BIOTROPIA Vol. 30 No. 2, 2023: 147 - 157 DOI: 10.11598/btb.2023.30.2.1765 147 PRODUCTION OPTIMIZATION, PARTIAL PURIFICATION, AND THROMBOLYTIC ACTIVITY EVALUATION OF PROTEASE OF Bacillus cereus HSFI-10 AINUTAJRIANI AINUTAJRIANI1, SRI DARMAWATI1, DEWI SESWITA ZILDA2, MUHAMMAD ARDI AFRIANSYAH3, RAGIL SAPTANINGTYAS3 AND STALIS NORMA ETHICA1* 1Magister of Clinical Laboratory Science, Postgraduate Program, Universitas Muhammadiyah Semarang, Central Java, 50273, Indonesia 2Research Center for Deep Sea, Earth Sciences and Maritime Research Organization, National Research and Innovation Agency (BRIN), Jl. Pasir Putih Raya Pademangan, North Jakarta City, Jakarta, 14430, Indonesia 3Department of Medical Laboratory Technology, Faculty of Nursing and Health Sciences, Universitas Muhammadiyah Semarang, Central Java, 50273, Indonesia Received 17 June 2022 / Revised 2 March 2023 /Accepted 11 March 2023 ABSTRACT Cardiovascular disease is the primary cause of mortality in the world due to the formation of blood clots or thrombi in blood vessels. Bacterial proteases commonly function as thrombus dissolver agents in the pharmaceutical industry. Bacterial isolate HSFI-10 (Holothuria scabra Fermented Intestine-10) previously isolated from Rusip fermented sea cucumber had demonstrated thrombolytic activity. This study aimed to produce crude protease of HSFI-10 strain at an optimized incubation time and determine the thrombolytic activity of crude and dialysate proteases on A, B, AB, and O blood types. Isolate HSFI-10 was first molecularly identified and found to be Bacillus cereus with a homology level of 99.80% with Bacillus cereus strain ST06. The optimum crude enzyme was obtained after 48-h incubation with an activity of 222.52 U/mL, which increased to 438.84 U/mL after ammonium sulfate precipitation and dialysis. Clot lysis activity of crude enzymes was measured based on the gravimetry method on blood in the ABO system, showing results that ranged from 68.99% to 69.76%, while the dialysate ranged from 81.16% to 82.52%. In conclusion, partial purification of bacterial protease could increase both its specific and thrombolytic activities on human blood in the ABO system, with only 1% activity variability between A, B, AB, and O blood types. Keywords: Bacillus cereus HSFI-10, blood system, clot lysis, partial purification, thrombosis INTRODUCTION Thrombosis is the leading cause of global death in cardiovascular disease (CVD) (Scheres et al. 2018). Cardiovascular is a group of diseases related to the heart and blood vessels. According to the World Health Organization (WHO), this disease causes 31% of death worldwide. WHO predicts that by 2030, cardiovascular disease will continue to increase to more than 23.6 million people. The number is two times higher than the death rate from cancer (WHO 2017). Thrombosis causes blood to clot (forming a thrombus), so blood vessels in the heart and brain will be blocked (Martina et al. 2019). Thrombus occurs in the area of the injured vascular wall, thereby stimulating platelet adhesion in that direction. The attached platelets are activated and release Adenosine Diphosphate (ADP) and thromboxane A2 (TxA2), which causes other platelets to stick to the activated platelets. Platelet aggregation is strengthened with the help of clotting factors in the form of fibrin threads to form a hemostatic plug (Bartinelli et al. 2013; Durachim and Astuti, 2018). Thrombosis therapy can use anti- coagulant agents, anti-platelet drugs, fibrinolytic/thrombolytic enzymes, and surgery. However, these drugs have side effects such as headaches, urticaria (allergic reactions), increased clotting time, nausea, vomiting, bleeding complications, low fibrin specificity, and *Corresponding author, email: norma@unimus.ac.id BIOTROPIA Vol. 30 No. 2, 2023 148 relatively high price (Saxena et al. 2015; Krishnamurthy et al. 2018; Sharma et al. 2019). Research conducted by Hidayati et al. (2021) successfully isolated protease-producing bacteria from the digestive organs of the sand sea cucumber coded HSFI-10 to -12 (Holothuria