| 1. |
- 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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| 2. |
- Ademark, Pia, et al.
(författare)
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Multiple alpha-galactosidases from Aspergillus niger: purification, characterization, and substrate specificities
- 2001
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Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 29:6-7, s. 441-448
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Tidskriftsartikel (refereegranskat)abstract
- Enzymes with α-galactosidase activity are produced by many organisms, often in multiple forms. Here we compare the biochemical and hydrolytic properties of four major α-galactosidase forms (α-gal I-IV) that were purified from the culture filtrate of Aspergillus niger. α-Gal II, III and IV appear to be isoforms of the same enzyme, and N-terminal amino acid sequence data suggest that they are closely related or identical to A. niger AglB in family 27 of the glycosyl hydrolases. α-Gal I is a completely different enzyme that belongs to family 36. α-Gal I had an isoelectric point of 4.15 and appears to be a tetramer composed of four 94-kDa subunits. α-Gal II, III and IV were dimers with monomeric molecular masses of 64 kDa and isoelectric points of 4.5, 4.7 and 4.8, respectively. α-Gal II-IV were stable when incubated for 17 h at 50°C and pH 2–5, whereas α-gal I was most stable at pH 5–6. All enzymes had maximal catalytic activity at pH 4.5 and 60°C, and hydrolyzed melibiose, raffinose and stachyose. α-Gal II-IV also degraded galactomanno-oligosaccharides and released 66% of the galactose side groups from polymeric locust bean gum galactomannan. α-Gal I released galactose from locust bean gum only in combination with A. niger β-mannosidase. Kinetic experiments showed that α-gal I hydrolyzed p-nitrophenyl-α-Image-galactopyranoside and melibiose more efficiently than α-gal II-IV. The distinct hydrolytic and biochemical properties of α-gal I and α-gal II-IV further signifies the difference between α-galactosidases of family 27 and 36.
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| 3. |
- Ademark, Pia, et al.
(författare)
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Softwood hemicellulose-degrading enzymes from Aspergillus niger: Purification and properties of a β-mannanase
- 1998
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Ingår i: Journal of Biotechnology. - 1873-4863. ; 63:3, s. 199-210
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Tidskriftsartikel (refereegranskat)abstract
- The enzymes needed for galactomannan hydrolysis, i.e. β-mannanase, α-galactosidase and β-mannosidase, were produced by the filamentous fungus Aspergillus niger. The β-mannanase was purified to electrophoretic homogeneity in three steps using ammonium sulfate precipitation, anion-exchange chromatography and gel filtration. The purified enzyme had an isoelectric point of 3.7 and a molecular mass of 40 kDa. Ivory nut mannan was degraded mainly to mannobiose and mannotriose when incubated with the β-mannanase. Analysis by 1H NMR spectroscopy during hydrolysis of mannopentaose showed that the enzyme acts by the retaining mechanism. The N-terminus of the purified A. niger β-mannanase was sequenced by Edman degradation, and comparison with Aspergillus aculeatus β-mannanase indicated high identity. The enzyme most probably lacks a cellulose binding domain since it was unable to adsorb on cellulose.
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| 4. |
- Alkasrawi, Malek, et al.
(författare)
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The effect of Tween-20 on simultaneous saccharification and fermentation of softwood to ethanol
- 2003
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Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 33:1, s. 71-78
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Tidskriftsartikel (refereegranskat)abstract
- Simultaneous sacchatification and fermentation (SSF) of steam-pretreated wood constitutes an attractive process configuration for ethanol production from biomass. However, the high enzyme addition in SSF contributes to a high process cost. In this study we explore the effect of the non-ionic surfactant Tween-20 as an additive in SSE Tween-20 addition at 2.5 g/l had several positive effects on SSF: (i) the ethanol yield was increased by 8%; (ii) the amount of enzyme loading could be reduced by 50%, while maintaining a constant yield; (iii) the enzyme activity increased in the liquid fraction at the end of SSF, probably by preventing unproductive binding of the cellulases to lignin, which could facilitate enzyme recovery; (iv) the time required to attain maximum ethanol concentration was reduced. Surfactants as an additive in SSF can significantly lower the operational cost of the process. However, less expensive surfactants must be investigated. (C) 2003 Elsevier Science Inc. All rights reserved.
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| 5. |
- Antov, Mirjana, et al.
