Characteristics of Thermo-stable Serine Peptidase Vpr from Endophytic Bacillus cereus strain InaCC-B1657

Characteristics of Thermo-stable Serine Peptidase Vpr from Endophytic Bacillus cereus strain InaCC-B1657 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Characteristics of Thermo-stable Serine Peptidase Vpr from Endophytic Bacillus cereus strain InaCC-B1657 Aerma Hastuty, Wibowo Mangunwardoyo, Maggy Thenawijaya Suhartono, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4567703/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Bacterial endophytes are a promising source of novel fibrinolytic enzymes with unique metabolic pathways and desirable characteristics that may not be present from conventionally explored sources. This study aimed to characterize fibrinolytic enzymes from selected endophytic bacteria isolated from papaya ( Carica papaya L.) leaves and the genes encoding the enzymes. A strain BFP1 (InaCC-B1657) that showed the highest fibrinolytic activity was identified as Bacillus cereus based on the phylogenetic analysis of the 16S rRNA sequence. The enzyme exhibited optimum activity at 50°C and pH 7.0, and remained stable until 80°C and pH 6–10 for 24 h. The assay of metal ions and inhibitors on the fibrinolytic enzyme activity found that adding Cu 2+ stimulated, while Fe 2+ reduced the activity. PMSF and TPCK inhibited the enzyme activity, while adding EDTA and EGTA increased the activity. These suggest that the fibrinolytic enzymes belong to the serine protease group. Of the 21 proteases/peptidases determined from the 5,257,484 base pairs (bp) genome, minor extracellular protease Vpr and S8 family peptidase genes were found related to the fibrinolytic enzyme activity. The Vpr gene has a molecular weight of 98.5 kDa. The subtilase domain (peptidase S8 family) and the catalytic triad subtilase active sites (Asp204, His237, and Ser531) were detected. A prediction of physicochemical characteristics of the Vpr gene showed that the enzyme is hydrophilic and exhibited alkali-halo stability and thermo-stability over a broad range of temperatures. bacterium blood clot Carica papaya In-silico protease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Fibrinolytic enzymes act as thrombolytic agents by activating plasminogen to form plasmin to degrade fibrin, followed by breaking down thrombin. The accumulation of fibrin in the blood can cause myocardial infarction and other cardiovascular diseases (CVDs) (Hwang et al. 2007). Currently, the trend of heart and blood vessel disease is not only suffered by the elderly population but also affects many young people 75% of deaths are caused by heart and blood vessel disease. Before 2022, data from the World Health Organization (WHO) showed that about 17.9 million people die yearly due to CVDs. While advancements in pharmacological therapies for CVDs have been substantial, discovering drugs specifically to their target within the cardiovascular system remains a challenge. Searching for novel and specific metabolites or biologically active compounds for CVDs therapy is necessary to overcome this hurdle. Hence, over the past few decades, exploring novel fibrinolytic enzymes has become a hotspot for researchers in CVDs therapy. A promising application of bacterial-derived fibrinolytic enzymes prevents and treats vascular occlusion due to their advantages in cost-benefit ratio and large-scale production. Several genera of bacteria are known to have the potential to produce fibrinolytic enzymes with specific characteristics, such as Streptococcus (Buiting et al. 1990), Aeromonas (Cho et al. 2011), Lactic Acid Bacteria ( Lactococcus , Vagococcus , Weisella ) (Thokchom and Joshi 2014), Paenibacillus (Vijayaraghavan and Prakash 2014; Lu et al. 2010), Pseudoalteromonas (Vijayaraghavan et al. 2015), Serratia (Taneja et al. 2017), Streptomyces (Verma et al. 2018), Stenotrophomonas (Taneja et al. 2019), Bacillus (Leite et al. 2022), and Acinetobacter (Umay et al. 2023). These bacteria are typically found in soil, fermented foods, and as endophytes in various plant species. Among these resources, bacterial endophytes have the potential to produce fibrinolytic enzymes with specialized metabolic pathways and desirable properties that may not be present in commonly explored sources. These enzymes may possess unique characteristics, such as high fibrinolytic activity or stability under specific conditions, with unique characteristics. Research on fibrinolytic enzymes derived from bacteria, especially Bacillus , has been interesting for several decades (Bode et al. 1996; Leite et al. 2022). Several species of Bacillus , such as B. licheniformis (Buiting et al. 1990), B. cereus (Ghosh et al. 2009), B. subtilis (Choi et al. 2010), B. amyloliquefaciens (Heo et al. 2013), B. vallismortis (Cheng et al. 2015), B. circulans (Yogesh and Halami 2015), B. tequilensis (Xin et al. 2018), B. velezensis (Yang et al. 2020), and B. flexus (Al Farraj et al. 2020) have been reported producing fibrinolytic enzyme with high fibrin specificity. The fibrinolytic enzymes from Bacillus also have the potential to be developed as alternatives to indirect mechanisms of tissue plasminogen activator (tPA), streptokinase, and urokinase (Weng et al. 2017; Chen et al. 2018) and the direct mechanisms of thrombus lysis (plasmin use and nattokinase) (Ali and Bavisetty 2020). This is because t-PA, streptokinase, urokinase, plasmin use, and nattokinase have adverse side effects, such as allergic reactions and fatal complications that cause intracerebral bleeding in patients (Frias et al. 2021). In addition, these thrombolytic agents are also high-costs and low-specificity for fibrin. Therefore, the discovery of more effective fibrinolytic enzymes that do not cause side effects and are low-cost has become increasingly