| 1. |
- Christakopoulos, Paul, et al.
(författare)
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Purification and characterization of two low molecular mass alkaline xylanases from Fusarium oxysporum F3
- 1996
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 51:2, s. 181-189
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Tidskriftsartikel (refereegranskat)abstract
- Two low molecular mass endo-1,4-β-d-xylanases from Fusarium oxysporum were purified to homogeneity by gel-filtration and ion-exchange chromatography. They exhibit molecular masses of 20.8 (xylanase I) and 23.5 (xylanase II) kDa, and isoelectric points of 9.5 and 8.45–8.70, respectively. Both xylanases display remarkable pH (9.0) stability. At 40 to 55 °C xylanase II is more thermostable than xylanase I but less active on xylan. In contrast to xylanase I, xylanase II is able to hydrolyze 1-O-4-methylumbelliferyl-(β-d-glucopyranosyl)-β-d-xylopyranoside (muxg). Neither of these enzymes hydrolyze xylotriose. They bind on crystalline cellulose but not on insoluble xylan. Analysis of reaction mixtures by high pressure liquid chromatography revealed that both enzymes cleave preferentially the internal glycosidic bonds of xylopentaose and oat spelts xylan. Thus the purified enzymes appeared to be true endo-β-1,4-xylanases. The amino terminal sequences of xylanases I and II show no homology. Xylanase I shows high similarity with alkaline low molecular mass xylanases of family G/11.
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| 2. |
- Christakopoulos, Paul, et al.
(författare)
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Purification and mode of action of a low molecular mass endo-1,4-β-d-glucanase from Fusarium oxysporum
- 1995
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 39:1, s. 85-93
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Tidskriftsartikel (refereegranskat)abstract
- A low molecular mass (23.2 kDa) endo-1,4-β-d-glucanase from Fusarium oxysporum was purified to homogeneity by gel-filtration and ion-exchange chromatographies. The enzyme was optimally active at pH 6.0 and at 50 ° C. It had a pI value of 8.6 and was stable at 55 ° C for 1 h. It hydrolyzed carboxymethylcellulose, cello-oligosaccharides (Glcn) and 4-methylumbelliferylcello-oligosaccharides but did not hydrolyze cellobiose, p-nitrophenyl β-o-glucoside, p-nitrophenyl β-d-xyloside, Avicel, filter paper and xylan. Analysis of reaction mixtures by high pressure liquid chromatography revealed that this enzyme cleaved preferentially the internal glycoside bonds of higher cello-oligosaccharides. The enzyme also catalyzed the formation of transfer products in the presence of cellotriose, cellotetraose and 4-methylumbelliferylglucoside (MeUmbGlc).
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| 3. |
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| 4. |
- Kalogeris, E., et al.
(författare)
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Studies on the solid-state production of thermostable endoxylanases from Thermoascus aurantiacus : Characterization of two isozymes
- 1998
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Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 60:3, s. 155-163
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Tidskriftsartikel (refereegranskat)abstract
- Production of xylanases by the thermophilic fungus Thermoascus aurantiacus under solid state culture (SSC) was enhanced by optimization of the type of carbon and nitrogen source, inoculum type, moisture level and particle size of the carbon source. Under these conditions, yields as high as 6193 U g−1 of carbon source were obtained. Chromogenic (fluorogenic) 4-methylumbelliferyl-β-glycosides of xylose (MUX) and xylobiose (MUX2) were used to characterize xylanase multienzyme components, after separation by isoelectric focusing. The zymogram indicated one major and two minor xylanases and one β-xylosidase. The major (xylanase I) and one of the minor (xylanase II) xylanases were separated and characterized. Both xylanases exhibited remarkable thermostability.
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| 5. |
- Sterky, Fredrik, et al.