scabra Fermented Intestine-10). The crude enzyme of bacterial isolate HSFI-10 showed higher thrombolytic ability than the positive control Nattokinase (a commercial antithrombosis agent). However, the bacterial isolates were only known for their microscopic and macroscopic characteristics. In addition, the thrombolysis test does not explicitly mention blood groups A, B, AB, and O. In contrast, antigens A and B in the ABO system blood group impact hemostatic balance and respond differently to thrombolytic therapy (Separham et al. 2020; Mehic et al. 2020). Identification was done to determine bacterial species by amplifying the 16S rRNA gene using Polymerase Chain Reaction (PCR). This molecular diagnostic method is more sensitive, specific, efficient, and faster than manual identification (Shahi et al. 2018). Bacteria secrete proteases in the production medium. Each bacterium has a different enzyme production time depending on the type of bacteria (Sharma et al. 2015)—partial purification of the protease enzyme Bacillus sp. HSFI-10 can be done by precipitating enzymes using ammonium sulfate. This salt has a fairly high ionic strength, does not cause damage to proteins, and has a high solubility in water (Mukherjee, 2019). The remaining ammonium sulfate salt from the enzyme precipitate is removed by dialysis (Razzaq et al. 2019). This study aims to determine the type of bacteria based on the 16S rRNA gene sequence using the PCR method, to determine differences in protease enzyme activity based on the optimization of production time for 24, 48, and 72 Hours and to determine the description of A, B, AB, and O blood smears after the addition thrombolytic protease enzymes of Bacillus sp. HSFI-10. MATERIALS AND METHODS Bacterial Origin Bacterial strain HSFI-10 was previously isolated in an earlier study from sand sea cucumber H. scabra captured from its captivity at Marine Bio Industry Office (BBIL), The Indonesian Institute of Sciences (LIPI), Kodek Gulf Village, Lombok, West Nusa Tenggara (Figure 1). The isolate was then subcultured, and the purified colonies were further analyzed to identify and confirm its fundamental characteristics. Figure 1 Location of the captivity of sand sea cucumber H. scabra, which intestine was the source of HSFI bacteria including HSFI-10 strain: (red dot, coordinates 8°24'15.1"S 116°04'47.2"E) (Google Map 2022) Thrombolytic protease of Bacillus cereus HSFI-10 – Ainutajriani et al. 149 Procedures Bacterial subculture and crude protease production Bacterial subculture was done by cultivating a loop-full of HSFI-10 bacterial single colony Nutrient Agar (NA) medium and then incubated at 37℃ for 24 hours. A single colony of isolate HSFI-10 obtained was then tested to confirm its proteolytic activities by streaking it on skim milk agar (SMA) media and re-incubated for 24 hours at 37°C. The clear proteolytic zone formed around the colony's growth was observed (Fuad et al. 2020; Hidayati et al. 2021). Bacterial Molecular Identification by Cloning 16S rDNA Bacterial colonies of HSFI-10 were inoculated into the BHIB 1 ml medium. After incubation at 37°C for 2 × 24 hours. BHIB media was centrifuged for 1 min at 12000 rpm. DNA genome was extracted with a DNA extraction kit from Geneaid, namely Presto™ Mini gDNA Bacteria Kit. The amplification process used Go Tag Green Master Mix (Promega). The universal primers used were 27- F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492-R (5'-GGT TAC CTT GTT ACG ACT T-3'). Amplicon was visualized using the Major Science UV Transilluminator (Darmawati et al. 2015). Cloning of the 16S rRNA gene was done as previously reported by Darmawati (2015) using Plasmid Vector pTA2 transformed in E. coli Zymo 5α competent cells. Recombinants were screened using the blue-white method (Green & Sambrook 2021). Plasmids were isolated according to the previously reported method, with the results of recombinant DNA checked with electrophoresis on 0.8% agarose gel. Sequencing was carried out using ABI sequence programs with primers T7 and T3 5'- TAATACGACTCACTATAGGG-3' and 