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Kenji Yamamoto - One of the best experts on this subject based on the ideXlab platform.

  • Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans.
    The Journal of biological chemistry, 2016
    Co-Authors: Toshihiko Katoh, Takane Katayama, Yusuke Tomabechi, Nishikawa Yoshihide, Jyunichi Kumada, Yuji Matsuzaki, Kenji Yamamoto
    Abstract:

    Endo-β-N-acetylglucosaminidase M (Endo-M), an endoglycosidase from the fungus Mucor hiemalis, is a useful tool for chemoenzymatic synthesis of glycoconjugates, including glycoprotein-based therapeutics having a precisely defined glycoform, by virtue of its transglycosylation activity. Although Endo-M has been known to act on various N-glycans, it does not act on core-fucosylated N-glycans, which exist widely in mammalian glycoproteins, thus limiting its application. Therefore, we performed site-directed mutagenesis on Endo-M to isolate mutant enzymes that are able to act on mammalian-type core-α1,6-fucosylated glycans. Among the Endo-M mutant enzymes generated, those in which the tryptophan at position 251 was substituted with alanine or asparagine showed altered substrate specificities. Such mutant enzymes exhibited increased hydrolysis of a synthetic α1,6-fucosylated trimannosyl core structure, whereas their activity on the afucosylated form decreased. In addition, among the Trp-251 mutants, the W251N mutant was most efficient in hydrolyzing the core-fucosylated substrate. W251N mutants could act on the immunoglobulin G-derived core-fucosylated glycopeptides and human lactoferrin glycoproteins. This mutant was also capable of transferring the sialyl glycan from an activated substrate intermediate (sialyl glyco-oxazoline) onto an α1,6-fucosyl-N-acetylglucosaminyl biotin. Furthermore, the W251N mutant gained a glycosynthase-like activity when a N175Q substitution was introduced and it caused accumulation of the transglycosylation products. These findings not only give insights into the substrate recognition mechanism of glycoside hydrolase family 85 enzymes but also widen their scope of application in preparing homogeneous glycoforms of core-fucosylated glycoproteins for the production of potent glycoprotein-based therapeutics.

  • complete nmr assignment of a bisecting hybrid type oligosaccharide transferred by Mucor hiemalis endo β n acetylglucosaminidase
    Carbohydrate Research, 2016
    Co-Authors: Takashi Yamanoi, Toshiyuki Inazu, Yoshiki Oda, Kaname Katsuraya, Kenji Yamamoto
    Abstract:

    This study describes the complete nuclear magnetic resonance (NMR) spectral assignment of a bisecting hybrid-type oligosaccharide 1, transferred by Mucor hiemalis endo-β-N-acetylglucosaminidase (Endo-M). Through (1)H- and (13)C-NMR, DQF-COSY, HSQC, HMBC, TOCSY, and NOESY experiments, we determine the structure of the glycoside linkage formed by the Endo-M transglycosylation, i.e., the connection between GlcNAc and GlcNAc in oligosaccharide 1.

  • efficient glycosynthase mutant derived from Mucor hiemalis endo β n acetylglucosaminidase capable of transferring oligosaccharide from both sugar oxazoline and natural n glycan
    Journal of Biological Chemistry, 2010
    Co-Authors: Midori Umekawa, Takayuki Higashiyama, Wei Huang, Hisashi Ashida, Kenji Yamamoto, Laixi Wang
    Abstract:

    Abstract Endo-M, an endo-β-N-acetylglucosaminidase from Mucor hiemalis, is a family 85 glycoside hydrolase. This enzyme is unique in that it can transfer en bloc the oligosaccharide of various types of N-glycans onto different acceptors, and thereby it enzymatically generates diverse glycoconjugates. In this study, we performed mutational and kinetic studies focusing on a key catalytic asparagine 175 of Endo-M. We have shown that most of the Asn-175 mutants had significantly diminished hydrolysis activity but acted as glycosynthases capable of using synthetic sugar oxazoline for transglycosylation. Our results confirm the critical role of this asparagine residue in promoting the formation of an oxazolinium ion intermediate in the first step of the substrate-assisted catalysis. Interestingly, the N175Q mutant was found to possess dramatically enhanced glycosynthase-like activity with sugar oxazoline in comparison with N175A and a transglycosidase-like activity with “natural” N-glycan as well. These results also implicated the significance of amide side chain in the asparagine 175 of Endo-M for promoting oxazoline transglycosylation in the second step of the catalysis. The highly efficient syntheses of glycopeptides/glycoproteins by N175Q combined with synthetic sugar oxazolines or natural N-glycan substrates were exemplified. In addition, we also identified several previously unknown residues that seem to play a role in the catalysis of Endo-M.

