米麴菌之麩胺酸脫羧酶gad8基因選殖、表現與特性分析

麩胺酸脫羧酶(Glutamate decarboxylase ;GAD ;EC 4.1.1.15) 可將麩胺酸 (Glutamate)轉化為γ-胺基丁酸 (γ-aminobutyric acid ;GABA)，從米麴菌選殖出gad8和gad8(T’)基因，利用大腸桿菌及畢赤酵母菌來表達，並以HisTrap FF管柱純化且利用比色法進行酵素活性分析。gad8和gad8(T’)之cDNA全長分別為1545 bp和1650 bp，其蛋白質分子量約為58.7 kDa與62.5 kDa，將其核苷酸與A.oryzae RIB40比對，其相同性為99.7 %和93.4 %，而胺基酸比對結果相同性為100%和93.3%。gad8和gad8(T’)基因選殖嵌入pET28a表現載體以E.coli BL21(DE3)為宿主進行表達，在16 ℃下表達，有大量水溶性蛋白的產生，經由親和性管柱純化後，酵素在60 ℃時有最高的活性為47768和28353 U/mg。gad8基因選殖嵌入pPICZαB表現載體中，以Pichia pastoris x33為宿主進行表達，在其上清液有並發現GAD8蛋白。此外，利用重組GAD8所生產之多株抗體來測試gad8基因於米麴菌本身的轉譯情形，利用西方墨點法來辨識米麴菌蛋白合成。

關鍵字

米麴菌；麩胺酸脫羧酶γ-胺基丁酸；畢赤酵母菌

並列摘要

The glutamate decarboxylase (GAD), which catalyzed α-decarboxylation of glutamate to produce γ-aminobutyric acid (GABA). The GAD-encoding gad8 and gad8(T’) gene from Aspergillus oryzae BCRC32288 with introns is 1712 and 1817 bp, were cloned and sequenced. The A.oryzae BCRC32288 was obtained from Bioresource Collection and Research Center (BCRC), Food Industry Research & Development Institute, Hsinchu, Taiwan. The proteins were collected for purification by HisTrap FF column, which were expressed in Escherichia coli and Pichia pastoris. The GABA-forming activity of the GAD8 was determined by the colorimetric assay. The two genes gad and gad(T’) without intron consist of 1545 and 1650 bp from A.oryzae BCRC32288, while the calculated molecular weight of the GAD8 and GAD8(T’) proteins was about 58.66 kDa and 62.5 kDa, respectively. The gad8 and gad8(T’ ) nucleotide identities of A.oryzae BCRC32288 with those of A.oryzae RIB40 were 99.7 % and 93.4 %, while amino acid identities were 100% and 93.3%. When the gene gad8 and gad8(T’) was cloned into pET28a and expressed in E.coli BL21(DE3), the best activity which purification of GAD8 and GAD8(T’) was 47768 and 28353 U/mg. The gene gad8 was cloned into pPICZαB and expressed in Pichia pastoris x33 ,but that didn’t find GAD8 expresstion. In addition, the crude GAD was obtained from A.oryzae, and was identified by the western blot.

並列關鍵字

Aspergillus oryzae ； Pichia pastoris ； glutamate decarboxylase (GAD) ； γ-aminobutyric acid (GABA)

參考文獻

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米麴菌之麩胺酸脫羧酶gad8基因選殖、表現與特性分析

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