5'- CCCTTTAGTGAGGGTTAATT-3' as previously reported (Darmawati 2015). Next, bioinformatics DNA sequencing results are analyzed using bioinformatics devices, then processed manually and matched with data in www.ncbi.nih.gov through the BLAST program (Ethica et al. 2018). Optimization of Bacterial Crude Protease Production Colony of Bacillus sp. HSFI-10, which has proteolytic abilities inoculated in minimally synthetic medium (MSM) (NaCl 0.1%, K2HPO4 0.1%, ammonium sulfate 0.7%, MgSO4.7H2O 0.01%, yeast extract 0.05%, skim milk 1%) and incubated at 37°C for 24,48 and 72 h (Farooq et al. 2021). The extraction of thrombolytic protease enzymes is done by bacterial culture centrifugation at 10,000 rpm, where supernatant was regarded as a crude enzyme. It further measured the enzyme activity of the Bergmeyer and Grab method (1983) using a spectrophotometer at λ 600 nm. Partial Purification of Bacterial Protease Partial purification was initiated by precipitation using 70% ammonium sulfate, as much as 400 g added slowly in 1 L of crude enzymes as previously described (Natsir et al. 2015). Cellophane bags were initially soaked in hot water at 60°C for 2 mins, then replaced with 0.2% sulfuric acid as previously reported. Enzymes obtained from the deposition of ammonium sulfate were put in cellophane bags. Both ends of the cellophane bag are tied, then the cellophane bag is soaked with a 500-700 ml phosphate buffer of 0.05 M pH 7 at ± 4°C for 2 h. The soaking buffer is replaced every 2 hours until all salts are separated (Abbas et al. 2018). Specific Enzyme and Clot Lysis Activity Assay The protease-specific activity was measured using the modified Bergmeyer & Grab (1983) (Natsir et al. 2015; Si & Jang 2018). In vitro, blood clot lysis tests were conducted on blood from 4 volunteers, each with blood types A, B, AB, and O, in 6 of the 1.5 mL microtubes previously weighed. Each tube was coded 1-6, tube 1 (negative control), tube 2 (positive control), tube 3 (blood type A), tube 4 (blood type B), tube 5 (blood type AB), and Tube 6 (blood type O), and filled with 600 μL blood of each. As previously reported, gravimetry determined clot lysis percentage (Fuad et al. 2020; Hidayati et al. 2021; Prasad et al. 2006). Proteolytic Activity Test A single colony of Bacillus sp. bacteria. HSFI- 10 obtained from the results of bacterial purification in NA media tested its proteolytic abilities. Bacteria that grow on NA media were streaked on skim milk agar (SMA) media and incubated for 24 h at 37°C then the clear zone BIOTROPIA Vol. 30 No. 2, 2023 150 formed around the growth of the colony was measured (Fuad et al. 2020; Hidayati et al. 2021). Optimization of Crude Enzyme Protease Production Time from Bacillus cereus Culture Colony of Bacillus sp. HSFI-10 with proteolytic abilities was inoculated in minimal synthetic medium (MSM) incubated at 37°C for 24, 48, and 72 h. Extraction of thrombolytic protease enzymes was done by centrifugation at 10,000 rpm for 20 mins at 4°C. The obtained supernatant was regarded as a crude enzyme, and its activity was further measured by Bergmeyer & Grab method (1983) using a spectrophotometer at λ=600 nm (Farooq et al. 2021). Partial Purification of Crude Protease 70% of ammonium sulfate (400 g) was inserted slowly in 1 L of crude enzymes in cold conditions until dissolved for 2 hours. The enzyme solution was kept in the refrigerator to precipitate the enzymes overnight. The enzyme solution was concentrated at 4°C at 10,000 rpm for 30 mins, and the formed pellet was kept. The pellet was then rushed with a 15 ml Tris-Cl buffer of 0.05 M pH 8 (Natsir et al. 2015). Cellophane bags were prepared by soaking in hot water and 0.2% sulfuric acid solution before use. Enzymes obtained from the deposition of ammonium sulfate are put in cellophane bags. Dialysis was conducted as previously described until all salts were separated (Abbas et al. 2018). Enzyme-Specific Activity Assay The protease-specific activity was measured using the modified Bergmeyer & Grab (1983) Folin reagent method (Natsir et al. 2015; Si and Jang 2018). Clot Lysis (Thrombolysis) Activity In Vitro Assay In vitro blood clot lysis tests before and after enzyme purification were conducted on blood taken from 4 volunteers, each with blood types A, B, AB, and O. Six 1.5-mL-microtube tubes that had been weighed were prepared. Each tube was coded 1-6, tube 1 (negative control), tube 2 (positive control), tube 3 (blood type A), tube 4 (blood type B), tube 5 (blood type AB), and tube 6 (blood type O). The percentage of % blood clots was determined following the previously reported method and was conducted in duplicates (Prasad et al. 2006; Fuad et al. 2020; Hidayati et al. 2021). The effect of blood clot lysis was also observed microscopically by the Eustrek (removal) technique according to the May Grunwald-Giemsa mixed method (Geneser 1994). Results were observed with magnification under a Dino-Lite digital microscope and documented with a camera (Ethica et al. 2018). RESULTS AND DISCUSSION Bacterial Subculture and Morphology Characteristics Bacillus sp. HSFI-10 has a circular shape, edge (entire), size (3 mm), milk-white color, convex elevation, and smooth consistency (Figure 2A)— Bacillus sp. HSFI-10 has a short life in NA media (Figure 2B), a colony life of approximately 4 days of storage at cold temperatures, characterized by colony colors beginning to fade and cannot grow back on the new NA medium. Figure 2 Characteristics of Bacillus sp. HSFI-10 in Agar Nutrient (NA) (A) Colonies after 24-h incubation, (B) after stored for 4 days. C. on skim milk agar media with Lugol-staining (clear zone showing proteolytic activity) Thrombolytic protease of Bacillus cereus HSFI-10 – Ainutajriani et al. 151 Figure 3 Similar characteristics of Bacillus sp. HSFI-10 on Blood Agar Plate media. (A) compared to (B) B. cereus indicates β-hemolysis (Milojevic et al. 2019) Gram staining showed the rod shape was Gram-positive, lined, and had spores. B. cereus could produce a clear zone on the 7th day of cultivation on Skim Milk Agar (SMA) media with a diameter of 36 mm. SMA contains casein as a protease enzyme substrate. Bacterial growth in blood agar plate (BAP) media indicates a β- hemolysis pattern, characterized by an area of clear zone around the colony (Figure 3) (Bottone 2010; Lu et al. 2018). As seen in Figure 3, the macroscopic characteristics of bacteria on BAP media were evaluated by the shape, color, size, edge, and elevation of the colony as well as discoloration in the media (e. g. hemolysis in the medium for blood) (Pitt et al. 2012). The result aligned with the Mogrovejo et al. (2020) study reporting that Bacillus cereus can disintegrate red blood cells (forming a β-hemolysis pattern on BAP). B. cereus HSFI-10 morphology and properties reported in this study following the results reported by Hidayati et al. (2021). The proteolytic ability test aims to determine bacteria that have the potential to produce proteases, characterized by the formation of a clear zone around the bacterial colony (Assaf et al. 2020). The clear zone produced by proteolytic bacteria occurs due to protease activity which breaks the peptide bonds of casein in skim milk medium by breaking the CO-NH peptide bond with the entry of water into the molecule, thereby releasing amino acids (Baehaki et al. 2011; Ethica et al. 2018; Artha et al. 2019). Bacterial Molecular Identification DNA extract of Bacillus sp. HSFI-10 has a concentration of 232.6 ng/μl and a purity of 1.82. The purity of extracted DNA was high if the absorbance ratio (λ260/λ280) was 1.83. If the absorbance ratio is less than 1.8, proteins still contaminate DNA. If greater than 1.8, the DNA is contaminated with RNA (Gupta 2019). The genomic 16S rRNA gene cloning technique obtained the full-length 16S rRNA gene sequence (Green & Sambrook 2021). The cloning process was done by ligating, transforming, and isolating recombinant DNA from transformants (Green & Sambrook 2021). Recombinant DNA was then transformed in Escherichia coli Zymo 5α bacterial cells and grown on a solid medium. The success of cloning could be seen through white