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Mieczyslawa I Boguś - One of the best experts on this subject based on the ideXlab platform.
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Conidiobolus coronatus induces oxidative stress and autophagy response in galleria mellonella larvae
PLOS ONE, 2020Co-Authors: Michalina Kazek, Agata Kaczmarek, Anna Katarzyna Wronska, Mieczyslawa I BoguśAbstract:Cell homeostasis requires the correct levels of reactive oxygen species (ROS) to be maintained as these regulate the proliferation and differentiation of cells, and control the immune response and inflammation. High levels of ROS can cause oxidative stress, leading to protein, lipid and DNA damage, or even cell death. Under physiological conditions, the rate of autophagy remains stable; however, it can be accelerated by a number of exogenous stimuli such as oxidative stress, starvation or hypoxia, leading to cell death. The present paper examines the effect of Conidiobolus coronatus infection on the immune response, oxidative stress processes and autophagy in the greater wax moth, Galleria mellonella. Fungal infection was found to result in the disorganization of the cytoskeleton of the larval immune cells and the enhancement of oxidative defense processes. Lipid peroxidation and autophagy were also induced in the hemocytes. Our findings show that G. mellonella is an ideal model for exploring immune mechanisms.
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harman and norharman metabolites of the entomopathogenic fungus Conidiobolus coronatus entomophthorales affect the serotonin levels and phagocytic activity of hemocytes insect immunocompetent cells in galleria mellonella lepidoptera
Cell & Bioscience, 2019Co-Authors: Anna Katarzyna Wronska, Mieczyslawa I BoguśAbstract:Although the β-carboline alkaloids harman and norharman are considered as plant metabolites, they can also be secreted by fungi such as the entomopathogen Conidiobolus coronatus. Norharman and harman are also known to be reversible competitive monamine oxidase inhibitors, which increase serotonin concentrations in tissues. In addition, these alkaloids are able to bind to serotonin receptors, an important immune regulatory molecule in both vertebrates and invertebrates. In insects, serotonin modulates hemocyte phagocytosis, nodule formation and the populations of hemocyte classes. The present study examines whether harman and norharman may influence the phagocytic activity of insect hemocytes by regulating serotonin levels. Significantly greater serotonin levels and hemocyte phagocytic activity were observed after 24 h of exposure to food contaminated with harman and norharman. Similar responses were noticed 1 h after topical application or addition to in vitro hemocyte cultures. Observations and measurements performed 24 h later revealed decreased responses, suggesting decomposition and/or exertion of alkaloids and/or serotonin. Harman and norharman influenced the activity of Galleria mellonella plasmatocytes and the granulocyte cytoskeleton. Disturbances in hemocyte network formation, abnormal cell shape, naked nuclei, cell aggregates, fragments of disintegrated cells, interrupted cell membrane continuity and actin condensation in cells were observed. Our findings may have a considerable impact on research concerning insect physiology, parasitology, immunology and biocontrol of pests. They confirm for the first time that harman and norharman (metabolites of the entomopathogenic fungus C. coronatus) elevate serotonin levels in G. mellonella hemocytes, thus potentially stimulating their phagocytic activity. Our studies shed light on the mechanisms underlying the interaction between innate insect immune responses and entomopathogen metabolites.
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harman and norharman metabolites of entomopathogenic fungus Conidiobolus coronatus entomopthorales disorganize development of galleria mellonella lepidoptera and affect serotonin regulating enzymes
PLOS ONE, 2018Co-Authors: Anna Katarzyna Wronska, Agata Kaczmarek, Mieczyslawa I Boguś, Michalina KazekAbstract:: Naturally occurring entomopathogenic fungi such as Conidiobolus coronatus are important regulatory factors of insect populations. GC-MS analysis of fungal cell-free filtrates showed that C. coronatus synthesizes two β- carboline alkaloids: harman and norharman. Significantly higher levels of both alkaloids are produced by C. coronatus in minimal postincubation medium than in rich medium. The beta-carboline alkaloids may have an effect on the nervous system of insects and their behavior. Harman and norharman were applied to Galleria mellonella larvae (a parasite of honeybees) either topically or mixed with food. Larvae received alkaloids in three concentrations: 750, 1000 or 1250 ppm. The effect on the survival and further development of larvae was examined. Both harman and norharman delayed pupation and adult eclosion, and inhibit total monoamine oxidase activity. In addition, they increased the serotonin concentration and decreased the monoamine oxidase A level in the heads of the moths. It is likely that the alkaloids were metabolized by the insects, as their effect wore off 24 hours after topical application. This is the first study to show that C. coronatus produces alkaloids. Its aim was to identify the actions of β-carboline alkaloids on insect development and serotonin-regulating enzymes. Knowledge of the potential role of harman and norharman in the process of fungal infection might lead to the development of more effective and environmentally-friendly means of controlling insect pests.