  • mutants of Mucor hiemalis endo β n acetylglucosaminidase show enhanced transglycosylation and glycosynthase like activities
    Journal of Biological Chemistry, 2008
    Co-Authors: Midori Umekawa, Wei Huang, Hisashi Ashida, Laixi Wang, Kiyotaka Fujita, Kenji Yamamoto
    Abstract:

    Abstract Endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M), a family 85 glycoside hydrolase, acts on the β1,4 linkage of N,N′-diacetylchitobiose moiety in the N-linked glycans of glycoproteins and catalyzes not only the hydrolysis reaction but also the transglycosylation reaction that transfers the releasing sugar chain to an acceptor other than water to form a new glycosidic linkage. The transglycosylation activity of Endo-M holds a great promise for the chemo-enzymatic synthesis and glyco-engineering of glycoproteins, but the inherent hydrolytic activity for product hydrolysis and low transglycosylation have hampered its broad applications. This paper describes the site-directed mutagenesis on residues in the putative catalytic region of Endo-M to generate mutants with superior transglycosylation activity. Two interesting mutants were discovered. The Y217F mutant was found to possess much enhanced transglycosylation activity and yet much diminished hydrolytic activity in comparison with the wild-type Endo-M. Kinetic analyses revealed that the Km value of Y217F for an acceptor substrate 4-methylumbelliferyl-β-l-N-acetylglucosaminide was only one-tenth of that of the wild-type, implicating a much higher affinity of Y217F for the acceptor substrate than the wild-type. The other mutant, N175A, acts like a glycosynthase. It was found that mutation at Asn175“knocked out” the hydrolytic activity, but the mutant was able to take the highly active sugar oxazolines (the transition state mimics) as donor substrates for transglycosylation. This is the first glycosynthase derived from endo-β-N-acetylglucosaminidases that proceed via a substrate-assisted mechanism. Our findings provide further insights on the substrate-assisted mechanism of GH85. The usefulness of the novel glycosynthase was exemplified by the efficient synthesis of a human immunodeficiency virus, type 1 (HIV-1) glycopeptide with potent anti-HIV activity.

  • synthesis of mono glucose branched cyclodextrins with a high inclusion ability for doxorubicin and their efficient glycosylation using Mucor hiemalis endo β n acetylglucosaminidase
    Bioorganic & Medicinal Chemistry Letters, 2005
    Co-Authors: Takashi Yamanoi, Kenji Yamamoto, Kenji Osumi, Eri Akaike, Kiyotaka Fujita, Yoshiki Oda, Maki Tsutsumida, Naomichi Yoshida, Natsumi Kobayashi
    Abstract:

    The mono-glucose-branched cyclodextrins having an appropriate spacer between the beta-cyclodextrin and a glucose moiety were synthesized from beta-cyclodextrin and arbutin. They had the significantly high association constants for doxorubicin, the anticancer agent, in the range of 10(5)-10(6)M(-1), and worked as highly reactive glycosyl acceptors for the transglycosylation reaction by endo-beta-N-acetylglucosaminidase of Mucor hiemalis to produce sialo-complex type oligosaccharide-branched cyclodextrins in the high yields of 65-67%.

Kiyotaka Fujita - One of the best experts on this subject based on the ideXlab platform.

  • mutants of Mucor hiemalis endo β n acetylglucosaminidase show enhanced transglycosylation and glycosynthase like activities
    Journal of Biological Chemistry, 2008
    Co-Authors: Midori Umekawa, Wei Huang, Hisashi Ashida, Laixi Wang, Kiyotaka Fujita, Kenji Yamamoto
    Abstract:

