Inhibitors of polygalacturonase and laccase of Botrytis cinerea and their application to the control of this fungus

EDTA, calcium chloride, and two siderophores (rhodotorulic acid produced by Rhodotorula glutinis BNM 0524, and enterochelin from the bacterium Rahnella aquatilis BNM 0523) were evaluated as possible inhibitors of polygalacturonase (PG) and laccase (LC) from Botrytis cinerea. The aim was to apply them to the control of this pathogen, taking into account the fact that these enzymes are related to the invasion and installation of the fungus in the host. Two B. cinerea Pers.:Fr strains (BNM 0527 and BNM 0528) were used. Enzyme activities were measured in the supernatant of 7-day-old cultures. EDTA, calcium chloride, rhodotorulic acid, or enterochelin were added in the reaction mixture. Laccase activity from two strains was more affected by enterochelin (70-80% inhibition) than by the other compounds, while polygalacturonase was more inhibited (45% inhibition) by calcium chloride. The inhibitors were added to the growth medium and after 7 days of culture, the activities of the enzymes were measured in the supernatants. The production of PG and LC in both strains was lower when enterochelin or calcium chloride was added. In the third step, when the inhibitors were tested on apple, all them provided both effects, preventive and curative, against infections caused by B. cinerea, with EDTA and rhodotorulic acid exhibiting more preventive effects while calcium chloride and enterochelin provided more control of pre-existing infections (curative effect), coinciding with their ability to inhibit the production of polygalacturonase and laccase.     

Comparison of Kinetic Properties Between Plant and Fungal Amine Oxidases

Abstract

Kinetic properties of novel amine oxidases isolated from a mold Aspergillus niger AKU 3302 were compared to those of typical plant amine oxidase from pea seedling (EC 1.4.3.6). Pea amine oxidase showed highest affinity with diamines, such as putrescine and cadaverine, while fungal enzymes oxidized preferably n-hexylamine and tyramine. All enzymes were inhibited by carbonyl reagents, copper chelating agents, some substrate analogs and alkaloids, but there were quite significant differences in the sensitivity and inhibition modes. Aminoguanidine, which strongly inhibited pea amine oxidases showed only little effect on fungal enzymes. Substrate analogs such as 1,5-diamino-3-pentanone and l-amino-3-phenyl-3-propanone, which were potent competitive inhibitors of pea amine oxidases, inhibited fungal enzymes much more weakly and non competitively. Also various alkaloids behaving as competitive inhibitors of pea amine oxidases inhibited the fungal enzymes non competitively. Very surprising was the potent inhibition of fungal enzymes by artificial substrates of pea amine oxidases, E- and Z-1,4-diamino-2-butene. The relationships between the different inhibition modes and possible binding at the active site are discussed.     

Effect of pectin on pectinases in autolysis of Botrytis cinerea

Pectic activity in autolyzed cultures of Botrytis cinerea in a medium with and without pectin was similar, but in the medium with pectin maximal activities occurred in younger cultures. The pectic activities found were polygalacturonase, polymethylgalacturonase, endo activity (pectin as substrate) and pectin lyase. The molecular weights of polygalacturonase, polymethylgalacturonase and endo activity (pectin as substrate) were 36000, 33000 and 30200 daltons respectively, and the molecular weight of pectin lyase was 18200 daltons. By gel electrophoresis four different pectic activities were detected, three in the top of the gel and one in the bottom. Two enzymes were characterized, the polygalacturonase activity (first band in the top) inhibited by Ca⁺⁺ and the pectin lyase activity (in the bottom) which was not inhibited by Ca⁺⁺. These enzymes are not induced by the presence of pectin in the medium during degradation of Botrytis cinerea.     