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cuticular fatty acids of galleria mellonella lepidoptera inhibit fungal enzymatic activities of pathogenic Conidiobolus coronatus
PLOS ONE, 2018Co-Authors: Anna Katarzyna Wronska, Agata Kaczmarek, Emilia Wloka, Michalina Kazek, Mieczyslawa I Boguś, Katarzyna ZalewskaAbstract:The entomopathogenic fungus Conidiobolus coronatus produces enzymes that may hydrolyze the cuticle of Galleria mellonella. Of these enzymes, elastase activity was the highest: this figure being 24 times higher than NAGase activity 553 times higher than chitinase activity and 1844 times higher than lipase activity. The present work examines the differences in the hydrolysis of cuticles taken from larvae, pupae and adults (thorax and wings), by C. coronatus enzymes. The cuticles of the larvae and adult thorax were the most susceptible to digestion by proteases and lipases. Moreover, the maximum concentration of free N-glucosamine was in the hydrolysis of G. mellonella thorax. These differences in the digestion of the various types of cuticle may result from differences in their composition. GC-MS analysis of the cuticular fatty acids isolated from pupae of G. mellonella confirmed the presence of C 8:0, C 9:0, C 12:0, C 14:0, C 15:0, C 16:1, C 16:0, C 17:0, C 18:1, C 18:0, with C 16:0 and C 18:0 being present in the highest concentrations. Additional fatty acids were found in extracts from G. mellonella imagines: C 10:0, C 13:0, C 20:0 and C 20:1, with a considerable dominance of C 16:0 and C 18:1. In larvae, C 16:0 and C 18:1 predominated. Statistically significant differences in concentration (p≤0.05) were found between the larvae, pupae and imago for each fatty acid. The qualitative and quantitative differences in the fatty acid composition of G. mellonella cuticle occurring throughout normal development might be responsible for the varied efficiency of fungal enzymes in degrading larval, pupal and adult cuticles.
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cuticle hydrolysis in four medically important fly species by enzymes of the entomopathogenic fungus Conidiobolus coronatus
Medical and Veterinary Entomology, 2017Co-Authors: Mieczyslawa I Boguś, Agata Kaczmarek, Michalina Kazek, Katarzyna Zalewska, A Wrońska, Ligęza M żuber, Marek GołębiowskiAbstract:. Entomopathogenic fungi infect insects via penetration through the cuticle, which varies remarkably in chemical composition across species and life stages. Fungal infection involves the production of enzymes that hydrolyse cuticular proteins, chitin and lipids. Host specificity is associated with fungus–cuticle interactions related to substrate utilization and resistance to host-specific inhibitors. The soil fungus Conidiobolus coronatus (Constantin) (Entomophthorales: Ancylistaceae) shows virulence against susceptible species. The larvae and pupae of Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae), Calliphora vomitoria (Linnaeus), Lucilia sericata (Meigen) (Diptera: Calliphoridae) and Musca domestica (Linnaeus) (Diptera: Muscidae) are resistant, but adults exposed to C. coronatus quickly perish. Fungus was cultivated for 3 weeks in a minimal medium. Cell-free filtrate, for which activity of elastase, N-acetylglucosaminidase, chitobiosidase and lipase was determined, was used for in vitro hydrolysis of the cuticle from larvae, puparia and adults. Amounts of amino acids, N-glucosamine and fatty acids released were measured after 8 h of incubation. The effectiveness of fungal enzymes was correlated with concentrations of compounds detected in the cuticles of tested insects. Positive correlations suggest compounds used by the fungus as nutrients, whereas negative correlations may indicate compounds responsible for insect resistance. Adult deaths result from the ingestion of conidia or fungal excretions.
