| 1. |
- Anasontzis, George E., et al.
(författare)
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Metabolic Engineering of Fusarium oxysporum to Improve Its Ethanol-Producing Capability
- 2016
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Fusarium oxysporum is one of the few filamentous fungi capable of fermenting ethanol directly from plant cell wall biomass. It has the enzymatic toolbox necessary to break down biomass to its monosaccharides and, under anaerobic and microaerobic conditions, ferments them to ethanol. Although these traits could enable its use in consolidated processes and thus bypass some of the bottlenecks encountered in ethanol production from lignocellulosic material when Saccharomyces cerevisiae is used-namely its inability to degrade lignocellulose and to consume pentoses-two major disadvantages of F. oxysporum compared to the yeast-its low growth rate and low ethanol productivity-hinder the further development of this process. We had previously identified phosphoglucomutase and transaldolase, two major enzymes of glucose catabolism and the pentose phosphate pathway, as possible bottlenecks in the metabolism of the fungus and we had reported the effect of their constitutive production on the growth characteristics of the fungus. In this study, we investigated the effect of their constitutive production on ethanol productivity under anaerobic conditions. We report an increase in ethanol yield and a concomitant decrease in acetic acid production. Metabolomics analysis revealed that the genetic modifications applied did not simply accelerate the metabolic rate of the microorganism; they also affected the relative concentrations of the various metabolites suggesting an increased channeling toward the chorismate pathway, an activation of the γ-aminobutyric acid shunt, and an excess in NADPH regeneration
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| 2. |
- Christakopoulos, Paul, et al.
(författare)
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Enhancement of pH-stability of a low molecular mass endoglucanase from Fusarium oxysporum by protein pegylation
- 1998
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Ingår i: Carbohydrate Research. - 0008-6215 .- 1873-426X. ; 314:1-2, s. 95-99
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Tidskriftsartikel (refereegranskat)abstract
- The stability of the low molecular mass endoglucanase (23.2 kDa) from Fusarium oxysporum at alkaline pH was enhanced by chemical modification. Two distinct types of amino acid-specific modifiers were used. The first, either cyanuric chloride activated polyethylene glycol (CC–PEG) or polyethylene glycol succinimidyl succinate active ester (SS–PEG), react (more or less specifically) with protein amino groups. The second type, maleimide polyethylene glycol (Mal–PEG), is specific for cysteinyl residues. The enzyme lost almost all of its activity when modified with CC–PEG, whereas no inactivation was observed with SS–PEG and Mal–PEG. The modified endoglucanase showed remarkably enhanced alkaline pH stability. When acting upon cello-oligosaccharides and 4-methylumbelliferyl cello-oligosaccharides, the enzyme preferentially cleaved the internal glycosidic bonds. The modified enzymes mediated a decrease in the viscosity of carboxymethyl cellulose (CMC) associated with the release of only small amounts of reducing sugar. Thus, the modified enzyme retains the endo character of the native enzyme
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| 3. |
- Christakopoulos, Paul, et al.
(författare)
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Purification and mode of action of an alkali-resistant endo-1,4-β-glucanase from Bacillus pumilus
- 1999
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 364:1, s. 61-66
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Tidskriftsartikel (refereegranskat)abstract
- Alkaline endo-1,4-β-d-glucanase was secreted byBacillus pumilusgrown in submerged culture on a combination of oat spelt xylan and corn starch as carbon sources. The enzyme was purified to homogeneity by Sephacryl S-200 and Q-Sepharose column chromatography. The protein corresponded to molecular mass and pIvalues of 67 kDa and 3.7, respectively. The enzyme was optimally active at pH 7.0–8.0 and 60°C and retained 50% of its optimum activity at pH 12. The most notable characteristic of the endoglucanase was its high stability up to pH 12 for 20 h at 30°C. The enzyme hydrolyzed carboxymethylcellulose (CMC) and cello-oligosaccharides but was inactive on cellobiose, cellotriose, Avicel, xylan, 4-nitrophenyl-β-d-glucoside, 4-nitrophenyl-β-d-cellobioside, and 4-nitrophenyl-β-d-xyloside. Analysis of reaction mixtures by HPLC revealed that the enzyme produced almost exclusively cellotriose when acted on CMC and appeared to hydrolyze cello-oligosaccharides by successively releasing cellotriose. The use of 4-methylumbelliferyl cello-oligosaccharides and the determination of bond cleavage frequency revealed that the enzyme preferentially hydrolyzed the third glycosidic bond adjacent to the glycon. The enzyme mediated a decrease in the viscosity of CMC associated with a release of only small amounts of reducing sugar. The enzyme activity was not inhibited by metal ions, surfactants, and chelating agents used as components of laundry detergents.