(författare)
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Direct sequencing of bacterial artificial chromosomes (BACs) and prokaryotic genomes by biotin-capture PCR
- 1998
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Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 60:1-2, s. 119-129
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Tidskriftsartikel (refereegranskat)abstract
- Determination of unknown DNA sequences adjacent to known segments is an important task in genome-related research. We have applied the methodology of biotin-capture PCR for direct sequencing of bacterial artificial chromosomes (BACs) and bacterial genomes. The strategy involves extension of a biotinylated primer from a known locus into unknown regions of the template to yield single-stranded DNA, which is immobilised onto paramagnetic beads. An arbitrary primer initiates extension from the unknown region and back towards the known locus. The arbitrary primer contains a universal primer 'handle', which is utilised for subsequent amplification. The PCR products are then directly sequenced by solid-phase or cycle sequencing. The fact that BACs or bacterial chromosomes can be sequenced without prior purification or subcloning might be useful in numerous applications, such as gap-filling, sequencing of regulatory regions upstream known genes and determination of intron/exon-boundaries.
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| 6. |
- Karlsson, Eva Nordberg, et al.
(författare)
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Enzymatic specificity and hydrolysis pattern of the catalytic domain of the xylanase Xyn1 from Rhodothermus marinus
- 1998
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Ingår i: Journal of Biotechnology. - 0168-1656. ; 60:1-2, s. 23-25
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Tidskriftsartikel (refereegranskat)abstract
- The catalytic domain of a xylanase from Rhodothermus marinus was produced in Escherichia coli. The catalytic domain belongs to glycosyl hydrolase family 10. The produced protein has a 22-amino acid leader peptide followed by a 411-amino acid truncated xylanase. The molecular mass was 48 kDa and the recombinant xylanase had a pI of 4.9. The pH and temperature optima for activity were determined to be 7.5 and 80°C, respectively. At that temperature the enzyme had a half-life of 1 h 40 min. An addition of 1 mM calcium stabilized the activity of the enzyme at 80°C. The xylanase had its highest specific activity on oat spelt xylan but was active also on other xylans and to a limited extent on some other polysaccharides (soluble glucans). No exo- or endo-cellulase activity was observed. Hydrolysis of xylo-oligomers and oat spelt xylan was studied and the predominant products of hydrolysis were xylobiose and xylotriose. The enzyme was inactive on xylobiose, xylotriose and on the soluble fraction from oat spelt xylan. The R. marinus xylanase is shown to have a strong preference for internal linkages and is therefore classified as an endo-xylanase. Copyright (C) 1998 Elsevier Science B.V.
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| 9. |
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| 10. |
- Ademark, Pia, et al.
(författare)
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Hydrolytic properties of a beta-mannosidase purified from Aspergillus niger. J. Biotechnol. 75: 281-289.
- 1999
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Ingår i: Journal of Biotechnology. - 1873-4863. ; 75:2-3, s. 281-289
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Tidskriftsartikel (refereegranskat)abstract
- A β-mannosidase was purified to homogeneity from the culture filtrate of Aspergillus niger. A specific activity of 500 nkat mg−1 and a 53-fold purification was achieved using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The isolated enzyme has an isoelectric point of 5.0 and appears to be a dimer composed of two 135-kDa subunits. It is a glycoprotein and contains 17% N-linked carbohydrate by weight. Maximal activity was observed at pH 2.4–5.0 and at 70°C. The β-mannosidase hydrolyzed β-1,4-linked manno-oligosaccharides of degree of polymerization (DP) 2–6 and also released mannose from polymeric ivory nut mannan and galactomannan. The Km and Vmax values for p-nitrophenyl-β-Image-mannopyranoside were 0.30 mM and 500 nkat mg−1, respectively. Hydrolysis of Image-galactose substituted manno-oligosaccharides showed that the β-mannosidase was able to cleave up to, but not beyond, a side group. An internal peptide sequence of 15 amino acids was highly similar to that of an Aspergillus aculeatus β-mannosidase belonging to family 2 of glycosyl hydrolases.
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