大豆血紅蛋白於大腸桿菌之異源表現

Abstract

大豆血紅蛋白為植物根瘤進行固氮作用的必要成份，是固氮植物與細菌共生關係的一環。近年來，植物性飲食廣受討論，越來越多公司積極開發植物性肉品，而大豆血紅蛋白是使用來作為增加植物肉食品中的風味和色澤的成分。本研究是使用全合成大豆血紅蛋白基因，轉殖至大腸桿菌異源表現，並測試重組大腸桿菌生產大豆血紅蛋白的能力，同時以Pyridine hemochromagen assay測定樣品中大豆血紅蛋白的含量，來探討透過Heme中心鐵離子和Heme前驅物的添加，是否能增加大豆血紅蛋白的產量。接著，以5L發酵槽配合pH stat細胞高密度培養，提高大豆血紅蛋白的產量。本研究成功建立生產大豆血紅蛋白的重組大腸桿菌，而實驗結果顯示，Hinton’s flask振盪培養，單純以IPTG誘導的產量為4.61±0.15 μM的Heme濃度；而FeCl_3的添加可以產生達6.05±0.12 μM的Heme濃度；5-ALA的添加則可以產生達7.68±0.78 μM的Heme濃度，兩者皆能有效提高大豆血紅蛋白的產量。而5L發酵槽配合pH stat細胞高密度培養，誘導後經5-ALA的添加，產量在48小時來到最高可達16.08 μM，相較於搖瓶的產量提高近20倍。本研究結果有利於後續以原核或真核系統發酵生產大豆血紅蛋白作為植物肉添加劑的研究開發。

Leghemoglobin is an essential ingredient for nitrogen fixation by root nodules and is a part of the symbiotic relationship between plants and the nitrogen fixing bacteria. In recent years, plant-based diets have become more and more widely discussed, and more and more companies are developing plant-based meat products. In this scenario, leghemoglobin is used as an ingredient to increase the flavor and color of plant-based meat products. In this study, the synthetic leghemoglobin gene was used to transformed into Escherichia coli for heterologous expression, and the ability of transformed Escherichia coli to produce leghemoglobin was tested. Moreover, whether the addition of Heme center iron ion or its precursors can increase the production of leghemoglobin. Then, a 5L fermentation tank with pH stat cell high-density culture was used to increase the yield of leghemoglobin. In this study, an E. coli transformant for leghemoglobin production was successfully established, and the results showed that, in the shaking culture of Hinton's flask, the Heme concentration reached 4.61±0.15 μM induced by 0.25 mM IPTG alone. The addition of FeCl_3 can increase production of Heme concentration to 0.61±0.01 μM, and the addition of 5-ALA can produce Heme concentrations up to 0.77±0.08 μM, both of which can effectively increase the production of leghemoglobin. The 5L fermentation tank was used with the high-density culture of pH stat cells. After induction, the addition of 5-ALA increase the production of leghemoglobin to 16.08 μM in 48 hours, which was nearly 20 times higher than that of the Hinton's flask. The results of this study are beneficial to the subsequent researches and the development of prokaryotic or eukaryotic fermentation production of soybean hemoglobin as a plant meat additive.