Thermostable isomerase processes for Biotechnology
The TIPs project focuses on the provision of novel thermostable isomerases from thermophilic microorganisms and metagenomes and their biotechnological applications. Isomers are molecules with identical atomic composition but with different structural characteristics. Different isomers can show very distinctive function. The formation of isomers often reduces the productivity of biotechnological and chemical processes because only one of the two or more isomers is utilized in biocatalytic reactions (reducing the final efficiency to below 50%). Isomerases are enzymes catalyzing the conversion between different types of isomers. Using the appropriate isomerase enzyme in the industrial process will increase production efficiency resulting in 100% conversion of a racemic substrate to the product. Thermostable isomerases are desired because they possess high resistance and durability, are able to withstand harsh industrial process conditions, including heating and organic solvents. Elevated temperatures can also enhance substrate accessibility and solubility. The proposed project includes comparative bioinformatic analyses of sequence data to identify different classes of thermostable isomerases of industrial interest. Recently developed genomic enzymology tools will be employed for mining the databases. Both sequence similarity networks (SSN) and genome neighbourhood networks (GNN) allow protein sequences to be gathered into clusters that represent families with only a single function. Further phylogenetic analysis of these isofunctional clusters will help to select the most promising candidates. These cherry-picked enzyme candidates will then be cloned and recombinantly produced. The expression bias will be minimized with codon-optimized synthetic genes, a multiple vector system for high-throughput expression analysis as well as the use of several streamlined expression hosts. Purified enzymes will be then functionally and structurally characterized and further optimized towards their biotechnological application. Three types of isomerases will be targeted: sugar isomerases (to produce new desirable sugars for calorie-free sweeteners and as building blocks for drugs), disulfide isomerases (to improve protein folding and stability of industrial enzymes), and chalcone isomerases (involved in the transformation of flavonoids, secondary metabolites of importance as natural colorants, anti-oxidants, anti-microbial and anti-inflammatory agents). Durable isomerases will allow new opportunities for green, competitive and sustainable biotechnological processes that can replace conventional chemical synthesis.
| Main Authors: | , , , |
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| Format: | póster de congreso biblioteca |
| Published: |
University of Pretoria
2017-08-27
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| Online Access: | http://hdl.handle.net/10261/163488 http://dx.doi.org/10.13039/501100002347 http://dx.doi.org/10.13039/501100005416 http://dx.doi.org/10.13039/501100010198 |
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| Summary: | The TIPs project focuses on the provision of novel thermostable isomerases from thermophilic microorganisms and metagenomes and their biotechnological applications. Isomers are molecules with identical atomic composition but with different structural characteristics. Different isomers can show very distinctive function. The formation of isomers often reduces the productivity of biotechnological and chemical processes because only one of the two or more isomers is utilized in biocatalytic reactions (reducing the final efficiency to below 50%). Isomerases are enzymes catalyzing the conversion between different types of isomers. Using the appropriate isomerase enzyme in the industrial process will increase production efficiency resulting in 100% conversion of a racemic substrate to the product. Thermostable isomerases are desired because they possess high resistance and durability, are able to withstand harsh industrial process conditions, including heating and organic solvents. Elevated temperatures can also enhance substrate accessibility and solubility.
The proposed project includes comparative bioinformatic analyses of sequence data to identify different classes of thermostable isomerases of industrial interest. Recently developed genomic enzymology tools will be employed for mining the databases. Both sequence similarity networks (SSN) and genome neighbourhood networks (GNN) allow protein sequences to be gathered into clusters that represent families with only a single function. Further phylogenetic analysis of these isofunctional clusters will help to select the most promising candidates. These cherry-picked enzyme candidates will then be cloned and recombinantly produced. The expression bias will be minimized with codon-optimized synthetic genes, a multiple vector system for high-throughput expression analysis as well as the use of several streamlined expression hosts. Purified enzymes will be then functionally and structurally characterized and further optimized towards their biotechnological application.
Three types of isomerases will be targeted: sugar isomerases (to produce new desirable sugars for calorie-free sweeteners and as building blocks for drugs), disulfide isomerases (to improve protein folding and stability of industrial enzymes), and chalcone isomerases (involved in the transformation of flavonoids, secondary metabolites of importance as natural colorants, anti-oxidants, anti-microbial and anti-inflammatory agents). Durable isomerases will allow new opportunities for green, competitive and sustainable biotechnological processes that can replace conventional chemical synthesis. |
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