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| 2. |
- Karlsson, Johan, et al.
(författare)
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Enzymatic properties of the low molecular mass endoglucanases Cel12A (EG III) and Cel45A (EG V) of Trichoderma reesei
- 2002
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Ingår i: Journal of Biotechnology. - 1873-4863. ; 99:1, s. 63-78
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Tidskriftsartikel (refereegranskat)abstract
- Trichoderma reesei produces five known endoglucanases. The most studied are Cel7B (EG I) and Cel5A (EG II) which are the most abundant of the endoglucanases. We have performed a characterisation of the enzymatic properties of the less well-studied endoglucanases Cel12A (EG III), Cel45A (EG V) and the catalytic core of Cel45A. For comparison, Cel5A and Cel7B were included in the study. Adsorption studies on microcrystalline cellulose (Avicel) and phosphoric acid swollen cellulose (PASC) showed that Cel5A, Cel7B, Cel45A and Cel45Acore adsorbed to these substrates. In contrast, Cel12A adsorbed weakly to both Avicel and PASC. The products formed on Avicel, PASC and carboxymethylcellulose (CMC) were analysed. Cel7B produced glucose and cellobiose from all substrates. Cel5A and Cel12A also produced cellotriose, in addition to glucose and cellobiose, on the substrates. Cel45A showed a clearly different product pattern by having cellotetraose as the main product, with practically no glucose and cellobiose formation. The kinetic constants were determined on cellotriose, cellotetraose and cellopentaose for the enzymes. Cel12A did not hydrolyse cellotriose. The kCat values for Cel12A on cellotetraose and cellopentaose were significantly lower compared with Cel5A and Cel7B. Cel7B was the only endoglucanase which rapidly hydrolysed cellotriose. Cel45Acore did not show activity on any of the three studied cello-oligosaccharides. The four endoglucanases' capacity to hydrolyse @b-glucan and glucomannan were studied. Cel12A hydrolysed @b-glucan and glucomannan slightly less compared with Cel5A and Cel7B. Cel45A was able to hydrolyse glucomannan significantly more compared with @b-glucan. The capability of Cel45A to hydrolyse glucomannan was higher than that observed for Cel12A, Cel5A and Cel7B. The results indicate that Cel45A is a glucomannanase rather than a strict endoglucanase.
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- Karlsson, Johan, et al.
(författare)
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Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei
- 2001
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 268:24, s. 6498-6507
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Tidskriftsartikel (refereegranskat)abstract
- There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing β-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and β-glucan. The endoglucanase activity on CMC and β-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.
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| 4. |
- Teleman, Anita, et al.
(författare)
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Characterization of O-acetyl-(4-O-methylglucurono)xylan isolated from birch and beech
- 2002
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Ingår i: Carbohydrate Research. - 0008-6215 .- 1873-426X. ; 337:4, s. 373-377
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Tidskriftsartikel (refereegranskat)abstract
- The structures of water-soluble birch and beech xylans, extracted from holocellulose using dimethyl sulfoxide, were determined employing 1H and 13C NMR spectroscopy together with chemical analysis. These polysaccharides were found to be O-acetyl-(4-O-methylglucurono)xylans containing one 4-O-methylglucuronic acid substituent for approximately every 15 D-xylose residues. The average degree of acetylation of the xylose residues in these polymers was 0.4. The presence of the structural element → 4)[4-O-Me-α-D-GlcpA-(1 → 2)][3-O-Ac]-β-D-Xylp-(1 → was demonstrated. Additional acetyl groups were present as substituents at C-2 and/or C-3 of the xylopyranosyl residues. Utilizing size-exclusion chromatography in combination with mass spectroscopy, the weight-average molar masses (and polydispersities) were shown to be 8000 (1.09) and 11,100 (1.08) for birch and beech xylan, respectively.
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