Cloning and comparison of phylogenetically related chitinases from Listeria monocytogenes EGD and Enterococcus faecalis V583.
OBJECTIVE
To compare enzymatic activities of two related chitinases, ChiA and EF0361, encoded by Listeria monocytogenes and Enterococcus faecalis, respectively.


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-beta-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 beta-D-N, N', N''-triacetylchitotriose and carboxy-methyl-chitin-Remazol Brilliant Violet but not towards 4-nitrophenyl N-acetyl-beta-D-glucosaminide. Chitinolytic specificities of the enzymes were supported by their inactivity towards the substrates 4-nitrophenyl beta-D-cellobioside and peptidoglycan. The pH and temperature profiles for catalytic activities were relatively similar for both the enzymes.


CONCLUSIONS
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.


CONCLUSIONS
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.
Leisner
J J
JJ
Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C, Denmark. jjl@life.ku.dk
Larsen
M H
MH
Ingmer
H
H
Petersen
B O
BO
Duus
J Ø
JØ
Palcic
M M
MM
D000595:Amino Acid Sequence; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D013293:Enterococcus faecalis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity
D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases