Simultaneous purification and immobilization of d-hydantoinase on the immobilized metal affinity membrane via coordination bonds

• Published in: Biochemical engineering journal Vol. 61; pp. 20 - 27
• Main Authors: Ko, Yi-Miao, Chen, Chih-I, Shieh, Chwen-Jen, Liu, Yung-Chuan
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.02.2012; Elsevier
• Subjects: Biological and medical sciences; Biotechnology; d-Hydantoinase; Enzyme immobilization; Enzyme purification; Enzyme stability; Fundamental and applied biological sciences. Psychology; General aspects; Immobilization techniques; Immobilized metal affinity membrane; Methods. Procedures. Technologies; Immobilized metal affinity membrane; Enzyme immobilization; Enzyme purification; Enzyme stability; d-Hydantoinase; Purification; Enzyme; Dihydropyrimidinase; Immobilization; Hydrolases; Affinity; Membrane; Metal
• ISSN: 1369-703X; 1873-295X
• DOI: 10.1016/j.bej.2011.11.013

This study constructs the immobilized metal affinity membrane (IMAM) via coupling of epichlorohydrin, iminodiacetic acid, and nickel ion on the regenerated cellulose membrane. The d-hydantoin-hydrolyzing enzyme (DHTase) harboring a poly-His tagged residue was used as a model protein immobilized on the prepared IMAM. Various immobilization conditions were examined based on the yield of N-carbamoyl-d-p-hydroxyphenylglycine in batch reactions. The immobilization conditions were studied and the optimal conditions are as follows. By employing an IMAM with nickel ion of 155.5±5μmol/disc immersed in 0.1M Tris–HCl buffer pH 8 (with 0.8M sodium chloride) and immobilized time of 14h, a DHTase activity of 4.2±0.3U/disc was obtained. The immobilized DHTase membrane can achieve a larger pH and thermal tolerant range than that of free enzyme. Meanwhile, the stability test showed that 99% of enzyme activity could be retained after being repeated 15-times. The storage test also displayed 99% enzyme preservation after 7 weeks of storage.