药用级微生物谷氨酰胺转胺酶的纯化工艺研究

doi:10.13523/j.cb.20140409

中国生物工程杂志

2014, Vol. 34

Issue (4): 53-58 DOI: 10.13523/j.cb.20140409

技术与方法

药用级微生物谷氨酰胺转胺酶的纯化工艺研究

裴正培¹, 李博², 张伟¹, 高红亮¹, 常忠义¹, 金明飞¹, 鲁伟³, 步国建³

1. 华东师范大学生命科学学院上海 200241;
2. 上海工会管理职业学院上海 201415;
3. 泰兴市东圣食品科技有限公司泰兴 225411

Large Scale Purification of Pharmaceutical Grade Microbial Transglutaminase from Streptoverticillium mobaraense

PEI Zheng-pei¹, LI Bo², ZHANG Wei¹, GAO Hong-liang¹, CHANG Zhong-yi¹, JIN Ming-fei¹, LU Wei³, BU Guo-jian³

1. School of Life Science of East China Normal University, Shanghai 200241, China;
2. Shanghai Vocational Management College of Trade Union, Shanghai 201415, China;
3. Taixing Dongsheng Food and Technology Limited Company, Taixing 225411, China

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摘要：

为了提高谷氨酰胺转胺酶的纯度和扩展在医药领域的应用，探索了一种适合工业化生产的、安全高效的微生物谷氨酰胺转胺酶纯化方法。轮枝链霉菌发酵后，经离心10 000 r/min 4℃除去菌体，调节发酵液电导率至4.1mS/cm和pH6.0后，以直线流速60cm/h通过SP Sepharose FF阳离子交换层析柱对目的蛋白高选择性和高载量地捕获，再通过phenyl sepharose 6 FF（high sub）疏水层析柱进行精细纯化。纯化后经SDS-PAGE鉴定纯度达到95%以上，HPLC分析纯度> 99%。鲎试剂测定内毒素含量为0.013EU/ml，达到中国药典中血制品要求的低于0.15EU/ml标准。

关键词： 谷氨酰胺转胺酶; 离子交换层析; 疏水层析; 纯化

Abstract:

A safe efficient microbial transglutaminase purification method applied for industrial manufacture was introduced in order to improve the purity of transglutaminase and extend its applications for pharmaceuticals. Cultures was centrifuged at 10 000 r/min 4℃ to remove the cells before adjusting conductivity to 4.1 mS/cm and pH to 5.5 for purification. The sample was purified by cation-exchange chromatography at a linear velocity of 60 cm/h in the first step. In this step SP Sepharose FF as a packing provided high selectivity and high loading for the target protein. After primary purification, the sample was further processed/isolated with hydrophobic chromatography packed with phenyl Sepharose 6 FF (high sub). SDS-PAGE pattern manifested a purity of over 95% and HPLC analysis indicated a purity of above 99% were achieved after this two purification step method. LPS of the purified sample was determined at 0.013 EU/ml with tachypleus amebocyte lysate, met the blood products requirement (<0.15 EU/ml) provided in Chinese Pharmacopoeia. Thus the method described/established is efficient and practical in microbial transglutaminase purification.

Key words: Transglutaminase Ion-exchange chromatography Hydrophobic chromatography Purification

收稿日期: 2014-02-14 出版日期: 2014-04-25

ZTFLH:

Q555+.6

基金资助:

质检公益性行业科研专项项目资助项目（201310255）

通讯作者: 金明飞 E-mail: mfjin@bio.ecnu.edu.cn

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引用本文:

裴正培, 李博, 张伟, 高红亮, 常忠义, 金明飞, 鲁伟, 步国建. 药用级微生物谷氨酰胺转胺酶的纯化工艺研究[J]. 中国生物工程杂志, 2014, 34(4): 53-58.

PEI Zheng-pei, LI Bo, ZHANG Wei, GAO Hong-liang, CHANG Zhong-yi, JIN Ming-fei, LU Wei, BU Guo-jian. Large Scale Purification of Pharmaceutical Grade Microbial Transglutaminase from Streptoverticillium mobaraense. China Biotechnology, 2014, 34(4): 53-58.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140409 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I4/53

[1] 陈佳,金明飞,谭玉静,等. NaCl 对轮枝链霉菌产谷氨酰胺转胺酶的促进作用研究. 中国生物工程杂志,2013,33(002):54-58. Chen J, Jin M F, Tan Y J, et al. Enhancement of transglutaminase production in Streptoverticillium mobaraensis as achieved by treatment with NaCl. China Biotechnology,2013,33(2):54-58.

