Preparation of carboxylated magnetic particles for the efficient immobilization of C-terminally lysine-tagged Bacillus stearothermophilus aminopeptidase II.
This article reports the synthesis and use of surface-modified iron oxide particles for the simultaneous purification and immobilization of Bacillus stearothermophilus aminopeptidase II (BsAPII) tagged C-terminally with either tri- or nona-lysines (BsAPII-Lys(3/9)). The carboxylated magnetic particles were prepared by the simple co-precipitation of Fe(3+)/Fe(2+) in aqueous medium and then subsequently modified with adipic acid. Transmission electron microscopy (TEM) micrographs showed that the carboxylated magnetic particles remained discrete and had no significant change in size after binding BsAPIIs. Wild-type enzyme and BsAPII-Lys(3) could be purified to near homogeneity by the carboxylated magnetic particles, but it was not easy to elute the adsorbed BsAPII-Lys(9) from the matrix. Free BsAPII, BsAPII-Lys(3), and BsAPII-Lys(9) were active in the temperature range 50-70 degrees C and all had an optimum of 50 degrees C, whereas the optimum temperature and thermal stability of BsAPII-Lys(3) and BsAPII-Lys(9) were improved as a result of immobilization. The immobilized BsAPII-Lys(9) could be recycled ten times without a significant loss of the enzyme activity and had a better stability during storage than BsAPII. Owing to its high efficiency and cost-effectiveness, this magnetic adsorbent may be used as a novel purification-immobilization system for the positively charged enzymes.
Huang
Cheng-Liang
CL
Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi, Taiwan.
Cheng
Wei-Chun
WC
Yang
Jia-Ci
JC
Chi
Meng-Chun
MC
Chen
Jiau-Hua
JH
Lin
Hong-Ping
HP
Lin
Long-Liu
LL
D000626:Aminopeptidases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D052203:Ferrosoferric Oxide / Q000737:chemistry*; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D013696:Temperature
D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D000626:Aminopeptidases; D052203:Ferrosoferric Oxide