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Improving a bacterial pyranose 2-oxidase from Arthrobacter siccitolerans through directed evolution
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"Pyranose 2-oxidases (P2Ox) are flavoproteins that catalyze the oxidation of several aldopyranoses to yield the corresponding 2-keto-aldoses with concomitant reduction of O2 to H2O2 and are enzymes that show many biotechnological applications. Recently, a bacterial P2Ox from Arthrobacter siccitolerans (AsP2Ox) was characterized for the first time since bacteria grow faster as compared to fungi and have well-established genetic and molecular biological tools allowing for higher enzyme production yields. Directed evolution has proven to be a powerful approach to improve enzyme efficiency and robustness required for biotechnological applications. Therefore, in this work the optimization and validation of critical steps of directed evolution was performed, namely mutagenesis protocols, cell growth, lysis and high-throughput enzymatic assays. One round of evolution through error prone PCR was performed and a total of 25 000 clones were screened to find variants with improved activity for D-glucose and dioxygen. One hit variant, 2C9, was identified, showing higher activity than wild-type and containing two non-synonymous (A35T and F300V) and one synonymous mutation (Q343Q).(...)."
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flavoproteins hydrogen peroxide forming enzymes directed evolution catalytic efficiency