Degradation of endocrine disrupting chemicals by genetic transformants with two lignin degrading enzymes in <Emphasis Type="Italic">Phlebia tremellosa</Emphasis>

A white rot fungus Phlebia tremellosa produced lignin degrading enzymes, which showed degrading activity against various recalcitrant compounds. However, manganese peroxidase (MnP) activity, one of lignin degrading enzymes, was very low in this fungus under various culture conditions. An expression vector that carried both the laccase and MnP genes was constructed using laccase genomic DNA of P. tremellosa and MnP cDNA from Polyporus brumalis. P. tremellosa was genetically transformed using the expression vector to obtain fungal transformants showing increased laccase and MnP activity. Many transformants showed highly increased laccase and MnP activity at the same time in liquid medium, and three of them were used to degrade endocrine disrupting chemicals. The transformant not only degraded bisphenol A and nonylphenol more rapidly but also removed the estrogenic activities of the chemicals faster than the wild type strain.     

Changes in Sapwood of Roots of Norway Spruce, Attacked by Fomes annosus

Acetone extracts of sapwood and reaction zone of spruce roots attacked by Fomes annosus, collected in February, June and October, were separated into resinous and phenolic fractions. The fractions were further separated by column, thin layer and gas liquid chromatography, followed by biological tests, using Fomes annosus and other rot fungi. The reaction zone contained quantitatively less light petroleum soluble compounds than the sapwood but more acids. The phenolic content was about ten times higher in the reaction zone than in the sapwood. Nine lignans and one simple phenol (4-methylcatechol) were identified and quantitatively estimated in the reaction zone. The resinous fraction of the extract from the reaction zone as well as some of the lignans and 4-methylcatechol inhibited fungal growth, in some cases followed by detoxification and continued growth. The predominant lignan, hydroxymatairesinol, had no effect on Fomes annosus or five other wood degrading fungi. About 15 unidentified phenols were observed, some of them probably of importance as inhibitors, either alone or in combination with other phenols.     

Screening for Ligninolytic Enzyme Production by Diverse Fungi from Tunisia

Summary This work represents the first report on the ability of autochthonous fungi from Tunisia to produce ligninolytic enzymes. Three hundred and fifteen fungal strains were isolated from different Tunisian biotopes. These fungal strains were firstly screened on solid media containing Poly R-478 or ABTS as indicator compounds that enabled the detection of lignin-modifying enzymes as specific color reactions. Of the 315 tested strains, 49 exhibited significant ABTS-oxidation activity expressed within the first week of incubation and only 18 strains decolorized the Poly R-478. Liquid cultivations and laccase, manganese peroxidase and lignin peroxidase activity assays of positive strains confirmed that eight efficient enzyme producers were found in the screening. These strains were attributed to the most closely related species using PCR amplification and sequencing of the internal transcribed spacer ‘ITS’ regions of the ribosomal DNA. The identification results showed fungal genera such as Oxyporus, Stereum and Trichoderma which have been only rarely reported as ligninolytic enzyme producers in the literature. Culture conditions and medium composition were optimized for the laccase producer Trametes trogii CTM 10156. This optimization resulted in high laccase production, 367 times more than in non-optimized conditions and which reached 110 U ml^-1 within 15 days of incubation.     

Spatiotemporal Patterns of Laccase Activity in Interacting Mycelia of Wood-Decaying Basidiomycete Fungi

Abstract Interspecific fungal interactions are important ecological processes, whereas their physiological mechanisms are little understood. The aim of this work was to study how activity of fungal extracellular laccase was changed across mycelia during interactions between white- and brown-rot basidiomycetes from different wood decay stages. Qualitative assay of eight species interacting with each other in all combinations showed four spatial patterns of laccase activity: (I) laccase activity present both in contact zone and mycelium, (II) laccase activity only in contact zone, (III) laccase activity in mycelium but not in contact zone, (IV) no laccase activity. Presence of laccase activity only in the contact zone was more frequent than expected from random samples associated with mycelia that replaced other ones. On the other hand, the presence of laccase activity in the mycelium but not in the contact zone was only attributed to fungal species that were replaced by their antagonists. After one month, laccase activity was distributed over mycelia more homogeneously than after 6 days of interactions. In interacting mycelia, laccase activity was higher than in control and increasing with time. Saprotrophic fungi from late successional stages of wood decay generally had higher laccase activity than early succession saprotrophic and pathogenic fungi. The qualitative assays were confirmed by quantitative assay of total laccase activity. Significance of the results in antagonistic fungal interactions as well as in the processes of hyphal tip growth and mycelium senescence is discussed. Received: 6 October 1999; Accepted: 1 February 2000; Online Publication: 5 May 2000     

