對Rhodosporidium toruloides D型胺基酸氧化酶過氧化氫耐受性的改善

IMPROVEMENT IN TOLERANCE TO HYDROGEN PEROXIDE OF RHODOSPORIDIUM TORULOIDES D-AMINO ACID OXIDASE

張永昆(Yung-Kun Chang)

指導教授 ： 官宜靜

大同大學/工程學院/生物工程學系所/碩士(2006年)

摘要

D-amino acid oxidase (DAO)為工業上應用於是製造頭孢菌素類抗生素之先驅物7-aminocephalosporanic acid的重要酵素。DAO在轉化受質後，會產生副產物H2O2，將其某些胺基酸殘基氧化而使之失活。由於 methionine的氧化常於蛋白質傷害中扮演重要角色，因此本研究藉SOEing (splicing by overlapped extension)方法進行點突變，將Rhodosporidium toruloides和Candida boidinii DAO上的methionine residues轉變成leucine residues，以提高其對H2O2之耐受性而延長作用能力。
以單點突變產生R.. toruloides DAO的突變蛋白M59L 、M88L和M213L以及C. boidinii DAO的突變蛋白 M151L和M221L。經檢測R.. toruloides DAO的三個突變蛋白的比活性依次為101.9、84.1和106.7 U/mg，其中M59L和M213L的比活性較wild type DAO的98.6 U/mg為高。此外，酵素動力學分析顯示R.. toruloides M59L以 D-alanine為受質時的kcat/Km值達39.9 s-1 mM-1，高於wild type DAO的kcat/Km值35.7 s-1 mM-1。另方面，C. boidinii突變蛋白卻皆未檢測出任何活性。
H2O2耐受性的分析顯示，R.. toruloides DAO的三個突變蛋白中，M59L和M213L對H2O2耐受性較wild type DAO略佳，M213L又略優於M59L。以100 mM H2O2處理20分鐘後，M59L、M213L和wild type DAO分別殘餘7.88%、12.36%和0.66%的活性。

關鍵字

過氧化氫 ； D型胺基酸氧化酶 ； 點突變

並列摘要

D-amino acid oxidase (DAO, EC1.4.3.3) is a key enzyme for the production of 7-aminocephalosporanic acid, the precursor of semi-synthetic cepham antibiotics. It was found to be inactivated by H2O2 generated from its own catalysis. Methionine oxidation usually plays an important role in protein damage. Therefore, using the SOEing (splicing by overlapped extension) method, the methionine residues in Rhodosporidium toruloides and Candida boidinii DAO were replaced by leucine residues to improve their resistance to H2O2.
Three R. toruloides mutant DAOs M59L, M88L, and M213L as well as two C. boidinii mutant DAOs were produced. The specific activities of R. toruloides mutant DAOs M59L, M88L, and M213L were 101.9, 84.1, and 106.7 U/mg, respectively. The former two were higher than that of wild type DAO (98.6 U/mg). The kinetic analysis also showed that M59L had a kcat/Km of 39.9 s-1 mM-1 which was better than that of wild type DAO (35.7 s-1 mM-1). On the other hand, C. boidinii mutant DAOs didn’t show any detectable activity.
The resistance to H2O2 of M59L and M213L were slightly better than that of wild type DAO. The treatment with 100 mM H2O2 for 20 minutes resulted in a decrease in the specific activities of M59L, M213L, and wild type DAO by 7.88, 12.36 and 0.66%, respectively.

並列關鍵字

D-amino acid oxidase ； SOEing ； H2O2

參考文獻

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