[2] 王莉,常忠义,李平作. 转谷氨酰胺酶基因在大肠杆菌中的克隆表达. 中国生物工程杂志,2004,24(11):56-60. Wang L, Chang Z Y,Li P Z. Cloning and expression of transglutaminase gene in Escherichia coli. China Biotechnology,2004,24(11):56-60.

[3] Ando H, Adachi M, Umeda K, et al. Purification and characteristics of a novel transglutaminase derived from microorganisms. Agricultural and Biological Chemistry,1989,53(10):2613-2617.

[4] Jaros D, Partschefeld C, Henle T, et al. Transglutaminase in dairy products: chemistry, physics, applications. Journal of Texture Studies,2006,37(2):113-155.

[5] Liu Y, Kopelman D, Wu L Q, et al. Biomimetic sealant based on gelatin and microbial transglutaminase: an initial in vivo investigation. Journal of Biomedical Materials Research Part B: Applied Biomaterials,2009,91(1):5-16.

[6] Zhu Y, Tramper J. Novel applications for microbial transglutaminase beyond food processing. Trends in Biotechnology,2008,26(10):559-565.

[7] Marquez G R, Di Pierro P, Esposito M, et al. Application of transglutaminase-crosslinked whey protein/pectin films as water barrier coatings in fried and baked foods. Food and Bioprocess Technology,2013:1-9.

[8] Rossa P N, de Sá E M F, Burin V M, et al. Optimization of microbial transglutaminase activity in ice cream using response surface methodology. LWT-Food Science and Technology,2011,44(1):29-34.

[9] 谈丹,陈雄,黄敬华,等. 微生物谷氨酰胺转胺酶对大鼠创伤愈合作用研究. 中国生物工程杂志,2007,27(9):85-90. Tan D, Chen X, Huang J H, et al. Efficacy of microbial transglutaminase on wound healing in rats. China Biotechnology,2007,27(9):85-90.

[10] 尹少谦,王淼. Streptomyces hygroscopicus谷氨酰胺转胺酶的分离纯化及其性质研究. 食品与发酵工业,2005,31(3):26-29. Yin S Q,Wang M. Purification and characterization of transglutaminase from Streptomyces hygroscopicus. Food and Fermentation Industries,2005,31(3):26-29.

[11] Gerber U, Jucknischke U, Putzien S, et al. A rapid and simple method for the purification of transglutaminase from Streptoverticillium mobaraense. Biochemical Journal,1994,299(Pt 3):825.

[12] Grossowicz N, Wainfan E, Borek E, et al. The enzymatic formation of hydroxamic acids from glutamine and asparagine. Journal of Biological Chemistry,1950,187(1):111-125.

[13] Yu S, Geng J, Zhou P, et al. Application of a new hybrid organic-inorganic monolithic column for efficient deoxyribonucleic acid purification. Analytica Chimica Acta,2008,611(2):173-181.

[14] 彭燕,张玲莉,王宗春. 显色基质法检测乳酸左氧氟沙星氯化钠注射液细菌内毒素含量. 中国药师,2013(10):1597-1599. Peng Y, Zhang L L,Wang Z C. Chromogenic substrate assay within levofloxacin lactate and sodium chloride injection of bacterial endotoxin.China Pharmacist,2013(10):1597-1599.

[15] Zotzel J, Pasternack R, Pelzer C, et al. Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step. European Journal of Biochemistry,2003,270(20):4149-4155.

[16] 黄亮,张东旭,刘和,等. 微生物谷氨酰胺转胺酶制备过程中蛋白酶的抑制研究. 食品与发酵工业,2005,31(12):8-11. Huang L,Zhang D X, Liu H, et al. Inhibition of the protease in the preparation of microbial transglutaminase. Food and Fermentation Industries,2005,31(12):8-11.

[17] 杨晓燕,周哲敏,堵国成,等. 吸水链霉菌谷氨酰胺转胺酶分离纯化方法改进及结构研究. 生物技术通报,2011,4:40. Yang X Y, Zhou Z M, Du G C, et al. Improvement of purification method and study on structure of transglutaminase from Streptomyces hygroscopicus. Biotechnology Bulletln,2011,4,4:40.

[18] 鲁时瑛,周楠迪,堵国成,等. 谷氨酰胺转胺酶的分离纯化及酶学性质. 无锡轻工大学学报,2002,21(6):546-568. Lu S Y, Zhou N D, Du G C, et al. Purification of transglutaminase and properties of crude enzyme. Journal of Wuxi University of Light Industry,2002,21(6):546-568.

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