Production and regulation of lignocellulose-degrading enzymes of <Emphasis Type="Italic">Poria</Emphasis>-like wood-inhabiting basidiomycetes

The wood-decomposing fungal species Antrodia macra, A. pulvinascens, Ceriporiopsis aneirina, C. resinascens and Dichomitus albidofuscus were determined for production of laccase (LAC), Mn peroxidase (MnP), lignin peroxidase (LiP), endo-l,4-P-β-glucanase, endo-l,4-β-xylanase, cellobiohydrolase, 1,4-β-glucosidase and 1,4-β-xylosidase. The results confirmed the brown-rot mode of Antrodia spp. which did not produce the activity of LAC and MnP. The remaining species performed detectable activity of both enzymes while no strain produced LiP. Significant inhibition of LAC production by high nitrogen was found in all white-rot species while only MnP of D. albidofuscus was regulated in the same way. The endoglucanase and endoxylanase activities of white-rotting species were inhibited by glucose in the medium while those of Antrodia spp. were not influenced by glucose concentration. The regulation of enzyme activity and bio-mass production can vary even within a single fungal genus.     

Physiological characterization of opine-utilizing rhizobacteria for traits related to plant growth-promoting activity

Thirty-two strains of opine-utilizing rhizobacteria were evaluated for physiological traits which have been related to plant growth-promoting activity. Tests included antibiosis against two bacterial and eight fungal pathogens of potato (Solanum tuberosum L.), production of hydrogen cyanide and fluorescent pigment production. On average, 71 and 12% of the bacteria inhibited the growth of Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens, respectively. The growth of Botrytis sp. was inhibited by 62% of the bacteria, and half of these produced an inhibition zone of more than 7 mm in diameter. Fusarium solani, Colletotrichum coccodes, Phoma exigua, Verticillium dahliae, F. oxysporum, V. albo-atrum and F. sambucinum were antagonized by 43, 34, 31, 25, 19, 18, and 12% of the bacteria, respectively. Only four strains produce hydrogen cyanide. The inhibition of a plant pathogen was not correlated to the production of fluorescent pigment. No strain produced a hypersensitive reaction whereas only three strains induced soft-rot and two produced polygalacturonase. Some opine-utilizing rhizobacteria were strong inhibitors of all plant pathogens, while most were active against specific plant pathogens.     

Comparative study of the degradation of flax shive by strains of Pleurotus

Summary Among the four strains of Pleurotus tested, hybrid strain A was found to degrade flax shive faster than the other three strains. Strain A produced more polygalacturonase, pectinlyase, cellulase and laccase enzymes than the other three strains of Pleurotus. Maximum activities of laccase and polysaccharide degrading enzymes were correlated with high weight loss, reduction in the yield of lignin and holocellulose and the degree of polymerisation of holocellulose. The addition of crude extract of flax shive on unextracted-shive increased the production of primordia of all the four strains, but it did not increase the degradation of flax shive. Flavonoid type compounds were detected in the crude extract of flax shive and they may be responsible for the increase in the production of mushroom primordia.     

Characterization of proteolytic enzymes of the midgut and excreta of the biting fly Stomoxys calcitrans

Proteolytic enzymes were characterized in the midgut and the excreta of the stable fly Stomoxys calcitrans (L) with proteins, synthetic substrates, and inhibitors. Inhibition studies suggested trypsinlike activity in sugar-fed fly midguts, whereas excreta and blood-fed fly guts exhibited other proteases. Trypsinlike activity in midguts removed 20 and 30 h after a blood meal increased from 20% to 50% of the total proteolytic enzymes present. Trypsinlike activity was inhibited with human sera, trypsin-specific inhibitors, and a protein isolated from the stable fly thorax. When human albumin and globulin fractions were incubated with trypsinlike enzymes isolated from the midgut and excreta, the albumin fraction was less inhibitory than the globulin fractions and was readily hydrolyzed by the proteolytic enzymes. These results may indicate that the proteolytic enzymes produce an abortive complex with the globulin fractions of the sera. Such a complex may explain the temporary inhibition of proteolysis by the blood meal. Soybean trypsin inhibitor fed to stable flies caused 50% inhibition in proteolytic activity in the midguts of sugar-fed stable flies and 25% inhibition in the midguts of blood-fed stable flies. Complete inhibition of proteolytic enzyme activity was achieved only in vitro. pH profiles of proteolytic enzyme activity isolated from the excreta of blood-fed stable flies indicated that several proteolytic enzymes were excreted.     

