[1]高 品,赵 杰,韦光绪,等.枯草芽孢杆菌醛缩酶的克隆表达、纯化、酶学性质研究及应用[J].南京师范大学学报(自然科学版),2017,40(02):76.[doi:10.3969/j.issn.1001-4616.2017.02.013]
　Gao Pin,Zhao Jie,Wei Guangxu,et al.Expression,Purification,Biochemical Characterization andApplication of Recombinant Bacillus subtilis Aldolase[J].Journal of Nanjing Normal University(Natural Science Edition),2017,40(02):76.[doi:10.3969/j.issn.1001-4616.2017.02.013]
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枯草芽孢杆菌醛缩酶的克隆表达、纯化、酶学性质研究及应用()
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《南京师范大学学报》（自然科学版）[ISSN:1001-4616/CN:32-1239/N]

卷:
第40卷
期数:
2017年02期
页码:
76
栏目:
·生命科学·
出版日期:
2017-06-29

文章信息/Info

Title:
Expression,Purification,Biochemical Characterization andApplication of Recombinant Bacillus subtilis Aldolase
文章编号:
1001-4616(2017)02-0076-07
作者:
高 品; 赵 杰; 韦光绪; 殷志敏
南京师范大学生命科学学院,生物化学与生物制品研究所,江苏省分子与医学生物技术重点实验室,江苏 南京 210023
Author(s):
Gao Pin; Zhao Jie; Wei Guangxu; Yin Zhimin
School of Life Sciences,Nanjing Normal University,Institute of Biochemistry and Biological Products,Jiangsu Key Laboratory for Molecular and Medical Biotechnology,Nanjing 210023,China
关键词:
FBA; 分离纯化; 酶学性质; FDP; 单酶法
Keywords:
FBA; separation and purification; biochemical characterization; FDP; DNPH-FBA method
分类号:
Q819
DOI:
10.3969/j.issn.1001-4616.2017.02.013
文献标志码:
A
摘要:
果糖-1,6二磷酸醛缩酶是糖酵解途径中的关键酶,可催化果糖-1,6二磷酸(FDP)分解成磷酸二羟丙酮(DAP)和三磷酸甘油醛(G3P)的可逆反应. 本文首次克隆枯草芽孢杆菌中的果糖-1,6二磷酸醛缩酶基因(fba),构建原核重组表达载体,利用Ni-NTA柱对重组枯草芽孢杆菌果糖-1,6二磷酸醛缩酶(FBA)进行分离纯化和酶学性质研究,构建表达载体pET28a(+)-fba转化入BL21(DE3)宿主内,在30 ℃下诱导表达,通过SDS-PAGE检测FBA纯度和分子量,FBA分子量约为35 kDa. 最适反应条件为pH 7.5、35 ℃,金属离子K+、Na+、Fe2+对该酶有较好的激活作用,而Zn2+、Mn2+、Ca2+、Mg2+对该酶有一定的抑制作用. 基于FBA可专一性分解FDP的基本原理,建立了快速、准确测定酵母发酵液中FDP含量的单酶法. 通过单酶法测定FDP含量的样品回收率为99.54%,相对标准偏差为0.427%,测定结果基本不受样品中其他成分干扰. T检验结果表明,单酶法和多酶法对发酵液中FDP含量的检测结果无显著差异,与紫外分光光度法测定的结果存在显著差异,这说明单酶法可以准确测定发酵液中FDP的含量. 单酶法测定FDP含量具有快速、准确、灵敏的特点,不仅适合于成分复杂的发酵液中FDP含量的测定,也具有广泛的应用价值.
Abstract:
Fructose 1,6-biphosphate aldolase(FBA)is glycolytic enzyme that catalyze the reversible conversion of fructose-1,6-disphosphate(FDP)to glyceraldehyde-3-phosphate(G3P)and dihydroxyacetone phosphate(DAP). A fba gene(fba)from Bacillius subtilis encoding a monofunctional FBA was firstly cloned and expressed in Escherichia coli(E.coli). The recombinant FBA was purified by Ni-NTA column. Protein expression were induced under 30 ℃ and analyzed by 12% SDS-PAGE. The recombinant FBA molecular weight was about 35 kDa. The optimal reaction temperature and pH of this recombinant enzyme were 35 ℃ and 7.5,respectively. Furthermore,the effect of metal ions on recombinant enzyme was determined. K+,Na+and Fe2+ could promote the activity of this enzyme while Zn2+,Mn2+,Ca2+ and Mg2+had a strong inhibitory effect on it. Based on the specificity of FBA in catalyzing FDP to G3P and DAP,we set up a method that can be used for determination of FDP concentration in the fermentation broth by using 2,4-dinitrophenylhydrazine(DNPH)as substrate and recombinant FBA as enzyme. Compared with multienzyme method and spectrophotometry method,the result of this method showed that the recovery rate of FDP was 99.54% and the relative standard deviation was 0.427%. T-test showed that there was no significant difference between the result of multienzyme method and DNPH-FBA method. This result means that DNPH-FBA method can be used as a fast and simple way to detect FDP concentration in FDP mass production process.

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期:2016-12-25.
基金项目:江苏省科技厅前瞻性研究项目(BY2013001-03).
通讯联系人:殷志敏,教授,博士生导师,研究方向:生物化学及细胞生物学. E-mail:yinzhimin@njnu.edu.cn
更新日期/Last Update: 2017-06-30