Fermentation of a Yeast Producing A. Niger Glucose Oxidase: Scale-Up, Purification and Characterization of the Recombinant Enzyme

Baetselier, Annie De; Vasavada, Amit; Dohet, P.; Ha-Thi, V.; Beukelaer, Michèle De; Erpicum, T.; Clerck, Laura De; Hanotier, J.; Rosenberg, Steven

doi:10.1038/nbt0691-559

Cited by 47 publications

(18 citation statements)

References 12 publications

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“…In our study, the level of expression of GLO under control of the GAP promoter was five to six times lower (Table 1), and the level of expression obtained with the GAP promoter when glycerol was used as a carbon source was approximately 10 times higher than the level of expression obtained when glucose was used as a carbon source in an otherwise similar medium. Previously published findings suggest that the GOD gene can be expressed in yeast quite efficiently (4,8,15), and this suggestion was supported by our study. We observed almost 10-fold-higher levels of GOD (30 to 40 mg/liter) than of GLO (4 to 5 mg/liter) when the two genes were expressed under control of the same promoter (GAP) and under the same cultivation conditions.…”

Section: Resultssupporting

confidence: 80%

Cloning and Characterization of Gluconolactone Oxidase of Penicillium cyaneo-fulvum ATCC 10431 and Evaluation of Its Use for Production of d -Erythorbic Acid in Recombinant Pichia pastoris

Salusjärvi¹,

Kalkkinen

Miasnikov³

2004

Appl Environ Microbiol

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A D-erythorbic acid-forming soluble flavoprotein, gluconolactone oxidase (GLO), was purified from Penicillium cyaneo-fulvum strain ATCC 10431 and partially sequenced. Peptide sequences were used to isolate a cDNA clone encoding the enzyme. The cloned gene (accession no. AY576053) exhibits high levels of similarity with the genes encoding other known eukaryotic lactone oxidases and also with the genes encoding some putative prokaryotic lactone oxidases. Analysis of the coding sequence of the GLO gene indicated the presence of a typical secretion signal sequence at the N terminus of GLO. No other targeting or anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria. Experimental evidence, including the N-terminal sequence of mature GLO and data on glycosylation and localization of the enzyme in native and recombinant hosts, supports this analysis. The GLO gene was expressed in Pichia pastoris, and recombinant GLO was produced by using the strong methanol-induced AOX1 promoter. In order to evaluate the suitability of purified GLO for production of D-erythorbic acid, we immobilized it on N-hydroxysuccinimide-activated Sepharose and found that the immobilized GLO retained full activity during immobilization but was rather unstable under reaction conditions. Our results show that both soluble and immobilized forms of GLO can, in principle, be used for production of D-erythorbic acid from D-glucono-␦-lactone or (in combination with glucose oxidase and catalase) from glucose. We also demonstrated the feasibility of glucose-D-erythorbic acid fermentation with recombinant strains coexpressing GLO and glucose oxidase genes, and we analyzed problems associated with construction of efficient D-erythorbic acid-producing hosts.D-Erythorbic acid (D-EA) (D-araboascorbic acid, isovitamin C) is a C-5 epimer of L-ascorbic acid. It has chemical properties very similar to those of ascorbic acid but very low vitamin C activity. D-EA and erythorbates have been used as food antioxidants for many years, particularly in processed meat and soft drinks. Currently, D-EA is manufactured by a two-step process; glucose is first converted into D-2-ketogluconic acid by fermentation, and this is followed by chemical lactonization similar to the lactonization of L-2-ketogulonic acid in the Reichstein process. In the early 1960s, direct conversion of glucose into D-EA by Penicillium fungi was discovered (48). This discovery was followed by an extensive mutagenesis and selection program that resulted in strains capable of converting glucose into D-EA with a yield of approximately 40% over a week-long fermentation (44, 56). These process parameters are still not sufficient to make the direct fermentation of glucose into D-EA economically feasible. In the last decade, much research effort has been spent on development of biotechnology-based processes for L-ascorbic acid manufacture (12,40). Most of the studies aimed at a two-s...

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Section: Resultssupporting

confidence: 80%

Cloning and Characterization of Gluconolactone Oxidase of Penicillium cyaneo-fulvum ATCC 10431 and Evaluation of Its Use for Production of d -Erythorbic Acid in Recombinant Pichia pastoris

Salusjärvi¹,

Kalkkinen

Miasnikov³

2004

Appl Environ Microbiol

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“…Therefore medium dilution to reduce salt concentrations [13] or ultrafiltration with following dialysis [17] or adsorption on quartz sand or Al 2 O 3 [18] is sufficient to condition a sample for the IEC. There is a method that solely uses filtration techniques (cross-flow filtration, microfiltration, ultrafiltration and diafiltration) without resorting to chromatographic methods to recover the rGOx from large volume batch fermentation [19,20]. Thus, this approach is time consuming although is compromisingly efficient and capable of treating large amounts of fermentation medium.…”

Section: Introductionmentioning

confidence: 99%

Purification of glucose oxidase from complex fermentation medium using tandem chromatography

Zakhartsev

Momeu

2007

Journal of Chromatography B

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“…Overexpression of proteins was accomplished following Frederick et al (23). The Ϸ320-kDa WT and H516A, purified as described (24,25), exhibited identical migration patterns on SDS and native 7.5% polyacrylamide gels.…”

mentioning

confidence: 99%

Catalysis of electron transfer during activation of O ₂ by the flavoprotein glucose oxidase

Roth

Klinman

2002

Proc. Natl. Acad. Sci. U.S.A.