necessary in the last few decades, along with the increase in CVDs cases. In this study, screening, isolation, and optimization of fibrinolytic enzymes from bacterial endophytes isolated from papaya leaves ( C. papaya ) were carried out to discover fibrinolytic enzymes with specific characteristics. The strain showing fibrinolytic activity was identified using molecular phylogenetic analysis based on the 16S rRNA sequence. Materials and Methods Bacterial Endophyte Isolation Mature and healthy leaves of C. papaya were collected from the Cibinong Science and Technology Campus of the National Research and Innovation Agency of Indonesia (BRIN), West Java (Latitude: approximately 6° 28’ 54.01" S; Longitude: approximately 106° 51’ 15.01" E; Elevation: 130 m.). Isolation of bacteria was conducted on a Nutrient Agar (NA) medium using the surface sterilization method described by (Shukla and Wahla 2019). Proteolytic and Fibrinolytic Activity Screening Screening of proteolytic activity was carried out using the agar diffusion method on Skim Milk Agar (SMA), and the fibrinolytic screening on fibrin plate agar (FPA) (Taneja et al. 2017). Fibrin and protein hydrolysis index (FHI and PHI) values were calculated as follow: $$\text{H}\text{y}\text{d}\text{r}\text{o}\text{l}\text{y}\text{s}\text{i}\text{s} \text{I}\text{n}\text{d}\text{e}\text{x}= \frac{\text{C}\text{l}\text{e}\text{a}\text{r} \text{Z}\text{o}\text{n}\text{e} \text{D}\text{i}\text{a}\text{m}\text{e}\text{t}\text{e}\text{r}}{\text{C}\text{o}\text{l}\text{o}\text{n}\text{y} \text{o}\text{f} \text{W}\text{e}\text{l}\text{l} \text{D}\text{i}\text{a}\text{m}\text{e}\text{t}\text{e}\text{r}}$$ The highest positive protease and fibrinolytic activity isolates were deposited at the Indonesian Culture Collection (InaCC). Bacterial Endophyte Identification A 48-h bacterial colony on NA medium was used for the DNA extraction using the Geneaid Presto™ Mini gDNA Bacteria Kit. The primer pairs used in the amplification of the 16S rRNA gene region were 27F (Ludwig et al. 1993), 1492R (Wilson et al. 1990), 357F (Kim et al. 2011), and 907R (Muyzer et al. 1995). PCR amplification was carried out using the following conditions: pre-denaturation at 94°C for 2 min, followed by 30 cycles (94°C for 2 min of denaturation, 55°C for 1 min of annealing, 72°C for 1 min of extension, and final extension at 72°C for 10 min). The quality of the PCR product was determined by electrophoresis using 1.5% agarose gel. The PCR products were sent to FirstBASE (Malaysia) for sequencing. A newly nucleotide sequences were edited in ChromasPro v2.6.6 and aligned with the homologous sequences obtained from the NCBI GenBank through BLASTN program. Multiple sequence alignment was conducted with MEGA v11 software (Tamura et al. 2021). The Bayesian phylogeny analysis was reconstructed in BEAST v2.7.6 using default parameters (Douglas et al. 2022). The chain length was set to 1,000,000, the tree was sampled every 1,000 steps, the trace log was set to 100, and the screening was adjusted to 1,000. The results were analyzed with Tracer v1.7 (Rambaut et al. 2018). The burn-in percentage was set to 10, the posterior probability (PP) was limited to 0.75 in TreeAnnotator v1.10, and the PP support for the specified branches was displayed in the DensiTree v2 (Bouckaert and Heled 2014). Fibrinolytic Activity Assay A total of 2 mL of bacterial inoculant (OD value 1.0) was added to 100 mL of fermentation medium (peptone 0.5 g, dextrin 2 g, yeast extract 0.15 g, KH 2 PO 4 0.4 g, Na 2 HPO 4 0.04 g, CaCO 3 0.3 g, distilled water 100 mL), then incubated in a shaker incubator for 48 h, 37°C, at 120 rpm. The fermentation product was centrifuged at 10,000 rpm and 4°C, for 10 min. The centrifuged supernatant was used as a crude enzyme. A total of 0.4 mL of 0.5% (w/v) fibrinogen solution was added to 1.4 mL of 50 mM Tris-HCl buffer, pH 7.8, then incubated at 37°C for 5 min. A 0.1 mL of thrombin (20 U/mL) was further added and incubated at 37°C for 10 min, then 0.1 mL of enzyme solution was added and incubated again at 37°C for 60 min. Inactivation of the enzyme was conducted by adding 1 mL of 0.2 M TCA (trichloroacetic acid) and centrifuged at 10,000 rpm for 5 min at 4°C, then the fibrinolytic enzyme activity was measured using a spectrophotometer at 275 nm. The fibrinolytic activity was calculated as follows (Cupp-Enyard 2008): where: A: Total volume (mL) of assay B: Time of assay (min) as per Unit definition C: Volume of enzyme (mL) of enzyme used D: Volume (mL) used in colorimetric determination Crude Fibrinolytic Enzyme Production Production of fibrinolytic enzymes was carried out using the following composition: peptone 5 g, dextrin 20 g, yeast extract 1.5 g, KH 2 PO 4 4 g, Na 2 HPO 4 0.4 g, CaCO 3 3 g, and 1000 mL of distilled water. A 2% bacterial inoculum with an OD (optical density) value of 1.0 was added into 50 mL of medium, then incubated at 30°C for 48 h. After the incubation, the inoculum in the 50 mL medium was added into 2 L of production medium and incubated at 30°C, 150 rpm for 96 h. Bacterial inoculum was separated from the enzyme by centrifugation at 13,000 rpm, 4°C, and 10 min. The final supernatant was filtered using a cellulose acetate membrane with 0.22 µm pores and stored at 4°C for purification. Partial Purification and SDS-PAGE Analysis The supernatant was first precipitated using 70% of (NH 4 ) 2 SO 4 (ammonium sulphate) at 4°C, then left overnight in a slow stirrer. It was then centrifuged at 13,000 rpm, 10 min, and at 4°C. The Precipitate was further dissolved using phosphate buffer (50 mM, pH 7.0), and continued with dialysis using a 10 kDa cut-off cellulose membrane. The resulting enzyme was diluted 100 times. A total of 5 mL of dialysate was applied in a Hi-Trap Q FF ion exchange column connected to an FPLC system - AKTA Pure, equilibrated using phosphate buffer (50 mM, pH 7.0). A protein was eluted using 1 M NaCl solution in phosphate buffer (50 mM, pH 7.0) at a 2 mL/min flow rate. The protein content was measured at 595 nm, and the fibrinolytic enzyme activity at 275 nm. Fractions with activity were concentrated using a centric Amicon® Ultra Centrifugal Filter 10 kDa MWCO. Samples were stored at -20°C. The molecular weight and homogeneity of the pure enzyme were determined using SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) (Gallagher and Wiley 2008). Effect of pH, Temperature, Metal Ions, and Inhibitors An assay on the effect of pH and temperature on the fibrinolytic enzyme activity refers to the method published by Lee et al. (2005). A total of 0.1 mL of enzyme solution was added and incubated for 30 minutes with temperature variations of 30, 40, 50, 60, 70, and 80°C. The effects of metal ions and inhibitors on the enzyme activity were conducted using the method described by Liu et al. (2017). Several monovalent and divalent metal ions such as CaCl 2 , CoCl 2 , CuSO 4 , FeCl 2 , HgCl 2 , KCl, MgSO 4 , MnSO 4 , and ZnSO 4 were used in this study. In addition, ethylene diamine tetra-acetic acid (EDTA), ethylene glycol-bis-(β-aminoethyl ether)-N, N, N′, N′-tetra-acetic acid (EGTA), phenyl methyl sulfonyl fluoride (PMSF), and N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) [25] were used in the inhibition assay. Whole Genome Sequencing Genomic DNA was extracted from the 48-h isolates using the Quick-DNA MagBead Kit (Zymo Research). DNA quality and concentrations were estimated by NanoDrop Spectrophotometer and Qubit Fluorometer. Amplicons were prepared for nanopore sequencing using the ONT Native Barcoding Expansion Kits. Library preparations were conducted using Kits from Oxford Nanopore Technology. The libraries were multiplexed on FLO-MIN106 flow cells and run on the GridION X5. GridION sequencing was operated by MinKNOW V23.04.5. Base calling was performed using Guppy V6.5.7 with high accuration mode (Wick et al. 2019). The quality of reads was visualized using NanoPlot (De Coster et al. 2018). Filtering was performed by Filtlong and De novo Assembly was performed using Flye V2.8.3. Mapping and Genome polishing were done four times with Minimap2 (Li 2018), Racon (v.1.5.0), and three times polishing with Medaka V1.7.2. The closely related species of the assembled sequence and genome completeness were determined using dfast-qc V0.4.2 and BUSCO V5.4.4 (Simão et al. 2015). The quality of the assembled sequence was determined using QUAST V5.0.2 (Gurevich et al. 2013). Genome visualization was conducted using Circos software (Krzywinski et al. 2019). Identification of Gene Encoding Fibrinolytic, Prediction of Functional Sites, Physicochemical Properties, and Subcellular Localization Genome annotation and the full-length gene encoding fibrinolytic identification were performed using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) service system ( https://www.bv-brc.org/ ). Genomic mapping of fibrinolytic proteins was performed using Proksee (CG-View) software ( https://cgview.ca/ ). Homologous sequences of the amino acids fibrinolytic gene protein were obtained from the BLASTP program. In addition, a similarity searching of the fibrinolytic gene protein to the type amino acids sequence was also conducted using the BLAST tool for sequence similarity searching of the Universal Protein Resource (UniProt) ( https://www.uniprot.org/blast ). A prediction about the protein family, catalytic domain, and active sites related to the gene responsible for producing the fibrinolytic enzyme was conducted using ScanProsite ( https://prosite.expasy.org/scanprosite/ ) (Sigrist et al. 2016). In addition, the physical and chemical properties of the gene-encoded fibrinolytic enzyme were evaluated using the ProtParam assessment tool available on the ExPASy server ( http://web.expasy.org/protparam/ ). The subcellular localization of the gene-encoded fibrinolytic enzyme was determined using PSORTb v.3.0 ( https://www.psort.org/psortb/ ) (Yu et al. 2010). In-Silico Structural Modeling The protein structure of the minor extracellular protease Vpr gene in this study was predicted using two methods, including SWISS-MODEL ( https://swissmodel.expasy.org/ ) (Waterhouse et al. 2018) and I-TASSER ( https://zhanggroup.org/I-TASSER/ ) (Yang et al. 2014; Yang and Zhang 2015). Both methods use homology modeling techniques to create a protein structure model based on the sequence of amino acid residues. The SWISS-MODEL automatically builds the predicted protein model by searching for protein model templates using BLAST (Camacho et al. 2009), HHblits (Steinegger et al. 2019), and AlphaFold DB (Váradi et al. 2022). The best template is chosen based on sequence coverage, sequence similarity, and GMQE (Global Model Quality Estimate) score. This template is then used to predict the protein model. In addition, I-TASSER suggests the protein model result along with the predicted secondary structure, predicted solvent accessibility, predicted normalized B-factor, C-score, TM-score, and other parameters. The Ramachandran Plot (RC Plot) was utilized to validate the protein structures. GenBank Submission The 16S rRNA and alkaline protease gene sequences were submitted to GenBank under the accession numbers LC804381 and PP530334 (protein_id="WXU37559"), respectively. The whole genome data was submitted to NCBI GenBank (Accession Number: JBCEZT000000000) with the BioProject ID PRJNA1090300. Results Screening of Proteolytic and Fibrinolytic Activity Out of 25 different endophytic bacterial strains tested, bacterial endophyte strain BFP1 (Culture collection accession number: InaCC-B1657) exhibited a high fibrin hydrolysis index (FHI) (fibrinolytic activity) of 2.60 