| 1. |
- Dimarogona, M, et al.
(författare)
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The structure of a GH10 xylanase from Fusarium oxysporum reveals the presence of an extended loop on top of the catalytic cleft
- 2012
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 68:7, s. 735-742
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Tidskriftsartikel (refereegranskat)abstract
- Xylanase enzymes have been the focus of considerable research in recent decades owing to their extensive use in a variety of biotechnological applications. Previous structural studies of a number of GH10 xylanases revealed that all GH10 family members have the (β/α)8-barrel fold and their catalytic site is conserved. The structure of a new GH10 xylanase from Fusarium oxysporum (FoXyn10a) was determined at 1.94 Å resolution from crystals belonging to the tetragonal space group P41212 with five molecules per asymmetric unit. Comparison of the structure of FoXyn10a with previously determined structures of GH10 family members indicated that most of the differences were located in the loop regions between the ordered secondary-structure elements of the barrel, as expected. However, alignment of FoXyn10a with sequence and structural homologues denoted an atypically long loop connecting strand β6b and helix 6 that was only present in one other GH10 xylanase, the structure of which is not known. This structural feature may be of functional importance, with potential implications in the catalytic efficiency of the enzyme.
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| 2. |
- Kalogeris, E, et al.
(författare)
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Performance of an intermittent agitation rotating drum type bioreactor for solid-state fermentation of wheat straw
- 2003
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Ingår i: Bioresource Technology. - 0960-8524 .- 1873-2976. ; 86:3, s. 207-213
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Tidskriftsartikel (refereegranskat)abstract
- A laboratory bioreactor, designed for solid-state fermentation of thermophilic microorganisms, was operated for production of cellulases and hemicellulases by the thermophilic fungus Thermoascus aurantiacus. The suitability of the apparatus for the effective control of important operating variables affecting growth of microbes in solid-state cultivation was determined. Application of the optimum conditions found for the moisture content of the medium, growth temperature and airflow rate produced enzyme yields of 1709 U endoglucanase, 4 U cellobiohydrolase, 79 U β-glucosidase, 5.5 U FPA, 4490 U xylanase and 45 U β-xylosidase per g of dry wheat straw. The correlation between microorganism growth and production of enzymes was efficiently described by the Le Duy kinetic model. © 2002 Elsevier Science Ltd. All rights reserved.
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| 3. |
- Kourtoglou, E., et al.
(författare)
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Purification, characterization and mass spectrometric sequencing of transaldolase from Fusarium oxysporum
- 2008
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Ingår i: Process Biochemistry. - : Elsevier BV. - 1359-5113 .- 1873-3298. ; 43:10, s. 1094-1101
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Tidskriftsartikel (refereegranskat)abstract
- Transaldolase (FoTal) was purified to homogeneity from the fungus Fusarium oxysporum. The native enzyme revealed a monomeric structure with molecular mass of 36 kDa. This FoTal depicted an optimal pH of 7.5 using imidazole buffer, while loss of activity was observed with Tris/HCl buffer. The optimal temperature was between 40 and 45 °C and the enzyme became unstable at temperatures above 50 °C. The isoelectric point of the purified enzyme was 4.5. The kinetics of the purified enzyme is consistent with a Ping Pong mechanism. The Km values for d-erythrose-4-phosphate and d-fructose-6-phosphate were 0.49 and 6.66 mM, while the kcat values were estimated at 4114 and 4151 min-1, respectively. LC-MS/MS analysis provided peptide mass and sequence information that facilitated primary structure confirmation, allowing us to identify the FoTal gene (foxg_03074) from the genome of F. oxysporum.
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| 4. |
- Topakas, E., et al.
(författare)
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Bioconversion of ferulic acid into vanillic acid by the thermophilic fungus Sporotrichum thermophile
- 2003
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Ingår i: Lebensmittel-Wissenschaft + Technologie. - 0023-6438 .- 1096-1127. ; 36:6, s. 561-565
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Tidskriftsartikel (refereegranskat)abstract
- Sporotrichum thermophile is capable of promoting the formation of vanillic acid during ferulic acid degradation. Ferulic acid metabolism by S. thermophile apparently occurred via the propenoic chain degradation and the formation of 4-hydroxy-3-methoxystyrene (4-vinylguaiacol) was observed which was presumably metabolized to vanillic acid. Guaiacol was detected in addition to the above-mentioned intermediates, usually as a result of nonoxidative decarboxylation of vanillic acid. The bioconversion of ferulic into vanillic acid was affected by the amount of ferulic acid that was treated and the carbon source on which the biomass was grown. Under optimum conditions vanillic acid production from ferulic acid by S. thermophile attained very high levels of 4798 mg/L with a molar yield of 35%
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| 5. |
- Topakas, E., et al.
