Commensal bacterium Clostridium paraputrificum J4 produces several extracellular chitinolytic enzymes including a 62 kDa chitinase Chit62J4 active toward 4-nitrophenyl N,N'-diacetyl-β-d-chitobioside (pNGG). We characterized the crude enzyme from bacterial culture fluid, recombinant enzyme rChit62J4, and its catalytic domain rChit62J4cat. This major chitinase, securing nutrition of the bacterium in the human intestinal tract when supplied with chitin, has a pH optimum of 5.5 and processes pNGG with Km = 0.24 mM and kcat = 30.0 s-1. Sequence comparison of the amino acid sequence of Chit62J4, determined during bacterial genome sequencing, characterizes the enzyme as a family 18 glycosyl hydrolase with a four-domain structure. The catalytic domain has the typical TIM barrel structure and the accessory domains-2x Fn3/Big3 and a carbohydrate binding module-that likely supports enzyme activity on chitin fibers. The catalytic domain is highly homologous to a single-domain chitinase of Bacillus cereus NCTU2. However, the catalytic profiles significantly differ between the two enzymes despite almost identical catalytic sites. The shift of pI and pH optimum of the commensal enzyme toward acidic values compared to the soil bacterium is the likely environmental adaptation that provides C. paraputrificum J4 a competitive advantage over other commensal bacteria.

• Klíčová slova
• adaptation to the environment, chitinase, exochitinase, glycosyl hydrolase family 18, human commensal bacterium,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• chitin metabolismus   MeSH
• chitinasy chemie   genetika   metabolismus   MeSH
• Clostridium růst a vývoj   izolace a purifikace   metabolismus   MeSH
• katalytická doména MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• střevní mikroflóra MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• chitin MeSH
• chitinasy MeSH
• rekombinantní proteiny 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
• Názvy látek
• acetylglukosaminidasa MeSH
• bakteriální proteiny MeSH
• chitinasy 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
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• molekulová hmotnost MeSH
• stabilita enzymů MeSH
• teplota MeSH
• ultrafiltrace metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetylglukosaminidasa MeSH
• bakteriální proteiny MeSH
• chitin MeSH
• chitinasy MeSH

The novel chitinolytic bacterium Clostridium beijerinckii strain JM2 was isolated from the stool of healthy volunteers supplied daily per orally with 3 g of chitosan. The bacterium grown on colloidal chitin produced a complete array of chitinolytic enzymes. Significant activities of endochitinase, exochitinase and chitosanase were excreted into the medium (301, 282 and 268 nkat/microg protein, respectively). The high cellular activity of N-acetyl-beta-glucosaminidase (NAGase) and chitosanase were detected (732.4 and 154 nkat/microg protein, respectively). NAGase activity represented the main activity associated with the cellular fraction. The activities of both enzymes tested increased from 20 to 50 degrees C; the optimum reaction temperature estimated being 50 degrees C. Endochitinase as well as NAGase showed an activity in the pH interval of 4.0-8.0; the optimum pH values were 6.5 and 6.0, respectively. The extracellular endochitinase complex consisted of six isoenzymes with molar mass of 32-76 kDa; in the cellular fraction five bands with molar mass of 45-86 kDa were detected. Exochitinase activity was demonstrated in the form of three bands (with molar mass of 30-57 kDa), NAGase activity displayed one band of 45 kDa.

• MeSH
• acetylglukosaminidasa metabolismus   MeSH
• chitin metabolismus   MeSH
• chitinasy metabolismus   MeSH
• chitosan aplikace a dávkování   MeSH
• Clostridium klasifikace   enzymologie   růst a vývoj   izolace a purifikace   MeSH
• feces mikrobiologie   MeSH
• glykosidhydrolasy metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• stabilita enzymů MeSH
• teplota MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetylglukosaminidasa MeSH
• chitin MeSH
• chitinasy MeSH
• chitosan MeSH
• chitosanase MeSH Prohlížeč
• glykosidhydrolasy MeSH

Growth of 6 bacterial strains representing dominant members of the human colonic microflora was measured in the presence of 0.025, 0.05 and 0.5 % chitosan (from shrimp shells, with a 97 % final degree of deacetylation). The effect of chitosan was variable and dependent on bacterial species. The most susceptible to chitosan were bacteria belonging to genera Bacteroides and Clostridium (91-97% growth inhibition). On the other hand, Roseburia sp., Eubacterium sp. and Faecalibacterium sp. were more resistant (63-83 % inhibition of growth). Chitosan can thus be considered as one of the means for influencing the bacterial population in the human colon.

• MeSH
• anaerobní bakterie účinky léků   růst a vývoj   izolace a purifikace   MeSH
• antibakteriální látky farmakologie   MeSH
• chitosan metabolismus   farmakologie   MeSH
• kolon mikrobiologie   MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• chitosan MeSH

The main representatives of bacteria in the human colon were investigated by specific PCR and denaturing gradient gel electrophoresis (DGGE). Prevalent in both cases were species of Bifidobacterium, Clostridium, Bacteroides, Faecalibacterium and Eubacterium. Simultaneously, cellulolytic bacteria were isolated from the human feces. The largest proportion was represented by ruminococcus-like isolates. Their presence was confirmed both by PCR and DGGE methods; the latter one was able to give more comprehensive data about the composition of bacterial population in the human colon chyme.

