聚對苯二甲酸乙二醇酯(PET)在廢棄之塑膠所佔比例與日愈增，造成嚴重的環境問題，如何回收PET 的研究課題也日益迫切。最近，Ideonella sakaiensis PETase 被發現有顯著的降解 PET的能力。而將聚乙二醇(PEG)共價連接於生物活性分子上，已被證實用能延長生物分子藥物在體內循環時間，提高穩定性和降低免疫抗原性。本論文研究以聚乙二醇來修飾PETase 之N端氨基和賴氨酸側鏈，期望聚乙二醇修飾後之PETase能提升其對PET水解能力。首先來自 Ideonella sakaiensis 的PETase 在大腸桿菌中被表達，經金屬親和層析純化為分子量30 kDa 的可溶蛋白質，而末端含有醛基的mPEG-CHO則是以TEMPO 催化 mPEG氧化而得，利用Schiff鹼反應則可將mPEG-CHO接於PETase上，聚乙二醇修飾後之PETase仍保有PETase催化活性，也提升PET水解後MHET和TPA的產量。聚乙二醇修飾也提升了PETase的熱穩定性，可提高溫度至50℃，最適水解 pH也可提升至pH 9。此外，本論文也研究添加 mPEG 對 PET 表面的潤濕性及PETase對 PET 水解的效應，以了解聚乙二醇化 PETase 提升 PET 水解的可能機制。

Polyethylene terephthalate (PET) is a recyclable thermoplastic polyester. It is one of major plastic waste in urban area. Its recycling is in critical need. Ideonella sakaiensis PETase recently demonstrated the notable ability to degrade PET. PEGylation, covalent attachment of polyethylene glycol to bioactive molecules is one of the leading approaches to prolong the pharmacokinetics, improve the stability and to reduce the immunogenicity of therapeutic proteins. In this work, site specific PEGylation of PETase was demonstrated. N-terminal and lysine side chain amine reduction reaction was employed to perform PEGylation of PETase. TEMPO-catalyzed oxidation of mPEG was prepared to obtain mPEG-CHO as activated PEG. Recombinant PETase from Ideonella sakaiensis was successfully expressed in E.coli and purified as a soluble protein with molecular weight ± 30 kDa by immobilized metal affinity chromatography (IMAC). PETase was PEGylated successfully via Schiff base reaction with 1:10 weight ratio of PETase to mPEG-CHO. PEGylation increased hydrolysis products MHET and TPA production from PET powder (64.8% CrI) in a mild reaction condition (30℃, pH 8) as compared with native PETase. Furthermore, PEGylation enhanced PETase thermal stability that PET hydrolysis could be carried out under elevated temperature of 50℃. Stabilizing effect on optimum reaction pH was also achieved by PEGylation that pH 9 could be used for PET hydrolysis. Moreover, wettability on PET surface and crowding effect on PET hydrolysis due to the addition of mPEG were also studied to understand the possible mechanism between of PEGylated PETase and PET substrate.

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