Developing a Novel Enzyme Immobilization Process by Activation of Epoxy Carriers with Glucosamine for Pharmaceutical and Food Applications

Immacolata Serra,Marco Terreni,Marina Simona Robescu,Marco Esti,Cinzia Calvio,Claudio Lombardelli,Massimo Pregnolato,Ilaria Benucci,Teodora Bavaro

doi:10.3390/catal9100843

Abstract

In this paper, we describe the development of an efficient enzyme immobilization procedure based on the activation of epoxy carriers with glucosamine. This approach aims at both creating a hydrophilic microenvironment surrounding the biocatalyst and introducing a spacer bearing an aldehyde group for covalent attachment. First, the immobilization study was carried out using penicillin G acylase (PGA) from Escherichia coli as a model enzyme. PGA immobilized on glucosamine activated supports has been compared with enzyme derivatives obtained by direct immobilization on the same non-modified carriers, in the synthesis of different 3′-functionalized cephalosporins. The derivatives prepared by immobilization of PGA on the glucosamine-carriers performed better than those prepared using the unmodified carriers (i.e., 90% versus 79% cefazolin conversion). The same immobilization method has been then applied to the immobilization of two other hydrolases (neutral protease from Bacillus subtilis, PN, and bromelain from pineapple stem, BR) and one transferase (γ-glutamyl transpeptidase from Bacillus subtilis, GGT). Immobilized PN and BR have been exploited in the synthesis of modified nucleosides and in a bench-scale packed-bed reactor for the protein stabilization of a Sauvignon blanc wine, respectively. In addition, in these cases, the new enzyme derivatives provided improved results compared to those previously described.

Highlights

The biocatalytic use of enzymes provides the possibility to develop efficient processes with low environmental impact in several fields ranging from pharma to food [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]
The critical role of the microenvironment has been extensively investigated with regard to the enzyme penicillin G acylase (PGA, EC number 3.5.1.11) from Escherichia coli, one of the most studied enzymes for biocatalytic applications, largely used in pharma industries for the synthesis of β-lactam antibiotics [17,24,25,26]
Afterwards, reactive aldehyde groups were introduced by oxidation of the glucosamine-coated carrier with periodate

Summary

Introduction

The biocatalytic use of enzymes provides the possibility to develop efficient processes with low environmental impact in several fields ranging from pharma to food [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]. Covalent immobilization of enzymes [17,18,19] is increasingly used to improve operational performances, to ensure the recovery and the reuse of the biocatalysts and to avoid contamination of the final product with residual proteins [20,21,22]. In this context, the selection of the most appropriate immobilization method and carrier material is crucial when designing a biocatalytic process as it strongly depends on the type and conditions of the process as well as on the enzyme. Acrylic carriers are largely employed for industrial applications thanks to their optimal mechanical properties, the low cost, and the rapid and simple immobilization procedure

Methods

Results

Conclusion