In vitro effects of alginate lyase SG4 + produced by Paenibacillus lautus alone and combined with antibiotics on biofilm formation by mucoid Pseudomonas aeruginosa.

Alginate is a major extra polymeric substance in the biofilm formed by mucoid Pseudomonas aeruginosa. It is the main proven perpetrator of lung infections in patients suffering from cystic fibrosis. Alginate lyases are very important in the treatment of cystic fibrosis. This study evaluated the role of standalone and in conjugation, effect of alginate lyase of SG4 + isolated from Paenibacillus lautus in enhancing in vitro bactericidal activity of gentamicin and amikacin on mucoid P. aeruginosa. Using Response Surface Methodology (RSM) alginate lyase SG4 + production was optimized in shake flask and there 8.49-fold enhancement in enzyme production. In fermenter, maximum growth (10.15 mg/ml) and alginate lyase (1.46 International Units) production, 1.71-fold was increased using Central Composite Design (CCD). Further, fermentation time was reduced from 48 to 20 h. To the best of our knowledge this is the first report in which CCD was used for fermenter studies to optimize alginate lyase production. The Km and Vmax of purified enzyme were found to be 2.7 mg/ml and 0.84 mol/ml-min, respectively. The half-life (t 1/2) of purified alginate lyase SG4 + at 37 °C was 180 min. Alginate lyase SG4 + in combination with gentamicin and amikacin eradiated 48.4- 52.3% and 58- 64.6%, alginate biofilm formed by P. aeruginosa strains, respectively. The study proves that alginate lyase SG4 + has excellent exopolysaccharide disintegrating ability and may be useful in development of potent therapeutic agent to treat P. aeruginosa biofilms.

Postharvest Treatment of Hydrogen Sulfide Delays the Softening of Chilean Strawberry Fruit by Downregulating the Expression of Key Genes Involved in Pectin Catabolism.

Hydrogen sulfide (H2S) plays several physiological roles in plants. Despite the evidence, the role of H2S on cell wall disassembly and its implications on fleshy fruit firmness remains unknown. In this work, the effect of H2S treatment on the shelf-life, cell wall polymers and cell wall modifying-related gene expression of Chilean strawberry (Fragaria chiloensis) fruit was tested during postharvest storage. The treatment with H2S prolonged the shelf-life of fruit by an effect of optimal dose. Fruit treated with 0.2 mM H2S maintained significantly higher fruit firmness than non-treated fruit, reducing its decay and tripling its shelf-life. Additionally, H2S treatment delays pectin degradation throughout the storage period and significantly downregulated the expression of genes encoding for pectinases, such as polygalacturonase, pectate lyase, and expansin. This evidence suggests that H2S as a gasotransmitter prolongs the post-harvest shelf-life of the fruit and prevents its fast softening rate by a downregulation of the expression of key pectinase genes, which leads to a decreased pectin degradation.

Increases in alginate production and transcription levels of alginate lyase (alyA1) by control of the oxygen transfer rate in Azotobacter vinelandii cultures under diazotrophic conditions

BACKGROUND: Alginates are polysaccharides used in a wide range of industrial applications, with their functional properties depending on their molecular weight. In this study, alginate production and the expression of genes involved in polymerization and depolymerization in batch cultures of Azotobacter vinelandii were evaluated under controlled and noncontrolled oxygen transfer rate (OTR) conditions. RESULTS: Using an oxygen transfer rate (OTR) control system, a constant OTR (20.3 ± 1.3 mmol L 1 h 1 ) was maintained during cell growth and stationary phases. In cultures subjected to a controlled OTR, alginate concentrations were higher (5.5 ± 0.2 g L 1 ) than in cultures under noncontrolled OTR. The molecular weight of alginate decreased from 475 to 325 kDa at the beginning of the growth phase and remained constant until the end of the cultivation period. The expression level of alyA1, which encodes an alginate lyase, was more affected by OTR control than those of other genes involved in alginate biosynthesis. The decrease in alginate molecular weight can be explained by a higher relative expression level of alyA1 under the controlled OTR condition. CONCLUSIONS: This report describes the first time that alginate production and alginate lyase (alyA1) expression levels have been evaluated in A. vinelandii cultures subjected to a controlled OTR. The results show that automatic control of OTR may be a suitable strategy for improving alginate production while maintaining a constant molecular weight.

