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Characterization of a Recombinant d-Allulose 3-epimerase from Thermoclostridium caenicola with Potential Application in d-Allulose Production

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Abstract

In recent years, with the increasing public health awareness, low-calorie rare sugars have received more attention on a global scale. d-Allulose, the C-3 epimer of d-fructose, is a representative rare sugar. It displays high sweetness and excellent physiological functions, but only provides a caloric value of 0.4 kcal/g. d-Allulose 3-epimerase (DAEase) is indispensable in d-allulose production. In this study, a putative DAEase from Thermoclostridium caenicola was identified and characterized. The novel T. caenicola DAEase displayed maximum activity at pH 7.5 and 65 °C in the presence of 1 mM Co2+. The half-life (t1/2) at 50 °C was 13.6 h, and the melting temperature (Tm) was 62.4 °C. It was strictly metal-dependent, and the addition of Co2+ remarkably enhanced its thermostability, with a 5.4-fold increase in t1/2 value at 55 °C and 4.8 °C increase in Tm. Furthermore, DAEase displayed high relative activity (89.0%) at a weakly acidic pH 6.5 and produced 139.8 g/L d-allulose from 500 g/L d-fructose, achieving a conversion ratio of 28.0%. These findings suggest that T. caenicola DAEase is a promising biocatalyst for the production of d-allulose.

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Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 31801583), the Natural Science Foundation of Jiangsu Province (No. BK20180607), the Key Technology R&D Program of Jiangsu Province (BE2019629), Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-KJGG-003), and Key-Area Research and Development Program of Guangdong Province (2020B020226007).

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Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, People’s Republic of China

    Jiajun Chen, Ding Chen, Mengyu Ke, Shengyuan Ye, Xinyu Wang, Wenli Zhang & Wanmeng Mu

  2. International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, 214122, Jiangsu, China

    Wanmeng Mu

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  1. Jiajun Chen
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  2. Ding Chen
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  6. Wenli Zhang
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Contributions

JJC: conceptualization, experimentation, investigation, methodology and wrote original manuscript. DC: formal analysis, corrected the original manuscript and data processing and data curation. MYK, SYY, and XYW: performed the experiments and analyzed the data. WLZ and WMM: supervise the project, gave major comments, reviewed the manuscript and project administration.

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Correspondence to Wenli Zhang.

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Chen, J., Chen, D., Ke, M. et al. Characterization of a Recombinant d-Allulose 3-epimerase from Thermoclostridium caenicola with Potential Application in d-Allulose Production. Mol Biotechnol 63, 534–543 (2021). https://doi.org/10.1007/s12033-021-00320-z

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