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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2011, Vol. 31 Issue (03): 76-80    
技术与方法     
超滤膜分离纯化珊瑚藻溴过氧化物酶
程小飞1, 章表明2,3, 薛松2, 肖通虎1
1. 宁波大学材料科学与化学工程学院 宁波 315211;
2. 中国科学院大连化学物理研究所 大连 116023;
3. 中国科学院研究生院 北京 100049
Isolation and Purification of Bromoperoxidase from Corallina Officinalis Using Ultrafiltration Membrane
CHENG Xiao-fei1, ZHANG Biao-ming2,3, XUE Song2, XIAO Tong-hu1
1. Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211, China;
2. Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China;
3. Graduate University of Chinese Academy of Sciences, Beijing 100049, China
 全文: PDF(660 KB)   HTML
摘要:

利用超滤膜对珊瑚藻中溴过氧化物酶(EC 1.11.1.18,分子质量740kDa)进行分离纯化,对膜的截留分子质量、操作压力、起始蛋白质浓度、搅拌速率、pH、离子强度等条件进行了优化。超滤分离纯化最优条件为:截留分子质量为100kDa的聚偏氟乙烯(PVDF)膜,操作压力为0.02MPa,搅拌速率为600r/min,起始蛋白质浓度为0.1g/L,pH=6.0。对粗酶液先进行热沉淀纯化,再进行超滤纯化,溴过氧化物酶被纯化了21倍,比活为212U/mg,酶活回收率为96%。

关键词: 聚偏氟乙烯(PVDF)膜超滤纯化溴过氧化物酶    
Abstract:

A novel, simple and highly efficient process for purifying vanadium bromoperoxidase from Corallina officinalis is developed, which is consist of two steps: crude enzyme extraction was heated at 70℃ for 2h, and then was ultrafiltered with PVDF membrane. The ultrafiltration conditions were optimized, and the optimized conditions are: MWCO=100kDa, 0.02MPa pressure, 600r/min stirring, 0.12mg/ml initial protein concentration and pH=6.0. The crude extraction purified with heat treatment and then ultrafitration results a 21-fold purification, with a 96% overall yield and 212U/mg specific activity.

Key words: Vanadium Bromoperoxidase    Polyvinylidenefluoride (PVDF) membrane    Ultrafiltration    Purification
收稿日期: 2010-11-19 出版日期: 2011-04-01
ZTFLH:  Q814.1  
基金资助:

国家"973"计划(2009CB724700)、中国科学院知识创新工程重要方向项目 (KSCX2-YW-G-073)、浙江省自然科学基金 (Y407353)、浙江省公益技术研究项目(2010C31099)资助项目

通讯作者: 程小飞     E-mail: xthci@sina.com
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引用本文:

程小飞, 章表明, 薛松, 肖通虎. 超滤膜分离纯化珊瑚藻溴过氧化物酶[J]. 中国生物工程杂志, 2011, 31(03): 76-80.

CHENG Xiao-fei, ZHANG Biao-ming, XUE Song, XIAO Tong-hu. Isolation and Purification of Bromoperoxidase from Corallina Officinalis Using Ultrafiltration Membrane. China Biotechnology, 2011, 31(03): 76-80.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I03/76

[1] Dembitsky V M. Oxidation, epoxidation and sulfoxidation reactions catalysed by haloperoxidases. Tetrahedron, 2003, 59(26): 4701-4720.
[2] Andersson M, Willetts A, AIlenmark S. Asmmetric sulfoxidation catalyzed by a vanadium-containing bromopemxidase. J Org Chem, 1997, 62(24): 8455-8458.
[3] Butler A. Vanadium haloperoxidases. Curr Opin Chem Biolog, 1998, 2(2): 279-285.
[4] Moore C A, Okuda R K. Bromopemxidase activity in 94 speciesof marine algae. J Nat Toxins, 1996, 5(1): 295-305.
[5] Manthey J A, Hager L P. Purification and properties of bromoperoxidase from Penicillus capitatus. J Biol Chem, 1981, 256(21): 11232-11238.
[6] Sheffield D J, Harry T, Smith A J, et al. Purification and characterization of the vanadium bromoperoxidase from the macroalga Corallina officinalis. Phytochemistry, 1993, 32(1): 21-26.
[7] 陈全省, 赵杰文, 岳鹏翔. 中空纤维超滤膜分离菜籽饼粕中蛋白质的实验. 农业机械学报, 2006, 37(8): 1-4. Chen Q S, Zhao W J, Yue Q P. Transactions of the Chinese Society for Agricultural Machinery, 2006, 37(8): 1-4.
[8] 王健, 张雪霞, 王海燕. 超滤法提纯万古霉素的应用. 生物加工过程, 2010, 8(4): 17-21. Wang J, Zhang X X, Wang H Y. Chinese Journal of Bioprocess Engineering, 2010, 8(4): 17-21.
[9] Wan J B, Liu J G, Lu J R, et al. Isolation and purification of superoxide dismutase from garlic using two-stage ultrafiltration. Journal of Membrane Science, 2010, 352(1): 231-238.
[10] Liu J G, Lu J R, Zhao X B, et al. Separation of glucose oxidase and catalase using ultrafiltration with 300-kDa polyethersulfone membranes. Journal of Membrane Science, 2007, 299(1): 222-228.
[11] 薛长湖, 管华诗, 于宜法. 现代生物工程分离提取技术在海洋生物资源开发上的应用. 生物工程进展, 1996, 16(6): 49-53. Xue C H, Guan H S, Yu Y F. China Biotechnology, 1996, 16(6): 49-53.
[12] Lin S H, Hung C L, Juang R S. Effect of operating parameters on the separation of proteins in aqueous solutions by dead-end ultrafiltration. Desalination, 2008, 234(1): 116-125.
[13] 刘大川, 杨国燕, 翁利荣. 超滤膜法制备大豆分离蛋白工艺研究. 中国油脂, 2003, 28(11): 30-34. Liu D C, Yang G Y, Weng L R. China Oils and Fats, 2003, 28(11): 30-34.
[14] Mukai Y, Iritani E, Murase T. Fractionation characteristics of binary protein mixture by ultrafiltration. Sep Sci Tech, 1998, 33 (2): 169-185.
[15] Zhang B M,Cao X P,Cheng X F, et al. Efficient purification with high recovery of vanadium bromoperoxidase from Corallina officinalis. Biotechnol Lett,2011,33(1):545-548.
[16] Itoh N, Izumi Y. Characterization of nonheme type bmmpemxidase in Corallina pilulifera.J Biol Chem, 1986, 26l(1): 5194-5200.
[17] Lowry O H, Rosebrough N J, Lewisfarr A. Protein measurement with the Folin phenol reagent. J Biol Chem, 195l, 193(1): 265-275.
[18] 汪家政, 范明. 蛋白质技术手册. 北京: 科学出版社, 2000, 77-122. Wang J Z, Fan M. Protein Technical Manual. Beijing:Science Press, 2000, 77-122.
[19] Michail N I, Aandrew R D, Amanda A B, et al. Crystal structure of dodecameric vanadiumdependent bromoperoxidase from the red algae Corallina officinalis. J Mol Biol, 2000, 299(1):1035-1049.

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