and a protein hydrolysis index (PHI) (proteolytic activity) of 1.73, suggesting its ability to break down both fibrin and protein. This was demonstrated by the clear zones around the colony of the bacterial strain (Fig. 1a-d). Identification of Bacterial Endophyte In the phylogenetic analysis of strain InaCC-B1657, a total of 19 homologous Bacillus sequences were included from the BLAST results. Bacillus megaterium strain IAM 13418 (GenBank Accession number: KJ569088.1) was selected as an outgroup (Fig. 1d). The Bayesian inference analysis showed that all homologous B. cereus group (s.lat.) sequences nested in the same clade with 100% PP. Sequence of strain InaCC-B1657 was also nested in the same clade with B. cereus strain ATCC 14579 (NR074540.1) to form a monophyletic clade with 88% PP. This indicates that strain InaCC-B1657 belongs to B. cereus s.lat. The finding of this study represents the first documentation of B. cereus s.lat., an endophytic bacterium isolated from C. papaya , as a potential producer of fibrinolytic enzymes from Indonesia. Crude Fibrinolytic Enzyme Activity and Partial Purification Based on initial observations on the crude enzyme of fibrinolytic activity, the B. cereus strain InaCC-B1657 exhibited the highest activity, reaching 36.61 U/mL (Fig. 2a) with a specific activity of 0.807 U/µg protein at 24 h (Fig. 2b). Enzyme purification was further performed on crude enzyme obtained from B. cereus strain InaCC-B1657. The process involved enzyme protein precipitation using (NH 4 ) 2 SO 4 at varying concentrations, ranging from 40–100%. The study demonstrated that the highest enzyme activity was achieved at 70% (NH 4 ) 2 SO 4 saturation, yielding a fibrinolytic enzyme activity of 7.47 U/mL (Fig. 2c). In this study, only one peak of fibrinolytic activity was observed in the eluate of the primary fraction with fibrinolytic activity applied to the Hi-Trap Q FF column. The fractions 18 to 25 showed fibrinolytic activity, with the 20th fraction exhibiting the highest level (3.12 U/mL). This fraction had a protein content of 0.00133 mg/mL (Fig. 2d-e). This final stage of purification showed impressive results, with a purity level 2.89 times higher and an activity recovery of 8.52%, with a specific activity of 2,345.86 U/mg protein (Table 1 ). These results suggest that the B. cereus strain InaCC-B1657 is a promising candidate for producing fibrinolytic enzyme protein owing to its highly specific activity. Using SDS-PAGE, the purified fibrinolytic enzyme from B. cereus strain InaCC-B1657 was found to be 98.53096 kDa (Fig. 2f). This is in close accordance with the molecular weight of the amino acids calculated from the fibrinolytic gene (GenBank Accession Number: PP530334, 98.502 kDa) (Table 3 ). Table 1 Summary of purification results for fibrinolytic enzyme from B. cereus strain InaCC-B1657 Steps Total Protein (mg/mL) Total Activity (U/mL) Specific Activity (U/mg prot.) Recovery (%) Fold Purification (x fold) Crude extract 0.04519 36.61 810.13 100 1 70% Ammonium sulphate precipitation 0.0035 7.47 2,134.19 20.40 2.63 Hi-Trap Q FF 0.00133 3.12 2,345.86 8.52 2.89 Activity of Fibrinolytic Enzyme at Different pH and Temperature Enzymes have specific temperature and pH requirements for optimal function. This study found that the fibrinolytic enzyme was active at all temperatures tested (30–90°C), with its highest activity occurring at 50°C (Fig. 3a). The fibrinolytic enzyme also demonstrated optimal activity at pH 7.0 and remained stable within the pH range of 6.0–10.0 (Fig. 3b). These results suggest that the fibrinolytic enzyme produced is most active at a temperature of 50°C and a pH of 7.0, with activity measured at 28.68 U/mL (Fig. 3a) and 32.10 U/mL (Fig. 3b), respectively. Additionally, after being incubated for 24 h, the enzyme retained 89.6, 88, 85.6, and 75.3% of its relative activity at 60, 70, 80, and 90°C, respectively, of that measured at 50°C (Fig. 3a). Similarly, after being incubated for 24 h at different pH conditions, the enzyme retained 74.4% of its activity at pH 6, 90.7% of its activity at pH 8, 85% of its activity at pH 9, and 75.1% of its activity at pH 10 (Fig. 3b). Therefore, this study shows that the fibrinolytic enzyme produced by B. cereus strain InaCC-B1657 is thermostable and functions optimally under neutral to basic pH conditions. Effect of Ion Metals and Inhibitors This study found that adding Cu 2+ metal ion (10 mM) increased the activity of fibrinolytic enzymes to 53.11 U/mL, while the presence of Fe 2+ ion (10 mM) reduced the activity to 3.10 U/mL (Fig. 3c). Inhibitor analysis showed that PMSF (5 mM) and TPCK (5 mM) also inhibited the enzymes activity while adding EDTA (5 mM) and EGTA (5 mM) increased the activity (Fig. 3d). The addition of EDTA and EGTA increased the activity of enzymes to 30.03 U/mL (9.6-fold) and 32.67 U/mL (10.5-fold), respectively, from 3.12 U/mL. The Genome of Bacillus cereus strain InaCC-B1657 A current study identified a gene in the B. cereus strain InaCC-B1657 that produces a fibrinolytic enzyme called the Vpr gene (Fig. 4). The B. cereus strain InaCC-B1657 genome (GB Accession Number: JBCEZT000000000) comprises a circular chromosome containing 5,257,484 base pairs (bp). The N50 of the assembled genome was found to be 5,183,747 bp. The genome has an average G + C content of 35.33% and 5,364 genes (Table 2 ). Among these genes, 5,211 were predicted to be coding sequences (CDSs), consisting of 5,085 CDSs with protein and 126 CDSs without protein. Additionally, 107 tRNAs, 42 rRNAs, and five ncRNAs were detected. Through a BLASTp of the NCBI GenBank, it has been discovered that there is a close relationship between the current fibrinolytic-encode gene and S8 serine peptidase of B. cereus and B. thuringiensis isolates. The BLASTp results of the Vpr gene of