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| 4. |
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| 5. |
- Hatzinikolaou, Dimitris G., et al.
(författare)
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Cell bound and extracellular glucose oxidases from Aspergillus niger BTL : evidence for a secondary glycosylation mechanism
- 2007
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Ingår i: Applied Biochemistry and Biotechnology. - : Springer Science and Business Media LLC. - 0273-2289 .- 1559-0291. ; 142:1, s. 29-43
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Tidskriftsartikel (refereegranskat)abstract
- Two glucose oxidase (GOX) isoforms where purified to electrophoretic homogeneity from the mycelium extract (GOXI) and the extracellular medium (GOXII) of Aspergillus niger BTL cultures. Both enzymes were found to be homodimers with nonreduced molecular masses of 148 and 159 kDa and pI values of 3.7 and 3.6 for GOXI and GOXII, respectively. The substrate specificity and the kinetic characteristics of the two GOX forms, as expressed through their apparent Km values on glucose, as well as pH and T activity optima, were almost identical. The only structural difference between the two enzymes was in their degrees of glycosylation, which were determined equal to 14.1 and 20.8% (w/w) of their molecular masses for GOXI and GOXII, respectively. The above difference in the carbohydrate content between the two enzymes seems to influence their pH and thermal stabilities. GOXII proved to be more stable than GOXI at pH values 2.5, 3.0, 8.0, and 9.0. Half-lives of GOXI at pH 3.0 and 8.0 were 8.9 and 17.5 h, respectively, whereas the corresponding values for GOXII were 13.5 and 28.1 h. As far as the thermal stability is concerned, GOXII was also more thermostable than GOXI as judged by the deactivation constants determined at various temperatures. More specifically, the half-lives of GOXI and GOXII, at 45°C, were 12 and 49 h, respectively. These results suggest A. niger BTL probably possesses a secondary glycosylation mechanism that increases the stability of the excreted GOX.
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| 6. |
- Hatzinikolaou, Dimitris G., et al.
(författare)
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Modeling of the simultaneous hydrolysis-ultrafiltration of whey permeate by a thermostable beta-galactosidase from Aspergillus niger
- 2005
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Ingår i: Biochemical engineering journal. - : Elsevier BV. - 1369-703X .- 1873-295X. ; 24:2, s. 161-172
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Tidskriftsartikel (refereegranskat)abstract
- A wild type strain of Aspergillus niger, denoted as BTL, produced elevated levels of β-galactosidase when grown in a low cost medium that contained wheat bran as the sole carbon and energy source. The enzyme was collected, concentrated and partially purified from the culture supernatant. Its kinetic and stability properties were thoroughly examined towards its potential use for the hydrolysis of acid whey permeate lactose. The β-galactosidase of A. niger BTL showed increased pH and thermal stability, with activation energy for the first order deactivation constant equal to 180 kJ/mol at pH 3.5. Lactose hydrolysis by the enzyme was described by Michaelis–Menten kinetics with competitive inhibition only from galactose. An integrated process, concerning the simultaneous hydrolysis–ultrafiltration of whey lactose that incorporated the specific kinetic properties of the β-galactosidase was developed and modeled. The model proved very successful in predicting the behavior of a continuous laboratory hydrolysis–ultrafiltration set up, specifically designed for that purpose. The validated model was finally used in a number of computer simulations in order to investigate the effect of the various process parameters on the overall system performance.
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| 7. |
- Mamma, Diomi, et al.
(författare)
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Biochemical characterization of the multi-enzyme system produced by Penicillium decumbens grown on rutin
- 2004
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Ingår i: Food biotechnology. - 0890-5436 .- 1532-4249. ; 18:1, s. 1-18
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Tidskriftsartikel (refereegranskat)abstract
- Penicillium decumbens produced a set of enzymes, including a monoxygenase and two glycosidases, which degrade rutin, a nontoxic flavonoid glycoside, to water-soluble products. The monoxygenase (quercetinase) cleaves the heterocyclic ring in quercetin, the aglycone part of rutin. The glycosidases (alpha-L-rhamnosidase and beta-glucosidase) hydrolyze the bonds between quercetin and rutinose, and between glucose and rhamnose, the constituent monosaccharides of rutinose. Simultaneous production of the three enzymes was optimized following the examination of a number of culture conditions. Maximum enzyme activities were observed when the fungus was grown at 30 °C with an initial pH of 7.0, using 8.0 g/L rutin and 9.0 g/L di-ammonium hydrogen phosphate as carbon and nitrogen sources, respectively. The enzymes were purified to electrophoretic homogeneity by a series of consecutive chromatographic steps including anion and cation exchange as well as gel filtration. The purified quercetinase revealed an apparent tetrameric structure, with a reduced molecular mass of 45 kDa. alpha-L-Rhamnosidase showed an apparent molecular mass of 58 kDa and the purified beta-glucosidase was a tetramer exhibiting a reduced molecular mass of 120 kDa.
