Endopolygalacturonase Production Research Articles

Effect of codon optimization and promoter choice on recombinant endo-polygalacturonase production in Pichia pastoris

Pectinase is one of the most widely used enzymes in different fields of food industry for different purposes, such as clarification of fruit juice, extraction of vegetable oil and saccharification of agricultural substrates. The aim of this study is to produce recombinant pectinase and evaluate the effect of codon optimization and promoter selection on production.In this study, the gene encoding pectinase in Aspergillus niger was optimized according to the codon usage of Pichia pastoris. Within the scope of the study, codon-optimized and native (non-codon-optimized) pectinase genes were transferred to P. pastoris X33 strain and expressed under the regulation of methanol-inducible AOX1 and ethanol-inducible ADH2 promoter.As a result of the study, the promoter and codon combination which exhibited the highest production level was determined as the ADH2 and codon-optimized pectinase yielding an enzyme activity of 42.33 U/mL. The best producer clone was cultured in 400 mL media in 2 L shake-flask and the enzyme was purified by His-tag method. The optimum working conditions of the purified enzyme was 50 °C and pH 5.0, and after incubation at 60 °C for 1 -h, enzyme activity was maintained at 60% level. The Michelis–Menten constant (Km) and maximal velocity (Vmax) of the purified recombinant pectinase were 6.9 mg/mL and 67.57 μmol/mg/min, respectively.

Characterization of a novel thermophilic endopolygalacturonase produced by Bacillus licheniformis IEB-8

Endopolygalacturonases characterized until now have either low working temperatures, working pH in acidic range, high Michaelis–Menten constant (Km), or a high production cost. These characteristics are a hurdle in the industrial applications of these endopolygalacturonases. The purpose of this work was to characterize a novel endopolygalacturonase produced by Bacillus licheniformis IEB-8. Phylogenetic analysis of Bacillus licheniformis IEB-8 showed that the isolate was unique. Citrus peels were used as the only nutrient source for the growth of Bacillus licheniformis IEB-8, allowing a cheap production of endopolygalacturonase. All the synthetic carbon sources showed a negative impact on the production of endopolygalacturonase, while ammonium sulfate enhanced its production. Among different metal ions, Zn+2 showed a negative effect while Mg+2 and Ca+2 did not have any significant effect on the endopolygalacturonase activity. A Lineweaver-Burk plot was prepared for the characterization of the kinetic parameters including Km and Vmax, which were 0.45 mg/mL and 285.7 µM/min, respectively. A comprehensive comparison of the endopolygalacturonase from this study with the available literature indicated that it is better than the reported and commercially available endopolygalacturonases in having the optimum working temperature of 55 °C, a low Km of 0.57 mg/mL, and pH of 7 to 8, which indicated its novelty.

Accelerated Bioprocess Development of Endopolygalacturonase-Production with Saccharomyces cerevisiae Using Multivariate Prediction in a 48 Mini-Bioreactor Automated Platform.

Mini-bioreactor systems enabling automatized operation of numerous parallel cultivations are a promising alternative to accelerate and optimize bioprocess development allowing for sophisticated cultivation experiments in high throughput. These include fed-batch and continuous cultivations with multiple options of process control and sample analysis which deliver valuable screening tools for industrial production. However, the model-based methods needed to operate these robotic facilities efficiently considering the complexity of biological processes are missing. We present an automated experiment facility that integrates online data handling, visualization and treatment using multivariate analysis approaches to design and operate dynamical experimental campaigns in up to 48 mini-bioreactors (8–12 mL) in parallel. In this study, the characterization of Saccharomyces cerevisiae AH22 secreting recombinant endopolygalacturonase is performed, running and comparing 16 experimental conditions in triplicate. Data-driven multivariate methods were developed to allow for fast, automated decision making as well as online predictive data analysis regarding endopolygalacturonase production. Using dynamic process information, a cultivation with abnormal behavior could be detected by principal component analysis as well as two clusters of similarly behaving cultivations, later classified according to the feeding rate. By decision tree analysis, cultivation conditions leading to an optimal recombinant product formation could be identified automatically. The developed method is easily adaptable to different strains and cultivation strategies, and suitable for automatized process development reducing the experimental times and costs.

Detection of growth rate-dependent product formation in miniaturized parallel fed-batch cultivations.

