Production Of Exo-polygalacturonase Research Articles

Exo-polygalacturonase production enhancement by Piriformospora indica from sugar beet pulp under submerged fermentation using the response surface methodology.

This study proposed a novel and cost-effective approach to enhance and optimize the exo-polygalacturonase from P. indica, a root endophytic fungus. In the current investigation, the impact of ammonium sulfate, sugar beet pulp (SBP), and glucose as variables on induction of exo-polygalacturonase from P. indica was optimized using the central composite design (CCD) of response surface methodology (RSM) under submerged fermentation (SmF). Additionally, determination of the exo-polygalacturonase molecular weight and in situ analysis was performed. The optimal reaction conditions, which resulted in the highest enzyme activity, were observed in the following conditions: ammonium sulfate (4g/L), SBP (20g/L), and glucose (60g/L). Under the optimized condition, the maximum enzyme activity reached 19.4U/ml (127U/mg), which increased by 5.84 times compared to non-optimized conditions. The exo-polygalacturonase molecular weight was estimated at 60 KDa. In line with the bioinformatic analysis, the exo-polygalacturonase sequence of P. indica showed similarity with Rhizoctonia solani's and Thanateporus cucumeris. These results indicated that SBP acts as a cheap and suitable inducer of exo-polygalacturonase production by P. indica in submerged cultivation. The outcome of this study will be useful for industries to decrease environmental pollution with cost-effective approaches.

Production of microalgae-derived exo-polygalacturonase for fruit juice clarification

Due to the growing demand for commercial enzymes derived from renewable and sustainable resources with higher efficacy, algal biomass has gained more attention. Thus, the production of exo-polygalacturonase (PGase) from Bacillus subtilis under submerged fermentation (SmF) using algal feedstocks as a carbon source in fermentation medium and its potential use for the clarification of apple and carrot juices with the optimization of the enzyme loading, pH, and time using Box–Behnken response surface methodology (RSM) were prompted. The maximum PGase activity of 510.61 ± 7.86 U/L was achieved at pH 5.0 after 48 h at 40°C using 0.5% (w/v) pectin, 0.1% (w/v) yeast extract, 0.02% (w/v) MgSO47H2O, 0.02% (w/v) K2HPO4, 0.3% (w/v) glucose, and 0.583% (w/v) microalgae hydrolysate as a fermentation medium. PGase production without glucose was favored by SmF with a shake flask working volume of 100 ml (248.90 ± 4.58 U/L) as compared to a bioreactor with a working volume of 1 L (192.99 ± 0.84) for 48 h. Both carrot and apple juices were treated with the crude PGase (248.9 U/L) at different concentrations (0.1–0.5%), temperature (30–50°C), and time (30–120 min), and the maximal clarification conditions were obtained as 0.3% (w/v) enzyme concentration at 50°C for 30 min exposure time. This study revealed that microalgae-derived PGase in crude form could be effectively used for clarification of the juices.

Exo-polygalacturonase production from agro-waste by Penicillium fellutanum and insight into thermodynamic, kinetic, and fruit juice clarification

Exo-polygalacturonase production from agro-waste by Penicillium fellutanum and insight into thermodynamic, kinetic, and fruit juice clarification

High-yield intracellular production of an exo-polygalacturonase enzyme via heterologous expression of Penicillium notatum gene in Saccharomyces cerevisiae

Exo-polygalacturonase (Exo-PG) is one of the most important members of the pectinolytic group of enzymes with immense applications in the food industry. The present work was undertaken to investigate the cloning, expression, and transformation of an Exo-PG gene in yeast Saccharomyces cerevisiae to achieve the high titre of Exo-PG from Penicillium notatum. For this, the Exo-PG gene from P. notatum was cloned into BamHI and XbaI digested pYES2 plasmid with GAL1 promoter, and heterologously expressed in S. cerevisiae. The recombinant yeast cells were cultivated at 30°C in shake flask fermentation using minimal media without uracil, in the presence of ampicillin (100 µg/mL), following the addition of 2.0% galactose as an expression inducer. Results revealed that the yeast was a good expression host, and successfully produced 6.67 U/mL of the recombinant enzyme into the culture media after 24 h of induction; under longer induction time, the activity was decreased. The secreted Exo-PG exhibited two strong bands with an approximate molecular weight of 20 - 25 kDa and 70 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, thus indicating a dimeric protein. In conclusion, the results demonstrated that the gene was successfully expressed, thus resulting in high-yield intracellular production of Exo-PG.