(författare)
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Production of phenolics from corn cobs by coupling enzymic treatment and solid state fermentation
- 2004
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Ingår i: Engineering in Life Sciences. - : Wiley. - 1618-0240 .- 1618-2863. ; 4:3, s. 283-286
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Tidskriftsartikel (refereegranskat)abstract
- A two-stage process that combined solid-state fermentation (SSF) and subsequent enzymic treatment was used in order to release p-coumaric (p-CA) and ferulic acid (FA) from corn cobs. Sporotrichum thermophile was grown on corn cobs under SSF conditions, and the production of cinnamoyl esterases and xylanases was studied over 7 days. The time course of enzyme production showed a maximum activity of 1483 nkat/g, 0.3 nkat/g and 0.067 nkat/g for xylanase, feruloyl esterase, and p-coumaroyl esterase, respectively. The importance of the moisture level of the growth substrate was discussed. After SSF, the fermented substrate was directly exposed to autohydrolysis and the in situ produced multienzyme system was successfully used for the partial degradation of cell wall components and the liberation of p-CA and FA. A yield of 0.85 g/kg and 0.38 g/kg FA and p-CA, respectively, of dry matter of corn cobs was obtained.
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| 6. |
- Dimarogona, M., et al.
(författare)
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Lignin boosts the cellulase performance of a GH-61 enzyme from Sporotrichum thermophile
- 2012
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Ingår i: Bioresource Technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; 110, s. 480-487
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Tidskriftsartikel (refereegranskat)abstract
- An enzyme belonging to the glycoside hydrolase family 61 from the thermophilic fungus Sporotrichum thermophile, was functionally expressed in the methylotrophic yeast Pichia pastoris under the transcriptional control of the alcohol oxidase (AOX1) promoter. The enzyme hydrolyzed barley beta-glucan, carboxymethyl cellulose, lichenan, wheat arabinoxylan and birchwood xylan showing optimal activity at pH 8 and 65 degrees C. A 2:1 mixture of Celluclast 1.5 L and StCel61a was capable of increasing the degree of spruce conversion by 42%. The use of substrates with varying lignin content permitted the detection of a dependence of the enhancing capacity of StCel61a on the radical scavenging capacity of the different lignocellulosics. In the presence of a reductant, StCel61a boosted the efficiency of a mixture of purified cellulases (EGII, CBHI, beta-GLUC) by 20%. The synergistic activity exhibited by StCel61a and its dependence on reducing substances provide guidelines for process design towards the production of economically viable bioethanol.
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| 7. |
- 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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| 8. |
- Montanier, Cedric, et al.
(författare)
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The Active Site of a Carbohydrate Esterase Displays Divergent Catalytic and Noncatalytic Binding Functions
- 2009
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Ingår i: PLoS Biology. - : Public Library of Science (PLoS). - 1545-7885. ; 7:3, s. 687-697
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Tidskriftsartikel (refereegranskat)abstract
- Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of "gene sharing,'' where the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the biocatalyst.
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| 9. |
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| 10. |
- Panagiotou, Gianni, 1974, et al.
(författare)
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Identification of NADH kinase activity in filamentous fungi and structural modelling of the novel enzyme from Fusarium oxysporum
- 2008
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Ingår i: Process Biochemistry. - : Elsevier BV. - 1359-5113 .- 1873-3298. ; :43, s. 1114-1120
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Tidskriftsartikel (refereegranskat)abstract
- ATP-NADH kinase phosphorylates NADH to produce NADPH at the expense of ATP. The present study describes Fusarium oxysporum NADH kinase (ATP:NADH 2'-phosphotransferase, EC 2.7.1.86), a novel fungal enzyme capable of synthesizing NADPH using NADH as the preferred diphosphonicotinamide (diphosphopyridine) nucleotide donor. NADH kinase was highly purified (~66-fold) and the enzyme was found to be a homodimeric with a subunit of Mr 72,000. Isoelectric focusing in the pH range of 3.0-9.5 of the purified NADH kinase yielded a pI value of about 5.6. The Km values of NADH kinase for NADH and ATP were found to be 0.13 and 2.59 mM, respectively. Prediction of the secondary structure of the protein was performed in the PSIPRED server while modelling the three-dimensional (3D) structure was accomplished by the use of the HH 3D-structure prediction server.
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