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Molecular analysis of a fibrin-degrading enzyme from Bacillus subtilis K2 isolated from the Indonesian soybean-based fermented food moromi

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Abstract

The screening of proteolytic and fibrinolytic bacteria from moromi (an Indonesian soybean-based fermented food) yielded a number of isolates. Based on morphological and biochemical analyses and sequencing of the 16S rRNA gene, the isolate that exhibited the highest proteolytic and fibrinolytic activity was identified as Bacillus subtilis K2. The study was performed to analyze molecular characteristic of a fibrin-degrading enzyme from B. subtilis K2. BLASTn analysis of the nucleotide sequence encoding this fibrinolytic protein demonstrated 73.6% homology with the gene encoding the fibrin-degrading enzyme nattokinase of the B. subtilis subsp. natto, which was isolated from fermented soybean in Japan. An analysis of the putative amino-acid sequence of this protein indicated that it is a serine protease enzyme with aspartate, histidine, and serine in the catalytic triad. This enzyme was determined to be a 26-kDa molecule, as confirmed with a zymogram assay. Further bioinformatic analysis using Protparam demonstrated that the enzyme has a pI of 6.02, low instability index, high aliphatic index, and low GRAVY value. Molecular docking analysis using HADDOCK indicated that there are favorable interactions between subtilisin K2 and the fibrin substrate, as demonstrated by a high binding affinity (ΔG: − 19.4 kcal/mol) and low Kd value (6.3E-15 M). Overall, the study concluded that subtilisin K2 belong to serine protease enzyme has strong interactions with its fibrin substrate and fibrin can be rapidly degraded by this enzyme, suggesting its application as a treatment for thrombus diseases.

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Acknowledgements

This work was supported by Master Program of Education Leading to Doctoral Degree for Excellent Graduates (PMDSU), Ministry of Research, Technology and Higher Education of the Republic of Indonesia (RISTEKDIKTI) [Grant No. 025/E3/2017]. This research was supported By Research Grant for acceleration of PhD graduate (Fathma Syahbanu) from the Ministry of Higher Education 2015–2020 to Bogor Agricultural University (IPB) with Prof. Maggy T Suhartono as the Principle Investigator and Major Advisor.

Funding

The author(s) received financial support for the research and/or publication from Master Program of Education Leading to Doctoral Degree for Excellent Graduates (PMDSU), Ministry of Research, Technology and Higher Education of the Republic of Indonesia (RISTEKDIKTI) [Grant No. 025/E3/2017].

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Authors and Affiliations

  1. Department of Food Science and Technology, IPB University (Bogor Agricultural University), Dramaga, P.O. BOX 220, Bogor, Indonesia

    Fathma Syahbanu, Puspo Edi Giriwono & Maggy T. Suhartono

  2. Dexa Laboratories of Biomolecular Sciences, Dexa Medica Jababeka, Cikarang, Indonesia

    Raymond R. Tjandrawinata

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  1. Fathma Syahbanu
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Contributions

FS: writing-original draft preparation, editing, methodology, software; PEG: data curation, validation, bioinformatic methodology; RRT: investigation, data curation, editing: MTS: supervision, conceptualization, methodology, validation.

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Correspondence to Maggy T. Suhartono.

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The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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The bacteria as source of the gene and enzyme was isolated from local soy fermented food. The data taken for result and discussion were original and came from the experimets stated in the methodology. This research did not involve the type of work that could be a threat to public health, does not produce any biological agents or toxins.

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This research did not use any animal or human subject. All methodology applied were in accordance with the reference cited.

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Syahbanu, F., Giriwono, P.E., Tjandrawinata, R.R. et al. Molecular analysis of a fibrin-degrading enzyme from Bacillus subtilis K2 isolated from the Indonesian soybean-based fermented food moromi. Mol Biol Rep 47, 8553–8563 (2020). https://doi.org/10.1007/s11033-020-05898-2

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