Extracellular Polygalacturonase Research Articles

Induction of Extracellular Lytic Enzymes from Aureobasidium pullulans

Aims: The objective of this work was to analyze the production of some extracellular lytic enzymes of the fungus Aureobasidium pullulans.
 Methodology: The fungus was isolated from Valencian orange and was cultivated on Mathur medium modified with polygalacturonic acid (2% w/v) or xylan (2% w/v) as a carbon source, and they were incubated at 28°C, with constant stirring at 100 rpm, and at different times, the supernatant was harvested by filtration, and the extracellular lytic activity was determined by the Nelson method modified by Somogy, as well as the extracellular protein by the Lowry method.
 Results: The production of extracellular polygalacturonase and xylanase was induced, finding that the former has an optimal induction time at 7 days, with glutamic acid as a nitrogen source, and is stable at 4°C and 28°C and an optimal temperature and activity times of 60-80°C and 4 hours, while xylanase presents an optimal induction time of 9 days, with glutamic acid as nitrogen source, and very stable at 4 and 28°C, with optimal temperature and time of activity of 28°C and 8 hours.
 Conclusion: The fungus exhibits both polygalacturonase and xylanase activity. The polygalacturonase activity has a maximum induction time at 7 days, while xylanase has an optimal induction time at 9 days of incubation at 28°C. The xylanase activity was very stable at 4°C and 28°C, as it retains an activity of 100% at both temperatures after five days of incubation, while polygalacturonase retains 82.5% of its initial activity at 4°C, and 56.7% at 28°C.

Biorefinery of exhausted olive pomace through the production of polygalacturonases and omega-3 fatty acids by Crypthecodinium cohnii

The market for pectinolytic enzymes is continually expanding. In addition to the food, animal feed, textile and paper industries, pectinases have recently found application in the field of biorefinery. However, the industrial production of these enzymes is associated with high costs. Thus, the search for new source of microbial enzymes with low culture requirements, or from new substrates that allow the development of cost-effective and ecofriendly processes is of utmost importance. In this study, an unusual microbial source was postulated to produce pectinases from an uncommon agro-waste substrate. This study is the first report that reveals the production of pectinases by Crypthecodinium cohnii using as culture media liquors obtained after organosolv pretreatment of exhausted olive pomace (EOP). Results showed that using EOP as substrate, this strain was able to produce 42.15 ± 0.41 U/mL of extracellular polygalacturonase. Additionally, the valorization of EOP as a substrate to produce pectinases can be enhanced through the extraction of lipids rich in omega-3, making it a cost-effective and zero-waste process.

Toward Enhancing the Enzymatic Activity of a Novel Fungal Polygalacturonase for Food Industry: Optimization and Biochemical Analysis.

The review of literature and patents shows that enhancing the polygalacturonase (PG) production and activity are still required to fulfill the increasing demands. A dual optimization process, which involved Plackett-Burman design (PBD), with seven factors, and response surface methodology, was applied to optimize the production of extracellular PG enzyme produced by a novel strain of Aspergillus flavus isolated from rotten orange fruit. The fungal PG was purified and biochemically characterized. Three variables (harvesting time, pH and orange pomace concentration), that were verified to be significant by the PBD analysis, were comprehensively optimized via Box-Behnken design. According to this optimization, the highest PG activity (4073 U/mL) was obtained under pH 7 after 48 h using 40 g/L orange pomace as a substrate, with enhancement in PG activity by 51% compared to the first PBD optimization step. The specific activity of the purified PG was 1608 U/mg with polygalacturonic acid and its molecular weight was 55 kDa. The optimum pH was 5 with relative thermal stability (80%) at 50˚C after 30 min. The PG activity improved in the presence of Cu2+ and Ca2+, while Ba2+, Fe2+ and Zn2+ greatly inhibited the enzyme activity. The obvious Km and Vmax values were 0.8 mg/mL and 2000 μmol/min, respectively. This study is a starting point for initial research in the field of optimization and characterization of A. flavus PG. The statistical optimization of A. flavus PG and its biochemical characterization clearly revealed that this fungal strain can be a potential producer of PG which has a wide range of industrial applications.

