Preparation of carboxylated magnetic particles for the efficient immobilization of C-terminally lysine-tagged Bacillus stearothermophilus aminopeptidase II

Abstract

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.

Surface-Functionalized Hyperbranched Poly(Amido Acid) Magnetic Nanocarriers for Covalent Immobilization of a Bacterial γ-Glutamyltranspeptidase

“…Thus, the immobilization process decreased the substrate affinity of Bl GGT significantly and may also have hindered the accessibility of the reactants to the active site. These results are consistent with the kinetic observations of other enzymes immobilized on magnetic nanomaterials [4,5,28].…”

“…Figure 1 displays TEM micrographs and magnetization curves for the free HBPAA-γ-Fe 2 O 3 magnetic NPs and those covalently linked to Bl GGT. The HBPAA-γ-Fe 2 O 3 magnetic NPs had a size distribution of 6–11 nm, significantly smaller than that (20–30 nm) of corresponding pristine γ-Fe 2 O 3 prepared in the absence of HBPAA (Figure S1) [3,28]. The particle size distribution from a statistical sample of 150 particles in five TEM micrographs revealed a mean diameter of 8.3 ± 1.1 nm (Figure 1c).…”

Covalent Immobilization of Bacillus licheniformis γ-Glutamyl Transpeptidase on Aldehyde-Functionalized Magnetic Nanoparticles

“…These observations indicate that the immobilization process significantly reduces the substrate affinity of Bl GGT and may also hinder the accessibility of reactants to the active site. These results are consistent with the kinetic observations of other enzymes immobilized on magnetic nanomaterials [37,43,46–48]. …”

MHJ_0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae

“…MHJ_0461 can be classified as belonging to the M17 family of proteases by the presence of the NTDAEGRL motif and a C-terminal catalytic domain with highly conserved metal binding sites. These binding residues are coordinated to two divalent ions which act as positively charged electrophilic catalysts that complex an oxygen atom at a scissile peptide bond, thus facilitating the nucleophilic attack of an additionally coordinated water molecule leading to peptide bond cleavage [ 54 ]. Our data show that greatest activity across all substrates tested was achieved with the addition of manganese cations and was also increased considerably in the presence of cobalt and magnesium.…”