Publications by category
Journal articles
De Rose SA, Kuprat T, Isupov MN, Reinhardt A, Schönheit P, Littlechild JA (2021). Biochemical and Structural Characterisation of a Novel D-Lyxose Isomerase from the Hyperthermophilic Archaeon Thermofilum sp.
Frontiers in Bioengineering and Biotechnology,
9Abstract:
Biochemical and Structural Characterisation of a Novel D-Lyxose Isomerase from the Hyperthermophilic Archaeon Thermofilum sp.
A novel D-lyxose isomerase has been identified within the genome of a hyperthermophilic archaeon belonging to the Thermofilum species. The enzyme has been cloned and over-expressed in Escherichia coli and biochemically characterised. This enzyme differs from other enzymes of this class in that it is highly specific for the substrate D-lyxose, showing less than 2% activity towards mannose and other substrates reported for lyxose isomerases. This is the most thermoactive and thermostable lyxose isomerase reported to date, showing activity above 95°C and retaining 60% of its activity after 60 min incubation at 80°C. This lyxose isomerase is stable in the presence of 50% (v/v) of solvents ethanol, methanol, acetonitrile and DMSO. The crystal structure of the enzyme has been resolved to 1.4–1.7 A. resolution in the ligand-free form and in complexes with both of the slowly reacting sugar substrates mannose and fructose. This thermophilic lyxose isomerase is stabilised by a disulfide bond between the two monomers of the dimeric enzyme and increased hydrophobicity at the dimer interface. These overall properties of high substrate specificity, thermostability and solvent tolerance make this lyxose isomerase enzyme a good candidate for potential industrial applications.
Abstract.
De Rose SA, Finnigan W, Harmer NJ, Littlechild JA, consortium TH, Bettina S, Christopher B, Christina S, Benjamin M, N. IM, et al (2021). Production of the Extremolyte Cyclic 2,3-Diphosphoglycerate Using Thermus thermophilus as a Whole-Cell Factory. Frontiers in Catalysis, 1
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
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.
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.
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
2021
De Rose SA, Kuprat T, Isupov MN, Reinhardt A, Schönheit P, Littlechild JA (2021). Biochemical and Structural Characterisation of a Novel D-Lyxose Isomerase from the Hyperthermophilic Archaeon Thermofilum sp.
Frontiers in Bioengineering and Biotechnology,
9Abstract:
Biochemical and Structural Characterisation of a Novel D-Lyxose Isomerase from the Hyperthermophilic Archaeon Thermofilum sp.
A novel D-lyxose isomerase has been identified within the genome of a hyperthermophilic archaeon belonging to the Thermofilum species. The enzyme has been cloned and over-expressed in Escherichia coli and biochemically characterised. This enzyme differs from other enzymes of this class in that it is highly specific for the substrate D-lyxose, showing less than 2% activity towards mannose and other substrates reported for lyxose isomerases. This is the most thermoactive and thermostable lyxose isomerase reported to date, showing activity above 95°C and retaining 60% of its activity after 60 min incubation at 80°C. This lyxose isomerase is stable in the presence of 50% (v/v) of solvents ethanol, methanol, acetonitrile and DMSO. The crystal structure of the enzyme has been resolved to 1.4–1.7 A. resolution in the ligand-free form and in complexes with both of the slowly reacting sugar substrates mannose and fructose. This thermophilic lyxose isomerase is stabilised by a disulfide bond between the two monomers of the dimeric enzyme and increased hydrophobicity at the dimer interface. These overall properties of high substrate specificity, thermostability and solvent tolerance make this lyxose isomerase enzyme a good candidate for potential industrial applications.
Abstract.
De Rose SA, Finnigan W, Harmer NJ, Littlechild JA, consortium TH, Bettina S, Christopher B, Christina S, Benjamin M, N. IM, et al (2021). Production of the Extremolyte Cyclic 2,3-Diphosphoglycerate Using Thermus thermophilus as a Whole-Cell Factory. Frontiers in Catalysis, 1
De Rose SA, Harmer N, Littlechild J (2021). Production of the extremolyte cyclic 2,3-diphosphoglycerate using Thermus thermophilus as a whole-cell factory.
Abstract:
Production of the extremolyte cyclic 2,3-diphosphoglycerate using Thermus thermophilus as a whole-cell factory
Osmolytes protect microbial cells against temperature, osmolarity and other stresses. The osmolyte cyclic 2,3-diphosphoglycerate, originally isolated from the thermophilic archaeon Methanothermus fervidus, naturally protects cellular proteins under extreme conditions. The biosynthetic pathway for cyclic 2,3-diphosphoglycerate has been introduced into the thermophilic bacterium Thermus thermophilus. The two enzymes in this synthetic pathway, 2-phosphoglycerate kinase and cyclic diphosphoglycerate synthetase, were incorporated into a newly designed modular BioBricks vector. The expression of this two-enzyme cascade resulted in the whole cell production of cyclic 2,3 diphosphoglycerate. In vivo production of cyclic 2,3-diphosphoglycerate was confirmed by mass spectrometry to a concentration up to 650 µM. This study demonstrates the feasibility of using this well studied thermophilic bacterium as a host in a whole-cell factory approach to produce cyclic 2,3 diphosphoglycerate. This raises the potential for commercialisation of cDPG for cosmetic and healthcare applications. Our work demonstrates the potential of Thermus thermophilus as an alternative host for other high value small organic molecules of industrial interest.
Abstract.
De Rose S, Harmer N, Littlechild J, Finnigan W (2021). Production of the extremolyte cyclic 2,3-diphosphoglycerate using Thermus thermophilus as a whole-cell factory- DATASET.
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
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.
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.
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.
