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Molecular cloning, expression, purification, and characterization of Bacillus subtilis hydrolyzed ginsenoside Rc of α-L-arabinofuranosidase in Escherichia coli

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Abstract

The α-L-arabinofuranosidase enzyme plays a crucial role in the degradation of ginsenosides. In this study, we successfully cloned and expressed a novel α-L-arabinofuranosidase bsafs gene (1503 bp, 501 amino acids, 55 kDa, and pI = 5.4) belonging to glycosyl hydrolase (GH) family 51 from Bacillus subtilis genome in Escherichia coli BL21 cells. The recombinant protein Bsafs was purified using Ni2+ sepharose fastflow affinity chromatography and exhibited a specific activity of 2.91 U/mg. Bsafs effectively hydrolyzed the α-L-arabinofuranoside at C20 site of ginsenoside Rc to produce Rd as the product. The Km values for hydrolysis of pNP-α-L-arabinofuranoside (pNPαAraf) and ginsenoside Rc were determined as 0.74 and 4.59 mmol/L, respectively; while the Vmax values for these substrates were found to be 24 and 164 μmol/min/mg, respectively; furthermore, the Kcat values for these enzymes were calculated as 22.3 and 1.58 S−1 correspondingly.

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Data availability

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.

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Funding

This work is supported by Special Basic Cooperative Research Programs of Yunnan Provincial Undergraduate Universities’ Association (Grant NO. 202301BA070001-081), Yunnan Fundamental Research Projects (Grant NO. 202301AU070012), Special Basic Cooperative Research Innovation Programs of Qujing Science and Technology Bureau & Qujing Normal University (Grant NO. KJLH2023ZD06, KJLH2022YB07).

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Authors and Affiliations

  1. Yunnan Engineering Research Center of Fruit Wine, Qujing Normal University, Qujing, 655011, People’s Republic of China

    Ling Zhu & Jian Cai

  2. School of Chemical Biology and Environment, Yuxi Normal University, Yuxi, 653100, People’s Republic of China

    Yuchen Wang

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  1. Ling Zhu
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  2. Yuchen Wang
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  3. Jian Cai
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Contributions

L. Z and Y. C. W conceived the study and supervised the project. J C cloned and expressed Bsafs. L. Z tudy of the enzymatic activity of Bsafs. L. Z wrote the main manuscript. All authors participated in preparation of the manuscript.

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Correspondence to Yuchen Wang or Jian Cai.

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Communicated by PANKAJ BHATT.

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Zhu, L., Wang, Y. & Cai, J. Molecular cloning, expression, purification, and characterization of Bacillus subtilis hydrolyzed ginsenoside Rc of α-L-arabinofuranosidase in Escherichia coli. Arch Microbiol 206, 181 (2024). https://doi.org/10.1007/s00203-024-03902-y

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  • DOI: https://doi.org/10.1007/s00203-024-03902-y

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