TY - JOUR
T1 - One step purification-immobilization of fuculose-1-phosphate aldolase, a class II DHAP dependent aldolase, by using metal-chelate supports
AU - Ardao, Inés
AU - Benaiges, M. Dolors
AU - Caminal, Gloria
AU - Álvaro, Gregorio
PY - 2006/6/1
Y1 - 2006/6/1
N2 - 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.
AB - 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.
KW - DHAP dependent aldolase
KW - Enzyme immobilization
KW - Fuculose-1-phosphate aldolase (FucA)
KW - Immobilized metal-chelate affinity chromatography (IMAC)
KW - Metal-enzyme
KW - One step purification-immobilization of enzymes
U2 - 10.1016/j.enzmictec.2005.09.001
DO - 10.1016/j.enzmictec.2005.09.001
M3 - Article
SN - 0141-0229
VL - 39
SP - 22
EP - 27
JO - Enzyme and Microbial Technology
JF - Enzyme and Microbial Technology
IS - 1
ER -