28 results on '"Kaper, Thijs"'
Search Results
2. Identification of acceptor substrate binding subsites +2 and +3 in the amylomaltase from Thermus thermophilus HB8
- Author
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Kaper, Thijs, Leemhuis, Hans, Uitdehaag, Joost C.M., van der Veen, Bart A., Dijkstra, Bauke W., van der Maarel, Marc J.E.C., and Dijkhuizen, Lubbert
- Subjects
Gram-negative bacteria -- Chemical properties ,Gram-negative bacteria -- Structure ,Glycosides -- Chemical properties ,Glycosides -- Structure ,Binding proteins -- Structure ,Binding proteins -- Chemical properties ,Biological sciences ,Chemistry - Abstract
The characterization of amylomaltase from the hyperthermophilic bacterium Thermus thermophilus HB8 (Tt AMase), the roles of the putative catalytic residues and the identification of acceptor subsites +2 and +3 in the active site are reported. The results have shown that the main catalytic machinery is the same for glycoside hydrolase family 77 (GH77) and GH13, whereas the properties of acceptor subsite +2 has distinguished the two families.
- Published
- 2007
3. Activity and stability of hyperthermophilic enzymes: a comparative study on two archaeal β-glycosidases
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Pouwels, Jeroen, Moracci, Marco, Cobucci-Ponzano, Beatrice, Perugino, Giuseppe, van der Oost, John, Kaper, Thijs, Lebbink, J.H.G., de Vos, W.M., Ciaramella, Maria, and Rossi, M.
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- 2000
- Full Text
- View/download PDF
4. Single amino acid residue changes in subsite − 1 of inulosucrase from Lactobacillus reuteri 121 strongly influence the size of products synthesized
- Author
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Ozimek, Lukasz K., Kralj, Slavko, Kaper, Thijs, van der Maarel, Marc J. E. C., and Dijkhuizen, Lubbert
- Published
- 2006
5. Hypocrea jecorina CEL6A protein engineering
- Author
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Lantz Suzanne E, Goedegebuur Frits, Hommes Ronald, Kaper Thijs, Kelemen Bradley R, Mitchinson Colin, Wallace Louise, Ståhlberg Jerry, and Larenas Edmundo A
- Subjects
Fuel ,TP315-360 ,Biotechnology ,TP248.13-248.65 - Abstract
Abstract The complex technology of converting lignocellulose to fuels such as ethanol has advanced rapidly over the past few years, and enzymes are a critical component of this technology. The production of effective enzyme systems at cost structures that facilitate commercial processes has been the focus of research for many years. Towards this end, the H. jecorina cellobiohydrolases, CEL7A and CEL6A, have been the subject of protein engineering at Genencor. Our first rounds of cellobiohydrolase engineering were directed towards improving the thermostability of both of these enzymes and produced variants of CEL7A and CEL6A with apparent melting temperatures above 70°C, placing their stability on par with that of H. jecorina CEL5A (EG2) and CEL3A (BGL1). We have now moved towards improving CEL6A- and CEL7A-specific performance in the context of a complete enzyme system under industrially relevant conditions. Achievement of these goals required development of new screening strategies and tools. We discuss these advances along with some results, focusing mainly on engineering of CEL6A.
