Cloning and Characterization of a Novel N-acetylglucosaminidase (AtlD) from Enterococcus faecalis.

Abstract

The atlD gene from an Enterococcus faecalis strain isolated from a Mexican artisanal cheese was cloned, sequenced and expressed in Escherichia coli in order to perform a biochemical characterization. A partial amino acid sequence of the heterologous protein was obtained by LC-MS/MS, and it corresponded to a novel peptidoglycan hydrolase designated AtlD. Its molecular mass was 62-75 kDa, as determined by SDS-PAGE, zymography, Western blot, and exclusion chromatography. Electrofocusing rendered an isoelectric point (pI) of 4.8. It exhibited N-acetylglucosaminidase activity, with an optimal pH and temperature between 6-7 and 50°C, respectively. It retained 85% activity with NaCl at 1,000 mM, but it was susceptible to divalent ions, particularly Zn2+. It showed antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, and enterococcal strains of clinical origin. Due to the fact that it showed activity versus pathogenic bacteria, and because of its capabilities under ionic strength, temperature, and pH values present in food matrices, it could be applied as an additive in the food industry. This study will aid in the design of new antibacterial agents of natural origin to combat food-borne diseases, and it could be used as an industrial or hospital hygiene agent as well.

• Authors: Carlos Eduardo Serrano-Maldonado, Israel García-Cano, Augusto González-Canto, Eliel Ruiz-May, Jose Miguel Elizalde-Contreras, Maricarmen Quirasco
• Journal: Journal of molecular microbiology and biotechnology (J Mol Microbiol Biotechnol) Vol. 28 Issue 1 Pg. 14-27 ( 2018) ISSN: 1660-2412 [Electronic] Switzerland
• PMID: 29510391 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2018 S. Karger AG, Basel.

Chemical References

• Anti-Bacterial Agents
• Bacterial Proteins
• Recombinant Proteins
• Acetylglucosaminidase

Topics

• Acetylglucosaminidase (chemistry, genetics, pharmacology)
• Amino Acid Sequence
• Anti-Bacterial Agents (pharmacology)
• Bacterial Proteins (genetics)
• Cheese (microbiology)
• Chromatography, Liquid
• Cloning, Molecular (methods)
• Enterococcus (drug effects)
• Enterococcus faecalis (enzymology, genetics, metabolism)
• Enzyme Stability
• Escherichia coli (genetics)
• Food Industry
• Food Microbiology
• Foodborne Diseases
• Gene Expression Regulation, Bacterial
• Genes, Bacterial (genetics)
• Hydrogen-Ion Concentration
• Isoelectric Point
• Listeria monocytogenes (drug effects)
• Molecular Weight
• Recombinant Proteins (chemistry, genetics)
• Staphylococcus aureus (drug effects)
• Substrate Specificity
• Tandem Mass Spectrometry
• Temperature