Overexpression and Biochemical Characterization of an Endo-α-1,4-polygalacturonase from Aspergillus nidulans in Pichia pastoris .

Pectinases have many applications in the industry of food, paper, and textiles, therefore finding novel polygalacturonases is required. Multiple sequence alignment and phylogenetic analysis of AnEPG (an endo-alpha-1,4-polygalacturonase from Aspergillus nidulans) and other GH 28 endo-polygalacturonases suggested that AnEPG is different from others. AnEPG overexpressed in Pichia pastoris was characterized. AnEPG showed the highest activity at pH 4.0, and exhibited moderate activity over a narrow pH range (pH 2.0-5.0) and superior stability in a wide pH range (pH 2.0-12.0). It displayed the highest activity at 60 degrees C, and retained >42.2% of maximum activity between 20 and 80 degrees C. It was stable below 40 degrees C and lost activity very quickly above 50 degrees C. Its apparent kinetic parameters against PGA (polygalacturonic acid) were determined, with the K-m and k(cat) values of 8.3 mg/mL and 5640 mu mol/min/mg, respectively. Ba2+ and Ni2+ enhanced activity by 12.2% and 9.4%, respectively, while Ca2+, Cu2+, and Mn2+ inhibited activity by 14.8%, 12.8%, and 10.2% separately. Analysis of hydrolysis products by AnEPG proved that AnEPG belongs to an endo-polygalacturonase. Modelled structure of AnEPG by I-TASSER showed structural characteristics of endo-polygalacturonases. This pectinase has great potential to be used in food industry and as feed additives.