| 1. |
- Henriksson, Gunnar, et al.
(författare)
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Endoglucanase 28 (Cel12A), a new Phanerochaete chrysosporium cellulase.
- 1999
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Ingår i: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 259:1-2, s. 88-95
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Tidskriftsartikel (refereegranskat)abstract
- A 28-kDa endoglucanase was isolated from the culture filtrate of Phanerochaete chrysosporium strain K3 and named EG 28. It degrades carboxymethylated cellulose and amorphous cellulose, and to a lesser degree xylan and mannan but not microcrystalline cellulose (Avicel). EG 28 is unusual among cellulases from aerobic fungi, in that it appears to lack a cellulose-binding domain and does not bind to crystalline cellulose. The enzyme is efficient at releasing short fibres from filter paper and mechanical pulp, and acts synergistically with cellobiohydrolases. Its mode of degrading filter paper appears to be different to that of endoglucanase I from Trichoderma reesei. Furthermore, EG 28 releases colour from stained cellulose beads faster than any other enzyme tested. Peptide mapping suggests that it is not a fragment of another known endoglucanases from P. chrysosporium and peptide sequences indicate that it belongs to family 12 of the glycosyl hydrolases. EG 28 is glycosylated. The biological function of the enzyme is discussed, and it is hypothesized that it is homologous to EG III in Trichoderma reesei and the role of the enzyme is to make the cellulose in wood more accessible to other cellulases.
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| 2. |
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| 3. |
- Bollok, Monika, et al.
(författare)
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Production of poplar xyloglucan endotransglycosylase using the methylotrophic yeast Pichia pastoris
- 2005
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Ingår i: Applied Biochemistry and Biotechnology. - : Springer Science and Business Media LLC. - 0273-2289 .- 1559-0291. ; 126, s. 61-77
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Tidskriftsartikel (refereegranskat)abstract
- The gene XET16A encoding the enzyme xyloglucan endotransglycosylase (XET) from hybrid aspen (Populus tremula x tremuloides Mich) was transformed into Pichia pastoris GS115 and the enzyme was secreted to the medium. The influence of process conditions on the XET production, activity, and proteolytic degradation were examined. Inactivation of XET occurred in the foam, but could be decreased significantly by using an efficient antifoam. Rich medium (yeast extract plus peptone) was needed for product accumulation, but not for growth. The proteolytic degradation of the enzyme in the medium was substantially decreased by also adding yeast extract and peptone to the glycerol medium before induction with methanol. Decreasing the fermentation pH from 5.0 to 4.0 further reduced the proteolysis. The specific activity was further improved by production at 15 degrees C instead of 22 degrees C. In this way a XET production of 54 mg/L active enzyme could be achieved in the process with a specific activity of 18 Unit/mg protein after a downstream process including centrifugation, micro- and ultrafiltration, and ion exchange chromatography.
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| 4. |
- Henriksson, Hongbin, et al.
(författare)
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Cellobiohydrolase 58 (P.c. Cel 7D) is complementary to the homologous CBHI (T.r. Cel 7A) in enantioseparations
- 2000
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Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 898:1, s. 63-74
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Tidskriftsartikel (refereegranskat)abstract
- Cellobiohydrolase 58 (EC 3.2.1.91, pc. Cel 7D) from Phanerochaete chrysosporium was immobilized on silica and the resulting material, CBH 58-silica, was then used as a chiral stationary phase (CSP) in liquid chromatographic separations of enantiomers. The enantioselectivities obtained on CBH 58-silica were compared with those on CBH I-silica (a phase based on a corresponding cellulase from Trichoderma reesei). CBH 58-silica displayed higher selectivity than CBH I-silica for the more hydrophilic compounds, such as atenolol and metoprolol, although great similarities in chiral separation of beta -adrenergic antagonists were found between the two phases. None of the acidic compounds tested could be resolved on the CBH 58 phase. Moreover, the solutes were retained more on the CBH 58 phase in general, indicating an improved application potential in bioanalysis. Addition of cellobiose or lactose, both of which are inhibitors of cellulases, To the mobile phase impaired the enantioselectivity, indicating an overlap of the enantioselective and catalytic sites. The chiral analytes also functioned as competitive inhibitors and their inhibition constants were determined.