Anna Katarzyna Wronska - One of the best experts on this subject based on the ideXlab platform.
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the interaction between cuticle free fatty acids ffas of the cockroaches blattella germanica and blatta orientalis and hydrolases produced by the entomopathogenic fungus Conidiobolus coronatus
PLOS ONE, 2020Co-Authors: Agata Kaczmarek, Mieczyslawa I Boguś, Emilia Wloka, Anna Katarzyna Wronska, Anna Krawiel, Michalina Kazek, Katarzyna Zalewska, Katarzyna Klocinskabialy, Martyna Sobocinska, Aleksandra GliniewiczAbstract:The interactions between entomopathogenic fungi and insects serve a classic example of a co-evolutionary arms race between pathogens and their target host. The cuticle, site of the first contact between insects and entomopathogenic fungus, is an important defensive barrier against pathogens. It is covered by a layer of lipids that appears to play a key role in these processes and cuticular free fatty acid (FFA) profiles are consider as a determinant of susceptibility, or resistance, to fungal infections. These profiles are species-specific. The cockroaches Blattella germanica (Blattodea: Blattidae) and Blatta orientalis (Blattodea: Ectobiidae) are unsusceptible to the soil fungus Conidiobolus coronatus (Entomophthorales: Ancylistaceae) infection, therefore we studied the profiles of FFAs in order to understand the defensive capabilities of the cockroaches. The fungus was cultivated for three weeks in minimal medium. Cell-free filtrate was obtained, assayed for elastase, N-acetylglucosaminidase, chitobiosidase and lipase activity, and then used for in vitro hydrolysis of the cuticle from wings and thoraces of adults and oothecae. The amounts of amino acids, N-glucosamine and FFAs released from the hydrolysed cuticle samples were measured after eight hours of incubation. The FFA profiles of the cuticle of adults, and the wings, thoraces and oothecae of both species were established using GC-MS and the results were correlated with the effectiveness of fungal proteases, chitinases and lipases in the hydrolyzation of cuticle samples. Positive correlations would suggest the existence of compounds used by the fungus as nutrients, whereas negative correlations may indicate that these compounds could be engaged in insect defence.
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heat shock proteins hsp 90 70 60 and 27 in galleria mellonella lepidoptera hemolymph are affected by infection with Conidiobolus coronatus entomophthorales
PLOS ONE, 2020Co-Authors: Anna Katarzyna Wronska, Mieczyslawa I BoguśAbstract:Invertebrates are becoming more popular models for research on the immune system. The innate immunity possessed by insects shows both structural and functional similarity to the resistance displayed by mammals, and many processes occurring in insect hemocytes are similar to those that occur in mammals. The humoral immune response in insects acts by melanization, clotting and the production of reactive oxygen species and antimicrobial peptides, while the cellular immunity system is based on nodulation, encapsulation and phagocytosis. An increasingly popular insect model in biological research is Galleria mellonella, whose larvae are sensitive to infection by the entomopathogenic fungus Conidiobolus coronatus, which can also be dangerous to humans. One group of factors that modulate the response of the immune system during infection in mammals are heat shock proteins (HSPs). The aim of this study was to investigate whether infection by C. coronatus in G. mellonella hemolymph is accompanied by an increase of HSP90, HSP70, HSP60 and HSP27. Larvae (five-day-old last instar) were exposed for 24 hours to fully-grown and sporulating fungus. Hemolymph was collected either immediately after termination of exposure (F24) or 24 hours later (F48). The concentration of the HSPs in hemolymph was determined using ELISA. Immunolocalization in hemocytes was performed using fluorescence microscopy and flow cytometry. HSP90, HSP70, HSP60 and HSP27 were found to be present in the G. mellonella hemocytes. HSP60 and HSP90 predominated in healthy insects, with HSP70 and HSP27 being found in trace amounts; HSP60 and HSP27 were elevated in F24 and F48, and HSP90 was elevated in F48. The fungal infection had no effect on HSP70 levels. These findings shed light on the mechanisms underlying the interaction between the innate insect immune response and entomopathogen infection. The results of this innovative study may have a considerable impact on research concerning innate immunology and insect physiology.
