Molecular Characterization of Bacterial Fibrinolytic Proteins from Indonesian Traditional Fermented Foods
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Molecular Characterization of Bacterial Fibrinolytic Proteins from Indonesian Traditional Fermented Foods Eni Purwaeni1 · Catur Riani1 · Debbie Soefie Retnoningrum1
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract 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. Keywords DFE G169A · Fibrin specificity · Fibrinogen degradation · Ion resistance · NK variants Abbreviations NatTK Recombinant nattokinase from bacteria in dried tauco (with amino acid variation D41N and V192A) NatOC Recombinant nattokinase from bacteria in yellow oncom (with amino acid variation V4F, D41N and V192A) NatWT Recombinant nattokinase NatBS Nattokinase from Bacillus subtilis, AAO65246 (NCBI) DFE Douchi fibrinolytic enzyme, ACA34903 (NCBI) Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10930-020-09897-x) contains supplementary material, which is available to authorized users. * Debbie Soefie Retnoningrum [email protected] 1
Laboratory of Pharmaceutical Biotechnology, School of Pharmacy, Bandung Institute of Technology, Jalan Ganesha 10, Bandung 40132, West Java, Indonesia
DFEG169A DFE with glycine substitution in position 169 with alanine SDS Sodium dodecyl sulphate EDTA Ethylenediaminetetraacetic acid PMSF Phenylmethylsulfonyl fluoride CN Coordination number
1 Introduction Cardiovascular diseases (CVDs), such as acute myocardial infarction, per
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