Purification
and Kinetic Parameters of a Streptomyces olivaceoviridis Protein Which
Binds N-acetylglucosamine and Chitin Oligomers
XIAO Xiang，WANG Feng-Ping*，SCHREMPF Hildgund
( The Applied Genetics of Microorganisms, Faculty of Biology/Chemistry,
University of Osnabrueck, Barbarastr. 11, D49069 Osnabrueck, Germany
)
Abstract A specific
substrate binding protein is located within the membrane of Streptomyces
olivaceoviridis mycelia. After Triton extraction of the membrane, two forms
of the protein (46.0 kD and 47.5 kD) were purified to apparent homogeneity by
consecutive anionic exchange chromatographies. The results of competetion
binding assay suggest that the 47.5 kD protein differs from the 46 kD form by a
lipid anchor. Both specifically interacted with N-acetylglucosamine and
chitin oligomers (C2 to C6), but not with cellobiose nor glucose. Using surface
plasmon resonance, the kinetic parameters of the 46 kD form of the binding
protein were determined. This protein showed a very high affinity for N-acetylglucosamine
(Kd=8.29×10－9 mol/L) and for chitobiose (Kd=2.88×10－8 mol/L). Varying equilibrium
dissociation constants in the micromole range were determined for chitotetraose
(Kd=4.5×10－6 mol/L), chitopentaose (Kd=1.03×10－6 mol/L) and chitohexaose (Kd=3.81×10－6 mol/L)， and the lowest one was for
chitotriose (Kd=1.95×10－5 mol/L). Comparisons of the
dissociation and association rate constants indicated that the interaction of
this protein with each ligand was controlled by the association rate. N
terminal sequence indicated that this protein might belong to an ABC
transporter system.
Key words chitooligomer binding protein； competetion binding assay； surface plasmon resonance； dissociation rate
*Corresponding author: Tel,
86-592-2195236； Fax， 86-592-2085376； e-mail， [email protected]