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| 5. |
- Henriksson, Hongbin
(författare)
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Chiral recognition on cellulases : The active sites of the enzymes are involved
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Two cellobiohydrolases (CBH I and CBH II) and endoglucanase I (EG I) fromTrichoderma reesei, and CBH 58 from Phanerochaete chrysosporium were immobilized onsilica as chiral stationary phases (CSPs) in HPLC for enantioseparation of a group ofpharmaceutical compounds. CBH I and CBH 58 wore excellent chiral selectors for β-adrenergic blocking agents, whereas CBH II displayed good enantioselectivity for someother compounds The CBHs share the common feature that their active sites are located intunnels. EG I, with an open groove-shaped active site, had much poorer enantioselectivitythan CBH I, even though these proteins are highly homologous, indicating that a tunnel-shaped active site is important for the chiral recognition ability of the cellulase.The chiral compounds separated on the cellulase-CSPs can influence the catalyticactivity of the cellulases as either inhibitors (in most cases) or activators (some cases inCBH II). The β-blockers were competitive inhibitors of CBH I, EG I and CBH 58, and theirinhibition constants showed that the stronger inhibitors were also the enantiomers that weremore retained on the cellulase-CSPs. The consistency of the enzyme kinetics with thechromatography revealed that the active sites of the cellulases coincided with the chiralrecognition sites. The impairment of the enantioselectivities of the catalytically deficientmutants of CBH I (E212Q, D214N and E217Q) paralleled the loss of their enzymaticactivities, suggesting that the catalytic carboxylate residues (Glu212, Asp214 and Glu217)were directly involved in the binding and the chiral discrimination. Amidation of all thecarboxylate groups of CBH I-CSP abolished both the enantioselectivity and the enzymaticactivity of the CSP, but these properties could be protected from the modification by thepresence of cellobiose that binds in the active site. The crystal structure of the catalyticdomain of CBH I with bound (S)-propranolol revealed, for the first time, the bindingbetween an enantiomer and a protein used as chiral selector at a molecular level.Using cellobiose or lactose as selective competitors in chromatography, the totalbinding sites of the enantiomers on the cellulase-CSPs could be resolved into two classes,namely enantioselective sites that were blocked by the competitor and non-selective sitesthat were unaffected. Their relationship is well described by a mathematical model.
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| 9. |
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| 10. |
- Kallas, Åsa M., et al.
(författare)
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Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula x tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris
- 2005
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Ingår i: Biochemical Journal. - 0264-6021. ; 390, s. 105-113
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Tidskriftsartikel (refereegranskat)abstract
- The cDNA encoding a xyloglucan endotransglycosylase, PttXET16A, from hybrid aspen (Populus tremula x tremuloides) has been isolated from an expressed sequence tag library and expressed in the methylotrophic yeast Pichia pastoris. Sequence analysis indicated a high degree of similarity with other proteins in the XTH (xyloglucan transglycosylase/hydrolase) gene subfamily of GH16 (glycoside hydrolase family 16). In addition to the conserved GH16 catalytic sequence motif, PttXET16A contains a conserved N-glycosylation site situated proximal to the predicted catalytic residues. MS analysis indicated that the recombinant PttXET16A expressed in P. pastoris is heterogeneous due to the presence of variable N-glycosylation and incomplete cleavage of the a-factor secretion signal peptide. Removal of the N-glycan by endoglycosidase H treatment did not influence the catalytic activity significantly. Similarly, site-directed mutagenesis of Asn(93) to serine to remove the N-glycosylation site resulted in an enzyme which was comparable with the wild-type enzyme in specific activity and thermal stability but had clearly reduced solubility. Hydrolytic activity was detected neither in wild-type PttXET16A before or after enzymatic deglycosylation nor in PttXET16A N93S (Asn(93) -> Ser) mutant.
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| 11. |
- Master, Emma, et al.