Extracellular laccase from Monilinia fructicola: isolation,primary characterization and application

Laccases are enzymes belonging to the family of blue copper oxidases. Due to their broad substrate specificity, they are widely used in many industrial processes and environmental bioremediations for removal of a large number of pollutants. During last decades, laccases attracted scientific interest also as highly promising enzymes to be used in bioanalytics. The aim of this study is to obtain a highly purified laccase from an efficient fungal producer and to demonstrate the applicability of this enzyme for analytics and bioremediation. To select the best microbial source of laccase, a screening of fungal strains was carried out and the fungus Monilinia fructicola was chosen as a producer of an extracellular enzyme. Optimal cultivation conditions for the highest yield of laccase were established; the enzyme was purified by a column chromatography and partially characterized. Molecular mass of the laccase subunit was determined to be near 35 kDa; the optimal pH ranges for the highest activity and stability are 4.5–5.0 and 3.0–5.0, respectively; the optimal temperature for laccase activity is 30°C. Laccase preparation was successfully used as a biocatalyst in the amperometric biosensor for bisphenol A assay and in the bioreactor for bioremediation of some xenobiotics.     

Increasing <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> laccase production by culture medium optimization and copper/lignin synergistic induction

Laccases have great biotechnological potential in diverse industries as they catalyze the oxidation of a broad variety of chemical compounds. Production of laccases by basidiomycetes has been broadly studied as they secrete the enzymes, grow on cheap substrates, and they generally produce more than one isoenzyme (constitutive and/or inducible). Laccase production and isoenzyme profile can be modified through medium composition and the use of inducers. The objective of this work was to increase laccase production by Pleurotus ostreatus CP-50 through culture medium optimization and the simultaneous use of copper and lignin as inducers. Increased fungal growth was obtained through the use of a factorial fractional experimental design 2^6–2 where the influence of the nature and concentration of carbon and nitrogen sources was assessed. Although specific laccase production (U/mg biomass) decreased when malt extract medium was supplemented with carbon and nitrogen sources, fungal growth and laccase volumetric activity increased four and sixfold, respectively. The effect of media supplementation with copper and/or lignin on laccase production by P. ostreatus CP-50 was studied. A positive synergistic effect between copper and lignin was observed on laccase production. Overall, the use of an optimized medium and the simultaneous addition of copper and lignin improved growth, laccase volumetric activity, and process productivity by 4-, 60-, and 10-fold, respectively.     

Inhibition of P-Enolpyruvate Carboxykinase and of Glyconeogenesis in Tetrahymena by 3-Mercaptopicolinic Acid*

SYNOPSIS. Tetrahymena grown overnight in deep cultures were incubated for 1 hr with [1-¹⁴C]labeled substrates in the presence or absence of 3-mercaptopicolinic acid (3-MPA). 3-MPA inhibited appearance of label in glycogen from bicarbonate, acetate, pentanoate, octanoate, and succinate, but not from glycerol or glucose. In vitro assays of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase activity showed that both enzymes were about equally distributed between the particulate and cytosol fractions. 3-MPA inhibited phosphoenolpyruvate carboxykinase from both the cytoplasmic and particulate fractions, but had no effect on phosphoenolpyruvate carboxylase from either location. These results suggest that the in vivo effects of this drug are due to inhibition of glyconeogenesis at this site.     

