Hexose oxidase from Chondrus crispus: improved purification using perfusion chromatography

An improved method for purifying hexose oxidase (D-hexose: O-2 1-oxidoreduc tase, EC 1.1.3.5) from the marine red alga Chondrus crispus is described fo r obtaining enzyme suitable for structural characterization and use in bioc onversion of lactose to lactobionic acid. This involved extracting enzyme f rom finely ground lyophilized tissue in sodium phosphate buffer (pH 7) cont aining 20% ammonium sulfate, eliminating the previously used solvent extrac tion and protease treatments, and by applying Poros perfusion chromatograph y media to achieve rapid separations of high resolution. Primary separation of contaminating phycobiliproteins and carrageenans was achieved using Por os DEAE-50. Sequential HPLC purification steps using Poros HP2 and Poros HQ were followed by Sephacryl S200 h chromatography. Enzyme activity was dete rmined with a peroxidase-coupled assay using 2,2'-azino-bis (3-ethylbenzthi azoline-6 sulfonic acid) substrate. A final specific activity of 69 U/mg wa s obtained, representing a 100-fold purification with an activity recovery of about 10%. A native size of approximately 117,000 Da was determined by s ize exclusion chromatography, and SDS-PAGE: revealed the presence of 38,000 and 29,000 Da polypeptides that appear to be derived from a 65,000 Da subu nit. Further properties of the enzyme are described. (C) 2001 Elsevier Scie nce Inc. All rights reserved.