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Structure‐function relationships in immobilized chymotrypsin catalysis
Author(s) -
Clark Douglas S.,
Bailey James E.
Publication year - 2002
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.10438
Subject(s) - active site , chemistry , immobilized enzyme , electron paramagnetic resonance , enzyme , sepharose , enzyme assay , chymotrypsin , spin probe , electron paramagnetic resonance spectroscopy , organic chemistry , biochemistry , nuclear magnetic resonance , trypsin , physics , membrane
Specific activities and the amounts of active immobilized enzyme were determined for several different preparations of α‐chymotrypsin immobilized on CNBr‐activated Sepharose 4B. Electron paramagnetic resonance (EPR) spectroscopy of free and immobilized enzyme with a spin label coupled to the active site was used to probe the effects of different immobilization conditions on the immobilized enzyme active site configuration. Specific activity of active enzyme decreased and rotational correlation time of the spin label increased with increasing immobilized enzyme loading. Enzyme immobilized using an intermediate six‐carbon spacer arm exhibited greater specific activity and spin label mobility than directly coupled enzyme. The observed activity changes due to immobilization were completely consistent with corresponding active site structure alterations revealed by EPR spectroscopy. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 539–549, 2002.