Chemical modification of organic compounds by biological systems is a powerful method to synthesize valuable chemicals, as it provides good yield, requires mild conditions and shows different types of selectivity. Hundreds of reactions and enzymes have been identified, but there is still a great need to discover new types of biocatalysts. Anaerobic bacteria possess unique metabolic pathways which make them a perfect target for screening for new industrially useful enzymatic activities.Clostridium sporogenes, a Gram-positive obligately anaerobic bacterium, was previously shown to catalyze two types of biocatalytic reduction, the reduction of amides to amines in the presence of hydrogen and hydrogenation of unsaturated carbon-carbon double bonds of activated alkenes. The first aim of this project was to optimize the reaction conditions for reduction of benzamide to benzylamine. Previous attempts to optimize this reaction were unsuccessful because of the poor method for benzylamine detection. The method was improved by changing the equipment and the conditions of analysis, and the benzylamine detection limit was significantly enhanced. Unfortunately, even this did not allow progress in the project since no benzylamine formation was observed. Further optimization of the reaction such as testing cells from different physiological stages of growth and improvement of the hydrogen delivery system is required.The second reaction provided by C. sporogenes, reduction of nitroalkenes, was proposed to be catalysed by enoate reductase enzyme, however previous attempts to purify this enzyme were unsuccessful. As an alternative strategy to identify the enzyme, C. sporogenes enoate reductase knock out mutants were prepared. The mutants were not able to reduce cinnamic acid, an intermediate of phenylalanine fermentation in the Stickland reaction, which had been proposed to be converted by enoate reductase. Moreover, mutated C. sporogenes showed decreased biomass production in media containing different energy sources such as glucose or phenylalanine. The mutants were not able to reduce (E)-1-nitro-2-phenylpropene, which confirmed the hypothesis that enoate reductase was responsible for reduction of this substrate. On the other hand, reduction of (E)-1-phenyl-2-nitropropene was still possible, but with a lower yield compared to the wild type strain. It is possible that this substrate was reduced by two independent enzymes, and that enoate reductase is one of them. In the future the gene encoding enoate reductase will be cloned and the enzyme will be overexpressed in E. coli to produce a potential new biocatalyst for industrial biotransformation. Moreover, the mutant will be used for purification of the enzyme responsible for reduction of (E)-1-phenyl-2-nitropropene.
|Date of Award||31 Dec 2010|
- The University of Manchester
|Supervisor||Gillian Stephens (Supervisor) & John Gardiner (Supervisor)|
Biocatalytic reduction using Clostridium sporogenes
Mordaka, P. (Author). 31 Dec 2010
Student thesis: Master of Philosophy