    Abstract Endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M), a family 85 glycoside hydrolase, acts on the β1,4 linkage of N,N′-diacetylchitobiose moiety in the N-linked glycans of glycoproteins and catalyzes not only the hydrolysis reaction but also the transglycosylation reaction that transfers the releasing sugar chain to an acceptor other than water to form a new glycosidic linkage. The transglycosylation activity of Endo-M holds a great promise for the chemo-enzymatic synthesis and glyco-engineering of glycoproteins, but the inherent hydrolytic activity for product hydrolysis and low transglycosylation have hampered its broad applications. This paper describes the site-directed mutagenesis on residues in the putative catalytic region of Endo-M to generate mutants with superior transglycosylation activity. Two interesting mutants were discovered. The Y217F mutant was found to possess much enhanced transglycosylation activity and yet much diminished hydrolytic activity in comparison with the wild-type Endo-M. Kinetic analyses revealed that the Km value of Y217F for an acceptor substrate 4-methylumbelliferyl-β-l-N-acetylglucosaminide was only one-tenth of that of the wild-type, implicating a much higher affinity of Y217F for the acceptor substrate than the wild-type. The other mutant, N175A, acts like a glycosynthase. It was found that mutation at Asn175“knocked out” the hydrolytic activity, but the mutant was able to take the highly active sugar oxazolines (the transition state mimics) as donor substrates for transglycosylation. This is the first glycosynthase derived from endo-β-N-acetylglucosaminidases that proceed via a substrate-assisted mechanism. Our findings provide further insights on the substrate-assisted mechanism of GH85. The usefulness of the novel glycosynthase was exemplified by the efficient synthesis of a human immunodeficiency virus, type 1 (HIV-1) glycopeptide with potent anti-HIV activity.

  • synthesis of mono glucose branched cyclodextrins with a high inclusion ability for doxorubicin and their efficient glycosylation using Mucor hiemalis endo β n acetylglucosaminidase
    Bioorganic & Medicinal Chemistry Letters, 2005
    Co-Authors: Takashi Yamanoi, Kenji Yamamoto, Kenji Osumi, Eri Akaike, Kiyotaka Fujita, Yoshiki Oda, Maki Tsutsumida, Naomichi Yoshida, Natsumi Kobayashi
    Abstract:

    The mono-glucose-branched cyclodextrins having an appropriate spacer between the beta-cyclodextrin and a glucose moiety were synthesized from beta-cyclodextrin and arbutin. They had the significantly high association constants for doxorubicin, the anticancer agent, in the range of 10(5)-10(6)M(-1), and worked as highly reactive glycosyl acceptors for the transglycosylation reaction by endo-beta-N-acetylglucosaminidase of Mucor hiemalis to produce sialo-complex type oligosaccharide-branched cyclodextrins in the high yields of 65-67%.

  • molecular cloning of Mucor hiemalis endo β n acetylglucosaminidase and some properties of the recombinant enzyme
    Archives of Biochemistry and Biophysics, 2004
    Co-Authors: Kiyotaka Fujita, Hidehiko Kumagai, Kazuo Kobayashi, Akihiro Iwamatsu, Makoto Takeuchi, Kenji Yamamoto
    Abstract:

    Endo-M, endo-beta-N-acetylglucosaminidase from Mucor hiemalis, is known as a useful enzyme for the synthesis of neoglycopeptides due to its transglycosylation activity. We cloned the Endo-M gene encoding a putative 744 amino acids, which shows high identity to glycoside hydrolase family 85 endo-beta-N-acetylglucosaminidases. The gene encoding Endo-M was expressed in protease-deficient Candida boidinii with a molecular mass of 85 kDa as a monomeric form. Recombinant Endo-M could liberate both high-mannose type and biantennary complex type oligosaccharides from glycopeptides, which was same as the native enzyme. The Km and Kcat values for DNS-Man6GlcNAc2Asn were 0.51 mM and 8.25 s(-1), respectively. Recombinant Endo-M also exhibited transglycosylation activity toward high-mannose type and biantennary complex type oligosaccharides, which were transferred to alcohols, monosaccharides, oligosaccharides, and glycosides. To investigate about the catalytically essential amino acids of Endo-M, site-directed mutagenesis was performed, and it was found that mutants E177G and E177Q completely abolished the hydrolytic activity and W228R partially abolished the transglycosylation activity.

  • Mucor hiemalis endo β n acetylglucosaminidase can transglycosylate a bisecting hybrid type oligosaccharide from an ovalbumin glycopeptide
    Carbohydrate Research, 2004
    Co-Authors: Kenji Osumi, Kenji Yamamoto, Yoshitaka Makino, Eri Akaike, Takashi Yamanoi, Mamoru Mizuno, Midori Noguchi, Toshiyuki Inazu, Kiyotaka Fujita
    Abstract:

    Abstract We found that the recombinant endo -β- N -acetylglucosaminidase of Mucor hiemalis (Endo-M) expressed in Candida boidinii had the transglycosylation activity of transferring a bisecting hybrid-type oligosaccharide from an ovalbumin glycopeptide to the acceptor ( p -nitrophenyl 2-acetamido-2-deoxy-β- d -glucopyranoside) in a good yield of 43%.

