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- Henriksson, Gunnar, et al.
(author)
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Endoglucanase 28 (Cel12A), a new Phanerochaete chrysosporium cellulase.
- 1999
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In: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 259:1-2, s. 88-95
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Journal article (peer-reviewed)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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| 3. |
- Bollok, Monika, et al.
(author)
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Production of poplar xyloglucan endotransglycosylase using the methylotrophic yeast Pichia pastoris
- 2005
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In: Applied Biochemistry and Biotechnology. - : Springer Science and Business Media LLC. - 0273-2289 .- 1559-0291. ; 126, s. 61-77
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Journal article (peer-reviewed)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.
(author)
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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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In: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 898:1, s. 63-74
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Journal article (peer-reviewed)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
(author)
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Chiral recognition on cellulases : The active sites of the enzymes are involved
- 2000
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Doctoral thesis (other academic/artistic)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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| 10. |
- Kallas, Åsa M., et al.
(author)
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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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In: Biochemical Journal. - 0264-6021. ; 390, s. 105-113
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Journal article (peer-reviewed)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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