Characterization of a novel Salmonella Typhimurium chitinase which hydrolyzes chitin, chitooligosaccharides and an N-acetyllactosamine conjugate

Tanja Larsen, Bent O. Petersen, Birgit G. Storgaard, Jens Øllgaard Duus, Monica M. Palcic, Jørgen J. Leisner

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

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 1H-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-(CH2)8CONH(CH2)2NHCO-tetramethylrhodamine, a model substrate for LacNAc terminating glycoproteins and glycolipids.
Original languageEnglish
JournalGlycobiology
Volume21
Issue number4
Pages (from-to)426-436
ISSN0959-6658
DOIs
Publication statusPublished - 2011
Externally publishedYes

Keywords

  • Enterobacteriaceae
  • family 18 hydrolase
  • kinetics
  • N-acetyllactosamine
  • NMR

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