Quorum Sensing Signaling Molecules Positively Regulate c-di-GMP Effector PelD Encoding Gene and PEL Exopolysaccharide Biosynthesis in Extremophile Bacterium Acidithiobacillus thiooxidans.

Acidithiobacillus species are fundamental players in biofilm formation by acidophile bioleaching communities. It has been previously reported that Acidithiobacillus ferrooxidans possesses a functional quorum sensing mediated by acyl-homoserine lactones (AHL), involved in biofilm formation, and AHLs naturally produced by Acidithiobacillus species also induce biofilm formation in Acidithiobacillus thiooxidans. A c-di-GMP pathway has been characterized in Acidithiobacillus species but it has been pointed out that the c-di-GMP effector PelD and pel-like operon are only present in the sulfur oxidizers such as A. thiooxidans. PEL exopolysaccharide has been recently involved in biofilm formation in this Acidithiobacillus species. Here, by comparing wild type and ΔpelD strains through mechanical analysis of biofilm-cells detachment, fluorescence microscopy and qPCR experiments, the structural role of PEL exopolysaccharide and the molecular network involved for its biosynthesis by A. thiooxidans were tackled. Besides, the effect of AHLs on PEL exopolysaccharide production was assessed. Mechanical resistance experiments indicated that the loss of PEL exopolysaccharide produces fragile A. thiooxidans biofilms. qRT-PCR analysis established that AHLs induce the transcription of pelA and pelD genes while epifluorescence microscopy studies revealed that PEL exopolysaccharide was required for the development of AHL-induced biofilms. Altogether these results reveal for the first time that AHLs positively regulate pel genes and participate in the molecular network for PEL exopolysaccharide biosynthesis by A. thiooxidans.

Refined sorghum flours precooked by extrusion enhance the integrity of the colonic mucosa barrier and promote a hepatic antioxidant environment in growing Wistar rats.

The effects of precooked-refined sorghum flour consumption on antioxidant status, lipid profile, and colonic and bone health were evaluated. Twenty-four male Wistar rats were fed with control diet (C), or red or white precooked-refined sorghum based diets (SD) for 60 days. The intake of SD was lower than that of C, but the efficiency of all diets was similar. Rats fed with SD showed lower feces excretion, cecal pH and enzyme activities (ß-glucosidase, ß-glucuronidase and mucinase) than C. White SD improved intestinal architecture, cell proliferation and apoptosis, upregulated ZO1 and occludin tight junction proteins and stimulated goblet cell differentiation, enhancing the integrity of the mucosa barrier in both proximal and distal colonic mucosa in a better way than red SD. Consumption of SD significantly decreased serum triglyceride levels compared with the C diet. The mineral content of the right femur was not different among diets. The liver enzyme activities (superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase) did not show differences among diets. Liver reducing power and reduced glutathione/oxidize glutathione ratio were higher for animals consuming SD than C. It can be concluded that the consumption of precooked refined sorghum flours still has beneficial effects for health, mainly at the colonic level, despite the lower phenolics and fibre contents of refined flours with respect to whole grain flours.

Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP.

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.

Saccharification of Brown Macroalgae Using an Arsenal of Recombinant Alginate Lyases: Potential Application in the Biorefinery Process.

Alginate lyases (endo and exo-lyases) are required for the degradation of alginate into its constituting monomers. Efficient bioethanol production and extraction of bioactives from brown algae requires intensive use of these enzymes. Nonetheless, there are few commercial alginate lyase preparations, and their costs make them unsuitable for large scale experiments. A recombinant expression protocol has been developed in this study for producing seven endo-lyases and three exo-lyases as soluble and highly active preparations. Saccharification of alginate using 21 different endo/exo-lyase combinations shows that there is complementary enzymatic activity between some of the endo/exo pairs. This is probably due to favorable matching of their substrate biases for the different glycosidic bonds in the alginate molecule. Therefore, selection of enzymes for the best saccharification results for a given biomass should be based on screens comprising both types of lyases. Additionally, different incubation temperatures, enzyme load ratios, and enzyme loading strategies were assessed using the best four enzyme combinations for treating Macrocystis pyrifera biomass. It was shown that 30°C with a 1:3 endo/exo loading ratio was suitable for all four combinations. Moreover, simultaneous loading of endo-and exo-lyases at the beginning of the reaction allowed maximum alginate saccharification in half the time than when the exo-lyases were added sequentially.

Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression

Abstract Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL) if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG) that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different) that were also studied. The mutants were analyzed for pectinases production on pectinase-agar plates and five mutants and two sectors showing larger clearing zones than the wild type were selected for quantitative assay. Although PL production higher than the wild type has been found, phenotype instability was observed for most of the mutants and, after transfers to nonselective medium, the DG resistance was no longer present. Only mutants M03 and M04 were stable maintaining the DG-resistance phenotype. When growing for 120 h in liquid medium containing glucose with or without pectin, both mutants showed higher PL production. In the presence of glucose as sole carbon source, the mutant M03 produced 7.8-fold more PL than the wild type. Due its phenotypic stability and PL overproduction, the mutant M03 presents potential for industrial applications.

Nf-GH, a glycosidase secreted by Naegleria fowleri, causes mucin degradation: an in vitro and in vivo study.

AIM: The aim of this work was to identify, characterize and evaluate the pathogenic role of mucinolytic activity released by Naegleria fowleri. MATERIALS & METHODS: Zymograms, protease inhibitors, anion exchange chromatography, MALDI-TOF-MS, enzymatic assays, Western blot, and confocal microscopy were used to identify and characterize a secreted mucinase; inhibition assays using antibodies, dot-blots and mouse survival tests were used to evaluate the mucinase as a virulence factor. RESULTS: A 94-kDa protein with mucinolytic activity was inducible and abolished by p-hydroxymercuribenzoate. MALDI-TOF-MS identified a glycoside hydrolase. Specific antibodies against N. fowleri-glycoside hydrolase inhibit cellular damage and MUC5AC degradation, and delay mouse mortality. CONCLUSION: Our findings suggest that secretory products from N. fowleri play an important role in mucus degradation during the invasion process.

Different responses in the expression of alginases, alginate polymerase and acetylation genes during alginate production by Azotobacter vinelandii under oxygen-controlled conditions.

Alginate production and gene expression of genes involved in alginate biosynthesis were evaluated in continuous cultures under dissolved oxygen tension (DOT) controlled conditions. Chemostat at 8% DOT showed an increase in the specific oxygen uptake rate [Formula: see text] from 10.9 to 45.3 mmol g-1 h-1 by changes in the dilution rate (D) from 0.06 to 0.10 h-1, whereas under 1% DOT the [Formula: see text] was not affected. Alginate molecular weight was not affected by DOT. However, chemostat at 1% DOT showed a downregulation up to 20-fold in genes encoding both the alginate polymerase (alg8, alg44), alginate acetylases (algV, algI) and alginate lyase AlgL. alyA1 and algE7 lyases gene expressions presented an opposite behavior by changing the DOT, suggesting that A. vinelandii can use specific depolymerases depending on the oxygen level. Overall, the DOT level have a differential effect on genes involved in alginate synthesis, thus a gene expression equilibrium determines the production of alginates of similar molecular weight under DOT controlled.

Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression.

Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL) if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG) that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different) that were also studied. The mutants were analyzed for pectinases production on pectinase-agar plates and five mutants and two sectors showing larger clearing zones than the wild type were selected for quantitative assay. Although PL production higher than the wild type has been found, phenotype instability was observed for most of the mutants and, after transfers to nonselective medium, the DG resistance was no longer present. Only mutants M03 and M04 were stable maintaining the DG-resistance phenotype. When growing for 120h in liquid medium containing glucose with or without pectin, both mutants showed higher PL production. In the presence of glucose as sole carbon source, the mutant M03 produced 7.8-fold more PL than the wild type. Due its phenotypic stability and PL overproduction, the mutant M03 presents potential for industrial applications.

