| 1. |
- Abou Hachem, Maher, et al.
(författare)
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Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase : Cloning, expression and binding studies
- 2000
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021. ; 345:1, s. 53-60
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Tidskriftsartikel (refereegranskat)abstract
- The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichia coli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na+ and Ca2+ ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and β-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities. Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities. The binding of the modules was shown to be dominated by enthalpic forces. The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module. This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose.
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| 2. |
- 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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| 3. |
- 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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| 4. |
- Bartonek Roxå, Eva
(författare)
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RECOMBINANT PEROXIDASES AND XYLANASES: I. Cloning and production of a peroxidase from horseradish II. Characterisation of functional domains of thermostable xylanases from Rhodothermus marinus
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Part I: Plant peroxidases are useful in many areas of application e.g. as tracer enzymes for clinical applications and in biosensors as well as for industrial purposes like in waste water treatment and bleaching processes. A gene encoding a new, neutral horseradish peroxidase isoenzyme, HRP-n, was isolated. The encoded protein shows only 50% sequence identity with the classical HRP-C and lacks the C-terminal propeptide found in HRP-C. The cDNA encoding HRP-n was expressed in Escherichia coli but was found to be growth inhibiting and under certain circumstances also toxic. HRP-n was then successfully produced in the Baculovirus/insect cell system. Preliminary results indicate that HRP-n differs from the classical HRP-C with regard to catalytic properties. Differences between HRP-n and HRP-C are found in the peripheral Phe residues which are reported to guard the entrance to the exposed heme, suggesting difference in their properties with respect to substrate specificity. Part II: Xylanases are enzymes involved in plant cell wall hydrolysis. Many of them have a modular structure, with functional domains separated by linker sequences. The most common domains are the catalytic domains (CDs), and the cellulose or carbohydrate binding domains (CBDs). CBDs are proposed to enhance the efficiency of hydrolysis of crystalline and complex substrates. Xylanases have a recognised potential as modifying enzymes in the pulp and paper, food and feed, and textile industries. Some of these processes are harsh and require robust enzymes that can withstand both high temperatures and extremes of pH. Such requirements can be fulfilled by enzymes from thermophiles. The xyn1 encoded xylanase (Xyn1) from the thermophilic bacterium Rhodothermus marinus, is a modular multidomain enzyme, containing two putative CBDs repeated in tandem. The full length xylanase and the two CBDs were produced separately in E. coli and were subsequently characterised. Our results show that Xyn1 represents a new, unique combination of thermostable binding domains, classified into CBD family IV, with affinity for xylan, in addition to amorphous cellulose and soluble glycans.
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