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Cloning and comparison of phylogenetically related chitinases from Listeria monocytogenes EGD and Enterococcus faecalis V583
Author(s) -
Leisner J.J.,
Larsen M.H.,
Ingmer H.,
Petersen B.O.,
Duus J.Ø.,
Palcic M.M.
Publication year - 2009
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2009.04420.x
Subject(s) - listeria monocytogenes , enterococcus faecalis , enzyme , microbiology and biotechnology , biology , bacteria , food science , biochemistry , escherichia coli , gene , genetics
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‐β‐ d ‐chitobioside with K m values of 1·6 and 2·1 mmol l −1 , respectively, and k cat values of 21·6 and 6·5 s −1 . The enzymes also showed activity towards 4‐nitrophenyl β‐ d ‐ N , N ′, N ″‐triacetylchitotriose and carboxy‐methyl‐chitin‐Remazol Brilliant Violet but not towards 4‐nitrophenyl N‐ acetyl‐β‐ d ‐glucosaminide. Chitinolytic specificities of the enzymes were supported by their inactivity towards the substrates 4‐nitrophenyl β‐ 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.