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Study On The Immobilization Of Esterase BioH And Rhizomucor Miehei Lipase

Posted on:2015-05-11Degree:MasterType:Thesis
Country:ChinaCandidate:R R LiFull Text:PDF
GTID:2180330431490347Subject:Chemical Engineering
Abstract/Summary:PDF Full Text Request
In this paper, two kinds of functional magnetic nanosupports were prepared, one was modified with acyl azide groups on its surface (Pol(EA/AA)-MNPs), the other was modified with acyl azide groups and hydroxyl groups (Pol(EA/AA-VAc/HO)-MNPs). The esterase BioH from Escherichia coli was covalently immobilized onto their surface, respectively, obtaining two types of immobilized esterase BioHs. One was immobilized onto the surface of the functional magnetic nanoparticles modified by acyl azide groups, namely IMBioH1,the other was immobilized onto the surface of the functional magnetic nanoparticles modified by acyl azide groups and hydroxyl groups, namely IMBioH2. The effect of the suface property of carrier on the protein loading of immobilized enzyme, the retention activity of enzyme, enzyme kinetics and the linking manner of esterase BioH were determined. The thermo-/pH-stability of the immobilized esterase BioH onto the surface of the functional magnetic nanoparticles modified by acyl azide groups and hydroxyl groups were investigated, in comparison with those of the free enzyme. In addition, Pol(EA/AA)-MNPs, Pol(EA/AA-VAc/HO)-MNPs, IMBioH1and IMBioH2were characterized. The studies confirmed the presence of the acyl azide groups of Pol(EA/AA)-MNPs, with a mean size of121nm, and the presence of both acyl azide and hydroxyl groups of Pol(EA/AA-VAc/HO)-MNPs, with a mean size of119nm. The mean sizes of IMBioH’and IMBioH2were219nm and235nm, respectively. The retention activity of IMBioH2(70%) was higher than that of IMBioH1(30%), so as protein loading of immbilized esterase BioH (99mg/g and53mg/g for IMBioH2and IMBioH1). Meanwhile, the Km of the BioH immobilized on the hydrophilic-modified nanosupport was a little higher than that of the free esterase BioH, and was far lower than the BioH immobilized on the nanosupport without hydrophilic modification. For the BioH immobilized on the hydrophilic-modified nanosupport, the recovery activity remained80%of the original activity after10times of recycling. Besides, the comparatively high activity retention, improved thermo-/pH-stability and good reusability of the immobilized enzyme indicate that oriented covalent immobilization is an efficient method for immobilizing esterase BioH.Besides, the Rhizomucor miehei lipase from Escherichia coli was immobilized onto the surface of the functional magnetic nanosupport and resins. The surface of the former support was modified with acyl azide groups and hydroxyl groups, the latter supports included BN-AN, D101and NKA-9. The functional magnetic nanopartcles and the resin BN-AN immobilized the Rhizomucor miehei lipase by covalent bonding, the resin D101and NKA-9attached lipase by adsorption. For the lipase immobilized on the hydrophilic-modified magnetic nanosupport, the recovery activity remained61%of the original activity after10times of recycling. The protein loading of the immobilized lipase onto the nanosupport without hydrophilic modification. The specific activities of the immbilized lipase onto the resin of BN-AN, D101and NKA-9were122U/g,225U/g#1188U/g, respectively. The thermo-/pH-stability of the immobilized Rhizomucor miehei lipase were also determined and compared to those of the free enzyme. Improved thermo-/pH-stability and good reusability were found for the immobilized enzyme.
Keywords/Search Tags:covalent bonding, oriented immobilization, magnetic nanoparticles, resin, esterase BioH, lipase
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