Rapid Thermostabilization of Bacillus thuringiensis Serovar Konkukian 97–27 Dehydroshikimate Dehydratase through a Structure-Based Enzyme Design and Whole Cell Activity Assay
| Autor: | Emily N. Schmidt, David T. Fox, Andrew T. Koppisch, Kellan B. Finney, Charlie E. M. Strauss, Gustavo M. Canales, Ramesh K. Jha, Theresa L. Kern, Lucas B. Harrington |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Protein Conformation Mutant Bacillus thuringiensis Biomedical Engineering Biology Protein Engineering Serogroup Biochemistry Genetics and Molecular Biology (miscellaneous) Green fluorescent protein law.invention 03 medical and health sciences Bacterial Proteins law Enzyme Stability Escherichia coli Genomic library Hydro-Lyases chemistry.chemical_classification Genomic Library Temperature General Medicine Protein engineering biology.organism_classification Molecular biology High-Throughput Screening Assays Kinetics 030104 developmental biology Enzyme chemistry Biochemistry Genes Bacterial Dehydratase Mutation Recombinant DNA Synthetic Biology |
| Zdroj: | ACS Synthetic Biology. 6:120-129 |
| ISSN: | 2161-5063 |
| DOI: | 10.1021/acssynbio.6b00159 |
| Popis: | Thermostabilization of an enzyme with complete retention of catalytic efficiency was demonstrated on recombinant 3-dehydroshikimate dehydratase (DHSase or wtAsbF) from Bacillus thuringiensis serovar konkukian 97–27 (hereafter, B. thuringiensis 97–27). The wtAsbF is relatively unstable at 37 °C, in vitro (t1/237 = 15 min), in the absence of divalent metal. We adopted a structure-based design to identify stabilizing mutations and created a combinatorial library based upon predicted mutations at specific locations on the enzyme surface. A diversified asbF library (∼2000 variants) was expressed in E. coli harboring a green fluorescent protein (GFP) reporter system linked to the product of wtAsbF activity (3,4-dihydroxybenzoate, DHB). Mutations detrimental to DHSase function were rapidly eliminated using a high throughput fluorescence activated cell sorting (FACS) approach. After a single sorting round and heat screen at 50 °C, a triple AsbF mutant (Mut1), T61N, H135Y, and H257P, was isolated and characterized... |
| Databáze: | OpenAIRE |
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