Characterization of a novel Salmonella Typhimurium chitinase which hydrolyzes chitin, chitooligosaccharides and an N-acetyllactosamine conjugate.
Salmonella contain genes annotated as chitinases; however, their chitinolytic activities have never been verified. We now demonstrate such an activity for a chitinase assigned to glycoside hydrolase family 18 encoded by the SL0018 (chiA) gene in Salmonella enterica Typhimurium SL1344. A C-terminal truncated form of chiA lacking a putative chitin-binding domain was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3) with an N-terminal (His)(6) tag. The purified enzyme hydrolyzes 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside, 4-nitrophenyl β-D-N,N',N″-triacetylchitotriose and carboxymethyl chitin Remazol Brilliant Violet but does not act on 4-nitrophenyl N-acetyl-β-D-glucosaminide, peptidoglycan or 4-nitrophenyl β-D-cellobioside. Enzyme activity was also characterized by directly monitoring product formation using (1)H-nuclear magnetic resonance which showed that chitin is a substrate with the release of N,N'-diacetylchitobiose. Hydrolysis occurs with the retention of configuration and the enzyme acts on only the β-anomers of chitooligosaccharide substrates. The enzyme also released N-acetyllactosamine disaccharide from Galβ1 → 4GlcNAcβ-O-(CH(2))(8)CONH(CH(2))(2)NHCO-tetramethylrhodamine, a model substrate for LacNAc terminating glycoproteins and glycolipids.
Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Grønnegårdsvej 15, 1870 Frederiksberg C., Denmark.
Tanja
T
Larsen
Bent O
BO
Petersen
Birgit G
BG
Storgaard
Jens Ø
JØ
Duus
Monica M
MM
Palcic
Jørgen J
JJ
Leisner
D000606:Amino Sugars / Q000378:metabolism*; D002686:Chitin / Q000737:chemistry / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D057075:Enzyme Assays; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology*; D013696:Temperature
D000606:Amino Sugars; D009844:Oligosaccharides; D011994:Recombinant Proteins; D002686:Chitin; C000458:N-acetyllactosamine; D002688:Chitinases