title: Biocatalytic activity of recombinant human β-mannosidase immobilized onto magnetic nanoparticles for bioprocess.
abstract: Recombinant human β-mannosidase (rhMANB) is an important glycosidase enzyme that degrades mannose-linked glycoproteins and mannan polysaccharides. rhMANB was purified and covalently immobilized onto magnetic nanoparticles. The immobilization of the enzyme was confirmed by Fourier-transform infrared spectroscopy (FTIR) and magnetic nanoparticles linked immunosorbent assay (MagLISA). Antibodies against rhMANB were raised, purified and characterized for MagLISA. The binding of rhMANB onto magnetic nanoparticles was found to be 65%. The V( max ) and K( m ) of immobilized rhMANB was observed 3.0-fold higher and 2.024-fold lower, respectively, as compared to unbound rhMANB. The stability and activity of immobilized enzyme was observed at different pH, temperature, and after storage at 4°C. Metal chelators (oxalic acid, citric acid, and ascorbic acid) did not affect the enzyme activity of immobilized enzyme, whereas ethylenediamine tetraacetic acid reduced the activity. The results obtained from thin-layer chromatography indicate that immobilized rhMANB is more efficient than the unbound form to hydrolyze mannobiose, mannotriose, mannotetraose, mannopentose, galactoglucomannan, and locust bean gum. Magnetic nanoparticles suspended gel-permeation chromatography showed that 29% locust bean gum hydrolyzed efficiently during flow in the column. The immobilization of rhMANB will be a good process for gelling and saccharification of mannan polymers at industrial scale.
authors.affiliation: Institute of Biochemistry and Biotechnology, Quaid-i-Azam Campus University of the Punjab, Lahore, Pakistan. samra201@yahoo.com
authors.forename: Zahoor Qadir
authors.initials: ZQ
authors.lastname: Samra
authors.forename: Nadia
authors.initials: N
authors.lastname: Dar
authors.forename: Muhammad Amin
authors.initials: MA
authors.lastname: Athar
mesh_terms: D055162:Biocatalysis; D002614:Chelating Agents / Q000737:chemistry; D002855:Chromatography, Thin Layer; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008280:Magnetics; D053758:Nanoparticles / Q000737:chemistry*; D009844:Oligosaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002614:Chelating Agents; D004800:Enzymes, Immobilized; D009844:Oligosaccharides; D011994:Recombinant Proteins; D044902:beta-Mannosidase