DEVELOPMENT OF BIOREACTOR SYSTEM FOR OLIGOPEPTIDE SYNTHESIS COMBINED WITH A SIMULATED MOVING BED ADSORBER

结合模拟移动床吸附器的寡肽合成生物反应器系统的开发

基本信息

批准号：
07555563
负责人：
NAKANISHI Kazuhiro
金额：
$ 1.09万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

In this study, we aimed to construct an effective bioreactor for synthesis of aspartame precursor, as a model oligopeptide in an organic solvent, using thermolysin immobilized onto Amberlite XAD-7. First, we investigated the factors affecting the synthesis of the asparatme precursor using an immobilized enzyme. We studied a synthetic rate, particularly in a mixed organic solvent with ethyl acetate and tert-amyl alcohol. Using a mixed solvent of 33% tert-amyl alcohol and 67% ethyl acetate, the activity and stability of the immobilized thermolysin were high enough to conduct a continuous reaction. With increasing the content of tert-amyl alcohol, the stability of the immobilized enzyme was enhanced. The reason for the high activity and stability of the immobilized thermolysin in the presence of tert-amyl alcohol was studied from the viepoint of stability of calcium bound on the enzyme molecule, a stabilizing factor for thermolysin. An optimum water content for the synthesis of asparatame … More precursor was found to be 4-6 % in the mixed organic solvent. The concentration of L-phenyllalanine methyl ester (PheOMe) and N- (benzy loxy carbonyl) -L-as partic acid (Z-Asp) was optimized. The concentration of PheOMe was set to 200 mM and that of Z-Asp was 40 mM.We could also conduct a continuous reaction in a column reactor at 40ﾟC.The conversion at the outlet of the reactor with respect of Z-Asp was around 99% with a space velocity of 4.9 (1/h). Based on this result, we aimed to develop a bioreactor system combined with a simulated moving bed absorber. Namely, at the outlet of the bioreactor, the Z-Asp concentration was negligible low with asparatame precursor Z-Asp PheOMe of around 40 mM and PheOMe of 160 mM.Thus, we could recycle PheOMe to the bioreactor with continuous separation of PheOMe and Z-AspPheOMe using a simulated mooving bed adsorber. We found Dowex MWA-1 as a suitable adsorbent and analyzed adsorption/desorption behaviors of PheOMe and Z-AspPheOMe. We also calculated a separation performance for a simulated moving bed adosorber. We also showed a possible bioreactor system which could be used for an industrial production. Finally, we briefly compared an enzymatic synthesis and chemical synthesis. Less

在这项研究中，我们旨在使用固定在琥珀酸盐XAD-7上的热溶素构建有效的Aspartame前体合成的生物反应器，作为有机溶液中的模型寡肽。首先，我们研究了使用固定的酶影响芦被前体合成的因素。我们研究了合成速率，尤其是在乙酸乙酯和丁醇的混合有机溶液中。使用33％Tert-氨基醇和67％乙酸乙酯的混合溶液，固定的热素蛋白的活性和稳定性足够高以进行连续反应。随着Tert-氨基醇的含量增加，固定酶的稳定性得到了增强。从钙分子上结合的钙结合的稳定性的Viepoint中研究了固定热蛋白质的高活性和稳定性的原因，这是热溶素的稳定因子。在混合有机溶液中发现了一种优化的水含量来合成asparatame…更多的前体含量为4-6％。优化了L-苯基丙氨酸甲酯（Pheome）和N-（Benzy loxy carbonyl）-L-AS颗粒酸（Z-ASP）的浓度。植体的浓度设置为200 mm，Z-ASP的浓度为40 mm。我们还可以在40°C的柱反应器中连续反应。Z-ASP的出口处的转化率约为99％，空间速度为4.9（1/h）。基于此结果，我们旨在开发一种生物反应器系统，并结合模拟的移动床吸收器。也就是说，在生物反应器的出口上，Z-Asp的浓度可忽略不计，而天源前体前体Z-Asp Pheome约为40毫米约40毫米，而160 mm的腓骨则可以通过使用模拟的Moiver的Moiver dedssorbercore的床位恢复到生物反应器中，并连续地将其恢复到生物反应器中。我们发现Dowex MWA-1是合适的吸附剂，并分析了Pheome和Z-Asppheome的吸附/解吸行为。我们还计算了模拟移动床吸附者的分离性能。我们还展示了可能用于工业生产的生物反应器系统。最后，我们简要比较了一种酶促合成和化学合成。较少的