Saccharomyces cerevisiae is a popular expression system for recombinant proteins. In most cases, production processes are performed as carbon-limited fed-batch cultures to avoid aerobic ethanol formation. Especially for constitutive expression systems, the specific product formation rate depends on the specific growth rate. The development of optimal feeding strategies strongly depends on laboratory-scale cultivations, which are time and resource consuming, especially when continuous experiments are carried out. It is therefore beneficial for accelerated process development to look at alternatives. In this study, S. cerevisiae AH22 secreting a heterologous endo-polygalacturonase (EPG) was characterized in microwell plates with an enzyme-based fed-batch medium. Through variation of the glucose release rate, different growth profiles were established and the impact on EPG secretion was analyzed. Product formation rates of200-400U(gxh)-1 were determined. As a reference, bioreactor experiments using the change-stat cultivation technique were performed. The growth-dependent product formation was analyzed over dilution rates of D = 0.01-0.35 with smooth change of D at a rate of 0.003 h-2. EPG production was found to be comparable with a qp of 400 U (gx h)-1 at D = 0.27 h-1. The presented results indicate that parallel miniaturized fed-batch cultures can be applied to determine product formation profiles of putative production strains. With further automation and parallelization of the concept, strain characterization can be performed in shorter time.

Cloning, Characterization, and Functional Analysis of theEPG1-2Gene: A New Allele Coding for an Endopolygalacturonase in Kluyveromyces marxianus

A new allele, the EPG1-2 gene, which codes for an endopolygalacturonase in Kluyveromyces marxianus CECT1043, has been cloned. The gene has 1086 bp and the protein 362 amino acids, one more than the previously described Epg1p. Epg1-2p shows a high degree of similarity with the polygalacturonases of fungi and yeasts. The sequences common to all of the polygalacturonases of prokaryotes, fungi, and higher plants are also conserved in Epg1-2p. The EPG1-2 gene has been expressed in Pichia pastoris , and, when fused with the signal peptide of the alpha-factor of Saccharomyces cerevisiae , the protein is properly secreted into the media. The recombinant enzyme does not appear to be fully glycosylated by P. pastoris or not glycosylated in the same manner as in K. marxianus, but it maintains the same optimum temperature (55 degrees C) and pH (4.5) and the same stability at different temperatures and pH values as the native enzyme, also showing the same hydrolytic behavior. The recombinant strain produces 200-fold more enzyme than the wild-type strain of K. marxianus, making it a yeast of potential industrial interest for the production of endopolygalacturonase for the food industry.

Overexpression of a Gene Encoding a Catabolite Repression Element in Alternaria citri Causes Severe Symptoms of Black Rot in Citrus Fruit

ABSTRACT A gene (AcCreA) encoding a catabolite repression element (CreA) with (two zinc fingers of the Cys(2)His(2) type was isolated from the postharvest fungal pathogen Alternaria citri. The AcCreA overexpression mutant AcOEC2 of A. citri showed normal growth on pectin medium and on segments of peel or the juice sac area from citrus fruit. Production of endopolygalacturonase, an essential virulence factor of this pathogen, was similar in AcOEC2 and the wild type in pectin-containing media. However, addition of glucose to the medium showed that carbon catabolite repression of endopolygalacturonase gene (Acpg1) expression, as well as endopolygalacturonase production, was lost in AcOEC2. The wild-type strain of A. citri causes rot mainly in the central axis of citrus fruit without development of rotting in the juice sac area; however, AcOEC2 caused severe black rot symptoms in both the central axis and juice sac areas. These results indicate that AcCreA-mediated catabolite repression controls the virulence or infection of this pathogen, and that the wild-type A. citri does not cause symptoms in the juice sac area due to carbon catabolite repression by sugars in the juice of the juice sac area.

Use of two different carriers in a packed bed reactor for endopolygalacturonase production by a yeast strain

A packed bed reactor (PBR) design was tested for the purpose of continuous pectinase production with yeasts, as a possible alternative to the traditional batch process using fungal cultures. Two different carriers – a porous glass (Siran) and a cellulosic carrier obtained from spent grains (barley) – were used to immobilize Kluyveromyces marxianus CCT 3172, a yeast strain secreting endopolygalacturonase. To improve cell distribution throughout the column, part of the outflow was recycled. Cell loads of 0.204 and 0.247 g biomass/g carrier were obtained at the top and bottom of the PBR with spent grains, respectively. Using the PBR with Siran as the immobilization support, 0.071 g biomass/g carrier was the biomass load at the top of the column while at the bottom a value of 0.147 g biomass/g carrier was found. The highest value for pectinase volumetric productivity ( P V = 1.68 U/ml h) was achieved in the PBR with Siran for a D = 0.260 h −1 and a glucose concentration on the inlet of S in = 40 g/l. Both carriers were suitable for pectinase production. The best results were obtained with a high and uniform biomass concentration in the column, together with high dilution rates and total glucose consumption.