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- 2010
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- View/download PDF
6. Fluorescence resonance energy transfer sensors for quantitative monitoring of pentose and disaccharide accumulation in bacteria
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Looger Loren L, Lager Ida, Kaper Thijs, Chermak Diane, and Frommer Wolf B
- Subjects
Fuel ,TP315-360 ,Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background Engineering microorganisms to improve metabolite flux requires detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. These sensors have been applied successfully in mammalian and plant cells but potentially could also be used to monitor steady-state levels of metabolites in microorganisms using fluorimetric assays. Sensors for hexose and pentose carbohydrates could help in the development of fermentative microorganisms, for example, for biofuels applications. Arabinose is one of the carbohydrates to be monitored during biofuels production from lignocellulose, while maltose is an important degradation product of starch that is relevant for starch-derived biofuels production. Results An Escherichia coli expression vector compatible with phage λ recombination technology was constructed to facilitate sensor construction and was used to generate a novel fluorescence resonance energy transfer sensor for arabinose. In parallel, a strategy for improving the sensor signal was applied to construct an improved maltose sensor. Both sensors were expressed in the cytosol of E. coli and sugar accumulation was monitored using a simple fluorimetric assay of E. coli cultures in microtiter plates. In the case of both nanosensors, the addition of the respective ligand led to concentration-dependent fluorescence resonance energy transfer responses allowing quantitative analysis of the intracellular sugar levels at given extracellular supply levels as well as accumulation rates. Conclusion The nanosensor destination vector combined with the optimization strategy for sensor responses should help to accelerate the development of metabolite sensors. The new carbohydrate fluorescence resonance energy transfer sensors can be used for in vivo monitoring of sugar levels in prokaryotes, demonstrating the potential of such sensors as reporter tools in the development of metabolically engineered microbial strains or for real-time monitoring of intracellular metabolite during fermentation.
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- 2008
- Full Text
- View/download PDF
7. The catalytic potency of β-glucosidase from Pyrococcus furiosus in the direct glucosylation reaction
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Mattheus de Roode, B., van der Meer, Tjebbe D., Kaper, Thijs, Franssen, Maurice C.R., van der Padt, Albert, van der Oost, John, and Boom, Remko M.
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- 2001
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8. Amylomaltase of Pyrobaculum acrophilum IM2 produces thermoreversible starch gels
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Kaper, Thijs, Dijkhuizen, Lubbert, Talik, Boguslawa, Ettema, Thijs J., Bos, Herman, and van der Maarel, Marc J.E.C.
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Genetic code -- Research ,Gene expression -- Research ,Escherichia coli -- Genetic aspects ,Genetic research ,Biological sciences - Abstract
The amylomaltase-encoding gene from the hyperthermophilic archaeon Pyrobaculum acrophilum IM2 was cloned and expressed in Escherichia coli, and the gene product (PyAMase) was characterized. Acting on gelatinized food-grade potato starch, PyAMase produced a thermoreversible starch product with gelatin-like properties.
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- 2005
9. Activity of hyperthermophilic glycosynthases is significantly enhanced at acidic pH
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Perugino, Giuseppe, Trincone, Antonio, Giordano, Assunta, Oost, John van der, Kaper, Thijs, Rossi, Mose, and Moracci, Marco
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Catalysts -- Physiological aspects ,Catalysts -- Composition ,Biosynthesis -- Analysis ,Hydrogen-ion concentration -- Physiological aspects ,Acids ,Glycosylation -- Analysis ,Biochemistry -- Research ,Biological sciences ,Chemistry - Abstract
The authors have investigated hyperthermophilic glycosynthases. They report that these glycosynthases demonstrate k (sub)cat values which are similar to those of the wild-type enzymes, and suggest that the reactivation conditions can modify the ionization state of the resudues which act as the acid/base catalyst.
