【作者】 王燕；

【导师】 张凤宝；

【作者基本信息】 天津大学 ， 化学工程， 2003， 博士

【摘要】 本文首次以氨基酰化酶为酶源拆分非天然氨基酸—N-乙酰-DL-苯甘氨酸。实验首先以固定化米曲霉菌体为酶源在间歇反应器中进行初步拆分，进而利用拆分效率相对较高的固定化酶在连续床中进行最后拆分。本文采用理论分析与实验研究相结合的方法，综合运用数学建模、过程模拟等多种手段，对氨基酰化酶拆分N-乙酰-DL-苯甘氨酸的酶解体系进行了较全面系统的研究，内容涉及菌体的培养、菌体和酶的固定化及其性质表征、酶分子的作用机理及酶促反应动力学、反应器的模型计算等方面，主要包括1通过对各种不同类型培养基的比较，筛选出了适宜的液体培养基—豆汁培养基，得到了菌体培养的适宜条件：30℃，pH值自然，转速130rpm，接种量4％，培养40h。该菌体用于拆分N-乙酰-DL-苯甘氨酸时菌体的比酶活可达1600-1800U·g-1。以甲醛为交联剂，惰性蛋白明胶为包埋剂和活性保护剂，对米曲霉菌体进行固定化。在正交实验的基础上利用人工神经网络得到了较优的固定化条件：菌体与固定液的用量比(固液比)为1:8，其中固定液中明胶浓度为0.5%，甲醛浓度为0.5%，固定化时间为1.5h。在此条件下制得的固定化菌体比酶活达到1500U·g-1，酶活保留率超过80%。固定化后米曲霉菌体热稳定性明显增强，在连续反应器中的操作半衰期可达77d。最佳反应条件pH值从7.0变为8.0、温度由52℃变为63℃，最适反应条件明显变宽。Co2+浓度为1×10-3mol·L-1时对固定化菌体的激活作用达到最强。2研究了固定化米曲霉菌体拆分N-乙酰-DL-苯甘氨酸的的催化特性。当底物浓度大于200mmol·L-1时，产生底物抑制。产物乙酸和L-苯甘氨酸（L-PG）分别产生非竞争性抑制和竞争性抑制。在37℃条件下，固定化菌体的米氏常数、底物抑制常数、产物乙酸的抑制常数和L-苯甘氨酸的抑制常数分别为19.8mmol·L-1、225.5mmol·L-1、38.7mmol·L-1和20.23mmol·L-1。3从酶反应动力学基本方程出发，考虑各种抑制物的作用特点，提出了新的分子作用机理，建立了新的动力学方程。固定化菌体在间歇反应器中的实验数据表明，当底物浓度较低时，该方程与Warburton方程和Cleland方程均能很好地与实验结果相吻合；随着底物浓度的增大和反应的进行，除新的动力学方程仍能很好地与实验结果吻合外，其它两个方程与实验结果的偏差越来越大，表明本文提出的分子作用机理更为合理。 4采用结构与DEAE-Sephadex极其相似的大孔弱碱性树脂DEAE-E/H作为氨基酰化酶的固定化载体。考察了影响固定化结果的因素，确定了适宜的固定化条<WP=4>件：自由酶液浓度为120U·ml-1，pH值6.5左右，温度为室温。在此条件下制得的固定化氨基酰化酶比酶活可达1200-1500U·g-1，酶活保留率超过60%，与文献报道的DEAE-Sephadex固定化效果相当，固定化后酶的稳定性大大提高。固定化酶的米氏常数、底物抑制常数、产物乙酸和苯甘氨酸的抑制常数和分别为5.668mmol·L-1、763mmol·L-1、400mmol·L-1和10.0mmol·L-1。5 对固定床反应器中固定化氨基酰化酶连续拆分N-乙酰-DL-苯甘氨酸的反应进行模拟，用推广的割线法求解模型，模型的计算值与实验结果吻合较好。最后所得产品D-苯甘氨酸的旋光度为= -157o （C=1，1mol·L-1 HCl），产品的光学纯度为99.4%。理论收率达到65.1%。更多还原

【Abstract】 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 =<WP=6>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更多还原