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-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.


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]. As one of the most complex polysaccharides in plant cell walls, pectin exists in a variety of structures such as homogalacturonan (HG), xylogalacturonan, rhamnogalacturonan I and rhamnogalacturonan II, among which HG is the most abundant (<65%) [2,3].

Sequence Alignment and Phylogenetic Analysis of AnEPG with Other GH 28 endo-PGs
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]. According to the genome sequence of Aspergillus nidulans FGSC A4, the gene (Gene ID: 2868744) encoding a hypothetical protein (GenBank accession number AN8327.2) belonging to the GH 28 family, was named as AnEPG (endo-α-1,4-polygalacturonase from Aspergillus nidulans) in the current study. A 19-residue signal peptide with a putative processing site (VMA-TP) was identified using the SignalP 4.1 server. The potential Oand N-glycosylation sites were predicted to be Thr27, Thr28, Ser30, Thr32, Ser38, and Asn154, Asn192, Asn371, respectively.
Structural models of AnEPG based on homologous enzymes were obtained by the I-TASSER server [32]. Five top ranking 3D models were generated. Each model was validated based on C-score (confidence score), TM-score (template modeling score), RMSD (the root-mean-square deviation), and cluster density. In general, models with C-score > −1.5 have a correct fold [32]. Model 1 had the highest C-score (1.69) value reflecting a model of better quality (TM-score = 0.95 ± 0.05 and RMSD = 3.1 ± 2.2 Å) (Figure 1). Similarly to homologous GH28 endo-PGs [30,31,33], the predicted three-dimensional structure of AnEPG was a right handed parallel β-helix with 12 (10 complete) turns, in which the β-strands were separated by turns that consisted of either a sharp bend or a loop ( Figure 1). Based on structural and sequence alignment of AnEPG and AaEPG from Aspergillus aculeatus and pga II from Aspergillus niger, residues Asp194, Asp215, Asp216, and His237 of AnEPG, which are the equivalent of Asp159, Asp180, Asp181, and His202 of AaEPG, and Asp180, Asp201, Asp202, and His223 of pga II, were predicted to be involved in catalysis ( Figure 1) ( Figure S1, Supplemental files) [30,33]. Cys369 and Cys378, three of which (Cys39-Cys57, Cys217-Cys233, Cys345-Cys350) are highly conserved among GH 28 endo-PGs ( Figure S1, Supplemental files). Structural models of AnEPG based on homologous enzymes were obtained by the I-TASSER server [32]. Five top ranking 3D models were generated. Each model was validated based on C-score (confidence score), TM-score (template modeling score), RMSD (the root-mean-square deviation), and cluster density. In general, models with C-score > −1.5 have a correct fold [32]. Model 1 had the highest C-score (1.69) value reflecting a model of better quality (TM-score = 0.95 ± 0.05 and RMSD = 3.1 ± 2.2 Å) (Figure 1). Similarly to homologous GH28 endo-PGs [30,31,33], the predicted threedimensional structure of AnEPG was a right handed parallel -helix with 12 (10 complete) turns, in which the -strands were separated by turns that consisted of either a sharp bend or a loop ( Figure  1). Based on structural and sequence alignment of AnEPG and AaEPG from Aspergillus aculeatus and pga II from Aspergillus niger, residues Asp194, Asp215, Asp216, and His237 of AnEPG, which are the equivalent of Asp159, Asp180, Asp181, and His202 of AaEPG, and Asp180, Asp201, Asp202, and His223 of pga II, were predicted to be involved in catalysis ( Figure 1) ( Figure S1, Supplemental files) [30,33]. and AaEPG (PDB ID: 1IA5) (green) [33]. The amino acid sequence identity between AnEPG and AaEPG was 57.8%.

Determination of Kinetic Parameters of AnEPG
The kinetic parameters of recombinant AnEPG against CMC were determined. Since saturation was not achieved even when high PGA concentrations were used, the deduced kinetic parameters were apparent ( Figure 5). The apparent Km and Vmax values of AnEPG towards PGSA were 8.3  2.2 mg/mL and 5640  300 mol/min/mg, respectively.

Determination of Kinetic Parameters of AnEPG
The kinetic parameters of recombinant AnEPG against CMC were determined. Since saturation was not achieved even when high PGA concentrations were used, the deduced kinetic parameters were apparent ( Figure 5). The apparent K m and V max values of AnEPG towards PGSA were 8.3 ± 2.2 mg/mL and 5640 ± 300 µmol/min/mg, respectively.

Effects of Divalent Metal Ions on Enzyme Activity
The effects of divalent metal ions on AnEPG activity were examined ( Figure 6). Ba 2+ and Ni 2+ upregulated the pectinolytic activity of AnEPG by 12.2% and 9.4%, respectively, while Ca 2+ , Cu 2+ and Mn 2+ decreased the activity of AnEPG by 14.8%, 12.8%, and 10.2% separately. Other divalent metal ions did not show an obvious influence on the catalytic activity of AnEPG. According to the modelled structure of the AaEPG-octagalacturonate complex [33], 17 residues, which are possibly involved in interaction with substrate, were identified, including Lys105, His110, Gln128, Asp150, His156, Asn157, Asp162, Asp180, Asn186, Ser208, His202, Arg212, Asp231, Arg235, Lys237, Lys61, Tyr270. Based on sequence and structural alignment of AaEPG (PDB ID: 1IA5) and modelled AnEPG, 15 residues are completely conserved, and only two residues Asp231 and Lys261 of AaEPG, corresponding to Val266 and Asp296 of AnEPG, are not conserved. The difference between these two residues might lead to different K m values of AnEPG and AaEPG, which needs further investigation.