INHIBITION OF AN EXTRACELLULAR POLYGALACTURONASE FROM GEOTRICHUM CANDIDUM BY A PROTEINACEOUS INHIBITOR ISOLATED FROM LEMON FRUITS

An extracellular polygalacturonase [EC 3.2.1.15], named PG-543, was purified from the culture of Geotrichum candidum IEV 543, a phytopathogenic fungi isolated from infected lemon. The molecular mass of the enzyme was estimated to be 26 kDa by SDS-PAGE. The pH optimum determined was 5.5; the enzyme showed high stability in the pH range between 4.0 and 6.0. The temperature optimum was 37 ºC. Furthermore, a proteinaceous inhibitor of the polygalacturonase (PG) named PGIP was isolated from lemon albedo. This protein was able to maintain the inhibitory activity in a wide range of pH and temperature. The maximal interaction between PG-PGIP was reached after 20 min of contact. PGIP did not show toxic effect on Artemia salina in the concentration range that was active on PG enzyme. Also, the proteinaceous inhibitor did not show mutagenic nor phytotoxic effects. Our results suggest that, the PGIP isolated from lemon could be used as a natural product to decrease the propagation of pathogenic fungi responsible for postharvest diseases in citrus by the inhibition of hydrolytic enzymes secreted by pathogens.

Optimization of cultural conditions for polygalacturonase production by a newly isolated Aspergillus fumigatus R6 capable of retting kenaf

Sequential optimization strategy was used to enhance the production of extracellular polygalacturonase by a newly isolated Aspergillus fumigatus R6 using rice bran as a substrate in solid state conditions. Three significant variables influencing the polygalacturonase production were identified as initial moisture level, temperature and incubation time (P<0.0001). The model established by face-centered central composite design was significant (P<0.05) with high R2 (0.98). The model validity was verified and the optimum conditions were at an initial moisture level of 49.6%, 33°C and 129h of incubation time with the maximum polygalacturonase activity of 565U/g, resulted in 2.65 fold increase in polygalacturonase activity compare to the unoptimized conditions. Kenaf stem treated with A. fumigatus R6 polygalacturonase enzyme at 72h produced high strength of kenaf bast fibers (287MPa) with high Young’s modulus (10404MPa) and the color is in satisfactory.

Purification and characterization of exo-polygalacturonase from Zygoascus hellenicus V25 and its potential application in fruit juice clarification.

The purification and characterization of the extracellular polygalacturonase from Zygoascus hellenicus V25 submerged culture using orange peel waste were investigated. This polygalacturonase, with a molecular weight of 75.28 kDa, was purified to 16.89 purification fold with a recovery of 18.46% and specific activity of 2469.77 U/mg protein by ammonium sulfate precipitation, DEAE cellulose chromatography, and Sephadex G-100 gel filtration. The enzyme exhibited maximum activity at 60°C and pH 5.0 and was stable over a wide range of pH levels (3.0-11.0). Moreover, enzyme activity was enhanced by Cu2+ and cysteine, whereas it was strongly inhibited by Hg2+. The extent of enzymatic hydrolysis was negatively correlated with the degree of pectin esterification. Km and Vmax values of the polygalacturonase were 5.44 mg/mL and 61.73 μmol/(min·mg), respectively. The polygalacturonase was applied in the juice clarification of four fruits, and results showed that the percentage transmittance at 660 nm increased by 3.51, 4.36, 8.04, and 12.2%.

