Characterization of a novel Salmonella typhimurium chitinase which hydrolyzes chitin, chitooligosaccharides and an N-acetyllactosamine conjugate
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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-ß-D-chitobioside, 4-nitrophenyl ß-D-N,N',N¿-triacetylchitotriose and carboxymethyl chitin Remazol Brilliant Violet but does not act on 4-nitrophenyl N-acetyl-ß-D-glucosaminide, peptidoglycan or 4-nitrophenyl ß-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 ß-anomers of chitooligosaccharide substrates. The enzyme also released N-acetyllactosamine disaccharide from Galß1 ¿ 4GlcNAcß-O-(CH(2))(8)CONH(CH(2))(2)NHCO-tetramethylrhodamine, a model substrate for LacNAc terminating glycoproteins and glycolipids.
Original language | English |
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Journal | Glycobiology |
Volume | 21 |
Issue number | 4 |
Pages (from-to) | 426-436 |
Number of pages | 11 |
ISSN | 0959-6658 |
DOIs | |
Publication status | Published - 2011 |
- Amino Sugars, Chitin, Chitinase, Cloning, Molecular, Enzyme Assays, Hydrogen-Ion Concentration, Hydrolysis, Kinetics, Magnetic Resonance Spectroscopy, Molecular Structure, Oligosaccharides, Phylogeny, Recombinant Proteins, Salmonella typhimurium, Temperature
Research areas
ID: 37583241