生物素作为微生物的生长因子,对生长速率、细胞膜通透性、代谢产物的生成等方面具有重要作用。为提高黄色短杆菌产L-亮氨酸产量,降低副产物生成,在30 L发酵罐水平研究了在培养基中添加20、50、80、120 μg/L四种不同质量浓度生物素,对黄色短杆菌产L-亮氨酸的影响。结果表明:培养基中添加50 μg/L生物素,黄色短杆菌发酵44 h,L-亮氨酸的产量最高,达到60 g/L,糖酸转化率为22%,副产物L-丙氨酸的质量浓度为8 g/L。在最适生物素浓度下,发酵36 h后,采用膜偶联间歇透析发酵工艺,发酵周期延长至56 h,L-亮氨酸的糖酸转化率为25%,较普通发酵工艺约提高13.6%,副产物L-丙氨酸的浓度降低约71.3%,L-亮氨酸的总产量提高了16.7%。研究结果对提高糖利用率、降低副产物、提高生产效率等方面具有重要意义。
As a growth factor of microorganisms, biotin plays important roles in growth rate, cell membrane permeability, and metabolites production etc. In order to increase the yield of L-leucine produced by Brevibacterium flavum and reduce the formation of by-products, different concentrations of biotin (20, 50, 80, 120 μg/L) were added to the medium in a 30 L bioreactor. The results showed that the yield of L-leucine produced by B. flavum reached the highest (60 g/L) in the medium that contained 50 μg/L biotin and fermented for 44 h. Moreover, the glucose conversion rate was 22% and the concentration of by-product L-alanine was 8 g/L. At the optimal biotin concentration, the membrane coupled intermittent dialysis fermentation was used after 36 h fermentation, and the fermentation period was extended to 56 h. As a result, the glucose conversion rate reached 25%, which was about 13.6% higher than that of common fermentation process, and the concentration of L-alanine was about 71.3% lower. Furthermore, the production of L-leucine increased by 16.7%. Overall, the results are of great significance to improve glucose utilization, reduce by-products, and improve the production of L-leucine.
[1] 孟仕平, 丁玉,黄姗姗. L-亮氨酸的生理功能和分离纯化技术[J]. 食品工业科技, 2011, 32(4): 397-400.
[2] VOGT M, HAAS S, KLAFFL S, et al. Pushing product formation to its limit: metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction [J]. Metabolic Engineering, 2014, 22(3): 40-52.
[3] PARK J H, LEE S Y. Fermentative production of branched chain amino acids: a focus on metabolic engineering [J]. Applied Microbiology & Biotechnology, 2010, 85(3): 491-506.
[4] LAYMAN D K. The role of leucine in weight loss diets and glucose homeostasis [J]. Journal of Nutrition, 2003, 133(1): 261S.
[5] ROTTH USER B, KRAUS G, SCHMIDT P C. Optimization of an effervescent tablet formulation containing spray dried L-leucine and polyethylene glycol 6000 as lubricants using a central composite design [J]. European Journal of Pharmaceutics & Biopharmaceutics, 1998, 46(1): 85.
[6] 张伟国, 程功. 微生物发酵法生产L-亮氨酸的研究进展[J]. 食品与生物技术学报, 2015, 34(2): 113-120.
[7] D'ESTE M, ALVARADO-MORALES M, ANGELIDAKI I. Amino acids production focusing on fermentation technologies-A review [J]. Biotechnology Advances, 2018, 36(1): 14-25.
[8] MCMAHON R J. Biotin in metabolism and molecular biology [J]. Annual Review of Nutrition, 2002, 22(1): 221-239.
[9] LI Y, SUN L, FENG J, et al. Efficient production of alpha-ketoglutarate in the gdh deleted Corynebacterium glutamicum by novel double-phase pH and biotin control strategy [J]. Bioprocess And Biosystems Engineering, 2016, 39(6): 967-976.
[10] WEN J, XIAO Y, LIU T, et al. Rich biotin content in lignocellulose biomass plays the key role in determining cellulosic glutamic acid accumulation by Corynebacterium glutamicum [J]. Biotechnology for Biofuels, 2018, 11(1): 132.
