Overview
Research group links
Research
Research projects
Project HotSolute
Thermophilic organisms are composed of both bacterial and archaeal species. The enzymes isolated from these species and from other extreme habitats are more robust to temperature, organic solvents and proteolysis. They often have unique substrate specificities and originate from novel metabolic pathways. Thermophiles as well as their stable enzymes (‘thermozymes’) are receiving increased attention for biotechnological applications.
The proposed project will establish thermophilic in vitro enzyme cascades as well as two new chassis, the thermophilic bacterium Thermus thermophilus (Tth, 65-75°C, pH 7.0) and the thermoacidophilic archaeon Sulfolobus acidocaldarius (Saci, 75-80°C, pH 2-4), as new thermophilic, bacterial and archaeal platforms for the production of novel high added-value products, i.e. ‘extremolytes’. Extremolytes are small molecular compatible solutes found naturally in the cells of thermophilic species that accumulate in the cell in response to multiple environmental stresses and stabilize cellular components (including proteins, membranes). Extremolytes offer an amazing so far unexploited potential for industrial applications including food, health, consumer care and cosmetics. However, their production in common mesophilic organisms (i.e. yeast, E. coli) is currently hampered by the hyperthermophilic origin of the respective metabolic pathways requiring a thermophilic cell factory.
http://hotsolute.com/
https://fairdomhub.org/projects/108
Publications
Key publications | Publications by category | Publications by year
Publications by category
Journal articles
James P, Isupov MN, De Rose SA, Sayer C, Cole IS, Littlechild JA (2020). A ‘Split-Gene’ Transketolase from the Hyper-Thermophilic Bacterium Carboxydothermus hydrogenoformans: Structure and Biochemical Characterization.
Frontiers in Microbiology,
11 Full text.
Yilmazer B, Isupov MN, De Rose SA, Bulut H, Benninghoff JC, Binay B, Littlechild JA (2020). Structural insights into the NAD+-dependent formate dehydrogenase mechanism revealed from the NADH complex and the formate NAD+ ternary complex of the Chaetomium thermophilum enzyme. Journal of Structural Biology, 212(3), 107657-107657.
Cutlan R, De Rose S, Isupov MN, Littlechild JA, Harmer NJ (2020). Using enzyme cascades in biocatalysis: Highlight on transaminases and carboxylic acid reductases.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics,
1868(2), 140322-140322.
Full text.
Ferrandi EE, Sayer C, De Rose SA, Guazzelli E, Marchesi C, Saneei V, Isupov MN, Littlechild JA, Monti D (2018). New thermophilic α/β class epoxide hydrolases found in metagenomes from hot environments.
Frontiers in Bioengineering and Biotechnology,
6(OCT).
Abstract:
New thermophilic α/β class epoxide hydrolases found in metagenomes from hot environments
Two novel epoxide hydrolases (EHs), Sibe-EH and CH65-EH, were identified in the metagenomes of samples collected in hot springs in Russia and China, respectively. The two α/β hydrolase superfamily fold enzymes were cloned, over-expressed in Escherichia coli, purified and characterized. The new EHs were active toward a broad range of substrates, and in particular, Sibe-EH was excellent in the desymmetrization of cis-2,3-epoxybutane producing the (2R,3R)-diol product with ee exceeding 99%. Interestingly these enzymes also hydrolyse (4R)-limonene-1,2-epoxide with Sibe-EH being specific for the trans isomer. The Sibe-EH is a monomer in solution whereas the CH65-EH is a dimer. Both enzymes showed high melting temperatures with the CH65-EH being the highest at 85°C retaining 80% of its initial activity after 3 h thermal treatment at 70°C making it the most thermal tolerant wild type epoxide hydrolase described. The Sibe-EH and CH65-EH have been crystallized and their structures determined to high resolution, 1.6 and 1.4 Å, respectively. The CH65-EH enzyme forms a dimer via its cap domains with different relative orientation of the monomers compared to previously described EHs. The entrance to the active site cavity is located in a different position in CH65-EH and Sibe-EH in relation to other known bacterial and mammalian EHs.
Abstract.
Full text.
de Rose SA, Novak H, Dowd A, Singh S, Lang DA, Littlechild J (2017). Stabilization of a lipolytic enzyme for commercial application.
Catalysts,
7(3).
