title: Chitinolytic enzymes from bacterium inhabiting human gastrointestinal tract -- critical parameters of protein isolation from anaerobic culture.
abstract: The object of this study are chitinolytic enzymes produced by bacterium Clostridium paraputrificum J4 isolated from the gastrointestinal tract of a healthy human. In particular, we focus on the development of purification protocols, determination of properties of the enzymes and their activity profiles. The process of bacteria cultivation and isolation of chitinolytic complex of enzymes showing specific activities of endo-, exo-chitinase and N-acetyl-β-glucosaminidase was optimized. A range of various purification procedures were used such as ultrafiltration, precipitation, chromatographic separations (ion-exchange, size exclusion, chromatofocusing) in altered combinations. The optimal purification protocol comprises two or three steps. Individual samples were analyzed by SDS/PAGE electrophoresis and after renaturation their activity could be detected using zymograms. Mass spectroscopy peptide fragment analysis and MALDI analysis of the purest samples indicate presence of endochitinase B (molecular mass about 85 kDa) and of 60-kDa endo- and exochitinases.
authors.affiliation: Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Praha, Czech Republic. duskova@imc.cas.cz
authors.forename: Jarmila
authors.initials: J
authors.lastname: Dušková
authors.forename: Galina
authors.initials: G
authors.lastname: Tishchenko
authors.forename: Evgenia
authors.initials: E
authors.lastname: Ponomareva
authors.forename: Jiří
authors.initials: J
authors.lastname: Šimůnek
authors.forename: Ingrid
authors.initials: I
authors.lastname: Koppová
authors.forename: Tereza
authors.initials: T
authors.lastname: Skálová
authors.forename: Andrea
authors.initials: A
authors.lastname: Štěpánková
authors.forename: Jindřich
authors.initials: J
authors.lastname: Hašek
authors.forename: Jan
authors.initials: J
authors.lastname: Dohnálek
mesh_terms: D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000302:isolation & purification / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology* / Q000254:growth & development; D046508:Culture Techniques / Q000379:methods; D041981:Gastrointestinal Tract / Q000382:microbiology*; D006026:Glycoside Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D006596:Hexosaminidases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans
chemical_list: D001426:Bacterial Proteins; D002686:Chitin; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; C064499:exo-beta-D-glucosaminidase; C019868:chitosanase; D002688:Chitinases