Analysis of Hydrolysis Products of PGA by AnEPG
TLC (thin-layer chromatography) analysis of the soluble sugars released from PGA by AnEPG indicated that digalacturonate and other oligogalacturonates were produced at the initial stage (20 min) and accumulated as hydrolysis continued (Figure 7). The monomer galacturonate was observed after 3 h. The presence of galacturonate released from oligogalacturonates by AnEPG suggested that it was a typical endo-acting enzyme, which preferentially cleaved the internal glycosidic bonds of oligogalacturonates and pectin/pectate.

Protein Overexpression
Protein expression was induced with 0.5% (v/v) methanol in baffled flasks (100 ml BMMY) for four days. The supernatants were precipitated with 80% (NH 4 ) 2 SO 4 , and the precipitated proteins were redissolved in buffer A (50 mM Tris/HCl, pH 8.0, 0.5 M NaCl). The protein concentration was determined by the Bradford method using bovine serum albumin as a standard.

Enzyme Activity Assay
All enzyme assays were done in triplicate. Endo-α-1,4-polygalacturonase activity was determined by measuring the amount of reducing sugars released from PGA (polygalacturonic acid) through the DNS (3,5-dinitrosalicylic acid) method [36]. D-(+)-galacturonic acid was used as a standard. Enzymatic reactions were performed in the presence of 0.5% PGA (w/v) in 50 mM B & R (Britton and Robinson) buffer at 37 • C for 15 min. One unit (U) of endo-α-1,4-polygalacturonase activity toward PGA was defined as the amount of protein required to release 1 µmol of reducing sugar per min under standard assay conditions, and specific activity was defined as units mg -1 protein.

Determination of Optimal pH and pH Stability
The optimal pH of AnEPG was determined in 50 mM B & R buffer at 37 • C and pH between 2.0 and 12.0, and all enzymatic reactions were incubated for 15 min. The pH stability was estimated by first preincubating PGA in 50 mM B&R buffer at different pH values (pH 2.0 -12.0) at 4 • C for 2, 24, and 120 h respectively. The residual activities were then determined at 37 • C and optimal pH, and the percentage of the residual activity at different time points and pH values against the initial one was calculated.

Determination of Optimal Temperature and Thermal Stability
The optimal temperature of AnEPG was determined in 50 mM B & R buffer (pH 4.0) between 20 and 80 • C, and all enzymatic reactions were incubated for 15 min. To determine the thermal stability of AnEPG, it was pre-incubated for varied time intervals (15 min to 2 h) at pH 6.0, and 30, 40, and 50 • C, respectively. The residual activities were measured at 37 • C and optimal pH, and the percentage of the residual activity at different time points and temperatures against the original one was calculated.

Determination of Kinetic Parameters
Endo-α-1,4-polygalacturonase activity was measured at 37 • C using PGA as substrate at concentrations ranging from 0.2% to 2% (w/v) in 50 mM B & R buffer (optimal pH). The release of reducing sugars was quantified after being incubated for 5 min, and kinetic parameters were determined based on the Michaelis-Menten equation.

Effects of Divalent Metal Ions on Enzyme Activity
The effects of divalent metal ions on the catalytic activity of AnEPG were determined in the presence of various divalent metals (Pb(CH 3 COO) 2 , NiSO 4 , MnSO 4 , CuSO 4 , BaCl 2 , ZnSO 4 , CoCl 2 , CaCl 2 , MgCl 2 , and FeSO 4 ). Since phosphate in B & R buffer may interfere with the enzyme assay, 100 mM sodium acetate (optimal pH) was used. The percentage of the activity in the presence of different divalent metal ions against the control without metal ions was calculated.

Thin Layer Chromatography Analysis of Hydrolysis Products of PGA by AnEPG
To demonstrate the mode of action of AnEPG, hydrolysis of PGA (0.5%) by AnEPG (0.89 µg/mL) was carried out in 50 mM B & R buffer (optimal pH) at 37 • C for 5 min, 10 min, 20 min, 30 min, 1 h, 2 h, 3 h, and 24 h, respectively, and a control in the absence of AnEPG was also set up. The hydrolysis products of PGA by AnEPG were analyzed by TLC (thin-layer chromatography). Samples were spotted on the silica gel plate and the TLC plate was placed in the mixture (butan-1-ol:acetic acid:water (9:4:7 (v/v) as the mobile phase [19]. At the end of migration, products were visualized by heating at 105 • C for 5 min after spraying the plates with 10% sulfuric acid.

Conclusions
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.
In summary, multiple sequence alignment and phylogenetic analysis of AnEPG and homologous GH 28 endo-PGs suggested that AnEPG is a new endo-polygalacturonase. AnEPG was overexpressed in Pichia pastoris and characterized in detail. AnEPG showed the highest activity at pH 4.0 and 60 • C.
It was very stable between pH 2.0 and pH 12.0. AnEPG exhibited high stability below 40 • C and was unstable above 50 • C. The apparent K m and k cat values of AnEPG against PGA (polygalacturonic acid) were 8.3 mg/mL and 5640 µmol/min/mg respectively. Ba 2+ and Ni 2+ showed some stimulatory effects on AnEPG, while AnEPG was inhibited by Ca 2+ , Cu 2+ , and Mn 2+ . The structural characteristics of endo-polygalacturonases were demonstrated by structure modelling of AnEPG. This pectinase could be potentially used in the beverage industry and/or other fields requiring acidic endo-polygalacturonases.