Heterologous expression of a Penicillium purpurogenum pectin lyase in Pichia pastoris and its characterization.

Lignocellulose is the major component of plant cell walls and it represents a great source of renewable organic matter. One of lignocellulose constituents is pectin. Pectin is composed of two basic structures: a 'smooth' region and a 'hairy' region. The 'smooth' region (homogalacturonan) is a linear polymer of galacturonic acid residues with α-(1→4) linkages, substituted by methyl and acetyl residues. The 'hairy' region is more complex, containing xylogalacturonan and rhamnogalacturonans I and II. Among the enzymes which degrade pectin (pectinases) is pectin lyase (E.C. 4.2.2.10). This enzyme acts on highly esterified homogalacturonan, catalysing the cleavage of α-(1→4) glycosidic bonds between methoxylated residues of galacturonic acid by means of ß-elimination, with the formation of 4,5-unsaturated products. In this work, the gene and cDNA of a pectin lyase from Penicillium purpurogenum have been sequenced, and the cDNA has been expressed in Pichia pastoris. The gene is 1334 pb long, has three introns and codes for a protein of 376 amino acid residues. The recombinant enzyme was purified to homogeneity and characterized. Pectin lyase has a molecular mass of 45 kDa as determined by SDS-PAGE. It is active on highly esterified pectin, and decreases 40% the viscosity of pectin with a degree of esterification ≥85%. The enzyme showed no activity on polygalacturonic acid and pectin from citrus fruit 8% esterified. The optimum pH and temperature for the recombinant enzyme are 6.0 and 50 °C, respectively, and it is stable up to 50 °C when exposed for 3 h. A purified pectin lyase may be useful in biotechnological applications such as the food industry where the liberation of toxic methanol in pectin degradation should be avoided.

Construction of a Kluyveromyces lactis ku80⁻ host strain for recombinant protein production: extracellular secretion of pectin lyase and a streptavidin-pectin lyase chimera.

In several organisms used for recombinant protein production, integration of the expression cassette into the genome depends on site-specific recombination. In general, the yeast Kluyveromyces lactis shows low gene-targeting efficiency. In this work, two K. lactis ku80⁻ strains defective in the non-homologous end-joining pathway (NHEJ) were constructed using a split-marker strategy and tested as hosts for heterologous gene expression. The NHEJ pathway mediates random integration of exogenous DNA into the genome, and its function depends on the KU80 gene. KU80-defective mutants were constructed using a split-marker strategy. The vectors pKLAC1/Plg1 and pKLAC1/cStpPlg1 were used to evaluate the recovered mutants as hosts for expression of pectin lyase (PNL) and the fusion protein streptavidin-PNL, respectively. The transformation efficiency of the ku80⁻ mutants was higher than the respective parental strains (HP108 and JA6). In addition, PNL secretion was detected by PNL assay in both of the K. lactis ku80⁻ strains. In HP108ku80⁻/cStpPlg1 and JA6ku80⁻/Plg1 cultures, the PNL extracellular specific activity was 551.48 (±38.66) and 369.04 (±66.33) U/mg protein. This study shows that disruption of the KU80 gene is an effective strategy to increase the efficiency of homologous recombination with pKLAC1 vectors and the production and secretion of recombinant proteins in K. lactis transformants.

Alginate lyase and ciprofloxacin co-immobilization on biopolymeric microspheres for cystic fibrosis treatment.

A new formulation is described based on biopolymeric microspheres containing alginate lyase (AL) and ciprofloxacin (Cip) for sustainable oral delivery in CF patients. Alginate (ALG) and high-methoxyl pectin (HMP) are selected as the biopolymers to develop a composite matrix. ALG microspheres coated with HMP and ALG-HMP blend are gelled in water/organic solvents mixtures, obtaining Cip encapsulations from 46.0 to 100.0%. ALG-HMP shows a Cip sustainable release profile and is able to encapsulate 90.0% of AL, showing 76.0% enzyme activity after release under simulated intestinal conditions. The developed system is a promising delivery carrier to treat chronic infection of Pseudomonas aeruginosa and to reduce the viscoelasticity of the mucus accumulated into intestine of CF patients.

