Expression and characterization of endochitinase C from Serratia marcescens BJL200 and its purification by a one-step general chitinase purification method.
In this study we cloned, expressed, purified, and charaterized chitinase C1 from Serratia marcescens strain BJL200. As expected, the BJL200-ChiC1 amino acid sequence of this strain was highly similar to sequences of ChiC1 identified in two other strains of S. marcescens. BJL200-ChiC1 was overproduced in E. coli by the T7 expression system, and purified by a one-step hydrophobic interaction chromatography (HIC) with phenyl-sepharose. BJL200-ChiA and BJL200-ChiB had an approximately 30-fold higher k(cat) and 15 fold-lower K(m) than BJL200-ChiC1 for the oligomeric substrate 4-methylumbelliferyl-beta-D-N-N'-N''-triacetylchitotrioside, while BJL200-ChiC1 was 10-15 times faster than BJL200-ChiB and BJL200-ChiA in degrading the polymeric substrate CM-chitin-RBV. BJL200-ChiC1 degradation of beta-chitin resulted in a range of different chito-oligosaccharides (GlcNAc)(2) (main product), GlcNAc, (GlcNAc)(3), (GlcNAc)(4), and (GlcNAc)(5), indicating endo activity. The purification method used for BJL200-ChiC1 in this study is generally applicable to family 18 chitinases and their mutants, including inactive mutants, some of which tend to bind almost irreversibly to chitin columns. The high specificity of the interaction with the (non-chitinous) column material is mediated by aromatic residues that occur in the substrate-binding clefts and surfaces of the enzymes.
Synstad
Bjørnar
B
Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, PO Box 5003, N-1432 Aas, Norway.
Vaaje-Kolstad
Gustav
G
Cederkvist
F Henning
FH
Saua
Silje F
SF
Horn
Svein J
SJ
Eijsink
Vincent G H
VG
Sørlie
Morten
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis; D012706:Serratia marcescens / Q000201:enzymology*; D013379:Substrate Specificity
D009844:Oligosaccharides; D002686:Chitin; D002688:Chitinases