Secretory Expression and Characterization of an Acidic Endo-Polygalacturonase from Aspergillus niger SC323 in Saccharomyces cerevisiae

Abstract

The endo-polygalacturonase gene (endo-pgaA) was cloned from DNA of Aspergillus niger SC323 using the cDNA synthesized by overlapping PCR, and successfully expressed in Saccharomyces cerevisiae EBY100 through fusing the α-factor signal peptide of yeast. The fulllength cDNA consists of 1,113 bp and encodes a protein of 370 amino acids with a calculated molecular mass of 38.8 kDa. After induction by galactose for 48 h, the activity of recombinant endo-PgaA in the culture supernatant can reach up to 1,448.48 U/mg. The recombinant protein was purified to homogeneity by ammonium sulfate precipitation and gel filtration column chromatography and subsequently characterized. The optimal pH and temperature of the purified recombinant enzyme were 5.0 and 50℃, respectively. The Michaelis-Menten constant (K_m) and maximal velocity (V_max) of the enzyme for pectin were 88.54 μmol/ml and 175.44 μmol/mg/min, respectively. The enzyme activity was enhanced by Ca²⁺, Cu²⁺, and Na⁺, and strongly inhibited by Pb²⁺ and Mn²⁺. The pectin hydrolysates were mainly galacturonic acid and other oligo-galacturonates. Therefore, these characteristics suggest that the recombinant endo-PgaA may be of potential use in the food and feed industries.

Introduction

Pectin, an acidic polysaccharide whose basic structural repeats are α-1,4-linked-D-galacturonic acid, is widely found in the middle lamella and primary cell wall of plants [22]. As an important renewable resource, pectin has great potential applications in the biomedical, food, agricultural, and other industries [18]. The degradation of pectin is mainly based on pectinase, including pectin methyl esterases (PME, E.C. 3.1.1.11), pectin lyases (PL, E.C. 4.2.2.10), exopolygalacturonase (exo-PG, E.C. 3.2.1.67), and endopolygalacturonase (endo-PG, E.C. 3.2.2.15) [13, 24]. As one of the four most highly produced commercial enzymes in the world, pectinase is widely used in foods, wines, the environment, medicines, textiles, and detergents [26]. Among various pectinases, endo-polygalacturonase has become the focus of concern owing to its wide application in fruit juice clarification and preparation of wine as well as functional oligo-galacturonates of pectin [6, 20, 25].

So far, the endo-polygalacturonase gene (endo-PG) has been successfully cloned from several bacteria and fungi such as Erwinia sp., Bacillus sp., Pseudomonas sp., Aspergillus sp., and Botrytis sp., and further expressed in relative protein expression systems, respectively [7]. The studies on production of endo-polygalacturonase mainly focus on the Aspergillus sp., especially that of Aspergillus niger, which has a long history of safe using in industrial-scale endopolygalacturonase production and has been commercialized [11]. In spite of this, the activity and yield of the native enzyme are not high enough for industrial applications and it is very difficult to purify [5, 11]. Escherichia coli (E. coli) as an effective heterologous expression system has been widely applied, but it is not a good candidate for the expression of endo-polygalacturonase from Aspergillus niger because the fusion protein cannot be folded correctly in the prokaryotic cell, and also fails to secrete to the outside of the cell [17]. As a safe eukaryotic host for heterogeneous expression, Saccharomyces cerevisiae is also widely used in gene cloning and overexpression and it is an important applied strain in practical production. However, there is little research on the secretory expression of the endo-polygalacturonase gene in Saccharomyces cerevisiae [29].

In this work, an endo-polygalacturonase-encoding gene (endo-pgaA) was cloned from Aspergillus niger SC323 and efficiently expressed in Saccharomyces cerevisiae EBY100. The purification and characterization of the recombinant endo-PgaA were investigated. Its high activity and stability at a wide pH range make endo-PgaA potentially useful in application of pectin hydrolysis or bioactive pectin oligosaccharides production.