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- 2003
10. Structural studies of a glycoside hydrolase family 3 β‐glucosidase from the model fungus Neurospora crassa.
- Author
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Karkehabadi, Saeid, Hansson, Henrik, Mikkelsen, Nils Egil, Kim, Steve, Kaper, Thijs, Sandgren, Mats, and Gudmundsson, Mikael
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GLYCOSIDASES ,HYDROLYSIS ,LIGNOCELLULOSE ,GLUCOSIDASES ,NEUROSPORA crassa - Abstract
The glycoside hydrolase family 3 (GH3) β‐glucosidases are a structurally diverse family of enzymes. Cel3A from Neurospora crassa (NcCel3A) belongs to a subfamily of key enzymes that are crucial for industrial biomass degradation. β‐Glucosidases hydrolyse the β‐1,4 bond at the nonreducing end of cellodextrins. The hydrolysis of cellobiose is of special importance as its accumulation inhibits other cellulases acting on crystalline cellulose. Here, the crystal structure of the biologically relevant dimeric form of NcCel3A is reported. The structure has been refined to 2.25 Å resolution, with an Rcryst and Rfree of 0.18 and 0.22, respectively. NcCel3A is an extensively N‐glycosylated glycoprotein that shares 46% sequence identity with Hypocrea jecorina Cel3A, the structure of which has recently been published, and 61% sequence identity with the thermophilic β‐glucosidase from Rasamsonia emersonii. NcCel3A is a three‐domain protein with a number of extended loops that deepen the active‐site cleft of the enzyme. These structures characterize this subfamily of GH3 β‐glucosidases and account for the high cellobiose specificity of this subfamily. The glycoside hydrolase family 3 β‐glucosidase Cel3A from Neurospora crassa (NcCel3A) is highly specific for cellobiose. The crystal structure of NcCel3A has been solved to 2.25 Å resolution. The structure is a dimer and exhibits a high degree of N‐glycosylation. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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11. Oxygen Activation by Cu LPMOs in Recalcitrant Carbohydrate Polysaccharide Conversion to Monomer Sugars.
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Meier, Katlyn K., Jones, Stephen M., Kaper, Thijs, Hansson, Henrik, Koetsier, Martijn J., Karkehabadi, Saeid, Solomon, Edward I., Sandgren, Mats, and Kelemen, Bradley
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- 2018
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12. Single amino acid residue changes in subsite -1 of inulosucrase from Lactobacillus reuteri 121 strongly influence the size of products synthesized
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Ozimek, Lukasz K., Kralj, Slavko, Kaper, Thijs, Maarel, Marc J.E.C. van der, Dijkhuizen, Lubbert, Groningen Biomolecular Sciences and Biotechnology, Host-Microbe Interactions, and Molecular Microbiology
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FRUCTOSYLTRANSFERASE ,GLYCOSYL HYDROLASES ,REUTERI ,Lactobacillus reuteri ,SITE-DIRECTED MUTAGENESIS ,BACILLUS-SUBTILIS LEVANSUCRASE ,DIAZOTROPHICUS ,CRYSTAL-STRUCTURE ,ENZYMATIC-PROPERTIES ,inulosucrase ,processivity ,ENZYMES ,STREPTOCOCCUS-MUTANS - Abstract
Bacterial fructansucrase enzymes belong to glycoside hydrolase family 68 and catalyze transglycosylation reactions with sucrose, resulting in the synthesis of fructooligosaccharides and/or a fructan polymer. Significant differences in fructansucrase enzyme product specificities can be observed, i.e. in the type of polymer (levan or inulin) synthesized, and in the ratio of polymer versus fructooligosaccharide synthesis. The Lactobacillus reuteri 121 inulosucrase enzyme produces a diverse range of fructooligosaccharide molecules and a minor amount of inulin polymer [with β(2–1) linkages]. The three-dimensional structure of levansucrase (SacB) of Bacillus subtilis revealed eight amino acid residues interacting with sucrose. Sequence alignments showed that six of these eight amino acid residues, including the catalytic triad (D272, E523 and D424, inulosucrase numbering), are completely conserved in glycoside hydrolase family 68. The other three completely conserved residues are located at the -1 subsite (W271, W340 and R423). Our aim was to investigate the roles of these conserved amino acid residues in inulosucrase mutant proteins with regard to activity and product profile. Inulosucrase mutants W340N and R423H were virtually inactive, confirming the essential role of these residues in the inulosucrase active site. Inulosucrase mutants R423K and W271N were less strongly affected in activity, and displayed an altered fructooligosaccharide product pattern from sucrose, synthesizing a much lower amount of oligosaccharide and significantly more polymer. Our data show that the -1 subsite is not only important for substrate recognition and catalysis, but also plays an important role in determining the size of the products synthesized.