colonies (clones carrying recombinant DNA). The white colony indicates the LacZ region on the plasmid is inactive because it has been inserted by the inserted gene so that the galactose present in the media cannot be hydrolyzed by the -galactosidase enzyme which, if active, will form blue colonies (Sambrook et al. 1989; Darmawati 2015)— results of the 16S rRNA gene cloning of Bacillus sp. HSFI-10 is displayed in Figure 4, with an amplicon size of 1517 bp. BIOTROPIA Vol. 30 No. 2, 2023 152 Figure 4 The complete sequence of the 16S rRNA Bacillus sp. HSFI-10 obtained from cloning and sequencing 16S rRNA gene The consensus was made on the forward and reversed 16S rDNA sequences using the Bio Edit program (Hall 2004). The 16S rRNA gene sequence data saved in FASTA format were then analyzed and matched with the data available in the Gene Bank Basic Local Alignment Search Tool (BLAST). The results of BLAST analysis of Bacillus sp. HSFI-10 16S rRNA gene fragments showed a homology level of 99.80% with Bacillus cereus strain ST06 (Acc. No.: MH475925.1). Optimization of Crude Production Time for Protease Enzymes from Bacterial Culture Crude enzyme production was optimized with various incubation times of 24, 48, and 72 hours. The results of this study indicate that Bacillus cereus HSFI-10 can produce the most optimum crude enzyme at 48 hours with an enzyme activity of 222.52 U/mL. It has been reported that protease production was proportional to bacterial growth. In the early stages of the growth curve, bacteria produced few proteases and optimum production in the stationary phase (Ahmadpour and Yakhchali 2017; Pagarra et al. 2020; Suleiman et al. 2020). Partial Purification of Protease and Specific Activity Assay The protease activity of B. cereus HSFI-10 was calculated using the equation from Hidayati et al. (2021): Y = 0.0019 X + 0.0092. Y = absorbance, while X = enzyme activity (U/mL). Enzyme activity was expressed as the number of tyrosine amino acids released by the casein substrate per unit of time under test conditions (Zainuddin et al. 2020). Results of specific activity assay on crude and dialysate protease of B. cereus HSFI-10 are shown in Table 1. Table 1 Absorbance and specific activity data of crude and dialysate protease of B. cereus HSFI-10 Protease extract Absorbance at λ=600 nm at incubation time (h)*: Enzyme activity (U/mL) at incubation time (h)*: 24 48 72 24 48 72 Crude 0,264 0,433 0,346 134,105 222,526 146,000 Dialysate 0,843 438,842 *All tests were conducted in duplicate Thrombolytic protease of Bacillus cereus HSFI-10 – Ainutajriani et al. 153 Based on data in Table 3, it could be inferred that ammonium sulfate precipitation followed by dialysis could increase the enzyme-specific activity by almost doubling from 222.526 U/m to 438.842 U/mL. These results are to the research of Mothe and Sultanpuram (2016), reporting that enzymes purified by dialysis had high activity compared to crude enzymes, where the level of enzyme purity could reach 2.23 times higher. Clot Lysis Activity Assay on Crude and Partially Purified Protease Crude and dialysate enzyme B. cereus HSFI-10 could lyse ABO blood clot better than Nattokinase as control (Figure 5). A higher percentage of clot lysis characterized it. The % blood clot lysis of each sample is shown in Table 2. Nattokinase is a serine protease enzyme with fibrinolytic and antithrombotic activity. This ability can be used for cardiovascular treatment. Several studies have stated that Nattokinase can thin the blood and dissolve blood clots in experimental animals and humans (Gallelli et al. 2021; Chen et al. 2022). This study shows that crude enzymes and dialysate can lyse blood clots better than Nattokinase (Figure 5). Hence, the protease enzyme from B. cereus HSFI-10 bacteria can be used as a substitute for Nattokinase. As seen in Table 2, part of the limitation of this preliminary study is that the thrombolysis ability test was conducted only in duplicates with the objective of screening. One of the reasons is that the gravimetry test, first described by Prasad et