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Molecular Characterization of Bacterial Fibrinolytic Proteins from Indonesian Traditional Fermented Foods
Purwaeni E.a, Riani C.a, Retnoningrum D.S.a
a Laboratory of Pharmaceutical Biotechnology, School of Pharmacy, Bandung Institute of Technology, Bandung, 40132, Indonesia
[vc_row][vc_column][vc_row_inner][vc_column_inner][vc_separator css=”.vc_custom_1624529070653{padding-top: 30px !important;padding-bottom: 30px !important;}”][/vc_column_inner][/vc_row_inner][vc_row_inner layout=”boxed”][vc_column_inner width=”3/4″ css=”.vc_custom_1624695412187{border-right-width: 1px !important;border-right-color: #dddddd !important;border-right-style: solid !important;border-radius: 1px !important;}”][vc_empty_space][megatron_heading title=”Abstract” size=”size-sm” text_align=”text-left”][vc_column_text]© 2020, Springer Science+Business Media, LLC, part of Springer Nature.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.[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Author keywords” size=”size-sm” text_align=”text-left”][vc_column_text]Amino Acid Substitution,Bacterial Proteins,Calcium,Cations, Divalent,Cations, Monovalent,Cloning, Molecular,Coenzymes,Escherichia coli,Fermented Foods and Beverages,Fibrinogen,Fibrinolysis,Gene Expression,Genetic Vectors,Humans,Hydrogen-Ion Concentration,Potassium,Recombinant Proteins,Sodium,Subtilisins,Temperature[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Indexed keywords” size=”size-sm” text_align=”text-left”][vc_column_text]DFE G169A,Fibrin specificity,Fibrinogen degradation,Ion resistance,NK variants[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Funding details” size=”size-sm” text_align=”text-left”][vc_column_text][{‘$’: ‘The authors would like to thank Wangsa Tirta Ismaya for productive discussion of structural analysis and Rahmat Muliadi for technical assistance. This research was funded by Science and Technology Research Grant (STRG) from the Indonesian Toray Science Foundation (ITSF) and the RISBIN IPTEKDOK (Riset Pembinaan Ilmu Pengetahuan dan Teknologi Kedokteran) grant from the Health Research and Development Agency in the Ministry of Health, Republic Indonesia.’}, {‘$’: ‘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 .’}][/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”DOI” size=”size-sm” text_align=”text-left”][vc_column_text]https://doi.org/10.1007/s10930-020-09897-x[/vc_column_text][/vc_column_inner][vc_column_inner width=”1/4″][vc_column_text]Widget Plumx[/vc_column_text][/vc_column_inner][/vc_row_inner][/vc_column][/vc_row][vc_row][vc_column][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][/vc_column][/vc_row]