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Enhancing the thermostability of d-allulose 3-epimerase from Clostridium cellulolyticum H10 via directed evolution

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Abstract

d-allulose, the epimer at C-3 position of d-fructose, is a low-calorie functional rare sugar, which is regarded as one of the most potential sweeteners. At present, the main production method of d-allulose is epimerization of d-fructose by d-allulose 3-epimerase (DAE). However, industrial applications of DAE are still limited by its poor thermostability. Herein, directed evolution was applied to improve the thermostability of DAE from Clostridium cellulolyticum H10 (CcDAE). Two optimal mutants D281G and C289R, exhibiting 13.80-fold and 13.88-fold t1/2 values as that of wild type at 65 ℃, respectively, were obtained. To further enhance the thermostability, the triple mutant A107P/D281G/C289R was constructed after combination of mutants D281G, C289R, and previously identified thermostability-enhanced mutant A107P. The Tm and optimal temperature of triple mutant were increased by 14.39 ℃ and 5 ℃, respectively, compared to the wild type, meanwhile, the half-life of triple mutant was 58.85-fold as that of wild type at 65 ℃. Furthermore, the conversion rate of triple mutant was increased from 24.76% of wild type to 27.53% using 300 g/L d-fructose as substrate at 70 ℃. The effectiveness of directed evolution was verified and the triple mutant with enhanced thermostability had great application value in the large-scale production of d-allulose.

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References

  1. Chen J, Chen D, Ke M, Ye S, Wang X, Zhang W, Mu W. Characterization of a recombinant d-allulose 3-epimerase from Thermoclostridium caenicola with potential application in d-allulose production. Mol Biotechnol. 2021;63(6):534–43. https://doi.org/10.1007/s12033-021-00320-z.

    Article  CAS  PubMed  Google Scholar 

  2. Nagata Y, Kanasaki A, Tamaru S, Tanaka K. d-psicose, an epimer of d-fructose, favorably alters lipid metabolism in Sprague-Dawley rats. J Agric Food Chem. 2015;63(12):3168–76. https://doi.org/10.1021/jf502535p.

    Article  CAS  PubMed  Google Scholar 

  3. Chen J, Huang W, Zhang T, Lu M, Jiang B. Anti-obesity potential of rare sugar d-psicose by regulating lipid metabolism in rats. Food Funct. 2019;10(5):2417–25. https://doi.org/10.1039/c8fo01089g.

    Article  CAS  PubMed  Google Scholar 

  4. Wang Y. Molecular modification, expression optimization and stability study on d-psicose. 2021. Jiangnan University

  5. Zhang W, Yu S, Zhang T, Jiang B, Mu W. Recent advances in d-allulose: Physiological functionalities, applications, and biological production. Trends Food Sci Technol. 2016;54:127–37. https://doi.org/10.1016/j.tifs.2016.06.004.

    Article  CAS  Google Scholar 

  6. Kim H, Hyun E, Kim Y, Lee Y, Oh D. Characterization of an Agrobacterium tumefaciens d-psicose 3-epimerase that converts d-fructose to d-psicose. Appl Environ Microbiol. 2006;72(2):981–5. https://doi.org/10.1128/AEM.72.2.981-985.2006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zhang W, Zhang Y, Huang J, Chen Z, Zhang T, Guang C, Mu W. Thermostability improvement of the d-allulose 3-epimerase from Dorea sp. CAG317 by site-directed mutagenesis at the interface regions. J Agric Food Chem. 2018;66(22):5593–5601. https://doi.org/10.1021/acs.jafc.8b01200

  8. Su L, Sun F, Liu Z, Zhang K, Wu J. Highly efficient production of Clostridium cellulolyticum H10 d-psicose 3-epimerase in Bacillus subtilis and use of these cells to produce d-psicose. Microb Cell Fact. 2018;17(1):188. https://doi.org/10.1186/s12934-018-1037-1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Li C, Lin J, Guo Q, Zhang C, Du K, Lin H, Lin J. d-psicose 3-epimerase secretory overexpression, immobilization, and d-psicose biotransformation, separation and crystallization. J Chem Technol Biotechnol. 2018;93(2):350–7. https://doi.org/10.1002/jctb.5360.