  • transglycosylation reaction of Mucor hiemalis endo β n acetylglucosaminidase using sugar derivatives modified at c 1 or c 2 as oligosaccharide acceptors
    Carbohydrate Research, 2004
    Co-Authors: Takashi Yamanoi, Kenji Yamamoto, Kenji Osumi, Eri Akaike, Yoshiki Oda, Maki Tsutsumida, Kiyotaka Fujita
    Abstract:

    Abstract We investigated the transglycosylation reaction of the recombinant endo-β- N -acetylglucosaminidase from Mucor hiemalis (Endo-M) expressed in Candida boidinii using such sugar derivatives as N-acylated d -glucosamines, C -glucosyl derivatives, and a 2-O-glycosylated disaccharide as acceptors. We found that a variety of sugar derivatives modified at C-1 or C-2 could be used as acceptors for transglycosylation by Endo-M to create novel oligosaccharides.

Johannes Fritscher - One of the best experts on this subject based on the ideXlab platform.

  • Multimetal bioremediation and biomining by a combination of new aquatic strains of Mucor hiemalis
    Scientific Reports, 2019
    Co-Authors: Enamul Hoque, Johannes Fritscher
    Abstract:

    Here we describe a unique microbial biotechnology for simultaneous bioremediation and biomining of twelve ionic metals overcoming the obstacles of multimetal toxicity to microbes. After a thorough search of key microorganisms in microbiomes of many sulfidic springs in Bavaria (Germany) over an area of 200 km^2, we found three new strains EH8, EH10 and EH11 of Mucor hiemalis physiologically compatible and capable of multimetal-remediation and enrichment. We combined the multimetal-resistance, hyper-accumulation and elicitation power of EH8, EH10 and EH11 to develop a novel biotechnology for simultaneous removal, fractionation and enrichment of metal ions. As a first step we showed the intracellular fixing and deposition of mercury as nanospheres in EH8’s sporangiospores. Scanning Electron Microscopy-Energy-Dispersive X-Ray analysis revealed binding and precipitation of other applied metal ions as spherical nano-particles (~50–100 nm) at the outer electro-negative cellwall-surface of EH8, EH10 and EH11 sporangiospores. Microbiomes, germinated spores and dead insoluble cellwalls of these strains removed >81–99% of applied Al, Cd, Co, Cr, Cu, Hg, Ni, Pb, U, and Zn simultaneously and furthermore enriched precious Ag, Au and Ti from water all within 48 h, demonstrating the potential of new biotechnologies for safe-guarding our environment from metal pollution and concentrating precious diluted, ionic metals.

  • ecology adaptation and function of methane sulfidic spring water biofilm microorganisms including a strain of anaerobic fungus Mucor hiemalis
    MicrobiologyOpen, 2017
    Co-Authors: Enamul Hoque, Johannes Fritscher
    Abstract:

    Ecological aspects, adaptation, and some functions of a special biofilm and its unique key anaerobic fungus Mucor hiemalis strain EH11 isolated from a pristine spring (Kunzing, Bavaria, Germany) are described. The spring's pure nature is characterized by, for example, bubbling methane, marine-salinity, mild hydrothermal (~19.1°C), sulfidic, and reductive-anoxic (Eh: −241 to −253 mV, O2: ≤ 0.1 mg/L) conditions. It is geoecologically located at the border zone between Bavarian Forest (crystalline rocky mountains) and the moor-like Danube River valley, where geological displacements bring the spring's water from the deeper layers of former marine sources up to the surface. In the spring's outflow, a special biofilm with selective microorganisms consisting of archaea, bacteria, protozoa (ciliate), and fungus was found. Typical sulfidic-spring bryophyta and macrozoobenthos were missing, but many halo- and anaerotolerant diatoms and ciliate Vorticella microstoma beside EH11 were identified. Phase contrast and scanning electron microscopy revealed the existence of a stabilizing matrix in the biofilm formed by the sessile fungal hyphae and the exopolysaccharide substance (EPS) structures, which harbors other microorganisms. In response to ecological adaptation pressure caused by methane bubbles, EH11 developed an atypical spring-like hyphal morphology, similar to the spiral stalk of ciliate V. microstoma, to rise up with methane bubbles. For the first time, it was also demonstrated that under strict anaerobic conditions EH11 changes its asexual reproduction process by forming pseudosporangia via hyphal cell divisions as well as switching its metabolism to chemoautotrophic bacteria-like anaerobic life using acetate as an e-donor and ferrihydrite as an e-acceptor, all without fermentation. EH11 can be suggested to be useful for the microbial community in the Kunzing biofilm not only due to its physical stabilization of the biofilm's matrix but also due to its ecological functions in element recycling as well as a remover of toxic metals.

