Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2016, Vol. 36 Issue (8): 73-79    DOI: 10.13523/j.cb.20160810
技术与方法     
重组毕赤酵母(Pichia pastoris)高产Lunasin的发酵工艺优化
康国凯, 冯国栋, 曹坤琳, 陈正军, 葛向阳
华中农业大学 农业微生物学国家重点实验室 武汉 430070
Optimization for High Production Fermentation of Lunasin from Recombinant Pichia pastoris
KANG Guo-kai, FENG Guo-dong, CAO Kun-lin, CHEN Zheng-jun, GE Xiang-yang
State Key Laboratory of Agricultural Microbiology Huazhong Agricultural University, Wuhan 430070, China
 全文: PDF(801 KB)   HTML
摘要：

Lunasin是一个最初从大豆中分离，具有43个氨基酸的多肽，具有抗高血压，抗氧化活性，预防癌症，抗炎和降胆固醇的活性。为了实现Lunasin的高效生产，对重组毕赤酵母 GS115 LN诱导阶段甲醇补料策略（溶氧反馈补料，指数-恒速补料）以及诱导温度进行了优化，并且在此基础上对双碳源（山梨醇、甘露醇）和甲醇混合补料策略以及复合氮源与甲醇混合补料策略进行了优化。研究表明，最优补料策略以及诱导温度分别为指数-恒速补料、24℃，最终在流加1%大豆蛋白胨以及0.02%天冬氨酸条件下，Lunasin表达量最高，达到3.27 g/L，是单一甲醇诱导的1.27倍。
关键词： 重组毕赤酵母;  Lunasin;  高密度发酵;  工艺优化    
Abstract:

Lunasin is a 43 amino acid polypeptide originally isolated from soybean with bioactive properties such as antihypertensive, antioxidant activity, cancer prevention and therapy, anti-inflammation, hypocholesterolemic activity, anti obesity and immunomodulation. In order to achieve efficient production of Lunasin, the methanol fed methods (DO feedback fedding and index-constant speed feeding) and induction temperature of recombinant Pichia pastoris GS115 LN were investigated. Furthermore, the feeding strategies of mixed carbon sources during induction phase were investigated. The results show that the best feeding strategies and induced temperature were index-constant speed feeding, 24℃. Consequently,the highest expression of Lunasin was 3.27g/L by feeding 1% soy peptone and 0.02% aspartic acid,which was 1.27 times higher than the single methanol induction process.
Key words: High density fermentation    Process optimization    Recombinant Pichia pastoris    Lunasin
收稿日期: 2016-03-03 出版日期: 2016-08-25
ZTFLH:  Q815  
基金资助:

科技部支撑计划资助项目（2013BAD10B02）
通讯作者: 葛向阳     E-mail: gxy@mail.hzau.edu.cn
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　

引用本文:

康国凯, 冯国栋, 曹坤琳, 陈正军, 葛向阳. 重组毕赤酵母(Pichia pastoris)高产Lunasin的发酵工艺优化[J]. 中国生物工程杂志, 2016, 36(8): 73-79.

KANG Guo-kai, FENG Guo-dong, CAO Kun-lin, CHEN Zheng-jun, GE Xiang-yang. Optimization for High Production Fermentation of Lunasin from Recombinant Pichia pastoris. China Biotechnology, 2016, 36(8): 73-79.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160810    或    https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I8/73