172

281

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Two prototropic forms of glucose oxidase undergo aerobic oxidation reactions that convert FADH ؊ to FAD and form H2O2 as a product. Limiting rate constants of kcat͞KM(O2) ‫؍‬ (5.7 ؎ 1.8) ؋ 10 2 M ؊1 ⅐s ؊1 and kcat͞KM(O2) ‫؍‬ (1.5 ؎ 0.3) ؋ 10 6 M ؊1 ⅐s ؊1 are observed at high and low pH, respectively. Reactions exhibit oxygen-18 kinetic isotope effects but no solvent kinetic isotope effects, consistent with mechanisms of rate-limiting electron transfer from flavin to O2. Site-directed mutagenesis studies reveal that the pH dependence of the rates is caused by protonation of a highly conserved histidine in the active site. Temperature studies (283-323 K) indicate that protonation of His-516 results in a reduction of the activation energy barrier by 6.0 kcal⅐mol ؊1 (0.26 eV). Within the context of Marcus theory, catalysis of electron transfer is attributed to a 19-kcal⅐mol ؊1 (0.82 eV) decrease in the reorganization energy and a much smaller 2.2-kcal⅐mol ؊1 (0.095 eV) enhancement of the reaction driving force. An explanation is advanced that is based on changes in outer-sphere reorganization as a function of pH. The active site is optimized at low pH, but not at high pH or in the H516A mutant where rates resemble the uncatalyzed reaction in solution. F lavins are highly versatile enzyme cofactors that undergo electron and proton-coupled electron transfer reactions (1-3). As a result, flavoenzymes are involved in an array of chemical and photochemical processes from COH oxidations (4) to electron transport (5) to repair of cross-linked DNA (6). Glucose oxidase (GO) is a homodimeric protein found predominantly in fungi (National Center for Biotechnology Information, www.ncbi.nlm.nih.gov). Each protein subunit contains an equivalent of noncovalently bound FAD Ϸ15 Å below the surface (7). GO mediates net hydride transfer from the anomeric COH bond of glucose to FAD in the reductive half-reaction (8, 9) and the oxidation of reduced cofactor (FADH Ϫ ) by O 2 in the oxidative half-reaction, forming H 2 O 2 as a product. All evidence points toward a rate-limiting electron transfer step during FADH Ϫ oxidation as shown in Eq. 1 (10). This reaction involves the transfer of negative charge from cofactor to superoxide ion with no net change in charge at the active site.To date, most mechanistic studies of O 2 activation have focused on metalloenzymes. In such reactions, electron transfer and electrostatic stabilization often occur within a single step (ref. 11 and references therein), causing rates to approach the diffusion limit (12). Additionally, ligands control metal coordination geometries and modulate redox potentials, thereby tuning reactivity toward O 2 (13). Enzymes that use organic cofactors do not enjoy such advantages, but may use specialized protein environments to help overcome the kinetic and thermodynamic barriers associated with activation of O 2 .The physical characterization of the protein dielectric is an area of growing interest (14-18) and may provide the key to understanding how proteins like GO facilita...

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Fermentation of a Yeast Producing A. Niger Glucose Oxidase: Scale-Up, Purification and Characterization of the Recombinant Enzyme

Cited by 47 publications

References 12 publications

Cloning and Characterization of Gluconolactone Oxidase of Penicillium cyaneo-fulvum ATCC 10431 and Evaluation of Its Use for Production of d -Erythorbic Acid in Recombinant Pichia pastoris

Cloning and Characterization of Gluconolactone Oxidase of Penicillium cyaneo-fulvum ATCC 10431 and Evaluation of Its Use for Production of d -Erythorbic Acid in Recombinant Pichia pastoris

Purification of glucose oxidase from complex fermentation medium using tandem chromatography

Catalysis of electron transfer during activation of O ₂ by the flavoprotein glucose oxidase

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Fermentation of a Yeast Producing A. Niger Glucose Oxidase: Scale-Up, Purification and Characterization of the Recombinant Enzyme

Cited by 47 publications

References 12 publications

Cloning and Characterization of Gluconolactone Oxidase of Penicillium cyaneo-fulvum ATCC 10431 and Evaluation of Its Use for Production of d -Erythorbic Acid in Recombinant Pichia pastoris

Cloning and Characterization of Gluconolactone Oxidase of Penicillium cyaneo-fulvum ATCC 10431 and Evaluation of Its Use for Production of d -Erythorbic Acid in Recombinant Pichia pastoris

Purification of glucose oxidase from complex fermentation medium using tandem chromatography

Catalysis of electron transfer during activation of O 2 by the flavoprotein glucose oxidase

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Product

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Catalysis of electron transfer during activation of O ₂ by the flavoprotein glucose oxidase