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Characterization of an Intracellular Alkaline Serine Protease from Bacillus velezensis SW5 with Fibrinolytic Activity

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Abstract

ISP-SW5 is an intracellular alkaline serine protease gene from Bacillus velezensis SW5 that was heterologously expressed in Escherichia coli BL21 (DE3). Sequence analysis indicated that the ISP-SW5 gene has 960 bp open reading frame and encodes a protein of 319 amino acid residues. Three-dimensional structure of ISP-SW5 with the fibrinolytic activity from Bacillus velezensis was predicted by in silico analysis. Gly219 was the most likely active site for the fibrinolytic activity of ISP-SW5. The recombinant enzyme ISP-SW5 was purified by Ni–NTA Superflow Column. SDS-PAGE showed that this enzyme had a molecular mass of 34 kDa. The result of native-PAGE and N-terminal sequencing showed that the N-terminal propeptide of ISP-SW5 was cleaved during the maturation of protease. The optimum pH and temperature were 8.0 and 40 °C, respectively. Enzyme activity was markedly inhibited by PMSF and EDTA but enhanced by 5 mM Ca2+ and 2 mM Zn2+ by up to 143% and 115%, respectively. Additionally, ISP-SW5 retained 93%, 78%, and 49% relative enzyme activity after incubation with 0.5 M, 1 M and 2 M NaCl, respectively, at 4 °C for 12 h. The enzyme activity determined by casein as substrate was 1261 U/mg. ISP-SW5 could degrade fibrin at an activity of 3428 U/mg, and its properties reflect its potential application in developing a novel biological catalyst for efficient fibrin hydrolysis in medical treatment.

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Acknowledgements

This work was financially supported by the Natural Science Fund Project of Ningbo (2018A610337) and Public Welfare Project of Zhejiang (GG19C200003).

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Authors and Affiliations

  1. Laboratory of Food Biotechnology, College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo, 315832, People’s Republic of China

    Haining Yang, Yang Liu, Yuchang Ning, Changyu Wang, Xin Zhang, Peifang Weng & Zufang Wu

  2. Institute of Quality Standards and Testing Technology for Agro-Products, Fujian Academy of Agricultural Sciences, Fuzhou, 350003, People’s Republic of China

    Yang Liu

  3. Laboratory of Food Biotechnology, School of Food and Bioengineering, Henan University of Animal Husbandry and Economy, Zhengzhou, 450046, People’s Republic of China

    Yuchang Ning

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  1. Haining Yang
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  2. Yang Liu
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  3. Yuchang Ning
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  5. Xin Zhang
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  6. Peifang Weng
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Contributions

Conceived and designed the experiments: HY, YL, YN—Performed the experiments: HY, CW—Analyzed the data: HY, NL—Contributed reagents/materials/analysis tools: ZW, PW, XZ—Wrote the manuscript: HY—Revised and approved the final version of this work: HY ZW.

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Correspondence to Zufang Wu.

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Yang, H., Liu, Y., Ning, Y. et al. Characterization of an Intracellular Alkaline Serine Protease from Bacillus velezensis SW5 with Fibrinolytic Activity. Curr Microbiol 77, 1610–1621 (2020). https://doi.org/10.1007/s00284-020-01977-6

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  • DOI: https://doi.org/10.1007/s00284-020-01977-6