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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，我们的目的是构建一个有效的生物反应器，用于在有机溶剂中合成作为模型寡肽的甜菜碱前体，使用固定到Amberlite XAD-7上的嗜热菌蛋白酶。首先，我们研究了影响使用固定化酶合成的前体的因素。研究了在乙酸乙酯和叔戊醇混合溶剂中的合成速率。在33%叔戊醇和67%乙酸乙酯的混合溶剂中，固定化嗜热菌蛋白酶的活性和稳定性都很高，可以进行连续反应。随着叔戊醇含量的增加，固定化酶的稳定性增强。本文从固定化嗜热菌蛋白酶的稳定因素--钙离子的稳定性角度，探讨了固定化嗜热菌蛋白酶在叔戊醇存在下具有较高活性和稳定性的原因。合成阿帕坦的最佳含水量 ...更多信息发现前体在混合有机溶剂中为4- 6%。优化了苯丙氨酸甲酯（PheOMe）和N-苄氧羰基-L-天冬氨酸（Z-Asp）的浓度。PheOMe的浓度设定为200 mM，Z-Asp的浓度设定为40 mM。我们也可以在40 ℃下在柱反应器中进行连续反应。在反应器出口处，Z-Asp的转化率为约99%，空速为4.9（l/h）。基于此结果，我们的目的是开发一个生物反应器系统结合模拟移动床吸收器。也就是说，在生物反应器的出口处，Z-Asp浓度低到可忽略不计，其中阿帕坦前体Z-Asp PheOMe为约40 mM，PheOMe为160 mM。因此，我们可以使用模拟移动床吸附器将PheOMe再循环到生物反应器中，连续分离PheOMe和Z-AspPheOMe。考察了Dowex MWA-1对PheOMe和Z-AspPheOMe的吸附/脱附行为。我们还计算了模拟移动床吸附器的分离性能。我们还展示了一种可能的生物反应器系统，可用于工业生产。最后，我们简要地比较了酶法合成和化学合成。少