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Conidiobolus coronatus induces oxidative stress and autophagy response in galleria mellonella larvae
PLOS ONE, 2020Co-Authors: Michalina Kazek, Agata Kaczmarek, Anna Katarzyna Wronska, Mieczyslawa I BoguśAbstract:Cell homeostasis requires the correct levels of reactive oxygen species (ROS) to be maintained as these regulate the proliferation and differentiation of cells, and control the immune response and inflammation. High levels of ROS can cause oxidative stress, leading to protein, lipid and DNA damage, or even cell death. Under physiological conditions, the rate of autophagy remains stable; however, it can be accelerated by a number of exogenous stimuli such as oxidative stress, starvation or hypoxia, leading to cell death. The present paper examines the effect of Conidiobolus coronatus infection on the immune response, oxidative stress processes and autophagy in the greater wax moth, Galleria mellonella. Fungal infection was found to result in the disorganization of the cytoskeleton of the larval immune cells and the enhancement of oxidative defense processes. Lipid peroxidation and autophagy were also induced in the hemocytes. Our findings show that G. mellonella is an ideal model for exploring immune mechanisms.
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harman and norharman metabolites of the entomopathogenic fungus Conidiobolus coronatus entomophthorales affect the serotonin levels and phagocytic activity of hemocytes insect immunocompetent cells in galleria mellonella lepidoptera
Cell & Bioscience, 2019Co-Authors: Anna Katarzyna Wronska, Mieczyslawa I BoguśAbstract:Background Although the β-carboline alkaloids harman and norharman are considered as plant metabolites, they can also be secreted by fungi such as the entomopathogen Conidiobolus coronatus. Norharman and harman are also known to be reversible competitive monamine oxidase inhibitors, which increase serotonin concentrations in tissues. In addition, these alkaloids are able to bind to serotonin receptors, an important immune regulatory molecule in both vertebrates and invertebrates. In insects, serotonin modulates hemocyte phagocytosis, nodule formation and the populations of hemocyte classes. The present study examines whether harman and norharman may influence the phagocytic activity of insect hemocytes by regulating serotonin levels.
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harman and norharman metabolites of the entomopathogenic fungus Conidiobolus coronatus entomophthorales affect the serotonin levels and phagocytic activity of hemocytes insect immunocompetent cells in galleria mellonella lepidoptera
Cell & Bioscience, 2019Co-Authors: Anna Katarzyna Wronska, Mieczyslawa I BoguśAbstract:Although the β-carboline alkaloids harman and norharman are considered as plant metabolites, they can also be secreted by fungi such as the entomopathogen Conidiobolus coronatus. Norharman and harman are also known to be reversible competitive monamine oxidase inhibitors, which increase serotonin concentrations in tissues. In addition, these alkaloids are able to bind to serotonin receptors, an important immune regulatory molecule in both vertebrates and invertebrates. In insects, serotonin modulates hemocyte phagocytosis, nodule formation and the populations of hemocyte classes. The present study examines whether harman and norharman may influence the phagocytic activity of insect hemocytes by regulating serotonin levels. Significantly greater serotonin levels and hemocyte phagocytic activity were observed after 24 h of exposure to food contaminated with harman and norharman. Similar responses were noticed 1 h after topical application or addition to in vitro hemocyte cultures. Observations and measurements performed 24 h later revealed decreased responses, suggesting decomposition and/or exertion of alkaloids and/or serotonin. Harman and norharman influenced the activity of Galleria mellonella plasmatocytes and the granulocyte cytoskeleton. Disturbances in hemocyte network formation, abnormal cell shape, naked nuclei, cell aggregates, fragments of disintegrated cells, interrupted cell membrane continuity and actin condensation in cells were observed. Our findings may have a considerable impact on research concerning insect physiology, parasitology, immunology and biocontrol of pests. They confirm for the first time that harman and norharman (metabolites of the entomopathogenic fungus C. coronatus) elevate serotonin levels in G. mellonella hemocytes, thus potentially stimulating their phagocytic activity. Our studies shed light on the mechanisms underlying the interaction between innate insect immune responses and entomopathogen metabolites.