• MeSH
• Bacteria klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• Bacteroides klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• Bifidobacterium klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• celulasa biosyntéza   MeSH
• celulosa metabolismus   MeSH
• Clostridium klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• DNA bakterií analýza   izolace a purifikace   MeSH
• DNA fingerprinting MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• Eubacterium klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• geny rRNA genetika   MeSH
• kolon mikrobiologie   MeSH
• lidé MeSH
• polymerázová řetězová reakce MeSH
• ribozomální DNA analýza   izolace a purifikace   MeSH
• RNA ribozomální 16S genetika   MeSH
• Ruminococcus klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• celulasa MeSH
• celulosa MeSH
• DNA bakterií MeSH
• ribozomální DNA MeSH
• RNA ribozomální 16S MeSH

A strain isolated from the feces of takin was identified as Clostridium aminovalericum. In response to various types of chitin used as growth substrates, the bacterium produced a complete array of chitinolytic enzymes: chitinase ('endochitinase'), exochitinase, beta-N-acetylglucosaminidase, chitosanase and chitin deacetylase. The highest activities of chitinase (536 pkat/mL) and exochitinase (747 pkat/mL) were induced by colloidal chitin. Fungal chitin also induced high levels of these enzymes (463 pkat/mL and 502 pkat/mL, respectively). Crab shell chitin was the best inducer of chitosanase activity (232 pkat/mL). The chitinolytic enzymes of this strain were separated from culture filtrate by ion-exchange chromatography on the carboxylic sorbent Polygran 27. At pH 4.5, some isoforms of the chitinolytic enzymes (30% of total enzyme activity) did not bind to Polygran 27. The enzymes were eluted under a stepwise pH gradient (pH 5-8) in 0.1 mol/L phosphate buffer. At merely acidic pH (4.5-5.5), the adsorbed enzymes were co-eluted. However, at pH close to neutral values, the peaks of highly purified isoforms of exochitinases and chitinases were isolated. The protein and enzyme recovery reached 90%.

• MeSH
• acetylglukosaminidasa chemie   izolace a purifikace   metabolismus   MeSH
• amidohydrolasy chemie   izolace a purifikace   metabolismus   MeSH
• chitin metabolismus   MeSH
• chitinasy chemie   izolace a purifikace   metabolismus   MeSH
• chromatografie iontoměničová MeSH
• Clostridium enzymologie   izolace a purifikace   metabolismus   MeSH
• enzymová indukce MeSH
• feces mikrobiologie   MeSH
• glykosidhydrolasy chemie   izolace a purifikace   metabolismus   MeSH
• izoenzymy chemie   izolace a purifikace   metabolismus   MeSH
• kozy mikrobiologie   MeSH
• substrátová specifita MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetylglukosaminidasa MeSH
• amidohydrolasy MeSH
• chitin deacetylase MeSH Prohlížeč
• chitin MeSH
• chitinasy MeSH
• chitosanase MeSH Prohlížeč
• glykosidhydrolasy MeSH
• izoenzymy MeSH

The ability to produce extracellular chitosanase (EC 3.2.1.132) was found by plate assays in 18 (23%) out of 77 crystalliferous strains of Bacillus thuringiensis. The best chitosanase producer was selected after the growth chosen in a liquid medium with colloidal chitosan as carbon source. Enzyme production was optimized (a 4-d incubation at 32 degrees C with shaking in a medium of pH 6.5 with 4% colloidal chitosan) and the enzyme was partially characterized. This is the first report on the chitosanase of B. thuringiensis.

• MeSH
• Bacillus thuringiensis enzymologie   MeSH
• biologická kontrola škůdců MeSH
• chitin analogy a deriváty   metabolismus   MeSH
• chitosan MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• gelová chromatografie MeSH
• glykosidhydrolasy izolace a purifikace   metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• koncentrace vodíkových iontů MeSH
• molekulová hmotnost MeSH
• stabilita enzymů MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chitin MeSH
• chitosan MeSH
• chitosanase MeSH Prohlížeč
• glykosidhydrolasy MeSH
• inhibitory enzymů MeSH

Plastic waste disposal is a huge ecotechnological problem and one of the approaches to solving this problem is the development of biodegradable plastics. This review summarizes data on their use, biodegradability, commercial reliability and production from renewable resources. Some commercially successful biodegradable plastics are based on chemical synthesis (i.e. polyglycolic acid, polylactic acid, polycaprolactone, and polyvinyl alcohol). Others are products of microbial fermentations (i.e. polyesters and neutral polysaccharides) or are prepared from chemically modified natural products (e.g., starch, cellulose, chitin or soy protein).

• MeSH
• Bacteria metabolismus   MeSH
• biodegradace MeSH
• biotechnologie metody   MeSH
• celulosa chemie   metabolismus   MeSH
• houby metabolismus   MeSH
• plastické hmoty chemická syntéza   metabolismus   MeSH
• polyestery metabolismus   MeSH
• polymery MeSH
• rostliny metabolismus   MeSH
• škrob chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• celulosa MeSH
• plastické hmoty MeSH
• polyestery MeSH
• polymery MeSH
• škrob MeSH