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

• Authors: Jarmila Dušková , Galina Tishchenko , Evgenia Ponomareva , Jiří Šimůnek , Ingrid Koppová , Tereza Skálová , Andrea Štěpánková , Jindřich Hašek , Jan Dohnálek
• Languages of publication: EN

Abstracts

EN

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.

Keywords

EN

chitinolytic enzymes; anaerobic cultivation; protein isolation

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2011
• Volume: 58
• Issue: 2
• Pages: 261-263

Dates

published

2011

received

2011-03-05

revised

2011-05-12

accepted

2011-06-08

(unknown)

2011-06-13

Contributors

author

Jarmila Dušková

• Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Praha, Czech Republic

author

Galina Tishchenko

• Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Praha, Czech Republic

author

Evgenia Ponomareva

• Institute of Macromolecular Compounds, Russian Academy of Sciences, St. Petersburg, Russia

author

Jiří Šimůnek

• Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Praha, Czech Republic

author

Ingrid Koppová

• Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Praha, Czech Republic

author

Tereza Skálová

• Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Praha, Czech Republic

author

Andrea Štěpánková

• Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Praha, Czech Republic

author

Jindřich Hašek

• Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Praha, Czech Republic

author

Jan Dohnálek

• Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Praha, Czech Republic

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