Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.783944
Title: Thermostable enzymes important for industrial biotechnology
Author: Westlake, A.
Awarding Body: University of Exeter
Current Institution: University of Exeter
Date of Award: 2019
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Abstract:
The use of enzymes in technology is of increasing commercial interest due to their high catalytic efficiency and specificity and the lowering of manufacturing costs. Enzymes are also becoming more widely utilised because they are more environmentally friendly compared to chemical methods. Firstly, they carry out their reactions at ambient temperatures requiring less energy to achieve the high temperatures and pressures that many chemical methods require. Secondly, they can substitute for toxic chemical catalysts which need careful disposal. In this project two classes of enzymes of industrial interest from thermophiles were investigated, lactonase enzymes and 1-deoxy-D-xylulose 5-phosphate (DXP) synthases. A quorum sensing lactonase from Vulcanisaeta moutnovskia, a thermoacidophilic anaerobic crenarchaeon, was expressed in high levels in an Escherichia coli host, then purified and characterised with a range of industrially relevant substrates. These enzymes are of industrial interest for water treatment and bioreactors for their ability to prevent biofilm formation in bacteria. This enzyme showed different specificity to another well characterised quorum sensing lactonase from a thermophilic crenarchaeon, Sulfolobus solfataricus. Crystals of the native enzyme were obtained. Structural examination revealed that the V. moutnovskia lactonase possesses an α-helix obstructing a hydrophobic channel near the active site, whereas the S. solfataricus lactonase has a flexible loop leaving the hydrophobic channel unrestricted. As a result the acyl chains of substrates interact with surface residues of the α-helix in V. moutnovskia lactonase rather than sitting in the channel, so its activity is no longer restricted to substrates with long acyl chains. A gluconolactonase encoded by a thermophilic Planctomyces genome was cloned and expressed at high levels in an E. coli host, purified and successfully crystallised. The crystals had a space group of P 3 2 1 and diffracted to a resolution of 2.41 Å. This enzyme was intended to be used by an industrial partner for synthesis of metabolite standards for mass spectrometry and diagnostics. Attempts were made to clone, purify and express two other lactonases from thermophilic metagenomes obtained from terrestrial hot springs: an enol lactonase and a second quorum sensing lactonase. Homology modelling was used to create predicted structures for both of these enzymes. The quorum sensing lactonase showed a 2.5 Å difference in the position of a catalytic serine and a 3.1 Å difference in a catalytic histidine in comparison to a mesophilic homologue. The enol lactonase contained an aspartic acid in place of a catalytic serine found in a mesophilic homologue. A DXP synthase from an anaerobic Gram-negative bacterium Thermovibrio ammonificans was successfully cloned, over-expressed and purified. Crystals were successfully produced although these diffracted only to low resolution. A DXP synthase from an anaerobic Gram-positive bacterium Carboxydothermus hydroformans was successfully cloned, however the protein was expressed primarily in the insoluble fraction. Homology models were made for these two enzymes. Both enzymes showed strong similarity with mesophilic DXP synthases in terms of tertiary structure and positions of active site residues. Visual analysis revealed an increase of 15-20 % in the number of hydrophobic interactions within the enzymes and a high proportion of charged residues at the dimer interface, which would confer increased thermostability. The hope was to obtain high resolution diffraction data to assist in understanding what allows these enzymes to utilise pyruvate as a substrate compared to transketolase, a related enzyme, which uses hydroxypyruvate.
Supervisor: Aves, S. ; Littlechild, J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.783944  DOI: Not available
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