Natural substrate assay for chitinases using high-performance liquid chromatography: a comparison with existing assays.
The determination of kinetic parameters of chitinases using natural substrates is difficult due to low K(m) values, which require the use of low substrate concentrations that are hard to measure. Using the natural substrate (GlcNAc)(4), we have developed an assay for the determination of k(cat) and K(m)values of chitinases. Product concentrations as low as 0.5 microM were detected using normal-phase high-performance liquid chromatography (HPLC) with an amide 80 column (0.20 x 25 cm) using spectrophotometric detection at 210 nm. By means of this assay, k(cat) and K(m)values for chitinases A (ChiA) and B (ChiB) of Serratia marcescens were found to be 33+/-1s(-1) and 9+/-1 microM and 28+/-2s(-1) and 4+/-2 microM, respectively. For ChiB, these values were compared to those found with commonly used substrates where the leaving group is a (nonnatural) chromophore, revealing considerable differences. For example, assays with 4-methylumbelliferyl-(GlcNAc)(2) yielded a k(cat) value of 18+/-2s(-1) and a K(m) value of 30+/-6 microM. For two ChiB mutants containing a Trp --> Ala mutation in the +1 or +2 subsites, the natural substrate and the 4-methylumbelliferyl-(GlcNAc)(2) assays yielded rather similar K(m) values (5-fold difference at most) but showed dramatic differences in k(cat) values (up to 90-fold). These results illustrate the risk of using artificial substrates for characterization of chitinases and, thus, show that the new HPLC-based assay is a valuable tool for future chitinase research.
Krokeide
Inger-Mari
IM
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Aas, Norway.
Synstad
Bjørnar
B
Gåseidnes
Sigrid
S
Horn
Svein J
SJ
Eijsink
Vincent G H
VG
Sørlie
Morten
M
D001681:Biological Assay; D002686:Chitin / Q000737:chemistry* / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D012706:Serratia marcescens / Q000201:enzymology*; D013379:Substrate Specificity
D009844:Oligosaccharides; D002686:Chitin; C482346:ChiB protein, Serratia marcescens; D002688:Chitinases; C094590:chitinase ChiA