His-tagged recombinant fuculose-1-phosphate aldolase (FucA) from E. coli has been purified by immobilized metal-chelate affinity chromatography (IMAC) at gram scale. During this operation, there was a metal exchange between FucA and the affinity matrix, being the purification yields dependent on the metal nature, which was bound to affinity matrix. One step purification-immobilization of FucA has been carried out on metal-chelate support. The preparation of a FucA immobilized derivative, available to be used as catalyst in aldol addition reactions, has been accomplished in a single step starting from E. coli cell extracts. The best results were obtained with high density support containing Co2+. The immobilization yield was 100% and the immobilized derivative showed 63% of FucA activity initially offered to the support. The best derivative of immobilized FucA is 21-fold more stable than the soluble FucA in DMF/buffer (1:4) at 25 °C and it catalyzes aldol addition between S-Cbz-Alaninal and DHAP. © 2005 Elsevier Inc. All rights reserved.
|Journal||Enzyme and Microbial Technology|
|Publication status||Published - 1 Jun 2006|
- DHAP dependent aldolase
- Enzyme immobilization
- Fuculose-1-phosphate aldolase (FucA)
- Immobilized metal-chelate affinity chromatography (IMAC)
- One step purification-immobilization of enzymes