Identification and characterization of thermostable endo‐polygalacturonase II B from Aspergillus luchuensis

Abstract

Endo‐polygalacturonase II B (PgaB) from Aspergillus luchuensis was orthologous to endo‐polygalacturonase from Aspergillus niger with mutant sites Thr42Ser and Glu52Ala. Mature pgaB gene was cloned from the genomic DNA of A. luchuensis and secreted expressed with over 90% purity in Pichia Pastoris and reached 1.0 g/L after 144 hr culture. The recombinant PgaB was further purified by Ni‐NTA chromatography. Using polygalacturonic acid (PGA) as substrate, the optimal condition for PgaB activity was 40°C and pH 4.5, respectively. Km and Vmax of PgaB were 0.19 mmol/l and 103.58 μmol min−1 mg−1, respectively. The relative activity of PgaB remained more than 60% and 40% of maximum activity at 50 and 60°C for 7 hr. PgaB increased the light transmittance by 85% and showed high efficiency in juice clarification. The main product was galacturonic acid oligosaccharides with degrees of polymers (DP) 1–3. The PgaB is a potential pectinolytic enzyme in food industries. Practical applications Endo‐polygalacturonase II B (PgaB) was identified from Aspergillus luchuensis, a filamentous fungus widely used in food and beverage fermentation in East Asia. PgaB still kept its most activity at 60°C for 7 hr. Polygalacturonic acid (PGA) can be digested effectively by the PgaB and the main products are galacturonic acid oligosaccharides with degrees of polymers (DP) 1–3. PgaB shows high efficiency in juice clarification. The PgaB is a potential pectinolytic enzyme for the applications in food industries.

A review on pectinase properties, application in juice clarification, and membranes as immobilization support

“…and Fusarium oxysporum MTCC 1755 (OT-50 • C) showed substantial stability at optimum temperature for 2 h of incubation time (Dixit et al, 2013;Guan et al, 2020;He et al, 2018;Tan et al, 2020;Yadav et al, 2017;Zhou et al, 2017). Thermophile isolates can be a vital tool in high-temperature biochemical processes.…”

“…Pectic polymers occur in the middle lamella of plant cell walls, promoting the structural stability in plants and safeguarding them against phytopathogens (Wang et al., 2018). Pectinaceous compounds are colloidal polysaccharides of 1, 4 glycosidic linked α–D galacturonic acid monomers (Tan et al., 2020). The reactive groups like acetyl or methoxyl group substitute the hydrogen atom of galacturonic acid, causing the esterification of polysaccharide backbone.…”

“…To determine the thermal stability, an enzyme is exposed to a certain temperature for a varying period and the residual activity of the enzyme is estimated at standard assay conditions at the completion of incubation time. Aspergillus luchuensis endoPG (OT‐40°C), Datura stramonium PME (OT‐50°C), PL from Bacillus subtilis PB1 (OT‐50°C), Escherichia coli (OT‐50°C), PNL from A. niger (OT‐50°C), and Fusarium oxysporum MTCC 1755 (OT‐50°C) showed substantial stability at optimum temperature for 2 h of incubation time (Dixit et al., 2013; Guan et al., 2020; He et al., 2018; Tan et al., 2020; Yadav et al., 2017; Zhou et al., 2017). Thermophile isolates can be a vital tool in high‐temperature biochemical processes.…”

“…EndoPG from P. oxalicum CZ1028, Penicillium occitanis , and Pichia pastoris is stimulated in the presence of magnesium ions (Cheng et al., 2016; Sassi et al., 2016; Yang et al., 2011). However, A. luchuensis and S. purpureum endoPG activity degraded in the presence of magnesium ions (Carli et al., 2019; Tan et al., 2020). ExoPG also observed the same behavior in the presence of magnesium ions (Anand et al., 2017; Chen et al., 2014; Kant et al., 2013; Lu et al., 2016; Pagnonceli et al., 2019).…”

“…ExoPG also observed the same behavior in the presence of magnesium ions (Anand et al., 2017; Chen et al., 2014; Kant et al., 2013; Lu et al., 2016; Pagnonceli et al., 2019). Cations of cadmium, copper, lead, tin, mercury, and silver showed inhibitory effects on pectinases activity (Pagnonceli et al., 2019; Poturcu et al., 2017; Saoudi et al., 2015; Tan et al., 2020). The presence of Hg +2 oxidized the thiol group in cysteine residue near the enzyme's active site, hindering the enzymatic catalysis (Lu et al., 2016).…”