a strategy for immobilization‐stabilization of α‐chymotrypsin by multipoint covalent attachment of the enzyme, through its amino groups, to agarosealdehyde gels. We have studied the role of the main variables that control the intensity of these enzyme‐support multi‐interaction processes (surface density of aldehyde groups in the activated gel, contact time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives, etc.). In this way, we have prepared a number of very different chymotrypsinagarose derivatives. Our best derivatives, with the most intense multipoint attachment, were more stable than one‐point attached derivatives and were more than 60,000‐fold more stable than soluble enzyme in the absence of autolysis phenomena. In spite of the dramatic stabilization, the catalytic activity of these derivatives is little changed (they only lose 35% of intrinsic activity after this intense enzyme‐support multi‐interaction process). In addition, we have also demonstrated the very high capacity of 6% aldehyde‐agarose gels to immobilize pure chymotrypsin (40 mg enzyme/mL catalyst). Furthermore, we have been able to establish a clear correlation between enzyme‐support multipoint covalent attachment, stabilization against very different denaturing agents (heat, urea, organic cosolvents), and insensitivity of those immobilized chymotrypsin molecules to some activating agents.