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Characterization of a extreme thermostable fructose-1,6-bisphosphate aldolase from hyperthermophilic bacterium Aquifex aeolicus

Paper ID Volume ID Publish Year Pages File Format Full-Text
17685 42689 2009 6 PDF Available
Title
Characterization of a extreme thermostable fructose-1,6-bisphosphate aldolase from hyperthermophilic bacterium Aquifex aeolicus
Abstract

Fructose-1,6-bisphosphate (FBP) aldolase, is a glycolytic enzyme that catalyzes the reversible condensation reaction of FBP to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). The aldolase gene from Aquifex aeolicus was subcloned, overexpressed in E. coli and purified to 95% homogeneity. The purified enzyme was activated by high concentrations of NH4+ and low concentrations of Co2+. The native molecular weight of the purified FBP aldolase was identified as 67 kDa (dimer) by gel filtration chromatography. The enzyme exhibits optimum pH at 6.5 and temperature at 90 °C. Based on the kinetic characterizations, the apparent Km was calculated to be 4.4 ± 0.07 mM, while Vmax was found to be 100 ± 0.02 μM min−1 mg protein−1. The recombinant protein showed extreme heat stability; no activity loss was observed even at 100 °C for 2 h. In addition, the thermophilic enzyme also showed higher stability against several organic solvents viz. acetonitrile, 1,4-dioxane, and methanol. With higher stability against both heat and organic solvents than any other class II aldolase, the A. aeolicus FBP aldolase is an attractive enzyme for use as a biocatalyst for industrial applications.

Keywords
FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde-3-phosphate; MES, 2-[N-morpholino]ethansulfonic acid; DMSO, dimethyl sulfoxideAquifex aeolicus; Circular dichroism; Class II aldolase; Hyperthermophile; Thermostability
First Page Preview
Characterization of a extreme thermostable fructose-1,6-bisphosphate aldolase from hyperthermophilic bacterium Aquifex aeolicus
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 45, Issue 4, 7 October 2009, Pages 261–266
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering