Saccharides and their derivatives are typical polar analytes without a suitable UV-chromophore that are nowadays analyzed by HPLC (high-performance liquid chromatography) under HILIC (hydrophilic interaction liquid chromatography) mode. Usually an evaporative light scattering detector (ELSD) is utilized which, however, gives a nonlinear response. A procedure to overcome the problem of mutarotating (time-varying) analytes recorded with such a nonlinear response detector is described. The procedure was applied for determination of glucosamine in two commercially available pharmaceutical formulations containing the common inorganic ions that the detector gives a response to. Under optimized conditions, both the anomers of glucosamine were separated and could be determined separately. Owing to the short retention time of the analyte (a run time <4 min) and relatively slow kinetics of the anomeric conversion (equilibration time 2.5 h), mutarotation could be monitored and corresponding rate constants calculated.
Enzymatic depolymerization of chitosan, a β-(1,4)-linked polycationic polysaccharide composed of d-glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) provides a possible route to the exploitation of chitin-rich biomass. Complete conversion of chitosan to mono-sugars requires the synergistic action of endo- and exo- chitosanases. In the present study we have developed an efficient and cost-effective chitosan-degrading enzyme cocktail containing only two enzymes, an endo-attacking bacterial chitosanase, ScCsn46A, from Streptomyces coelicolor, and an exo-attacking glucosamine specific β-glucosaminidase, Tk-Glm, from the archaeon Thermococcus kodakarensis KOD1. Moreover, we developed a fast, reliable quantitative method for analysis of GlcN using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The sensitivity of this method is high and less than 50 pmol was easily detected, which is about 1000-fold better than the sensitivity of more commonly used detection methods based on refractive index. We also obtained qualitative insight into product development during the enzymatic degradation reaction by means of ElectroSpray Ionization-Mass Spectrometry (ESI-MS).
- MeSH
- bakteriální proteiny metabolismus MeSH
- beta-glukosidasa metabolismus MeSH
- chitosan chemie MeSH
- chromatografie iontoměničová metody MeSH
- glukosamin analýza chemie MeSH
- glykosidhydrolasy metabolismus MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- Streptococcus enzymologie MeSH
- substrátová specifita MeSH
- Thermococcus enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- elektrická vodivost MeSH
- elektroforéza kapilární metody MeSH
- financování organizované MeSH
- glukosamin analýza MeSH
- kalibrace MeSH
- léčivé přípravky chemie MeSH
- potravní doplňky analýza MeSH
- referenční standardy MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- validační studie MeSH
- MeSH
- autolýza MeSH
- buněčná stěna fyziologie metabolismus účinky léků MeSH
- buněčné dělení MeSH
- centrifugace MeSH
- dusík analýza MeSH
- elektronová mikroskopie MeSH
- fosfor analýza MeSH
- glukosa analýza metabolismus MeSH
- glukosamin analýza MeSH
- hexosy analýza farmakologie MeSH
- kultivační média MeSH
- laktáty metabolismus MeSH
- lyofilizace MeSH
- mannitol farmakologie MeSH
- mannosa analýza MeSH
- osmotická fragilita MeSH
- polysacharidy biosyntéza MeSH
- Saccharomyces cytologie metabolismus růst a vývoj účinky léků MeSH
- MeSH
- experimentální sarkom enzymologie krev MeSH
- glukosamin analýza MeSH
- hlen metabolismus MeSH
- hyaluronoglukosaminidasa krev MeSH
- krev MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- nádory dělohy chirurgie MeSH
- nádory děložního čípku chirurgie MeSH
- nádory enzymologie krev MeSH
- sarkom Yoshidův enzymologie krev MeSH
- tyrosin krev MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
