| 1. |
- Karlsson, Eva Nordberg, et al.
(författare)
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Cloning and sequence of a thermostable multidomain xylanase from the bacterium Rhodothermus marinus
- 1997
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Ingår i: Biochimica et Biophysica Acta - Gene Structure and Expression. - 0167-4781. ; 1353:2, s. 118-124
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Tidskriftsartikel (refereegranskat)abstract
- The gene (xyn1) encoding a Rhodothermus marinus xylanase has been cloned and expressed in Escherichia coli. The gene comprises 5 different domains in an unusual combination. The cellulose binding domains (CBDs) encoded by xyn1 are repeated in tandem at the N-terminus and show similarity with the CBD family IV. The xyn1-gene is the first example encoding a CBD family IV in combination with a xylan hydrolyzing catalytic domain of the glycosyl hydrolase family 10.
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| 2. |
- Nordberg Karlsson, Eva, et al.
(författare)
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Evidence for substrate binding of a recombinant thermostable xylanase originating from Rhodothermus marinus
- 1998
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Ingår i: FEMS Microbiology Letters. - 0378-1097. ; 168:1, s. 1-7
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Tidskriftsartikel (refereegranskat)abstract
- The xyn1 encoded 5 domain xylanase from the thermophilic bacterium Rhodothermus marinus binds specifically to xylan, β-glucan and amorphous but not crystalline cellulose. Our results show that the binding is mediated by the full length xylanase, but not by the catalytic domain only. Based on similarities concerning both predicted secondary structure and binding specificity found with one cellulose binding domain of CenC from Cellulomonas fimi, we suggest that the binding is mediated by the two N-terminally repeated domains. Copyright (C) 1998 Federation of European Microbiological Societies.
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| 3. |
- Karlsson, Eva Nordberg, et al.
(författare)
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Enzymatic specificity and hydrolysis pattern of the catalytic domain of the xylanase Xyn1 from Rhodothermus marinus
- 1998
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Ingår i: Journal of Biotechnology. - 0168-1656. ; 60:1-2, s. 23-25
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Tidskriftsartikel (refereegranskat)abstract
- The catalytic domain of a xylanase from Rhodothermus marinus was produced in Escherichia coli. The catalytic domain belongs to glycosyl hydrolase family 10. The produced protein has a 22-amino acid leader peptide followed by a 411-amino acid truncated xylanase. The molecular mass was 48 kDa and the recombinant xylanase had a pI of 4.9. The pH and temperature optima for activity were determined to be 7.5 and 80°C, respectively. At that temperature the enzyme had a half-life of 1 h 40 min. An addition of 1 mM calcium stabilized the activity of the enzyme at 80°C. The xylanase had its highest specific activity on oat spelt xylan but was active also on other xylans and to a limited extent on some other polysaccharides (soluble glucans). No exo- or endo-cellulase activity was observed. Hydrolysis of xylo-oligomers and oat spelt xylan was studied and the predominant products of hydrolysis were xylobiose and xylotriose. The enzyme was inactive on xylobiose, xylotriose and on the soluble fraction from oat spelt xylan. The R. marinus xylanase is shown to have a strong preference for internal linkages and is therefore classified as an endo-xylanase. Copyright (C) 1998 Elsevier Science B.V.
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| 4. |
- Nordberg Karlsson, Eva, et al.
