Chitinolytic enzymes from bacterium inhabiting human gastrointestinal tract - critical parameters of protein isolation from anaerobic culture

0363003 - ÚMCH 2012 RIV PL eng J - Journal Article
Dušková, Jarmila - Tishchenko, Galina - Ponomareva, E. - Šimůnek, Jiří - Koppová, Ingrid - Skálová, Tereza - Štěpánková, Andrea - Hašek, Jindřich - Dohnálek, Jan
Chitinolytic enzymes from bacterium inhabiting human gastrointestinal tract - critical parameters of protein isolation from anaerobic culture.
Acta Biochimica Polonica. Roč. 58, č. 2 (2011), s. 261-263. ISSN 0001-527X. E-ISSN 1734-154X
R&D Projects: GA ČR GA310/09/1407; GA ČR GA305/07/1073
Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50450515
Keywords : chitinolytic enzymes * anaerobic cultivation * protein isolation
Subject RIV: EE - Microbiology, Virology
Impact factor: 1.491, year: 2011
http://www.actabp.pl/pdf/2_2011/261.pdf

The object of this study are chitinolytic enzymes produced by bacterium Clostridium paraputrificum 14 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 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.
Permanent Link: http://hdl.handle.net/11104/0199145