Kirstin Eley - Academia.edu (original) (raw)

Papers by Kirstin Eley

Research paper thumbnail of A novel β-xylosidase structure from Geobacillus thermoglucosidasius: the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds

Acta crystallographica. Section D, Biological crystallography, 2014

Geobacillus thermoglucosidasius is a thermophilic bacterium that is able to ferment both C6 and C... more Geobacillus thermoglucosidasius is a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular β-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding a G. thermoglucosidasius β-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed in Escherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7 Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6 Å) structure of the enzyme-substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lac...

Research paper thumbnail of Theoretical analysis of chronoamperometric transients in electrochemical machining and characterization of titanium 6/4 and inconel 718 alloys

An analytical expression is derived for the current±time transient for electrochemical machining ... more An analytical expression is derived for the current±time transient for electrochemical machining (ECM) using a planar tool and workpiece con®guration. This is obtained as a function of such parameters as the initial interelectrode gap, applied voltage, electrolytic conductivity, valency, density and feed rate. Good theoretical ®ts to experimental data are found for the alloys titanium 6/4 (Ti6/4) and Inconel 718 (In718) using both sodium chloride and sodium nitrate electrolytes, demonstrating the applicability of this theory. The values of the electrolytic molar conductivity obtained for chloride and nitrate are consistent with the expected conductivity obtained from molar conductivity measurements. The mean valency values obtained for Ti6/4 and In718 are 3X5 AE 0X2 and 3X0 AE 0X2, respectively. The fraction of the applied voltage used to drive the electrochemical surface reactions, V 0 , has also been obtained. The variation in V 0 between alloys when using the same electrolyte and also for each alloy when using dierent electrolytes is attributed to dierences in the thermodynamics of the removal of the metal from the surface metal oxide. For In718 using chloride electrolyte, an increase in V 0 is observed at higher applied voltages, consistent with a change in the electrochemical dissolution reaction. Analysis of the variation of V 0 at low applied voltages throughout the current±time transient has enabled the current±voltage characteristics of these surfaces electrochemical reactions to be determined, indicating Tafel behaviour. These data show this analysis to be a powerful methodology for understanding and measuring ECM characteristics under realistic ECM conditions.

Research paper thumbnail of Biosynthesis of the 2-Pyridone Tenellin (I) in the Insect Pathogenic Fungus Beauveria bassiana

ChemInform, 2007

Other natural products U 0800 Biosynthesis of the 2-Pyridone Tenellin (I) in the Insect Pathogeni... more Other natural products U 0800 Biosynthesis of the 2-Pyridone Tenellin (I) in the Insect Pathogenic Fungus Beauveria bassiana. -(ELEY, K. L.; HALO, L. M.; SONG, Z.; POWLES, H.; COX*, R. J.; BAILEY, A. M.; LAZARUS, C. M.; SIMPSON, T. J.; ChemBioChem 8 (2007) 3, 289-297; Sch. Chem., Univ. Bristol, Cantock's Close, Bristol BS8 1TS, UK; Eng.) -Mischke 25-198

Research paper thumbnail of Biosynthesis of the 2-Pyridone Tenellin in the Insect Pathogenic FungusBeauveria bassiana

ChemBioChem, 2007

Other natural products U 0800 Biosynthesis of the 2-Pyridone Tenellin (I) in the Insect Pathogeni... more Other natural products U 0800 Biosynthesis of the 2-Pyridone Tenellin (I) in the Insect Pathogenic Fungus Beauveria bassiana. -(ELEY, K. L.; HALO, L. M.; SONG, Z.; POWLES, H.; COX*, R. J.; BAILEY, A. M.; LAZARUS, C. M.; SIMPSON, T. J.; ChemBioChem 8 (2007) 3, 289-297; Sch. Chem., Univ. Bristol, Cantock's Close, Bristol BS8 1TS, UK; Eng.) -Mischke 25-198

Research paper thumbnail of Microbial responses to solvent and alcohol stress

Biotechnology Journal, 2008

Research paper thumbnail of Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

Applied Microbiology and Biotechnology, 2014

This study reports the expression, purification, and kinetic characterization of a pyruvate decar... more This study reports the expression, purification, and kinetic characterization of a pyruvate decarboxylase (PDC) from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120 μM at pH 5), high catalytic efficiency (4.75×10 5 M −1 s −1 at pH 5), a pH opt of approximately 4.5 and an in vitro temperature optimum at approximately 55°C. Due to in vitro thermostablity (approximately 40 % enzyme activity retained after 30 min at 65°C), this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius for ethanol production. Initial studies using a variety of methods failed to detect activity at any growth temperature (45-55°C). However, the application of codon harmonization (i.e., mimicry of the heterogeneous host's transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45°C (as determined by enzyme activity, Western blot, mRNA detection, and ethanol productivity). Here, we describe the first successful expression of PDC in a true thermophile. Yields as high as 0.35±0.04 g/g ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription/translation coupling.