Identification and characterization of the biotechnological potential of a wild strain of Paraconiothyrium sp

The isolation and characterization of fungal strains from poorly described taxa allows undercover attributes of their basic biology useful for biotechnology. Here, a wild fungal strain (CMU‐196) from recently described Paraconiothyrium genus was analyzed. CMU‐196 was identified as Paraconiothyrium brasiliense by phylogenetic analysis of the rDNA internal transcribed spacer region (ITS). CMU‐196 metabolized 57 out of 95 substrates of the Biolog FF microplates. Efficient assimilation of dextrins and glycogen indicates that CMU‐196 is a good producer of amylolytic enzymes. It showed a remarkably assimilation of α‐d ‐lactose, substrate described as inducer of cellulolytic activity but poorly assimilated by several fungi. Metabolically active mycelium of the strain decolorized broth supplemented with direct blue 71, Chicago sky blue and remazol brilliant blue R dyes. The former two dyes were also well removed from broth by mycelium inactivated by autoclaving. Both mycelia had low efficiency for removing fuchsin acid from broth and for decolorizing wastewater from the paper industry. CMU‐196 strain showed extracellular laccase activity when potato dextrose broth was supplemented with Cu⁺², reaching a maximum activity of 46.8 (±0.33) U L^?1. Studied strain antagonized phytopathogenic Colletotrichum spp. fungi and Phytophthora spp. oomycetes in vitro, but is less effective towards Fusarium spp. fungi. CMU‐196 antagonism includes overgrowing the mycelia of phytopathogens and growth inhibition, probably by hydrosoluble extracellular metabolites. The biotechnological potential of strain CMU‐196 here described warrants further studies to have a more detailed knowledge of the mechanisms associated with its metabolic versatility, capacity for environmental detoxification, extracellular laccase production, and antagonism against phytopathogens. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:846–857, 2018     

Comparative Characterization of Methods for Removal of Cu(II) from the Active Sites of Fungal Laccases

Copper-containing sites of laccases isolated from the Basidiomycetes Coriolus hirsutus and Coriolus zonatus were characterized by optical methods and EPR spectroscopy. Methods for preparation of fungal laccase derivatives free from type 2 copper ions were compared. The data of EPR spectroscopy and spectrophotometric titration of copper sites showed that only a modified method based on the use of bathocuproine as a chelator for type 2 copper yielded laccase derivatives completely free from type 2 copper. The original enzymes can be reconstituted from the derivatives by dialysis under anaerobic conditions, resulting in complete recovery of native conformation of the protein molecule and the structure of the copper-containing site.     

Reactivity of bacterial and fungal laccases with lignin under alkaline conditions

The ability of Streptomyces ipomoea laccase to polymerize secoisolariciresinol lignan and technical lignins was assessed. The reactivity of S. ipomoea laccase was also compared to that of low redox fungal laccase from Melanocarpus albomyces using low molecular mass p-coumaric, ferulic and sinapic acid as well as natural (acetosyringone) and synthetic 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) mediators as substrates. Oxygen consumption measurement, MALDI-TOF MS and SEC were used to follow the enzymatic reactions at pH 7, 8, 9 and 10 at 30 °C and 50 °C. Polymerization of lignins and lignan by S. ipomoea laccase under alkaline reaction conditions was observed, and was enhanced in the presence of acetosyringone almost to the level obtained with M. albomyces laccase without mediator. Reactivities of the enzymes towards acetosyringone and TEMPO were similar, suggesting exploitation of the compounds and low redox laccase in lignin valorization under alkaline conditions. The results have scientific impact on basic research of laccases.     

Soil and entomopathogenic fungi with potential for biodegradation of insecticides: degradation of flubendiamide in vivo by fungi and in vitro by laccase

Purpose

Flubendiamide is a highly toxic and persistent insecticide that causes loss of insect muscle functions leading to paralysis and death. The objective was to screen for filamentous fungi in soils where insecticides had been applied, to isolate entomopathogenic fungi from insect larva (Anticarsia gemmatalis) that infest soybean crops, and to use these in biodegradation of insecticides.

Method

Filamentous fungi were isolated from soils, and growth inhibition was evaluated on solid medium containing commercial insecticides, Belt® (flubendiamide) and Actara® (thiamethoxam). A total of 133 fungi were isolated from soil and 80 entomopathogenic fungi from insect larva. Based on growth inhibition tests, ten soil fungi, 2 entomopathogenic fungi, and Botryosphaeria rhodina MAMB-05 (reference standard) were selected for growth on commercial insecticides in solid media. Fungi were grown in submerged fermentation on media containing commercial insecticides and assayed for laccase activity.

