Comparative studies of chitinases A and B from Serratia marcescens.
Serratia marcescens produces several chitinolytic enzymes, including chitinase A (ChiA) and chitinase B (ChiB). In this study, ChiB was purified to homogeneity using a newly developed protocol based on hydrophobic interaction chromatography. Subsequently, characteristics of ChiB and of the hitherto only partly characterized ChiA were determined and compared. Pure ChiA and ChiB shared several characteristics such as a broad pH optimum around pH 5.0-6.0, and a temperature optimum between 50 and 60 degrees C. Both enzymes were fairly stable, with half-lives of more than 10 d at 37 degrees C, pH 6.1. Analyses of the degradation of various N-acetylglucosamine oligomers, fluorogenic substrates and colloidal chitin showed that both enzymes cleave chitobiose [(GlcNAc)2] from (GlcNAc)n and thus possess an exo-N,N'-diacetylchitobiohydrolase activity. Both enzymes were also capable of producing monomers from longer (GlcNAc)n substrates, indicating that they also have an endochitinase (ChiA) or exo-N,N',N"-triacetylchitotriohydrolase (ChiB) activity. Kinetic analyses with 4-methylumbelliferyl-beta-D-N,N'-diacetylchitobioside, an analogue of (GlcNAc)3, showed cooperative kinetics for ChiA, whereas for ChiB normal hyperbolic kinetics were observed. ChiA had a higher specific activity towards chitin than ChiB and synergistic effects on the chitin degradation rate were observed upon combining the two enzymes. These results, together with the results of sequence comparisons and previous studies of the cellular localization of the two chitinases in S. marcescens indicate possible roles for ChiA and ChiB in chitin breakdown.
Brurberg
M B
MB
Department of Biotechnological Sciences, Agricultural University of Norway, Norway. mbruberg@bioslave.uio.no
Nes
I F
IF
Eijsink
V G
VG
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D002686:Chitin / Q000737:chemistry; D002688:Chitinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
D007527:Isoenzymes; D009844:Oligosaccharides; D002686:Chitin; D002688:Chitinases