- Published
- 2006
13. A novel thermoreversible gelling product made by enzymatic modification of starch
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Van Der Maarel, Marc J E C, Capron, Isabelle, Euverink, Gerrit Jan W, Bos, Herman Th, Kaper, Thijs, Binnema, Doede J., Steeneken, Peter A M, and Biobased Ingredients and Materials
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gelatin ,gelatine ,potato starch ,food and beverages ,aardappelzetmeel - Abstract
Amylomaltases or D-enzyme (4-α-glucanotransferases; E.C. 2.4.1.25) are carbohydrate-active enzymes that catalyze the transfer of glucan units from one α-glucan to another in a disproportionation reaction. These enzymes are involved in starch metabolism in plants or maltose/glycogen metabolism in many microorganisms. The amylomaltase of the hyperthermophilic bacterium Thermus thermophilus HB8 was overproduced in Escherichia coli, partially purified and used to modify potato starch. The action of amylomaltase caused the disappearance of amylose and the broadening of the side-chain length distribution in amylopectin, which resulted in a product with both shorter and longer side chains than in the parent starch. Amylomaltase-treated potato starch showed thermoreversible gelation at concentrations of 3% (w/v) or more, thus making it comparable to gelatin. Because of its animal origin, gelatin is not accepted by several consumer groups. Therefore, the amylomaltase-treated potato starch might be a good plant-derived substitute for gelatin. ? 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
- Published
- 2005
14. Structural and functional studies of the glycoside hydrolase family 3 β-glucosidase Cel3A from the moderately thermophilic fungus Rasamsonia emersonii.
- Author
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Gudmundsson, Mikael, Hansson, Henrik, Karkehabadi, Saeid, Larsson, Anna, Stals, Ingeborg, Kim, Steve, Sunux, Sergio, Fujdala, Meredith, Larenas, Edmund, Kaper, Thijs, and Sandgren, Mats
- Subjects
GLYCOSIDASES ,BIOMASS ,OLIGOMERS - Abstract
The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. β-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the β-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the β-glucosidase from H. jecorina (HjCel3A) with the β-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 β-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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15. Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase.
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LOO, SUZANNE WOLTERINK-VAN, SIEMERINK, MARCO A. J., PERRAKIS, GEORGIOS, KAPER, THIJS, KENGEN, SERVÉ W. M., and VAN DER OOST, JOHN
- Published
- 2009
16. Nanosensor Detection of an Immunoregulatory Tryptophan Influx/Kynurenine Efflux Cycle.
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Kaper, Thijs, Looger, Loren L., Takanaga, Hitomi, Platten, Michael, Steinman, Lawrence, and Frommer, Wolf B.
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GENETICS , *TRYPTOPHAN , *KYNURENINE , *AMINO acids , *QUINOLINIC acid - Abstract
Genetically encoded FRET nanosensors were developed to measure cytosolic tryptophan steady-state levels and flux in real time. [ABSTRACT FROM AUTHOR]
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- 2007
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17. Persistence of Tertiary Structure in 7.9 M Guanidinium Chloride: The Case of Endo-β-1,3-glucanase from Pyrococcus furiosus.
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Chiaraluce, Roberta, van der Oost, John, Lebbink, Joyce H.G., Kaper, Thijs, and Consalvi, Valerio
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- 2002
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18. Improved oligosaccharide synthesis by protein engineering of β-glucosidase CelB from hyperthermophilic Pyrococcus furiosus.
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Hansson, Therese, Kaper, Thijs, van der Oost, John, de Vos, Willem M., and Adlercreutz, Patrick
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- 2001
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19. Comparative structural analysis and substrate specificity engineering of the hyperthermostable....
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Kaper, Thijs and Lebbink, Joyce H.G.
