Thermostable variants of pyranose 2-oxidase showing altered substrate selectivity for glucose and galactose

Spadiut, Oliver,Nguyen, Tien-Thanh,Haltrich, Dietmar

, p. 3465 - 3471 (2010)

The homotetrameric flavoprotein pyranose 2-oxidase (P2Ox) has several proposed biotechnological applications, among others as a biocatalyst for carbohydrate transformations toward higher-value products. To improve some of the catalytic properties of P2Ox from Trametes multicolor, we selected a semirational enzyme engineering approach, namely, saturation mutagenesis of the amino acid His 450 located at a pivotal point of the active site loop and subsequent screening of the libraries thus obtained for improved activity with the sugar substrate D-galactose. A variant with improved catalytic characteristics identified was H450G, which showed a significant, 3.6-fold decrease In KM together with a 1.4-fold increase in κcat for its substrate D-galactose and an overall improvement in the catalytic efficiency by a factor of 5. By combining H450G with other amino acid replacements, we obtained the P2Ox variants H450G/V546C and H450G/E542K/V546C, which can be of interest for applications in food industry due to their increased activity with D-galactose, high activity with D-glucose, and considerably increased stability for the latter variant. While the His-tagged recombinant wild-type enzyme strongly prefers D-glucose to D-galactose as its substrate, H450G/E542K/V546C converts both sugars, which are found in lactose hydrolysates, concomitantly, as was shown by laboratory-scale biotransformation experiments. The 2-keto sugars thus obtained can conveniently be reduced to the corresponding ketoses D-fructose and D-tagatose. 2010 American Chemical Society.

CONVENIENT, LABORATORY PROCEDURE FOR PRODUCING SOLID D-arabino HEXOS-2-ULOSE (D-GLUCOSONE)

Liu, Te-ning E.,Wolf, Beverly,Geigert, John,Neidleman, Saul L.,Chin, Janice D.,Hirano, David S.

, p. 151 - 158 (1983)

The production of solid D-arabino-hexos-2-ulose (D-glucosone) from D-glucose by use of an enzyme, pyranose-2-oxidase (EC 1.1.3.10), is described.The enzyme is extracted from the mycelia of Polyporus obtusus, partially purified, and then immobilized on activated CH-Sepharose 4B.The enzymic conversion of D-glucose into D-glucosone is simple and convenient, and provides a product free from residual D-glucose.Lyophilization of the filtered reaction-solution yields the product, solid D-glucosone.Assay methods have been developed for monitoring the enzymic reaction and evaluating the purity of the final product.

CLEAVAGE OF D-arabino-HEXOS-2-ULOSE AND GLYOXAL WITH HYDROGEN PEROXIDE

Vuorinen, Tapani

, p. 319 - 326 (1984)

The kinetics of the cleavage of D-arabino-hexos-2-ulose (1) and of glyoxal (2) with hydrogen peroxide in alkaline water and in 44percent (w/w) ethanol-water solutions (pOH 0.5-5) were studied over a temperature range of -25 to +25 deg C.The relative rate of the competing reactions of 1 with the cleavage in 0.03-1M sodium hydroxide was determined from the rate of formation of hydrogen peroxide in the oxidation of D-glucose to 1 with 2-anthraquinonesulfonic acid in the presence of oxygen at 25 and 40 deg C.The cleavages of both 1 and 2 were firs-order with respect to hydrogen peroxide, and also to hydroxyl ion at low alkalinities.The rate of cleavage of 1 reached a maximum at pOH ca. 2.5, whereas the competing reactions of 1 and the cleavage of 2 were constantly accelerated with increasing hydroxyl-ion concentration.Unlike 2, compound 1 was cleaved more rapidly in ethanol-water than in water.The activation energies of the cleavage of 1 and 2, and the competing reactions of 1, were 49, 57, and 65 kJ.mol-1, respectively.

Pina,Tatum

, p. 265 (1967)

AN IMPROVED PREPARATION OF 3-DEOXY-D-erythro-HEXOS-2-ULOSE VIA THE BIS(BENZOYLHYDRAZONE) AND SOME RELATED CONSTITUTIONAL STUDIES

Madson, Michael A.,Feather, Milton S.

