Expression of pectate lyase A from Aspergillus nidulans in Bacillus subtilis

Abstract

Pectate lyases catalyze the eliminative cleavage of de-esterified pectin, which is a major component of the primary cell walls of many higher plants. The gene encoding Aspergillus nidulans pectate lyase A (PelA) without signal peptide was fused with a six-His tag at its C-terminus in the expression vector VBSzqx containing Sac b signal peptide and p43 promotor. The VBSzqx-pelA plasmid was transformed into Bacillus subtilis, an expression host that is safe, free of any endotoxin, has a potential for large-scale production of foreign proteins in food industry. PelA was successfully expressed, and effectively purified using a Ni²⁺-nitrilotriacetate-agarose column. Shaken in flasks, the medium produced the maximum enzyme production of 620 U ml⁻¹ medium at 30°C, 200 rpm, and starting pH 7.0. Higher enzyme production (985 U ml⁻¹ medium) was reached in 2.5-l fermentor at 30°C, pH 7.0, 500 rpm, and 2.0 l min⁻¹ airflow rate. The expressed PelA exhibited its optimal activity at pH 8.5 and 50°C. The V _max and K _m of recombinant PelA were 77 μmol min⁻¹ mg⁻¹ and 0.50 mg ml⁻¹.