B. cereus strain InaCC-B1657 (915 amino acids) showed a 100% similarity with S8 family serine peptidase of B. cereus (Accession number: WP_048538152.1) (max score: 1864, total score: 1864, query coverage: 100%). This result agrees with the current biochemical analysis, which has revealed a high level of inhibition with serine protease inhibitors (PMSF and TPCK), confirming that the Vpr gene of B. cereus strain InaCC-B1657 is a serine protease. A BLAST result from the UniProt protein sequence database showed 97.8% similarity to the minor extracellular protease Vpr gene of type species of B. cereus strain ATCC 14579, 93.1% to minor extracellular protease Vpr gene of the B. anthracis strain ATCC 14578, and 76.6% to the peptidase S8 gene of B. manliponensis strain JCM 15802. Table 2 Genome information of B. cereus strain InaCC-B1657 Feature Observation Size (total length) (bp) 5,257,484 G + C content (%) 35.33% Number of contigs 2 Total genes 5,364 tRNAs 107 rRNAs 42 ncRNAs 5 CDSs 5,211 CDSs (with protein) 5,085 CDSs (without protein) 126 Total pseudo genes 126 Hypothetical Protein 2,355 (N50 (bp) 5,183,747 Number of N’s per 100 kbp 0 Prediction of Functional Sites, Physicochemical Properties, and Subcellular Localization In Vpr gene of B. cereus InaCC-B1657, the Subtilase domain (peptidase S8) was predicted to be located at amino acid position 182–591 (compared to PROSITE entry PS51892). This prediction included the catalytic triad subtilase, which is composed of Subtilase_Asp204, Subtilase_His237, and Subtilase_Ser531, predicted with a score of 44.621. The physicochemical characterization of the B. cereus InaCC-B1657 Vpr gene with the sequences of five serine proteases from different Bacillus species is shown in Table 3 . The molecular weight ranged from 98.5–99.2 kDa, with the Vpr gene of B. cereus InaCC-B1657 weighting 98.5 kDa, which was consistent with the weight detected in vitro by SDS-PAGE. The pI scores, which determine the pH at which a molecule or surface carries no net electrical charge, ranged from 5.33 to 6.15, with the Vpr gene of B. cereus InaCC-B1657 scoring 6.15. The predicted instability index was < 40 (29.18–31.03), indicating that the protein was stable. The aliphatic index of the Vpr gene of B. cereus InaCC-B1657 was 85.05, indicating its enhanced thermo-stability over a broad range of temperatures. These findings agreed with the biochemical characterization, which showed that the fibrinolytic enzyme of B. cereus InaCC-B1657 was active at all temperatures tested (30–90°C), with its highest activity at 50°C. The number of negatively charged amino acids (Asp + Glu) was higher than the positively charged amino acids (Arg + Lys) for all sequences, with the Vpr gene of B. cereus InaCC-B1657 having a ratio of 95/87, indicating its alkalo-halo stability. Furthermore, the GRAVY (Grand Average of Hydropathy) index ranged from − 0.370 to -0.343, with the Vpr gene of B. cereus InaCC-B1657 having an index of -0.353, which suggests that it is a hydrophilic serine protease enzyme that exhibits strong interaction with water. A subcellular localization analysis showed that the Vpr gene of B. cereus InaCC-B1657 is situated beyond the confines of the cell. The enzyme is known to be responsible for the breakdown of fibrin in blood clots, and its localization outside the cell suggests that it can act on external substrates. The PSORTb score of 9.98 further supports the extracellular localization of the enzyme (Table 4 ). Table 3 Physicochemical properties of the Vpr gene from B. cereus strain InaCC-B1657 compared to similar serine proteases from Bacillus species obtained from NCBI GenBank database and UniProt Reference Number Organism N Amino Acids MW Asp + Glu /Arg + Lys Theoritical pI GRAVY Index Instability Index Aliphatic Index B. cereus strain InaCC-B1657 915 98502.20 95/87 6.15 -0.353 29.74 85.05 WP_048538152.1 B. cereus 915 98502.20 95/87 6.15 -0.353 29.74 85.05 Q818B5 B. cereus strain ATCC 14579 917 99047.83 97/88 6.11 -0.361 30.50 85.82 WP_086407785.1 Bacillus thuringiensis 915 98687.43 96/88 6.15 -0.358 30.07 85.26 A0A6H3AKS4 B. anthracis strain ATCC 14578 917 98751.42 98/87 5.86 -0.343 29.18 86.03 A0A073JYX1 B. manliponensis strain JCM 15802 915 99266.52 102/79 5.33 -0.370 31.03 83.77 Table 4 Subcellular localization of the gene-encoded fibrinolytic enzyme of B. cereus strain InaCC-B1657 using PSORTb Localization PSORTb Score Cytoplasmic 0.00 Cytoplasmic Membrane 0.00 Cell Wall 0.02 Extracellular 9.98 Final Prediction: Extracellular 9.98 In-Silico Structural Modeling A BLAST result from UniProtKB database shows protein with accession number A0A0D0PQG3.1 as the most homolog sequence. Therefore, this sequence was used as a template. Because the status of the A0A0D0PQG3.1 protein is obsolete in the database, we selected an identical amino acid sequence from the database (Accession number: A0A9X0JT16) as a template protein. According to SWISS-MODEL's evaluation, the A0A9X0JT16 was then chosen for modeling due to its superior sequence identity and GMQE ratings. Using the AlphaFold DB technique, the alignment revealed a 98.8% match with the template in a span of 917 residues. Furthermore, the model generated with the A0A9X0JT16 template showed a GMQE score of 0.89 in the SWISS-MODEL analysis, signifying a high-quality estimate within the 0 to 1 range. Based on the template protein on the SWISS-MODEL analysis, the active sites were predicted at Asp204, His237, and Ser531 (Fig. 5a). Furthermore, the predicted protein model demonstrates structural stability, with over 95% of its amino acid residues situated within the favoured region and over 99% within the allowed region of the Ramachandran plot, suggesting an accurate representation of the natural conformation of the protein. In the protein model prediction using I-TASSER