发酵条件对天冬氨酸转氨酶重组菌高效表达与质粒稳定性的影响
Effects of Fermentation Conditions on High Expression and Plasmid Stability in Recombinant Bacteria Producing Aspartate Transaminase
DOI: 10.12677/BP.2022.123021, PDF,    国家科技经费支持
作者: 宫长斌, 何冰芳, 曾 杨, 张 霞, 谢宁昌：南京工业大学生物化学工程实验教学示范中心，江苏 南京
关键词: 质粒稳定性；天冬氨酸转氨酶；条件优化；工程菌发酵； Plasmid Stability； Aspartate Aminotransferase； Condition Optimization； Engineering Bacteria Fermentation
摘要: 通过对以pET-22b质粒为载体，大肠杆菌BL21为宿主构建的产天冬氨酸转氨酶的重组菌BL21/pET-aspC，质粒稳定性与重组菌产酶活性的发酵条件优化，对比了不同发酵条件对基因工程菌菌体生长、质粒稳定性和转氨酶酶活性的影响，得到较工程菌常用LB培养基更利于工程菌生长的培养基CSN。考察了该培养基主要成份玉米浆、酵母粉和柠檬酸钠对重组菌生长及质粒稳定性的影响。优化后，该发酵体系的发酵成本为LB培养基的10%，外源基因的质粒稳定性得到明显改善，重组菌生物量与天冬氨酸转氨酶活性显著提高。优化后培养体系成本低廉，发酵稳定，适于基因工程菌的大规模工业化生产发酵使用。
Abstract: Using the recombinant strain Bl21/pet-AspC producing aspartate aminotransferase which was constructed with pET-22b as the vector and E. coli Bl21 as the host, the stability of plasmid and the most suitable fermentation conditions for the production of enzyme were studied. By comparing the relationship between cell growth, plasmid stability, transaminase activity and medium composition of genetically engineered bacteria, the CSN medi-um, which is more favorable for the growth of engineering bacteria than the LB medium commonly used for engineering bacteria, was obtained. The effects of corn syrup, yeast powder and sodium citrate on the growth of recombinant bacteria and the stability of plasmid were investigated. The fermentation cost of the new medium was 10% of that of LB medium. The stability of plasmid of for-eign gene, the biomass of recombinant bacteria and the activity of aspartate aminotransferase were significantly improved. The optimized culture system has low cost and stable fermentation, which is suitable for large-scale industrial production and fermentation of genetic engineering bacteria.
文章引用：宫长斌, 何冰芳, 曾杨, 张霞, 谢宁昌. 发酵条件对天冬氨酸转氨酶重组菌高效表达与质粒稳定性的影响[J]. 生物过程, 2022, 12(3): 186-197. https://doi.org/10.12677/BP.2022.123021

参考文献

[1] 张清华, 李莎, 廉政, 等. 利用DNA重组技术生产L-苯丙氨酸的研究[J]. 天津农学院学报, 2020, 27(2): 38-43.
[2] Chao, Y.P., Lai, Z.J., Chen, P., et al. (1999) Enhanced Conversion Rate of L-Phenylalanine by Coupling Reactions of Aminotransferases and Phosphoenolpyruvate Carboxykinase in Escherichia Coli K-12. Biotechnology Pro-gress, 15, 453-458. [Google Scholar] [CrossRef] [PubMed]
[3] Chao, Y.P., Lo, T.-E., Luo, N.S. (2000) Selective Production of L-Aspartic Acid and L-Phenylalanine by Coupling Reactions of Aspartase and Aminotransferase in Esche-richia coli. Enzyme and Microbial Technology, 27, 19-25. [Google Scholar] [CrossRef] 
[4] 邵宇, 张显, 胡孟凯, 等. 重组大肠杆菌全细胞催化合成L-苯乳酸[J]. 食品与发酵工业, 2021, 47(14): 1-8.
[5] 喻国策, 焦瑞身, 王骥程, 王树青. 大肠杆菌E. coli HB101(pBR322)高密度培养过程质粒的稳定性[J]. 过程工程学报, 2001(2): 185-188.
[6] 张琳琳. 重组蛋白包涵体变复性过程优化及折叠机制探索[D]: [硕士学位论文]. 上海: 华东理工大学, 2011.
[7] 张若兰, 刘庆国, 王敏, 等. 枯草芽孢杆菌碱性蛋白酶基因在酿酒酵母中的表达和应用[J]. 食品与发酵工业, 2018, 44(7): 76-81.
[8] 张刚, 杨光, 裴海生, 等. 质粒平均分配基因parDE对甲酸脱氢酶NADH再生系统稳定性的影响[J]. 过程工程学报, 2008(2): 345-349.
[9] 翟成一, 徐岩, 聂尧, 等. 产普鲁兰酶重组大肠杆菌质粒稳定性的研究[J]. 工业微生物, 2015, 45(2): 13-20.
[10] Johan, P. (2002) Multieveled Selection on Plasmid Replication. Genetics, 161, 1373-1384. [Google Scholar] [CrossRef] [PubMed]
[11] 杨涛, 陈坚, 方芳, 等. 多铜氧化酶在大肠杆菌中的分泌表达[J]. 过程工程学报, 2020, 20(10): 1210-1217.
[12] 杨晓玫, 师尚礼. 红、黄、绿三种颜色荧光质粒导入大肠杆菌中的稳定性表达[J]. 甘肃农业大学学报, 2018, 53(3): 193-198.