Mieczyslawa I Boguś - One of the best experts on this subject based on the ideXlab platform.
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the interaction between cuticle free fatty acids ffas of the cockroaches blattella germanica and blatta orientalis and hydrolases produced by the entomopathogenic fungus Conidiobolus coronatus
PLOS ONE, 2020Co-Authors: Agata Kaczmarek, Mieczyslawa I Boguś, Emilia Wloka, Anna Katarzyna Wronska, Anna Krawiel, Michalina Kazek, Katarzyna Zalewska, Katarzyna Klocinskabialy, Martyna Sobocinska, Aleksandra GliniewiczAbstract:The interactions between entomopathogenic fungi and insects serve a classic example of a co-evolutionary arms race between pathogens and their target host. The cuticle, site of the first contact between insects and entomopathogenic fungus, is an important defensive barrier against pathogens. It is covered by a layer of lipids that appears to play a key role in these processes and cuticular free fatty acid (FFA) profiles are consider as a determinant of susceptibility, or resistance, to fungal infections. These profiles are species-specific. The cockroaches Blattella germanica (Blattodea: Blattidae) and Blatta orientalis (Blattodea: Ectobiidae) are unsusceptible to the soil fungus Conidiobolus coronatus (Entomophthorales: Ancylistaceae) infection, therefore we studied the profiles of FFAs in order to understand the defensive capabilities of the cockroaches. The fungus was cultivated for three weeks in minimal medium. Cell-free filtrate was obtained, assayed for elastase, N-acetylglucosaminidase, chitobiosidase and lipase activity, and then used for in vitro hydrolysis of the cuticle from wings and thoraces of adults and oothecae. The amounts of amino acids, N-glucosamine and FFAs released from the hydrolysed cuticle samples were measured after eight hours of incubation. The FFA profiles of the cuticle of adults, and the wings, thoraces and oothecae of both species were established using GC-MS and the results were correlated with the effectiveness of fungal proteases, chitinases and lipases in the hydrolyzation of cuticle samples. Positive correlations would suggest the existence of compounds used by the fungus as nutrients, whereas negative correlations may indicate that these compounds could be engaged in insect defence.
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The composition of lipid profiles in different developmental stages of Dermestes ater and Dermestes maculatus and their susceptibility to the entomopathogenic fungus Conidiobolus coronatus
Phytoparasitica, 2020Co-Authors: Magdalena Cerkowniak, Mieczyslawa I Boguś, Piotr Stepnowski, Marek GołębiowskiAbstract:Insects from the Dermestidae family ( Dermestes ater and Dermestes maculatus ) are synanthropic insects, which are household, agricultural and warehouse pests. Their lipidomics and the insects’ ability to use compounds present in their bodies to protect them against pathogens are not fully understood. Therefore, the purpose of this work was to determine the composition of compounds present in the bodies of two insect species, Dermestes ater and Dermestes maculatus, by the MALDI technique. Several free fatty acids and acylglycerols were found to be present as a result of the research. Significant differences in the composition and number of identified compounds have been shown, depending on the tested species and on the development stage. In lipids of D. ater, a greater variety of free fatty acids were found than in those of the second species. Biological studies have determined the high resistance of both species of Dermestidae to fungal infection with Conidiobolus coronatus . These results provide baseline data for further studies on the possible role of lipids.
