Variovorax paradoxus ISO1 D型胺基酸氧化酶基因大量表現之研究

Abstract

D型胺基酸氧化酶 (D-amino acid oxidase, EC1.4.3.3, 以下簡稱DAAO ) 具有高度之立體專一性，能夠催化D型胺基酸之脫氨反應，產生alpha-酮酸與過氧化氫。本研究由前人自Variovorax paradoxus ISO1中選殖出的ORF5基因，經由序列分析比對，發現ORF5全長為1251 bp，帶有416個胺基酸，分子量為 45 kDa，與Ralstonia eutropha之putative Glycine/D-amino acid oxidase有51% 的最高相似度。將ORF5此段基因利用不同的表現載體與宿主細胞表現系統，以得到最適之表現條件。在pET21a表現系統中，以大腸桿菌BL21(DE3)pLysS為表現宿主，培養於2X YT培養基，在25℃，0.5 mM IPTG，誘導6小時，以100 mM D-Alanine為基質，可得到DAAO酵素比活性 451 mU/mg ; 而以此為表現條件再添加2% DL-Glutamic acid 時，可提高將近兩倍的DAAO酵素比活性為880 mU/mg。在此條件下，大部分之pET21a-DAAO表現蛋白仍然存在於不可溶部份，只有少部份為可溶性蛋白質，故不利於DAAO之大量純化。在pET41a之表現系統，以大腸桿菌BL21(DE3)pLysS為表現宿主，採用與上述相同表現條件培養及誘導菌體，結果發現所表現之GST-DAAO 融合蛋白幾乎全為可溶性蛋白質，而其DAAO酵素比活性為 450 mU/mg。在蛋白質純化方面，GST-DAAO經由GSTrap column回收，其純化倍率大約為1.93倍，DAAO酵素比活性為790 mU/mg，再經過thrombin 或是enterokinase等酵素截切之後，其比活性可再提高兩倍。探討經由enterokinase及thrombin截切後之DAAO酵素的基質專一性，發現此DAAO對 Glycine有最高的比活性，其次是D-alanine，約為Glycine比活性的25%。推測此酵素有可能為甘胺酸氧化酶的一種。除此之外，此酵素尚對D-Aspartic acid以及D-Arginine有微量的比活性，對於其他受測試之D型或L型的胺基酸均不作用。

D-amino acid oxidase (DAAO, EC 1.4.3.3) stereospecifically catalyzes the oxidative reaction of the amino group of D-amino aicd to produce its related alpha-keto acid and the H2O2. This study is to characterize an open reading frame ORF5 sequence from Variovorax paradoxus ISO1 and overexpression its protein to do further study. The ORF5 sequence was analyzed by NCBI web, and found it has 1251 bp, 415 amino acids, 45 kDa molecular weight and a high identity with Glycine/DAAO of Ralstonia eutropha. We subclone the sequence into different expression system and investigate the optimal expression conditions. In pET21a system with host E. coli BL21(DE3)pLysS, the best expression condition is induced at 25℃ for 6hr with 0.5 mM IPTG, and the specific activity of pET21a-DAAO is 451 mU/mg. In the same condition with adding 2% DL-Glu to the medium, the specific activity of pET21a-DAAO raised to 880 mU/mg. Since most of the pET-DAAO are inclusion body fraction, we try to use another expression vector that was pET41a system. In pET41a expression system with host E. coli BL21(DE3)pLysS, induction as the same condition, the GST-DAAO fusion protein is almost totally in soluble form and rear in insoluble fraction, and the specific activity of GST-DAAO is 450 mU/mg. After running through the GSTrap column purification of GST-DAAO fusion protein, the purification fold is about 1.93. The activity of purify GST-DAAO cleavaged by thrombin and enterokinase is 2 fold to GST-DAAO fusion protein. The substrate specificity of DAAO has the highest specific activity to Glycine, and the second is D-Alanine, and D-Aspartic acid and D-Arginine have the fewer specific activity.