Active site titration as a tool for the evaluation of immobilization procedures of penicillin acylase.
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.
Laboratory of Organic Chemistry and Catalysis, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands.
Luuk M
LM
Van Langen
Michiel H A
MH
Janssen
Natasja H P
NH
Oosthoek
Sílvia R M
SR
Pereira
Vytas K
VK
Svedas
Fred
F
van Rantwijk
Roger A
RA
Sheldon
D000421:Alcaligenes / Q000201:enzymology*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000032:analysis* / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D010399:Penicillin Amidase / Q000032:analysis* / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D012680:Sensitivity and Specificity; D017186:Titrimetry / Q000379:methods*
D004800:Enzymes, Immobilized; D011108:Polymers; C065843:Eupergit C; D010399:Penicillin Amidase