Comparative proteomic analyses reveal that Gnt2-mediated N-glycosylation affects cell wall glycans and protein content in Fusarium oxysporum

Protein N-glycosylation is a ubiquitous post-translational modification that contributes to appropriate protein folding, stability, functionality and localization. N-glycosylation has been identified as an important process for morphogenesis and virulence in several fungal pathogens including Fusarium oxysporum. Here we conducted comparative chemical and proteome-based analyses to better understand the physiological changes associated with protein hypo-N-glycosylation in F. oxysporum N-glycosyltransferase Gnt2-deficient mutant. The results suggest that lack of functional Gnt2 alters the size of galactofuranose chains in cell wall glycans, resulting in polysaccharides with a broad range of polymerization degrees and differential protein glycosylation patterns. Functional Gnt2 is necessary for normal conidium size and morphology and wild-type hyphal fusion rates. Hypo-N-glycosylation in ∆gnt2 mutant results in enhanced oxidative stress resistance and reduced levels of proteins involved in cell wall organization, biogenesis and remodelling. Deletion of gnt2 gene led to accumulation of trafficking vesicles at hyphal tips, reduced secretion of extracellular proteins related to detoxification of antifungal compounds and degradation of plant cell walls, and lowered extracellular polygalacturonase activity. Altogether, the results confirm that Gnt2-mediated N-glycosylation plays a crucial role in morphogenesis and virulence, and demonstrate that Gnt2 is essential for protein function, transport and relative abundance in F. oxysporum.

Alkaline and thermostable polygalacturonase from Streptomyces halstedii ATCC 10897 with applications in waste waters

Abstract Pectin degrading enzymes with polygalacturonase (PG) activity hydrolyze α-(1,4) glycosidic bonds of polysaccharides present in higher plants. In the current study one hundred bacterial strains were screened for extracellular PG activity using an inductive culture medium. Optimization of fermentation conditions for Streptomyces halstedii ATCC 10897 was conducted using experimental designs. The maximum enzymatic activity obtained was 3.489 U/mL and 98.0% of viscosity reduction after 12 h of fermentation using soy peptone as unique source of carbon and nitrogen. PG from S. halstedii ATCC 10897 showed high thermal stability, an approximate molecular weight of 48 kDa and its optimum conditions for catalytic reaction were 50 °C and pH 12.0. This study reveals that alkaline PG is a useful enzyme for depectinization in alkaline pulping mill and papermaking waste waters.

Cell-wall polysaccharide composition and glycanase activity of Silene vulgaris callus transformed with rolB and rolC genes.

The aim of this research is to investigate the effects of the Agrobacterium rhizogenes rol genes on the composition of cell-wall polysaccharides and glycanase activity in the campion callus. The expression of the rolC gene reduces the yield of campion pectin, while the expression of the rolB or rolC gene inhibits the volumetric production of both pectin and intracellular arabinogalactan. The rol genes are involved in regulating the activity of glycanases and esterases, thereby contributing to the modification of polysaccharide structures, their molecular weight (Mw) and the degree of pectin methyl esterification (DE). The increase in pectin arabinose residue appears to be connected to a decrease in intracellular and extracellular α-l-arabinofuranosidase activity in transgenic campion calluses. In transgenic calluses expressing the rolB and rolC genes, the increase in pectin galactose residue is likely due to a decrease in β-galactosidase activity. The decrease in the Mw of pectin and its d-galacturonic acid content appears to be connected to an increase in extracellular polygalacturonase activity. Finally, the increase in pectinesterase activity causes a decrease in the DE of pectin. Thus, the expression of rolB and rolC genes in campion callus has a considerable effect on pectin's sugar composition, DE and Mw, while it appears to have an insignificant influence on intracellular and extracellular arabinogalactans.