 

Materials and Methods

Strains, Plasmids, and Reagents

The Aspergillus niger SC323 was isolated from pomelo peel samples of Chengdu, China and has been deposited with Sichuan Type Culture Collection (SCTCC), Sichuan University, Chengdu, China, under Accession No. SCTCC 2012102001. Escherichia coli DH5α was stored in this laboratory. The pESC-LEU plasmid and Saccharomyces cerevisiae EBY100 (MATa ura 3-52 trp 1 leu 2Δ1 his3Δ200 pep4: HIS3 prb 1Δ1.6R can 1 GAL) were purchased from Invitrogen (Carlsbad, CA, USA) and used for heterologous expression. The PMD19-T plasmid for sequencing was purchased from TaKaRa. Pectin from apple peel and galacturonic acid were purchased from Ruji Biology Technology Co, China and Sigma, respectively. All other chemicals used in the study were of analytical grade and commercially available.

Media and Culture Conditions

Czapek medium (CM) containing 3% sucrose, 0.3% NaNO3, 0.1% K2HPO4, 0.05% MgSO4·7H2O, 0.05% KCl, 0.001% Fe2SO4, and 1.5% agar was used to cultivate Aspergillus niger SC323 at 28℃. Luria–Bertani (LB) medium consisting of 1% NaCl, 1% peptone, and 0.5% yeast extract was used to cultivate E. coli DH5α at 37℃. YPD medium (yeast extract peptone dextrose), MDT (minimal dextrose plates with tryptophan), and YNB-CAA medium (yeast nitrogen base with casamino amino acids) with 2% glucose or galactose were used for the growth, selection, and expression of recombinant protein in S. cerevisiae according to the manufacturer’s instructions (https://tools.lifetechnologies.com/content/sfs/manuals/pyd1_man.pdf). The 1% pectin solution as substrate for the enzyme and hydrolysis product assay was prepared as follows: 1 g of pectin was dissolved in 100 ml of buffer (0.1 M citric acid and Na2HPO4, pH 4.5) and kept at 4℃. For plates, 1.5 % agar was added unless otherwise specified. All other chemicals used in the present study were of analytical grade.

Preparation of Genomic DNA

A. niger 323 mycelia were inoculated into CM liquid medium at 28℃ and 180 rpm for 2 days, and then the mycelia were collected with a sterile gauze and washed with sterile water. Genomic DNA was extracted by the modified CTAB method [16]. S. cerevisiae EBY100 cells were inoculated into YPD medium at 30℃ and 200 rpm for 24 h. The yeast cells were obtained by centrifugation and washed, and then resuspended with sterile water. The genomic DNA in the supernate was achieved by centrifugation after treatment with alternate freezing and thawing.

Cloning and Sequencing

Based on our previous finding, the complete endo-polygalacturonase (endo-pgaA) gene from A. niger has an intron with 50 bp in the sequence. It was amplified with the primer sets PgaA-F1/PgaAR1 and PgaA-F2/PgaA-R2 (Table 1). The α-factor gene of yeast signal peptide was amplified from S. cerevisiae EBY100 with the primer sets α-factor-F/α-factor-R (Table 1) for secretory expression of endo-PgaA protein in S. cerevisiae. The three DNA fragments were recovered and used as templates for each other to amplify the whole secretory sequence by overlapping PCR using primer sets α- factor-F/PgaA-R2. The PCR conditions were as follows: 94℃ for 5 min; 30 cycle of 98℃ for 15 sec, 55℃ for 10 sec, and 72℃ for 15 sec; and 72℃ for 50 sec. The PCR products were purified and cloned into the pMD19-T vector and transferred into E. coli DH5α for sequencing. The obtained positive plasmid was named pMD19-T-α-factor-PgaA.

Table 1.Primers used in this study.

Construction of Expression Vector

The obtained plasmid pMD19-T-α-factor-PgaA and pESC-LEU expression vector were digested with BamHI and ApaI, respectively. The recovered α-factor-PgaA and pESC-LEU nucleotide products were ligated with T4 ligase at 22℃ for 1 h and transferred into E. coli DH5α. Large quantities of pESC-LEU-α-factor-endo-PgaA plasmid were extracted for transformation into S. cerevisiae EBY100 using the TIANprep Mini Plasmid Kit (Tiangen Biotech, Beijing, China).

Expression and Purification of the Recombinant Endo-PgaA

The pESC-LEU-α-factor-PgaA plasmid was transformed into competent S. cerevisiae strain EBY100 with the lithium acetate method according to the pYD1Yeast Display Vector Kit (Invitrogen). Transformants were cultured on MDT plates and incubated at 30℃ for 2-4 days until single colonies appeared. Leu+ phenotype transformants were chosen and confirmed by yeast colony PCR [1]. Positive transformants were grown in 2% glucose-containing YNB-CAA medium at 30℃ with constant shaking at 200 rpm overnight, and then switched to YNB-CAA medium containing 2% galactose at 20℃ with shaking at 200 rpm for about 48 h for induction of expression.