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| 8. |
- Mamma, Diomi, et al.
(författare)
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Biodegradation of phenol by acclimatized Pseudomonas putida cells using glucose as an added growth substrate
- 2004
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Ingår i: Journal of Environmental Science and Health. Part A. - 1093-4529 .- 1532-4117. ; 39:8, s. 2093-2104
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Tidskriftsartikel (refereegranskat)abstract
- Biodegradation of phenol, a pollutant derived from many industrial processes, was achieved through acclimatized Pseudomonas putida cells. The strategy to overcome the inhibitory effect of phenol on microbial growth involved the addition of glucose, a conventional carbon source. A factorial experimental design was employed in order to optimize the initial phenol and glucose concentrations. The optimum conditions found were applied in 2-lt bioreactors. The development of acclimatized cells and the use of glucose as an added growth substrate resulted in a significant phenol degradation rate of 60.7 mg L−1 h−1 with a complete removal of 1200 mg L−1 phenol.
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| 9. |
- Kourtoglou, Elisavet, et al.
(författare)
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Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum
- 2011
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Ingår i: Enzyme and microbial technology. - : Elsevier BV. - 0141-0229 .- 1879-0909. ; 48:3, s. 217-224
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Tidskriftsartikel (refereegranskat)abstract
- The phosphoglucomutase gene from a wild type Fusarium oxysporum strain (F3), was homologously expressed, under the control of the constitutive promoter of gpdA of Aspergillus nidulans. The transformant produced elevated levels of phosphoglucomutase activity compared to the wild type, a fact that facilitated the subsequent purification procedure. The enzyme (FoPGM) was purified to homogeneity applying three anion exchange and one gel filtration chromatography steps. The native enzyme revealed a monomeric structure with a molecular mass of 60 kDa, while the isoelectric point was 3.5. FoPGM was active in pH ranged from 6.0 to 8.0, with an optimum using 3-(N-morpholino)propanesulfonic acid buffer at 7.0, while loss of activity was observed when phosphate buffer was used in the above mentioned pH range. The optimal temperature for activity was 45 °C but the enzyme became unstable at temperatures above 40 °C. FoPGM requires the presence of a divalent cation for its function with maximum activity being obtained with Co2+. The apparent Km for Co2+ was found to be 10 μM. The enzyme was also active with other divalent metal ions such as Mn2+, Mg2+, Ni2+ and Ca2+ but to a lesser extent. The following kinetic constants were determined: vmax, 0.74 μmol mgprotein−1 min−1; kcat, 44.2 min−1; Km(G1P), 0.10 mM; Km(G1,6diP), 1.03 μM; kcat/Km(G1P), 443 mM−1 min−1 and kcat/Km(G1,6diP), 42,860 mM−1 min−1. The enzyme was considered to follow a Ping Pong substituted enzyme or enzyme isomerization mechanism.
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| 10. |
- Mamma, Diomi, et al.
(författare)
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Biochemical and catalytic properties of two intracellular β-glucosidases from the fungus Penicillium decumbens active on flavonoid glucosides
- 2004
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Ingår i: Journal of Molecular Catalysis B. - : Elsevier BV. - 1381-1177 .- 1873-3158. ; 27:4-6, s. 183-190
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Tidskriftsartikel (refereegranskat)abstract
- In the presence of rutin as sole carbon source, Penicillium decumbens produces two intracellular β-glucosidases named GI and GII, with molecular masses of 56,000 and 460,000 Da, respectively. The two proteins have been purified to homogeneity. GI and GII composed of two and four equal sub-units, respectively and displayed optimal activity at pH 7.0 and temperature 65–75 °C. Both β-glucosidases were competitively inhibited by glucose and glucono-δ-lactone. GI and GII exhibited broad substrate specificity, since they hydrolyzed a range of (1,3)-, (1,4)- and (1,6)-β-glucosides as well as aryl β-glucosides. Determination of kcat/Km revealed that GII hydrolyzed 3–8 times more efficiently the above-mentioned substrates. The ability of GI and GII to deglycosylate various flavonoid glycosides was also investigated. Both enzymes were active against flavonoids glycosylated at the 7 position but GII hydrolyzed them 5 times more efficiently than GI. Of the flavanols tested, both enzymes were incapable of hydrolyzing quercetrin and kaempferol-3-glucoside. The main difference between GI and GII as far as the hydrolysis of flavanols is concerned, was the ability of GII to hydrolyze the quercetin-3-glucoside.
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