(författare)
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Affinity partitioning of a Cellulomonas fimi beta-mannanase with a mannan-binding module in galactomannan/starch aqueous two-phase system
- 2006
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1123:1, s. 53-59
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Tidskriftsartikel (refereegranskat)abstract
- A new approach in affinity separations was studied by partitioning of Cellulomonas fimi beta-mannanase (EC 3.2.1.78) containing a mannan-binding module in galactomannan/hydroxypropyl starch aqueous two-phase system. Comparison was made with a truncated version of C. fimi beta-mannanase which lacked the mannan-binding module. Results showed that affinity partitioning of the beta-mannanase was achieved due to biospecificity of the mannan-binding module towards the top phase containing galactomannan. Experiments were conducted at pH 8 to prevent enzyme degradation of the phase containing galactomannan. Removal of the top phase polymer was accomplished by beta-mannanase degradation allowed by shifting to the optimal pH 6. In the combination with the genetic fusion of any given protein to the mannan-binding module, the results envision a general procedure for primary affinity recovery of such fusion proteins.
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| 6. |
- Bergfeldt, K, et al.
(författare)
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Phase Separation Phenomena and Viscosity Enhancements in
- 1995
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Ingår i: Macromolecules. - : American Chemical Society (ACS). - 0024-9297 .- 1520-5835. ; 28:9, s. 3360-3370
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Tidskriftsartikel (refereegranskat)abstract
- Interactions between poly(styrenesu1fonate) (PSS) and poly(acry1ic acid) (PA) in aqueous solution have been studied, with and without added salt, and at various degrees of neutralization (a) of PA. Equilibrium phase diagrams have been determined, and the viscosities of monophasic mixtures have been measured. Both types of experiments reveal striking effects of a on the PA-PSS interactions. Salt-free mixtures with fully or partially neutralized poly(acry1ic acid) phase separate segregatively, except at very low a where, instead, an association between PA and PSS occurs. The association is evidenced by a dramatically increased viscosity, relative to solutions of PA or PSS alone, in semidilute mixtures. Addition of salt (1 M NaC1) results in an increased two-phase area at all a, and in the appearance of an associative phase separation for non-neutralized PA. The qualitative phase behavior observed in the presence of salt can be generated by calculations using the Flory-Huggins theory, if it is assumed that both the PSS-PA and the PA-solvent interactions change monotonically with a in a specified fashion. All experimental observations, and the theoretical modeling, suggest that the effective PA-PSS interaction changes (over a narrow interval of a) from an attraction at very low a to a repulsion at higher a.
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| 7. |
- Berggren, Kristina, et al.
(författare)
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Effects of salts and the surface hydrophobicity of proteins on partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide-propylene oxide copolymers
- 1995
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 718:1, s. 67-79
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Tidskriftsartikel (refereegranskat)abstract
- The partitioning of five well-characterised model proteins, bovine serum albumin (BSA), lysozyme, [beta ]-lactoglobulin A, myoglobin and cytochrome c, in aqueous two-phase systems has been studied. As top phase polymers PEG (polyethylene glycol, 100% EO) and the thermoseparating ethylene oxide (EO)-propylene oxide (PO) random copolymers, Ucon 50-HB-5100 (50% EO, 50% PO) and EO30PO70 (30% EO, 70% PO), respectively, were used. The top phase polymers are increasing in hydrophobicity with increasing content of PO. Reppal PES 200 (hydroxypropyl starch) was used as the bottom phase polymer. Phase diagrams for Reppal PES 200-PEG and Reppal PES 200-EO30PO70 two-phase systems were determined. The partitioning of four salts with different hydrophobicity, and also the effect of the salts on protein partitioning in these systems, was studied. It was found that the partitioning of the salts followed the Hofmeister series. The partitioning of proteins with low surface hydrophobicity, myoglobin and cytochrome c, was little affected by hydrophobic polymers and salts. However, the partitioning of a protein with higher surface hydrophobicity, lysozyme, was strongly affected when polymer hydrophobicity was increased and a hydrophobic counterion was used. A protein with a relatively hydrophobic surface can be partitioned to a phase containing a thermoseparating EO-PO copolymer by using a hydrophobic counterion. The partitioning of lysozyme and cytochrome c in the polymer-water system formed after temperature-induced phase separation was also examined. Both proteins partitioned exclusively to the water phase. A separation of the protein and polymer was obtained by temperature-induced phase separation on the isolated phase containing the EO-PO copolymer. The partitioning data also indicated that the hydroxypropyl starch polymer had a weak negative charge.
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| 8. |
- Berggren, Kristina, et al.