    Article  CAS  Google Scholar 

  10. Chen J, Chen D, Chen Q, Xu W, Zhang W, Mu W. Computer-aided targeted mutagenesis of Thermoclostridium caenicola d-allulose 3-epimerase for improved thermostability. J Agric Food Chem. 2022;70(6):1943–51. https://doi.org/10.1021/acs.jafc.1c07256.

    Article  CAS  PubMed  Google Scholar 

  11. Zhu Z, Li L, Zhang W, Li C, Mao S, Lu F, Qin HM. Improving the enzyme property of d-allulose 3-epimerase from a thermophilic organism of Halanaerobium congolense through rational design. Enzyme Microb Technol. 2021;149: 109850. https://doi.org/10.1016/j.enzmictec.2021.109850.

    Article  CAS  PubMed  Google Scholar 

  12. Zhao J, Chen J, Wang H, Guo Y, Li K, Liu J. Enhanced thermostability of d-psicose 3-epimerase from Clostridium bolteae through rational design and engineering of new disulfide bridges. Int J Mol Sci. 2021;22(18):10007. https://doi.org/10.3390/ijms221810007.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhang W, Jia M, Yu S, Zhang T, Zhou L, Jiang B, Mu W. Improving the thermostability and catalytic efficiency of the d-psicose 3-epimerase from Clostridium bolteae ATCC BAA-613 using site-directed mutagenesis. J Agric Food Chem. 2016;64(17):3386–93. https://doi.org/10.1021/acs.jafc.6b01058.

    Article  CAS  PubMed  Google Scholar 

  14. Mao S, Cheng X, Zhu Z, Chen Y, Li C, Zhu M, Liu X, Lu F, Qin H. Engineering a thermostable version of d-allulose 3-epimerase from Rhodopirellula baltica via site-directed mutagenesis based on B-factors analysis. Enzyme Microb Technol. 2020;132: 109441. https://doi.org/10.1016/j.enzmictec.2019.109441.

    Article  CAS  PubMed  Google Scholar 

  15. Choi J, Ju Y, Yeom S, Oh D. Improvement in the thermostability of d-psicose 3-epimerase from Agrobacterium tumefaciens by random and site-directed mutagenesis. Appl Environ Microbiol. 2011;77(20):7316–20. https://doi.org/10.1128/AEM.05566-11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Bosshart A, Hee C, Bechtold M, Schirmer T, Panke S. Directed divergent evolution of a thermostable d-tagatose epimerase towards improved activity for two hexose substrates. ChemBioChem. 2015;16(4):592–601. https://doi.org/10.1002/cbic.201402620.

    Article  CAS  PubMed  Google Scholar 

  17. Li C, Zhang W, Wei C, Gao X, Mao S, Lu F, Qin HM. Continuous spectrophotometric assay for high-throughput screening of predominant d-Allulose 3-epimerases. J Agric Food Chem. 2021;69(39):11637–45. https://doi.org/10.1021/acs.jafc.1c04716.

    Article  CAS  PubMed  Google Scholar 

  18. Armetta J, Berthome R, Cros A, Pophillat C, Colombo B, Pandi A, Grigoras I. Biosensor-based enzyme engineering approach applied to psicose biosynthesis. Synth Biol (Oxf). 2019;4(1):ysz028. http://doi.org/https://doi.org/10.1093/synbio/ysz028

  19. Krauss U, Jaeger KE, Eggert T. Rapid sequence scanning mutagenesis using in silico oligo design and the Megaprimer PCR of whole plasmid method (MegaWHOP). Methods Mol Biol. 2010;634:127–35. https://doi.org/10.1007/978-1-60761-652-8_9.