An analysis of exo-polygalacturonase bioprocess in submerged and solid-state fermentation by Pleurotus ostreatus using pomelo peel powder as carbon source

BackgroundAs there has been an increasing trend in the effective utilization of plant and crop residues for microbial transformation into a desired product, an attempt was made to compare of exo-polygalacturonase production using logistic and Luedeking-Piret kinetic model by Pleurotus ostreatus in submerged (smf) or solid-state fermentation (ssf) using pomelo peel powder, an agro-forestry residue as carbon substrate. ResultsCultures grown in submerged fermentation produced a peak of exo-polygalacturonase activity as 6160 Ul-1 on the 4th day of culture as compared with 2410 Ul-1 on the 5th day of fermentation by solid-state fermentation. The enzyme yield coefficient (YE/X) is of higher value in smf vs. ssf system (YE/X = 1.05 × 103 vs. 0.622 × 103) indicating the more efficient product yield in smf as compared with ssf. The plots derived fromλ versusζ clearly demonstrate that the secondary product destruction is higher in smf than in ssf. ConclusionP. ostreatus performs much better in submerged fermentation as compared with solid-state fermentation in respect to exo-polygalacturonase production although ssf technique produced a more thermo-stable exo-polygalacturonase in crude extract, which is highly desirable in various industrial applications.

In Situ and Contact Immobilization/Adhesion of Penicillium brasilianum in Polyurethane Foam for Production of Exo-Polygalacturonase

The present work aimed to immobilize Penicillium brasilianum fungus in situ or by contact in polyurethane foam and assess the production of exo-polygalacturonase with the immobilized fungus while e...

SCREENING OF FACTORS INFLUENCING EXO-POLYGALACTURONASE PRODUCTION BY ASPERGILLUS NIGER ATCC 120120 USING TWO-LEVEL FRACTIONAL FACTORIAL DESIGN

Exo-polygalacturonase was produced by Aspergillus niger ATCC 120120 in a solid-state fermentation using Nephrolepis biserrata leaves. Factors affecting the production of exo-polygalacturonase were determined using a two-level fractional factorial design. The screening process for six factors; pH, incubation time, temperature, pectin concentration, inoculum size and moisture content, that influence the production of exo-polygalacturonase by A. niger was performed. The result of variance analysis (ANOVA) suggested that there were four statistically significant (P < 0.005) factors in the production of exo-polygalacturonase by A. niger. These factors were incubation time, temperature, pectin concentration and moisture content. The statistical analysis shows that the linear mathematical model is significant with coefficient of determination (R2) value of 0.9711. The optimum production of exo-polygalacturonase obtained using the model in this study was at 40.00 U/g.

OPTIMIZATION AND CHARACTERIZATION OF EXO-POLYGALACTURONASE BY Aspergillus niger CULTURED VIA SOLID STATE FERMENTATION

Polygalacturonases represent an important member of pectinases group of enzymes with immense industrial applications. The activity of exo-polygalacturonase produced by Aspergillus niger was studied in solid state fermentation (SSF) using Nephrolepis biserrata leaves as substrate. Central composite design (CCD) was used to optimize four significant variables resulted from the screening process that has been initially analyzed for the production of exo-polygalacturonase which are incubation time, temperature, concentration of pectin and moisture content. The optimum exo-polygalacturonase production obtained was 54.64 U/g at 120 hours of incubation time, temperature at 340C, 5.0 g/L of pectin concentration and 75.26% of moisture content. For partial characterization of exo-polygalacturonase, the optimum temperature and pH were obtained at 50°C and pH 4.0, respectively. SDS-PAGE analysis showed that molecular weight of exo-polygalacturonase were 35 and 71 kDa. This study has revealed a significant production of exo-polygalacturonase by A. niger under SSF using cheap and easily available substrate and thus could found immense potential application in industrial sectors and biotechnology

Production and characterization of Penicillium brasilianum pectinases with regard to industrial application

