Properties of Immobilised Penicillin G Acylase in β-Lactam Antibiotic Synthesis

141 Pages·2006·0.8 MB·English

Properties of Immobilised Penicillin G
Acylase in β-Lactam Antibiotic Synthesis

Michiel Janssen
Picture at the cover (© M.H.A. Janssen):
The Saipem 7000, a semi submersible crane and pipelaying (J-lay) dynamic positioning
vessel moored nearby the EMO area of the Rotterdam Europoort. Dimensions: length, 198
m; width, 87 m; depth (to main deck), 45 m. Maximum crane lifting capacity: 14,000
tonnes. Maximum crane lowering capacity: 450 m below sea level. In 2002 the Saipem
7000 lay pipes in the Black Sea between Russia and Turkey (Blue Stream project) to then a
record depth of 2,150 m.
Properties of Immobilised Penicillin G
Acylase in β-Lactam Antibiotic Synthesis

Proefschrift

ter verkrijging van de graad van doctor
aan de Technische Universiteit Delft,
op gezag van de Rector Magnificus prof. dr. ir. J.T. Fokkema,
voorzitter van het College voor Promoties,
in het openbaar te verdedigen op
maandag 22 mei 2006 om 12:30 uur

door

Michiel Hubertus Arnold JANSSEN

ingenieur in de bioprocestechnologie
geboren op 21 mei 1974 te Cuijk
Dit proefscrift is goedgekeurd door de promotor:
Prof. dr. R.A. Sheldon

Toegevoegd promotor:
Dr. ir. F. van Rantwijk

Samenstelling promotiecommissie:

Rector Magnificus, Technische Universiteit Delft, voorzitter
Prof. dr. R.A. Sheldon, Technische Universiteit Delft, promotor
Dr. ir. F. van Rantwijk, Technische Universiteit Delft, toegevoegd promotor
Prof. dr. A. Bruggink, Radboud Universiteit Nijmegen, DSM
Prof. dr. L. Fischer, Universität Hohenheim
Prof. dr. V.K. Švedas, Lomonosov Moscow State University
Prof. dr. ir. L.A.M. van der Wielen Technische Universiteit Delft
Dr. M.C.R. Franssen Wageningen Universiteit
em. Prof. dr. ir. H. van Bekkum Technische Universiteit Delft, reservelid

The research described in this thesis was financially supported by DSM and the Netherlands Ministry of
Economic Affairs.

ISBN 90-9020754-6

© 2006 by M.H.A. Janssen

All rights reserved. No part of the material protected by this copyright notice may be reproduced or utilised
in any form or by any means, electronic or mechanical, including photocopying, recording or by any
information storage and retrieval system, without written permission from the copyright holder.
To my parents

Contents

Abbreviations and symbols xi

1. Introduction to β-lactam antibiotics 1
1.1 β-Lactam antibiotcs 2
1.2 Developments in β-lactam antibiotic synthesis 4
1.3 Enzymatic developments 11
Appendix 1.1: ACV synthase 13
References and notes 14

2. Penicillin G acylase: Structure and enzymatic β-lactam antibiotic
synthesis 17
2.1 Penicillin G acylase 18
2.1.1 Discovery and occurrence 18
2.1.2 Classification 18
2.1.3 Structure and catalytic machinery 19
2.2 Enzymatic synthesis of β-lactam antibiotics 22
2.2.1 Thermodynamic and kinetic controll 22
2.2.2 Coupling procedures 24
2.2.3 Penicillin acylase kinetics 27
2.2.4 Immobilisation of penicillin acylase 31
2.2.5 Increasing the efficiency through additives 33
2.3 Motivation, objective, and outline of the thesis 35
References and notes 36

3. Evaluation of the performance of immobilised penicillin G acylase
using active-site titration 43
3.1 Introduction 44
3.2 Results and discussion 46
vii
Contents
3.2.1 Immobilisation of penicillin acylase on Eupergit C 46
3.2.2 Active-site titration 47
3.2.3 Diffusion limitation effects 48
3.2.4 Calculating the minimum substrate bulk concentration 49
3.2.5 Calculation of the zero substrate concentration radius 52
3.2.6 Diffusion limitations and product inhibition 54
3.2.7 Cephalexin synthesis 55
3.3 Conclusions 61
3.4 Materials and methods 61
3.4.1 Materials 61
3.4.2 Immobilisation 62
3.4.3 Hydrolysis of penicillin G 62
3.4.4 Synthesis of cephalexin 63
3.4.5 Active-site titration 63
Acknowledgements 63
References 64

4. Properties and immobilisation of penicillin G acylase on
macroporous acrylic supports 67
4.1 Introduction 68
4.2 Results and discussion 69
4.2.1 Immobilisation on Eupergit C 250 L 70
4.2.2 Immobilisation on Sepabeads FP-EP 71
4.2.3 Immobilisation on Sepabeads FP-EA and FP-HA 72
4.2.4 Active-site titration 73
4.2.5 Cephalexin synthesis at low substrate concentrations 75
4.2.6 Cephalexin synthesis at high substrate concentrations 77
4.3 Conclusions 79
4.4 Materials and methods 80
4.4.1 General 80
viii
Contents
4.4.2 Immobilisation of penicillin acylase 81
4.4.3 Crushing of immobilised penicillin acylase catalysts 82
4.4.4 Hydrolysis of penicillin G 82
4.4.5 Active-site titration 82
4.4.6 Synthesis of cephalexin 83
Acknowledgements 83
References 84

5. Fluoride burst active-site titration of penicillin G acylase using
a fluoride ion selective electrode 85
5.1 Introduction 86
5.2 Results and discussions 88
5.2.1 Fluoride burst active-site titration 88
5.2.2 Indirect active-site titration and comparison 94
5.3 Conclusions 96
5.4 Materials and methods 98
5.4.1 Materials 98
5.4.2 Hydrolysis of penicillin G 99
5.4.3 Fluoride burst active-site titration 99
5.4.4 Indirect active-site titration 100
Acknowledgements 100
References and notes 100

6. Dendrimer-activated supports for the immobilisation of
penicillin G acylase 103
6.1 Introduction 104
6.2 Results and discussion 107
6.2.1 Immobilisation of dendrimer and enzyme 107
6.2.2 Synthesis of ampicillin and cephalexin 108
6.3 Conclusions 111
ix
Contents
6.4 Materials and methods 111
6.4.1 Materials 111
6.4.2 Hydrolysis of penicillin G 111
6.4.3 Polyacrylamide gel dispersion polymerisation 112
6.4.4 Astramol-polyacrylamide gel beads immobilisation 112
6.4.5 Starburst/Astramol-Assemblase immobilisation 113
6.4.6 Astramol-polyketone immobilisation 113
6.4.7 Synthesis of ampicillin and cephalexin 114
Acknowledgements 114
References 114

Summary 117
Samenvatting 120
Dankwoord 124
List of publications 127
Curriculum vitae 129
x