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

Abstract

Cereal Chem. 75(1):51-57Hexose 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′-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. MATERIALS AND METHODS ChemicalsGOX was obtained from Aspergillus niger (Grindamyl S 757, Danisco Ingredients, Denmark). Maltotriose and maltotetraose were obtained from Megazyme, Ireland. Peroxidase, odianisidine, nitroblue tetrazolium, phenazine methosulphate, and endoproteinase Lys-C were obtained from Sigma Chemical Co., St. Louis, MO. Phenyl Sepharose FF, chelating Sepharose FF, Source Q, Superdex 200, Mono P HR 5/5, isoelectrofocusing (IEF), and molecular weight standards were from Pharmacia, Sweden. The 8-16% tris-glycine gels used for electrophoresis and the precast 3-10 IEF gels were from Novex, San Diego, CA. The inorganic chemicals used were analytical grade. A Vydac C18 column was obtained from The Separation Group, CA. A Brownlee C2 column was obtained from Applied Biosystems. A Dionex PA2 column was from Dionex. Assay for HOX ActivityThe assay of Sullivan and Ikawa (1973) was scaled down to be run in microtiter plates. The absorbance was read in a microplate reader. The HOX assay method was also used for the assay of GOX. One unit is defined as the amount of enzyme that catalyzes the production of 1 µmole of H 2 O 2 per minute at 25°C, pH 6.3, and at a substrate concentration of 50 mM D-glucose. The assay was used to determine the Michaelis-Menten constant (K m ) and maximum velocity (V max ) for D-glucose, D-galactose, cellobiose, maltose, lactose, xylose, and arabinose, respectively. The data were fitted by nonlinear regression to v = V max S/(K m + S), where S is substrate concentration and K m is the concentration giving half maximum velocity. The values were determined by a curve-fitting computer program, EZ-FIT (Perella 1988). Determination of Protein and CarbohydrateThe protein concentration ...

Effect of Enzymatic Preparations and Baking Methods on Selected Parameters of Wheat-Oat Bread

“…Ich zadaniem jest wzmocnienie siatki glutenowej, osłabionej działaniem niskich temperatur. Najczęściej używana jest oksydaza glukozowa [15,30]. W wyniku działania tego enzymu w cieście powstaje nadtlenek wodoru, który powoduje utlenia-nie wolnych grup hydrosulfidowych (-SH) cysteiny do mostków disulfidowych (-S-S-) cystyny [1,31].…”

Dough rheology and bread quality of supplemented flours Reología de la masa y calidad del pan de harinas suplementadas

“…The addition of both an oxidative enzyme, such as glucose oxidase and the ascorbic acid determined the opposite effect. The strengthening action of glucose oxidase (especially at the highest concentration) was related to the hydrogen peroxide produced during reaction that promoted the formation of disulphide linkages in gluten protein (Poulsen & Hostrup, 1998). The mechanism proposed for the improver action of the ascorbic acid (L-threo-ascorbic acid, in particular) is based on the assumption that the dehydroascorbic acid formed inhibits the cleavage of the intermolecular SS bonds of gluten proteins that happens in presence of reduced glutathione (Grosch & Wieser, 1999).…”

“…Gluten cross-linking enzymes improve the ability of dough to entrap gas and the bread crumb texture and volume promoting the formation of covalent bonds between polypeptide chains (Gujral & Rosell, 2004) through mechanisms such as oxidative coupling of thiols, cross-link of tyrosine residues mediated by hydrogen peroxide and acyltransfers between aminoacid residues (Gerrard, 2002). When added to a dough, oxidant enzymes such as hexose oxidases induce the formation of disulphide bridges between proteins and the gelation of pentosans, increasing dough strength and bread volume (Poulsen & Hostrup, 1998). Amylases, pentosanases, xylanases are hydrolytic enzymes able to change physicochemical and structural properties of polysaccharides, making dough softer and viscous and increasing the availability of fermentable sugars.…”

Enhancing the Rheological Performance of Wheat Flour Dough with Glucose Oxidase, Transglutaminase or Supplementary Gluten

“…At low GO concentrations, the strain-hardening index tends to increase because of the dough's increased resistance to flow at larger strains, while the viscosity curve at small strains remains relatively unaffected. Low GO concentrations have indeed been reported to be beneficial for the bread volume (Poulsen and Høstrup 1998;Vemulapalli et al 1998;Bonet et al 2006;Dagdelen and Gocmen 2007), and this improvement is clearly reflected in the strain-hardening index as its value increases significantly (from 17.0 ± 1.6 to 23.3 ± 4.5). By contrast, the use of excessive amounts of GO (> 0.015 -0.100 U/g flour, depending on the flour characteristics) often proves counterproductive, as the gluten network becomes too stiff to allow a proper leavening of the dough (Bonet et al 2006;Steffolani et al 2010;Decamps et al 2012).…”

“…The GO enzyme is able to alter the structure and functional properties of the gluten network by creating additional covalent disulphide cross-links through the intermediary action of H 2 O 2 (Poulsen and Høstrup 1998;Decamps et al 2013). In the presence of O 2 , GO catalyses the oxidation of β-D-glucose into gluconic acid and H 2 O 2 (Bankar et al 2009).…”