Bacillus tequilensis Isolated from Fermented Intestine of Holothuria Scabra Produces Fibrinolytic Protease with Thrombolysis Activity

Hidayati, Nur; Nurrahman, Nurrahman; Fuad, Hayatun; Munandar, Hendra; Zilda, Dewi Seswita; Ernanto, Aditya Rahman; Samiasih, Amin; Oedjijono, Oedjijono; Ethica, Stalis Norma

doi:10.1088/1755-1315/707/1/012008

Cited by 10 publications

(11 citation statements)

References 21 publications

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“…The aim was to determine whether bacteria have the potential to produce proteases or not. The presence of proteolytic activity is indicated by the appearance of a clear zone around the bacteria [13,16] as shown in Figure 6. The clear zone formed around the colony was due to the ability of bacterial protease to degrade the casein substrate contained in the skim milk agar media.…”

Section: Proteolytic Testmentioning

confidence: 99%

“…The PCR process consisted of initial denaturation at 95 for 4 min, then followed by 40 cycles of denaturation at 95 ℃ for 30 s each cycle, annealing at 55 ℃ for 35 s, extension at 72 ℃ for 45 s. The PCR results were examined using 2 % agarose gel electrophoresis based on the appearance of a single band at 1,500 bp. Amplicon visualization using Major Science UV Transluminator [16,17]. The cloning of the 16s rRNA gene using pTA2 vector and E. coli Zymo 5α was carried out in several steps as reported by Gao et al [18].…”

Section: Bacterial Subculturementioning

confidence: 99%

“…After obtaining a single isolate resulting from bacterial purification on NA media, the next step was to test the production of protease enzymes in Skim Milk Agar (SMA) producer media. Bacteria growing on NA media were scratched on SMA media and incubated for 24 h at 37 ℃ and then observed the formation of a clear zone around the colony growth [13,16,23].…”

Section: Isolation Of Protease-producing Bacteriamentioning

confidence: 99%

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Optimization of Crude Protease Production from Bacillus thuringiensis HSFI-12 and Thrombolytic Activity Its Enzyme Dialysate

Zafrida

Ethica²,

Ernanto³

et al. 2022

Trends Sci

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Cardiovascular disease (CVD) is among the leading causes of death in the world caused by thrombosis. Thrombosis is the formation of excessive blood clots on the walls of blood vessels called thrombus. This could lead to fatal blockage of the heart muscle or brain. Thrombosis can be treated using enzyme-type drugs such as thrombolytic proteases. There have been many studies of bacteria producing thrombolytic enzymes isolated from fermented foodstuffs. However, commercial production of bacterial enzymes to fulfill the need of thrombolytic agents is still limited, causing high price of CVD therapy. Bacillus sp. HSFI-12 (Holothuria scabra Fermented Intestine-12) isolated from the fermented intestine of sea cucumber Holothuria scabra had been reported to have a competitive thrombolytic activity to the commercial Nattokinase. This study aimed to determine bacterial optimum incubation time based on enzyme activity after bacterium molecular identification was done. It also aimed to obtain the dialysate of bacterial crude protease and then determine the thrombolytic activity of the obtained dialysate. Thrombolytic activity was tested on 4 different types of blood (A, B, AB and O). Results of molecular identification showed that strain HSFI-12 shared similarity level of 99.80 %, with Bacillus thuringiensis. Activity of crude protease from the incubated cultures peaked at 48 h of incubation with activity of 191.5 U/mL The activity of concentrated protease after precipitation and dialysis process (dialysate) was 2-times higher by 355,7 U/mL. The percentage of lysis of blood clots produced by crude blood groups A, B, AB and O showed a range of values of 66.423 - 67.656 % while those that produce dialysate are 77.564 - 78.861 %. As conclusion, the best (optimized) incubation time to produce crude enzyme from B. thuringiensis HSFI-12 with the highest activity was 48 h. The process of concentrating the crude thrombolytic protease of B. thuringiensis HSFI-12 increases both activity and thrombolytic ability of the bacterial enzyme. HIGHLIGHTS The most optimum incubation time for protease production from Bacillus thuringiensis is 48 h Partial purification on bacterial crude protease through resulted in 86 % increased activity Partial purification on bacterial crude protease resulted in 17 % increased blood clot lysis ability Clot lysis ability of both crude and dialysate proteases are higher than that of Nattokinase (NK) GRAPHICAL ABSTRACT

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Section: Proteolytic Testmentioning

confidence: 99%

Section: Bacterial Subculturementioning

confidence: 99%

Section: Isolation Of Protease-producing Bacteriamentioning

confidence: 99%

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Optimization of Crude Protease Production from Bacillus thuringiensis HSFI-12 and Thrombolytic Activity Its Enzyme Dialysate

Zafrida

Ethica²,

Ernanto³

et al. 2022

Trends Sci

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“…On the Fibrin Agar (FA) medium, 4 wells were made and 100 l mL of sterile distilled water was added to each well, Nattokinase standard, crude, and dialysate respectively. The brin media was incubated at 37°C for 24 h (Hidayati et al, 2021c) . Fibrinolytic activity was indicated by the presence of a clear zone around the well and was determined by measuring the brinolytic enzyme activity index by calculating the diameter of the clear zone divided by the diameter of the colony (Archna et al, 2015) .…”

