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Rational Substitution of Surface Acidic Residues for Enhancing the Thermostability of Thermolysin

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Abstract

Solvent-exposed acidic/amide residue (Asp/Glu or Asn/Gln) exerts great effects on the thermostability of protein; however, experimental attempts appear to be time-consuming, so a more scientific, simple, and effective rational strategy is necessary. In this study, molecular dynamic (MD) simulation was performed to analyze two surface acidic residues (Asp37 and Glu119) of thermolysin (TLN) in mediating its thermostability. Root-mean-square-deviation (RMSD) was calculated to evaluate the thermosensitivity effect by acidic/amide substitutions. The wild-type TLN and three mutants (TLM1, TLM2, and TLM) presented significantly different thermostability effect. Four profiles of RMSD values demonstrated that the thermal insensitivity of variants were TLM2 > TLM > TLN > TLM1. As expected, the thermostability and half-life (at 60 °C) behavior of enzyme variants showed the same trends with the computational predictions, and it was worth noting that the half-life of TLM2 showed 3.1-fold longer than that of wild-type. The T m and T 50 of TLM2 were 9 and 7 °C higher, respectively, than that of wild-type enzyme. Rational substitution of acidic/amide residue in regulation of thermostability using MD simulation would be an efficient approach for instructional design to improve the thermostability.

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Acknowledgments

This work was supported by the National Program on Key Basic Research Project (2011CBA00807), the National High Technology Research and Development Key Program of China (2012AA022205), and KYZZ_0228 from Jiangsu Province.

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Authors and Affiliations

  1. College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, 30 Puzhunan Road, Nanjing, 211816, China

    Fucheng Zhu, Yu Zhuang, Bin Wu & Bingfang He

  2. School of Pharmaceutical Sciences, Nanjing Tech University, 30 Puzhunan Road, Nanjing, 211816, China

    Bingfang He

  3. State Key Laboratory of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University, Nanjing, 210093, China

    Jiahuang Li

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  1. Fucheng Zhu
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Correspondence to Jiahuang Li or Bingfang He.

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Zhu, F., Zhuang, Y., Wu, B. et al. Rational Substitution of Surface Acidic Residues for Enhancing the Thermostability of Thermolysin. Appl Biochem Biotechnol 178, 725–738 (2016). https://doi.org/10.1007/s12010-015-1905-7

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