[1]陈宏文,聂金峰,方柏山.克雷伯杆菌1,3-丙二醇氧化还原酶的分离纯化及其酶学性质[J].华侨大学学报(自然科学版),2009,30(1):62-66.[doi:10.11830/ISSN.1000-5013.2009.01.0062]
　CHEN Hong-wen,NIE Jin-feng,FANG Bai-shan.Purification and Characterization of 1,3-Propanediol Oxidoreductase from Klebsiella pneumoniae[J].Journal of Huaqiao University(Natural Science),2009,30(1):62-66.[doi:10.11830/ISSN.1000-5013.2009.01.0062]
点击复制

克雷伯杆菌1,3-丙二醇氧化还原酶的分离纯化及其酶学性质()
分享到：

《华侨大学学报（自然科学版）》[ISSN:1000-5013/CN:35-1079/N]

卷:
第30卷
期数:
2009年第1期
页码:
62-66
栏目:
出版日期:
2009-01-20

文章信息/Info

Title:
Purification and Characterization of 1,3-Propanediol Oxidoreductase from Klebsiella pneumoniae
文章编号:
1000-5013(2009)01-0062-05
作者:
陈宏文; 聂金峰; 方柏山
华侨大学工业生物技术福建省高等学校重点实验室
Author(s):
CHEN Hong-wen;  NIE Jin-feng;  FANG Bai-shan
Key Laboratory of Industrial Biotechnology of Fujian Province University, Huaqiao University, Quanzhou 362021, China
关键词:
1; 3-丙二醇氧化还原酶;  克雷伯杆菌;  甘油脱氢酶;  酶学性质;  纯化
Keywords:
1; 3-propanediol oxidoreductase;  Klebsiella pneumoniae;  characterization;  purification
分类号:
TQ426.97
DOI:
10.11830/ISSN.1000-5013.2009.01.0062
文献标志码:
A
摘要:
在有氧条件下,利用DEAE Sepharose Fast Flow弱阴离子离子交换层析和Blue Sepharose CL-6B亲和层析,同时分离并提纯克雷伯杆菌胞内的1,3-丙二醇氧化还原酶和甘油脱氢酶.研究表明,1,3-丙二醇氧化还原酶的纯化倍数为35.86倍,回收率为5.17%,该酶最适表观反应温度为57℃,最适反应pH值为9.5.在30℃及pH=8.0~10.0时,该酶具有良好的稳定性.在45℃和pH=9.5条件下,该酶以1,3-丙二醇和NAD+为底物,其米氏常数Km分别为15.8,0.2 mmol.L-1.1,3-丙二醇氧化还原酶对生理反应底物3-羟基丙醛活性最大,对其他醇类也有氧化能力.Mn2+对酶有显著激活作用,巯基保护剂能明显提高酶的活力.
Abstract:
1,3-propanediol oxidoreductase from Klebsiella pneumoniae was purified by DEAE sepharose fast flow ion-exchange chromatography and Blue Sepharose CL-6B affinity chromatography under aerobic conditions.A 35.86-fold purification was obtained with the recovery of 5.17% activity.The optimum temperature and pH of the enzyme activity were 57 ℃ and 9.5 of pH value.At 30 ℃ and at range of pH 8.0~10.0,1,3-propanediol oxidoreductase was stable.At 45 ℃ and pH 9.5 the Km for 1,3-propanediol and NAD+ were 15.8 mmol·L-1 and 0.2 mmol·L-1,respectively.The enzyme oxidized other alcohols such as pentanol,propanol,ethylene glycol and 1,4-butanediol,besides physiological substrate 3-hydroxypropionaldehyde.The enzyme was significantly activated by Mn2+.Reducing agents were able to enhence the activity of 1,3-propanediol oxidoreductase.

