| 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. |
- Abou-Hachem, Maher, et al.
(författare)
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Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus.
- 2002
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:18, s. 5720-5729
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Tidskriftsartikel (refereegranskat)abstract
- Calcium binding to carbohydrate binding module CBM4-2 of xylanase 10A (Xyn10A) from Rhodothermus marinus was explored using calorimetry, NMR, fluorescence, and absorbance spectroscopy. CBM4-2 binds two calcium ions, one with moderate affinity and one with extremely high affinity. The moderate-affinity site has an association constant of (1.3 +/- 0.3) x 10(5) M(-1) and a binding enthalpy DeltaH(a) of -9.3 +/- 0.4 kJ x mol(-1), while the high-affinity site has an association constant of approximately 10(10) M(-1) and a binding enthalpy DeltaH(a) of -40.5 +/- 0.5 kJ x mol(-1). The locations of the binding sites have been identified by NMR and structural homology, and were verified by site-directed mutagenesis. The high-affinity site consists of the side chains of E11 and D160 and backbone carbonyls of E52 and K55, while the moderate-affinity site comprises the side chain of D29 and backbone carbonyls of L21, A22, V25, and W28. The high-affinity site is in a position analogous to the calcium site in CBM4 structures and in a recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (T(m)) by approximately 23 degrees C at pH 7.5. No correlation between binding affinity and T(m) change was noted, as each of the two calcium ions contributes almost equally to the increase in unfolding temperature.
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| 3. |
- Abou-Hachem, Maher, et al.
(författare)
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Probing stability of the modular thermostable xylanase Xyn10A
- 2003
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Ingår i: Extremophiles. - : Springer Science and Business Media LLC. - 1433-4909 .- 1431-0651. ; 7:6, s. 483-491
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Tidskriftsartikel (refereegranskat)abstract
- The thermophilic bacterium Rhodothermus marinus produces a modular xylanase (Xyn10A) consisting of two N-terminal carbohydrate-binding modules (CBMs), followed by a domain of unknown function, and a catalytic module flanked by a fifth domain. Both Xyn10A CBMs bind calcium ions, and this study explores the effect of these ions on the stability of the full-length enzyme. Xyn10A and truncated forms thereof were produced and their thermostabilities were evaluated under different calcium loads. Studies performed using differential scanning calorimetry showed that the unfolding temperature of the Xyn10A was significantly dependent on the presence of Ca2+, and that the third domain of the enzyme binds at least one Ca2+. Thermal inactivation studies confirmed the role of tightly bound Ca2+ in stabilizing the enzyme, but showed that the presence of a large excess of this ion results in reduced kinetic stability. The truncated forms of Xyn10A were less stable than the full-length enzyme, indicative of module/domain thermostabilizing interactions. Finally, possible roles of the two domains of unknown function are discussed in the light of this study. This is the first report on the thermostabilizing role of calcium on a modular family 10 xylanase that displays multiple calcium binding in three of its five domains/modules.
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| 4. |
- Abou-Hachem, Maher, et al.
(författare)
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The modular organisation and stability of a thermostable family 10 xylanase
- 2003
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Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 21:5-6, s. 253-260
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Tidskriftsartikel (refereegranskat)abstract
- The thermophilic marine bacterium Rhodothermus marinus produces a modular family 10 xylanase (Xyn10A). It consists of two N-terminal family 4 carbohydrate binding modules (CBMs) followed by a domain of unknown function (D3), and a catalytic module (CM) flanked by a small fifth domain (D5) at its C-terminus. Several truncated mutants of the enzyme have been produced and characterised with respect to biochemical properties and stability. Multiple calcium binding sites are shown to be present in the two N-terminal CBMs and recent evidence suggests that the third domain of the enzyme also has the ability to bind the same metal ligand. The specific binding of Ca2+ was demonstrated to have a pronounced effect on thermostability as shown by differential scanning calorimetry and thermal inactivation studies. Furthermore, deletion mutants of the enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme. Finally, recent evidence indicates that the fifth domain of Xyn10A is a novel type of module mediating cell-attachment.
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| 5. |
- Blücher, Anna, et al.