Cell-free supernatant (500 ml) of the strain after induction was treated with ammonium sulfate to 85% saturation to precipitate the recombinant enzyme. The precipitated enzyme was then resuspended in 6.0 ml of eluant buffer (0.1 M citric acid and Na2HPO4, pH 4.5) and loaded onto an ENrich SEC 650 gel filtration column (polymethacrylate; Bio-Rad, USA; 10 cm × 300 cm). The enzyme was eluted with the same buffer system at a flow rate of 1 ml/min. The active fractions were pooled and analyzed for pectinase activity and characterization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a stacking gel of 5% acrylamide and a separation gel of 12% acrylamide as described by Laemmli [9]. The resolved proteins were visualized by staining with Coomassie Brilliant Blue R250 (Sigma, St. Louis, MO, USA)

Enzymatic Activity Assay

The crude activity of endo-PgaA was preliminarily analyzed by transparent zone for the positive transformants [17]. After induction, the fermentative broth of S. cerevisiae recombinant was centrifuged at 12,000 ×g for 2 min and the supernatant was collected for further analysis. First, 100 μl of the sample was added into the hole (0.5 cm diameter) of the 1% pectin (w/v) plate at 37℃ for 7-10 h. Then the plate was stained by 1% Congo Red (w/v) for 30 min and discolored by 1 mol/l NaCl for 1 h to observe the transparent zone.

The endo-PgaA activity was determined by using the dinitrosalicylic acid method [23] with galacturonic acid as the standard. Protein concentration was measured by the dye-binding method of Bradford, and bovine serum albumin was used as the standard [2]. One unit (U) of endo-PgaA activity was defined as the amount of enzyme that released reducing sugars equivalent to 1.0 μmol galacturonic acid per minute under standard conditions (pH 4.5, 40℃, 10 min). All reactions were performed in triplicate.

Characterization of Purified Recombinant Endo-PgaA

Pectin was used as the substrate for enzyme characterization. The optimum pH was determined at 40℃ in buffer (0.1 M citric acid and Na2HPO4) with pH ranging from 2.0 to 8.0. The enzyme stability at different pH values was performed with the same buffer system by measuring the residual activity enzyme. The optimal temperature was estimated by performing the standard assay at temperatures of 30-80℃. Thermal stability was measured by assessing the residual enzyme activity after incubation of the enzyme at the above temperatures for 30 min, respectively.

To determine the effects of metal ions on the activity of purified endo-PgaA, the enzyme solutions were incubated in the assay buffer containing 1 mM of metal ions (NaCl, KCl, LiCl, FeSO4, PbSO4, CoCl2, CuSO4, NiSO4, BaCl2, MgSO4, MnSO4, CaCl2, and ZnSO4) and test compounds, respectively [28]. The activity of the enzyme was then measured under standard conditions.

The Km and Vmax values for the purified enzyme were defined by Lineweaver-Burk plotting using activity assay at 50℃ in buffer (0.1 M citric acid and Na2HPO4, pH 5.0) with 1-20 mg/ml pectin as substrates.

Analysis of Hydrolysis Products

The 1.0% (w/v) pectin solutions in buffer (0.1 M citric acid and Na2HPO4, pH 4.5) were hydrolyzed by the recombinant endo-PgaA at 50℃ for 24 h with constant shaking. The hydrolysis products and standard oligo-galacturonates were analyzed by HPLC with a Sugar-pak I column (6.5 mm × 300 mm; Waters), 5 mM solution of sulfuric acid as mobile phase (0.6 ml/min), and injection volume of 25 μl. The column was maintained at 80℃. Sugar peaks were screened using a Shodex RI-101 refractive index detector.

Nucleotide Sequence Accession Number

The nucleotide sequence of the endo-polygalacturonase gene from A. niger 323 has been deposited in the GenBank database under the accession number KP265703.

