Improving the thermal stability of lactate oxidase by directed evolution
| Autor: | Yoshihito Yoshida, Makio Furuichi, Hiroki Kaneko, Jiro Shimada, Hirotaka Minagawa, N. Kenmochi |
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| Rok vydání: | 2006 |
| Předmět: |
Models
Molecular Streptococcaceae Mutant Biology medicine.disease_cause Protein Structure Secondary Mixed Function Oxygenases Cellular and Molecular Neuroscience Enzyme Stability medicine Genomic library Protein Structure Quaternary Molecular Biology Thermostability Pharmacology chemistry.chemical_classification Mutation Temperature Cell Biology Directed evolution Molecular biology DNA shuffling Enzyme Amino Acid Substitution Biochemistry chemistry Molecular Medicine Directed Molecular Evolution Aerococcus viridans |
| Zdroj: | Cellular and Molecular Life Sciences. 64:77-81 |
| ISSN: | 1420-9071 1420-682X |
| DOI: | 10.1007/s00018-006-6409-8 |
| Popis: | Lactate oxidase is used in biosensors to measure the concentration of lactate in the blood and other body fluids. Increasing the thermostability of lactate oxidase can significantly prolong the lifetime of these biosensors. We have previously obtained a variant of lactate oxidase from Aerococcus viridans with two mutations (E160G/V198I) that is significantly more thermostable than the wild-type enzyme. Here we have attempted to further improve the thermostability of E160G/V198I lactate oxidase using directed evolution. We made a mutant lactate oxidase gene library by applying error-prone PCR and DNA shuffling, and screened for thermostable mutant lactate oxidase using a plate-based assay. After three rounds of screening we obtained a thermostable mutant lactate oxidase, which has six mutations (E160G/V198I/G36S/T103S/A232S/F277Y). The half-life of this lactate oxidase at 70 degrees C was about 2 times that of E160G/V198I and about 36 times that of the wild-type enzyme. The amino acid mutation process suggests that the combined neutral mutations are important in protein evolution. |
| Databáze: | OpenAIRE |
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