(författare)
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Substrate-dependent production and some properties of a thermostable, α-galactosidase from Rhodothermus marinus
- 2000
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Ingår i: Biotechnology Letters. - 0141-5492. ; 22:8, s. 663-669
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Tidskriftsartikel (refereegranskat)abstract
- α-Galactosidase activity in Rhodothermus marinus is dependent on the composition of the growth media. A maximum of 46 μU g-1 cell dry weight was obtained using minimal medium with galactooligo- or polysaccharides as single carbon source. An enzyme hydrolysing both high and low molecular weight galacto-saccharides was partly purified from the cell fractions. The molecular weight was 200 kDa (native) and 50 kDa (monomer). It was optimally active at 85°C, with a half-life of 2 h at 75°C, and had a broad pH range (4-8).
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| 6. |
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| 7. |
- Crennell, Susan, et al.
(författare)
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The structure of Rhodothermus marinus Cel12A, a highly thermostable family 12 endoglucanase, at 1.8 Å resolution
- 2002
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Ingår i: Journal of Molecular Biology. - 1089-8638. ; 320:4, s. 883-897
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Tidskriftsartikel (refereegranskat)abstract
- Cellulose is one of the most abundant polysaccharides in nature and microorganisms have developed a comprehensive system for enzymatic breakdown of this ubiquitous carbon source, a subject of much interest in the biotechnology industry. Rhodothermus marinus produces a hyperthermostable cellulase, with a temperature optimum of more than 90 °C, the structure of which is presented here to 1.8 Å resolution. The enzyme has been classified into glycoside hydrolase family 12; this is the first structure of a thermophilic member of this family to have been solved. The β-jelly roll fold observed has identical topology to those of the two mesophilic members of the family whose structures have been elucidated previously. A Hepes buffer molecule bound in the active site may have triggered a conformational change to an active configuration as the two catalytic residues Glu124 and Glu207, together with dependent residues, are observed in a conformation similar to that seen in the structure of Streptomyces lividans CelB2 complexed with an inhibitor. The structural similarity between this cellulase and the mesophilic enzymes serves to highlight features that may be responsible for its thermostability, chiefly an increase in ion pair number and the considerable stabilisation of a mobile region seen in S. lividans CelB2. Additional aromatic residues in the active site region may also contribute to the difference in thermophilicity.
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| 8. |
- Gunnarsson, Lavinia Cicortas, 1977-, et al.
(författare)
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A carbohydrate binding module as a diversity-carrying scaffold
- 2004
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press. - 1741-0126 .- 1741-0134. ; 17:3, s. 213-221
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Tidskriftsartikel (refereegranskat)abstract
- The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2. Despite the small size of the library (1.6 x 10(6) clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 +/- 5.3 degrees C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.
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| 9. |
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| 10. |
- Nordberg Karlsson, Eva, et al.
(författare)
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Citrate synthase from Thermus aquaticus: a thermostable bacterial enzyme with a five-membered inter-subunit ionic network.
- 2003
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Ingår i: Extremophiles. - : Springer Science and Business Media LLC. - 1433-4909 .- 1431-0651. ; 7:1, s. 9-16
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Tidskriftsartikel (refereegranskat)abstract
- A bacterial thermostable citrate synthase has been analyzed to investigate the structural basis of its thermostability, and to compare such features with those previously identified in archaeal citrate synthases. The gene encoding the citrate synthase from Thermus aquaticus was identified from a gene library by screening with a PCR fragment amplified from genomic DNA using a primer based on the determined N-terminal amino acid sequence and a citrate synthase consensus primer. Apart from high sequence similarities with citrate synthase sequences within the Thermus/Deinococcus group, the analyzed enzyme has highest similarities with the enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The recombinant enzyme is a dimer with high specific activity. Compared to its thermoactivity (Topt at 80°C), the thermal stability of the enzyme is high, as judged from its Tm (101°C), and from irreversible thermal inactivation assays. Molecular modeling of the structure revealed an inter-subunit ion-pair network, comparable in size to the network found in the citrate synthase from P. furiosus; these networks are discussed in relation to the high thermal stability of these bacterial and archaeal enzymes.
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