Research paper thumbnail of Application of pheB as a Reporter Gene for Geobacillus spp., Enabling Qualitative Colony Screening and Quantitative Analysis of Promoter Strength

Applied and Environmental Microbiology, 2012

The pheB gene from Geobacillus stearothermophilus DSM6285 has been exploited as a reporter gene f... more The pheB gene from Geobacillus stearothermophilus DSM6285 has been exploited as a reporter gene for Geobacillus spp. The gene product, catechol 2,3-dioxygenase (C23O), catalyzes the formation of 2-hydroxymuconic semialdehyde, which can be readily assayed. The reporter was used to examine expression from the ldh promoter associated with fermentative metabolism.

Research paper thumbnail of Structure of a bifunctional alcohol dehydrogenase involved in bioethanol generation in Geobacillus thermoglucosidasius

Acta Crystallographica Section D Biological Crystallography, 2013

Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative mic... more Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative microorganisms. They catalyse the conversion of an acyl-coenzyme A to an alcohol via an aldehyde intermediate; this is coupled to the oxidation of two NADH molecules to maintain the NAD(+) pool during fermentative metabolism. The structure of the alcohol dehydrogenase (ADH) domain of an ADHE protein from the ethanol-producing thermophile Geobacillus thermoglucosidasius has been determined to 2.5 Å resolution. This is the first structure to be reported for such a domain. In silico modelling has been carried out to generate a homology model of the aldehyde dehydrogenase domain, and this was subsequently docked with the ADH-domain structure to model the structure of the complete ADHE protein. This model suggests, for the first time, a structural mechanism for the formation of the large multimeric assemblies or `spirosomes' that are observed for this ADHE protein and which have previously been reported for ADHEs from other organisms.

Research paper thumbnail of A novel β-xylosidase structure from Geobacillus thermoglucosidasius: the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds

Acta crystallographica. Section D, Biological crystallography, 2014

Geobacillus thermoglucosidasius is a thermophilic bacterium that is able to ferment both C6 and C... more Geobacillus thermoglucosidasius is a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular β-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding a G. thermoglucosidasius β-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed in Escherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7 Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6 Å) structure of the enzyme-substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lac...

Research paper thumbnail of Theoretical analysis of chronoamperometric transients in electrochemical machining and characterization of titanium 6/4 and inconel 718 alloys

An analytical expression is derived for the current±time transient for electrochemical machining ... more An analytical expression is derived for the current±time transient for electrochemical machining (ECM) using a planar tool and workpiece con®guration. This is obtained as a function of such parameters as the initial interelectrode gap, applied voltage, electrolytic conductivity, valency, density and feed rate. Good theoretical ®ts to experimental data are found for the alloys titanium 6/4 (Ti6/4) and Inconel 718 (In718) using both sodium chloride and sodium nitrate electrolytes, demonstrating the applicability of this theory. The values of the electrolytic molar conductivity obtained for chloride and nitrate are consistent with the expected conductivity obtained from molar conductivity measurements. The mean valency values obtained for Ti6/4 and In718 are 3X5 AE 0X2 and 3X0 AE 0X2, respectively. The fraction of the applied voltage used to drive the electrochemical surface reactions, V 0 , has also been obtained. The variation in V 0 between alloys when using the same electrolyte and also for each alloy when using dierent electrolytes is attributed to dierences in the thermodynamics of the removal of the metal from the surface metal oxide. For In718 using chloride electrolyte, an increase in V 0 is observed at higher applied voltages, consistent with a change in the electrochemical dissolution reaction. Analysis of the variation of V 0 at low applied voltages throughout the current±time transient has enabled the current±voltage characteristics of these surfaces electrochemical reactions to be determined, indicating Tafel behaviour. These data show this analysis to be a powerful methodology for understanding and measuring ECM characteristics under realistic ECM conditions.

