The present study aimed to purify and characterize one polygalacturonase from L. gongylophorus (PGaseLg), the symbiotic fungus of Atta sexdens. The enzyme was isolated by salting out of crude extract followed by two chromatographic steps. PGaseLG was identified with MS analysis and molecular exclusion chromatography revealed the monomeric nature of a protein with an estimated molecular weight of about 39 kDa. PGaseLg has an optimum temperature of 60 °C and optimum pH activity at 5.0. Using polygalacturonate as a substrate, the calculations of K M, V max and k cat were 0.65 mg·mL -1, 1800 μmol·min -1·mg -1 and 35.97 s -1, respectively. The enzyme was stable for more than 3 h at 50°C at pH 5.0; otherwise, at lower or higher pH values, the PGaseLg was less stable. The influence of several metals, EDTA and β-mercaptoethanol on enzyme activity was also determined. Thin layer chromatography (TLC) analyses indicated that PGaseLg is an exopolygalacturonase.
The symbiotic relationship of the leaf-cutter ant Atta sexdens with basidiomycete Leucoagaricus gongylophorus is an important Neotropical herbivorous system [
Biochemical issues are relevant in the intricate interdependence of mutualism [
Pectin is a major structural component of plant cell walls, forming a gel-like matrix that is particularly abundant at cell wall interfaces in the middle lamella region of leaves, where it regulates intercellular adhesion [
As parts of our efforts to better understand the pectinases expressed by L. gongylophorus, we identified a bifunctional polygalacturonase/xylanase [
All chemicals were of analytical grade; Milli-Q water was used throughout the experiments. Citric acid, formic acid, K3[Fe(CN)6], Na2S2O3, NH4HCO3, (NH4)2SO4, Na2HPO4, KCl, NH4Cl, MgCl2, CaCl2, FeCl3, MnCl2, CoCl2, CuSO4, NiCl2, ZnSO4, HgCl2, PbCl2, EDTA, β-mercaptoethanol, MES (2-[N-morpholino]ethanesulfonic acid), dithiothreitol (DTT), iodoacetamide (IAA), yeast nitrogen base (YNB), 3,5-dinitrosalicylic acid (DNS) and polygalacturonic acid were purchased from Sigma (St. Louis, MO). A Bradford method kit was purchased from Bio-Rad Laboratories (Hercules, CA) and trypsin gold, MS grade, from Promega Corporation (Madison, WI). A McIlvaine’s buffer solutions system (Citrate/phosphate, pH 5.0) was applied as a working buffer. The pH values were obtained by mixing Na2HPO4 0.2 mol∙L−1 and citric acid 0.1 mol∙L−1 at a suitable ratio.
The CCTI strain of L. gongylophorus (isolated from an A. sexdens nest) was donated by the Center of Studies on Social Insects (UNESP, Rio Claro, Brazil). The mold was cultivated at room temperature in stationary liquid cultures of an inductive medium (YNB 6.7 mg∙mL−1 and polygalacturonic acid 5.0 mg∙mL−1 in working buffer) [
Polygalacturonase activity was evaluated according to the Miller method for reducing sugar determination [
Polygalacturonase (PGaseLg) purification chromatographic procedures were carried out in the AKTA-FPLC system (GE Healthcare). The centrifugation through the purification procedures was conducted at 12,000 ×g for 20 min. at 4˚C. Dialyses, unless stated, were carried out against the working buffer at 4˚C.
(NH4)2SO4 salting out: prior to the chromatography columns, the stepwise precipitation with (NH4)2SO4 was useful in clarifying the crude extract. In the first step, ammonium sulfate was added to the crude enzyme solution up to 30% of saturation, and the insoluble contents were then removed by centrifugation and discharged. The supernatant received new additions of salt until the saturation reached 70%. The solution reposed overnight and was centrifuged to produce the precipitant within the interval from 30% to 70% of salt saturation. This pellet was solubilized in a 10 mL working buffer and dialyzed.