The objective of this study was to evaluate the production of pectinase by an isolated strain of Penicillium brasilianum in a bioreactor and to consider its potential for industrial applications (i.e. fruit juice). The optimization of production was achieved through experimental design. The maximum exo-polygalacturonase (Exo-PG) production in the bioreactor was 53.8 U mL−1 under the conditions of 180 rpm, an aeration rate of 1.5 vvm, 30 °C, pHinitial of 5.5, 5 × 106 spores mL−1, 32 g L−1 pectin, 10 g L−1 of yeast extract and 0.5 g L−1 magnesium sulfate and bioproduction for 36 h. The production of Exo-PG in the bioreactor was 1.3 times higher than that obtained in shake flasks, with aeration (1.5 vvm) and agitation (180 rpm) control. The crude enzyme complex, beyond the pectinolytic activity of Exo-PG (53.8 U mL−1), also contained activity pectin methylesterase (6.0 U mL−1) and pectin lyase (6.61 U mL−1). At a crude enzyme complex with a concentration of 0.5% (v/v), viscosity of peach juice was reduced by 11.66%, turbidity was reduced by 13.71% and clarification was increased by 26.92%. Based on the present results, we can conclude that the new strain of isolated P. brasilianum produced high amounts of pectinases in a bioreactor with mechanical agitation, and has the potential to be applied to in the clarification of juices.

Utilization of Orange Peel, Corn Steep Liquor, and Parboiled Rice Water in the Production of Polygalacturonase from Aspergillus niger

Abstract The objective of this work was to optimize the process parameters of exo-polygalacturonase production from Aspergillus niger ATCC 9642 via submerged fermentation using agro-industrial residues such as orange peel, corn steep liquor, and parboiled rice water and to do partial enzyme characterization. A complete second-order design was used for screening the most important factors and to maximize exo-polygalacturonase production. The maximum exo-polygalacturonase activity in our work was 18.5 U/mL (specific activity of 195.6 U/mg) at 120 h with concentrations of 80 g/L orange peel, 60 g/L corn steep liquor, and 450 g/L parboiled rice water, at 180 rpm, 30°C, pHinitial 5.5, and spore concentration 5x106 spores/mL. The highest storage stability was obtained at 348 h for 55°C and 808 h for −80°C (pH 5.5). The activation energy (Ed) for deactivation reaction was 1.05 KJ/mol, with a half-life (t1/2) of 63 h.

Utilization of orange peel, a food industrial waste, in the production of exo-polygalacturonase by pellet forming Aspergillus sojae.

The production of exo-polygalacturonase (exo-PG) from orange peel (OP), a food industrial waste, using Aspergillus sojae was studied in submerged culture. A simple, low-cost, industrially significant medium formulation, composed of only OP and (NH4)2SO4 (AS) was developed. At an inoculum size of 2.8×10(3) spores/mL, growth was in the form of pellets, which provided better mixing of the culture broth and higher exo-PG activity. These pellets were successfully used as an inoculum for bioreactors and 173.0U/mL exo-PG was produced. Fed-batch cultivation further enhanced the exo-PG activity to 244.0U/mL in 127.5h. The final morphology in the form of pellets is significant to industrial fermentation easing the subsequent downstream processing. Furthermore, the low pH trend obtained during this fermentation serves an advantage to fungal fermentations prone to contamination problems. As a result, an economical exo-PG production process was defined utilizing a food industrial by-product and producing high amount of enzyme.

Oxygen transfer coefficient and the kinetic parameters of exo-polygalacturonase production by Aspergillus flavipes FP-500 in shake flasks and bioreactor.