The pectate lyase encoded by the pecCl1 gene is an important determinant for the aggressiveness of Colletotrichum lindemuthianum.

Colletotrichum lindemuthianum is the causal agent of anthracnose in the common bean, and the genes that encode its cell-wall-degrading enzymes are crucial for the development of the disease. Pectinases are the most important group of cell wall-degrading enzymes produced by phytopathogenic fungi. The pecC1l gene, which encodes a pectate lyase in C. lindemuthianum, was isolated and characterized. Possible cis-regulatory elements and transcription factor binding sites that may be involved in the regulation of genetic expression were detected in the promoter region of the gene. pecCl1 is represented by a single copy in the genome of C. lindemuthianum, though in silico analyses of the genomes of Colletotrichum graminicola and Colletotrichum higginsianum suggest that the genome of C. lindemuthianum includes other genes that encode pectate lyases. Phylogenetic analysis detected two groups that clustered based on different members of the pectate lyase family. Analysis of the differential expression of pecCl1 during different stages of infection showed a significant increase in pecCl1 expression five days after infection, at the onset of the necrotrophic phase. The split-maker technique proved to be an efficient method for inactivation of the pecCl1 gene, which allowed functional study of a mutant with a site-specific integration. Though gene inactivation did not result in complete loss of pectate lyase activity, the symptoms of anthracnose were reduced. Analysis of pectate lyases might not only contribute to the understanding of anthracnose in the common bean but might also lead to the discovery of an additional target for controlling anthracnose.

Expression of alginases and alginate polymerase genes in response to oxygen, and their relationship with the alginate molecular weight in Azotobacter vinelandii.

The transcription of genes involved in alginate polymerization and depolymerization, as well as the alginase activity (extracellular and intracellular) under oxygen-limited and non oxygen-limited conditions in cultures of A. vinelandii, was studied. Two levels of dissolved oxygen tension (DOT) (1% and 5%, oxygen-limited and non-oxygen-limited, respectively) strictly controlled by gas blending, were evaluated in a wild type strain. In cultures at low DOT (1%), in which a high molecular weight alginate (1200 kDa) was synthesized, the transcription levels of alg8 and alg44 (genes encoding alginate polymerase complex), and algX (encoding a protein involved in polymer transport through periplasmic space) were considerably higher as compared to cultures conducted at 5% DOT, under which an alginate with a low MW (42 kDa) was produced. In the case of genes encoding for intracellular and extracellular alginases, the levels of these transcripts were higher at 1% DOT. However, intracellular and extracellular alginase activity were lower (0.017 and 0.01 U/mg protein, respectively) in cultures at 1% DOT, as compared with the activities measured at 5% DOT (0.027 and 0.052 U/mg protein for intracellular and extracellular maximum activity, respectively). The low alginase activity measured in cultures at 1% DOT and the high level of transcription of genes constituting alginate polymerase complex might be mechanisms by which oxygen regulates the production of alginates with a high MW.

Evaluation of a potential starter culture for enhance quality of coffee fermentation.

The coffee fermentation is characterized by the presence of different microorganisms belonging to the groups of bacteria, fungi and yeast. The objectives of this work were to select pectinolytic microorganisms isolated from coffee fermentations and evaluate their performance on coffee pulp culture medium. The yeasts and bacteria isolates were evaluated for their activity of polygalacturonase (PG), pectin lyase (PL) and pectin methylesterase (PME) and metabolites production. Among 127 yeasts isolates and 189 bacterial isolates, 15 were pre-selected based on their ability to produce PL and organic compounds. These isolates were strains identified as Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Candida parapsilosis, Pichia caribbica, Pichia guilliermondii and Saccharomyces cerevisiae. When cultivated in Coffee peel and pulp media in single culture or two by two mixed inocula, different behavior concerning to PME, PL and PG were found. The two principal components PC1 and PC2 accounted for 45.27 and 32.02 % of the total variance. UFLA CN727 and UFLA CN731 strains were grouped in the positive part of PC1 being characterized by 1,2-propanediol, hexanoic acid, decanoic acid, nonanoic acid and ethyl acetate. The UFLA CN448 and UFLA CN724 strains were grouped in the negative part of PC1 and were mainly characterized by guaiacol, butyric acid and citronellol. S. cerevisiae UFLACN727, P. guilliermondii UFLACN731 and C. parapsilosis UFLACN448 isolates are promising candidates to be tested in future studies as coffee starter cultures.