[1] 盖文丽, 颜冬菁, 王伟, 等. 生物活性肽Lunasin的原核表达和分离纯化. 现代生物医学进展, 2011, 11(5):805-807. Ge W L,Yan D J,Wang W,et al. Pro-karyotic expression and the separation and purification of bioactive peptides Lunasin. Progress in Modern Biomedicine, 2011, 11(5):805-807.
[2] Liu J, Jia S H, Kirberger M, et al. Lunasin as a promising health-beneficial peptide. European Review for Medical and Phar-macological Sciences, 2013, 18(14):2070-2075.
[3] 干飞, 薛承斌, 陈正望. 多肽Lunasin的生物活性和药理作用研究进展. 中国药房, 2015, 26(28):3993-3995. Gan F,Xue C B,Chen Z W. Advances in biological and pharmacological effects of polypeptide Lunasin. China Pharmacy, 2015, 26(28):3993-3995.
[4] Kyle S, Aggeli A, Ingham E, et al. Production of self-assembling biomaterials for tissue engineering. Trends in Biotechnology, 2009, 27(7):423-433.
[5] Kyle S, James K A, McPherson M J. Recombinant production of the therapeutic peptide Lunasin. Microbial Cell Factories, 2012, 11(28):1-8.
[6] Celik E, Çalik P, Oliver S G. Fed-batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co-substrate sorbitol. Yeast, 2009, 26(9):473-484.
[7] 钟秦, 崔有信. 酵母生产培养基指数流加的研究. 南京理工大学学报(自然科学版), 1988, 4:8. Zhong Q,Cui Y X. The study of yeast production medium exponential feed-ing. Journal of Nanjing University of Science and Technology, 1988, 4:8.
[8] 任海涛, 袁景淇, 邓建慧, 等. 毕氏酵母流加发酵过程的比生长速率控制. 上海交通大学学报, 2004, 38(5):799-801. Ren H T,Yuan J Q,Deng J H, et al. The specific growth rate control of Pichia pastoris fed fermentation process control. Journal of Shanghai Jiaotong University,, 2004, 38(5):799-801.
[9] Jin H, Liu G, Dai K, et al. Improvement of porcine interferon-α production by re-combinant Pichia pastoris via induction at low methanol concentration and low temperature. Applied Biochemistry and Biotechnology, 2011, 165(2):559-571.
[10] Inan M, Meagher M M. Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. Journal of Bioscience and Bioengineering, 2001, 92(6):585-589.
[11] Sears I B, O'Connor J, Rossanese O W, et al. A versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris. Yeast, 1998, 14(8):783-790.
[12] Jung Hee W, Yuan Yi L, Scott S, et al. In-creasing secretion of a bivalent anti-T-cell immunotoxin by Pichia pastoris. Applied & Environmental Microbiology, 2004, 70(70):3370-3376.
[13] Woo J H, Liu J S, Kang S H, et al. GMP production and characterization of the bivalent anti-human T-cell immunotoxin, A-dmDT390-bisFv(UCHT1) for phase I/Ⅱ clinical trials. Protein Expression & Purification, 2008, 58(1):1-11.
[14] Wang J, Nguyen V, Glen J, et al. Improved yield of recombinant merozoite surface protein 3(MSP3). Biotechnology & Bioengineering, 2005, 90(7):838-847.
[15] Zhao H L, Xue C, Wang Y, et al. Increasing the cell viability and heterologous protein expression of Pichia pastoris mutant deficient in PMR1 gene by culture condition optimization. Applied Microbiology & Biotechnology, 2008, 81(2):235-241.
[16] Khatri N K, Frank H. Impact of methanol concentration on secreted protein produc-tion in oxygen-limited cultures of recom-binant Pichia pastoris. Biotechnology & Bioengineering, 2006, 93(5):871-879.
[17] 周祥山, 范位民, 张元兴. 不同甲醇流加策略对重组毕赤酵母高密度发酵生产水蛭素的影响. 生物工程学报,2002, 18(3):348-351. Zhou X S, Fan W M, Zhang Y X. Effects of different methanol feeding strategy on hirudin production in high-density fer-mentation by recombinant Pichia pastoris. Chin J Biotech, 2002, 18(3):348-351.
[18] Lee J, Sang Y L, Park S, et al. Control of fed-batch fermentations. Biotechnology Advances, 1999, 17(1):29-48.
[19] Ohashi R, Mochizuki E, Suzuki T. A mini-scale mass production and separation system for secretory heterologous proteins by perfusion culture of recombinant Pichia pastoris using a shaken ceramic membrane flask. Journal of Bioscience & Bioengineering, 1999, 87(5):655-660.
[20] 吴丹, 储炬, 王永红,等. 甲醇浓度对毕赤酵母表达重组人复合α干扰素分离纯化得率的影响. 生物工程学报, 2011, 27(12):1789-1796. Wu D,Chu J, Wang Y H, et al. Influence of methanol concentration on purification recovery of consensus interferon-α produced by Pichia pastoris. Chin J Biotech, 2011, 27(12):1789-1796.
