青霉素酰化酶固定化技术和新型酶反应器的研究
文献类型：学位论文
| 作者 | 刘建国 |
| 学位类别 | 博士 |
| 答辩日期 | 2000-09 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 欧阳藩 |
| 关键词 | 青霉素 青霉素酰化酶 固定化 酶反应器 |
| 其他题名 | Immobilization of penicillin acylase and a novel enzyme bioreactor |
| 学位专业 | 生化工程 |
| 中文摘要 | 该文是根据国内固定技术的研究和应用仍然较为落后，青霉素工业生产中仍使用进口固定化酶的现状，提出研究课题的.具有研究内容如下：（1）青霉素酰化酶固定化载体.（2）固定化青霉素酰化酶.（3）固定化青霉素酰化酶连续反应过程.（4）外场作用下的固定化青霉素酰化酶酶反应器. |
| 英文摘要 | With the development of immobilizing technologies in enzymatic engineering, the enzymes produced by microorganisms, plants and animals play more and more important roles in industrial production, such as the semi-synthesis of penicillin. In which, the immobilized penicillin acylases are applied to produce a kind of very important precursor (6-APA) for the serai-synthesis of penicillin. At present, the immobilizing technologies of penicillin acylase are behind the western country in China. So it is urgent to accelerate these researches, which is the aim of these paper. In this article, the results were divided into four parts, and listed as follows respectively. 1. The study of the synthesis of carriers for immobilized penicillin acylase Four polymerization systems were studied in the paper, such as HEM A, Acrylamine, Vinyl acetate and spherical insoluble PVA cross-linked with p-Phthaldehyde. The synthesis of carriers was mainly based on the method of suspension polymerization, also combined with other methods, such as ontic polymerization etc. The carriers' inner surface, swelling degree in different solvents and porosity were determined. PVAs cross-linked with p-Phthaldehyde were used as materials to investigate the relationships between the hydrophilia/hydrophobicity of carriers and the amount of enzymes coupled on the carriers and the stability of immobilized BSA. The treating conditions, such as the content of benzealdehyde (which can react with hydroxyl radicals), treating time etc were considered. The results showed that the hydroxyl radicals content on the surface of the insoluble PVAs were gradually decreased with increasing of benzealdehyde concentration and its treating time, i.e., the percent of hydroxyl radicals was 45%, 25%, 10%,0% after the insoluble PVAs were treated at 50°C with 0.05N benzealdehyde for 15, 30, 60, 90min respectively. The insoluble PVAs contained different hydroxyl radicals were used to immobilize BSA. When the percent of hydroxyl radicals on the surface of insoluble PVAs was 40% or so, the amount of immobilized BSA reached the highest, and the immobilized BSA exhibited the highest stability. The higher hydrophobility, the more stability of immobilized BSA as the amount of hydroxyl radicals was at the range of 50-89%. So, it was concluded that the properties of immobilized BSA were related to the surface properties of carriers. 2. The immobilization of penicillin acylase The activation agents used were comprised of Epoxy chloride, p-Penzoquinone, p-Toluenesulfonyl Chloride, 4-(A-sulfatoethylsulfony I)-aniline ( also called SESA in short), 1,4-butanediol diglycidyl ether etc. SESA was proved to be an excellent activator. Above all, a kind of carrier was found exhibiting higher activity and stability. Its activity was up to 435units/g (wet). The loss of activity was only 10% after repeated using for ten batches, and the recovered activity of penicillinacyalse was 32.8%. The type of catalysis mechanism of immobilized penicillin acylase belonged to Uni-Bi type and three mechanism equations were obtained according to the hypothesis of Michaelis-Menten and Briggs-Haldane. Deduced from the King-Altaian method, the equation proved to agree with the practical hydrolysis process of PGK The equation could be expressed as follows: where v was the reaction rate, Vf the forward reaction rate , Vmax the maximum reaction rate; Ks was the dissolving constand, Kiq> Kip was the inhibition constant of PAA and 6-APA respectively, [S], [Q] and [P] was the equilibrium concentration of PenG, PAA and 6-APA respectively. The study of dynamics of immobilized penicillin acylase showed that the immobilized penicillin acylase exhibited less sensitive to the change of environmental pH and temperature. Its optimum of pH and temperature were 8.5 and 37 °C , and Km> KjSi Kjp and Kjq obtained equaled to 40.22mmol/L,342.23mmol/L,44.82mmol/L and 82.94mmol/L respectively. The substrate inhibition was the strongest comparised with the inhibition from PAA and 6-APA; On the contrary, 6-APA has least inhibition of the activity of immobilized pencillin acylase. 3. The continuous reaction engineering of immobilized penicillin acylase It was found that the hydrolysis of different initial concentration exhibited the similar hydrolysis rate when the amount of immobilized enzyme per mol of substrate was equal. And some curves were even overlapped. So it is feasible to be adopted higher initial concentration of substrate if the amount of immobilized enzyme per mol of substrate is higher. The results similar to above were also found in the study of CSTR of immobilized penicillin acylase. According to the results, higher inlet concentration of substrate, higher amount of immobilized enzyme and flux were also feasible. 4. The study of ultrasonic bioreactor of immobilized penicillin acylase It was found that the activity of the immobilized penicillin acylase could be increased up to 1.5-1.75 times control after the enzymes in phosphate buffer (pH7.5) without the presence of the PenG were treated with 0.25-1.2W/ml of ultrasound for 3min. When the same intensity of ultrasound was applied in the hydrolysis of PenG, the hydrolysis rate of PenG could be increased 5.6 times. The studies of mechanisms of ultrasound on immobilized penicillin acylase showed a certain intensity of ultrasound could increase the diffusion rate of substrates and products. And it also could alter the configuration of enzymes coupled on the surface of carriers, therefore, increase the immobilized penicillin acylase tolerance of higher pH value and environmental temperature. In the stirring batch enzyme reactor, the ultrasound should be adopted discontinuous operational model. The operational conditions was as follows: (1) the intensity of 20KHz ultrasound was 0.4~0.6W/ml; (2) the duration of each cycle was 0.5~lmin; (3) the intervals of two sequent cycles was 3min.In the loop reactor, the ultrasound should be adopted continuous operational model. The intensity of ultrasound could be adopted 48W in a 5.5L of loop reactor. The magnetic field had no effect on the activity of free pencillin acylase, and could increase 1.33-1.7 times the activity of immobilized penicillin acylase at the magnetic intensity 91.4Oe~273.9Oe; When the magnetic intensity was over 365.3Oe, the hydrolysis rate was inhibited to some extent. In addition, it was not apt to be applied in enzyme reactors because the apparatus was complex and could produce greater thermal effect. |
| 语种 | 中文 |
| 公开日期 | 2013-09-26 |
| 页码 | 182 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1889]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 刘建国. 青霉素酰化酶固定化技术和新型酶反应器的研究[D]. 中国科学院研究生院. 2000. |
入库方式： OAI收割
来源：过程工程研究所
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