Gel filtrationchromatography: 2 mL of the salted-out 30% - 70% active fractions were loaded onto a Superdex S-75 (GE Healthcare) column (16 × 600 mm) pre-equilibrated with a working buffer with NaCl 100 mmol∙L−1. Elution was carried out with the same buffer at a flow rate of 1.0 mL∙min−1. The PGaseLg activity- eluted fractions were pooled and dialyzed against a 20 mmol∙L−1 acetate buffer, pH 5.0.
Cation exchange chromatography: 1 mL of the Superdex S-75 active fractions pool in a 20 mmol∙L−1 acetate buffer was applied onto a SPFF-Sepharose column (GE healthcare) pre-equilibrated with the same acetate buffer. The column was washed with 10 mL of the acetate buffer at a flow rate of 1 mL∙min−1, and the elution was with a NaCl linear gradient from 0 to 290 mmol∙L−1 in the same buffer. The PGaseLg activity was determined in desalted fractions, and the active peak was pooled for characterization.
Each step of enzyme purification was followed by electrophoresis in 15% (v/v) polyacrylamide gel in denaturing conditions (SDS-PAGE), as described by Laemmli [
The temperature effect on enzyme activity was determined at 30˚C, 40˚C, 45˚C, 50˚C, 55˚C, 60˚C, 65˚C, 75˚C and 80˚C using polygalacturonic acid as the substrate. Experiments were expressed in enzyme activity (U) versus temperature plot. Based on the temperature assay, it was possible to calculate activation energy.
Thermal inactivation was evaluated by incubating the purified PGaseLg at 30˚C, 40˚C and 50˚C. Reaction vials were kept, at pH 5.0, under mild agitation for 10 hours in the experimented temperature. At each 30 min., 50 μL aliquots were gathered and submitted to ice bath, and the enzyme residual activity (U%) was determined at 60˚C and pH 5.0. The residual activity was plotted as a function of the incubation temperature.
The optimal pH value of PGaseLg activity was determined by assaying the purified enzyme at 60˚C in the working buffer at pH values from 2.5 to 6.5 (slope of 0.5). Activities (U) were plotted against pH values.
To evaluate the influence of pH on the stability of the enzyme, it was kept for six hours at 50˚C in the working buffer at pH values of 3.0, 4.0, 5.0, 6.0 and 7.0 or the Tris/glycine buffer for pH 8.0. The residual activities of 50 μL aliquots were determined at optimal activity conditions. The determined half-lives of each experiment were plotted versus pH treatment.
The PGaseLg kineticconstant (KM) was determined with ten substrate concentrations ranging from 0.10 to 5.0 mg∙mL−1 of galacturonic acid or 74% esterified pectin. KM and Vmax values were calculated using the double reciprocal Lineweaver-Burk plots.
The effect of a number of metal ions and other reagents on enzyme activity in the assay medium was tested. Pure PGaseLg was assayed in the presence of KCl, NH4Cl, MgCl2, CaCl2, FeCl3, MnCl2, CuSO4, CoCl2, NiCl2, ZnSO4, PbCl2, HgCl2, EDTA and β-mercaptoethanol at 5.0 mmol∙L−1. The assays were conducted with the 50 mmol∙L−1 MES buffer, pH 5.5. Before the assay, the enzyme solution was dialyzed against this same buffer. Residual activity was determined in triplicate at each treatment.
The SDS-PAGE running was used for molecular mass determination, and the oligomerization state determination of PGaseLg was performed by loading the enzyme on a Superose 12 HR column (10 × 30 cm) calibrated with bovine serum albumin (BSA) as molecular mass standard.
Thin layer chromatography (TLC) analyses of polygalacturonic acid and 74% esterified pectin hydrolysis products were performed on heat-inactivated samples from overnight digestion, at 40˚C in the working buffer. Aliquots of respective pectic hydrolysate were 10 times concentrated and spotted on 8 × 10 cm silicagel 60G aluminum sheets (Merck, Germany). Mono-, di-, tri- and polygalacturonic acid were applied as standard. The chromatography was performed using the ascending method; the mobile phase consisted of a 5:3:2 mixture of n-butanol: H2O: acetic acid. For visualization of the spots, the dried plate was sprayed with 10% sulfuric acid in methanol followed by heating at 105˚C for 5 min.
