Cloning and comparison of phylogenetically related chitinases from Listeria monocytogenes EGD and Enterococcus faecalis V583

J. J. Leisner, M. H. Larsen, H. Ingmer, B. O. Petersen, Jens Øllgaard Duus, M. M. Palcic

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Aims: To compare enzymatic activities of two related chitinases, ChiA and EF0361, encoded by Listeria monocytogenes and Enterococcus faecalis, respectively. Methods and Results: The chiA and EF0361 genes were amplified by PCR, cloned and expressed with histidine tags, allowing easy purification of the gene products. ChiA had a molecular weight as predicted from the amino acid sequence, whereas EF0361 was 1840 Da lower than expected because of C-terminal truncation. The ChiA and EF0361 enzymes showed activity towards 4-nitrophenyl N,N¢-diacetyl-b-d-chitobioside with Km values of 1Æ6 and 2Æ1 mmol l)1, respectively, and kcat values of 21Æ6 and 6Æ5 s)1. The enzymes also showed activity towards 4-nitrophenyl b-d-N, N¢, N¢¢-triacetylchitotriose and carboxy-methyl-chitin-Remazol Brilliant Violet but not towards 4-nitrophenyl N-acetyl-b-d-glucosaminide. Chitinolytic specificities of the enzymes were supported by their inactivity towards the substrates 4-nitrophenyl b-d-cellobioside and peptidoglycan. The pH and temperature profiles for catalytic activities were relatively similar for both the enzymes.
Conclusion: The ChiA and EF0361 enzymes show a high degree of similarity in their catalytic activities although their hosts share environmental preferences only to some extent.
Significance and Impact of the Study: This study contributes to an understanding of the chitinolytic activities by L. monocytogenes and Ent. faecalis. Detailed information on their chitinolytic systems will help define potential reservoirs in the natural environment and possible transmission routes into food-manufacturing plants.

Original languageEnglish
JournalJournal of Applied Microbiology
Volume107
Issue number6
Pages (from-to)2080-2087
ISSN1364-5072
DOIs
Publication statusPublished - 2009
Externally publishedYes

Keywords

  • hitinase
  • Enterococcus
  • Family 18 glycosyl hydrolase
  • Kinetics
  • Listeria
  • Substrate specificity

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