Section: Fibrinolytic Activity Assay Of Protease Of Bacillus Thuringi...mentioning

confidence: 99%

Characteristics and Substrate Specificity of Semi-Purified Bacterial Protease ofBacillusthuringiensisHSFI-12 with Potential as Antithrombotic Agent

Ferdiani

Zilda²,

Afriansyah

et al. 2023

Sci. Technol. Indones

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Commercial proteases, such as Nattokinase (NK), Staphylokinase (SAK), and Streptokinase (SK) play an important role in the destruction of thrombus, the main cause of death in cardiovascular disease. The latest technology combining enzymes with certain drugs is the target of new research in the thrombolytic area. The first step is to develop protease from Bacillus thuringiensis HSFI-12 bacteria as an antithrombotic agent, characterization of the bacterial enzyme is necessary. This study aims to determine the specificity of protease from Bacillus thuringiensis HSFI-12 to explore its potential as an antithrombotic agent in terms of anticoagulant and fibrinolytic activities. The molecular weight and specificity of bacterial protease were determined with a zymographic method with casein as substrate. Bacillus thuringiensis HSFI-12 was first cultured on Nutrient Agar (NA) media and then on Skim Milk Agar (SMA) media. The obtained crude protease from Skim Milk Broth (SMB) was then concentrated as dialysate. Both crude and dialysate proteases were tested for their specific activity, as well as anticoagulant and fibrinolytic activities. Next, the dialysate’s molecular weight and specificity on the casein substrate were investigated using the zymographic method. As result, protease activity in crude form is lower than that in dialysate, which was 0.5570 ± 0,004 U/mL and 2.1767 ± 0,005 U/mL, respectively. The molecular weight of the obtained bacterial protease was between 117 – 133 kDa and the enzyme is capable of degrading casein as shown on the zymogram. Overall, both crude and dialysate proteases of Bacillus thuringiensis HSFI-12 show potential as an antithrombotic agent for exhibiting anticoagulant and antiplatelet activities. Yet, it could not exhibit direct fibrinolytic activity implying the possibility that the enzyme plays a role as a plasminogen activator, which can dissolve fibrin by activating plasmin.

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“…Bacillus tequilensis HSFI-5 is a bacterial isolate obtained from the fermented intestine of the sand sea cucumber, Holothuria scabra. The sea cucumber was isolated from sand sea cucumber from Kodek Gulf Village, Lombok, West Nusa Tenggara.Strain HSFI-5 was found to have the ability to produce fibrinolytic protease, which is an important characteristic of an antithrombotic agent(11,12).Another important characteristic of the protease of B. tequilensis that underlines its potential as an antithrombotic agent as clot lysis activity. This study represents novelty as the anticoagulant activity of the protease from B. tequilensis HSFI-5 has not yet been tested.…”

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confidence: 99%

In Vitro Anticoagulant Activity of Crude Protease of Bacillus tequilensis HSFI-5

Ethica,

Raharjo,

Zilda

et al. 2023

IJMLST

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obtained from the fermented intestine of Holothuria scabra (sand sea cucumber). Strain HSFI-5 had been reported to be able to produce proteases, which had shown several characteristics of an antithrombotic agent, i.e., fibrinolytic and clot-lysis activities. However, its anticoagulation activity test is yest to be done. This study aimed to determine the anticoagulant activity of the crude protease HSFI-5 in vitro. The study design was a completely randomized design with a sample size of 90 calculated using the Federer formula. The material used was crude protease from B. tequilensis in skim milk broth. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and plasma recalcification time (PRT) were carried out to test the anticoagulant activity. Citrated platelet poor plasma samples were divided into positive control, normal control, direct examination with crude enzyme in volumes of 50 and 100 µL and pre-incubation at 37ºC for 5, 10, and 15 min with crude enzyme volumes of 50 and 100 µL. The data normality was tested with the Kolmogorov-Smirnov test and the different tests were analyzed by one-way ANOVA with the Post hoc LSD test. The results of one-way ANOVA both on PT, aPTT, and PRT examinations showed that there was a significant difference between the treatment groups (p<0.05). The longest results of PT, aPTT, and PRT are positive controls, and the shortest results are normal controls for PT, and 15’ 50 group for aPTT and PRT. It is clear that crude protease B. tequilensis HSFI-5 exhibits anticoagulant as well as thrombolytic action, raising the possibility that it could function as an antithrombotic drug.

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Bacillus tequilensis Isolated from Fermented Intestine of Holothuria Scabra Produces Fibrinolytic Protease with Thrombolysis Activity

Cited by 10 publications

References 21 publications

Optimization of Crude Protease Production from Bacillus thuringiensis HSFI-12 and Thrombolytic Activity Its Enzyme Dialysate

Optimization of Crude Protease Production from Bacillus thuringiensis HSFI-12 and Thrombolytic Activity Its Enzyme Dialysate

Characteristics and Substrate Specificity of Semi-Purified Bacterial Protease ofBacillusthuringiensisHSFI-12 with Potential as Antithrombotic Agent

In Vitro Anticoagulant Activity of Crude Protease of Bacillus tequilensis HSFI-5

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