Improved activity and thermostability of (S)-aminotransferase by error-prone polymerase chain reaction for the production of a chiral amine
The evolution of a mesophilic aminotransferase, isolated from Athrobacter citreus, to a thermostable aminotransferase was accomplished via error-prone PCR. After three rounds of mutagenesis, a mutant was generated that decreased the biocatalyst loading 3-fold. This improved biocatalyst was engineered further and a new mutant was isolated that was capable of the same performance with 5-fold reduction in biocatalyst loading. Overall, the best mutant (#6) enabled a 3-fold reduction in biocatalyst loading, almost a 5-fold increase in product concentration, and a 5-fold reduction in process cycle time. Through these rounds of mutagenesis enzyme specific activity improved from 5.9 to 1582.8 IU/g with an overall improvement in product yield due to reduced biocatalyst loading. The new mutants were also able to operate at temperatures greater than 50 °C for an extended period of time. A simple cost model was developed to describe the impact of enzyme improvement on product cost. © 2007 Elsevier B.V. All rights reserved.
Biochemical Engineering Journal
Improved activity and thermostability of (S)-aminotransferase by error-prone polymerase chain reaction for the production of a chiral amine.
Biochemical Engineering Journal,
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