(författare)
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Efficient production of truncated thermostable xylanases from Rhodothermus marinus in Escherichia coli fed-batch cultures
- 1999
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Ingår i: Journal of Bioscience and Bioengineering. - 1389-1723. ; 87:5, s. 598-606
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Tidskriftsartikel (refereegranskat)abstract
- A cultivation strategy for the production of two truncated thermostable recombinant xylanases (Xyn1ΔN and Xyn1ΔNC) was developed. Fed-batch cultivations of Escherichia coli strain BL21(DE3) with a controlled exponential glucose feed led to high specific production of the recombinant proteins. Addition of complex nutrients (e.g. Tryptone Soya Broth (TSB)) to the media were shown to increase both the specific growth rate during the production phase and the production per cell. The final cellmass concentration depended on the time of induction in relation to both the feed- start and the expected time at which the cultivation had to be terminated due to oxygen transfer limitations or cell lysis. The gene used for the genetic constructions (encoding Xyn1ΔN and Xyn1ΔNC) was originally isolated from Rhodothermus marinus. Recombinant protein expression was controlled by the T7 lac-promoter and induced in the fed-batch phase at low glucose concentrations by the single addition of either lactose or isopropyl-thio-β-D-galactoside (IPTG). In lactose-induced cells, the production of recombinant xylanase was delayed for approximately 30 min in comparison with those induced with IPTG, but the specific product levels were comparable at 3 h after induction. At this time, approximately 35% of the intracellular protein content was constituted by recombinant xylanase. Under the cultivation conditions used, production of the shorter deletion derivative (Xyn1ΔNC) led to nonspecific leakage and cell lysis, starting 1.5 or 2 h after induction with IPTG or lactose, respectively. At 3 h after induction, 50% of the produced protein (Xyn1ΔNC) was found in the culture medium. This was not the case for the longer protein (Xyn1ΔN), where only 10% of the xylanase leaked into the medium.
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| 5. |
- Nordberg Karlsson, Eva
(författare)
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Xylan degradation by the thermophilic bacterium Rhodothermus marinus. Characterization and function of a thermostable xylanase
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A gene encoding a multidomain xylanase from the thermophilic bacterium Rhodothermus marinus has been cloned, sequenced and produced in Escherichia coli . The gene product was found to be a 109 kDa protein consisting of five domains. The catalytic domain and the two polysaccharide binding domains (PBD, previously called carbohydrate binding domains) have been cloned and produced separately for functional studies. The catalytic domain is classified under glycosyl hydrolase family 10, a family in which 3D-structure determined representatives exhibit 8-fold alpha/beta(TIM)-barrels. It catalyses endo-hydrolysis of xylans and to a lower extent mixed linkage (1-3,4) ß-glucans. Hydrolysis has been observed using oligosaccharides with a degree of polymerisation of 4 or higher. The two polysaccharide binding domains are likely displaying folds based on ß-strands. Their binding specificities to soluble substrates are corresponding well to observed substrate specificity of the catalytic domain. Among insoluble substrates they display binding to both amorphous cellulose and xylan. Both the binding level and thermal stability were markedly increased by additions of calcium ions. The three functionally characterized domains have transition temperatures for unfolding above, or long half-lives within, the growth temperatures (55-77 °C) of the organism R. marinus. The efficiency in enzyme aided bleaching of kraft pulps was tested for three catalytically active forms of the enzyme. All forms lead to brightness gains although at different extents. The gain was found to be dependent on both enzyme construction and pulp characteristics. The trial with an enzyme construct lacking the two N-terminal PBDs was most successful. A strategy for production in E. coli was also developed for two enzyme forms. The results show that production in the selected host-vector system was dependent on both cultivation mode, medium composition and type of inducer. Most efficient production was obtained in fed-batch cultivations in the presence of complex nutrients. Under these conditions the two alternative inducers, IPTG/lactose, resulted in approximately equal amounts of the produced recombinant proteins.
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| 6. |
- Åkesson, Mats, et al.
(författare)
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On-line Detection of Acetate Formation in Escherichia coli Cultures using Dissolved Oxygen Responses to Feed Transients
- 1999
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Ingår i: Biotechnology and Bioengineering. - 1097-0290. ; 64:5, s. 590-598
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant protein production in Escherichia coli can be significantly reduced by acetate accumulation. It is demonstrated that acetate production can be detected on-line with a standard dissolved oxygen sensor by superimposing short pulses to the substrate feed rate. Assuming that acetate formation is linked to a respiratory limitation, a model for dissolved oxygen responses to transients in substrate feed rate is derived. The model predicts a clear change in the character of the transient response when acetate formation starts. The predicted effect was verified in fed-batch cultivations of E. coli TOPP1 and E. coli BL21(DE3), both before and after induction of recombinant protein production. It was also observed that the critical specific glucose uptake rate, at which acetate formation starts, was significantly decreased after induction. On-line detection of acetate formation with a standard sensor opens up new possibilities for feedback control of substrate feeding.
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