A novel carbohydrate:acceptor oxidoreductase from Microdochium nivale

Abstract

A Microdochium nivale carbohydrate:acceptor oxidoreductase was purified, cloned, heterologously expressed, and characterized. The gene encoding the protein showed one intron, and the ORF showed a sequence with low homology (# 25% identity or 65% similarity) to other known flavin-containing carbohydrate oxidases. The maturation of the protein required the cleavage of a tetrameric propeptide in addition to an 18 amino-acid signal peptide. The enzyme was found to have a relative molecular mass of 55 000 Da, an isoelectric point of 9, and one FAD per protein. It could oxidize mono-, oligo-, or polymeric saccharides, and transfer their electrons to O 2 or other acceptors. When d-glucose served as electrondonating substrate, an activity of 2 s 21 was observed at pH 5.5 and 23 8C. Among various oligosaccharides, the enzyme preferred tetrameric dextrins, indicating a favorable interaction of four linked glucose units with the substrate pocket. The unique structure and ability of oxidizing oligo/ polymeric saccharides suggest a promising prospect of this enzyme for various industrial/medicinal applications. Keywords:oxidoreductase; flavin; carbohydrate; Microdochium.Carbohydrate oxidases are a family of flavin-or Cu-containing enzymes that catalyze the oxidation of the primary or secondary alcohol in various saccharides with the concomitant reduction of O 2 to H 2 O 2 . These enzymes are widely distributed and play important roles in various metabolism steps. Extensive physical and chemical characterizations, including X-ray crystallography and sitedirected mutagenesis, have been carried out to elucidate the fundamental aspects of the structure-function relationship of these enzymes [1±3].Recently, carbohydrate oxidases are receiving increased attention as potential diagnostic reagents or industrial biocatalysts [3±8]. Attractive applications include (a) biotransformation of glycopolymers (glycolipids, glycoproteins, polysaccharides) into desirable materials such as sweeteners, flavourants, or paper strength additives; (b) in situ generation of oxidant H 2 O 2 for uses in food manufacturing, dyeing or bleaching of lignocellulolytic/ keratin materials, detergent, or waste-water treatment; (c) utilization of economic and stable carbohydrates, rather than expensive and unstable NAD(P)H, as electron-donor for various biocatalyses; (d) construction of biosensors for blood sugar, O 2 , or other substances; and (e) biosynthesis of functional/chiral pharmaceutical molecules. The high substrate specificity, mild operation conditions, and environmental-friendliness of bio-oxidative systems hold advantages over conventional chemical systems that are often hard to control, nonspecific, costly, or hazardous.We report here the identification, cloning, expression, and characterization of a novel carbohydrate:acceptor oxidoreductase from Microdochium nivale (MnCO). We found that this flavin-containing enzyme differs significantly from other functional analogs [such as glucose oxidase (GO; EC 1.1.3.4)] as demonstrated by its low sequence...

Structural Analysis of the Catalytic Mechanism and Stereoselectivity in Streptomyces coelicolor Alditol Oxidase,

“…Oxidation of sugars and their derivatives results in changed chemical and physical properties such as altered reductive power, solubility, gel strength, swelling, and chelation characteristics. Oxidized carbohydrates are applied, for example, as thickeners or emulsifiers in the food industry, as water binders in the paper industry, as metal chelators, or as antioxidizing agents in organ preservation (2). These compounds are also used as building blocks in chemical synthesis routes, as precursors for further chemical modification or for conjugation to other biomolecules (3).…”

Optimization of Lactobionic Acid Production byAcetobacter orientalisIsolated from Caucasian Fermented Milk, “Caspian Sea Yogurt”

“…[1][2][3] It has been reported to increase intestinal calcium absorption, 4,5) suggesting that it might be possible to use LacA for health benefit in foods. There have been a number of studies focusing on lactose-oxidizing bacteria, [6][7][8][9][10][11] fungi, [12][13][14][15][16][17] and red algae. 18) Most of these organisms, however, are difficult to use in the production of LacA for foods.…”

“…Therefore, fungal enzymes, carbohydrate: acceptor oxidoreductase, [12][13][14] glucooligosaccharide oxidase, 16,17) and cellobiose dehydrogenase 15) have been studied in connection with the production of LacA. Other bacteria, Pseudomonas aeruginosa 6) and Burkholderia cepacia, [7][8][9] also have been studied for LacA production.…”

Production of Calcium Lactobionate by a Lactose-oxidizing Enzyme from <i>Paraconiothyrium</i> sp. KD-3

“…Some enzymes have been known to oxidize lactose. Such enzymes, cellobiose dehydrogenases (EC 1.1.99.18, CDH), 7,8) cellobiose: quinone oxidoreductases (EC 1.1.5.1, CBQ), 9,10) glucooligosaccharides oxidases (GOOX), 11,12) and carbohydrate: acceptor oxidoreductase (COX), 13) however, have not been studied from a viewpoint of calcium lactobionate production in detail. This may be due to their instability at acidic pH, and or low specificity on O2 as an electron acceptor.…”

“…The Paraconiothyrium enzyme lacking heme in the protein is discriminated from CDH, 7,8) which contains heme. CBQ, 9,10) GOOX 11,12) and COX, 13) however, have similar properties; for instance, a molecular mass of 55 60 kDa, a broad substrate specificity, and the presence of FAD as a prothetic group. On the other hand, CDH and CBQ have similar amino acid sequences.…”