Abstract
The hydantoinase process is applied for the industrial synthesis of optically pure amino acids via whole cell biocatalysis, providing a simple and well-established method to obtain the catalyst. Nevertheless, whole cell approaches also bear disadvantages like intracellular degradation reactions, transport limitations as well as low substrate solubility. In this work the hydantoinase and carbamoylase from Arthrobacter crystallopoietes DSM 20117 were investigated with respect to their applicability in a cell-free hydantoinase process. Both enzymes were heterologously expressed in Escherichia coli BL21DE3. Cultivation and induction of the hydantoinase under oxygen deficiency resulted in markedly higher specific activities and a further increase in expression was achieved by codon-optimization. Further expression conditions of the hydantoinase were tested using the microbioreactor system BioLector®, which showed a positive effect upon the addition of 3% ethanol to the cultivation medium. Additionally, the hydantoinase and carbamoylase were successfully purified by immobilized metal ion affinity using Ni Sepharose beads as well as by functionalized magnetic beads, while the latter method was clearly more effective with respect to recovery and purification factor. Immobilization of both enzymes via functionalized magnetic beads directly from the crude cell extract was successful and resulted in specific activities that turned out to be much higher than those of the purified free enzymes.
Highlights
The hydantoinase process (Fig. 1) is well established in industry for the biocatalytic production of enantiopure α-amino acids, especially α-d-amino acids like α-dphenylglycine and α-d-p-hydroxyphenylglycine
The use of whole cell biocatalysis is widespread in industrial biocatalysis due to the easy access to the biocatalyst, low production costs and simple separation of biocatalyst and product
The application of recombinantly expressed enzymes using well-established expression hosts like Escherichia coli allows an overexpression of the target enzyme as well as the attachment of tags for purification and immobilization (Chen 2012)
Summary
The hydantoinase process (Fig. 1) is well established in industry for the biocatalytic production of enantiopure α-amino acids, especially α-d-amino acids like α-dphenylglycine and α-d-p-hydroxyphenylglycine. They serve as side chains of the semisynthetic antibiotics ampicillin and amoxicillin Uncontrollable degradative side reactions may take place inside of the cells and especially for the hydantoinase process, the substrate solubility is relatively low (Slomka et al 2014). This points out that codon-optimization of the desired gene according to the host organism, for instance, is an effective tool to improve the expression and avoid misfolding and aggregation of the protein (Kurland and Gallant 1996)
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