Sliced and washed SDS-PAGE slabs were destained using freshly prepared K3[Fe(CN)6] 30 mmol∙L−1 and Na2S2O3 100 mmol∙L−1 mixed with a 1:1 ratio and added to cover the gel pieces. Afterward, the gels were washed until they were clear by ammonium bicarbonate 100 mmol∙L−1. The gels were emerged in acetonitrile and dried in a vacuum centrifuge. After the reduction with dithiothreitol 10 mmol∙L−1 in ammonium bicarbonate 50 mmol∙L−1, the samples were alkylated by iodoacetamide 55 mmol∙L−1 in NH4HCO3 50 mmol∙L−1. Alkylated peptides were digested by trypsin gold, mass spectrometry grade, in an ammonium bicarbonate buffer (25 mmol∙L−1, pH 8.0) and extracted by acetonitrile/formic acid 0.1% (40:60). Samples were desalted by ZipTip® and kept at 20˚C until the LC-MS/MS analysis.
Tryptic digested peptides were analyzed by online nanoflow LC-MS on an EASY-nLC II system (Thermo Scientific) connected to an LTQ-OrbitrapVelos instrument (Thermo Scientific) via a Proxeonnanoelectrospray ion source. Peptides were separated with a linear gradient from 0% to 60% acetonitrile (0.1% formic acid) on an analytical EASY-Column (10 cm, ID75 µm, 3 µm, C18-Thermo Scientific, 300 nL∙min−1) previously trapped in a pre-column EASY-Column (2 cm, ID100 µm, 5 µm, C18-Thermo Scientific). An LTQ-OrbitrapVelos mass spectrometer was operated using DDA (data-dependent acquisition) in positive ion mode. The 20th most intense precursor ions were selected for CID fragmentation. Full MS scans were performed with 60,000 full-width half-maximum (FWHM) nominal resolution settings (m/z range 400 - 1200, collision energy 35 eV, activation Qz of 0.250, activation time 10 ms). The minimum signal threshold was 15,000 counts, and for dynamic exclusion, 1 repeat count was considered with a duration of 30 s. The instrument was calibrated externally according to manufacturer’s instructions.
Polygalacturonic acid, added to the culture broth, was efficient in the PGaseLg induction whose purification was carried out as summarized in
Total activity (Utot) | Total protein (mg) | Specific activity (U) | Purification fold | Yield (%) | |
---|---|---|---|---|---|
Crude extract | 125.3 | 3.16 | 39.6 | 1.0 | 100.0 |
(NH4)2SO4 | 102.8 | 0.81 | 126.9 | 3.2 | 82.0 |
Gel filtration | 76.6 | 0.15 | 510.9 | 12.9 | 61.2 |
Cation exchange | 61.1 | 0.05 | 1221.4 | 30.8 | 48.8 |
mmol∙L−1), three isolated bands were eluted, and PGaseLg activity was detected in the first, corresponding to 85 mmol∙L−1 of salt concentration. An overall purification of up to 30.8-fold with a 48.8% recovery was achieved (
The homogeneity of the purified PGaseLg was demonstrated by the presence of one single protein band on SDS-PAGE stained with silver salts (
To calculate the molecular weight of the purified PGaseLg, the electrophoretic mobility against the logarithm molecular weights of known polypeptides was plotted and compared to the electrophoretic mobility of the PGaseLg. The calculated molecular weight of the PGaseLg was approximately 39.0 kDa. This molecular mass is in agreement with previously reported PGase from other sources, such as Rhizomucorpusillus [
The PGaseLg was identified in the SDS-PAGE gel; the band was excised from the gel and treated with trypsin, and the peptides were analyzed by online LC-MS nanoflow. The MS spectra were searched for on different databases with two different search engines and in-house Proteome Discoverer 1.4 software (Thermo, USA). Databases with different numbers of sequences were used to increase the protein identification confidence. The databases were downloaded by typing “Leucoagaricus” (202 protein sequences) as a keyword on both NCBI and Uniprot sites using the SEQUEST search engine (Proteome Discovery 1.4). The “Fungi_NCBI” (2,204,168 protein sequences) database was used directly from the MASCOT 2.2.4 search engine with NCBInr filtered by fungi taxonomy. The identified peptide sequences with significant sequence coverage with a polygalacturonase sequence of the L. gongylophorus are presented in
The database search of mass spectrometry analysis identified the PGaseLg with an annotated polygalacturonase at NCBI (Accession number 317468146) with a theoretical molecular weight of 37.034 kDa. Instead, in SDS-PAGE, a PGaseLg molecular weight of 39.04 kDa was observed. This fact may be explained by glycosylation, which is not annotated in the deposited L. gongylophorus polygalacturonase sequence, which is commonly assigned to other glycosidases.