 

Results

Cloning and Sequence Analysis

Using primer sets PgaA-F/PgaA-R1 and PgaA-F2/PgaA-R2, two DNA fragments amplified from genomic DNA of Aspergillus niger SC323 were obtained. A complete 1,113 bp sequence was amplified by overlapping PCR, and it began with an ATG codon and ended with a TAA codon after sequencing. The sequence exhibited 98% identity with the endo-polygalacturonase from Aspergillus niger JL-15 (GenBank: AGV40780). The ORF of the sequence was deduced to encode 370 amino acids with a calculated molecular mass of 38.8 kDa. A 19-residue signal peptide with a putative processing site (ALA-AP) was also identified using the SignalP 4.1 server. The mature protein showed the highest identity (99%) with the protein encoded by the Aspergillus niger JL-15 endo-polygalacturonase gene [11], suggesting that it was a new endo-polygalacturonase gene (Fig. 1).

Fig. 1.Multiple sequence alignment comparison of endo-PgaA with homologous sequences. GB_KP265703, A. niger 323; GB_AGV40780, A. niger JL-15; SP_Q9P4W4, A. niger N400; SP_Q8NK99, A. Kawachii IFO 4308; SP_Q9P358, A. awamori IFO 4033; and XP_001398000, A. niger CBS 513.88. The shadows with different color showed different similarity of amino acid residues. The predicted signal peptide was underlined in red; the potential N-glycosylation site was indicated with black circles; the two catalytic residues are shown with black inverted triangle; the activity region was in box.

Construction of Expression Plasmid

To facilitate easy purification, the protein was provided with a signal peptide causing it to be secreted out of the cell. The α-factor gene with 267 bp as an efficient signal peptide of yeast was amplified and fused with the PgaA DNA fragment, and a 1,380 bp fused DNA fragment (α-factor-endo-PgaA) was successfully assembled. The secretory expression plasmid pESC-LEU-α-factor-endo-PgaA was constructed and transformed into S. cerevisiae EBY100. After induction for 48 h by galactose, the endo-PgaA activities of 42 transformants were screened based on the endo-PgaA activity on 1% pectin plates. The transformant with highest endo-PgaA activity (11.4 U/ml) was obtained from shaker flask culture after induction with 2% (w/v) galactose for 48 h at 30℃ and 200 rpm, and was named EBY100/pESC-LEU-α-factor-endo-PgaA.

Purification and SDS-PAGE Analysis of Endo-PgaA

Recombinant endo-PgaA was successfully expressed in S. cerevisiae EBY100 and was purified to electrophoretic homogeneity by 85% ammonium sulfate precipitation and ENrich SEC 650 gel filtration (Table 2). The purified enzyme (specific activity of 1,448.48 U/mg) showed 42.62% recovery of the enzyme activity. The SDS-PAGE revealed that the molecular mass of endo-PgaA was about 45.0 kDa (Fig. 2), which was higher than the calculated value (38.8 kDa).

Table 2.Summary of purification of recombinant endo-PgaA.

Fig. 2.SDS–PAGE analysis of purified recombinant endo-PgaA. Lane M: standard protein molecular weight markers; Lane 1: Supernatant of S. cerevisiae EBY100 recombinant (pESC-LEU) induced by 2% D-galactose; Lane 2: Supernatant of S. cerevisiae EBY100 recombinant (pESC-LEU-α-factor-endo-PgaA) induced by 2% Dgalactose; Lane 3: purified recombinant endo-PgaA using ENrich SEC 650 gel filtration.

Characterization of the Recombinant Endo-PgaA

The biochemical characterization of the recombinant endo-PgaA was investigated. The activities of endo-PgaA at various pH values were measured by using pectin as the substrate. The reaction pHs were adjusted to 2.0-8.0 with buffer (0.1 M citric acid and Na2HPO4). The results showed that the pH optimum of recombinant endo-PgaA was at pH 5.0 (Fig. 3A). Moreover, the enzyme showed good stability within the pH range of 3.0-6.0 for different incubation times and over 65% of activity was retained (Fig. 3B).

Fig. 3.Effect of pH on the activity of recombinant endo-PgaA. (A) Optimum pH of the recombinant endo-PgaA; (B) pH tolerance of the recombinant endo-PgaA.

The optimal temperature for recombinant endo-PgaA activity was 50℃ (Fig. 4A). The thermal stability of recombinant endo-PgaA was measured by incubating the enzyme at 35-70℃ for different times, after which the residual activity was measured at 40℃ and pH 5.0. The recombinant endo-PgaA retained more than 50% of its activity after incubation for 60 min, but no activity remained after incubation at 70℃ for 2 h (Fig. 4B).