Alginate production and alg8 gene expression by Azotobacter vinelandii in continuous cultures.

Alginates are polysaccharides that are used as thickening agents, stabilizers, and emulsifiers in various industries. These biopolymers are produced by fermentation with a limited understanding of the processes occurring at the cellular level. The objective of this study was to evaluate the effects of agitation rate and inlet sucrose concentrations (ISC) on alginate production and the expression of the genes encoding for alginate-lyases (algL) and the catalytic subunit of the alginate polymerase complex (alg8) in chemostat cultures of Azotobacter vinelandii ATCC 9046. Increased alginate production (2.4 g l⁻¹) and a higher specific alginate production rate (0.1 g g⁻¹ h⁻¹) were obtained at an ISC of 15 g l⁻¹. Carbon recovery of about 100% was obtained at an ISC of 10 g l⁻¹, whereas it was close to 50% at higher ISCs, suggesting that cells growing at lower sucrose feed rates utilize the carbon source more efficiently. In each of the steady states evaluated, an increase in algL gene expression was not related to a decrease in alginate molecular weight, whereas an increase in the molecular weight of alginate was linked to higher alg8 gene expression, demonstrating a relationship between the alg8 gene and alginate polymerization in A. vinelandii for the first time. The results obtained provide a possible explanation for changes observed in the molecular weight of alginate synthesized and this knowledge can be used to build a recombinant strain able to overexpress alg8 in order to produce alginates with higher molecular weights.

Use of fruit residues for pectinase production by Aspergillus flavipes FP-500 and Aspergillus terreus FP-370.

AIMS: Utilization of fruit residues for pectinase production by two Aspergillus strains for recognizing the effects of some factors during fermentation and describing enzyme production kinetics. METHODS AND RESULTS: Pectinase production on several fruit residues was compared. The effects of three factors on the production of several pectinases were evaluated by a full factorial 2(k) experimental design. Higher activities were obtained on lemon peel. In both strains, acidic pH values and high carbon source concentration favoured exopectinase and endopectinase production, while higher pH values and low carbon source concentration promoted pectin lyase and rhamnogalacturonase production. Unstructured mathematical modelling provided a good description of pectinase production in a submerged batch culture. CONCLUSIONS: Fruit residues were very good substrates for pectinase production, and Aspergillus strains used showed a promising performance in submerged fermentation. Mathematical modelling was useful to describe growth and pectinase production. SIGNIFICANCE AND IMPACT OF THE STUDY: Lemon peel can be used as a substrate to obtain high pectinase titres by Aspergillus flavipes FP-500 and Aspergillus terreus FP-370. The factors that contributed to improve the yield were identified, which supports the possibility of using this substrate in the industrial production of these enzymes.

Enzymatic differences between the endophyte Guignardia mangiferae (Botryosphaeriaceae) and the citrus pathogen G. citricarpa.

The endophyte Guignardia mangiferae is closely related to G. citricarpa, the causal agent of citrus black spot; for many years these species had been confused with each other. The development of molecular analytical methods has allowed differentiation of the pathogen G. citricarpa from the endophyte G. mangiferae, but the physiological traits associated with pathogenicity were not described. We examined genetic and enzymatic characteristics of Guignardia spp strains; G. citricarpa produces significantly greater amounts of amylases, endoglucanases and pectinases, compared to G. mangiferae, suggesting that these enzymes could be key in the development of citrus black spot. Principal component analysis revealed pectinase production as the main enzymatic characteristic that distinguishes these Guignardia species. We quantified the activities of pectin lyase, pectin methylesterase and endopolygalacturonase; G. citricarpa and G. mangiferae were found to have significantly different pectin lyase and endopolygalacturonase activities. The pathogen G. citricarpa is more effective in pectin degradation. We concluded that there are significant physiological differences between the species G. citricarpa and G. mangiferae that could be associated with differences in pathogenicity for citrus plants.