Expression of the gene encoding oxalate decarboxylase from Bacillus subtilis and characterization of the recombinant enzyme

ABSTRACT The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. The nirA gene was overexpressed inEscherichia coli and was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated from A. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated from A. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.

Download Full-text

Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme

Biotechnology Research International ◽

10.1155/2012/951267 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 14

Author(s):

Dominic W. S. Wong ◽

Victor J. Chan ◽

Amanda A. McCormack ◽

Ján Hirsch ◽

Peter Biely

Keyword(s):

Glucuronic Acid ◽

Active Form ◽

Schizophyllum Commune ◽

Constitutive Expression ◽

Recombinant Enzyme ◽

Cloning And Expression ◽

Gene Encoding ◽

Glucuronoyl Esterase ◽

Esterase Gene

The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53 kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were Km 0.25 mM, Vmax 16.3 μM⋅min−1, and kcat 9.27 s−1 with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-β-D-xylopyranoside as the substrate.

Download Full-text