[21] Li J, Tang C, Shi H, et al. Cloning and optimized expression of a neutral endoglucanase gene (ncel5A) from Volvariella volvacea WX32 in Pichia pastoris. Journal of Bioscience & Bioen-gineering, 2011, 111(5):537-540.
[22] Dragosits M, Frascotti G, Bernardgranger L, et al. Influence of growth temperature on the production of antibody Fab frag-ments in different microbes:A host comparative analysis. Biotechnology Progress, 2012, 28(4):1114-1114.
[23] Batra G, Gurramkonda C, Nemani S K, et al. Optimization of conditions for secretion of Dengue virus type 2 envelope domain Ⅲ using Pichia pastoris. Journal of Bioscience & Bioengineering, 2010, 110(110):408-414.
[24] Gao M, Dong S, Yu R, et al. Improvement of ATP regeneration efficiency and opera-tion stability in porcine interferon-α pro-duction by Pichia pastoris under lower induction temperature. Korean Journal of Chemical Engineering, 2011, 28(6):1412-1419.
[25] Daly R, Hearn M T. Expression of het-erologous proteins in Pichia pastoris:a useful experimental tool in protein engi-neering and production. Journal of Mo-lecular Recognition, 2005, 18(2):119-138.
[26] Li Z, Xiong F, Lin Q, et al. Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris. Protein Expression and Purification, 2001, 21(3):438-445.
[27] Jia D, Liu L, Wang H, et al. Overproduc-tion of a truncated poly (vinyl alcohol) dehydrogenase in recombinant Pichia pastoris by low-temperature induction strategy and related mechanism analysis. Bioprocess and Biosystems Engineering, 2013, 36(8):1095-1103.
[28] Sears I B, O'Connor J, Rossanese O W, et al. A versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris. Yeast, 1998, 14(8):783-790.
[29] Walker G M. Yeast Physiology and Bio-technology. Hoboken,New Jersey:John Wiley & Sons, 1998.
[30] Eskitoros M S, Çalik P. Co-substrate mannitol feeding strategy design in semi-batch production of recombinant human erythropoietin production by Pichia pastoris. Journal of Chemical Technology and Biotechnology, 2014, 89(5):644-651.
[31] Ramón R, Ferrer P, Valero F. Sorbitol co-feeding reduces metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris. Journal of Biotechnology, 2007, 130(1):39-46.
[32] Jahie M,Gustavsson M,Jansen A K,et al.Anabsis and control of proteolysis of a fusion protein in Pichia pastoris fed-batchprocesses.Bioteehnol,2003,102(1):45-53.
[33] He X, Liu N, Li W, et al. Inducible and constitutive expression of a novel thermostable alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 in Pichia pastoris and characterization of the re-combinant enzyme. Enzyme and Microbial Technology, 2008, 43(1):13-18.
[1] 张文玉,魏东升,钱江潮. 共表达PDI1、MDH1和HAC1基因对重组毕赤酵母分泌表达葡糖氧化酶的影响 *[J]. 中国生物工程杂志, 2019, 39(10): 24-33.
[2] 黄燕,孙益荣,吴敬,宿玲恰. 重组Humicola insolens角质酶的高密度发酵优化 *[J]. 中国生物工程杂志, 2019, 39(1): 63-70.
[3] 孙帆,宿玲恰,张康,吴敬. D-阿洛酮糖 3-差向异构酶在枯草芽孢杆菌中的高效表达及固定化细胞研究 *[J]. 中国生物工程杂志, 2018, 38(7): 83-88.
[4] 姚韧辉, 董卓, 李会. Gibberella intermedia C2转化4-雄甾烯-3、17-二酮的研究[J]. 中国生物工程杂志, 2017, 37(3): 73-77.
[5] 张宇萌, 童梅, 陆小冬, 米月, 徐晨, 姚文兵. 提高大肠杆菌可溶性重组蛋白表达产率的研究进展[J]. 中国生物工程杂志, 2016, 36(5): 118-124.
[6] 武婕, 张晓雪, 余河水, 李薇, 贾宇平, 郭江玉, 张丽娟, 宋新波. 毕赤酵母工程菌高密度发酵研究与进展[J]. 中国生物工程杂志, 2016, 36(1): 108-114.
[7] 夏烨, 黄惟巍, 杨旭, 孙鹏艳, 姚月婷, 王世杰, 刘存宝, 孙文佳, 白红妹, 姚宇峰, 马雁冰. 利用不同碳源进行毕赤酵母高密度发酵及TEF-1启动子指导下的HPV16_L1蛋白表达[J]. 中国生物工程杂志, 2015, 35(10): 39-43.
[8] 罗漫杰, 谢渊, 钱志刚, 冯雁, 杨广宇. 超嗜热酯酶EST2在不同宿主中的异源高效表达研究[J]. 中国生物工程杂志, 2014, 34(12): 36-44.
[9] 刘爱军, 史守坤, 李兰, 王萍, 王伟, 贾军巧, 王泽建, 李海东, 庄英萍, 张嗣良. 基于活细胞量测量的利福霉素发酵过程氮源优化策略[J]. 中国生物工程杂志, 2014, 34(10): 73-78.
[10] 刘会影, 薛冬桦, 潘安龙, 徐洪章, 叶小金, 孙国英. 微生物油脂酯化工艺优化[J]. 中国生物工程杂志, 2013, 33(3): 92-98.
[11] 智晓燕, 汪小锋, 孙永川, 柯锋, 代敏, 闫云君. 多拷贝毕赤酵母重组菌表达GCL摇瓶优化和高密度发酵[J]. 中国生物工程杂志, 2012, 32(02): 82-89.
[12] 汪小锋, 申旭光, 赵鹤云, 孙永川, 纪昌涛, 闫云君. 带His-tag的解脂耶氏酵母脂肪酶Lip2在毕赤酵母中的表达及纯化[J]. 中国生物工程杂志, 2011, 31(04): 53-59.
[13] 黄静, 史建明, 霍文婷, 徐庆阳, 谢希贤, 温廷益, 陈宁. NH4+对L-色氨酸发酵的影响[J]. 中国生物工程杂志, 2011, 31(03): 55-60.
[14] 邓永康1,吴民泸2,刘盛邦1,杜林方1,伍黎黎1,李曼1,孟延发1. 乳糖诱导重组尿酸酶基因在大肠杆菌中的表达[J]. 中国生物工程杂志, 2009, 29(07): 74-79.
[15] 张广敏1,2,王炜1,3,包慧芳1,3,张晓琳1,2,詹发强,1,3,林瑞峰1,3. 植物乳杆菌Lp-2的高密度发酵[J]. 中国生物工程杂志, 2009, 29(06): 68-73.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会