| 1. |
- Ademark, Pia, et al.
(författare)
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Cloning and characterisation of Aspergillus niger genes encoding an alpha-galactosidase and a beta-mannosidase involved in galactomannan degradation
- 2001
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 268:10, s. 2982-2990
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Tidskriftsartikel (refereegranskat)abstract
- α-Galactosidase (EC 3.2.1.22) and β-mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an α-galactosidase (AglC) and a β-mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N-glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. nigerα-galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild-type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno-oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.
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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. |
- Collén, Anna, et al.
(författare)
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Extraction of endoglucanase I (Cel7B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)phosphate aqueous two-phase system
- 2002
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Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 943:1, s. 55-62
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Tidskriftsartikel (refereegranskat)abstract
- Endoglucanases (EGI) (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei are industrially important enzymes. Thus, there is a great need for development of a primary recovery method suitable for large-scale utilization. In this study we present a concept applicable for large-scale purification of an EGI fusion protein by one-step extraction in a poly(ethylene glycol) PEG-sodium/potassium phosphate aqueous two-phase system. EGI is a two-module enzyme composed of an N-terminal catalytic module and a C-terminal cellulose binding module (CBM) separated by a glycosylated linker region. Partitioning of six different EGI constructs, containing the C-terminal extensions (WP)(2), (WP)(4) or the amphiphilic protein hydrophobin I (HFB) of T. reesei instead of the CBM were studied to evaluate if any of the fusions could improve the partition coefficient sufficiently to be suitable for large-scale production. All constructs showed improved partitioning in comparison to full length EGI. The (WP)(4) extensions resulted in 26- to 60-fold improvement of partition coefficient. Consequently, a relative minor change in amino acid sequence on the two-module protein EGI improved the partition coefficient significantly in the PEG 4000-sodium/potassium phosphate system. The addition of HFBI to EGI clearly enhanced the partition coefficient (K=1.2) in comparison to full-length EGI (K=0.035). Partitioning of the construct with (WP)(4) fused to the catalytic module and a short sequence of the linker [EGI(core-P5)(WP)(4)] resulted in the highest partition coefficient (K=54) and a yield of 98% in the PEG phase. Gel electrophoresis showed that the construct with the (WP)(4) tag attached after a penta-proline linker could be purified from the other bulk proteins by only a single-step separation in the PEG 4000-sodium/potassium phosphate system. This is a major improvement in comparison with the previously studied model (ethylene oxide-propylene oxide)-dextran system. Hence, this construct will be suitable for further optimization of the extraction of the enzyme in a PEG 4000-sodium/potassium phosphate system from culture filtrate.
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| 4. |
- Collén, Anna, et al.
(författare)
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Genetic engineering of the Trichoderma reesei endoglucanase I (Cel7B) for enhanced partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide-propylene oxide copolymers
- 2001
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Ingår i: Journal of Biotechnology. - 1873-4863. ; 87:2, s. 179-191
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Tidskriftsartikel (refereegranskat)abstract
- Endoglucanases (endo-1,4---glucan-4-glucanohydrolase, EC 3.2.1.4) are industrially important enzymes. In this study endoglucanase I (EGI or Cel7B) of the filamentous fungi Trichoderma reesei has been genetically engineered to investigate the influence of tryptophan rich peptide extensions (tags) on partitioning in an aqueous two-phase model system. EGI is a two-domain enzyme and is composed of a N-terminal catalytic domain and a C-terminal cellulose binding domain, separated by a linker. The aim was to find an optimal tag and fusion position, which further could be utilised for large scale extractions. Peptide tags of different length and composition were attached at various localisations of EGI. The fusion proteins were expressed from T. reesei with the use of the gpdA promoter from Aspergillus nidulans. Variations in secreted levels between the engineered proteins were obtained. The partitioning of EGI in an aqueous two-phase system composed of a thermoseparating ethylene oxide–propylene oxide random copolymer (EO50PO50) and dextran, could be significantly improved by relatively minor genetic engineering. The (Trp-Pro)4 tag added after a short stretch of the linker, containing five proline residues, gave in the highest partition coefficient of 12.8. The yield in the top phase was 94%. The specific activity was 83% of the specific activity of unmodified EGI on soluble substrate. The efficiency of a tag fused to a protein is shown by the tag efficiency factor (TEF). A hypothetical TEF of 1.0 would indicate full tag exposure and optimal contribution to the protein partitioning by the fused tag. The location of the fusion point after the sequence of five proline residues in the linker of EGI is the most beneficial in two-phase separation. The highest TEF (0.97) was obtained with the (Trp-Pro)2 tag at this position, indicating full exposure and intactness of the tag. However, the peptide tag composed of (Trp-Pro)4 improved the partition properties the most but had lower TEF in comparison to (Trp-Pro)2
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| 5. |
- Collén, Anna, et al.
