Characterization of Fibrinolytic Precipitate from Bacillus subtilis

Isolated from A Traditional Indonesian Beverage, Tuak

Yudith B. Spagnoli, Ryan Hardika, Maggy T. Suhartono, Yanti
Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia

INTRODUCTION So, it was considered to be a relatively thermolabile

According to World Health Organization
RESULTS AND DISCUSSION and alkalophilic enzyme.

(WHO 2011), there were 30% deaths caused ISOLATION OF BACTERIA

of cardiovascular disease. One possible
cause is the accumulation of fibrin in blood Figure 3 Zymogram profiles of TBO2-24h (left) and TBO2-48h (right) precipitates
vessel (thrombosis) that can not be were mixed with solvents. 1 1% v/v ethanol; 2 5% v/v ethanol; 3 1% v/v
hydrolysed in balance (Kamath & Lip 2003). methanol; 4 5% methanol; 5 1% hexane; 6 5% hexane; 7 control.
Figure 1 The growth of bacteria at Luria Agar medium (left) and Skim Milk
Tissue-type plasminogen activator (t-PA) The presence of solvent did not affect to enzyme
Agar (right). activity. Stabilization of the enzyme was due to its
and urokinase are still widely used in
thrombolytic therapy today, but their Isolate number 2 and 4 have proteolytic activity. precipitation in organic solvent that made it possible
expensive prices, low specificity to fibrin, Then, bacterial isolate 2 was further studied for for its stereo configuration to be retained and thus
microbial precipitate production and biochemical also its activity. The other reason is the presence of
short half live and undesirable side effects,
characterization. Fibrinolytic is one of enzyme that disulfide bonds can stabilize proteins by reducing the
such as the risk for internal hemorrhage
belongs to serine protease group which hydrolysis entropy of the denatured state (Hassanein et al.
within the intestinal tract when orally protein by acylation and deacylation reaction 2011).
administrated, prompt researchers to (Zou & Zhang 2011).
search for cheaper and safer resources FIBRINOLYTIC ASSAY
(Peng et al. 2005; Sugimoto et al. 2007). PRODUCTION OF CRUDE ENZYME Table 4 Comparison of fibrinolytic activity from crude enzyme and precipitate
This research was aimed to produce and Table 1 Concentration of cell bacteria in TBO2-24h and TBO2-48h cultures Specific Activity (U)
Samples
characterize fibrinolytic precipitate from Optical Crude Precipitate
Samples Density [Protein] (mg mL-1) TBO2-24h 687.5 323.529
Bacillus subtilis isolated from tuak. Tuak is a
TBO2-24h 1.124 0.9459 TBO2-48h 468.75 294.117
traditional fermented beverage that is
TBO2-48h 1.192 0.5683
made by fermenting the sugary sap of Based on enzyme activity to fibrin, precipitate had
coconut tree (Cocos nucifera) Isolate TBO2 was grown in Skim Milk Broth medium lower activity than crude enzyme.
(Chandrasekhar et al. 2012). to produce crude enzyme. The time of incubation If compared between fibrinogen and fibrin as
was divided into 24h and 48h. TBO2-24h culture substrates, precipitate has higher activity to
resulted in fewer number of cells, but higher fibrinogen. So, the enzyme can be used as a drug to
protein concentration compared to TBO2-48h. This treat hyperfibrinogenemia (Machlus et al. 2011).
is in accordance with study of Pant et al. (2015)
METHODS said that protease production increased gradually
Isolation of protease-producing bacteria from 0 to 36h, at which it was maximal, then
decreased with time.
Production of crude enzyme
The time of incubation was divided into 24h and 48h PRECIPITATION OF CRUDE ENZYME
Table 2 Comparison of protein concentration between crude enzyme and Figure 4 Optimal temperature offibrinolytic enzyme from precipitate TBO2-24h
precipitate (left) and TBO2-48h (right).
Precipitation of crude enzyme
The enzyme was precipitated by acetone 70% [Protein] (mg mL-1) Fibrinolytic enzyme worked optimally at temperature
Samples
Crude Precipitate 37ºC. This is in accordance with Afifah et al. 2014 said
TBO2-24h 0.95 1.29 that fibrinolytic enzyme worked optimally at
Fibrinogenolytic assay Fibrinolytic assay TBO2-48h 0.57 0.59 temperature less than 60ºC.
• Activity and • Activity assay by Precipitate had higher protein concentration than
specificity assay fibrin plate method crude enzyme, because precipitation with MOLECULAR WEIGHT OF PRECIPITATE
• Optimal • Optimal acetone can decrease solubility of protein and
temperature and temperature was caused enhancement of interaction between
pH were characterized by protein molecules (Nejadi et al. 2014).
characterized by fibrin plate method
zymographic assay FIBRINOGENOLYTIC ASSAY
• Solvent resistance Table 3 Comparison of crude enzyme and precipitate activity and specificity Figure 5 SDS-PAGE profile of precipitate enzyme. 1-2 Low Molecular Weight
was characterized against fibrinogen (LMW) protein marker; 3-5 precipitate enzyme.
by zimographic UA (U mL-1) AS (U mg-1) Protein with molecular weight 23-24 kDa was
assay Samples
Crude Precipitate Crude Precipitate identified as β-1,3-1,4-endoglucanase precursor. The
TBO2-24h 0.0217 5.04 0.0229 3.907 28 kDa protein was identified as a subtilisin-type
Molecular weight of precipitate TBO2-48h 0.0993 4.49 0.1747 7.572
protease with fibrinolytic activity. the 45-47 kDa might
Determination of molecular weight of precipitate was performed with be a homolog of YnfF, a precursor of dimethyl
SDS-PAGE Precipitate has higher activity (UA) and specificity
(AS) than crude enzyme, because precipitate has sulfoxide reductase (Kim et al. 2009).
higher protein content than crude enzyme.
Identification of bacteria IDENTIFICATION OF BACTERIA
Fibrinolytic bacteria identification using 16S ribosomal RNA gene
amplification and DNA sequencing

Figure 2 Zymogram profile of optimal temperature (left) and pH (right) of

TBO2 precipitate. Figure 6 BLAST result of 16S rRNA gene sequence from isolate TBO2.
The enzyme worked optimally at 50ºC; pH 8 and Isolate TBO2 was identified as Bacillus subtilis with a
enzyme was unstable under acidic conditions. similarity degree of 99%.

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the increase level of fibrinogen plasma and the formation of fibrin clot