Debbie Soefie Retnoningrum

Bacterial Fibrinolytic Enzyme Coding Sequences from Indonesian Traditional Fermented Foods Isolated Using Metagenomic Approach and Their Expression in Escherichia Coli

ABSTRACT Fibrinolytic proteins play an important role in treatment of cardiovascular disease. This study aimed to express coding sequences of new bacterial fibrinolytic protein from Indonesian traditional fermented foods obtained by a PCR-based metagenomic approach and to test their biological activity. New amino acid variations were found in nattokinase (NAT) at several positions i.e. D41N and V192A from dried Tauco, V4F, D41N, and V192A from yellow Oncom but not in Douchy Fibrinolytic Enzyme (DFE) from Terasi. The NAT and DFE recombinant versions were overproduced in Escherichia coli BL21(DE3) using pET16b(+). Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis analysis showed all clones produced mature soluble fibrinolytic proteins of 28 kDa. All recombinant proteins displayed caseinolytic and fibrinolytic activities. In conclusion, metagenomic approach can be applied to obtain and to express coding sequences of new variant bacterial fibrinolytic protein as active soluble mature form in E. coli.

Leu169Trp substitution in MnSOD from Staphylococcus equorum created an active new form of similar resistance to UVC irradiation

Manganese superoxide dismutase from Staphylococcus equorum (MnSODSeq) maintains its activity after up to 45u202fminutes of UVC radiation. The enzyme occurs in a dimeric form that likely contributes to its activity and stability. Therefore, maintaining the dimeric form could be a way to improve the enzymes stability. One of the main interactions for dimer formation occurs between Tyr168 and His31, of which the latter is also involved in the enzymatic reaction. UVC radiation may cause alterations in the electronic structure of the phenolic ring in the Tyr168 side chain: this may disrupt the Tyr168-His31 pairing and lead to enzyme instability and/or activity loss. In this report, a Leu169Trp substitution was carried out to protect the Tyr168 residue by introducing an amino acid with an aromatic side chain for better photon absorption of the UV light. Interestingly, although the substitution appeared to have a minor effect on enzyme stability and activity upon UVC irradiation, the melting temperature (TM) of the Leu169Trp mutant was different. Unlike the native protein, the TM of the mutant had not changed after UV irradiation. Thus, our effort to extend the resistance to UVC radiation was not successful, but we have discovered a biologically active new form. The present finding provides evidence that MnSODSeq maintains most of its activity and resistance to UVC irradiation as long as the dimer and its glutamate-bridge are intact, despite an alteration that destabilizes its monomeric structure. The present finding further unravels the relationship between the structure of the enzyme and its activity. Furthermore, the results may provide further insight in how to modify the enzyme to improve its characteristics for application in medicine or cosmetics.