To evaluate and compare the sensitivity of Exo-PG production and kinetic parameters of Aspergillus flavipes FP-500 to oxygen transfer condition in shake flasks and bioreactor. Aspergillus flavipes FP-500 was grown on pectin as carbon source in shake flasks and bioreactor at different oxygen transfer conditions. The volumetric coefficient of oxygen transfer (kLa) was modified by changing both, the flask size/medium volume ratio and the agitation speed. Higher biomass concentration, Exo-PG activity, maximum specific growth rate and yield coefficient were obtained in bioreactor at higher kLa value. A strong correlation was found between biomass, Exo-PG activity and growth-associated product coefficient to kLa in bioreactor but does not in shake flasks. The mathematical model provided a good description of growth, pectin consumption and Exo-PG production in submerged batch cultures carried out in bioreactor. Biomass concentration, Exo-PG activity and their kinetics of Aspergillus flavipes FP-500 were strongly influenced by oxygen transfer condition and cultivation system. Significance and Impact of Study The production of enzymes by fungal fermentation is strictly aerobic and understanding the influence of oxygen transfer condition on the production kinetic is of vital importance in order to design, optimize and translate bioprocesses to industrial scale.

Enhanced production of exo-polygalacturonase from agro-based products by Aspergillus sojae

Aspergillus sojae has been previously shown to produce exo-polygalacturonase (exo-PG) in synthetic media, where the potential of the organism to utilize agricultural substrates was not considered so far. In this study, the utilization of agro-based products was taken into account in the enhanced production of exo-PG using an A. sojae mutant by applying statistical design methods. Complex sources (orange peel, wheat bran, and corn meal), simple sugar sources (glucose, maltrin, and sugar beet syrup), and two phosphate salts were screened using D-optimal design method. Orange peel yielded the highest exo-PG activity with all simple sugars and phosphate sources. According to the results of response surface methodology (RSM), the optimum concentrations of orange peel, sugar beet syrup, and (NH4)2SO4 were found to be 10, 60, and 8 g L-1, respectively. The exo-PG activity under these conditions was 145.4 U m L-1 in shake flask cultures. In bioreactor studies enzyme production was induced at low pH values; thus highest production was obtained under uncontrolled pH conditions, in which the pH dropped to 2.0 in 72 h. As a result high exo-PG could be produced by an A. sojae mutant using a cost-effective medium containing agro-industrial substrates. Another important advantageous outcome was the low optimal pH, which is especially desired in industrial fermentations prone to contamination problems. In fact this highlights the easy adaptation of this fermentation to industrial scales.

Production of exopolygalacturonase from wheat flour by Aspergillus awamori in submerged and surface culture fermentation

Pectinases are a group of hydrolytic enzymes that degrade pectic substances. They are produced by a variety of microorganisms including filamentous fungi. Studies were carried out on the production of exoplygalacturonase (Exo-PGase) using wheat flour by Aspergillus awamori in submerged fermentation (SmF) and surface culture fermentation (SCF). Higher titres of was observed when medium was supplements with carbon (4 g/L Pectin for both SmF and SCF) and nitrogen (ammonium sulphate 6 g/L for both SmF and SCF) sources. SCF proved to be a better process than SmF for Exo-PGase production, giving the highest enzyme production rate in 96 h. Exo-PGase produced by A. awamori presented optimum activity at pH 5 for SmF and 5.5 for SCF. Use of continuous system for Exo-PGase production obtained a better results than the batch system. Key words: Exopolygalacturonase, Aspergillus awamori, wheat flour, surface culture fermentation, submerged fermentation, continuous production.

Optimization of Exo-Polygalacturonase Production from Orange Peel by Aspergillus sojae

Optimization of Exo-Polygalacturonase Production from Orange Peel by Aspergillus sojae

Exo-polygalacturonase production by Bacillus subtilis CM5 in solid state fermentation using cassava bagasse

The purpose of this investigation was to study the effect of Bacillus subtilis CM5 in solid state fermentation using cassava bagasse for production of exo-polygalacturonase (exo-PG). Response surface methodology was used to evaluate the effect of four main variables, i.e. incubation period, initial medium pH, moisture holding capacity (MHC) and incubation temperature on enzyme production. A full factorial Central Composite Design was applied to study these main factors that affected exo-PG production. The experimental results showed that the optimum incubation period, pH, MHC and temperature were 6 days, 7.0, 70% and 50°C, respectively for optimum exo-PG production.

