Evaluation of the performance of immobilized penicillin G acylase using active-site titration.
Penicillin G acylase from Escherichia coli was immobilized on Eupergit C with different enzyme loading. The activity of the immobilized preparations was assayed in the hydrolysis of penicillin G and was found to be much lower than would be expected on the basis of the residual enzyme activity in the immobilization supernatant. Active-site titration demonstrated that the immobilized enzyme molecules on average had turnover rates much lower than that of the dissolved enzyme. This was attributed to diffusion limitations of substrate and product inhibition. Indeed, when the immobilized preparations were crushed, the activity increased from 587 U g-1 to up to 974 U g-1. The immobilized preparations exhibited up to 15% lower turnover rates than the dissolved enzyme in cephalexin synthesis from 7-ADCA and D-(-)-phenylglycine amide. The synthesis over hydrolysis ratios of the immobilized preparations were also much lower than that of the dissolved enzyme. This was partly due to diffusion limitations but also to an intrinsic property of the immobilized enzyme because the synthesis over hydrolysis ratio of the crushed preparations was much lower than that of the dissolved enzyme.
Janssen
Michiel H A
MH
Laboratory of Organic Chemistry and Catalysis, Delft University of Technology, Julianalaan 136, 2628 BL, Delft, The Netherlands.
van Langen
Luuk M
LM
Pereira
Silvia R M
SR
van Rantwijk
Fred
F
Sheldon
Roger A
RA
D000951:Antigens, Neoplasm; D002384:Catalysis; D015815:Cell Adhesion Molecules; D004058:Diffusion; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006868:Hydrolysis; D008562:Membrane Glycoproteins / Q000138:chemical synthesis* / Q000378:metabolism; D008956:Models, Chemical; D010399:Penicillin Amidase / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism*; D010406:Penicillins / Q000378:metabolism*; D010664:Phenylmethylsulfonyl Fluoride / Q000378:metabolism / Q000494:pharmacology; D011108:Polymers / Q000737:chemistry; D012680:Sensitivity and Specificity; D017186:Titrimetry / Q000379:methods
D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D004800:Enzymes, Immobilized; D008562:Membrane Glycoproteins; D010406:Penicillins; D011108:Polymers; D010664:Phenylmethylsulfonyl Fluoride; C065843:Eupergit C; D010399:Penicillin Amidase