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標題: Simultaneous purification and immobilization of d-hydantoinase on the immobilized metal affinity membrane via coordination bonds
作者: Ko, Yi-Miao
Chen, Chih-I
Shieh, Chwen-Jen
Liu, Yung-Chuan
關鍵字: d-Hydantoinase;Immobilized metal affinity membrane;Enzyme immobilization;Enzyme purification;Enzyme stability
Project: Biochemical Engineering Journal, Volume 61, page(s) 20– 27.
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-dp-
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 ± 5 !mol/disc immersed in 0.1 M
Tris–HCl buffer pH 8 (with 0.8 M sodium chloride) and immobilized time of 14 h, a DHTase activity of
4.2 ± 0.3 U/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.
ISSN: 1369-703X
DOI: 10.1016/j.bej.2011.11.013
Appears in Collections:化學工程學系所

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