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Characterization of a Novel Salmonella Typhimurium Chitinase Which Hydrolyzes Chitin, Chitooligosaccharides and an N-acetyllactosamine Conjugate

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 Ø Duus, Monica M Palcic, Jørgen J Leisner

Glycobiology. 2011 Apr;21(4):426-36.

PMID: 21062783

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 (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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP7699389	4-Nitrophenyl β-D-N,N′,N′′-triacetylchitotriose		7699-38-9	Price

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