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1 752 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 孙启星等 / 基于 ph 调节和有机氮源流加调控补料分批发酵过程提高 ε- 聚赖氨酸产量 May 25, 2015, 31(5): Chin J Biotech, All rights reserved 生物育种与工艺优化 ph ε- 孙启星, 陈旭升, 任喜东, 郑根成, 毛忠贵 ,,,. ph ε-., 2015, 31(5): Sun QX, Chen XS, Ren XD, et al. Enhanced ε-poly-l-lysine production through ph regulation and organic nitrogen addition in fed-batch fermentation. Chin J Biotech, 2015, 31(5): 摘要 : 为了解决 ε- 聚赖氨酸 (ε-pl) 补料分批发酵过程中后期 ε-pl 产率下降的问题, 提出了在补料阶段利用调节 ph 值和流加有机氮源 ( 酵母粉 ) 两种手段来提高 ε-pl 产率利用上述两种策略, 实现 ε-pl 平均产率分别达到 4.62 g/(l d) 和 5.16 g/(l d), 较未调控补料分批发酵 ( 典型补料分批发酵 ) 分别提高了 27.3% 和 42.15%; 同时, 实现 ε-pl 产量分别达到 g/l 和 g/l, 较未调控补料分批发酵分别提高了 27.4% 和 42.48% 进一步细胞活性染色和关键酶活性分析发现, 两种策略均能显著提高细胞活力和关键酶活性该研究结果表明, 在发酵中后期通过调节 ph 值和流加酵母粉两种措施能够显著增强细胞活性和产生菌代谢能力, 从而达到提高 ε-pl 产率和产量的目的 : ε- 聚赖氨酸, 细胞活力, 链霉菌 M-Z18, 补料分批发酵 Enhanced ε-poly-l-lysine production through ph regulation and organic nitrogen addition in fed-batch fermentation Qixing Sun, Xusheng Chen, Xidong Ren, Gencheng Zheng, and Zhonggui Mao Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi , Jiangsu, China Abstract: During the production of ε-poly-l-lysine (ε-pl) in fed-batch fermentation, the decline of ε-pl synthesis often occurs at middle or late phase of the fermentation. To solve the problem, we adopted two strategies, namely ph shift and feeding yeast extract, to improve the productivity of ε-pl. ε-pl productivity in fermentation by ph shift and feeding yeast extract achieved 4.62 g/ (L d) and 5.16 g/ (L d), which were increased by 27.3% and 42.2% compared with the control ε-pl Received: September 16, 2014; Accepted: November 26, 2014 Supported by: Jiangsu Key Project of Scientific and Technical Supporting Program (No. BE ), the Cooperation Project of Jiangsu Province Among Industries, Universities and Institutes (No. BY ). Corresponding author: Xusheng Chen. Tel: ; chenxs@jiangnan.edu.cn Zhonggui Mao. Tel: ; maozg@jiangnan.edu.cn (No. BE ) (No. BY )

2 孙启星等 / 基于 ph 调节和有机氮源流加调控补料分批发酵过程提高 ε- 聚赖氨酸产量 753 fed-batch fermentation, respectively. Meanwhile, ε-pl production enhanced g/l and g/l in 192 h with these two strategies, increased by 27.4% and 42.48% compared to the control, respectively. ε-pl production could be improved at middle or late phase of fed-batch fermentation by ph shift or feeding yeast extract. Keywords: ε-poly-l-lysine, cell viability, Streptomyces sp. M-Z18, fed-batch fermentation ε- (ε-poly-l-lysine ε-pl) L- α-cooh ε-nh 2 ε-pl [1-2] [3] ε-pl [4] ε-pl Kahar Streptomyces albulus S410 ( S410) ph 192 h ε-pl 48.3 g/l [5] Shih Shen S. albulus IFO d ε-pl 5.2 g/l [6] Jia S. albulus TUST2 120 h ε-pl 20.2 g/l [7] Liu [8] Streptomyces sp. GIM8 192 h ε-pl 23.4 g/l ε-pl ph 173 h ε-pl g/l [9-10] ε-pl ε-pl ph ( ) ε-pl ε-pl ε-pl 1 材料与方法 1.1 菌株和培养基 Streptomyces sp. M-Z18 ε-pl [10] 1.2 培养方法 [9] 5 L 1) ε-pl [10] 2) ph ε-pl 96 h ph h ph ph 3.8 ε-pl 3) ε-pl 80 h 3.13 ml/h (150 g/l) 144 h ε-pl 1.3 测定方法 (DCW) ε-pl [11] (PEPC) (ASPK) (CS) 6- (G6PDH) [12] Bacstain-CTC rapid staining kit [13] 2 结果与分析 2.1 不同策略调控 ε-pl 补料分批发酵过程参数分析 ε-pl Streptomyces sp. M-Z18 ε-pl 1A h ε-pl (110 h ) ε-pl 1D 24 h ε-pl ε-pl h 7.05 g/(l d) h ε-pl 1.52 g/(l d) ε-pl 3.63 g/(l d) ε-pl 29 g/l ( 1A) ε-pl cjb@im.ac.cn

