Previously, the crude extracts of recombinant Nattokinase (NK) variants i.e. NatTK and NatOC and one wild type Douchi Fibrinolytic Enzyme (DFE) from Indonesian traditional fermented foods has been shown to demonstrate fibrinolytic activity. Both NKs contain substitutions of D41N, V192A and 252-RLQHTLEALSTM-263 but NatOC has additional V4F. In the present study, the effects of amino acid substitutions in NK variants and G169A in DFE on their enzyme characteristics were evaluated. Pure proteins were obtained using two sequential steps chromatography using ion exchange and a gel filtration columns. Their activities were determined with fibrin plate, fibrin zymography, fibrinogen hydrolysis, and chromogenic assays. The fibrinogen degradation profile of the wild type NK (NatWT) was different to the NK variants but similar to DFEs. Optimum activity of all the NKs and DFEs was achieved at 50 °C while the optimum pH for NatWT/DFEs and NK variants were 8 and 7, respectively. DFEG169A exhibited higher fibrinogen degradation rate and fibrin specific activity than DFE. PMSF inhibited all the NKs and DFEs while SDS and EDTA caused lower activity. The NK variants were more resistant towards Na+ and Ca2+ but more sensitive to K+. The amino acid substitutions in NK variants alter their fibrinogen degradation profile, optimum working pH, working pH range, and resistance to some ions. Substitutions in NK variants likely promote structural changes, particularly with the binding mode of the calcium ion cofactor. The results provide a beneficial basis for future development of fibrino(gen)olytic proteins with improved properties for cardiovascular diseases therapy.
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Recombinant nattokinase from bacteria in dried tauco (with amino acid variation D41N and V192A)
Recombinant nattokinase from bacteria in yellow oncom (with amino acid variation V4F, D41N and V192A)
Nattokinase from Bacillus subtilis, AAO65246 (NCBI)
Douchi fibrinolytic enzyme, ACA34903 (NCBI)
DFE with glycine substitution in position 169 with alanine
Sodium dodecyl sulphate
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The authors would like to thank Wangsa Tirta Ismaya for productive discussion of structural analysis and Rahmat Muliadi for technical assistance. This research was funded by Science and Technology Research Grant (STRG) from the Indonesian Toray Science Foundation (ITSF) and the RISBIN IPTEKDOK (Riset Pembinaan Ilmu Pengetahuan dan Teknologi Kedokteran) grant from the Health Research and Development Agency in the Ministry of Health, Republic Indonesia.
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Purwaeni, E., Riani, C. & Retnoningrum, D.S. Molecular Characterization of Bacterial Fibrinolytic Proteins from Indonesian Traditional Fermented Foods. Protein J 39, 258–267 (2020). https://doi.org/10.1007/s10930-020-09897-x
- DFE G169A
- Fibrin specificity
- Fibrinogen degradation
- Ion resistance
- NK variants