    Article  CAS  PubMed  Google Scholar 

  20. Farnoosh G, Khajeh K, Mohammadi M, Hassanpour K, Latifi AM, Aghamollaei H. Catalytic and structural effects of flexible loop deletion in organophosphorus hydrolase enzyme: A thermostability improvement mechanism. Journal of Biosciences. 2020;45(1). https://doi.org/10.1007/s12038-020-00026-5

  21. Schwede T, Kopp J, Guex N, Peitsch M. SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res. 2003;31(13):3381–5. https://doi.org/10.1093/nar/gkg520.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kumar S, Dangi A, Shukla P, Baishya D, Khare S. Thermozymes: adaptive strategies and tools for their biotechnological applications. Bioresour Technol. 2019;278:372–82. https://doi.org/10.1016/j.biortech.2019.01.088.

    Article  CAS  PubMed  Google Scholar 

  23. Niu C, Zhu L, Zhu P, Li Q. Lysine-based site-directed mutagenesis increased rigid beta-sheet structure and thermostability of mesophilic 1,3–1,4-beta-glucanase. J Agric Food Chem. 2015;63(21):5249–56. https://doi.org/10.1021/acs.jafc.5b00480.

    Article  CAS  PubMed  Google Scholar 

  24. Sokalingam S, Raghunathan G, Soundrarajan N, Lee S. A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein. PLoS ONE. 2012;7(7): e40410. https://doi.org/10.1371/journal.pone.0040410.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Xu Z, Cen Y, Zou S, Xue Y, Zheng Y. Recent advances in the improvement of enzyme thermostability by structure modification. Crit Rev Biotechnol. 2020;40(1):83–98. https://doi.org/10.1080/07388551.2019.1682963.

    Article  CAS  PubMed  Google Scholar 

  26. Chen C, Su L, Xu F, Xia Y, Wu J. Improved thermostability of maltooligosyltrehalose synthase from Arthrobacter ramosus by directed evolution and site-directed mutagenesis. J Agric Food Chem. 2019;67(19):5587–95. https://doi.org/10.1021/acs.jafc.9b01123.

    Article  CAS  PubMed  Google Scholar 

  27. Tu T, Luo H, Meng K, Cheng Y, Ma R, Shi P, Huang H, Bai Y, Wang Y, Zhang L, Yao B. Improvement in thermostability of an Achaetomium sp. strain Xz8 endopolygalacturonase via the optimization of charge-charge interactions. Appl Environ Microbiol. 2015;81(19):6938–44. https://doi.org/10.1128/AEM.01363-15

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Acknowledgements

The authors are grateful for the financial support from the National Natural Science Foundation of China (NSFC) (32101884) and Natural Science Foundation of Jiangsu Province (BK20190586).

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Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu Province, China

    Shuhan Liu, Yifan Wang, Demin Kong, Jing Wu & Zhanzhi Liu

  2. School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu Province, China

    Shuhan Liu, Yifan Wang, Demin Kong, Jing Wu & Zhanzhi Liu

  3. International Joint Laboratory On Food Safety, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu Province, China

    Shuhan Liu, Yifan Wang, Demin Kong, Jing Wu & Zhanzhi Liu

Authors
  1. Shuhan Liu
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  2. Yifan Wang
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  3. Demin Kong
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  4. Jing Wu
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  5. Zhanzhi Liu
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Contributions

SL Conceptualization, Methodology, Data curation, Visualization, Writing-original draft; YW Methodology, Visualization; DK Methodology; JW Conceptualization, Supervision, Writing-Review & Editing; ZL Conceptualization, Funding acquisition, Supervision, Writing-Review & Editing.

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Correspondence to Jing Wu or Zhanzhi Liu.

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Conflict of interest the authors declare that they have no conflicts of interest.

Supplementary Information

Supplementary Information: SDS-PAGE of CcDAE and mutants, the probability distribution of distance between Arg289 in chain A and Glu275 and Arg 274 in chain B.

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Liu, S., Wang, Y., Kong, D. et al. Enhancing the thermostability of d-allulose 3-epimerase from Clostridium cellulolyticum H10 via directed evolution. Syst Microbiol and Biomanuf 2, 685–694 (2022). https://doi.org/10.1007/s43393-022-00096-6

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  • DOI: https://doi.org/10.1007/s43393-022-00096-6

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