参考文献/References:

[1] AHRENS K, MENZEL K, ZENG A P. Kinetic dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture(Ⅲ):Enzymes and fluxes of glycerol dissimilation and 1, 3-propanediol formation [J]. Biotechnology and Bioengineering, 1998(5):544-552.
[2] 陈宏文, 王蔚, 方柏山. 克雷伯杆菌生产1, 3-丙二醇关键酶发酵条件研究 [J]. 高校化学工程学报, 2004(5):621-627.doi:10.3321/j.issn:1003-9015.2004.05.016.
[3] 陈宏文, 洪其文, 徐晶. 克雷伯杆菌有氧发酵利用甘油生产3-羟基丙醛的研究 [J]. 现代化工, 2004, (11):36-39.doi:10.3321/j.issn:0253-4320.2004.11.009.
[4] 陈宏文, 王蔚, 方柏山. 克雷伯杆菌生产1, 3-丙二醇关键酶酶活分析方法研究 [J]. 广州食品工业科技, 2004(3):5-9.doi:10.3969/j.issn.1673-9078.2004.03.002.
[5] 汪家政. 蛋白质技术手册 [M]. 北京:科学出版社, 2000.42-47.
[6] MALAOUI H, MARCZAK R. Separation and characterization of the 1, 3-propanediol and glycerol dehydrogenase activities from Clostridium butyricum E5 wild type and mutant D [J]. Journal of Applied Microbiology, 2001, (6):1006-1014.doi:10.1046/j.1365-2672.2001.01335.x.
[7] DANIEL R, BOENIGK R, GOTTSCHALK G. Purification of 1, 3-propanediol dehydrogenase from Citrobacter freundii and cloning, sequencing, and overexpression of the corresponding gene in Escherichia coli [J]. Journal of Bacteriology, 1995, (8):2151-2156.
[8] VEOGA-DA-CUNHA M, FOSTER M A. 1, 3-propanediol:NAD+ oxidoreductase of Lactobacillus brevis and Lactobacillus buchneri [J]. Applied and Environmental Microbiology, 1992.2005-2010.

相似文献/References:

[1]张光亚,陈迪,方柏山.1,3-丙二醇脱氢酶三级结构的模建[J].华侨大学学报(自然科学版),2004,25(4):426.[doi:10.3969/j.issn.1000-5013.2004.04.021]
　Zhang Guangya,Chen Di,Fang Baishan.Modeling the Tertiary Structure of 1,3-Propandiol Dehydrogenase[J].Journal of Huaqiao University(Natural Science),2004,25(1):426.[doi:10.3969/j.issn.1000-5013.2004.04.021]
[2]朱春杰,方柏山.微生物转化法生产1,3-丙二醇的研究进展[J].华侨大学学报(自然科学版),2009,30(5):481.[doi:10.11830/ISSN.1000-5013.2009.05.0481]
　ZHU Chun-jie,FANG Bai-shan.Progress on the Production of 1,3-Propanediol by Microbial Conversion[J].Journal of Huaqiao University(Natural Science),2009,30(1):481.[doi:10.11830/ISSN.1000-5013.2009.05.0481]
[3]王文研,张光亚.ELPs-PDOR融合基因表达条件的优化[J].华侨大学学报(自然科学版),2012,33(3):296.[doi:10.11830/ISSN.1000-5013.2012.03.0296]
　WANG Wen-yan,ZHANG Guang-ya.Optimization of Cultivation Conditions of ELPs-PDOR Fusion Gene[J].Journal of Huaqiao University(Natural Science),2012,33(1):296.[doi:10.11830/ISSN.1000-5013.2012.03.0296]
[4]罗菊香,王晓霞,方柏山,等.重组PDOR基因的表达与纯化及部分酶学性质[J].华侨大学学报(自然科学版),2009,30(2):166.[doi:10.11830/ISSN.1000-5013.2009.02.0166]
　LUO Ju-xiang,WANG Xiao-xia,FANG Bai-shan,et al.Expression,Purification and Characterization of 1,3-Propanediol Oxidoreductase from Klebsiella pneumoniae[J].Journal of Huaqiao University(Natural Science),2009,30(1):166.[doi:10.11830/ISSN.1000-5013.2009.02.0166]

备注/Memo

备注/Memo:
国家重点基础研究发展(973)计划项目(2007CB707804); 国家高技术研究发展(863)计划项目(2006AA-020103); 国家自然科学基金资助项目(20676048); 福建省自然科学基金计划资助项目(E0510018)
更新日期/Last Update: 2014-03-23