乳酸菌麩胺酸脫羧酶之基因選殖、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)，由乳酸菌Lactobacillus plantarum BCRC12251萃取其染色體DNA做為模板，利用PCR技術選殖出gad基因，利用大腸桿菌來表達，並以HisTrap FF管柱純化且利用比色法進行酵素活性分析。gad基因之DNA全長為1410 bp，預測蛋白質分子量約為59.7 kDa，將其核苷酸與Lactobacillus plantarum subsp. plantarum ST-III進行比對，其相同性為99.8%。gad基因選殖嵌入pET28a表現載體以E.coli BL21(DE3)為宿主進行表達，在20℃下誘導16小時後，有大量水溶性蛋白的產生，經由親和性管柱純化後，利用此純化蛋白製備多株抗體，透過西方墨點法成功辨識重組GAD蛋白及乳酸菌天然GAD蛋白。酵素在pH 3.2、60℃時有最佳活性，添加Ca2+、Mg2+、Mn2+有助於活性提升，Km值和Vmax值分別為18.13和0.035 mM/min，所測得比活性為8.33 U/mg。在乳酸菌培養液添加Sodium Glutamate、Mg2+、Mn2+培養乳酸菌所萃取出的酵素液，測得的活性會比較高。此外，重組GAD酵素及天然GAD酵素皆可在細胞外表現。

關鍵字

乳酸菌；麩胺酸脫羧酶γ-胺基丁酸

並列摘要

The glutamate decarboxylase (GAD), which catalyzed α-decarboxylation of glutamate to produce γ-aminobutyric acid (GABA). The gad gene was 1410 bp from Lactobacillus plantarum BCRC12251, were cloned and sequenced, while the calculated molecular weight of the GAD protein was about 59.7 kDa. The gad nucleotide identities of Lactobacillus plantarum BCRC12251 with Lactobacillus plantarum subsp. plantarum ST-III was 99.8 %, while amino acid identities was 99.6 %. The proteins were collected for purification by HisTrap FF column, which were expressed in Escherichia coli. The recombinant GAD and crude GAD were identified by the western blot with antibody. The best activity of purified of recombinant GAD was at pH 3.2, 60℃; Ca2+, Mg2+ and Mn2+ can improve activity. Km was 18.13 mM/min and Vmax was 0.035 mM/min. The activity of crude native GAD was improved after the addition of monosodium glutamate, Mg2+ and Mn2+. Besides, both recombinant GAD enzyme and crude GAD enzyme showed activities in cultural medium.

並列關鍵字

Lactic acid bacteria ； glutamate decarboxylase (GAD) ； γ-aminobutyric acid (GABA)

參考文獻

1. Ben, O. N., Ampe, F., Raimbault, M., Guyot, J. P. & Tailliez, P. (2000) Molecular diversity of lactic acid bacteria from cassava sour starch (Colombia), Syst Appl Microbiol. 23, 285–291.

2. Karahan, A. G., Kilic, G. B., Kart, A., Aloglu, H. S., Oner, Z., Aydemir, S., Erkus, O. & Harsa, S. (2010) Genotypic identification of some lactic acid bacteria by amplified fragment length polymorphism analysis and investigation of their potential usage as starter culture combinations in Beyaz cheese manufacture, J Dairy Sci. 93, 1–11.

3. Lucke, F.K. (2000) Utilization of microbes to process and preserve meat, Meat Sci. 56, 105–115.

4. Hwanhlem, N., Watthanasakphuban, N., Riebroy, S., Benjakul, S., H-Kittikun, A. & Maneerat, S. (2010) Probiotic lactic acid bacteria from Kung-Som: isolation, screening, inhibition of pathogenic bacteria, Intel J Food Sci Technol. 45, 594–601.

5. Komatsuzaki, N., Shima, J., Kawamoto, S., Momose, H. & Kimura, T. (2005) Production of gamma-aminobutyric acid (GABA) by Lactobacillus paracasei isolated from traditional fermented foods, Food Microbiol. 22, 497–504.

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乳酸菌麩胺酸脫羧酶之基因選殖、表現與特性分析

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