Abstract:
Stabilization of a lipolytic enzyme for commercial application
Thermomyces lanouginosa lipase has been used to develop improved methods for carrier-free immobilization, the Cross-Linked Enzyme Aggregates (CLEAs), for its application in detergent products. An activator step has been introduced to the CLEAs preparation process with the addition of Tween 80 as activator molecule, in order to obtain a higher number of the individual lipase molecules in the “open lid” conformation prior to the cross-linking step. A terminator step has been introduced to quench the cross-linking reaction at an optimal time by treatment with an amine buffer in order to obtain smaller and more homogenous cross-linked particles. This improved immobilization method has been compared to a commercially available enzyme and has been shown to be made up of smaller and more homogenous particles with an average diameter of 1.85 ± 0.28 µm which are 129.7% more active than the free enzyme. The CLEAs produced show improved features for commercial applications such as an improved wash performance comparable with the free enzyme, improved stability to proteolysis and a higher activity after long-term storage.
Abstract.
Publications by year
2020
James P, Isupov MN, De Rose SA, Sayer C, Cole IS, Littlechild JA (2020). A ‘Split-Gene’ Transketolase from the Hyper-Thermophilic Bacterium Carboxydothermus hydrogenoformans: Structure and Biochemical Characterization.
Frontiers in Microbiology,
11 Full text.
Yilmazer B, Isupov MN, De Rose SA, Bulut H, Benninghoff JC, Binay B, Littlechild JA (2020). Structural insights into the NAD+-dependent formate dehydrogenase mechanism revealed from the NADH complex and the formate NAD+ ternary complex of the Chaetomium thermophilum enzyme. Journal of Structural Biology, 212(3), 107657-107657.
Cutlan R, De Rose S, Isupov MN, Littlechild JA, Harmer NJ (2020). Using enzyme cascades in biocatalysis: Highlight on transaminases and carboxylic acid reductases.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics,
1868(2), 140322-140322.
Full text.
2018
Ferrandi EE, Sayer C, De Rose SA, Guazzelli E, Marchesi C, Saneei V, Isupov MN, Littlechild JA, Monti D (2018). New thermophilic α/β class epoxide hydrolases found in metagenomes from hot environments.
Frontiers in Bioengineering and Biotechnology,
6(OCT).
Abstract:
New thermophilic α/β class epoxide hydrolases found in metagenomes from hot environments
Two novel epoxide hydrolases (EHs), Sibe-EH and CH65-EH, were identified in the metagenomes of samples collected in hot springs in Russia and China, respectively. The two α/β hydrolase superfamily fold enzymes were cloned, over-expressed in Escherichia coli, purified and characterized. The new EHs were active toward a broad range of substrates, and in particular, Sibe-EH was excellent in the desymmetrization of cis-2,3-epoxybutane producing the (2R,3R)-diol product with ee exceeding 99%. Interestingly these enzymes also hydrolyse (4R)-limonene-1,2-epoxide with Sibe-EH being specific for the trans isomer. The Sibe-EH is a monomer in solution whereas the CH65-EH is a dimer. Both enzymes showed high melting temperatures with the CH65-EH being the highest at 85°C retaining 80% of its initial activity after 3 h thermal treatment at 70°C making it the most thermal tolerant wild type epoxide hydrolase described. The Sibe-EH and CH65-EH have been crystallized and their structures determined to high resolution, 1.6 and 1.4 Å, respectively. The CH65-EH enzyme forms a dimer via its cap domains with different relative orientation of the monomers compared to previously described EHs. The entrance to the active site cavity is located in a different position in CH65-EH and Sibe-EH in relation to other known bacterial and mammalian EHs.
Abstract.
Full text.
2017
de Rose SA, Novak H, Dowd A, Singh S, Lang DA, Littlechild J (2017). Stabilization of a lipolytic enzyme for commercial application.
Catalysts,
7(3).
Abstract:
Stabilization of a lipolytic enzyme for commercial application
Thermomyces lanouginosa lipase has been used to develop improved methods for carrier-free immobilization, the Cross-Linked Enzyme Aggregates (CLEAs), for its application in detergent products. An activator step has been introduced to the CLEAs preparation process with the addition of Tween 80 as activator molecule, in order to obtain a higher number of the individual lipase molecules in the “open lid” conformation prior to the cross-linking step. A terminator step has been introduced to quench the cross-linking reaction at an optimal time by treatment with an amine buffer in order to obtain smaller and more homogenous cross-linked particles. This improved immobilization method has been compared to a commercially available enzyme and has been shown to be made up of smaller and more homogenous particles with an average diameter of 1.85 ± 0.28 µm which are 129.7% more active than the free enzyme. The CLEAs produced show improved features for commercial applications such as an improved wash performance comparable with the free enzyme, improved stability to proteolysis and a higher activity after long-term storage.
Abstract.
Refresh publications
