Thermostability enhancement of l-glutamate oxidase from Streptomyces sp. NT1 by full consensus protein design
2022
Hayashi, Yuka | Nakamura, Masaya | Nakano, Shogo | Ito, Sohei | Asano, Yasuhisa | Sugimori, Daisuke
Thermostable l-glutamate oxidases (LGOXs) are desirable for use in l-glutamate (L-Glu) assay kits, enzymatic synthesis of α-ketoglutarate and for biosensor development. However, protein engineering efforts to improve thermostability often lead to a decrease in enzymatic activity. In this report, we aimed to enhance the thermostability (melting temperature, Tₘ) of a mesophilic LGOX from Streptomyces sp. NT1 (LGOXNT₁) without a reduction in activity by a sequence-based protein design approach, termed full consensus (Fc) protein design. Among the 690 amino acids of LGOXNT₁, 104 amino acids were substituted by the Fc protein design. The mutant gene was artificially synthesized and expressed in Escherichia coli BL21(DE3) cells. The Tₘ of the purified, recombinant LGOX mutant (FcLGOX) was determined to be ∼72 °C, which is an increase on the Tₘ of 65 °C for LGOXNT₁ and the highest among known LGOXs. Importantly, purified FcLGOX showed no loss of specific activity or substrate specificity after a 30-min incubation at 70 °C. Our findings provide a new approach to improve the thermostability of enzymes.
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