Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
3578 | Biochemical Engineering Journal | 2012 | 8 Pages |
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 ± 5 μmol/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.