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

Hua Xu,Xuebing Zhang,Zhimin Li,Pengfei Zhang,Yuchen Zhang,Zebin Liu,Yuguang Du,Jian-Jun Li

doi:10.3390/ijms21062100

Abstract

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-α-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 °C, and retained >42.2% of maximum activity between 20 and 80 °C. It was stable below 40 °C and lost activity very quickly above 50 °C. Its apparent kinetic parameters against PGA (polygalacturonic acid) were determined, with the Km and kcat values of 8.3 mg/mL and 5640 μmol/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.

Highlights

Pectin is one of the most important components in the middle lamella and cell wall of plants, and accounts for one-third of the dry weight of plant material [1,2,3]
Though many polysaccharide-degrading enzymes from A. nidulans were characterized, so far no endo-PGs from this fungus have been characterized in detail [28,29]
Modelling was performed by using I-TASSER [32], and three enzymes were used as templates, including endo-polygalacturonase from Aspergillus aculeatus (PDB ID: 1IA5 and 1IB4) [33], endo-polygalacturonase II from Aspergillus niger (PDB ID: 1CZF) [30], and endo-polygalacturonase from Colleotrichum lupini (PDB ID: 2IQ7) [31]. This newly characterized endo-polygalacturonases from A. nidulans exhibited moderate activity under acidic conditions and good stability over a wide range of pH and below 40 ◦C. This pectinase has great potential to be used in the fields where acidic endo-polygalacturonases are required

Summary

Introduction

Pectin is one of the most important components in the middle lamella and cell wall of plants, and accounts for one-third of the dry weight of plant material [1,2,3]. It plays multiple functions during plant growth, including morphogenesis, defense, cell adhesion, cell wall structure, cellular expansion, and so on [2,4]. Most characterized endo-PGs are from fungi, such as the species of Aspergillus and Penicillium. Xz8 [22], Talaromyces leycettanus JCM 12802 [23], and Thielavia arenaria XZ7 [24]

Methods

Results

Conclusion