Production, Purification, and Characterization of Polygalacturonase fromRhizomucor pusillusIsolated from Decomposting Orange Peels

A thermophilic fungal strain producing polygalacturonase was isolated after primary screening of 40 different isolates. The fungus was identified as Rhizomucor pusilis by Microbial Type Culture Collection (MTCC), Chandigarh, India. An extracellular polygalacturonase (PGase) from R. pusilis was purified to homogeneity by two chromatographic steps using Sephadex G-200 and Sephacryl S-100. The purified enzyme was a monomer with a molecular weight of 32 kDa. The PGase was optimally active at 55°C and at pH 5.0. It was stable up to 50°C for 120 min of incubation and pH condition between 4.0 and 5.0. The stability of PGase decreases rapidly above 60°C and above pH 5.0. The apparent K m and V max values were 0.22 mg/mL and 4.34 U/mL, respectively. It was the first time that a polygalacturonase enzyme was purified in this species. It would be worthwhile to exploit this strain for polygalacturonase production. Polygalacturonase from this strain can be recommended for the commercial production because of its constitutive and less catabolically repressive nature, thermostability, wide range of pH, and lower K m properties. However, scale-up studies are needed for the better output for commercial production.

Production, Purification, and Characterization of Polygalacturonase fromMucor circinelloidesITCC 6025

Mucor circinelloides produced an extracellular polygalacturonase enzyme, the production of which was enhanced when various production parameters were optimized. Maximum polygalacturonase (PGase) activity was obtained in 48 h at 30°C and pH 4.0 with pectin methyl ester (1% w/v) as carbon source and a combination of casein hydrolysate (0.1% w/v) and yeast extract (0.1% w/v) as nitrogen source. The enzyme was purified to homogeneity (13.3-fold) by Sephacryl S-100 gel-filtration chromatography. Its molecular weight was 66 kDa on SDS-PAGE. The enzyme was found to have K m and V max values of 2.2 mM and 4.81 IU/ml at 0.1% to 0.5% (w/v) concentration of the substrate. The addition of phenolic acids (0.05 mM), metal ions such as Mn+2, Co+2, Mg+2, Fe+3, Al+3, Hg+2, and Cu+2, and thiols had inhibitory effect on the enzyme. The enzyme showed maximum activity in the presence of polygalacturonic acid (0.1% w/v) at pH 5.5 and 42°C.

Purification and characterization of an exo-polygalacturonase from Pycnoporus sanguineus

The present work describes the purification and characterization of a novel extracellular polygalacturonase, PGase I, produced by Pycnoporus sanguineus when grown on citrus fruit pectin. This substrate gave enhanced enzyme production as compared to sucrose and lactose. PGase I is an exocellular enzyme releasing galacturonic acid as its principal hydrolysis product as determined by TLC and orcinol-sulphuric acid staining. Its capacity to hydrolyze digalacturonate identified PGase I as an exo-polygalacturonase. SDS-PAGE showed that PGase I is an N-glycosidated monomer. The enzyme has a molecular mass of 42 kDa, optimum pH 4.8 and stability between pH 3.8 and 8.0. A temperature optimum was observed at 50–60 °C, with some enzyme activity retained up to 80 °C. Its activation energy was 5.352 cal mol −1. PGase I showed a higher affinity towards PGA than citric pectin (Km = 0.55 ± 0.02 and 0.72 ± 0.02 mg ml −1, respectively). Consequently, PGase I is an exo-PGase, EC 3.2.1.82.

