| Immobilized enzyme (mycelium) technology, as a new technology, was used in various fields, and the optical resolution of amino acid was one of the major applications. In this paper, aminoacylase from Aspergillus oryzae was used to resolute N-Ac-DL-phenylglycine, which was the materials of semi-synthetic antibiotics. This work was divided into two parts. In the first part, immobilized mycelium pellet was used as biocatalyst in batch stir tank reactor, and the second was that immobilized enzyme was used to continuous resolution process in packed reactor. The dissertation covered the submerged culture of Aspergillus oryzae in liquid, the immobilization of pellets, the kinetics and reaction mechanism, the immobilization of aminoacylase, properties of immobilized enzyme and continuous reactor.Because bean medium had the greatest industrial application value among the media studied, the bean medium was selected for the shaking flask culture of pellet. The effects of culture time, shaking flask rate and inoculum size on enzyme activity and biomass were investigated. The optimum cultivation conditions were obtained: culture temperature was 30℃, shaking rate 130 rpm, inoculum size 4% and the culture time 40h. The specific activity of mycelium pellet was more than 1600-1800U·g-1. Moreover, the L-aminoacylase activities of W001 in vivo and in vitro were tested. The result showed that W001 was a cell-bound enzyme and discharged only a small quantity in the liquid medium during the period of growth. Aspergillus oryzae W001 mycelium pellets were immobilized by entrapping-cross-linking method with gelatin and formaldehyde. Based on the orthogonal design table L16(45), a four-layer artificial neural network (ANN) model with back-propagation of error (BP) was used to optimize the immobilization conditions. The optimal condition was obtained: 1g wet pellets were added into 8ml immobilization solution, containing 0.5% gelatin and 0.5% formaldehyde, and the immobilization time was 1.5h. The immobilized enzyme activity was 1500U·g-1 and the activity yield was more than 80%. Compared to free pellet, the optimum pH of reaction system changed from 7.0 to 8.0, temperature changed from 52℃ to 63℃. The operational half-life was 77d. In addition, Co2+ was proved to be a good activator for the immobilized pellet in the concentration of 1×10-3 mol·L-1. To determine the catalysis properties of the enzyme reaction, experiments were carried out. The result showed that there were substrate inhibition, competitive inhibition of L-phenylglycine and noncompetitive inhibition of acetic acid. For the immobilized pellet, the Michaelis-Menten constant and the inhibition constants of the substrate, L-phenylglycine and acetic acid at 37℃ were =19.8 mmol·L-1, =225.5 mmol·L-1, =20.23mmol·L-1 and =38.7mmol·L-1, respectively.Based on the theoretical analysis and experimental data, the kinetics and reaction mechanism of aminoacylase were analyzed in detail. In terms of the reaction mechanism of Ordered Uni-Bi, the Cleland Equation was deduced, and hence the Warburton Equation was obtained, which was widely used to describe the hydrolysis of penicillin with penicillin amidase. But the predictions by the two equations deviated from experiment data at high concentration of substrate. Taking into account substrate inhibition and product inhibition, a novel equation was established, which was in good agreement with the experiment data. And the essential relationship of the novel equation and other equations was discussed. Immobilization of aminoacylase was carried out by the method of ion combination. The novel carrier of DEAE-E/H was a kind of weakly basic macro porous anion exchanger, which was the first time to be used as the carrier of enzyme. The immobilization was carried out at room temperature, pH6.5 and the enzyme concentration 120U.ml-1. The preparation immobilized on DEAE-E/H showed satisfactory activity. The specific activity was up to 1200U·g-1-1500 U·g-1 and the activity yield was more than 60%, which had the same level as...
|
PDF Full Text Request