南京师大学报（自然科学版）2017，Vol.40Issue(2)：76-82,7.DOI:10.3969/j.issn.1001-4616.2017.02.013
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
Expression, Purification, Biochemical Characterization and Application of Recombinant Bacillus subtilis Aldolase
摘要
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.关键词
FBA/分离纯化/酶学性质/FDP/单酶法Key words
FBA/separation and purification/biochemical characterization/FDP/DNPH-FBA method分类
生物科学引用本文复制引用
高品,赵杰,韦光绪,殷志敏..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].南京师大学报（自然科学版）,2017,40(2):76-82,7.基金项目
江苏省科技厅前瞻性研究项目(BY2013001-03). （BY2013001-03）