Fig. 4.Effect of temperature on the activity of recombinant endo-PgaA. (A) Optimum temperature of the recombinant endo-PgaA; (B) Thermotolerance of the recombinant endo-PgaA.

The enzyme activity of the recombinant endo-PgaA in different metal ions is shown in Fig. 5. The activity was enhanced by Ca2+, Cu2+, Mg2+, Na+, Zn2+, K+, Fe2+, and Li+, and inhibited by Pb2+, Mn2+, Co2+, Ni2+, and Ba2+. The apparent Km and Vmax values of endo-PgaA for various concentrations of pectin as substrate were 88.54 μmol/ml and 175.44 μmol/mg/min, respectively.

Fig. 5.Effect of 13 different metal ions on the activity of the recombinant.

Analysis of Hydrolysis Product

The hydrolysis products of pectin by purified recombinant endo-PgaA were determined by HPLC. After 24 h incubation at 50℃ in pH 5.0 buffer, the major components of the hydrolysis products were mainly galacturonic acid (G), digalacturonic acid (G2), and trigalacturonic acid (G3) (Fig. 6).

Fig. 6.HPLC profile of pectin degradation by recombinant endo-PgaA. The positions of galacturonic acid (G), digalacturonate (G2), and trigalacturonate (G3) are shown.

 

Discussion

Fungous endo-polygalacturonases, especially those from Aspergillus niger, represent the most important enzymes with commercial applications in the food industry. However, it is complicated and difficult to purify the endo-polygalacturonase from the culture supernatant of A. niger. Heterologous expression is an important technology for producing industrial enzymes, such as cellulase, xylanase, pectinase, protease, and others [11, 21]. Saccharomyces cerevisiae is one of the safe and excellent expression hosts for basic research and industrial use because of its high cell densities in minimal media and effective modifications. Up to now, several endo-polygalacturonases from A. niger have been expressed in Pichia pastoris, but there is little report in S. cerevisiae [11, 12, 29].

In this paper, an endo-polygalacturonase gene coding for a protein of 370 amino acid residues and a predicted molecular mass of 38.8 kDa was cloned and sequenced from A. niger SC323. Analysis of the amino acid sequence indicated that it was a 19 residues signal peptide. The Asp207 and His229 residues in the catalytic sites were for its catalytic functions (Fig. 1). The amino acid sequence of the endo-PgaA exhibited 99%, 99%, 98%, and 98% identity with endo-polygalacturonases from A. niger N400 (Q9P4W4), JL-15 (AGV40780), A. kawachii IFO 4308 (Q8NK99), and A. awamori (Q9P358), respectively [11, 12, 15].

The endo-PgaA-encoding gene was successfully expressed in S. cerevisiae EBY100 and the recombinant enzyme showed high activity (1,448.48 U/mg). The optimum pH of endo-PgaA was 5.0, similar to that found in A. niger, but the tolerance range of pH (3.0-6.0) was wider than others [11, 15, 29]. The optimum temperature of endo-PgaA was 50℃, higher than most others in A. niger [4, 11, 12, 15]. The low Km and high Vmax values of endo-PgaA suggested it possessed higher catalytic efficiency and affinity to pectin [4].

Moreover, the products of recombinant endo-PgaA hydrolysis to pectin were mainly oligo-galacturonates (G, G2, and G3) that are similar to those of rePgaA from Aspergillus niger JL-15 [11], but the ratios of galacturonic acid (G) and digalacturonic acid (G2) were higher. This indicated that hydrolysis by our recombinant endo-PgaA was more complete. For the hydrolysate from pectin, oligogalacturonates can be utilized by the beneficial gastrointestinal microflora and to suppress the activity of entero putrefactive bacteria [11, 14, 19]. It is also reported that pectinase can induce the resistance of plants, and inactive pectinase can be used as a biopesticide for production and refreshment of green food, which will benefit food safety and environment protection [10]. The recombinant enzyme in this work was induced by galactose but not methanol, which can ensure safety of hydrolysis products when it is applied in the food and feed industries [11, 28]. For crude pectin embedded in cellulose, pectinase can soften the plant tissue due to the exposure of cellulose. Thus, the endo-PgaA might be combined with other hydrolases (such as pectinesterase, cellulase, hemicellulase, and amylase) for potential applications in many fields [28].