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KMID : 1094720090140050654
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Biotechnology and Bioprocess Engineering
2009 Volume.14 No. 5 p.654 ~ p.661
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Novel Synthesis of Mannosamine Conjugated Magnetic Nanoparticles for Purification and Stabilization of Human Lysosomal β-mannosidase
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Zahoor Qadir Samra
Muhammad Amin Athar
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Abstract
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Human β-mannosidase (MANB) was purified to homogeneity directly from lysosomes by using mannosamine conjugated magnetic (Fe3O4) nanoparticles, DE-52 cellulose, and sephadex G-200 chromatography. Fe3O4 nanoparticles were synthesized and utilized ammonia to attach the amino group on the nanoparticles. The particles were covalently attached with D-mannosamine by cross linker glutaraldehyde and confirmed by FTIR spectroscopy. In FTIR analysis, the peaks appeared at 2,356.6 cm-1 for -N = CH linkage and at 3,378.4 cm-1, 3,664.9 cm-1 for -OH groups confirmed the conjugation of D-mannosamine with Fe3O4 nanoparticles. Results showed a single band of 97 kDa of purified MANB in SDS-PAGE. The isoelectric point was 4.5 and the Km and Vmax values were 2.51 mM and 0.315 μM/min/mg, respectively. The purification fold was 329 with 68% yield. The optimal activity was at pH 5.0 and 75% activity was stable in 20% glycerol at 4oC. The enzyme activity was inhibited by Ni2+, Zn2+, Cd2+, Cu2+, Mo2+, Ag+1, iodoacetate, SDS, DMF, DMSO, ethanol, and acetone; slightly reduced by Pb2+, Co2+, EDTA, DTT, and β-mercaptoethanol. The activity was not affected by Mg2+, Mn2+, Sn2+, Ca2+, Fe3+, PMSF, Triton X-100, D-mannosamine, D-mannose, D-mannitol, D-glucose, and D-fructose. The homogeneity of MANB enzyme was further confirmed by 2D-PAGE and immunoblot. This is the first novel report of conjugation of D-mannosamine with Fe3O4 nanoparticles for purification of human MANB enzyme.
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KEYWORD
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Mannosamine, magnetic nanoparticles, FTIR, western blot, 2D-PAGE
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