Screening of fungal strains for pectinolytic activity: endopolygalacturonase production by Peacilomyces clavisporus 2A.UMIDA.1

Forty-six fungal strains, isolated from soil and samples of cacao ( Theobroma cacao L.) jelly, were screened for pectinase production. All the strains were positive for pectinase activity in a cup-plate assay, as evidenced by clear hydrolysation halos. Twenty-eight percentages presented considerable pectinolytic activity. The cultivation of the best strain ( Peacilomyces clavisporus 2A.UMIDA.1) by submerged and semi-solid fermentation, resulted in high quantities of endopolygalacturonase enzyme. The optimal temperature for fungal growth and endopolygalacturonase production by P. clavisporus 2A.UMIDA.1 in submerged culture was 30 °C. Formulations containing cará–roxo ( Dioscorea alata) were also employed as substrates for endopolygalacturonase production, but results were poor. Solid state fermentation proved to be a better process than submerged fermentation for endopolygalacturonase production, giving the highest enzyme production rate in 96 h. An attempt to scale up the solid state fermentation resulted in a decrease in enzyme production.

Evaluation of Filamentous Fungi and Inducers for the Production of Endo-Polygalacturonase by Solid State Fermentation

Aspergillus oryzae CCT 3940, Aspergillus awamori NRRL 3112 and a Trichoderma sp.) were compared for their capacity to produce endo-polygalacturonase (endo-PG) in solid state fermentation. Maximum pectinolytic activity was reached in 72 h of growth, the best two fungal strains being A. niger T0005007-2 and A. oryzae CCT 3940. Three types of commercial purified pectin and four of unprocessed pectin (tangerine, orange, Tahiti lime and sweet lime rind) were used to assess the effect of pectin on the production of endo-PG by A. niger T0005007-2. Maximum pectinolytic activity was achieved using 6 and 10% (w/w) of purified pectin as inducer. Depending on the origin of the commercial pectin used as inducer, maximum endo-PG levels varied from 223 to 876 units per gram of dry medium (one endo-PG unit (U) was defined as the quantity of enzyme which caused a reduction in viscosity of 50% in a 1% w/v solution of pectin in 30 min), indicating that care should be taken when choosing this component of the medium. When the crude pectins were used as inducers at the same concentration as purified pectin, maximum endo-PG activities were 250-300 units/g. However, by increasing the amount of Tahiti lime rind to 50% (w/w) maximum endo-PG was 919 U/g, thus opening up the possibility of a low cost medium for endo-PG production.

Dissolved oxygen threshold for the repression of endo-polygalacturonase production by Kluyveromyces marxianus

A detailed study was conducted in terms of the influence of dissolved oxygen (DO) and growth temperature related to the endo-polygalacturonase synthesis by the yeast Kluyveromyces marxianus CDBB-L-278. It was found that 3.3 mg of DO/l was the threshold for the repression of enzyme production. Growth temperature had no effect on the synthesis of this enzyme, but had an indirect effect due to changes in oxygen solubility. Growth rate was influenced by temperature; maintaining the dissolved oxygen constant at 3.3 mg of DO/l, the optimum growth temperature was 40°C. Pectin in the culture medium allowed higher endo-polygalacturonase production; even with 3.3 mg of DO/l, pectin de-repressed the enzyme production.