(författare)
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Genetically engineered peptide fusions for improved protein partitioning in aqueous two-phase systems - Effect of fusion localization on endoglucanase I of Trichoderma reesei
- 2001
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 910:2, s. 275-284
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Tidskriftsartikel (refereegranskat)abstract
- Genetic engineering has been used for fusion of the peptide tag, Trp-Pro-Trp-Pro, on a protein to study the effect on partitioning in aqueous two-phase systems. As target protein for the fusions the cellulase, endoglucanase I (endo-1,4-β-d-glucan-4-glucanohydrolase, EC 3.2.1.4, EGI, Cel7B) of Trichoderma reesei was used. For the first time a glycosylated two-domain enzyme has been utilized for addition of peptide tags to change partitioning in aqueous two-phase systems. The aim was to find an optimal fusion localization for EGI. The peptide was (1) attached to the C-terminus end of the cellulose binding domain (CBD), (2) inserted in the glycosylated linker region, (3) added after a truncated form of EGI lacking the CBD and a small part of the linker. The different constructs were expressed in the filamentous fungus T. reesei under the gpdA promoter from Aspergillus nidulans. The expression levels were between 60 and 100 mg/l. The partitioning behavior of the fusion proteins was studied in an aqueous two-phase model system composed of the thermoseparating ethylene oxide (EO)-propylene oxide (PO) random copolymer EO-PO (50:50) (EO50PO50) and dextran. The Trp-Pro-Trp-Pro tag was found to direct the fusion protein to the top EO50PO50 phase. The partition coefficient of a fusion protein can be predicted with an empirical correlation based on independent contributions from partitioning of unmodified protein and peptide tag in this model system. The fusion position at the end of the CBD, with the spacer Pro-Gly, was shown to be optimal with respect to partitioning and tag efficiency factor (TEF) was 0.87, where a fully exposed tag would have a TEF of 1.0. Hence, this position can further be utilized for fusion with longer tags. For the other constructs the TEF was only 0.43 and 0.10, for the tag fused to the truncated EGI and in the linker region of the full length EGI, respectively.
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| 6. |
- Collén, Anna, et al.
(författare)
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Primary recovery of a genetically engineered Trichoderma reesei endoglucanase I (Cel 7B) fusion protein in cloud point extraction systems.
- 2002
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 78:4, s. 385-394
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Tidskriftsartikel (refereegranskat)abstract
- Here we present data to demonstrate how partitioning of a hydrophilic enzyme can be directed to the hydrophobic detergent-enriched phase of an aqueous two-phase system by addition of short stretches of amino acid residues to the protein molecule. The target enzyme was the industrially important endoglucanase I, EGI (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei. We investigated the partitioning of three EGI variants containing various C-terminal peptide extensions including Trp-Pro motifs of different lengths and localizations. Additionally, a recently developed system composed of the thermoseparating copolymer HM-EOPO was utilized to study the effects of fusion tags. The addition of peptides containing tryptohan residues enhanced the partitioning of EGI to the HM-EOPO-rich phase. The system composed of a nonionic detergent (Agrimul NRE1205) resulted in the highest partition coefficient (K = 31) and yield (90%) with the construct EGI(core-P5)(WP)(4) containing (Trp-Pro)(4) after a short linker stretch. A recombinant strain of T. reesei Rut-C30 for large-scale production was constructed in which the fusion protein EGI(core-P5)(WP)(4) was expressed from the strong promoter of the cellulase gene cbh1. The fusion protein was successfully expressed and secreted from the fungus during shake-flask cultivations. Cultivation in a 28-L bioreactor however, revealed that the fusion protein is sensitive to proteases. Consequently, only low production levels were obtained in large-scale production trials.
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| 7. |
- Eriksson, Torny, et al.
(författare)
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Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module
- 2004
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:7, s. 1266-1276
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Tidskriftsartikel (refereegranskat)abstract
- The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations.
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| 8. |
- Hägglund, Per, et al.