A search for factors influencing etioplast–chloroplast transition

Chloroplast biogenesis in angiosperm plants requires the light-dependent transition from an etioplast stage. A key factor in this process is NADPH:protochlorophyllide oxidoreductase A (PORA), which catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide. In a recent study the chloroplast outer envelope channel OEP16 was described to be involved in etioplast to chloroplast transition by forming the translocation pore for the precursor protein of PORA [Pollmann et al. (2007) Proc Natl Acad Sci USA 104:2019-2023]. This hypothesis was based on the finding that a single OEP16.1 knockout mutant in Arabidopsis thaliana was severely affected during seedling de-etiolation and PORA protein was absent in etioplasts. In contrast, in our study the identical T-DNA insertion line greened normally and showed normal etioplast to chloroplast transition, and mature PORA was present in etioplasts [Philippar et al. (2007) Proc Natl Acad Sci USA 104:678-683]. To address these conflicting results regarding the function of OEP16.1 for PORA import, we analyzed several lines segregating from the original OEP16.1 T-DNA insertion line. Thereby we can unequivocally show that the loss of OEP16.1 neither correlates with impaired PORA import nor causes the observed de-etiolation phenotype. Furthermore, we found that the mutant line contains at least 2 additional T-DNA insertions in the genes for the extracellular polygalacturonase converter AroGP1 and the plastid-localized chorismate mutase CM1. However, detailed examination of the de-etiolation phenotype and a genomewide transcriptional analysis revealed no direct influence of these genes on etioplast to chloroplast transition in Arabidopsis cotyledons.

Purification and Partial Characterization of an Exo-polygalacturonase from Paecilomyces variotii Liquid Cultures

An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K(m) and V(max) values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 micromol/min/mg, respectively. PG was found to have temperature optimum at 65 degrees Celsius and was totally stable at 45 degrees Celsius for 90 min. Half-life at 55 degrees Celsius was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na(+1), K(+1), and Co(+2) (1 mM) and Cu(+2) (1 and 10 mM).

Polygalacturonase secreted by yeasts from Brazilian semi-arid environments

Microbial pectinolytic enzymes are known to play a commercially important role in a number of industrial processes. The objective of this study was to investigate the extracellular polygalacturonases of yeasts isolated from Brazilian semi-arid environments. Among the 250 colonies tested, only 33 produced extracellular polygalacturonases: Aureobasidium pullulans (18 isolates), Candida boidinii (one isolate), Trichosporonoides sp. (three isolates), Kluyveromyces marxianus (one isolate), Cryptococcus liquefaciens (one isolate), Pseudozyma sp. (four isolates), and yeast-like related to fungal endophyte (five isolates). The highest activity of polygalacturonase was observed in Pseudozyma sp. CCMB 300 (14.17±0.08 µmol acid galacturonic released/min/mg protein). This study shows the potential of yeasts and yeast-like organisms isolated from Brazilian semi-arid environments to produce pectinolytic enzymes.

Induction of β-1,3-glucanase in callus cultures in vitro

Sodium salicylate (NaSA) increased induction of both intracellular and extracellular beta-1,3-glucanases in calluses of campion and duckweed. NaSA concentrations from 30 to 100 mM were optimal for induction of intracellular glucanase in the campion callus, and for induction of extracellular glucanase the optimal concentration varied from 5 to 100 mM. The glucanase activity in the duckweed callus was lower than in the campion callus, and co-cultivation of the campion callus with Trichoderma harzianum mycelium increased the production of intracellular and extracellular beta-1,3-glucanases and polygalacturonase in the callus. Biosynthesis by T. harzianum of glucanases, extracellular polygalacturonase and xylanase, and of intracellular galactosidase was increased. The co-cultivation was accompanied by increased activity of intracellular acidic isoform of glucanase Glu-3 secreted by the callus cells into the medium, whereas NaSA activated in the callus culture the extracellular acidic isoform Glu-1 and extracellular basic isoform Glu-5. These data indicate the induction of these isoforms and the specificity of protective response of plant cells to different factors.

Two Xanthomonas Extracellular Polygalacturonases, PghAxc and PghBxc, Are Regulated by Type III Secretion Regulators HrpX and HrpG and Are Required for Virulence

Xanthomonas campestris pv. campestris, the causal agent of black rot disease, produces a suite of extracellular cell-wall degrading enzymes (CWDE) that are involved in bacterial virulence. Polygalacturonase (PG) is an important CWDE and functions to degrade the pectic layers of plant cell walls. Although previous studies have documented the virulence functions of PG in Erwinia and Ralstonia species, the regulation of PG genes still needs to be elucidated. In this study, we identified two novel PG genes (pghAxc and pghBxc) encoding functional PG from X. campestris pv. campestris 8004. The expressions of these two PG genes are regulated by the type III secretion regulators HrpX and HrpG and the global regulator Clp. These PG genes could be efficiently induced in planta and were required for the full virulence of X. campestris pv. campestris to Arabidopsis. In addition, these PG were confirmed to be secreted via the type II secretion system in an Xps-dependent manner.

