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

The effect of the administration of chitosan (CS) and chitooligosaccharides (COS) on rat fecal microbiota was analyzed in this study. The profile of total bacterial population was monitored during 3 weeks of CS or COS application using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons. Quantitative PCR was used for monitoring possible changes in the levels of total bacteria and the levels of individual bacterial groups: Bifidobacteria, Clostridium leptum, Enterobacteriaceae, Lactobacillus-Streptococcus-Enterobacter, and Bacteroides-Prevotella. The DGGE profiles revealed a high complexity and individuality of each tested subject, and variations in the composition of band pattern were observed. CS or COS per os administration changed the profile and structure of the microbial ecosystem of the gastrointestinal tract of healthy rats. COS have, in most cases, an opposite effect compared with CS; only the Bacteroides-Prevotella bacterial group and Enterobacteriaceae were influenced in the same way. The Bifidobacteria group was not influenced by the administration CS and COS.

• MeSH
• Bacteria klasifikace   genetika   izolace a purifikace   MeSH
• chitosan aplikace a dávkování   farmakologie   MeSH
• feces mikrobiologie   MeSH
• krysa rodu Rattus MeSH
• metagenom účinky léků   MeSH
• oligosacharidy aplikace a dávkování   farmakologie   MeSH
• potkani Wistar MeSH
• potravinářské přísady aplikace a dávkování   farmakologie   MeSH
• střeva mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chitosan MeSH
• oligosacharidy MeSH
• potravinářské přísady MeSH

Antibacterial effect of chitooligosaccharides (COS) and low molar mass chitosans (LMWC) is considered as one of the most important characteristics of chitosan (CS) hydrolysates. Here, we show the in vitro effect of different COS, LMWC, and CS on representative anaerobic bacteria isolated from human colon as a possibility of targeting modification of colonic microflora composition by supplementation of dietary CS products by humans. Specific growth rate of seven selected nonpathogenic anaerobic bacterial strains (Clostridium paraputrificum, Clostridium beijerinckii, Roseburia intestinalis, Bacteroides vulgatus, Bacteriodes thetaiotaomicron, Faecalibacterium prausnitzii and Blautia coccoides) was determined in the presence of 0.25 and 0.5% COS (2, 3, and 6 kDa), 0.025 and 0.05% of LMWC (10 and 16 kDa), and 0.025 and 0.1% of CS in vitro. The growth rate decreased in all strains in the presence of COS and LMWC in higher concentrations in comparison to control incubations. A relatively higher resistance to CS hydrolyzates was detected in R. intestinalis and F. prausnitzii, and more susceptible were bacteria belonging to Bacteoides sp. and Clostridium sp. The antimicrobial activity, minimum inhibitory concentrations (MIC), and minimal bactericidal concentrations (MBC) were determined. The antimicrobial activity increased with the degree of polymerization (DP). MIC ranged from 0.25 to 4.5% in dependence on bacterial strain and DP of CS/LMWC. MBC also decreased with DP. The most effective antimicrobial action was detected in LMWC with 16 kDa and CS. Weak antimicrobial activity was found in COS with small molecules (2 and 3 kDa).

• MeSH
• antibakteriální látky chemie   farmakologie   MeSH
• Bacteria klasifikace   účinky léků   genetika   izolace a purifikace   MeSH
• chitosan chemie   farmakologie   MeSH
• kolon mikrobiologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• molekulová hmotnost MeSH
• oligosacharidy chemie   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• chitosan MeSH
• oligosacharidy 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 crude fractions of chitooligosaccharides (COS) and low-molar-mass chitosans (LMWC) were prepared by enzyme hydrolysis of chitosan (CS). Specific growth rate of B. adolescentis, B. bifidum, B. breve, B. catenulatum, B. infantis and B. longum ssp. longum was determined in the presence of 0.025 and 0.5 % COS (<5 kDa), LMWC (5-10 kDa), and 0.025, 0.1 and 0.5% of CS, chitosan succinate and chitosan glutamate in vitro. Minimum inhibitory concentrations (MIC; assayed by colony counting on TPY agar plates) of COS-LMWC and CS ranged from 0.025% to 0.75% of CS-LMWC. The growth of all bifidobacterial strains in the presence of chitosan, its derivatives and LMWC decreased at a concentration of 0.025%; the bacterial growth was completely inhibited at a concentration of 0.5%. COS did not show any inhibitory effect, an increased growth rate was even observed in the case of B. bifidum, B. catenulatum and B. infantis.

• MeSH
• antibakteriální látky farmakologie   MeSH
• Bifidobacterium účinky léků   růst a vývoj   MeSH
• chitosan farmakologie   MeSH
• mikrobiální testy citlivosti MeSH
• oligosacharidy farmakologie   MeSH
• počet mikrobiálních kolonií MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• chitosan succinate MeSH Prohlížeč
• chitosan MeSH
• oligosacharidy 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