The amino acid sequence similarity search in the Swissprot database indicated that peptides were similar to polygalacturonase sequences in glycosyl hydrolase family 28. The four amino acid groups (NTD, DD, HG and RIK), presumably involved in catalysis, are conserved in these polygalacturonases in GH family 28 and are situated at AA 177 - 179, 199 - 200, 221 - 222 and 252 - 254, respectively (
Search database | Accession number | protein source | Sequence coverage (%) | Peptide coverage sequences | Modications |
---|---|---|---|---|---|
NCBI | 317468146 | L. gongylophorus | 21.05 | VAVNcGVGScTGTWNWSNLK | C5 (Carbamidomethyl); C10 (Carbamidomethyl) |
VSGGTTGKITNFNGITGFSQ | |||||
ITNFNGITGFSQ | |||||
ISmSGTFSNVK | M3 (Oxidation) | ||||
ISMSGTFSNVK | |||||
TDAAATGSTVTNITYSGNTATGcKR | C23 (Carbamidomethyl) |
Using polygalacturonic acid as a substrate, the optimum temperature for activity of the enzyme was 60˚C,
The effect of pH on the purified PGaseLg activity toward polygalacturonic acid was examined at 60˚C. As shown in
in the presence of stabilizers, and by phytopathogen Burkholderia cepacia, with an optimum pH of 3.5 [
The stability of an enzyme is an important parameter since maintenance of activity over a long period is important in designing pectinases reactors and also for reproducibility of data acquisition. With respect to temperature, in the absence of a substrate, PGaseLg showed >75% of the original activity at 30˚C and 40˚C, for 10 hours. At 50˚C, the enzyme lost 50% of its initial activity ~240 minutes (
Thermal stability contrasts with optimal activity; in the presence of a substrate, at 60˚C, the enzyme completely loses the activity in less than 15 minutes. The effect of substrate protection is demonstrated by the fast deactivation of the enzyme at its optimal temperature of reaction. The results showed that assay conditions of pH and temperature are not the best conditions for stability, which is in agreement with the fact that catalytic performance (activity) and stability of pectinases are quite different aspects [
In another experiment, PGaseLg was maintained at 50˚C at different pH values (3.0 to 8.0) for 360 minutes. Samples were taken at each 15 minutes until 120 minutes and then at every 30 minutes as exhibited in
The resulting profile, in
A typical Michaelian kinetic was observed for the hydrolysis of polygalacturonic acid and also for 74% esterified pectin at pH 5.0 and 60˚C by PGaseLg. When polygalacturonic acid was the substrate, the KM, Vmax and Kcat values were 0.65 mg∙mL−1, 1800 μmol∙min−1∙mg−1 and 35.97 s−1, respectively, while in the hydrolysis of 74% esterified pectin, the values of KM, Vmax and Kcat were 1.18 mg∙mL−1, 310 μmol∙min−1∙mg−1 and 6.19 s−1, respectively. From these results, it can be reported that PGaseLg has higher affinity toward polygalacturonic acid than esterified pectin.
The KM values of polygalacturonase from A. nainiana [
range of kinetic parameter values has been reported for polygalacturonases from various sources of microorganisms. This may be attributed to differences in assay procedures [
The susceptibility of PGaseLg to several cations, EDTA and β-mercaptoethanol at 1.0 and 5.0 mmol∙L−1 was investigated.
