Immobilization of penicillin G acylase onto chemically grafted nylon particles

https://doi.org/10.1016/S1381-1177(99)00122-8Get rights and content

Abstract

Nylon particles, grafted with diethylene glycol dimethacrylate (DGDA) using potassium persulphate as initiator, were treated with hexamethylene diamine (HMDA). The aminoalkylated particles were activated with glutaraldehyde and finally, penicillin G acylase (PA) was immobilized to these activated particles.

Both the conditions of the aminoalkylation- and the immobilization process were optimized. The hydrolysis of cephalexin was used as model conversion. The retention of activity of the immobilized enzyme was 12%. This value improved to 30% by adding phenyl acetic acid (PAA), as active-site protecting agent, to the enzyme solution. The results suggest formation of multi-point attachment between the enzyme and the matrix.

Introduction

Immobilized penicillin G acylase (PA) is one of the few immobilized enzymes applied on industrial scale for the production of semi-synthetic antibiotics [1], [2], [3], [4]. Many supports have been investigated for immobilization of the enzyme [5], [6], [7], [8], including nylon [9]. Since nylon has a few free end groups for covalent attachment of enzyme molecules, it must be pretreated to generate potentially reactive centers [10]. In spite of the great potential of the grafting technique to create reactive centers on copolymer matrices, very few papers were published about grafted nylon used for enzyme immobilization [11], [12], [13], [14], [15]. The value of graft copolymers is that a variety of matrices, possessing different physical, chemical and morphological characteristics, can be made. By careful selection of the matrix and the grafted monomer, it is possible to vary the hydrophilic/hydrophobic nature of the immobilized support that could improve enzyme activity and stability and also give easier handling and storage. The structure of the immobilized enzyme system, using graft copolymers, suggests that the enzyme is more available for reaction than is the case with enzyme entrapped in a gel where problems of diffusion of reactants and products can arise especially if the products are competitive inhibitors. Additionally, the number of reactive groups used for coupling the enzyme can be more closely controlled. Recently, the authors used nylon membranes grafted with different vinyl monomers to immobilize different enzymes including PA [16], [17].

In the present work, nylon particles, which are cheaper than membranes, grafted with diethylene glycol dimethacrylate (DGDA) monomer was used as matrix for the immobilization of PA to be used in stirred tank bioreactor which is more economical than the membrane one. The activation and the immobilization conditions were optimized and the efficiency of the immobilization process is reported on.

Section snippets

Chemicals

Nylon 6,6 pellets of 5 mm diameter, were used as solid support to be grafted with DGDA. Potassium persulphate (KPS) served as initiator for the grafting process. 1,6-Hexamethylene diamine (HMDA), 70% aqueous solution, was used as spacer between the grafted membrane and the enzyme. Glutaraldehyde (GA), 25% aqueous solution, was used as coupling agent for covalent binding of the enzyme to the activated HMDA-nylon beads. All the chemicals were purchased from Aldrich and used without any further

Optimization of the aminoalkylation process

Since the activity of the immobilized enzyme depends on the amount of functional groups created on the matrix, optimization of the conditions of the activation process, including aminoalkylation, glutaraldehyde activation and enzyme binding are required. The dependency of the catalytic activity of the immobilized enzyme on the concentration of HMDA, temperature and duration of the aminoalkylation process were studied first.

Conclusions

PA was immobilized on poly-diethyleneglycoldimethacrylate (pDGDA) grafted nylon of 8% grafting. After optimization of the factors affecting the activity of the immobilized enzyme, 12% of the activity is retained. Binding in the wrong orientation of the immobilized enzyme, multi-point attachment to the matrix and binding with groups in the active site, are the probable reasons, which cause the loss of activity. It was found that the increase of the amine-groups concentration on the matrix

Acknowledgements

The financial support of DSM-Anti-infectives and the Ministry of Economic Affairs of the Netherlands is kindly acknowledged.

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    Citation Excerpt :

    Each has its own advantages and disadvantages (Li, Li, Wang, & Lirui, 2008). The synthetic polymers such as, polyacrylonitrile (Godjevargova & Gabrovska, 2003) and nylon (MohyEldin, Schroen, Janssen, Mita, & Tramper, 2000), could be used for enzyme immobilization, but on contrary to the natural macromolecules (Krajewska, 2004) they have some disadvantages, as the imperfect biocompatibility and hydrophobicity. Entrapment immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane.

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1

Permanent address: Department of Polymers and Pigments, National Research Center, Dokki, Cairo, Egypt.

2

International Institute of Genetics and Biophysics of CNR, via Guglielmo Marconi, 12, 80125 Naples, Italy.

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