Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.587728
Title: What causes an enzyme to degrade during biocatalysis?
Author: Morris, P.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2013
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Abstract:
Biocatalysis continues to be a powerful tool for the efficient synthesis of optically pure pharmaceuticals that are difficult to access via conventional chemistry. The efficient application of biocatalysis requires the availability of suitable enzymes with high activity and stability under process conditions. However, the borderline stability of biocatalysts in many types of reaction media has often prevented or delayed their implementation for industrial-scale syntheses of fine chemicals and pharmaceuticals. Consequently, there is great interest in understanding the effects of solution conditions on protein stability, as well as in developing strategies to improve enzyme stability and activity in desired reaction media. The enzyme transketolase (TK; E.C. 2.2.1.1) from Escherichia coli is an important biocatalyst in stereo-specific carbon-carbon bond synethesis. The power of transketolase is further augmented when the bioconversion takes place in a multi-step biotransformation in which transketolase and transaminases are employed in series to create chiral amino alcohols from achiral substrates. These compounds are synthetically very useful in the production of a range of compounds with pharmaceutical application. Although many useful reactions have been reported for TK, many of the substrates and products are unstable or insoluble at the pH or temperature range for which the enzyme has optimum activity in aqueous media. Understanding the activity and structural stability of transketolase under bioprocess conditions will Biocatalysis continues to be a powerful tool for the efficient synthesis of optically pure pharmaceuticals that are difficult to access via conventional chemistry. The efficient application of biocatalysis requires the availability of suitable enzymes with high activity and stability under process conditions. However, the borderline stability of biocatalysts in many types of reaction media has often prevented or delayed their implementation for industrial-scale syntheses of fine chemicals and pharmaceuticals. Consequently, there is great interest in understanding the effects of solution conditions on protein stability, as well as in developing strategies to improve enzyme stability and activity in desired reaction media. The enzyme transketolase (TK; E.C. 2.2.1.1) from Escherichia coli is an important biocatalyst in stereo-specific carbon-carbon bond synethesis. The power of transketolase is further augmented when the bioconversion takes place in a multi-step biotransformation in which transketolase and transaminases are employed in series to create chiral amino alcohols from achiral substrates. These compounds are synthetically very useful in the production of a range of compounds with pharmaceutical application. Although many useful reactions have been reported for TK, many of the substrates and products are unstable or insoluble at the pH or temperature range for which the enzyme has optimum activity in aqueous media. Understanding the activity and structural stability of transketolase under bioprocess conditions will improve our capacity to comprehend and ultimately to engineer it to make it work at a broader range of pHs and temperatures, and also in the presence of organic co-solvents. This will potentially help to reduce process development times and also increase the stability and solubility of substrates and products. To provide further insight into the underlying causes of TK deactivation in process conditions, the effects of temperature, pH and organic solvents on the structure, stability, aggregation and activity of Escherichia coli transketolase were characterized in Chapters 3 and 4. The results provided useful information for the engineering of TK enzymes with improved thermostability or extreme pH tolerance and in organic solvent mixtures. For thermostability and tolerance to low pH, mutations may be usefully targeted towards regions of protein sequence predicted to have a high propensity for aggregation. For the retention of biocatalytic activity at high pH or temperatures, stabilisation of the cofactor binding loops were found to be an attractive target. By contrast, the results in aqueous-solvent mixtures instead implied that the solvent dependence of catalytic activity cannot be simply explained by only one mechanism such as active-site binding or the replacement of water molecules, and that the effect of different solvents on protein structure penetration, denaturation and aggregation must also be considered. In the final Chapter, mutagenesis was targeted to the cofactor binding loops to further evaluate their impact on thermal stability. one mutant was found that successfully improved the stability of E. coli transketolase at elevated temperatures, giving a 3 fold specific activity increase at 60 oC compared to wild-type TK.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.587728  DOI: Not available
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