3 754 ISSN CN /Q Chin J Biotech May 25, 2015 Vol.31 No.5 图 1 不同调控策略补料分批发酵生产 ε-pl 的过程参数 Fig. 1 ε-pl production in the fed-batch fermentation by Streptomyces sp. M-Z18 under different regulation strategies. (A) Typical ε-pl fed-batch fermentation process. (B) ε-pl fed-batch fermentation by ph shift. (C) ε-pl fed-batch fermentation by yeast extract addition. (D) ε-pl productivity in three fed-batch fermentations. DO: dissolved oxygen; DCW: dry cell weight. ε-pl [14-15] 1A 10 h ( ) ph ε-pl ph ε-pl ε-pl ph ( 5.0) ε-pl ε-pl ph ( 4.0) ε-pl [5] (30%) ph (ph 4.1) ph ph (ph 3.8) ε-pl 1B 100 h ph 4.1 ph 110 h 120 h ph ph h ph ε-pl (85 h) ε-pl ε-pl ph 4.1 ε-pl ph 3.8 ε-pl 1D 96 h ε-pl h ph ε-pl 120 h ph ε-pl ph ε-pl g/l 27.4% ε-pl [16] 1C 1C 80 h 120 h 10% 150 h 150 h ε-pl 180 h 1D 96 h ε-pl ε-pl g/l 5.16 g/(l d) 42.48% 42.15%

孙启星等 / 基于 ph 调节和有机氮源流加调控补料分批发酵过程提高 ε- 聚赖氨酸产量 755 2.

4 孙启星等 / 基于 ph 调节和有机氮源流加调控补料分批发酵过程提高 ε- 聚赖氨酸产量不同策略调控 ε-pl 补料分批发酵过程的菌体活力变化 CTC 2A 120 h 144 h ph 144 h (ph ) 168 h ( ) (Gray value) 2B 72 h 168 h ph 72 h 120 h 144 h ph 96 h 120 h ph ph 168 h h 70 ph 2.3 不同策略调控 ε-pl 补料分批发酵过程关键酶活性变化 ph ε-pl 3 ε-pl (PEPC CS G6PDH ASPK) h ε-pl 1D 2 ε-pl 24 h ph PEPC CS ASPK ε-pl L- G6PDH ph ph ( ph ) ε-pl ph ε-pl PEPC CS ph ε-pl ATP L- ph ε-pl 11.8% 图 2 不同策略调控 ε-pl 补料分批发酵过程细胞活力的变化 Fig. 2 The change of cell viability of Streptomyces sp. M-Z18 in fed-batch fermentation under different regulation strategies. cjb@im.ac.cn

5 756 ISSN CN /Q Chin J Biotech May 25, 2015 Vol.31 No.5 图 3 不同策略调控 ε-pl 补料分批发酵过程关键酶活性变化 Fig. 3 The change of key enzymatic activity of Streptomyces sp. M-Z18 in fed-batch fermentation under different regulation strategies. 3 结论 ε-pl ph ε-pl ε-pl g/l g/l ( ) 27.4% 42.5% CTC ε-pl ε-pl ε-pl REFERENCES [1] Shih IL, Shen MH, Van YT. Microbial synthesis of poly (ε-lysine) and its various applications. Bioresour Technol, 2006, 97(9): [2] Yoshida T, Nagasawa T. ε-poly-l-lysine: microbial production, biodegradation and application potential. Appl Microbiol Biotechnol, 2003, 62(1): [3] ε- 4 [EB/OL]. [ ]. s7890/201404/a93467d652c24a75a6de637abde31f30.shtml. [4] Yoshimitsu H, Kazuya Y. Amino-Acid Homopolymers Occurring in Nature. 3rd ed. New York: Springer-Verlag Berlin Heidelberg, 2010: [5] Kahar P, Iwata T, Hiraki J, et al. Enhancement of ε-polylysine production by Streptomyces albulus strain 410 using ph control. J Biosci Bioeng, 2001, 91(2): [6] Shih IL, Shen MH. Application of response surface methodology to optimize production of poly-ε-lysine by Streptomyces albulus IFO Enzyme Microb Technol, 2006, 39(1): [7] Jia SR, Wang GL, Sun YF, et al. Improvement of ε-poly-l-lysine production by Streptomyces albulus TUST2 employing a feeding strategy//the 3rd International Conference on Bioinformatics and Biomedical Engineering., Beijing, China[C]. 2009: 1 4. doi: /ICBBE [8] Liu SR, Wu Q, Zhang J, et al. Production of ε-poly-l-lysine by Streptomyces sp. using resin-based, in situ product removal. Biotechnol Lett, 2011, 33(8): [9] Chen XS, Tang L, Mao ZG, et al. Optimization of medium for enhancement of ε-poly-l-lysine production by Streptomyces sp. M-Z18 with glycerol as carbon source. Bioresour Technol, 2011, 102(2): [10] Chen XS, Li S, Liao LJ, et al. Production of ε-poly-l-lysine using a novel two-stage ph control strategy by Streptomyces sp. M-Z18 from glycerol. Bioprocess Biosyst Eng, 2011, 34(5): [11] Chen XS, Ren XD, Dong N, et al. Culture medium containing glucose and glycerol as a mixed carbon source improves ε-poly-l-lysine production by Streptomyces sp. M-Z18. Bioprocess Biosyst Eng, 2012, 35(3): [12] Li S, Chen XS, Dong C, et al. Combining genome shuffling and interspecific hybridization among Streptomyces improved ε-poly-l-lysine production. Appl Biochem Biotechnol, 2013, 169(1): [13] Xu J, Chen YQ, Mao ZG, et al. Effect of acetic acid on citric acid fermentation in an integrated citric aicd-methane fermentation process. Appl Biochem Biotechnol, 2014, 174(1): [14] Kahar P, Iwata T, Hiraki J, et al. Enhancement of ε-polylysine production by Streptomyces albulus strain 410 using ph control. J Biosci Bioeng, 2001, 91(2): [15] Zhang Y, Feng X, Xu H, et al. ε-poly-l-lysine production by immobilized cells of Kitasatospora sp. MY 5-36 in repeated fed-batch cultures. Bioresour Technol, 2010, 101(14): [16] Li Y. Principles and Technology of Fermentation Engineering. Beijing: Higher Education Press, 2007: (in Chinese)... :, 2007: ( 本文责编陈宏宇 )

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