Molecular analysis of a fibrin-degrading enzyme from Bacillus subtilis K2 isolated from the Indonesian soybean-based fer
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ORIGINAL ARTICLE
Molecular analysis of a fibrin‑degrading enzyme from Bacillus subtilis K2 isolated from the Indonesian soybean‑based fermented food moromi Fathma Syahbanu1 · Puspo Edi Giriwono1 · Raymond R. Tjandrawinata2 · Maggy T. Suhartono1 Received: 5 July 2020 / Accepted: 3 October 2020 © Springer Nature B.V. 2020
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. Keywords Bacillus subtilis · Bioinformatic · Molecular docking · Moromi · Serine protease
Introduction Moromi is an intermediate phase in soy sauce fermentation. The high protein content of soybean (35–38%) implies that this product has potential as a growth medium for various proteolytic microorganisms, including fibrin-degrading microorganisms. Wei et al. [1] reported that during spontaneous fermentation of soybean, a number of microorganisms, such as Zygosaccharomyces rouxii, Tetragenococcus halophilus, Bacillus subtilis, Bacillus pumilus, Bacillus
* Maggy T. Suhartono [email protected] 1
Department of Food Science and Technology, IPB University (Bogor Agricultural University), Dramaga, P.O. BOX 220, Bogor, Indonesia
Dexa Laboratories of Biomolecular Sciences, Dexa Medica Jababeka, Cikarang, Indonesia
2
licheniformis, and Bacillus amyloliquefaciens, which produce hydrolytic enzymes, can be observed. The alkaline serine protease subtilisin (EC 3.4.21.14) is the dominant extracellular protease of Bacillus species [2], and a number of forms of this enzyme have been reported, including nattoki
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