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Synthesis of Mimic Chymotrypsin Enzyme and It’s Immobilizition Research

Author: YuXiang
Tutor: YangWanShen
School: Sichuan Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: Magnetic nanoparticles beta - cyclodextrin Enzyme mimics Immobilized
CLC: Q814
Type: Master's thesis
Year: 2010
Downloads: 95
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Abstract


Analog enzyme. (Artificial Enzyme) are a class of synthetic supramolecular compound having a specific catalytic ability significantly higher than that of biological enzyme catalysis and environmental stability. Magnetic nanoparticles (magnetic nanoparticles, MNPs) based on nano-magnetic Fe3O4 or gamma-Fe2O3 nuclei polymer matrix or surfactant shell, the formation of a package or embedded with superparamagnetic new composite materials. In this study, beta-cyclodextrin as the parent material, the introduction of an imidazole analog enzyme having the activity of chymotrypsin was prepared in the 2-position carbon atom. And the carboxyl group of the hydroxyl groups of the cyclodextrin other sites (C3 or C6) with the laboratory to synthesize magnetic nanoparticles covalently coupled, with or without oleic acid modified with oleic acid-modified magnetic nanoparticles through hydrophobic interactions analog enzyme immobilization. Through the above two methods successfully enzyme chymotrypsin analog fixed to the magnetic nanoparticles surface, to obtain water-dispersible analog enzyme / magnetic nano-composites having a magnetically-responsive and can reuse. Main results are as follows: 1 Synthesis of C2 and C6-bit single-toluenesulfonate, then substituted imidazolyl single p-toluenesulfonate to give different analog enzyme chymotrypsin substitutable positions, C2-substituted imidazolyl The activity of analog kcat: 3.01 × 10-3s-, C6 substituted analog enzyme activity kcat: 2.7 x 10-4s-. C2-substituted imidazole-based analog magnetic immobilization of the enzyme. Carboxyl-functionalized magnetic nanoparticles immobilized chymotrypsin analog enzyme optimum conditions: take 1 mL (5mg/ml) the carboxyl group of magnetic nanoparticles in the aqueous solution was added to the reaction system, when the amount of enzyme 0.1g, fixed temperature to 25 ° C the immobilized for 36h, pH = 7.0, resulting carboxyl nanoparticles immobilized enzyme mimics the highest enzyme activity (kcat: 1.98 × 10-3S-); oleic acid of magnetic nanoparticles immobilized optimal : take 1 mL (10mg/mL) the oleic acid magnetic nanoparticles chloroform solution was added to the reaction system, 0.15g, to the amount of enzyme immobilization temperature of 40 ° C the immobilized for 36h, pH = 7.5 when , oleic acid obtained magnetic nanoparticles immobilized enzyme mimics the highest enzyme activity (kcat: 2.60 × 10-3S-). Immobilized product was characterized. Transmission electron microscopy identified carboxyl magnetic nanoparticles immobilized chymotrypsin mimic enzyme uniform distribution, particle size of 10nm, a small amount of agglomeration; oleic acid nanoparticles immobilized enzyme mimics monodisperse state, particle size of 25nm agglomeration phenomenon does not appear, the cladding layer structure. The infrared spectra cyclodextrin synthesis enzyme chymotrypsin analog successful fixed to the surface of the nanoparticles carboxyl nanoparticles and oleic acid. Magnetic immobilized chymotrypsin enzyme having a good analog magnetic response within 10s can be completely separated from the solution in a magnetic field generated by the permanent magnet. 4 carboxyl nanoparticles immobilized enzyme mimics optimum reaction temperature is 25 ℃, the optimum pH value of 7.0, was repeated 10 times, enzyme activity remains 43% of the initial enzyme activity; oleic acid-modified magnetic nanoparticles fixed simulation of the enzyme, the optimum reaction temperature of 40 ° C, the optimum pH value of 7.5, repeated 10 times, enzyme activity remains at 82% of the initial enzyme activity. By enzyme activity assay, oleic nano immobilized enzyme activity kcat: 2.60 x 10-3s-the above carboxy nanoparticles kcat: 1.98 × 10-3s-;, oleic acid nanoparticles in dispersion stability and pH stability of immobilized analog activity than the carboxyl group of nanoparticles immobilized the analog high enzyme activity, but less than the free enzyme activity kcat: 3.01 x 10-3s- Carboxyl nanoparticles immobilization method is simple, the immobilized enzyme of the synthesis cycle is short, while the oleic acid nanoparticles immobilized complex method, the synthesis reaction of the immobilized enzyme longer period.

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CLC: > Biological Sciences > Bioengineering ( Biotechnology ) > Enzyme Engineering
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