  • induction of glutathione s transferase in biofilms and germinating spores of Mucor hiemalis strain eh5 from cold sulfidic spring waters
    Applied and Environmental Microbiology, 2007
    Co-Authors: Enamul Hoque, Johannes Fritscher, Stephan Pflugmacher, Manfred Wolf
    Abstract:

    The occurrence and activation of glutathione S-transferase (GST) and the GST activities in biofilms in cold sulfidic spring waters were compared to the occurrence and activation of GST and the GST activities of the aquatic fungal strains EH5 and EH7 of Mucor hiemalis isolated for the first time from such waters. Using fluorescently labeled polyclonal anti-GST antibodies and GST activity measurements, we demonstrated that a high level of GST occurred in situ in natural biofilms and pure cultures of strain EH5. Measurement of microsomal and cytosolic soluble GST activities using different xenobiotic substrates, including 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy)propane, 1-iodo-2,4-dinitrobenzene, and fluorodifen, showed that the overall biotransforming abilities of biofilms were at least sixfold greater than that of strain EH5 alone. Increasing the level of sodium thiosulfate (STS) in the medium stimulated the microsomal and cytosolic GST activities with CDNB of strain EH5 about 44- and 94-fold, respectively, compared to the activities in the control. The induction of microsomal GST activity with fluorodifen by STS was strongly linear, but the initial strong linear increase in cytosolic GST activity with fluorodifen showed saturation-like effects at STS concentrations higher than approximately 1 mM. Using laser scanning confocal and conventional fluorescence microscopy, abundant fluorescently labeled GST proteins were identified in germinating sporangiospores of strain EH5 after activation by STS. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of at least two main GSTs (∼27.8- and ∼25.6-kDa subunits) in the cytosol of EH5, whereas the major 27.8-kDa subunit was the only GST in microsomes. We suggest that differential cellular GST expression takes place in strain EH5 depending on spore and hyphal development. Our results may contribute to our understanding of induction of GST by sulfurous compounds, as well as to the immunofluorescence visualization of GST in aquatic fungus and fungus-bacterium biofilms.

Enamul Hoque - One of the best experts on this subject based on the ideXlab platform.

  • Multimetal bioremediation and biomining by a combination of new aquatic strains of Mucor hiemalis
    Scientific Reports, 2019
    Co-Authors: Enamul Hoque, Johannes Fritscher
    Abstract:

    Here we describe a unique microbial biotechnology for simultaneous bioremediation and biomining of twelve ionic metals overcoming the obstacles of multimetal toxicity to microbes. After a thorough search of key microorganisms in microbiomes of many sulfidic springs in Bavaria (Germany) over an area of 200 km^2, we found three new strains EH8, EH10 and EH11 of Mucor hiemalis physiologically compatible and capable of multimetal-remediation and enrichment. We combined the multimetal-resistance, hyper-accumulation and elicitation power of EH8, EH10 and EH11 to develop a novel biotechnology for simultaneous removal, fractionation and enrichment of metal ions. As a first step we showed the intracellular fixing and deposition of mercury as nanospheres in EH8’s sporangiospores. Scanning Electron Microscopy-Energy-Dispersive X-Ray analysis revealed binding and precipitation of other applied metal ions as spherical nano-particles (~50–100 nm) at the outer electro-negative cellwall-surface of EH8, EH10 and EH11 sporangiospores. Microbiomes, germinated spores and dead insoluble cellwalls of these strains removed >81–99% of applied Al, Cd, Co, Cr, Cu, Hg, Ni, Pb, U, and Zn simultaneously and furthermore enriched precious Ag, Au and Ti from water all within 48 h, demonstrating the potential of new biotechnologies for safe-guarding our environment from metal pollution and concentrating precious diluted, ionic metals.

