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Rational Side-Chain Amino Acid Substitution in Firefly Luciferase for Improved Thermostability

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Abstract

Improving the thermostability of the luciferase from firefly (Photinus pyralis) needs to be solved to broaden its industrial applications. In this paper, molecular dynamic (MD) simulations were used to identify 4 amino acid substitutions (P183V, E325K, Q338V, and E354K) which might have a significant influence on the thermostability of luciferase. Root-mean-square deviation values were calculated to further evaluate the effect of these mutations on thermostability of the enzyme and demonstrated that the thermostability of the corresponding protein variants was in the order E354K > E325K > WT > P183V > Q338V. Following the MD simulation, the enzyme variants were expressed in a recombinant host, and the results showed that the t1/2, T50, and Tm of mutant E354K were increased 2.32-fold, and 4.5 and 3.3°C more compared with the wild type, respectively. MD simulations, as well as circular dichroism and fluorescence spectroscopy were further applied to elucidate the conformational differences between the wild-type and E354K luciferases. The results indicated that a possible explanation for the improved thermostability of E354K enzyme lies in the formation of a salt bridge between Lys354 and Glu311 and alteration of protein conformation.

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Authors and Affiliations

  1. School of Pharmaceutical Sciences, Nanjing Tech University, 211800, Nanjing, China

    Q. Xu & H. Huang

  2. College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, 211800, Nanjing, China

    M. Si, Z. Zhang & Z. Li

  3. College of Food Sciences and Light Industry, Nanjing Tech University, 211800, Nanjing, China

    L. Jiang

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  1. Q. Xu
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  2. M. Si
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  3. Z. Zhang
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  4. Z. Li
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  5. L. Jiang
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  6. H. Huang
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Correspondence to L. Jiang or H. Huang.

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Xu, Q., Si, M., Zhang, Z. et al. Rational Side-Chain Amino Acid Substitution in Firefly Luciferase for Improved Thermostability. Appl Biochem Microbiol 54, 584–590 (2018). https://doi.org/10.1134/S0003683819010204

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  • DOI: https://doi.org/10.1134/S0003683819010204

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