Publication no. C-1998-0106-01R |  VIEW ARTICLE

Purification and Characterization of a Hexose Oxidase with Excellent Strengthening Effects in Bread.

Charlotte Poulsen (1,2) and Pernille Bak Høstrup (1). (1) Danisco Ingredients, Enzyme Development, Edwin Rahrs Vej 38, 8220 Brabrand, Denmark (2) Corresponding author. E-mail: <G8CHP@DANISCO.DK> Cereal Chem. 75(1):51-57. Accepted September 29, 1997. Copyright 1998 by the American Association of Cereal Chemists, Inc.

Hexose oxidase (EC 1.1.3.5) (HOX) was purified 51-fold from the red algae Chondrus crispus, by several chromatography methods, including hydrophobic interaction, chelating Sepharose, anion exchange, gel filtration, and chromatofocusing. Purified HOX was subjected to native PAGE and activity staining with nitroblue tetrazolium. For HOX electroeluted out of the gel and digested with endoproteinase Lys-C, the internal peptide sequence determined was: D-P-G-Y-I-V-I-D-V-N-A-G-T-(V or P)-D-K-P-D-P-X. The molecular mass, determined by gel filtration, was 126 kDa, versus 65 kDa determined by SDS-PAGE. The pI was determined to 4.64 and 4.79 as a double band on an isoelectrofocusing gel. K(m) was determined to 2.7 mM for D-glucose, 3.6 mM for D-galactose, 20.2 mM for cellobiose, 43.7 mM for maltose, 90.3 mM for lactose, 102 mM for xylose, and 531 mM for arabinose. The oxidation of thiol groups in gluten was determined by using Ellman's reagent: 5,5(prime)-dithiobis (2-nitrobenzoic acid). The effect of HOX was compared to that of glucose oxidase. Both enzymes caused a dose-responsive reduction in the free thiol groups. Extensigraph measurements and baking tests confirmed that HOX caused increased dough strength and increased bread volume more efficiently than glucose oxidase used in the same dosage.