  • ecology adaptation and function of methane sulfidic spring water biofilm microorganisms including a strain of anaerobic fungus Mucor hiemalis
    MicrobiologyOpen, 2017
    Co-Authors: Enamul Hoque, Johannes Fritscher
    Abstract:

    Ecological aspects, adaptation, and some functions of a special biofilm and its unique key anaerobic fungus Mucor hiemalis strain EH11 isolated from a pristine spring (Kunzing, Bavaria, Germany) are described. The spring's pure nature is characterized by, for example, bubbling methane, marine-salinity, mild hydrothermal (~19.1°C), sulfidic, and reductive-anoxic (Eh: −241 to −253 mV, O2: ≤ 0.1 mg/L) conditions. It is geoecologically located at the border zone between Bavarian Forest (crystalline rocky mountains) and the moor-like Danube River valley, where geological displacements bring the spring's water from the deeper layers of former marine sources up to the surface. In the spring's outflow, a special biofilm with selective microorganisms consisting of archaea, bacteria, protozoa (ciliate), and fungus was found. Typical sulfidic-spring bryophyta and macrozoobenthos were missing, but many halo- and anaerotolerant diatoms and ciliate Vorticella microstoma beside EH11 were identified. Phase contrast and scanning electron microscopy revealed the existence of a stabilizing matrix in the biofilm formed by the sessile fungal hyphae and the exopolysaccharide substance (EPS) structures, which harbors other microorganisms. In response to ecological adaptation pressure caused by methane bubbles, EH11 developed an atypical spring-like hyphal morphology, similar to the spiral stalk of ciliate V. microstoma, to rise up with methane bubbles. For the first time, it was also demonstrated that under strict anaerobic conditions EH11 changes its asexual reproduction process by forming pseudosporangia via hyphal cell divisions as well as switching its metabolism to chemoautotrophic bacteria-like anaerobic life using acetate as an e-donor and ferrihydrite as an e-acceptor, all without fermentation. EH11 can be suggested to be useful for the microbial community in the Kunzing biofilm not only due to its physical stabilization of the biofilm's matrix but also due to its ecological functions in element recycling as well as a remover of toxic metals.

  • responses of the antioxidative and biotransformation enzymes in the aquatic fungus Mucor hiemalis exposed to cyanotoxins
    Biotechnology Letters, 2017
    Co-Authors: Evelyn Balsano, Enamul Hoque, Maranda Esterhuizenlondt, Stephan Pflugmacher Lima
    Abstract:

    To investigate antioxidative and biotransformation enzyme responses in Mucor hiemalis towards cyanotoxins considering its use in mycoremediation applications. Catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) in M. hiemalis maintained their activities at all tested microcystin-LR (MC-LR) exposure concentrations. Cytosolic glutathione S-transferase (GST) activity decreased with exposure to 100 µg MC-LR l−1 while microsomal GST remained constant. Cylindrospermopsin (CYN) at 100 µg l−1 led to an increase in CAT activity and inhibition of GR, as well as to a concentration-dependent GPx inhibition. Microsomal GST was inhibited at all concentrations tested. β-N-methylamino-l-alanine (BMAA) inhibited GR activity in a concentration-dependent manner, however, CAT, GPx, and GST remained unaffected. M. hiemalis showed enhanced oxidative stress tolerance and intact biotransformation enzyme activity towards MC-LR and BMAA in comparison to CYN, confirming its applicability in bioreactor technology in terms of viability and survival in their presence.

  • fungal pellets as potential tools to control water pollution strategic approach for the pelletization and subsequent microcystin lr uptake by Mucor hiemalis
    Journal of Applied Biology and Biotechnology, 2016
    Co-Authors: Evelyn Balsano, Enamul Hoque, Maranda Esterhuizenlondt, Stephan Pflugmacher
    Abstract:

    Microcystin-LR is one of the most prevalent and toxic secondary metabolites produced by cyanobacteria worldwide, causing global concerns because of its hazardousness to ecosystems and human health. Green Liver Systems® have been developed to purify contaminated water, however, system capacities need to be extended to allow season- and location independent applications. Therefore, mycoremediation using temperature resistant Mucor hiemalis in pellet morphology was considered. In submerged liquid cultures, fungal morphology is strain specific and strongly depends on the cultivation environment. One main focus of this work was the investigation of diverse factors influencing pelletization. Moreover, we translated the pellet product into an immediate application and studied its biosorption ability towards microcystin-LR. Our results showed that, pH was a key factor stimulating pellet formation of M. hiemalis and that inoculum concentration played an essential role as well. Final pellet size was limited by the available space in the flask. Microcystin-LR was extracted from exposed pellets and quantified via LC-MS/MS measurements. Our results report for the first time optimized pelletization of M. hiemalis and cyanotoxin uptake by these fungal pellets in liquid cultures.

