β-N-acetylglucosaminidases from the family 84 of glycoside hydrolases form a small group of glycosidases in eukaryotes responsible for the modification of nuclear and cytosolic proteins with O-GlcNAc, thus they are involved in a number of important cell processes. Here, the first fungal β-N-acetylglucosaminidase from Penicillium chrysogenum was expressed in Pichia pastoris and secreted into the media, purified and characterized. Moreover, homology modeling and substrate and inhibitor docking were performed to obtain structural information on this new member of the GH84 family. Surprisingly, we found that this fungal β-N-acetylglucosaminidase with its sequence and structure perfectly fitting to the GH84 family displays biochemical properties rather resembling the β-N-acetylhexosaminidases from the family 20 of glycoside hydrolases. This work helped to increase the knowledge on the scarcely studied glycosidase family and revealed a new type of eukaryotic β-N-acetylglucosaminidase.
- MeSH
- acetylglukosaminidasa chemie genetika izolace a purifikace metabolismus MeSH
- molekulární sekvence - údaje MeSH
- Penicillium chrysogenum enzymologie genetika MeSH
- Pichia genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika izolace a purifikace metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- simulace molekulového dockingu MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A strictly anaerobic mesophilic chitinolytic bacterial strain identified as Clostridium paraputrificum J4 was isolated from human feces. In response to various types of growth substrates, the bacterium produced an array of chitinolytic enzymes representing significant components of the J4 strain secretome. The excreted active proteins were characterized by estimating the enzymatic activities of endochitinase, exochitinase, and N-acetylglucosaminidase induced by cultivation in medium M-10 with colloidal chitin. The enzyme activities produced by J4 strain cultivated in medium M-10 with glucose were significantly lower. The spectrum of extracellularly excreted proteins was separated by SDS-PAGE. The chitinase variability was confirmed on zymograms of renatured SDS-PAGE. The enzymes were visualized under ultraviolet light by using 4-methylumbelliferyl derivatives of N-acetyl-β-D: -glucosaminide, N,N´-diacetyl-β-D: -chitobiose, or N,N´,N˝-triacetyl-β-D: -chitotriose for β-N-acetylglucosaminidase, chitobiosidase, or endochitinase activities, respectively. Protein components of the secretome were separated by 2D-PAGE analysis. The distinct protein bands were excised, isolated, and subsequently characterized by using MALDI-TOF/TOF tandem mass spectrometry. The final identification was performed according to sequence homology by database searching.
- MeSH
- 2D gelová elektroforéza MeSH
- acetylglukosaminidasa chemie genetika metabolismus MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- chitinasy chemie genetika metabolismus MeSH
- Clostridium chemie enzymologie genetika izolace a purifikace MeSH
- extracelulární prostor chemie enzymologie genetika MeSH
- feces mikrobiologie MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- transport proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Membrane diafiltration was used for separation of the extracellular complex of chitinolytic enzymes of C. paraputrificum J4 free from contaminants with molar mass higher than 100 kDa and lower than 30 kDa. The enzyme complex containing beta-N-acetylglucosaminidase (NAGase) and six endochitinases was concentrated on a membrane with cut-off 30 kDa. In this retentate, the NAGase/endochitinase specific activity was 13.5/6.5-times higher than in the initial culture filtrate. The proportion (in%) of endochitinases: 23 (90 kDa), 42 (86 kDa), 8 (72 kDa), 16 (68 kDa) and 8 (60 kDa) was calculated from their peak areas (determined by densitometry) in images of zymograms. NAGase (38 kDa) was less active and stable at pH lower than 4 and higher than 8 but it was more temperature-stable than endochitinases, especially at 40-60 degrees C. In contrast to endochitinases, the pH optimum of NAGase activity was shifted by ca. 0.7 pH units to the alkaline region. Extracellular NAGase together with six endochitinases secreted by C. paraputrificum J4 were separated by membrane diafiltration and characterized by molar mass, stability and activity in dependence on pH and temperature. The knowledge of composition of chitinolytic enzymes, their pH and temperature stability is useful for optimization of the separation process.
- MeSH
- acetylglukosaminidasa chemie izolace a purifikace metabolismus MeSH
- bakteriální proteiny chemie izolace a purifikace metabolismus MeSH
- chitin metabolismus MeSH
- chitinasy chemie izolace a purifikace metabolismus MeSH
- Clostridium enzymologie MeSH
- financování organizované MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- molekulová hmotnost MeSH
- stabilita enzymů MeSH
- teplota MeSH
- ultrafiltrace metody MeSH
- Check Tag
- lidé MeSH
The common feature of all chytridiomycetous fungi, aerobic as well as anaerobic, is an abundance of chitin in their cell wall. The genes coding for chitinases have therefore been widely used as phylogenetic markers in ascomycetes. As their utility for Chytridiomycetes has not been determined we chose the gene encoding an enzyme involved in chitin degradation and energy metabolism, the beta-(1,4)-N-acetylglucosaminidase (nag1). Primer pair Nag-forward and Nag-reverse was used to create PCR product from 5 strains of anaerobic and 7 strains of aerobic chytrids. However, Blast search of sequenced amplicons showed that these primers are specific only for fungus Emericella nidulans. Amino acid alignment of Nag1 proteins of fungal, protozoal and bacterial origin available in GenBank database was therefore performed. Five amino acid regions were found to be conserved enough to serve as a suitable domain for the design of a set of primers for the universal amplification of the nag1 gene in the Neocallimastigales fungi.
- MeSH
- acetylglukosaminidasa genetika chemie MeSH
- aerobióza MeSH
- anaerobióza MeSH
- Chytridiomycota enzymologie genetika MeSH
- DNA fungální analýza izolace a purifikace MeSH
- DNA primery genetika MeSH
- fungální proteiny genetika MeSH
- houby enzymologie genetika klasifikace růst a vývoj MeSH
- polymerázová řetězová reakce MeSH
- techniky amplifikace nukleových kyselin metody MeSH
- MeSH
- acetylglukosaminidasa chemie izolace a purifikace MeSH
- chitin chemie MeSH
- chitinasy chemie izolace a purifikace MeSH
- chromatografie iontoměničová metody MeSH
- Clostridium enzymologie izolace a purifikace MeSH
- feces mikrobiologie MeSH
- finanční podpora výzkumu jako téma MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
