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Cloning and Expression of Heparinase Gene from a Novel Strain Raoultella NX-TZ-3–15

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Abstract

Heparin is a class of highly sulfated, acidic, linear, and complex polysaccharide that belongs to the heparin/heparan sulfate (HS) glycosaminoglycans family. Enzymatic depolymerization of heparin by heparinases is a promising strategy for the production of ultra-low molecular weight heparins (ULMWHs) as anticoagulants. In the present study, a novel heparinase-producing strain Raoultella NX-TZ-3–15 was isolated and identified from soil samples. Herein, the heparinase gene MBP-H1 was cloned to the pBENT vector to enable expression in Escherichia coli. The optimized conditions made the activity of recombinant heparinase reach the highest level (2140 U/L). The overexpressed MBP-H1 was purified by affinity chromatography and a purity of more than 90% was obtained. The condition for biocatalysis was also optimized and three metal ions Ca2+, Co2+, and Mg2+ were utilized to activate the reaction. In addition, the kinetics regarding the new fusion heparinase was also determined with a Vm value of 11.29 μmol/min and a Km value of 31.2 μmol/L. In short, due to excellent Km and Vmax, the recombinant enzyme has great potential to be used in the clinic in medicine and industrial production of low or ultra-low molecule weight heparin.

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All data generated or analyzed during this study are included in this article.

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Funding

This work was supported by the National Key R&D Program of China (2021YFA0910800), the Natural Science Foundation of Guangdong Province (Grant No. 2022A1515012043), and the Special Funds for Development of Strategic Emerging Industries in Shenzhen (JCYJ20190808145613154, KQJSCX20180328100801771), and Shenzhen Science and Technology Program (ZDSYS20210623100800001).

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Author notes

  1. Yinyin Li and Yue Lin contributed equally to this work.

Authors and Affiliations

  1. College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, 518060, Guangdong, China

    Yinyin Li, Yue Lin, Yingzi Jiang & Liqing Zhao

  2. Health Science Center, Shenzhen University, Shenzhen, 518060, China

    Hafiza Mahreen Mehwish

  3. Laboratory of Animal Food Function, Graduate School of Agricultural Science, Tohoku University, Sendai, 980-8572, Japan

    Hafiza Mahreen Mehwish & Muhammad Shahid Riaz Rajoka

  4. Institute for Advanced Study, Shenzhen University, Shenzhen, 518060, China

    Liqing Zhao

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  1. Yinyin Li
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  2. Yue Lin
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  3. Yingzi Jiang
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  4. Hafiza Mahreen Mehwish
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  5. Muhammad Shahid Riaz Rajoka
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Contributions

Y. Lin: conceptualization, methodology, software; Y. Lin: conceptualization, methodology, software; Y. Jiang: visualization, investigation; H. M. Mehwish: data curation, writing-original draft preparation; M. S. R. Rajoka: software, validation, writing-original draft preparation. Liqing Zhao: supervision, funding, validation. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Muhammad Shahid Riaz Rajoka or Liqing Zhao.

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Li, Y., Lin, Y., Jiang, Y. et al. Cloning and Expression of Heparinase Gene from a Novel Strain Raoultella NX-TZ-3–15. Appl Biochem Biotechnol 194, 4971–4984 (2022). https://doi.org/10.1007/s12010-022-03917-5

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