透性化酪氨酸脱羧酶工程菌制备酪胺的研究

摘要

The development of whole cell biocatalysts with high cell-bound tyrosine decarboxylase (TDC) activity is beneficial to improve tyramine production efficiency and reduce cost. A recombinant Escherichia coli (Origami(DE3)-pET21a-tdc) strain was constructed by expressing TDC from Lactobacillus breνis CGMCC 1306 in this study. In order to increase membrane permeability and cell-bound TDC activity of Origami(DE3)-pET21a-tdc, the recombinant cells were treated with nine different organic solvents. The results show that the cell-bound TDC activity is related with the hydrophobicity of the organic solvents, and the solvents with high hydrophobicity (logP > 2.28) are effective in increasing cell-bound TDC activity of cells. Hexane is the most effective among the tested solvents, which could increase cell-bound TDC activity to 1.48 U.mg-1 under optimum conditions (1% hexane, 10 min). In addition, the treatment of Origami(DE3)-pET21b-tdc under optimum permeabilized conditions results in structural changes of the cell envelope, but it does not substantially affect overall morphology of recombinant cells. With 0.47 g.L-1 hexane-treated Origami(DE3)-pET21b-tdc as the biocatalyst, 18 g.L-1 L-tyrosine is almost totally (> 99%) converted to tyramine at pH 5.5, 40℃ after 4.5 h. These results allow systematic selection of suitable organic solvents for improving the cell-bound TDC activity of recombinant E. coli cells, and the whole-cell biocatalyst developed in this study is promising for industrial production of tyramine.%制备具有高表观酪氨酸脱羧酶(TDC)催化活力的全细胞催化剂对于酪胺生产效率的提升和成本的降低都至关重要.为此,研究构建了表达有Lactobacillus breνis CGMCC 1306 TDC基因的工程菌(Origami(DE3)-pET21a-tdc),并在此基础上,采用9种有机溶剂对该工程菌进行透性化处理,来提高其菌体表观催化活力.结果表明,菌体表观TDC催化活力的改善程度与有机溶剂的疏水性具有较强相关性,高疏水性(logP>2.28)的有机溶剂能有效提高菌体表观TDC催化活力,最有效的透性化溶剂己烷的最适处理条件为:1%己烷处理工程菌10 min,在此条件下菌体细胞被膜结构发生明显改变,但细胞整体形态仍保持完整,菌体表观TDC催化活力可达1.48 U.mg-1.在pH 5.5,40℃的条件下,0.47 g.L-1透性化OrigamiB(DE3)-pET21a-tdc菌体在4.5 h内可将18 g.L-1酪氨酸完全转化为酪胺.研究为系统地选择有机溶剂来提高TDC工程菌表观催化活性提供了参考,同时制备的透性化菌体显示了良好的工业应用前景.