  • induction of glutathione s transferase in biofilms and germinating spores of Mucor hiemalis strain eh5 from cold sulfidic spring waters
    Applied and Environmental Microbiology, 2007
    Co-Authors: Enamul Hoque, Johannes Fritscher, Stephan Pflugmacher, Manfred Wolf
    Abstract:

    The occurrence and activation of glutathione S-transferase (GST) and the GST activities in biofilms in cold sulfidic spring waters were compared to the occurrence and activation of GST and the GST activities of the aquatic fungal strains EH5 and EH7 of Mucor hiemalis isolated for the first time from such waters. Using fluorescently labeled polyclonal anti-GST antibodies and GST activity measurements, we demonstrated that a high level of GST occurred in situ in natural biofilms and pure cultures of strain EH5. Measurement of microsomal and cytosolic soluble GST activities using different xenobiotic substrates, including 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy)propane, 1-iodo-2,4-dinitrobenzene, and fluorodifen, showed that the overall biotransforming abilities of biofilms were at least sixfold greater than that of strain EH5 alone. Increasing the level of sodium thiosulfate (STS) in the medium stimulated the microsomal and cytosolic GST activities with CDNB of strain EH5 about 44- and 94-fold, respectively, compared to the activities in the control. The induction of microsomal GST activity with fluorodifen by STS was strongly linear, but the initial strong linear increase in cytosolic GST activity with fluorodifen showed saturation-like effects at STS concentrations higher than approximately 1 mM. Using laser scanning confocal and conventional fluorescence microscopy, abundant fluorescently labeled GST proteins were identified in germinating sporangiospores of strain EH5 after activation by STS. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of at least two main GSTs (∼27.8- and ∼25.6-kDa subunits) in the cytosol of EH5, whereas the major 27.8-kDa subunit was the only GST in microsomes. We suggest that differential cellular GST expression takes place in strain EH5 depending on spore and hyphal development. Our results may contribute to our understanding of induction of GST by sulfurous compounds, as well as to the immunofluorescence visualization of GST in aquatic fungus and fungus-bacterium biofilms.

Stephan Pflugmacher - One of the best experts on this subject based on the ideXlab platform.

  • uptake and biotransformation of pure commercial microcystin lr versus microcystin lr from a natural cyanobacterial bloom extract in the aquatic fungus Mucor hiemalis
    Biotechnology Letters, 2017
    Co-Authors: Maranda Esterhuizenlondt, Stephan Pflugmacher, Stefanie Hertel
    Abstract:

    To evaluate the remediation efficiency of Mucor hiemalis by comparing media elimination, uptake, and biotransformation of microcystin-LR with exposure to pure toxin versus a crude bloom extract. With exposure to the extract, the elimination rate of microcystin-LR from the media, which was 0.28 ng MC-LR l−1 h−1, was significantly higher compared to that achieved with exposure to the pure toxin (0.16 ng MC-LR l−1 h−1) after 24 h. However, intracellular breakdown of microcystin-LR was significantly lower in the extract exposed pellets compared to the pure toxin treated fungal pellets over time. This coincided with reduced intracellular glutathione S-transferase activity with crude extract exposure which could be responsible for the detection of only the glutathione conjugate of microcystin-LR. This paper signifies the importance of using laboratory exposure scenarios which resemble conditions in nature to fully understand and evaluate remediation efficiency. There is merit in using M. hiemalis for mycoremediation of cyanotoxins in surface waters.

  • mycoremediation of diclofenac using Mucor hiemalis
    Toxicological & Environmental Chemistry, 2017
    Co-Authors: Maranda Esterhuizenlondt, Annalena Hendel, Stephan Pflugmacher
    Abstract:

    ABSTRACTDiclofenac (DCF) is a pharmaceutical drug widely found in the aquatic environment, where it represents a persistent, anthropogenic hazard to all biota. Owing to the reported inefficiency of water treatment strategies to remove pharmaceuticals, the present study aimed to investigate the ability of the aquatic fungus Mucor hiemalis to take up and accumulate DCF. Cultures of M. hiemalis were exposed to varying concentrations of DCF (10, 25 and 50 µg/L) over a period of 144 h. In the presence of M. hiemalis, DCF concentrations in the media decreased by 95% within 24 h. This early removal was most likely due to extracellular metabolism of DCF, as low proportions of the pharmaceutical were found in the mycelium of the fungus, thereby excluding bioaccumulation as the main mode of removal. However, adsorption of DCF onto the surface of the M. hiemalis pellets cannot be excluded. Our study provides the first report of DCF remediation using M. hiemalis and is the first successful step towards a feasible and...