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heat shock proteins hsp 90 70 60 and 27 in galleria mellonella lepidoptera hemolymph are affected by infection with Conidiobolus coronatus entomophthorales
PLOS ONE, 2020Co-Authors: Anna Katarzyna Wronska, Mieczyslawa I BoguśAbstract:Invertebrates are becoming more popular models for research on the immune system. The innate immunity possessed by insects shows both structural and functional similarity to the resistance displayed by mammals, and many processes occurring in insect hemocytes are similar to those that occur in mammals. The humoral immune response in insects acts by melanization, clotting and the production of reactive oxygen species and antimicrobial peptides, while the cellular immunity system is based on nodulation, encapsulation and phagocytosis. An increasingly popular insect model in biological research is Galleria mellonella, whose larvae are sensitive to infection by the entomopathogenic fungus Conidiobolus coronatus, which can also be dangerous to humans. One group of factors that modulate the response of the immune system during infection in mammals are heat shock proteins (HSPs). The aim of this study was to investigate whether infection by C. coronatus in G. mellonella hemolymph is accompanied by an increase of HSP90, HSP70, HSP60 and HSP27. Larvae (five-day-old last instar) were exposed for 24 hours to fully-grown and sporulating fungus. Hemolymph was collected either immediately after termination of exposure (F24) or 24 hours later (F48). The concentration of the HSPs in hemolymph was determined using ELISA. Immunolocalization in hemocytes was performed using fluorescence microscopy and flow cytometry. HSP90, HSP70, HSP60 and HSP27 were found to be present in the G. mellonella hemocytes. HSP60 and HSP90 predominated in healthy insects, with HSP70 and HSP27 being found in trace amounts; HSP60 and HSP27 were elevated in F24 and F48, and HSP90 was elevated in F48. The fungal infection had no effect on HSP70 levels. These findings shed light on the mechanisms underlying the interaction between the innate insect immune response and entomopathogen infection. The results of this innovative study may have a considerable impact on research concerning innate immunology and insect physiology.
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harman and norharman metabolites of the entomopathogenic fungus Conidiobolus coronatus entomophthorales affect the serotonin levels and phagocytic activity of hemocytes insect immunocompetent cells in galleria mellonella lepidoptera
Cell & Bioscience, 2019Co-Authors: Anna Katarzyna Wronska, Mieczyslawa I BoguśAbstract:Background Although the β-carboline alkaloids harman and norharman are considered as plant metabolites, they can also be secreted by fungi such as the entomopathogen Conidiobolus coronatus. Norharman and harman are also known to be reversible competitive monamine oxidase inhibitors, which increase serotonin concentrations in tissues. In addition, these alkaloids are able to bind to serotonin receptors, an important immune regulatory molecule in both vertebrates and invertebrates. In insects, serotonin modulates hemocyte phagocytosis, nodule formation and the populations of hemocyte classes. The present study examines whether harman and norharman may influence the phagocytic activity of insect hemocytes by regulating serotonin levels.
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Diet influences the bacterial and free fatty acid profiles of the cuticle of Galleria mellonella larvae
2019Co-Authors: Michalina Kazek, Agata Kaczmarek, Anna Katarzyna Wronska, Mieczyslawa I BoguśAbstract:The evolutionary success of insects is arguably due to their ability to build up a complex, highly-adaptable and very effective defense system against numerous pathogens, including entomopathogenic fungi. This system relies on the humoral immune system and cellular defense reactions. The first line of defense against biological pathogens is a cuticle formed of several layers. The cuticular lipids may contain hydrocarbons, free fatty acids (FFA), alcohols, waxes, glycerides, aldehydes and sterols. Cuticular fatty acids may also play a role in defending against fungal invasion. Our present findings show that the diet of insects can have a significant effect on their sensitivity and defense response to pathogens; for example, while G. mellonella larvae fed on beeswax had a similar appearance to those reared on a semi-artificial diet, they possessed a different cuticular free fatty acid (FFA) profile to those fed on a semi-artificial diet, and were less sensitive to Conidiobolus coronatus infection. It is possible that the presence of heneicosenoic acid (C21:1) and other long-chain free fatty acids (C22:0, C24:0, C26:0), as well as Brevibacillus laterosporus bacteria, on the cuticle of larvae fed on beeswax, plays a protective role against fungal invasion. Insect pests represent a global problem. An understanding of the basic mechanisms underlying the fungal infection of insects might provide a clearer insight into their defenses, thus allowing the design of more effective, and environmentally-friendly, means of controlling them. The greater wax moth is an excellent model for the study of immunology resistance. Knowledge of the influence of diet on pathogen resistance in insects can be also useful for creating a model of human diseases caused by pathogens, such as Candia albicans.
