«Previous Article|Table of Contents|Next Article»

《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

Issue:

2017年02期

Page:

76-

Research Field:

·生命科学·

Publishing date:

Info

Title:

Expression,Purification,Biochemical Characterization andApplication of Recombinant Bacillus subtilis Aldolase

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

Keywords:

FBA; separation and purification; biochemical characterization; FDP; DNPH-FBA method

PACS:

Q819

DOI:

10.3969/j.issn.1001-4616.2017.02.013

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.

References:

[1] OGAWA H,SHIRAKI H,NAKAGAWA H. Study on the regulatory role of fructose-1,6-diphosphate in the formation of AMP in rat skeletal muscle. A mechanism for synchronization of glycolysis and the purine nucleotide cycle[J]. Biochem Biophys Res Commun,1976,68(2):524-528.
[2]PARK J Y,KIM E J,KWON K J,et al. Neuroprotection by fructose-1,6-bisphosphate involves ROS alterations via p38 MAPK/ERK[J]. Brain research,2004,1 026(2):295-301.
[3]GáMEZ A,ALVA N,ROIG T,et al. Beneficial effects of fructose 1,6-biphosphate on hypothermia-induced reactive oxygen species injury in rats[J]. European journal of pharmacology,2008,590(1/3):115-119.
[4]VAXILLAIRE M,CAVALCANTI-PROEN A C,DECHAUME A,et al. The common P446L polymorphism in GCKR inversely modulates fasting glucose and triglyceride levels and reduces type 2 diabetes risk in the DESIR prospective general French population[J]. Diabetes,2008,57(8):2 253-2 257.
[5]LIAN X Y,KHAN F A,STRINGER J L. Fructose-1,6-bisphosphate has anticonvulsant activity in models of acute seizures in adult rats[J]. Journal of neuroscience,2007,27(44):12 007-12 011.
[6]STEPANENKO B N,BOBROVA L N. Method of production of stable forms of sodium salt of fructose 1,6-diphosphate(sodium-DFF)[J]. Biokhimiia,1957,22(6):1 019-1 022.
[7]占达东,李开鸿. 果糖二磷酸钠的紫外吸光光度法测定[J]. 琼州学院学报,2004,11(2):38-39.
[8]MICHAEL G. D-Fructose 1,6-bisphosphate,dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate[M]//BEYGMEYER H U. Methods of enzymatic analysis. Germany:Verlag Chemic,1984.
[9]FUSHINOBU,NISHIMASU S,HATTORI H,et al. Structural basis for the bifunctionality of fructose-1,6-bisphosphate aldolase/phosphatase[J]. Nature,2011,478(7 370):538-541.
[10]STOLZENBERGER J,LINDNER S N,WENDISCH V F. The methylotrophic Bacillus methanolicus MGA3 possesses two distinct fructose 1,6-bisphosphate aldolases[J]. Microbiology,2013,159(Pt 8):1 770-1 781.
[11]TONKIN M L,HALAVATY A S,RAMASWAMY R,et al. Structural and functional divergence of the aldolase fold in Toxoplasma gondii[J]. J Mol Biol,2015,427(4):840-852.
[12]PROMPIPAK J,SENAWONG T,JOKCHAIYAPHUM K,et al. Characterization and localization of opisthorchis viverrini fructose-1,6-bisphosphate aldolase[J]. Parasitol Int,2016,S1383-5769(16):30 162-30 163.
[13]GIL’MIIAROVA F N,BABICHEV A V. Properties of fructose-1,6-diphosphate aldolase from human muscles in atherosclerosis[J]. Biull Eksp Biol Med,1982,93(3):35-36.
[14]DRBELI H,TRZECIAK A,GILLESSEN D,et al. Expression,purification,biochemical characterization and inhibition of recombinant Plasmodium falciparum aldolase[J]. Molecular and biochemical parasitology,1990,41(1 990):259-268.

Memo

Memo:

-

Common functions

Last Update: 2017-06-30