analysis, the I-TASSER predicted model tends to have residues with normalized B-factor values below zero, indicating a relatively stable model. The 3D structural visualization of this model has a C-score of -2.38, which falls within the acceptable confidence range of (-5, 2), where higher values denote more reliable models. Similar to results from the SWISS-MODEL analysis, the I-TASSER analysis indicates that the model aligns with the serine-type peptidase classification and retains proteolytic activity (Fig. 5b). In addition, I-TASSER analysis has also forecasted binding sites allowing COFACTOR and COACH, identifying residues with a probability score equal to or more than 75%, notably His237 and Ser531. Discussion This study unveils the discovery of B. cereus strain InaCC-B1657, an endophytic bacterium found in C. papaya , as a promising source of fibrinolytic enzymes from Indonesia. Notably, previous research has reported on the production of these enzymes by several strains of B. cereus from various sources such as soil, blood, and cattle dung (Deepak et al. 2010; Bajaj et al. 2013; Majumdar et al. 2015; Narasimhan et al. 2015, 2018; Biji et al. 2016; D’Souza et al. 2020; Sharma et al. 2020). Bacillus cereus is a member of the B. cereus group, which includes B. anthracis , B. thuringiensis , and B. mycoides . These bacteria have been extensively studied for their pathogenic potential, particularly concerning foodborne illnesses and food safety. In the genome of B. cereus strain InaCC-B1657, there are several extracellular proteases with significant virulence factors found, including Immune inhibitor A (InhA) metalloprotease, M6 family metalloprotease immune inhibitor InhA1, and M6 family metalloprotease immune inhibitor InhA2 (Table 5 ). This raises concerns about the safety of B. cereus strain InaCC-B1657 as a fibrinolytic enzyme producer. For further development, comprehensive risk assessments, including toxicity studies, are essential to determine if B. cereus strain InaCC-B1657 can be considered GRAS (Generally Recognized as Safe). The GRAS status is necessary because it provides a solid foundation for utilizing these bacteria in producing fibrinolytic enzymes for various applications, including thrombosis treatment. Table 5 Genes encoding proteases and their predicted compartmentalization in B. cereus strain InaCC-B1657 ( https://www.psort.org/psortb/results.pl ) Gene Start Gene End Strand Protease Localization Score Subcellular Localization 86632 84731 - Cell division-associated, ATP-dependent zinc metalloprotease FtsH 9.99 Cytoplasmic Membrane 182557 183732 + Serine protease, DegP/HtrA, do-like (EC 3.4.21.-) - Unknown 271138 271812 + Uncharacterized membrane zinc metalloprotease YwhC 10.00 Cytoplasmic Membrane 598232 599902 + Extracellular neutral protease B 9.73 Extracellular 637997 638710 + CPBP family intramembrane metalloprotease 10.00 Cytoplasmic Membrane 756569 752346 - S8 family serine peptidase 9.73 Extracellular 757208 756603 - Cell envelope-bound metalloprotease, Camelysin (EC 3.4.24.-) - Unknown 1235777 1233030 - minor extracellular protease VpR 9.98 Extracellular 1941462 1942703 + Serine protease, DegP/HtrA, do-like (EC 3.4.21.-) - Unknown 2433589 2435976 + Immune inhibitor A metalloprotease 9.98 Extracellular 2540600 2542987 + Immune inhibitor A metalloprotease 9.73 Extracellular 3176660 3177853 + S8 family peptidase 9.98 Extracellular 3326332 3324821 - Putative neutral metalloprotease - Unknown 3488700 3487753 - Intracellular serine protease 9.67 Cytoplasmic 3961206 3960109 - Membrane metalloprotease 10.00 Cytoplasmic Membrane 4107404 4105014 - M6 family metalloprotease immune inhibitor InhA1 9.98 Extracellular 4250342 4249074 - Uncharacterized metalloprotease YhfN 10.00 Cytoplasmic Membrane 4663850 4661451 - M6 family metalloprotease immune inhibitor InhA2 (EC 3.4.24.-) 9.98 Extracellular 4691160 4690561 - Cell envelope-bound metalloprotease, Camelysin (EC 3.4.24.-) - Unknown 4695656 4691415 - S8 family serine peptidase 9.73 Extracellular 4983418 4982711 - Zinc metalloprotease 7.50 Cytoplasmic The biochemical characteristics of the current fibrinolytic enzyme from the B. cereus strain InaCC-B1657 are sufficient to support further development (Figs. 3a-b). Several studies on the fibrinolytic enzymes from B. cereus also reported that the optimum activity occurs not only at high-temperature conditions but also at neutral to alkaline pH conditions (Table 6 ). The thermostable nature could facilitate its integration into industrial processes that often involve high-temperature and alkaline pH conditions treatments in the production of bioactive peptides, which are valuable in the pharmaceutical and nutraceutical industries. In order to develop highly efficient fibrinolytic enzymes, several factors that can affect their performance must be evaluated. These include pH, temperature, medium composition, inhibiting and promoting factors. These elements can significantly impact the effectiveness of the enzyme activity and, in several cases, are specifically associated with the activity of the specific enzyme group. This study found that PMSF, TPCK, and ion metal Fe2 + inhibited the fibrinolytic enzyme produced by the B. cereus strain InaCC-B1657 (Fig. 3c-d). These suggest that the enzyme belongs to the serine protease group because the PMSF, TPCK, and metal ion Fe2 + were reported as serine metalloprotease or serine protease inhibitors (Hazare et al. 2024). Serine protease (EC 3.4.21) cleaves peptide bonds similarly to other proteases. However, the serine residue in the active site (serine as a nucleophile) can coordinate many other essential functions through protein hydrolysis (Patel 2017). Serine proteases perform various functions, including protein metabolism, digestion, and blood clotting (Grant et al. 