Cloning, Expression, Purification and Characterization of Fructose-1,6-bisphosphate Aldolase from Anoxybacillus gonensis G2

Abstract

The fructose-1,6-bisphosphate aldolase gene from the thermophilic bacterium, Anoxybacillus gonensis G2, was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame coding for a 30.9 kDa protein of 286 amino acids. The amino acid sequence shared approximately 80-90% similarity to the Bacillus sp. class II aldolases. The motifs that are responsible for the binding of a divalent metal ion and catalytic activity completely conserved. The gene encoding aldolase was overexpressed under T7 promoter control in Escherichia coli and the recombinant protein purified by nickel affinity chromatography. Kinetic characterization of the enzyme was performed at 60 degrees C, and K(m) and V(max) were found to be 576 microM and 2.4 microM min(-1) mg protein(-1), respectively. Enzyme exhibits maximal activity at pH 8.5. The activity of enzyme was completely inhibited by EDTA.

Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphatase from the Facultative Ribulose Monophosphate Cycle Methylotroph Bacillus methanolicus

“…Their similar thermal stabilities and temperature optima correlate well with the physiology of thermophilic B. methanolicus, which is able to grow at temperatures of between 35°C and 60°C (1). It is peculiar that the optimal temperature, especially for GlpX C , in the in vitro activity assay was higher than the temperature at which the enzyme is stable, but this is not unprecedented and has also been described for both FBAs of B. methanolicus (10) and for the FBAs of Bacillus stearothermophilus (53) and Anoxybacillus gonensis (54). However, a diverse stability of the GlpX enzymes in vitro cannot be excluded.…”

Characterization of a novel xylose isomerase from Anoxybacillus gonensis G2T

“…This is the reason why XI is also known as glucose isomerase (Jensen and Rugh, 1987;De Raadt et al, 1994). The fact that the enzyme isomerizes xylose to xylulose means that it could be used industrially for producing ethanol from hemicellulose (Wang et al, 1980;Ertunga et al, 2007;Karaoglu et al, 2013). Xylose, one of the major fermentable sugars in nature, is, after glucose, the second most abundant sugar in lignocellulosic biomass.…”

“…The enzyme has been isolated from many microorganisms and is well studied (Chen, 1980;Schellenberg et al, 1984;Wilhelm and Hollenberg, 1985;Saari et al, 1987;Amore and Hollenberg, 1989;Kikuchi et al, 1990;Dekker et al, 1991). Besides its commercial importance, XI is also an ideal enzyme for studying structure-function relationships from an academic perspective (Ertunga et al, 2007;Karaoglu et al, 2013).…”

A new xylanase from thermoalkaline Anoxybacillus sp. E2 with high activity and stability over a broad pH range

“…Some enzymes with favorable properties from Anoxybacillus have been reported. For example, the fructose-1,6-bisphosphate aldolase (Ertunga et al 2007) and thermoalkaliphilic esterase (Colak et al 2005) from A. gonensis G2 exhibited maximal activity at pH 8.5 and 60°C, and pH 7.5 and 60°C, respectively. In this study, we isolated a thermostable and alkali-tolerant strain, Anoxybacillus sp.…”