(författare)
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Recombinant Expression and Enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides
- 2004
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:31, s. 10080-10089
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Tidskriftsartikel (refereegranskat)abstract
- PttCel9A is a membrane-bound, family 9 glycosyl hydrolase from Populus tremula x tremuloides that is upregulated during secondary cell wall synthesis. The catalytic domain of PttCel9A, Delta(1-105)PttCel9A, was purified, and its activity was compared to TfCel9A and TfCel9B from Thermobifida fusca. Since aromatic amino acids involved in substrate binding at subsites -4, -3, and -2 are missing in PttCel9A, the activity of TfCel9A mutant enzymes W256S, W209A, and W313G was also investigated. Delta(1-105)PttCel9A hydrolyzed a comparatively narrow range of polymeric substrates, and the preferred substrate was (carboxymethyl)cellulose 4M. Moreover, Delta(1-105)PttCel9A did not hydrolyze oligosaccharides shorter than cellopentaose, whereas TfCel9A and TfCel9B hydrolyzed cellotetraose and cellotriose, respectively. These data suggest that the preferred substrates of PttCel9A are long, low-substituted, soluble cellulosic polymers. At 30degreesC and pH 6.0, the k(cat) for cellohexaose of Delta(1-105)PttCel9A, TfCel9A, and TfCel9B were 0.023 +/- 0.001, 16.9 +/- 2.0, and 1.3 +/- 0.2, respectively. The catalytic efficiency (k(cat)/K-m) of TfCel9B was 39% of that of TfCel9A, whereas the catalytic efficiency of Delta(1-105)PttCel9A was 0.04% of that of TfCel9A. Removing tryptophan residues at subsites -4, -3, and -2 decreased the efficiency of cellohexaose hydrolysis by TfCel9A. Mutation of W313 to G had the most drastic effect, producing a mutant enzyme with 1% of the catalytic efficiency of TfCel9A. The apparent narrow substrate range and catalytic efficiency of PttCel9A are correlated with a lack of aromatic amino acids in the substrate binding cleft and may be necessary to prevent excessive hydrolysis of cell wall polysaccharides during cell wall formation.
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| 12. |
- Muñoz, Inés G., et al.
(författare)
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Family 7 cellobiohydrolases from Phanerochaete chrysosporium : Crystal structure of the catalytic module of Cel7D (CBH58) at 1.32 Å resolution and homology models of the isozymes
- 2001
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Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 314:5, s. 1097-1111
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Tidskriftsartikel (refereegranskat)abstract
- Cellobiohydrolase 58 (Cel7D) is the major cellulase produced by the white-rot fungus Phanerochaete chrysosporimn, constituting approximately 10% of the total secreted protein in liquid culture on cellulose. The enzyme is classified into family 7 of the glycosyl hydrolases, together with cellobiohydrolase I (Cel7A) and endoglucanase 1 (Cel7B) from Trichoderma reesei. Like those enzymes, it catalyses cellulose hydrolysis with net retention of the anomeric carbon configuration. The structure of the catalytic module (431 residues) of Cel7D was determined at 3.0 Angstrom resolution using the structure of Cel7A from T. reesei as a search model in molecular replacement, and ultimately refined at 1.32 Angstrom resolution. The core structure is a beta-sandwich composed of two large and mainly antiparallel beta-sheets packed onto each other. A long cellulose-binding groove is formed by loops on one face of the sandwich. The catalytic residues are conserved and the mechanism is expected to be the same as for other family members. The Phanerochaete Cel7D binding site is more open than that of the T. reesei cellobiohydrolase, as a result of deletions and other changes in the loop regions, which may explain observed differences in catalytic properties. The binding site is not, however, as open as the groove of the corresponding endoglucanase. A tyrosine residue at the entrance of the tunnel may be part of an additional subsite not present in the T. reesei cellobiohydrolase. The Cel7D structure was used to model the products of the five other family 7 genes found in P. chrysosporium. The results suggest that at least two of these will have differences in specificity and possibly catalytic mechanism, thus offering some explanation for the presence of Cel7 isozymes in this species, which are differentially expressed in response to various growth conditions.
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| 13. |
- Svensson, Ingrid, et al.
(författare)
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Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells
- 2005
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 69:1, s. 92-98
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Tidskriftsartikel (refereegranskat)abstract
- Concentrated conditioned medium (CM) fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni cells, eluting from a gel filtration column at around 10 kDa, were found to exhibit strong antibacterial activity against Bacillus megaterium and Escherichia coli. The B. megaterium cells incubated in the CM fraction from Sf9 cells rapidly lost viability: after 8 min the viability had decreased to 0.7%, as compared with the control. Addition of the CM fraction to E. coli cells resulted in a less drastic drop in viability: 65% viability was lost after 60 min of incubation. Further, exposure to the CM fraction caused a substantial leakage of intracellular proteins, as demonstrated by SDS-PAGE analysis. Cell lysis was confirmed by optical density measurements, microscopic investigations and flow cytometry. B. megaterium exposed to a CM fraction from T. ni cells lost 97% of their viability in about 40 min. Ubiquitin, thioredoxin and cyclophilin were identified in the antibacterial fraction from Sf9 cells by mass spectrometry and N-terminal amino acid sequencing. Other proteins in the fraction gave no matches in a database search. Since ubiquitin was shown not to cause the antimicrobial effect and thioredoxin and cyclophilin were likely not involved, the responsible agent may be an unknown protein, not yet registered in databases. The antimicrobial effect of the CM fraction from T. ni cells most probably comes from a lysozyme precursor protein.
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