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Cloning and Characterization of a Ginsenoside-Hydrolyzing α-L-Arabinofuranosidase, CaAraf51, From Cellulosimicrobium aquatile Lyp51

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Abstract

Moutai Jiuqu is a famous aromatic raw material of Maotai flavor liquor in China. It is brewed at high temperature and contains many kinds of bacteria, molds, and yeasts. There are many useful glycoside hydrolases in these microfloras, from which efficient glycoside hydrolases can be screened for biotransformation of natural saponins. In this study, an α-L-arabinofuranosidase gene (CaAraf51, 1524 bp, 507 amino acid, 55.07 kDa, and pI = 4.8) was cloned from Cellulosimicrobium aquatile Lyp51, which was isolated from the Maotai Jiuqu. The CaAraf51 was heterogeneously expressed in E. coli BL21 (DE3) and purified by N-terminal His-tag with the Ni2+-affinity column chromatography. The results show that purified CaAraf51 has a 6.8-fold purification factor and specific activity of 15 U/mg. Under optimal conditions (pH 5.0, temperature 40 °C), kinetic parameters Km of CaAraf51 for pNPαAraf and Rc were 1.1 and 0.57 mM, the Vmax were 25 and 6.25 μmol/min/mg, respectively. 90% of 0.87 mg Rc substrate can be transformed by 9.6 U purified CaAraf51 in 1 mL reaction system under suitable conditions (30 °C, pH 7.5 phosphate buffer, 1 h). In addition, we also tested the effects of metal ions and chemical agents on the activity of CaAraf51. According to systematically studied its function and enzymatic properties, CaAraf51 has excellent value and potential of biotransformation Rc into Rd.

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Acknowledgements

This study was funded by the National Natural Science Foundation of China (Grant Nos. 31760189 and 31200066); Natural Science Foundation of Yunnan Province of China (Grant No. 2012FB115), and Yunnan Provincial Education Department Research Fund Project (Grant No. 2019J0028). The authors thank the Editage of Cactus Communications for English editing service.

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  1. Sha-Sha Zuo and Yu-Chen Wang have contributed equally to this work

Authors and Affiliations

  1. National Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, Yunnan Institute of Microbiology, School of Life Sciences, Yunnan University, Kunming, 650091, Yunnan, China

    Sha-Sha Zuo, Yu-Chen Wang, Ling Zhu, Jiang-Yuan Zhao, Ming-Gang Li, Xiu-Lin Han & Meng-Liang Wen

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  1. Sha-Sha Zuo
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  2. Yu-Chen Wang
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Contributions

SSZ and YCW designed and performed the experiments. SSZ, YCW, LZ, JYZ, MGL, XLH, and MLW analyzed the data. XLH and MLW conceived and supervised the project. XLH and MLW wrote the paper with contributions from all authors. All authors read and approved the manuscript.

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Correspondence to Xiu-Lin Han or Meng-Liang Wen.

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Zuo, SS., Wang, YC., Zhu, L. et al. Cloning and Characterization of a Ginsenoside-Hydrolyzing α-L-Arabinofuranosidase, CaAraf51, From Cellulosimicrobium aquatile Lyp51. Curr Microbiol 77, 2783–2791 (2020). https://doi.org/10.1007/s00284-020-02078-0

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