The PGaseLg inactivation observed in the presence of Hg2+ and Ni2+ and the inhibitory effect of other tested cations are indicative of active site blockage by metal complexation; the recovery activity for these cations is ordered in the following sequence: Ca2+ < Cu2+ < Co2+ < Pb2+ < Fe3+ < Zn2+ < Ni2+ ≈ Hg2+.
Manganese promoted 34% of enzyme activation at low concentration, but at 5.0 mmol∙L−1, the activity dropped to about 11%, as is observed for the PGase of A. giganteus(35). Taken together, apart from the specific responses, these results are in consonance with the literature which characteristically exhibits the inhibition by ordinary metals and the deactivation by heavy metals [
EDTA poorly affected the PGaseLg activity at lower experimental concentrations but the effect was amplified at higher concentration, indicating metal complexation in the catalysis process. β-mercaptoetanol promoted a similar effect on enzyme, which is an expected result since the deduced amino acid sequence of the enzyme (
The TLC technique was applied to investigate the PGaseLg mechanism using the hydrolysis products from enzyme action on polygalacturonic acid and 74% esterified pectin. It was observed that from polygalacturonic acid digestion, the only soluble product released was monogalacturonic acid (
Even upon prolonged incubation, the action of the enzyme on 74% esterified pectin did not exhibit the same accumulation of the final products. This observation is due to interruption of exopolygalacturonase action on esterified residues of the substrate since methyl esters limit an exopolygalacturonase action.
This paper is the first report of the isolation to electrophoretic homogeneity of an acidic exopolygalacturonase
Additives | Residual activity (%) | |
---|---|---|
1 mmol∙L−1 | 5 mmol∙L−1 | |
Na+ (control) | 100 | 100 |
K+ | 117 ± 1.9 | 531 ± 4.6 |
115 ± 1.6 | 492 ± 5.0 | |
Mg2+ | 105 ± 1.6 | 206 ± 0.8 |
Ca2+ | 67 ± 2.0 | 63 ± 5.1 |
Fe3+ | 27 ± 2.5 | 12 ± 1.6 |
Mn2+ | 124 ± 2.2 | 89 ± 0.5 |
Cu2+ | 52 ± 3.9 | 39 ± 1.9 |
Co2+ | 43 ± 1.3 | 33 ± 4.8 |
Zn2+ | 20 ± 1.3 | 7 ± 3.6 |
Ni2+ | 1 ± 0.1 | 1 ± 3.5 |
Pb2+ | 50 ± 0.9 | 31 ± 0.8 |
Hg2+ | 0 ± 0 | 0 ± 0.0 |
β-mercaptoethanol | 107 ± 2.3 | 250 ± 0.0 |
EDTA | 108 ± 2.1 | 562 ± 5.0 |
secreted by L. gongylophorus. The 37 kDa enzyme was biochemically and biophysically characterized as a typical fungal mesophilic exopolygalacturonase.
Polygalacturonases have been listed as an important factor in maintaining leaf-cutting ants/fungus symbiosis and, thus, inhibiting their activities can lead to the development of cutting ants control systems. In order to contribute to the study of these enzymes as targets for inhibition, we have described a xylanase/polygalacturonase bifunctional enzyme [
This work was supported by a grant 2011/21955-3 from the Sao Paulo Research Foundation (FAPESP). Paulo R. Adalberto is supported by a grant from the Brazilian National Research Council (CNPq 167524/2013-5). We thank to Prof. Fernando C. Pagnocca (UNESP, Rio Claro) for provided L. gongylophorus.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Paulo R.Adalberto,Camilla C.Golfeto,Fernando G.Almeida,DouglasFerreira,Quezia B.Cass,Dulce H. F.Souza,Ariele C.Moreira, (2016) Characterization of an Exopolygalacturonase from Leucoagaricus gongylophorus, the Symbiotic Fungus of Atta sexdens. Advances in Enzyme Research,04,7-19. doi: 10.4236/aer.2016.41002