, p. 183 - 192 (1981)

Sugar osazones and glycosuloses rapidly and quantitatively react with hydroxylamine to produce oximes that give trimethylsilyl derivatives suitable for g.l.c. and mass spectral analysis.The reaction of D-glucose with benzoylhydrazine to give the bishydrazone of 3-deoxy-D-erythro-hexos-2-ulose (1) was re-investigated, together with the conversion of this compound to the hexosulose.Although by-products are produced in the reaction, including the bis(benzoylhydrazone) (osazone) of D-glucose, the major product is the monohydrate of the bis(benzoylhydrazone) of 1 (colorless).The anhydrous (yellow) form can be prepared from the monohydrate by crystallization from absolute ethanol and has quite different physical properties.Improvements of the original preparation are described that allow the preparation of the bishydrazone and its subsequent conversion to 1 via transhydrazonation in 44percent overall yield, and with no detectable contamination by D-glucose or D-glucosone.Evidence is presented that the previously reported cyclic form of the bis(benzoylhydrazone) of D-glucose is the bis(benzoylhydrazone) (monohydrate) of 1.

Synthesis of 2-Acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole

Sweeny, James G.,Ricks, Edith,Estrada-Valdes, Maria C.,Iacobucci, Guillermo A.,Long, Robert C.

, p. 1133 - 1134 (1985)

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Temperature decrease (30-25 °c) influence on bi-enzymatic kinetics of D-glucose oxidation

Ene, Manuela Diana,Maria, Gheorghe

, p. 19 - 24 (2012)

Previous batch experiments reported by Maria et al. [1] for d-glucose oxidation in the presence of pyranose 2-oxidase (P2Ox from Coriolus sp. expressed in E. coli) and catalase at 30 °C and optimal pH = 6.5 have been extended to a lower temperature of 25 °C. This modification influences the process performance in different ways, leading to a higher activity of catalase for decomposing H2O2 by-product, thus maintaining its concentration to negligible levels. While the presence of catalase has a favourable effect at 30 °C on prolonging P2Ox life-time, a quick P2Ox inactivation is observed at 25 °C due to the high levels of the resulted oxidative intermediates. While the P2Ox activity does not vary too much in the range of 25-30 °C, a significant decline of the main reaction rate with the increase of catalase/P2Ox ratio is reported for both temperatures. Estimated rate constants of a proposed kinetic model are compared to the literature data, being used to predict the favourable operating conditions for this complex bi-enzymatic system.

Integration of Enzymatic and Heterogeneous Catalysis for One-Pot Production of Fructose from Glucose

Sun, Jiankui,Li, Helong,Huang, Hongzhi,Wang, Bo,Xiao, Ling-Ping,Song, Guoyong

, p. 1157 - 1162 (2018/03/21)

The search for efficient routes for the production of fructose from biomass-derived glucose is of great interest and importance, as fructose is a highly attractive substrate in the conversion of cellulosic biomass into biofuels and chemicals. In this study, a one-pot, multistep procedure involving enzyme-catalyzed oxidation of glucose at C2 and Ni/C-catalyzed hydrogenation of d-glucosone at C1 selectively gives fructose in 77 % yield. Starting from upstream substrates such as α-cellulose and starch, fructose was also generated with similar efficiency and selectivity by the combination of enzymatic and heterogeneous catalysis. This method constitutes a new means of preparing fructose from biomass-derived substrates in an efficient fashion.

Enzymatic Cascade Catalysis for the Synthesis of Multiblock and Ultrahigh-Molecular-Weight Polymers with Oxygen Tolerance

Liu, Zhifen,Lv, Yue,An, Zesheng

supporting information, p. 13852 - 13856 (2017/10/09)

Synthesis of well-defined multiblock and ultrahigh-molecular-weight (UHMW) polymers has been a perceived challenge for reversible-deactivation radical polymerization (RDRP). An even more formidable task is to synthesize these extreme polymers in the presence of oxygen. A novel methodology involving enzymatic cascade catalysis is developed for the unprecedented synthesis of multiblock polymers in open vessels with direct exposure to air and UHMW polymers in closed vessels without prior degassing. The success of this methodology relies on the extraordinary deoxygenation capability of pyranose oxidase (P2Ox) and the mild yet efficient radical generation by horseradish peroxidase (HRP). The facile and green synthesis of multiblock and UHMW polymers using biorenewable enzymes under environmentally benign and scalable conditions provides a new pathway for developing advanced polymer materials.