利用重組嗜甲醇酵母菌 Pichia pastoris 生產納豆菌漆酶

Production of Bacillus subtilis natto laccase using recombinant Pichia pastoris

施韋伶(Wei-Ling Shih)

指導教授 ： 李昆達

國立臺灣大學/生命科學院/生化科技學系/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10448

摘要

漆酶屬於含銅的氧化酵素，能氧化多種芳香族化合物，以氧分子為電子的接受者，將氧分子還原成水，在工業上的應用相當廣。本實驗室先前將 Bacillus subtilis natto NTU18 的漆酶基因轉殖到 Pichia pastoris X33 持續表現型載體 pGAPZαA，成功的進行異源表現。本研究進一步使用誘導型 AOX1 啟動子之 pPICZαA 載體，以 P. pastoris 進行納豆菌漆酶之生產。在 Hinton 氏搖瓶中比較兩種啟動子的表現量，發現使用 AOX1 啟動子的表現量是 GAP 啟起動子的 1.95 倍。接著將　pPICZαA-laccase 電轉形進入 P. pastoris 的宿主菌株，分別為蛋白酶缺陷株　SMD1168H 和甲醇利用率較慢的菌株 KM71H，並和原生型菌株 X33 比較產量。結果可知，以 KM71H 轉形株表現漆酶可得最高活性 0.51 U/mL，為 X33 轉形株的 1.34 倍、 SMD1168H 轉形株的 2.21 倍。將此最高表現量的 KM71H 轉形株於 7 公升醱酵槽中進行細胞高密度培養，其細胞乾重可達 80.1 g/L，酵素活性可達 3.84 U/mL。重組酵素在含 10% 的有機溶劑 (乙醇、甲醇、DMSO 或丙酮)，仍保有 80-95% 的活性。將此酵素進行脫色試驗，發現其在 85℃ 對染劑具有優良的脫色能力，同時再藉由加入 8 mM 過氧化氫能再提升漆酶對染劑的脫色效果。

關鍵字

漆酶Pichia pastoris ； 納豆菌

並列摘要

Laccases are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. These enzymes have a great potential within a variety of industrial applications. In our previous study, the laccase gene isolated from the Bacillus subtilis natto NTU18 has been successfully expressed in the methylotrophic yeast Pichia pastoris with the constitutive GAP promoter. In this study, laccase gene were subcloned to pPICZalphaA vectors and expressed in P. pastoris under the control of the AOX1 promoter. The activity of laccase using the AOX1 promotor were 1.95 fold higher than that using the GAP promoter in Hinton’s flasks. Then, we compare the laccase productivity among three phenotype of host strains, wild type strain (X33), protease-deficient strain (SMD1168H) and methanol slow utilizing strains (KM71H) in Hinton’s flasks. The results show that using KM71H strains produced the hightest laccase activity 0.51 U/mL, which was 1.34 and 2.21 fold higher than using X33 and SMD1168H strains, respectively. By high cell density culture in the Bioflo110 fermentor with the strains KM71H, the dry biomass was 104.3 g/L, and the laccase activity reached 3.84 U/mL. The recombinant laccase retained 80-95% activity in 10% concentration of ethanol, methanol, DMSO and acetone. The recombinant laccase has been tested for its ability to decolourize synthetic dye. The results obtained that the optimal temperature for decolourization was 85℃, and adding 8mM H2O2 can increase its ability of decolourization.

並列關鍵字

Laccase ； Pichia pastoris ； Bacillus subtilis natto

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