(författare)
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A cellulose-binding module of the Trichoderma reesei @b-mannanase Man5A increases the mannan-hydrolysis of complex substrates
- 2003
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Ingår i: Journal of Biotechnology. - 1873-4863. ; 101:1, s. 37-48
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Tidskriftsartikel (refereegranskat)abstract
- Endo-@b-1,4-d-mannanases (@b-mannanase; EC 3.2.1.78) are endohydrolases that participate in the degradation of hemicellulose, which is closely associated with cellulose in plant cell walls. The @b-mannanase from Trichoderma reesei (Man5A) is composed of an N-terminal catalytic module and a C-terminal carbohydrate-binding module (CBM). In order to study the properties of the CBM, a construct encoding a mutant of Man5A lacking the part encoding the CBM (Man5A@DCBM), was expressed in T. reesei under the regulation of the Aspergillus nidulans gpdA promoter. The wild-type enzyme was expressed in the same way and both proteins were purified to electrophoretic homogeneity using ion-exchange chromatography. Both enzymes hydrolysed mannopentaose, soluble locust bean gum galactomannan and insoluble ivory nut mannan with similar rates. With a mannan/cellulose complex, however, the deletion mutant lacking the CBM showed a significant decrease in hydrolysis. Binding experiments using activity detection of Man5A and Man5A@DCBM suggests that the CBM binds to cellulose but not to mannan. Moreover, the binding of Man5A to cellulose was compared with that of an endoglucanase (Cel7B) from T. reesei.
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| 9. |
- Hägglund, Per, et al.
(författare)
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Degradation of Mannan I and II Crystals by Fungal endo-β-1,4-Mannanases and a β-1,4-Mannosidase Studied with Transmission Electron Microscopy
- 2001
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1526-4602 .- 1525-7797. ; 2:3, s. 694-699
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Tidskriftsartikel (refereegranskat)abstract
- We have used the endo-β-1,4-mannanase from Trichoderma reesei (Tr Man5A), the endo-β-1,4-mannanase from Aspergillus niger (An Man5A) and the exo-β-1,4-mannosidase from A. niger (An Mnd2A) to follow the enzymatic degradation of mannan I and II crystals. The degradation process was studied by transmission electron microscopy and also followed by analysis of the released soluble reducing sugars. The mannan crystals were degraded by the endo-β-1,4-mannanases and to a lesser extent by the exo-β-1,4-mannosidase. The observed hydrolysis pattern on mannan I crystals is fully consistent with the current view of the molecular structure of these crystals. The molecular organization of the mannan chains in mannan II crystals is less clear and the digestion results give some further information about the ultrastructure of mannan II. In addition, insight is provided into the mode of the enzymatic attack on the crystals of mannan I and mannan II.
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| 10. |
- Jacobs, Anna, Ph. D., et al.
(författare)
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Characterization of water-soluble hemicelluloses from spruce and aspen employing SEC/MALDI mass spectroscopy
- 2002
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Ingår i: Carbohydrate Research. - 1873-426X .- 0008-6215. ; 337:8, s. 711-717
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Tidskriftsartikel (refereegranskat)abstract
- Partly depolymerized hemicelluloses isolated from wood chips of spruce and aspen employing microwave treatment were resolved using size-exclusion chromatography (SEC) into oligo- and polysaccharide fractions containing components with a narrow range of sizes, as determined by MALDI mass spectroscopy. The degree of substitution with acetyl moieties (DS) was also calculated on the basis of the MALDI-MS spectra obtained prior to and following deacetylation. For spruce hemicelluloses, the low molecular mass fraction contained small arabino-4-O-methylglucuronoxylan oligosaccharides, with DP values ranging from 4 to ~20, separated primarily on the basis of their charge density. The fraction eluted last consisted of an O-acetyl-(galacto)glucomannan polysaccharide of peak-average DP value (DPp) 14. The degree of substitution with acetyl groups (DS) decreased with decreasing DP, a value DS of 0.39 being obtained for the fraction with DPp 12. For the aspen hemicelluloses, the SEC fractions eluted first contained an acidic O-acetyl-4-O-methylglucuronoxylan polysaccharide with DP ranging from 10 to ~28 and an average DS of ~0.75. The fractions eluted last consisted of oligosaccharide mixtures composed primarily of small neutral O-acetyl-xylooligosaccharides (DPp 6, DS 0.41), together with minor quantities of an O-acetyl-glucomannan.
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