Journal
ENZYME AND MICROBIAL TECHNOLOGY
Volume 39, Issue 1, Pages 22-27Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2005.09.001
Keywords
fuculose-1-phosphate aldolase (FucA); DHAP dependent aldolase; enzyme immobilization; immobilized metal-chelate affinity chromatography (IMAC); metal-enzyme; one step purification-immobilization of enzymes
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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 degrees C and it catalyzes aldol addition between S-Cbz-Alaninal and DHAP. (c) 2005 Elsevier Inc. All rights reserved.
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