Exo-polygalacturonase production by Bacillus subtilis CM5 in solid state fermentation using cassava bagasse

The purpose of this investigation was to study the effect of Bacillus subtilis CM5 in solid state fermentation using cassava bagasse for production of exo-polygalacturonase (exo-PG). Response surface methodology was used to evaluate the effect of four main variables, i.e. incubation period, initial medium pH, moisture holding capacity (MHC) and incubation temperature on enzyme production. A full factorial Central Composite Design was applied to study these main factors that affected exo-PG production. The experimental results showed that the optimum incubation period, pH, MHC and temperature were 6 days, 7.0, 70% and 50°C, respectively for optimum exo-PG production.

Exo-polygalacturonase production by Penicillium roqueforti on pumpkin oil cake in solid state fermentation

The feasibility of using pumpkin oil cake (PuOC), individual and in combination with wheat bran (WB), as substrate for the production of Exo-polygalacturonase (Exo-p) by starter culture Penicillium roqueforti in solid state fermentation (SSF) has been evaluated. The kinetics of enzyme production was investigated using PuOC alone in the range from 13 to 168 h, with moisture contents varying from 44% the ability to grow and produce Exo-p activity on this substrate, reaching a maximum value of 1451.75 U/g.d.w PuOC by the 5th day of fermentation. Fermentation experiments indicated that the water activity (aw) influenced the enzyme production. A medium with aw 0.932 and the fermentation time of 5 days were selected, as these conditions resulted in the highest pectolytic activity and were used for further investigation. A next step in this research was to examine the effect of the substrate combination, PuOC with wheat bran (WB), in different ratios. The addition of WB as carbon sources was found to have a significant influence on the enzymes yields. Exo-p activities were the highest with initial water activity of a w 0.932 and PuOC supplementation with WB (1:0.67).

Polygalacturonase production by Aspergillus awamori on wheat in solid-state fermentation.

The production of exo-polygalacturonase (exo-PG) and endo-PG by Aspergillus awamori grown on wheat in solid-state fermentation was studied. Endo- and exo-PG activities were detected after 24 h of inoculation. Glucose released from starch hydrolysis acted as a catabolite repressor for the exo-PG enzyme. In contrast, endo-PG production was not affected by glucose repression. When milled grains were used, the particle-size distribution and the chemical composition of the medium influenced the rate of micro-organism growth and therefore the trend followed by endo- and exo-PG production. However, these two parameters did not affect the maximum production of exo-PG and endo-PG. For one of the milled samples, three different moisture contents were used (50, 55, 60%). Moisture contents of 60% provide a higher yield of pectinases by A. awamori.

Primary structure and characterization of an exopolygalacturonase from Aspergillus tubingensis.

From the culture fluid of the hyphal fungus Aspergillus tubingensis, an exopolygalacturonase with a molecular mass of 78 kDa, an isoelectric point in the pH-range 3.7-4.4 and a pH optimum of 4.2 was purified. The enzyme has been characterized as an exopolygalacturonase [poly(1,4-alpha-D-galacturonide)galacturonohydrolase] that cleaves monomer units from the non-reducing end of the substrate molecule. K(m) and Vmax for polygalacturonic acid hydrolysis were 3.2 mg ml-1 and 3.1 mg ml-1 and 255 U mg-1 and 262 U mg-1 for the wild-type and recombinant enzymes, respectively. The kinetic data of exopolygalacturonase on oligogalacturonates of different degree of polymerization (2-7) were interpreted in terms of a subsite model to obtain more insight into catalysis and substrate binding. On oligogalacturonates of different degrees of polymerization (2-7), the Michaelis constant (K(m)) decreased with increasing chain length (n). The Vmax value increased with chain length up to n = 4, then reached a plateau value. The enzyme was competitively inhibited by galacturonic acid (Ki = 0.3 mM) as well as by reduced digalacturonate (Ki = 0.4 mM). The exopolygalacturonase gene (pgaX) was cloned by reverse genetics and shows only 13% overall amino acid sequence identity with A. niger endopolygalacturonases. The exopolygalacturonase is most related to plant polygalacturonases. Only four small stretches of amino acids are conserved between all known endogalacturonases and exopolygalacturonases. Expression of the pgaX gene is inducible with galacturonic acid and is subject to catabolite repression. A fusion between the promoter of the A. niger glycolytic gene encoding pyruvate kinase and the pgaX-coding region was used to achieve high level production of exopolygalacturonase under conditions where no endopolygalacturonases were produced.