Characterization of a novel Salmonella Typhimurium chitinase which hydrolyzes chitin, chitooligosaccharides and an N-acetyllactosamine conjugate
| Autor: | Bent O. Petersen, Tanja Larsen, Monica M. Palcic, Jens Ø. Duus, Birgit G. Storgaard, Jørgen J. Leisner |
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| Rok vydání: | 2010 |
| Předmět: |
Salmonella typhimurium
Magnetic Resonance Spectroscopy Disaccharide Oligosaccharides Chitin Biochemistry chemistry.chemical_compound Cloning Molecular Phylogeny Glycoside hydrolase family 18 Enzyme Assays Molecular Structure biology Hydrolysis Chitinases Temperature Substrate (chemistry) Amino Sugars Hydrogen-Ion Concentration Recombinant Proteins Enzyme assay carbohydrates (lipids) N-Acetyllactosamine Kinetics chemistry Chitinase biology.protein Peptidoglycan |
| Zdroj: | Glycobiology. 21:426-436 |
| ISSN: | 1460-2423 0959-6658 |
| DOI: | 10.1093/glycob/cwq174 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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