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|標題:||Simultaneous purification and immobilization of d-hydantoinase on the immobilized metal affinity membrane via coordination bonds|
Immobilized metal affinity membrane
|摘要:||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.|
|Appears in Collections:||化學工程學系所|
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