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GLUCOSIDASES , *BINDING sites , *MUTAGENESIS , *PHOSPHORYLATION - Abstract
Examines the structure and substrate specificity of beta-glucosidase (CelB) from the hyperthermophilic archeon Pyrococcus furiosus. Difference in substrate specificity between two types of glycosidases; Characterization of the purified mutant CelB enzyme through mutagenesis; Cell site substitutions of CelB to increase phosphorylated galactose activity.
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- 2000
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20. Improving low-temperature catalysis in the hyperthermostable Pyrococcus furiosus....
- Author
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Lebbink, Joyce H. G. and Kaper, Thijs
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GLUCOSIDASES , *CATALYSIS , *AMINO acid sequence - Abstract
Examines the improvement in low-temperature catalysis in Pyrococcus furiosus (CelB) by directed evolution. Selection of CelB variants with higher activity on p-nitrophenyl-beta-delta-glucopyranoside at room temperature; Accommodation of CelB on amino acids; Residues critical to the determination of thermostability, low-temperature activity and substrate recognition.
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- 2000
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21. Biochemical Characterization and Crystal Structures of a Fungal Family β-Glucosidase, Cel3A fromHypocrea jecorina.
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Karkehabadi, Saeid, Helmich, Kate E., Kaper, Thijs, Hansson, Henrik, Mikkelsen, Nils-Egil, Gudmundsson, Mikael, Piens, Kathleen, Fujdala, Meredith, Banerjee, Goutami, Scott-Craig, John S., Walton, Jonathan D., Phillips Jr., George N., and Sandgren, Mats
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GLUCOSIDASES , *OLIGOMERS , *HYPOCREACEAE , *FUNGI , *BIOCHEMICAL research - Abstract
Cellulase mixtures from Hypocrea jecorina are commonly used for the saccharification of cellulose in biotechnical applications. The most abundant β-glucosidase in the mesophilic fungus Hypocrea jecorina is HjCel3A, which hydrolyzes the β-linkage between two adjacent molecules in dimers and short oligomers of glucose. It has been shown that enhanced levels of HjCel3AinH. jecorina cellulase mixtures benefit the conversion of cellulose to glucose. Biochemical characterization of HjCel3A shows that the enzyme efficiently hydrolyzes (1,4)- as well as (1,2)-, (1,3)-, and (1,6)β-D-linked disaccharides. For crystallization studies, HjCel3A was produced in both H. jecorina (HjCel3A) and Pichia pastoris (Pp-HjCel3A).Whereas the thermostabilities of HjCel3A and Pp-HjCel3A are the same, Pp-HjCel3A has a higher degree of N-linked glycosylation. Here, we present x-ray structures of HjCel3A with and without glucose bound in the active site. The structures have a three-domain architecture as observed previously for other glycoside hydrolase family 3 β-glucosidases. Both production hosts resulted in HjCel3A structures that have N-linked glycosylations at Asn208 and Asn310. In H. jecorina-produced HjCel3A, a single N-acetylglucosamine is present at both sites, whereas in Pp-HjCel3A, the P. pastoris-produced HjCel3A enzyme, the glycan chains consist of 8 or 4 saccharides. The glycosylations are involved in intermolecular contacts in the structures derived fromeither host. Due to the different sizes of the glycosylations, the interactions result in different crystal forms for the two protein forms. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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22. Three-way Stabilization of the Covalent Intermediate in Amylomaltase, an α-Amylase-like Transglycosylase.
- Author
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Barends, Thomas R. M., Bultema, Jelle B., Kaper, Thijs, Van Der Maarel, Marc J. E. C., Dijkhuizen, Lubbert, and Dijkstra, Bauke W.
