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Toward a cell-free hydantoinase process : screening for expression optimization and one-step purification as well as immobilization of hydantoinase and carbamoylase

Slomka, Christin; Späth, Georg Paris; Lemke, Phillip; Skoupi, Marc; Niemeyer, Christof M.; Syldatk, Christoph; Rudat, Jens

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. ... mehr

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DOI: 10.5445/IR/1000071502
DOI: 10.1186/s13568-017-0420-3
Zitationen: 2
Web of Science
Zitationen: 2
Cover der Publikation
Zugehörige Institution(en) am KIT Institut für Bio- und Lebensmitteltechnik (BLT)
Institut für Biologische Grenzflächen (IBG)
Publikationstyp Zeitschriftenaufsatz
Publikationsjahr 2017
Sprache Englisch
Identifikator ISSN: 2191-0855
KITopen-ID: 1000071502
HGF-Programm 47.02.01 (POF III, LK 01)
Zellpopul.auf Biofunk.Oberflächen IBG-1
Erschienen in AMB express
Band 7
Heft 1
Seiten Art. Nr. 122
Bemerkung zur Veröffentlichung Gefördert durch den KIT-Publikationsfonds
Schlagwörter Amino acids, Enzyme catalysis, Hydantoinase process, Protein purification, Enzyme immobilization, Magnetic beads
Nachgewiesen in Web of Science
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