Recovery of Isoamylase Using an Aqueous Two-Phase System

本研究旨在探討水二相系統(aqueous two-phase system)於發酵液中萃取澱粉枝切酵素－異澱粉之可行性，希望在不影響酵素活性的環境中，提供一種有效的回收方法。試驗中，利用聚乙二醇(PEG)及監類離子所形成之水二相系統，藉由上、下層溶液極性的差異，使得異澱粉在二相溶液分佈會有所區別。聚乙二醇與不同監類離子溶液之試驗，其中以硫酸胺最適合含異澱粉的粗酵素液分層萃取使用，而且在部分異澱粉會集中於親水性較高之上層液體。當系統重量組成爲6%PEG1500及15%(NH4)2SO4時，酵素在上、下層間之分配係數(partition coefficient)可達40，濃縮之酵素活性為粗酵素液之5倍；隨著聚乙二醇濃度的增加，使得上、下層之體積比增加，降低酵素濃縮效果，然而相對應上層溶液所得之回收率可在80%以上。一般而言，隨著聚乙二醇分子量或濃度之增加，異澱粉在二相中的分佈，會由上層轉移至下層溶液，使得酵素活性之分配係數有下降之趨勢，此現象應是分子間疏水作用力與溶液之自由體積改變的結果。因此，實際操作時選擇較低分子量或低聚乙二醇濃度與硫酸胺所形成之水二相系統，有利於提高異澱粉的濃縮效果。

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並列摘要

This study attempted to recover starch debranching enzyme, isoamylase, from the fermentation broths by using an aqueous two-phase system. A series of poly (ethylene glycol) (PEG) combined with different salt systems were used to prepare the partition systems. In these experiments, the partition coefficient of isoamylase for the PEG1500(6%)/ (NH4)2SO4 (15%) system could be up to 40 when a crude enzyme solution with an isoamylase activity of 3386U/mL was used, and the enzyme activity was 5.0-fold concentrated in the top phase as compared to the crude enzyme solution. Based on the recovery and concentration fold of isoamylase, a pH around 4~5 was found to be the optimal condition for this system. The polymer design and combination of salts could be manipulated to achieve different volume ratios between the top and the bottom phases, and could also achieve the purpose of enzyme concentration. For the PEG1500(6%)/(NH4)2SO4 (15%) system, experimental results indicated a 6.0-fold volume reduction in the top phase as compared to that of the crude enzyme solution, and the isoamylase activity retention in the top phase was higher than 80%. With PEG systems of various molecular weights and concentrations, the partition coefficients of isoamylase tended to decrease with the increases of PEG molecular weight and concentration. These behaviors could be interpreted by the influence of hydrophobic interaction as well as free volume change.

並列關鍵字

Aqueous two-phase system ； Debranching enzyme ； Isoamylase ； Partition coefficient ； Extraction ； Volume ratio

國際替代計量

Recovery of Isoamylase Using an Aqueous Two-Phase System

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