[11] SHIIO I, OTSUKA S I, KATSUYA N. Effect of biotin on the bacterial formation of glutamic acid. II. Metabolism of glucose [J]. Journal of Biochemistry, 1962, 52(1): 108-116.
[12] EGGELING L, KRUMBACH K, SAHM H. L-glutamate efflux with Corynebacterium glutamicum: why is penicillin treatment or Tween addition doing the same?[J]. Journal of Molecular Microbiology & Biotechnology, 2001, 3(1): 67-68.
[13] TAKINAMI K, YOSHII H, TSURI H, et al. Biochemical effects of fatty acid and its derivatives on L-glutamic acid fermentation. 3. Biotin-Tween 60 relationship in accumulation of L-glutamic acid and growth of Brevibacterium lactofermentum [J]. Agric Biol Chem, 1965, 29: 351-359.
[14] NARA T, SAMEJIMA H, KINOSHITA S. Effect of penicillin on amino acid fermentation [J]. Journal of the Agricultural Chemical Society of Japan, 1964, 28(2): 120-124.
[15] RADMACHER E, STANSEN K C, BESRA G S, et al. Ethambutol, a cell wall inhibitor of Mycobacterium tuberculosis, elicits L-glutamate efflux of Corynebacterium glutamicum [J]. Microbiology, 2005, 151(5): 1 359-1 368.
[16] DELAUNAY S, GOURDON P, LAPUJADE P, et al. An improved temperature-triggered process for glutamate production with Corynebacterium glutamicum [J]. Enzyme And Microbial Technology, 1999, 25(8-9): 762-768.
[17] IKEDA M, MIYAMOTO A, MUTOH S, et al. Development of biotin-prototrophic and -hyperauxotrophic Corynebacterium glutamicum Strains[J]. Applied and Environmental Microbiology, 2013, 79(15): 4 586-4 594.
[18] JENS S, PETRA P W, CORINNA S K, et al. Characterization of the biotin uptake system encoded by the biotin-inducible bioYMN operon of Corynebacterium glutamicum [J]. BMC Microbiology, 2012, 12(1): 1-10.
[19] 马雷, 郑梦鸽,石拓,等. 谷氨酸棒杆菌发酵过程中亮氨酸浓度近红外模型的建立[J]. 生物技术通讯, 2016, 27(4): 501-506.
[20] 徐庆阳, 马雷,程立坤,等. 葡萄糖流加方式对黄色短杆菌生产L-亮氨酸的影响[J]. 食品与发酵工业, 2010, 36(8): 1-5.
[21] 户红通, 徐达,徐庆阳,等. 谷氨酸发酵过程膜偶联间歇透析发酵工艺研究[J]. 食品与发酵科技, 2018, 54(1): 9-13.
[22] 薛晓明, 吴振强,吴清平,等. 添加ATP和生物素优化ε-聚赖氨酸发酵的研究[J]. 中国酿造, 2012, 31(1): 72-76.
[23] CAO Y, DUAN Z, SHI Z. Effect of biotin on transcription levels of key enzymes and glutamate efflux in glutamate fermentation by Corynebacterium glutamicum [J]. World Journal of Microbiology & Biotechnology, 2014, 30(2): 461-468.
[24] HASHIMOTO K, KAWASAKI H, AKAZAWA K, et al. Changes in composition and content of mycolic acids in glutamate-overproducing Corynebacterium glutamicum [J]. Bioscience Biotechnology And Biochemistry, 2006, 70(1): 22-30.
[25] TAKINAMI K, YAMADA Y, OKADA H. Biochemical effects of fatty acid and its derivatives on L-glutamic acid fermentation [J]. Journal of the Agricultural Chemical Society of Japan, 2008, 30(7): 674-682.
[26] 谢希贤, 杜军,刘淑云,等. L-亮氨酸高产菌TGL8207的定向选育及其发酵过程研究[J]. 中国食品学报, 2009, 9(2): 29-33.
[27] 刘镇瑜, 刘子强,蔡萌萌,等. L-色氨酸发酵过程偶联膜分离提取工艺研究[J]. 生物技术通讯, 2017, 28(3): 313-318.
[28] 陈宁. 氨基酸工艺学(高校教材) [M]. 北京:中国轻工业出版社, 2007.