  • using aquatic fungi for pharmaceutical bioremediation uptake of acetaminophen by Mucor hiemalis does not result in an enzymatic oxidative stress response
    Fungal Biology, 2016
    Co-Authors: Maranda Esterhuizenlondt, Katrin Schwartz, Stephan Pflugmacher
    Abstract:

    Abstract The increasing anthropogenic pollution of aquatic environments and fresh water scarcity worldwide have prompted the development of low-cost and effective water treatment alternatives. One example of a highly released anthropogenic xenobiotics is acetaminophen (APAP), which has been detected in surface waters at concentrations as high as 5 μg L −1 . To date, traditional water treatment plants were unable to remove all pharmaceutical xenobiotics and as in the case with APAP, the breakdown products are toxic. Phytoremediation has proved to remove xenobiotics efficiently producing no toxic breakdown products, however, they are often restrained in their application range. Therefore, it was necessary to find alternate remediation tools to extend and complement the application ranges of existing bioremediation techniques. With the success of mycoremediation as well as the adaptability of fungi, Mucor hiemalis was investigated in terms of its APAP uptake capabilities. The investigation included the examination of concentration- and time-dependent uptake studies to examine the effects of each of these parameters independently. Additionally, the extracellular peroxidase activity of M. hiemalis was measured with exposure to APAP to evaluate possible breakdown and the antioxidative stress enzymes, catalase, glutathione peroxidase, and glutathione reductase, were assayed to investigate whether APAP caused oxidative stress. The results showed that M. hiemalis was able to internalize between 1 and 2 μg APAP per g dried fungal biomass when exposed to 5, 10, 50 and 100 ng mL −1 APAP for 24–48 h, but not beyond this time frame. Further, exposure to APAP did not result in elevated extracellular peroxidase activity or oxidative stress. The findings led to the conclusion that M. hiemalis could be integrated in bioremediation systems, for short-term degradation at low concentrations of APAP with effective management.

  • fungal pellets as potential tools to control water pollution strategic approach for the pelletization and subsequent microcystin lr uptake by Mucor hiemalis
    Journal of Applied Biology and Biotechnology, 2016
    Co-Authors: Evelyn Balsano, Enamul Hoque, Maranda Esterhuizenlondt, Stephan Pflugmacher
    Abstract:

    Microcystin-LR is one of the most prevalent and toxic secondary metabolites produced by cyanobacteria worldwide, causing global concerns because of its hazardousness to ecosystems and human health. Green Liver Systems® have been developed to purify contaminated water, however, system capacities need to be extended to allow season- and location independent applications. Therefore, mycoremediation using temperature resistant Mucor hiemalis in pellet morphology was considered. In submerged liquid cultures, fungal morphology is strain specific and strongly depends on the cultivation environment. One main focus of this work was the investigation of diverse factors influencing pelletization. Moreover, we translated the pellet product into an immediate application and studied its biosorption ability towards microcystin-LR. Our results showed that, pH was a key factor stimulating pellet formation of M. hiemalis and that inoculum concentration played an essential role as well. Final pellet size was limited by the available space in the flask. Microcystin-LR was extracted from exposed pellets and quantified via LC-MS/MS measurements. Our results report for the first time optimized pelletization of M. hiemalis and cyanotoxin uptake by these fungal pellets in liquid cultures.

  • lc ms ms method development for quantitative analysis of acetaminophen uptake by the aquatic fungus Mucor hiemalis
    Ecotoxicology and Environmental Safety, 2016
    Co-Authors: Maranda Esterhuizenlondt, Katrin Schwartz, Evelyn Balsano, Sandra C Kuhn, Stephan Pflugmacher
    Abstract:

    Acetaminophen is a pharmaceutical, frequently found in surface water as a contaminant. Bioremediation, in particular, mycoremediation of acetaminophen is a method to remove this compound from waters. Owing to the lack of quantitative analytical method for acetaminophen in aquatic organisms, the present study aimed to develop a method for the determination of acetaminophen using LC-MS/MS in the aquatic fungus Mucor hiemalis. The method was then applied to evaluate the uptake of acetaminophen by M. hiemalis, cultured in pellet morphology. The method was robust, sensitive and reproducible with a lower limit of quantification of 5 pg acetaminophen on column. It was found that M. hiemalis internalize the pharmaceutical, and bioaccumulate it with time. Therefore, M. hiemalis was deemed a suitable candidate for further studies to elucidate its pharmaceutical tolerance and the longevity in mycoremediation applications.