Vasanti V Deshpande - One of the best experts on this subject based on the ideXlab platform.
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optimization and scale up of production of alkaline protease from Conidiobolus coronatus
Process Biochemistry, 2005Co-Authors: Seeta R Laxman, Atul P Sonawane, Snehal V More, Meenakshi V Rele, Vitthal V Jogdand, Vasanti V DeshpandeAbstract:Abstract Alkaline protease secreted by Conidiobolus coronatus has been evaluated extensively in tanneries and finds application in pre-tanning operations in leather manufacture. It is important to produce the enzyme in inexpensive and optimized media on large scale for the process to be commercially viable. The present paper describes optimization of fermentation conditions in shake flasks and scale up of production to 100 L in fermentors. The production is complete in 2–3 days comparable to bacterial fermentations. The organism utilized several carbon sources such as starch, sucrose, lactose, glucose and fructose for production. Soyabean meal at an optimum concentration of 2–3% was found to be best inducer. Diammonium hydrogen phosphate, casamino acids and Hi-media peptone gave activities comparable to yeast extract. Preservation and stabilization studies showed that glycerol conferred considerable stabilization at room temperature while ammonium sulphate precipitated enzyme at 0.9 saturation was best with stability up to 2 years even at room temperature.
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a serine alkaline protease from the fungus Conidiobolus coronatus with a distinctly different structure than the serine protease subtilisin carlsberg
Archives of Microbiology, 1996Co-Authors: Sangita Uday Phadtare, Vasanti V DeshpandeAbstract:In view of the functional similarities between subtilisin Carlsberg and the alkaline protease fromConidiobolus coronatus, the biochemical and structural properties of the two enzymes were compared. In spite of their similar biochemical properties, e.g., pH optima, heat stability, molecular mass, pI, esterase activity, and inhibition by diisopropyl fluorophosphate and phenylmethlysulfonylfluoride, the proteases were structurally dissimilar as revealed by (1) their amino acid compositions, (2) their inhibition by subtilisin inhibitor, (3) their immunological response to specific anti-Conidiobolus protease antibody, and (4) their tryptic peptide maps. Our results demonstrate that although they are functionally analogous, theConidiobolus protease is structurally distinct from subtilisin Carlsberg. TheConidiobolus protease was also different from other bacterial and animal proteases (e.g. pronase, protease K, trypsin, and chymotrypsin) as evidenced by their lack of response to anti-Conidiobolus protease antibody in double diffusion and in neutralization assays. TheConidiobolus serine protease fails to obey the general rule that proteins with similar functions have similar primary sequences and, thus, are evolutionarily related. Our results strengthen the concept of convergent evolution for serine proteases and provide basis for research in evolutionary relationships among fungal, bacterial, and animal proteases.
