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

TY - JOUR

T1 - Characterization of a novel Salmonella typhimurium chitinase which hydrolyzes chitin, chitooligosaccharides and an N-acetyllactosamine conjugate

AU - Larsen, Tanja

AU - Petersen, Bent O.

AU - Storgaard, Birgit Groth

AU - Duus, Jens Ø.

AU - Palcic, Monica M.

AU - Leisner, Jørgen

PY - 2011/4

Y1 - 2011/4

N2 - 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 - d-chitobioside, 4-nitrophenyl -d-N,N′,N″-triacetylchitotriose and carboxymethyl chitin Remazol Brilliant Violet but does not act on 4-nitrophenyl N-acetyl - d-glucosaminide, peptidoglycan or 4-nitrophenyl -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 -anomers of chitooligosaccharide substrates. The enzyme also released N-acetyllactosamine disaccharide from Gal1 → 4GlcNAc-O-(CH2)8CONH(CH 2)2NHCO-tetramethylrhodamine, a model substrate for LacNAc terminating glycoproteins and glycolipids.

AB - 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 - d-chitobioside, 4-nitrophenyl -d-N,N′,N″-triacetylchitotriose and carboxymethyl chitin Remazol Brilliant Violet but does not act on 4-nitrophenyl N-acetyl - d-glucosaminide, peptidoglycan or 4-nitrophenyl -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 -anomers of chitooligosaccharide substrates. The enzyme also released N-acetyllactosamine disaccharide from Gal1 → 4GlcNAc-O-(CH2)8CONH(CH 2)2NHCO-tetramethylrhodamine, a model substrate for LacNAc terminating glycoproteins and glycolipids.

KW - Amino Sugars

KW - Chitin

KW - Chitinase

KW - Cloning, Molecular

KW - Enzyme Assays

KW - Hydrogen-Ion Concentration

KW - Hydrolysis

KW - Kinetics

KW - Magnetic Resonance Spectroscopy

KW - Molecular Structure

KW - Oligosaccharides

KW - Phylogeny

KW - Recombinant Proteins

KW - Salmonella typhimurium

KW - Temperature

U2 - 10.1093/glycob/cwq174

DO - 10.1093/glycob/cwq174

M3 - Journal article

C2 - 21062783

SN - 0959-6658

VL - 21

SP - 426

EP - 436

JO - Glycobiology

JF - Glycobiology

IS - 4

ER -