臺灣博碩士論文加值系統

玻尿酸（Hyaluronic acid）為一黏性多醣體，可利用微生物醱酵獲得，在醫藥美容方面應用範圍廣而具有高經濟價值，但其純化過程繁複造成生產成本增加，因此本研究主要目的為建立一個快速回收玻尿酸的系統以降低玻尿酸生產成本。
首先利用修飾完成，具正電性及疏水端的磁微粒吸附回收玻尿酸，探討在不同吸附條件下之最大吸附量。不同鹽濃度下之吸附情形說明親和配體（Ligand）與玻尿酸間可利用靜電作用吸附；不同溫度下之恆溫吸附結果證明磁微粒與玻尿酸間存在疏水作用力。在最佳吸附條件下，修飾過之磁微粒每克可回收7.72 mg的玻尿酸；應用在純化醱酵液中之玻尿酸，由於雜質非專一性吸附以及醱酵液中之鹽濃度而降低磁微粒吸附玻尿酸能力，每克磁微粒回收1.21 mg之玻尿酸。
根據抗菌實驗測試結果，選用的親和配體具有抑菌效果，能有效抑制S. zooepidemicus菌株生長情形。

Hyaluronic acid ( HA ) can be produced through microbial fermentation by using Group A and C streptococci. Traditionally, it is recovered and purified from the fermentation broth by adding Cetylpyridinium chloride (CPC) to form complex with HA. The complex forming effect between CPC and HA involve the hydrophobic and attractive electrostatic interaction. However, the HA recovered by the CPC complexing method may be contaminated with the fermentation media that lowers its purity. Besides, centrifugation or filtration has to be employed to recover the precipitated complex. Silica coated magnetite can be easily modified with a chemical 3-trimethoxy silyl propyl dimethyl octadecyl ammonium choloride (AGIS) which has very similar structure as CPC to become M-AGIS and employed to purify HA from fermentation broth.. By applying magnetic field of enough strength, M-AGIS can be totally recovered. The adsorption isotherm of HA to M-AGIS at pH 4, 37 0C can be described by Langmuir adsorption model with maximum adsorption capacity Qmax of 7.72 mg/g and dissociation constant Kd of 0.0165. Higher temperature favors the adsorption. HA can be eluted from the adsorbent M-AGIS with about 80 % yield by employing 1 M NaCl. By employing M-AGIS, the recovery and purification of HA directly from HA producing cells suspension has been demonstrated.

目錄
中文摘要……………………………………………………………………………Ⅰ
英文摘要……………………………………………………………………………Ⅱ
誌謝…………………………………………………………………………………Ⅲ
目錄…………………………………………………………………………………Ⅳ
圖目錄………………………………………………………………………………Ⅵ
表目錄………………………………………………………………………………Ⅷ


第一章 緒論………………………………………………………………………1

1.1 研究背景…………………………………………………………………1
1.2 研究內容簡介……………………………………………………………2

第二章 文獻回顧…………………………………………………………………4

2.1 玻尿酸簡介…………………………………………………………………4
2.2 玻尿酸之純化……………………………………………………………9
2.2.1載體修飾純化玻尿酸………………………………………………10
2.3 HA binding protein………………………………………………………13
2.4 AGIS之性質與應用………………………………………………………17

第三章 實驗內容……………………………………………………………………20

3.1實驗流程……………………………………………………………………20
3.2實驗材料……………………………………………………………………20
3.2.1菌株…………………………………………………………………20
3.3實驗藥品……………………………………………………………………20
3.3.1修飾疏水性纖維素………………………………………………21
3.3.2磁微粒修飾…………………………………………………………21
3.4培養基………………………………………………………………………22
3.5 實驗設備……………………………………………………………………22
3.6實驗方法……………………………………………………………………24
3.6.1牛血清蛋白吸附玻尿酸……………………………………………24
3.6.2葡萄糖醛酸（Glucoronic acid）濃度測定……………………………27
3.6.3利用疏水性纖維素純化玻尿酸……………………………………28
3.6.4含正電並具疏水性超磁微粒之製備………………………………30
3.6.5利用修飾磁微粒純化玻尿酸………………………………………31
3.6.6玻尿酸之脫附液……………………………………………………34
3.6.7 磁微粒抗菌性測試…………………………………………………35

第四章 結果與討論………………………………………………………………37

4.1 利用BSA吸附玻尿酸……………………………………………………37
4.2 利用疏水性纖維素純化玻尿酸…………………………………………41
4.3 利用疏水性超磁微粒吸附玻尿酸………………………………………44
4.4 M-AGIS抗菌性之測試……………………………………………………57

第五章 結論………………………………………………………………………61

參考文獻……………………………………………………………………………62

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