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Active site titration as a tool for the evaluation of immobilization procedures of penicillin acylase
Author(s) -
van Langen Luuk M.,
Janssen Michiel H. A.,
Oosthoek Natasja H. P.,
Pereira Sílvia R. M.,
Švedas Vytas K.,
van Rantwijk Fred,
Sheldon Roger A.
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.10280
Subject(s) - titration , active site , alcaligenes faecalis , chemistry , penicillin amidase , penicillin , chromatography , enzyme , immobilized enzyme , turnover number , active ingredient , specific activity , alcaligenes , biochemistry , organic chemistry , bacteria , antibiotics , biology , pseudomonas , genetics , bioinformatics
Native and immobilized preparations of penicillin acylase from Escherichia coli and Alcaligenes faecalis were studied using an active site titration technique. Knowledge of the number of active sites allowed the calculation of the average turnover rate of the enzyme in the various preparations and allowed us to quantify the contribution of irreversible inactivation of the enzyme to the loss of catalytic activity during the immobilization procedure. In most cases a loss of active sites as well as a decrease of catalytic activity per active site (turnover rate) was observed upon immobilization. Immobilization techniques affected the enzymes differently. The effect of increased loading of penicillin acylase on the average turnover rate was determined by active site titration to assess diffusion limitations in the carrier. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 224–228, 2002.