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Biotechnology and bioprocess engineering v.14 no.5, 2009년, pp.651 - 661   SCI SCIE
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Novel Synthesis of Mannosamine Conjugated Magnetic Nanoparticles for Purification and Stabilization of Human Lysosomal $\beta$-mannosidase

Samra, Zahoor Qadir    (Institute of Biochemistry and Biotechnology, Quaid-i-Azam Campus, University of Punjab   ); Athar, Muhammad Amin    (Institute of Biochemistry and Biotechnology, Quaid-i-Azam Campus, University of Punjab  );
  • 초록

    Human $\beta$ -mannosidase (MANB) was purified to homogeneity directly from lysosomes by using mannosamine conjugated magnetic ( $Fe_3O_4$ ) nanoparticles, DE-52 cellulose, and sephadex G-200 chromatography. $Fe_3O_4$ nanoparticles were synthesized and utilized ammonia to attach the amino group on the nanoparticles. The particles were covalently attached with Dmannosamine 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 $Fe_3O_4$ nanoparticles. Results showed a single band of 97 kDa of purified MANB in SDS-PAGE. The isoelectric point was 4.5 and the $K_m$ and Vmax values were 2.51 mM and $0.315\;{\mu}M/min/mg$ , 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 $4^{\circ}C$ . The enzyme activity was inhibited by $Ni^{2+}$ , $Zn^{2+}$ , $Cd^{2+}$ , $Cu^{2+}$ , $Mo^{2+}$ , $Ag^{+1}$ , iodoacetate, SDS, DMF, DMSO, ethanol, and acetone; slightly reduced by $Pb^{2+}$ , $Co^{2+}$ , EDTA, DTT, and $\beta$ -mercaptoethanol. The activity was not affected by $Mg^{2+}$ , $Mn^{2+}$ , $Sn^{2+}$ , $Ca^{2+}$ , $Fe^{3+}$ , 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 $Fe_3O_4$ nanoparticles for purification of human MANB enzyme.


  • 주제어

    Mannosamine .   magnetic nanoparticles .   FTIR .   western blot .   2D-PAGE.  

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