ISOLATION OF DNA ENCODING FIBRINOLYTIC PROTEINS FROM BACTERIA IN INDONESIAN FERMENTED FOODS BY USING METAGENOMIC APPROACH WITH THEIR PROTEIN CHARACTERIZATION

• Main Author: Purwaeni
• Format: Dissertations
• Language: Indonesia
• Subjects: Farmakologi dan terapeutik
• Online Access: https://digilib.itb.ac.id/gdl/view/32711
• Institution: Institut Teknologi Bandung

Fibrinolytic proteins have an important role in the treatment of cardiovascular diseases and could be produced by bacteria in traditional fermented foods such as Japanese Natto, Chinese Douci, and Korean Chungkook-jang. This study was conducted to screen DNA encoding bacterial fibrinolytic proteins from Indonesian fermented foods, to express them in the Escherichia coli system, and to characterize the proteins. The DNA coding sequence for bacterial fibrinolytic protein was obtained by Polymerase Chain Reaction (PCR) method using metagenomic approach. The PCR products were ligated to the pGEM-T Easy cloning vector and the identity of the recombinant vectors were confirmed. The DNA sequences of fibrinolytic protein were ligated into pET16b(+) expression vectors for protein overproduction in E. coli BL21(DE3) host cell. DNA encoding the reference fibrinolytic proteins (NAT-WT and DFEG169A) was constructed synthetically and inserted into pET16b(+). Protein overproduction was performed at 37°C by 0.1 mM IPTG induction. The proteins were purified by two purification steps using ionexchange chromatography and followed by gel filtration chromatography. Characterizations included proteolytic and fibrinolytic activities, the effects of pH, temperature, inhibitor, metal ions, and fibrin-specific tests on purified recombinant fibrinolytic proteins were carried out. The results of the DNA sequence analysis showed that the sequences of amino acid deduction had high homology to nattokinase (NAT, AAO65246) and 100% homology to Douci Fibrinolytic Enzyme (DFE, AAZ66858). Higher amino acid variations were found in NAT from dried Tauco (NAT-TK) and yellow Oncom (NAT-OC) at several positions i.e. D41N and V192A (NAT-TK), V4F, D41N, and V192A (NAT-OC) respectively. The results of the SDS-PAGE analysis showed the presence of 28 kDa mature fibrinolytic proteins in soluble form. Crude extracts containing mature recombinant fibrinolytic proteins showed protease activity when tested on casein and fibrin plates. The result of characterization of purified 28 kDa fibrinolytic protein showed that NAT-TK and NAT-OC degraded ?and ?fibrinogen fragments and had optimum protein activity iv at pH 7 and 50°C. NAT-WT only degraded ?fibrinogen fragment and had an optimum activity at pH 8 and temperature of 50°C. NAT-TK and NAT-OC displayed fibrinolytic activity about 80% at pH 8-9, while NAT-WT at pH 9-10. DFE and DFEG169A have optimum protein activity at pH 8 and 50°C. DFEG169A degraded ?, ?, and ?fibrinogen fragments faster than DFE and had high specific fibrin activity among other recombinant fibrinolytic proteins. These recombinant fibrinolytic proteins still had fibrinolytic activity in the presence of 0.1 mM SDS and EDTA and its activity decreased about 10% in the presence of Ca 2+ . This research has succesfully screened the DNA sequence encoding NAT and DFE from Indonesian traditional fermented foods and the mature form has been produced as active soluble recombinant proteins in E. coli system. These recombinant fibrinolytic proteins have various characters and can potentially be developed for the thrombosis therapy.