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Enhanced thermal stability of Pseudomonas aeruginosa lipoxygenase through modification of two highly flexible regions

  • Biotechnologically relevant enzymes and proteins
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Abstract

Lipoxygenase (LOX; EC 1.13.11.12) is an enzyme which is widely used in food industry to improve aroma and rheological or baking properties of foods. A series of studies have proven that the flexible regions negatively relates to the thermal stability of enzymes. In this study, two highly flexible regions, residues20–49 and residues201–206, were modified to improve the thermal stability of LOX from Pseudomonas aeruginosa. Deletion of the first 20 and 30 residues of the former region increased the thermal stability of the LOX by 1.3- and 2.1-fold, respectively. Although deletion of the residues201–206 led to a sharp reduction of both thermal stability and catalytic activity of the enzyme, the residue substitutions with the glycines (G204P, G206P, and G204P/G206P) or even glycine-rich linker (L6/PT) within this region increased the thermal stability of LOX by values ranging from 0.46- to 3.45-fold. To be noted, over 85 % of the specific activity was maintained in all thermally stabilized LOX mutants. Circular dichroism and fluorescence analysis showed that the overall secondary and tertiary structures were not significantly changed by these modifications. To the best of our knowledge, this is the first report on increasing the thermal stability of LOX by protein engineering without remarkably affecting the catalytic rate.

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Acknowledgments

We would like to thank Professor Byong H. Lee for critical reading and suggestions on the manuscript. This work was supported by the Major State Basic Research Development Program of China (973 Program, 2013CB733602), the National Natural Science Foundation of China (No. 31171639), the National High Technology Research and Development Program of China (No. 2011AA100905), Program for Changjiang Scholars and Innovative Research Team in University (No.IRT1135), and the Independent Innovation Program of Jiangnan University (JUSRP11215), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (Grant No. 12KJD550007).

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Authors and Affiliations

  1. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Xinyao Lu, Song Liu & Yue Feng

  2. School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Xinyao Lu, Song Liu, Yue Feng, Xiaoman Zhou, Guocheng Du & Jian Chen

  3. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China

    Jian Chen

  4. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Guocheng Du

  5. School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China

    Miao Wang

  6. School of Food Science and Engineering, Yangzhou University, Yangzhou, 225127, China

    Shengqi Rao

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  1. Xinyao Lu
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  2. Song Liu
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  3. Yue Feng
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Correspondence to Song Liu or Guocheng Du.

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Lu, X., Liu, S., Feng, Y. et al. Enhanced thermal stability of Pseudomonas aeruginosa lipoxygenase through modification of two highly flexible regions. Appl Microbiol Biotechnol 98, 1663–1669 (2014). https://doi.org/10.1007/s00253-013-5039-y

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  • DOI: https://doi.org/10.1007/s00253-013-5039-y

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