(författare)
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Genetic engineering of protein-peptide fusions for control of protein partitioning in thermoseparating aqueous two-phase systems
- 1999
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Ingår i: Biotechnology and Bioengineering. - 1097-0290. ; 62:2, s. 135-144
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Tidskriftsartikel (refereegranskat)abstract
- Genetic engineering has been used for the fusion of peptides, with different length and composition, on a protein to study the effect on partitioning in aqueous two-phase systems containing thermoseparating polymers. Peptides containing 2-6 tryptophan residues or tryptophan plus 1-3 lysine or aspartate residues, were fused near the C-terminus of the recombinant protein ZZT0, where Z is a synthetic IgG-binding domain derived from domain B in staphylococcal protein A. The partitioning behavior of the peptides and fusion proteins were studied in an aqueous two-phase system composed of dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer, EO30PO70. The zwitterionic compound beta-alanine was used to reduce the charge-dependent salt effects on partitioning, and to evaluate the contribution to the partition coefficient from the amino acid residues, Trp, Lys, and Asp, respectively. Trp was found to direct the fusion proteins to the EO-PO copolymer phase, while Asp and Lys directed them to the dextran phase. The effect of sodium perchlorate and triethylammonium phosphate on the partitioning of the fusion proteins was also studied. Salt effects were directly proportional to the net charge of the fusion proteins. Sodium perchlorate was found to be 3.5 times more effective in directing positively charged proteins to the EO-PO copolymer phase compared to the effect of triethyl ammonium phosphate on negatively charged proteins. An empirical correlation has been tested where the fusion protein partitioning is a result of independent contributions from unmodified protein, fused peptide, and salt effects. A good agreement with experimental data was obtained which indicates the possibility, by independent measurements of partitioning of target protein and fusion peptide, to approximately predict the fusion protein partitioning. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 135-144, 1999.
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| 9. |
- Berggren, K., et al.
(författare)
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Partitioning of peptides and recombinant protein-peptide fusions in thermoseparating aqueous two-phase systems : effect of peptide primary structure
- 2000
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Ingår i: Journal of Chromatography B. - 0378-4347. ; 743:1-2, s. 295-306
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Tidskriftsartikel (refereegranskat)abstract
- Genetic engineering has been used for fusion of peptides, with different length and composition, on a protein to study the effect on partitioning in an aqueous two-phase system. The system was composed of dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer, EO30PO70. Peptides containing tryptophan, proline, arginine or aspartate residues were fused at the C-terminus of the recombinant protein ZZ-cutinase. The aim was to find effective tags for the lipolytic enzyme cutinase for large-scale extraction. The target protein and peptide tags were partitioned separately and then together in the fusion proteins in order to gain increased understanding of the influence of certain amino acid residues on the partitioning. The salt K2SO4 was used to reduce the charge dependent salt effects on partitioning and to evaluate the contribution to the partition coefficient from the hydrophobic-hydrophilic properties of the amino acid residues. The effect of Trp on peptide partitioning was independent of the difference in primary structure for (Trp)n, (Trp-Pro)n, (Ala-Trp-Trp-Pro)n and was only determined by the number of Trp. The effect of the charged residues, Arg and Asp, was dependent on the surrounding residues, i.e. if they were situated next to Trp or not. The partitioning behaviour observed for the peptides was qualitatively and in some cases also quantitatively the same as for the fusion proteins. The effect of the salts sodium perchlorate and triethylammonium phosphate on the partitioning was also studied. The salt effects observed for the peptides were qualitatively similar to the effects observed for the fusion proteins.
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| 10. |
- Berggren, K., et al.
(författare)
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Peptide fusion tags with tryptophan and charged residues for control of protein partitioning in PEG-potassium phosphate aqueous two-phase systems
- 2000
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Ingår i: Bioseparation (Dordrecht). - 0923-179X .- 1573-8272. ; 9:2, s. 69-80
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Tidskriftsartikel (refereegranskat)abstract
- A partition study with peptides and recombinant proteins in poly(ethylene glycol)4000-potassium phosphate aqueous two-phase systems has been performed. The aim was to study to what extent the insertion of charged residues could affect protein partition in addition to the already observed effects of tryptophan residues. The model proteins used are based on a staphylococcal protein A derivative, Z, and modified by the insertion of peptide tags close to the C-terminus. The tags differed with respect to their content of both Trp, negatively (Asp) and positively charged (Lys) amino acid residues. The same partitioning trends were observed for the peptides and fusion proteins. The effect of Trp residues was to direct the partitioning towards the PEG phase. The insertion of two negatively charged (Asp) residues into a Trp(4)-tag enhanced the partition towards the PEG phase even more. The introduction of positively charged (Lys) residues in addition to Trp residues, on the other hand, pulled the peptide or protein towards the potassium phosphate phase. The partitioning of peptides gave a good qualitative picture of the effect of the peptide on partitioning when fused to the protein. The efficiencies of the tags were calculated based on partitioning of tags and fusion proteins, and tag efficiencies generally varied between 60 and 85%.
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