Abstract
Glycosaminoglycans (GAGs) are a family of complex polysaccharides involved in a diversity of biological processes, ranging from cell signaling to blood coagulation. Chondroitin sulfate (CS) and dermatan sulfate (DS) comprise a biologically important subset of GAGs. Two of the important lyases that degrade CS/DS, chondroitinase AC (EC 4.2.2.5) and chondroitinase B (no EC number), have been isolated and cloned from Flavobacterium heparinum. In this study, we outline an improved methodology for the recombinant expression and purification of these chondroitinases, thus enabling the functional characterization of the recombinant form of the enzymes for the first time. Utilizing an N-terminal 6× histidine tag, the recombinant chondroitinases were produced by two unique expression systems, each of which can be purified to homogeneity in a single chromatographic step. The products of exhaustive digestion of chondroitin-4SO4 and chondroitin-6SO4 with chondroitinase AC and dermatan sulfate with chondroitinase B were analyzed by strong-anion exchange chromatography and a novel reverse-polarity capillary electrophoretic technique. In addition, the Michaelis–Menten parameters were determined for these enzymes. With chondroitin-4SO4 as the substrate, the recombinantly expressed chondroitinase AC has a Km of 0.8 μM and a kcat of 234 s−1. This is the first report of kinetic parameters for chondroitinase AC with this substrate. With chondroitin-6SO4 as the substrate, the enzyme has a Km of 0.6 μM and a kcat of 480 s−1. Recombinantly expressed chondroitinase B has a Km of 4.6 μM and a kcat of 190 s−1 for dermatan sulfate as its substrate. Efficient recombinant expression of the chondroitinases will facilitate the structure–function characterization of these enzymes and allow for the development of the chondroitinases as enzymatic tools for the fine characterization and sequencing of CS/DS.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: Biochemical and Biophysical Research Communications
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