2007). Understanding the inhibitory effects of different agents like PMSF, TPCK, and metal ions on these enzymes is crucial for developing strategies to overcome these limitations and develop an efficient enzyme at an industrial level. For instance, metal chelators and other inhibitors, in fact, can help modulate enzyme activity (Akbar and Sharma 2017; Zhao et al. 2019). In addition, several factors also need to be considered to tackle the inhibitors problem, such as understanding their biochemical properties and implementation of gene manipulation methods such as DNA shuffling (Yao et al. 2022) and similar protocols. Table 6 Optimum pH and temperature for fibrinolytic enzyme activity of B. cereus strain InaCC-B1657 and another B. cereus Sources pH Temp. (°C) Authors B. cereus strain InaCC-B1657 7.0 50 This study B. cereus NK1 9.0 37 Deepak et al., 2010 B. cereus NS2 9.0 40 Bajaj et al., 2013 B. cereus IND1 8.0 60 Vijayaraghavan & Vincent, 2014 B. cereus IND5 8.0 50 Biji et al., 2016 B. cereus S8 10.0 70 Lakshmi et al., 2018 B. cereus SRM-001 7.0 37 Narasimhan et al., 2018 B. cereus RSA1 8.0 50 Sharma et al., 2020 B. cereus S46 8.0 40 D’Souza et al., 2020 The genes associated with fibrinolytic activity in Bacillus species are diverse and may encode various fibrinolytic enzymes and proteases. The expression and regulation of proteases/peptidases genes may influence the production, maturation, and activity of fibrinolytic enzymes in various Bacillus species. In this study, the genome sequence of B. cereus strain InaCC-B1657 showed 21 proteases/peptidases divided into two groups based on cellular localization, and five proteases are unknown (Table 5 ). Coding sequences of seven extracellular proteases were also found in the genes encoding proteases, including extracellular neutral protease B, minor extracellular protease Vpr, Immune inhibitor A metalloprotease, S8 family peptidase, S8 family serine peptidase, M6 family metalloprotease immune inhibitor InhA1, and M6 family metalloprotease immune inhibitor InhA2. Among these genes, the Vpr and S8 family peptidases are related to the fibrinolytic enzyme activity produced by Bacillus species (Kho et al. 2005; Yao et al. 2020; Syahbanu et al. 2022; Zhou et al. 2022). Both genes belong to serine protease. Proteases are classified into four categories based on the specific catalytic mechanisms and the nature of the active site residues, including serine protease, aspartate protease, cysteine proteases, and metalloproteases (Rao et al. 1998). The Vpr gene belongs to the minor serine protease group and was first discovered in B. subtilis (Sloma et al. 1991; Huang et al. 2022) and exhibited fibrinolytic enzyme activity (Ghosh et al. 2009; Choi et al. 2010; Yao et al. 2020; Chen et al. 2020; Syahbanu et al. 2022). Ghosh et al. (2009) reported that the Vpr gene has been implicated as a processor protease in the Bacillus system and has been shown to process other secretory pro-proteins and pro-peptides, including its role as a fibrinolytic enzyme (Kho et al. 2005). Yao et al. (2020) also demonstrated that the co-expression of VprSJ4 and aprESJ4 increased the fibrinolytic activity by 117% compared to aprESJ4 single expression in B. subtilis WB600, indicating the potential involvement of Vpr in fibrinolysis. In addition, a specific catalytic triad of Asp/His/Ser of the Vpr gene of B. cereus strain InaCC-B1657 indicates its potential involvement in proteolytic processes, including fibrinolysis. The physicochemical properties of the Vpr gene of B. cereus strain InaCC-B1657 in Table 3 also showed the overall hydrophobicity or hydrophilicity of a protein or peptide sequence through the GRAVY index measures. A negative GRAVY score of the Vpr gene indicates hydrophilicity of the gene. In addition, the instability index score of 29.74 for the stability of a protein suggests that the protein is predicted to be relatively stable (Gamage et al. 2009), which can be advantageous for its functional properties and potential applications. This condition is also supported by the high aliphatic index score (85.05), which contributes to the structure stability and potential of a protein for functional activity. Therefore, a high aliphatic index value appears to have a beneficial effect on enhancing the thermostability of globular proteins (Ikai 1980). Furthermore, a higher ratio of Asp + Glu residues compared to Arg + Lys residues (95/87) (Table 3 ) exhibited increased stability, while the presence of Arg + Lys residues can impact the folding process and overall stability of the protein (Meuzelaar et al. 2016). A higher ratio of Asp + Glu residues may also influence the electrostatic interactions of protein, potentially impacting its binding to substrates and other molecules (Cheng et al. 2012). The predicted protein model of the Vpr gene of B. cereus strain InaCC-B1657 shows a stable conformation since its amino acid residues are placed in the favorable region of more than 95% and the allowed region of more than 99% in the Ramachandran plot. The more residues occupying the allowed region correspond to a more stable protein model closely resembling the original experimental structure. This relationship can be attributed to energetically favorable conformations, as indicated by the allowed region, which is more likely to represent native-like protein structures (Laskowski et al. 2013). The 3D visualization of the current predicted protein model by I-TASSER analysis (C-score of -2.38) is still within the recommended range (-5, 2), where the higher value signifies a model with high confidence. The gene ontology of the predicted model by I-TASSER analysis shows that it can be grouped as a serine-type peptidase and has proteolysis activity. This is similar to the result predicted by SWISS-MODEL analysis. The SWISS-MODEL