- Subjects
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GLYCOSYLTRANSFERASES , *GLYCOSIDES , *PROTEINS , *CRYSTALLOGRAPHY , *HYDROLYSIS - Abstract
Amylomaltases are glycosyl hydrolases belonging to glycoside hydrolase family 77 that are capable of the synthesis of large cyclic glucans and the disproportionation of oligosaccharides. Using protein crystallography, we have generated a flip book movie of the amylomaltase catalytic cycle in atomic detail. The structures include a covalent glycosyl enzyme intermediate and a covalent intermediate in complex with an analogue of a co-substrate and show how the structures of both enzyme and substrate respond to the changes required by the catalytic cycle as it proceeds. Notably, the catalytic nucleophile changes conformation dramatically during the reaction. Also, Gln-256 on the 250s loop is involved in orienting the substrate in the +1 site. The absence of a suitable base in the covalent intermediate structure explains the low hydrolysis activity. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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23. High-resolution structure of a lytic polysaccharide monooxygenase from Hypocrea jecorina reveals a predicted linker as an integral part of the catalytic domain.
- Author
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Hansson, Henrik, Karkehabadi, Saeid, Mikkelsen, Nils, Douglas, Nicholai R., Kim, Steve, Lam, Anna, Kaper, Thijs, Kelemen, Brad, Meier, Katlyn K., Jones, Stephen M., Solomon, Edward I., and Sandgren, Mats
- Subjects
- *
MONOOXYGENASES , *HYPOCREACEAE , *CATALYTIC domains , *CELLULOSE , *HYDROLYSIS - Abstract
For decades, the enzymes of the fungus Hypocrea jecorina have served as a model system for the breakdown of cellulose. Three-dimensional structures for almost all H. jecorina cellulose- degrading enzymes are available, except for HjLPMO9A, belonging to the AA9 family of lytic polysaccharide monooxygenases (LPMOs). These enzymes enhance the hydrolytic activity of cellulases and are essential for cost-efficient conversion of lignocellulosic biomass. Here, using structural and spectroscopic analyses, we found that native HjLPMO9A contains a catalytic domain and a family-1 carbohydrate-binding module (CBM1) connected via a linker sequence. A C terminally truncated variant of HjLPMO9A containing 21 residues of the predicted linker was expressed at levels sufficient for analysis. Here, using structural, spectroscopic, and biochemical analyses, we found that this truncated variant exhibited reduced binding to and activity on cellulose compared with the full-length enzyme. Importantly, a 0.95-Å resolution X-ray structure of truncated HjLPMO9Arevealed that the linker forms an integral part of the catalytic domain structure, covering a hydrophobic patch on the catalytic AA9 module. We noted that the oxidized catalytic center contains a Cu(II) coordinated by two His ligands, one of which has a His-brace in which the His-1 terminal amine group also coordinates to a copper. The final equatorial position of the Cu(II) is occupied by a water-derived ligand. The spectroscopic characteristics of the truncated variant were not measurably different from those of full-length HjLPMO9A, indicating that the presence of the CBM1 module increases the affinity of HjLPMO9A for cellulose binding, but does not affect the active site. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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24. Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase.
- Author
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Wolterink-van Loo S, Siemerink MA, Perrakis G, Kaper T, Kengen SW, and van der Oost J
- Subjects
- Aldehyde-Lyases genetics, Amino Acid Sequence, Cold Temperature, Directed Molecular Evolution, Enzyme Stability, Models, Molecular, Protein Engineering, Sequence Alignment, Sulfolobus acidocaldarius metabolism, Aldehyde-Lyases chemistry, Sulfolobus acidocaldarius enzymology
- Abstract
Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95 degrees C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 degrees C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40-60 degrees C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.
- Published
- 2009
- Full Text
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25. Fluorescence resonance energy transfer sensors for quantitative monitoring of pentose and disaccharide accumulation in bacteria.
