Rhodosporidium toruloides D型胺基酸氧化酶和幾丁質結合域融合蛋白的生化性質

Biochemical properties of Rhodosporidium toruloides D-amino acid oxidase-chitin binding domain fusion protein

林陽森(Yang-Sen Lin)

指導教授 ： 官宜靜

大同大學/工程學院/生物工程學系所/碩士(2012年)

摘要

D型胺基酸氧化酶 (D-Amino acid oxidase；DAO) 工業上被應用於製造頭孢菌素類抗生素的前驅物7-amino cephalosporanic acid (7-ACA)。但DAO易因溫度、pH值改變和副產物H2O2氧化傷害，造成蛋白質結構改變、或FAD (flavin adenine dinucleotide)輔酶的脫落，而甚至失去活性。
本研究將Bacillus circulans WL-12幾丁質酶之幾丁質鍵結區域chitin-binding domain (CBD) 的對應基因片段和Rhodosporidium toruloides DAO (RtDAO) cDAO基因融合，並置於大腸桿菌表現生產具有C端CBD的RtDAO，以固定於幾丁質顆粒增加其穩定性。此RtDAO-CBD融合酵素因具有N端His tag，故可以金屬螯合層析純化後進行生化性質檢測。最適反應溫度和TM分別為35和45oC，最適反應pH則為8.0，最佳pH穩定度於pH 7.0，皆與RtDAO相似。當與10 mM H2O2反應時，RtDAO和RtDAO-CBD的半衰期分別為105和75分鐘，當以0.01 mg/ml濃度儲存於4 oC下時，兩者的半衰期分別為7、6天；經過15次重複結凍，RtDAO和RtDAO-CBD分別保有20和50%的殘餘活性。

關鍵字

幾丁質鍵結區域 ； D型胺基酸氧化酶 ； 幾丁質酶

並列摘要

D-Amino acid oxidase (DAO) has been applied industrially to produce the precusor for cephalosporin antibiotic, 7-amino cephalosporanic acid. However, DAO is vulnerable to changes in temperature, pH as well as oxidative damages by hydrogen peroxide, thus resulting in alteration of the protein structure, the loss of coenzyme FAD (flavin adenine dinucleotide), and even inactvation.
In this study, the corresponding gene fragment for chitinase from Bacillus circulans WL-12 chitin-binding domain (CBD) was fused to Rhodosporidium toruloides DAO (RtDAO) cDAO gene, and was then expressed in E coli to generate the RtDAO fusion protein with C-terminal CBD for immobilization on chitin beads to obtain better stability. Because of having N-terminal His tag, RtDAO-CBD fusion protein was purified by metal chelation chromatography for the analysis of biochemical properties. The optimal temperature and the Tm value were 35 and 45oC, respectively, while the optimal pH was at 8.0 and the best pH stability appeared at pH 7.0. These properties were all similar to those of RtDAO. When reaction with 10 mM hydrogen peroxide, the half-lives of RtDAO and RtDAO-CBD were 105 and 75 min, respectively. At the concentration of 0.01 mg/ml, their half-lives of storage at 4 oC were 7and 6days, respectively. After 15-cycles of freeze-and-thaw, the residual activities of RtDAO and RtDAO-CBD were 20 and 50%, respectively.

並列關鍵字

chitinase ； chitin-binding domain ； D-Amino acid oxidase

參考文獻

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