Characterization of a novel Salmonella typhimurium chitinase which hydrolyzes chitin, chitooligosaccharides and an N-acetyllactosamine conjugate
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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.
Originalsprog | Engelsk |
---|---|
Tidsskrift | Glycobiology |
Vol/bind | 21 |
Udgave nummer | 4 |
Sider (fra-til) | 426-436 |
Antal sider | 11 |
ISSN | 0959-6658 |
DOI | |
Status | Udgivet - 2011 |
ID: 37583241