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目的探讨N-乙酰鸟氨酸的转氨酶于钝齿棒杆菌当中,其精氨酸合成环节所起到的作用,并验证其酶学性质,从而为优化提高培养基的成分,以及发酵过程的工艺条件,和精氨酸的产量提供理论及实验依据。方法于精氨酸的高产菌株中,使其钝齿棒杆菌的染色体(SYPA5-5)扩增,从而获取ACOAT的编码基因-argD,其全长为1176bp,编码氨基酸为390个,均于C.crenatum SYPA与Escherichia coli BL21(DE3)上成功表达。选择Ni柱进行亲和层析并纯化后,能够得到重组的蛋白比酶活可达到108.2U/g,并研究其酶学性质。再构建重组的钝齿棒杆菌,以加强其精氨酸的合成途径中ACOAT的蛋白表达量,同时对重组的菌产精氨酸予以发酵分析。结果重组的钝齿棒杆菌同出发菌株进行对比,其胞内的ACOAT的酶活得到增强;并且重组菌中CCD1其精氨酸的平均产量达到了39.7g/L,其产酸最终提高了14.7%。结论通过研究,不仅对高产精氨酸其重组基因菌工程的构建提供以理论依据,而且还具有非常重要的指导意义。
Objective To investigate the role of N-acetylornithine transaminase in the synthesis of arginine from Corynebacterium crenulos and to verify its enzymatic properties so as to optimize the composition of the medium and the fermentation process Conditions, and arginine yield provide theoretical and experimental basis. Methods The arginine-producing strain (SYPA5-5) was used to amplify the gene encoding ACOAT-argD. The full-length cDNA was 1176 bp in length and contained 390 amino acids. .crenatum SYPA was successfully expressed on Escherichia coli BL21 (DE3). After selective Ni column affinity chromatography and purification, the recombinant protein can be obtained activity of 108.2U / g, and study its enzymatic properties. Recombinant Corynebacterium crenatum was reconstructed to enhance the protein expression of ACOAT in the arginine synthesis pathway. Fermentation analysis of recombinant arginine was also carried out. Results Compared with the original strain, Corynebacterium crenatum had an enhanced activity of intracellular ACOAT. The average arginine yield of CCD1 reached 39.7 g / L in the recombinant strain, and its acid production finally increased by 14.7 %. Conclusion Through the research, not only to provide a theoretical basis for the construction of recombinant arginine high-yield arginine, but also has very important guiding significance.