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Recombinant Express...
Recombinant Expression and Enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides
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- Master, Emma (författare)
- KTH,Bioteknologi
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- Rudsander, Ulla (författare)
- KTH,Bioteknologi
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Zhou, Welin (författare)
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- Henriksson, Hongbin (författare)
- KTH,Bioteknologi
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- Divne, Christina (författare)
- KTH,Bioteknologi,Industriell bioteknologi
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- Denman, Stuart (författare)
- KTH,Bioteknologi
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Wilson, David (författare)
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- Teeri, Tuula (författare)
- KTH,Bioteknologi
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(creator_code:org_t)
- 2004-07-15
- 2004
- Engelska.
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:31, s. 10080-10089
- Relaterad länk:
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http://pubs.acs.org/...
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- 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.
Ämnesord
- TEKNIK OCH TEKNOLOGIER -- Industriell bioteknik (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Industrial Biotechnology (hsv//eng)
Nyckelord
- CULTURED POPLAR CELLS; IN-GEL DIGESTION; CELLULOSE SYNTHESIS; THERMOMONOSPORA-FUSCA; ARABIDOPSIS-THALIANA; AGROBACTERIUM-TUMEFACIENS; CRYSTAL-STRUCTURE; ENDO-1
- 4-BETA-GLUCANASE; PROTEINS; KORRIGAN
- Bioengineering
- Bioteknik
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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