- Author
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Kaper T, Lager I, Looger LL, Chermak D, and Frommer WB
- Abstract
Background: Engineering microorganisms to improve metabolite flux requires detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. These sensors have been applied successfully in mammalian and plant cells but potentially could also be used to monitor steady-state levels of metabolites in microorganisms using fluorimetric assays. Sensors for hexose and pentose carbohydrates could help in the development of fermentative microorganisms, for example, for biofuels applications. Arabinose is one of the carbohydrates to be monitored during biofuels production from lignocellulose, while maltose is an important degradation product of starch that is relevant for starch-derived biofuels production., Results: An Escherichia coli expression vector compatible with phage lambda recombination technology was constructed to facilitate sensor construction and was used to generate a novel fluorescence resonance energy transfer sensor for arabinose. In parallel, a strategy for improving the sensor signal was applied to construct an improved maltose sensor. Both sensors were expressed in the cytosol of E. coli and sugar accumulation was monitored using a simple fluorimetric assay of E. coli cultures in microtiter plates. In the case of both nanosensors, the addition of the respective ligand led to concentration-dependent fluorescence resonance energy transfer responses allowing quantitative analysis of the intracellular sugar levels at given extracellular supply levels as well as accumulation rates., Conclusion: The nanosensor destination vector combined with the optimization strategy for sensor responses should help to accelerate the development of metabolite sensors. The new carbohydrate fluorescence resonance energy transfer sensors can be used for in vivo monitoring of sugar levels in prokaryotes, demonstrating the potential of such sensors as reporter tools in the development of metabolically engineered microbial strains or for real-time monitoring of intracellular metabolite during fermentation.
- Published
- 2008
- Full Text
- View/download PDF
26. Persistence of tertiary structure in 7.9 M guanidinium chloride: the case of endo-beta-1,3-glucanase from Pyrococcus furiosus.
- Author
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Chiaraluce R, Van Der Oost J, Lebbink JH, Kaper T, and Consalvi V
- Subjects
- Circular Dichroism, Enzyme Stability drug effects, Hot Temperature, Hydrogen-Ion Concentration, Protein Denaturation drug effects, Protein Folding, Protein Structure, Secondary drug effects, Protein Structure, Tertiary drug effects, Spectrometry, Fluorescence, Glucan Endo-1,3-beta-D-Glucosidase chemistry, Guanidine chemistry, Pyrococcus furiosus enzymology
- Abstract
The Pyrococcus furiosus endo-beta-1,3-glucanase belongs to the subfamily of laminarinase, which can be classified as "all beta proteins" as confirmed by deconvolution of far-UV CD and FTIR spectra. The persistence of a significant amount of tertiary structure in 7.9 M GdmCl, as indicated by near-UV CD spectroscopy, accompanied by a red-shift of the maximum fluorescence emission wavelength is a peculiar property of this hyperthermophilic endoglucanase. The possibility to observe tertiary structure elements under extremely denaturing conditions is notable and is limited to only a few examples. The unusual resistance toward guanidinium chloride denaturation is paralleled by a notable stability at extremely low pH and at high temperature. The analysis of the protein spectral properties indicates that the secondary structure elements are preserved down to pH 1.0 and up to 90 degrees C at pH 7.4 and pH 3.0. The study of the conditions that determine the persistence of residual structure at high denaturant concentration and the examination of these structures are particularly interesting because these state(s) may be preliminary or coincident with the coalescence of protein aggregates or to the formation of amyloid-like fibrils, and they may serve as seeds of protein folding.
- Published
- 2002
- Full Text
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27. DNA family shuffling of hyperthermostable beta-glycosidases.