Research paper thumbnail of Biosynthesis of the 2-Pyridone Tenellin (I) in the Insect Pathogenic Fungus Beauveria bassiana

ChemInform, 2007

Other natural products U 0800 Biosynthesis of the 2-Pyridone Tenellin (I) in the Insect Pathogeni... more Other natural products U 0800 Biosynthesis of the 2-Pyridone Tenellin (I) in the Insect Pathogenic Fungus Beauveria bassiana. -(ELEY, K. L.; HALO, L. M.; SONG, Z.; POWLES, H.; COX*, R. J.; BAILEY, A. M.; LAZARUS, C. M.; SIMPSON, T. J.; ChemBioChem 8 (2007) 3, 289-297; Sch. Chem., Univ. Bristol, Cantock's Close, Bristol BS8 1TS, UK; Eng.) -Mischke 25-198

Research paper thumbnail of Biosynthesis of the 2-Pyridone Tenellin in the Insect Pathogenic FungusBeauveria bassiana

ChemBioChem, 2007

Other natural products U 0800 Biosynthesis of the 2-Pyridone Tenellin (I) in the Insect Pathogeni... more Other natural products U 0800 Biosynthesis of the 2-Pyridone Tenellin (I) in the Insect Pathogenic Fungus Beauveria bassiana. -(ELEY, K. L.; HALO, L. M.; SONG, Z.; POWLES, H.; COX*, R. J.; BAILEY, A. M.; LAZARUS, C. M.; SIMPSON, T. J.; ChemBioChem 8 (2007) 3, 289-297; Sch. Chem., Univ. Bristol, Cantock's Close, Bristol BS8 1TS, UK; Eng.) -Mischke 25-198

Research paper thumbnail of Microbial responses to solvent and alcohol stress

Biotechnology Journal, 2008

Research paper thumbnail of Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

Applied Microbiology and Biotechnology, 2014

This study reports the expression, purification, and kinetic characterization of a pyruvate decar... more This study reports the expression, purification, and kinetic characterization of a pyruvate decarboxylase (PDC) from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120 μM at pH 5), high catalytic efficiency (4.75×10 5 M −1 s −1 at pH 5), a pH opt of approximately 4.5 and an in vitro temperature optimum at approximately 55°C. Due to in vitro thermostablity (approximately 40 % enzyme activity retained after 30 min at 65°C), this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius for ethanol production. Initial studies using a variety of methods failed to detect activity at any growth temperature (45-55°C). However, the application of codon harmonization (i.e., mimicry of the heterogeneous host's transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45°C (as determined by enzyme activity, Western blot, mRNA detection, and ethanol productivity). Here, we describe the first successful expression of PDC in a true thermophile. Yields as high as 0.35±0.04 g/g ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription/translation coupling.

Research paper thumbnail of Application of pheB as a Reporter Gene for Geobacillus spp., Enabling Qualitative Colony Screening and Quantitative Analysis of Promoter Strength

Applied and Environmental Microbiology, 2012

The pheB gene from Geobacillus stearothermophilus DSM6285 has been exploited as a reporter gene f... more The pheB gene from Geobacillus stearothermophilus DSM6285 has been exploited as a reporter gene for Geobacillus spp. The gene product, catechol 2,3-dioxygenase (C23O), catalyzes the formation of 2-hydroxymuconic semialdehyde, which can be readily assayed. The reporter was used to examine expression from the ldh promoter associated with fermentative metabolism.

Research paper thumbnail of Structure of a bifunctional alcohol dehydrogenase involved in bioethanol generation in Geobacillus thermoglucosidasius

Acta Crystallographica Section D Biological Crystallography, 2013

Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative mic... more Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative microorganisms. They catalyse the conversion of an acyl-coenzyme A to an alcohol via an aldehyde intermediate; this is coupled to the oxidation of two NADH molecules to maintain the NAD(+) pool during fermentative metabolism. The structure of the alcohol dehydrogenase (ADH) domain of an ADHE protein from the ethanol-producing thermophile Geobacillus thermoglucosidasius has been determined to 2.5 Å resolution. This is the first structure to be reported for such a domain. In silico modelling has been carried out to generate a homology model of the aldehyde dehydrogenase domain, and this was subsequently docked with the ADH-domain structure to model the structure of the complete ADHE protein. This model suggests, for the first time, a structural mechanism for the formation of the large multimeric assemblies or `spirosomes' that are observed for this ADHE protein and which have previously been reported for ADHEs from other organisms.