Production, purification and characterization of an endopolygalacturonase from Mucor rouxii NRRL 1894

An extracellular polygalacturonase (PGase) from Mucor rouxii NRRL 1894 was purified to homogeneity by two chromatographic steps using CM-Sepharose and Superdex 75. The purified enzyme was a monomer with a molecular weight of 43100 Da and a p I of 6. The PGase was optimally active at 35 °C and at pH 4.5. It was stable up to 30 °C and stability of PGase decrease rapidly above 60 °C. The extent of hydrolysis of different pectins was decreased with increasing of degrees of esterification. Except Mn 2+, all the examined metal cations showed inhibitory effects on the enzyme activity. The apparent K m and V max values for hydrolyze of polygalacturonic acid (PGA) were 1.88 mg/ml and 0.045 μmol/ml/min, respectively. The enzyme released a series of oligogalacturonates from polygalacturonic acid indicating that it had an endo-action. Its N-terminal sequence showed homologies with the endopolygalacturonase from the psychrophilic fungus Mucor flavus.

Purification and characterization of polygalacturonase produced by thermophilic Thermoascus aurantiacus CBMAI-756 in submerged fermentation

An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60-65 degrees C. The apparent K (m) with citrus pectin was 1.46 mg/ml and the V (max) was 2433.3 micromol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50 degrees C for 1 h and showed a half-life of 10 min at 60 degrees C. Polygalacturonase was stable at pH 5.0-5.5 and maintained 33% of initial activity at pH 9.0. Metal ions, such as Zn(+2), Mn(+2), and Hg(+2), inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing mono, di and tri-galacturonic acids within 10 min of hydrolysis.

Production d'enzyme polygalacturonase par des souches microbiennes isolées du lait cru et des olives noires et vertes

Forty microbial strains isolated from raw milk samples and black and green olives were grown in MP5 (mineral pectin 5) medium containing 0.5% lemon pectin. All strains synthesized an extracellular polygalacturonase. Rhodotorula sp. ONRh9 (0.44 U x mL(-1)) and Leuconostoc sp. LLn1 (0.16 U x mL(-1)), which had a more active polygalacturonase in MP5 medium, were studied in MAPG5 medium containing polygalacturonic acid. Highest biomass and polygalacturonase production by these two strains were observed for polygalacturonic acid concentrations of 10 g x L(-1) (Rhodotorula sp. ONRh9) and 5 g x L(-1) (Leuconostoc sp. LLn1) and for initial pH values of 6 (Rhodotorula sp. ONRh9) and 5.5 (Leuconostoc sp. LLn1). The two strains grown in fermenters in MAPG5 medium generated the following results: with controlled initial pH, Rhodotorula sp. produced maximum biomass (DO) and polygalacturonase (PG) after 20 h (DO, 3.86; PG, 0.24 U x mL(-1)) of growth, and this level was sustained until the end of the culture; Leuconostoc sp. LLn1 synthesized more cells and polygalacturonase between 4 h (DO, 1.80; PG, 0.17 U x mL(-1)) and 24 h (DO, 3.90; PG, 0.27 U x mL(-1)) of culture. With uncontrolled initial pH, the cultures produced maximum biomass and polygalacturonase after 20 h (DO, 3.30; PG, 0.26 U x mL(-1)) for Rhodotorula sp. ONRh9 and 10 h (DO, 2.84; PG, 0.17 U x mL(-1)) for Leuconostoc sp. LLn1.