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high activity alkaline protease from Conidiobolus coronatus ncl 86 8 20 enzyme production and compatibility with commercial detergents
Enzyme and Microbial Technology, 1993Co-Authors: Sangita U Phadatare, Vasanti V Deshpande, M C SrinivasanAbstract:Abstract Production of high alkaline protease activity (30 U ml-1) by Conidiobolus coronatus (NCL 86.8.20) in submerged culture and its application in commercial detergents is reported. The novel feature of the studies is the use of fungal protease in detergents. The rapidity of the enzyme production and the ease of preparation of microbe-free enzyme solution in case of C. coronatus are advantageous features. The economic feasibility of the enzyme for its application depends on the cost of its production. In order to obtain high and commercially viable yields of protease, it was essential to optimize fermentation medium for growth and production of protease from C. coronatus. Optimization of fermentation conditions revealed that the maximum protease production was obtained at 28°C and 200 rpm, for 48 h with a medium containing sucrose, (3%); ammonium nitrate, (0.38%); tryptone, (2%); potassium chloride, (0.2%); potassium dihydrogen phosphate, (0.1%); casein, (2%); zinc sulphate, (0.001%); and calcium chloride, (0.001%). The initial pH was 7.0–7.5, using 12% (v/v) preinduced inoculum grown in the same medium under similar conditions for 24 h. In order to assess the suitability of the enzyme for use in detergents, its stability over a range of alkaline pH and temperature and its compatibility with commercial detergents was determined. The enzyme was most stable between pH 6.0–8.5 and at temperatures up to 40°C. It was not inactivated in the presence of most of the detergents tested and appears to be potentially useful in washing powders.
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evidence for controlled autoproteolysis of alkaline protease a mechanism for physiological regulation of conidial discharge in Conidiobolus coronatus
FEBS Journal, 1992Co-Authors: Sangita U Phadatare, M C Srinivasan, Vasanti V DeshpandeAbstract:The alkaline serine protease of Conidiobolus coronatus was shown to be involved in its conidial discharge [Phadatare, S., Srinivasan, M. C., Deshpande, M. (1989) Arch. Microbiol. 153, 47–49]. To understand the regulation of conidial discharge, the mechanism of control of protease activity was investigated, which revealed the presence of two electrophoretically separable intracellular proteases (protease I and protease II). The formation of smaller and less-active protease II coincided with the decrease in conidial discharge. In order to trace the origin of protease II, the corresponding purified extracellular enzymes were compared with respect to their biochemical, physicochemical and immunological properties. The biochemical properties, such as optimum pH and temperature, stability, sensitivity to metal ions and substrate specificity were closely similar for both proteases. Amino acid analysis revealed that protease II is completely similar to protease I, though protease I contains an additional portion which is not contained in protease II. Western-blot ELISA, immunotitration and determination of antigenic valencies also revealed the structural similarity between the two proteases. Purified protease I showed partial degradation to protease II in vitro, the process being sensitive to phenylmethylsulfonyl fluoride, indicating its proteolytic nature. These results suggest that the formation of a less-active protease by autoproteolysis represents a novel means of physiological regulation of protease activity, which in turn regulates the conidial discharge in C. coronatus.
Robert W. Roberson - One of the best experts on this subject based on the ideXlab platform.
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hyphal tip growth and cytoplasmic characters of Conidiobolus coronatus zoopagomycota entomophthoromycotina
Mycologia, 2018Co-Authors: Karen E. Fisher, David Lowry, Isobel Romberger, Phakade Shange, Robert W. RobersonAbstract:ABSTRACTCharacteristics of hyphal structure and growth can provide insights into the mechanisms of polarized growth and support investigations of fungal phylogeny. To assist with the resolution of ...
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Hyphal tip growth and cytoplasmic characters of Conidiobolus coronatus (Zoopagomycota, Entomophthoromycotina).
Mycologia, 2018Co-Authors: Karen E. Fisher, David Lowry, Isobel Romberger, Phakade Shange, Robert W. RobersonAbstract:Characteristics of hyphal structure and growth can provide insights into the mechanisms of polarized growth and support investigations of fungal phylogeny. To assist with the resolution of evolutionary relationships of the zygomycetes, the authors used comparative bioimaging methods (light [LM] and transmission electron [TEM] microscopy) to describe selected subcellular characters of hyphal tips of Conidiobolus coronatus. Growing hyphae of C. coronatus contain Spitzenkorper (Spk). Spk are most commonly present in hyphae of Dikarya (Ascomycota and Basidiomycota) and are rarely reported in zygomycete hyphae, which possess an apical vesicle crescent (AVC). Such findings raise questions regarding the evolution of the Spk and its relationship with the AVC. Descriptions of additional subcellular characters (e.g., mitotic-phase spindle pole bodies, cytoplasmic behavior, organelle structure) are also presented.