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.
Original language | English |
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Pages (from-to) | 261-266 |
Number of pages | 6 |
Journal | Enzyme and Microbial Technology |
Volume | 45 |
Issue number | 4 |
DOIs | |
State | Published - 7 Oct 2009 |
Keywords
- Aquifex aeolicus
- Circular dichroism
- Class II aldolase
- Hyperthermophile
- Thermostability