- Author
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Kaper T, Brouns SJ, Geerling AC, De Vos WM, and Van der Oost J
- Subjects
- Amino Acid Sequence, Bacterial Proteins drug effects, Bacterial Proteins genetics, DNA chemistry, DNA genetics, Enzyme Stability, Gene Library, Glucose metabolism, Glucose pharmacology, Glycoside Hydrolases chemistry, Glycoside Hydrolases genetics, Glycoside Hydrolases metabolism, Imidazoles pharmacology, Kinetics, Lactose metabolism, Models, Molecular, Molecular Sequence Data, Protein Conformation, Pyridines pharmacology, Recombinant Proteins drug effects, Recombinant Proteins genetics, Sequence Homology, Amino Acid, Temperature, beta-Glucosidase drug effects, beta-Glucosidase genetics, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Protein Engineering methods, Recombinant Proteins chemistry, Recombinant Proteins metabolism, beta-Glucosidase chemistry, beta-Glucosidase metabolism
- Abstract
The structural compatibility of two hyperthermostable family 1 glycoside hydrolases, Pyrococcus furiosus CelB and Sulfolobus solfataricus LacS, as well as their kinetic potential were studied by construction of a library of 2048 hybrid beta-glycosidases using DNA family shuffling. The hybrids were tested for their thermostability, ability to hydrolyse lactose and sensitivity towards inhibition by glucose. Three screening rounds at 70 degrees C led to the isolation of three high-performance hybrid enzymes (hybrid 11, 18 and 20) that had 1.5-3.5-fold and 3.5-8.6-fold increased lactose hydrolysis rates compared with parental CelB and LacS respectively. The three variants were the result of a single crossover event, which gave rise to hybrids with a LacS N-terminus and a main CelB sequence. Constructed three-dimensional models of the hybrid enzymes revealed that the catalytic (betaalpha)(8)-barrel was composed of both LacS and CelB elements. In addition, an extra intersubunit hydrogen bond in hybrids 18 and 20 might explain their superior stability over hybrid 11. This study demonstrates that extremely thermostable enzymes with limited homology and different mechanisms of stabilization can be efficiently shuffled to form stable hybrids with improved catalytic features.
- Published
- 2002
- Full Text
- View/download PDF
28. Substrate specificity engineering of beta-mannosidase and beta-glucosidase from Pyrococcus by exchange of unique active site residues.
- Author
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Kaper T, van Heusden HH, van Loo B, Vasella A, van der Oost J, and de Vos WM
- Subjects
- Amino Acid Sequence, Base Sequence, DNA Primers, Hydrogen-Ion Concentration, Mannosidases chemistry, Molecular Sequence Data, Phylogeny, Protein Engineering, Sequence Homology, Amino Acid, Substrate Specificity, beta-Glucosidase chemistry, beta-Mannosidase, Mannosidases metabolism, Pyrococcus enzymology, beta-Glucosidase metabolism
- Abstract
A beta-mannosidase gene (PH0501) was identified in the Pyrococcus horikoshii genome and cloned and expressed in E. coli. The purified enzyme (BglB) was most specific for the hydrolysis of p-nitrophenyl-beta-D-mannopyranoside (pNP-Man) (Km: 0.44 mM) with a low turnover rate (kcat: 4.3 s(-1)). The beta-mannosidase has been classified as a member of family 1 of glycoside hydrolases. Sequence alignments and homology modeling showed an apparent conservation of its active site region with, remarkably, two unique active site residues, Gln77 and Asp206. These residues are an arginine and asparagine residue in all other known family 1 enzymes, which interact with the catalytic nucleophile and equatorial C2-hydroxyl group of substrates, respectively. The unique residues of P. horikoshii BglB were introduced in the highly active beta-glucosidase CelB of Pyrococcus furiosus and vice versa, yielding two single and one double mutant for each enzyme. In CelB, both substitutions R77Q and N206D increased the specificity for mannosides and reduced hydrolysis rates 10-fold. In contrast, BglB D206N showed 10-fold increased hydrolysis rates and 35-fold increased affinity for the hydrolysis of glucosides. In combination with inhibitor studies, it was concluded that the substituted residues participate in the ground-state binding of substrates with an equatorial C2-hydroxyl group, but contribute most to transition-state stabilization. The unique activity profile of BglB seems to be caused by an altered interaction between the enzyme and C2-hydroxyl of the substrate and a specifically increased affinity for mannose that results from Asp206.
- Published
- 2002
- Full Text
- View/download PDF
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