Induction by (alpha)-L-Arabinose and (alpha)-L-Rhamnose of Endopolygalacturonase Gene Expression in Colletotrichum lindemuthianum

The production of endopolygalacturonase (endoPG) by Colletotrichum lindemuthianum, a fungal pathogen causing anthracnose on bean seedlings, was enhanced when the fungus was grown in liquid medium with L-arabinose or L-rhamnose as the sole carbon source. These two neutral sugars are present in plant cell wall pectic polysaccharides. The endolytic nature of the enzyme was demonstrated by its specific interaction with the polygalacturonase-inhibiting protein of the host plant as well as by sugar analysis of the products released from its action on oligogalacturonides. Additional characterization of the protein was achieved with an antiserum raised against the pure endoPG of the fungus. Induction by arabinose and rhamnose was more prolonged and led to a level of enzyme activity at least five times higher than that on pectin. Northern blot experiments showed that this effect was correlated to the induction of a 1.6-kb transcript. A dose-response study indicated that the endoPG transcript level was already increased at a concentration of each sugar as low as 2.75 mM in the medium and was maximum at 55 mM arabinose and 28 mM rhamnose. Glucose, the main plant cell wall sugar residue which is also present in the apoplast, prevented endoPG gene expression, partially when added to pectin at concentrations ranging from 5 to 110 mM and totally when added at 55 mM to arabinose. Inhibition by glucose of the rhamnose-induced endoPG was correlated to nonuptake of rhamnose. This is the first report that arabinose and rhamnose stimulate endoPG gene expression in a fungus. The possible involvement of these various sugars on endoPG gene expression during pathogenesis is discussed.

Production of endopolygalacturonase fromRhizopus nigricans related to the evolution of growth substrate

Endopolygalacturonases production byRhizopus nigricans is studied. The enzymatic level present during the early growth stages depolymerized fully the substrate and afterwards the growth of the fungi, with the parallel production of endopolygalacturonase, began. The maximum level of enzymatic activity was 18 U/mL and was reached six days after the maximun metabolic activity of the fungi. The production of endopolygalacturonase finished when the carbon source was exhausted.

Endopolygalacturonase production from Kluyveromyces marxianus. I. Resolution, purification, and partial characterisation of the enzyme

The yeast Kluyveromyces marxianus has been shown to be a potential source of endopolygalacturonase for use in the food industry. However, uncertainty exists in the nature and spectrum of the enzymes secreted. In this study, four forms of endopolygalacturonase (1,4-α- d-galacosiduronate glycanohydrolase, EC 3.2.1.15) were isolated from the culture fluid of K. marxianus, strain NCYC 587. SDS polyacrylamide gel electrophoresis indicated that three of the isoenzymes had an M r of 49,600 and one of 46,700. The amino acid content of the isoenzymes was very similar and they all cross reacted strongly with antibodies raised against one of the purified enzymes. All the endopolygalacturonases were glycoproteins, the neutral sugar content varying between 2.3% and 13.8% .

The Role of Ethylene in Growth and Endopolygalacturonase Production by Sclerotium rolfsii

AbstractGrowth of and endopolygalacturonase production by Sclerotium rolfsii was better on a defined mineral medium than on a medium containing segments of tomato leaf petioles. The effect of treatment with ethylene (10μl/l) upon endopolygalacturonase activity with investigated at various stages of growth, in a mineral defined medium. Addition of ethylene to a 10 days‐old culture of S. rolfsii resulted in a decrease in activity by day 14, whereas the addition of ethylene to a 4, 6 and 8 days old cultures resulted in an increase in endopolygalacturonase activity. Ethylene seems to have little or no stimulating effect upon growth of S. rolfsii when applied after 8 days. However, inhibited fungal growth, after the addition of ethylene at earlier stages of growth, was obtained due to the depletion of oxygen from sealed culture flasks. Endopolygalacturonase was extracted and purified from control cultures after 14 days of growth. Fractionation of this enzyme protein on Sephadex G‐100 gel filtration columns resulted in two peaks of activity measured by the release of reducing sugars from polygalacturonic acid (PGA).

Studies on Pectic Enzymes of Microorganisms Part II

Conditions for the production of endo-polygalacturonase (endo-PG) with Aspergillus saitoi IAM 2217 in the submerged culture was examined. This strain was selected as the most potent producer of endo-PG. Endo-PG of this strain was produced in the absence of pectin, but the addition of pectin increased endo-PG activity when inoculated with proliferated mycelia. As far as examined with a modified Czapek medium (ordinary constituents+pectin and ammonium tartrate), the addition of organic nitrogen sources, such as corn steep liquor, markedly reduced the enzyme producibility. As for the carbon and nitrogen amount in the medium, sucrose: 4%, pectin: 2%, NaNO3: 1.15%, C/N=10, gave the best result among tested.