al. 2006, is semi-quantitative. The qualitative part of the assessment lies in whether the lyse blood is still present before weighing the step of the method. Thus, the more accurate thrombolysis test should be confirmed with the in vivo one involving precise crude and dialysate bacterial protease dosage followed by statistical analysis (Dewi et al. 2022). The effect of clot lysis on O blood cells before and after adding crude and dialysate enzymes was also observed under a microscope with 400× magnification, with results displayed in Figure 6. Figure 5 Crude protease enzyme thrombolysis test and dialysate on blood type O Table 2 Results of the Thrombolysis Ability Test for crude protease enzymes and dialysates in ABO blood type Blood Type Initial clot weight (g)* Final clot weight (g)* % Clot lysis* NK Crude Dialysate NK Crude Dialysate NK Crude Dialysate A 0.358 0.358 0.299 0.141 0.111 0.055 60.614 68.994 81.605 B 0.333 0.334 0.282 0.133 0.101 0.051 60.060 69.760 81.914 AB 0.290 0.290 0.276 0.116 0.088 0.052 60.000 69.655 81.159 O 0.369 0.368 0.349 0.145 0.113 0.061 60.704 69.293 82.521 *All measurement was conducted in duplicates NK: Nattokinase BIOTROPIA Vol. 30 No. 2, 2023 154 Figure 6 The observation results on protease clot lysis activity on O blood cells using a Dino-Lite Digital Microscope. (A) negative control, (B) positive control, (C) crude enzyme, (D) dialysate enzyme As seen in Figure 6, platelets undergoing aggregation and erythrocytes experience changes in shape and overlap (Figure 6A), indicating the occurrence of blood clots. While Figures 6B, C, and D show that platelets did not undergo aggregation, erythrocytes did not change in shape and are evenly distributed. The platelet condition was due to favorable control treatment, crude, and dialysate, which can lyse blood clots. These results align with the research of Fuad et al., 2020, where crude protease enzymes produced by Staphylococcus hominis HSFT-2, S. saprophyticus HSFT-11, and Bacillus aryabhattai HSFT-5 caused erythrocytes not to create, spread evenly and platelets did not aggregate. Our results showed that partial purification by ammonium sulfate precipitation and dialysis on produced bacterial crude protease is very beneficial. The partial purification could increase bacterial protease-specific activity from 222.52 U/mL to 438.84 U/mL. It could also increase clot lysis activity based on the gravimetry method on blood in the ABO system from 68.99% - 69.76% (crude protease) to 81.16% (crude 82.52% (dialysate protease). The next step will be enzyme purification techniques using various chromatography-based methods to maximize protease's specific and clot lysis activity from B. cereus HSFI-10 (Westphal & van Berkel 2021). In this study, bacterial proteases had a similar effect on the lysis of ABO blood clots regardless of their different types of agglutination. Despite the previously reported data that blood type did affect the formation of blood clots, it has been known that individuals with non-O blood type have been shown to have a higher risk of thrombus formation than individuals with blood type O. This was associated with levels of coagulation factors, especially von Willebrand factor (vWF), vWF levels 30% higher in individuals with non-O blood type compared with blood group O (Holle et al. 2020; Mohamed et al. 2020; Separham et al. 2020; Pendu et al. 2021). CONCLUSION The optimum production of crude enzyme of thrombolytic protease-producing bacterium, B. cereus HSFI-10, resulting in the highest specific activity, was at 48-h incubation. Partial purification of bacterial protease increased both its specific and thrombolytic activities in human blood of the ABO group system with only 1% activity variability between A, B, AB, and O blood types. ACKNOWLEDGMENTS The authors would like to express their sincere gratitude to the Center for Research and Community Service (LPPM) Universitas Muhammadiyah Semarang for financially supporting this research through an internal grant in 2021. 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