Result

Isolates JUSOLCL039 (soil), JUANT070 (insect), and MAMB-05 performed best, and were respectively inhibited by 48.41%, 75.97%, and 79.23% when cultivated on 35 g/L Actara®, and 0.0, 5.42%, and 43.39% on 39.04 g/L Belt®. JUSOLCL039 and JUANT070 were molecularly identified as Trichoderma koningiopsis and Neurospora sp., respectively. The three fungal isolates produced laccase constitutively, albeit at low activities. Fungal growth on pure flubendiamide and thiamethoxam resulted in only thiamethoxam inducing high laccase titers (10.16 U/mL) by JUANT070. Neurospora sp. and B. rhodina degraded flubendiamide by 27.4% and 9.5% in vivo, while a crude laccase from B. rhodina degraded flubendiamide by 20.2% in vitro.

Conclusion

This is the first report of fungi capable of degrading flubendiamide, which have applications in bioremediation.

     

Antimicrobial activity of saponin fractions of the leaves of Gymnema sylvestre and Eclipta prostrata

The antimicrobial activity of saponin fractions from the leaves of Gymnema sylvestre and Eclipta prostrata was evaluated against pathogenic bacteria and fungi in an in vitro condition. A series of concentrations of crude and pure saponin fractions were tested for antimicrobial activity by zone of inhibition method. The pure saponin fractions were found to be more effective against tested bacterial pathogens when compared to crude saponin fractions. The minimum inhibitory concentration (MIC) exhibited by the pure saponin fraction of G. sylvestre was found to be in the range of 600–1,200 mg/l against bacterial strains and 1,400 mg/l for fungal isolates. In the case of E. prostrata, the range was 1,000–1,200 mg/l for bacteria and 1,400 mg/l for fungal isolates. The susceptibility of bacterial pathogens for saponin fractions was in the order of P. aeruginosa, E. coli, S. typhi, K. pneumoniae, P. mirablis, S. aureus and for fungal pathogens A. fumigatus followed by A. niger and A. flavus. Whereas, A. niger was more susceptible to inhibition by E. prostrata saponin fractions, followed by A. flavus and A. fumigatus. The antimicrobial potential of saponin fractions was compared with antibiotics, Chloramphenicol and Amphotericin-B with respect to bacteria and fungi. The present study suggests that the saponin fractions G. sylvestre and E. prostrata possess significant antibacterial and antifungal activity. Our results further suggest that saponins of G. sylvestre and E. prostrata can be used as a potential fungicide against pathogenic fungi.     

Occurrence of laccases in lichenized ascomycetes of the Peltigerineae

Following our previous findings of high extracellular redox activity in lichens, the results of the work presented here identify the enzymes involved as laccases. Despite numerous data on laccases in fungi and flowering plants, this is the first report of the occurrence of laccases in lichenized ascomycetes. Extracellular laccase activity was measured in 40 species of lichens from different taxonomic groupings and contrasting habitats. Out of 20 species tested from suborder Peltigerineae, 18 displayed laccase activity, while activity was absent in species tested from other lichen groups. Identification of the enzymes as laccases was confirmed by the ability of lichen leachates to readily metabolize substrates such as 2,2′-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS), syringaldazine and o-tolidine in the absence of hydrogen peroxide, sensitivity of the enzymes to cyanide and azide, the enzymes having typical laccase pH and temperature optima, and an absorption spectrum with a peak at 614 nm. Desiccation and wounding stimulated laccase activity. Laccase activity was not increased after treatment with normal inducers of laccase synthesis, suggesting that they are constitutively expressed. Electrophoresis showed that the active form of laccase from Peltigera malacea was a tetramer with an unusually high molecular mass of 340 kDa and an isoelectric point (pI) of 4.7. The finding of abundant extracellular redox enzymes known to actively produce reactive oxygen species suggest that their roles may include increasing nutrient supply to lichens by delignification, and deterring pathogens by contributing to the oxidative burst. Furthermore, once released into the environment, they may participate in the carbon cycle by facilitating the breakdown or formation of humic substances.