and I-TASSER show conclusive results stating that the protein target was predicted as a serine-type peptidase of the S8 family peptidase. A serine-type peptidase is a group of proteases characterized by the presence of nucleophilic serine residues within the enzyme active site, which play a crucial role in initiating the catalytic activity by forming a bond with substrate protein (Antalis et al. 2010). In this study, the SWISS-MODEL identifies Asp204, His237, and Ser531 as pivotal contributors to catalytic activity, whereas I-TASSER highlights the significance of His237 and Ser531 in catalytic function. These residues comprise a catalytic triad of Asp-His-Ser, which is responsible for peptidase activity and constitutes a characteristic feature of serine-type peptidases. In addition, the secondary structure of Asp204, His237, and Ser531 predicted by the SWISS-MODEL was β-sheet pleated, α-helix, and α-helix, respectively. The secondary structure predicted by the I-TASSER appears to provide a more detailed description, wherein all three residues (Asp204, His237, and Ser531) exhibited coil structures. The predominance of α-helix structure within the catalytic triad area is in accordance with the Cryo-EM visualization results of the Vpr protein reported by Cheng et al. (2023), which indicates the catalytic region of Vpr protein is primarily characterized by α-helix structure. Conclusions This study determines the B. cereus strain InaCC-B1657, an endophyte from C. papaya leaves, as a potential candidate in fibrinolytic enzyme production. The optimal activity was found at 50°C and pH 7.0 and remained stable until 80°C and pH 6–10 for 24 h, indicating its thermo-stability and alkali-halo stability. Fe 2+ , PMSF, and TPCK inhibition to the fibrinolytic enzyme activity suggests that the fibrinolytic enzymes produced by B. cereus strain InaCC-B1657 belong to the serine protease group. The genome sequence of B. cereus strain InaCC-B1657 is 5,257,484 bp and comprises 21 proteases/peptidases, including the coding sequences of seven extracellular proteases. An in-silico study of the protein identifies a catalytic triad (Asp204, His237, and Ser531) as a pivotal contributor to catalytic activity. This triad is responsible for peptidase activity and constitutes a characteristic feature of serine-type peptidases. Declarations Acknowledgments This study was supported by a grant from the Hibah PUTI Doctoral Programme awarded to Prof. Dr. Wibowo Mangunwardoyo (No. NKB-3295/UN2.RST/HKP.05.00/2020), University of Indonesia. Indriawati and Fina Amreta Laksmi are thanked for laboratory assistance. Conflict of Interest The authors declares that there is no conflict of interest that are relevant to the content of this manuscript. This manuscript has not been published, and it is not under consideration for publication anywhere else. Authors Contribution Conceptualization, methodologies, research experiment, data analysis, writing the draft and final version, AH; Protein purification and genome analysis, NR; In-Silico study, PRF; Research supervision and writing review, WM, MTS, and IH. All authors have approved this manuscript. Data Availability Data will be made available on request. Informed Consent Informed consent has been obtained from all participants for both sample collection and data publication in the research study. References Akbar SM, Sharma HC (2017) Alkaline serine proteases from Helicoverpa armigera : potential candidates for industrial applications. 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Curr Microbiol 77:1610-1621. https://doi.org/10.1007/s00284-020-01977-6 Yao Z, Meng Y, Le HG, Lee SJ, Jeon HS, Yoo JY, Kim HJ, Kim JH (2020) Cloning of a Novel Vpr Gene Encoding a Minor Fibrinolytic Enzyme from Bacillus subtilis SJ4 and the Properties of Vpr. J Microbiol Biotechnol 30(11):1720-1728. https://doi.org/10.4014/jmb.2006.06014 Yao Z, Jeon HS, Yoo JY, Kang YJ, Kim MJ, Kim TJ, Kim JH (2022) DNA Shuffling of aprE Genes to Increase Fibrinolytic Activity and Thermostability. Journal of Microbiology and Biotechnology 32:800-807. https://doi.org/10.4014/jmb.2202.02017 Yogesh D, Halami PM (2015) A fibrin degrading serine metallo protease of Bacillus circulans with α-chain specificity. Food Biosci 11:72-78. https://doi.org/10.1016/2Fj.fbio.2015.04.007 Yu NY, Wagner JR, Laird MR, Melli G, Rey S, Lo R, Dao P, Sahinalp SC, Ester M, Foster LJ, Brinkman FS (2010) PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 26:1608-1615. https://doi.org/10.1093/bioinformatics/btq249 Zhao A, Li Y, Leng C, Wang P, Li Y (2019) Inhibitory Effect of Protease Inhibitors on Larval Midgut Protease Activities and the Performance of Plutella xylostella (Lepidoptera: Plutellidae). Frontiers in Physiology 9:1963. https://doi.org/10.3389/fphys.2018.01963 Zhou Y, Chen H, Yu B, Chen G, Liang Z (2022) Purification and characterization of a fibrinolytic enzyme from marine Bacillus velezensis z01 and assessment of its therapeutic efficacy in vivo. Microorganisms 10(5):843. https://doi.org/10.3390/microorganisms10050843 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 13 Jun, 2024 Submission checks completed at journal 13 Jun, 2024 First submitted to journal 12 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4567703","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":314138179,"identity":"aa33265d-ec26-4a38-8f8f-4b26225821f0","order_by":0,"name":"Aerma Hastuty","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYLCCBAMJOQMGxgYwh40hgQgtHyosjEnTwjjjTEXiBiRL8Svnb+99upm3TSJ9u/ThtgcMv2wY+NgJaJE4c9zsNlBL7s6+xHYDxr40BjaeBwRcdSONDaxlwxnGNgnGnsMMbBIEbJG//wysJd2AaC0GN9jYbs44I5EA1sLwgwgthmfS2G58qJAw3NkD1JLYkMZD0C9yx4+x3UgwqJM352F/JvHhj42cfDsBW1BBYhsDDynqQeAPqRpGwSgYBaNgJAAAatZA9PI/bzcAAAAASUVORK5CYII=","orcid":"","institution":"National Research and Innovation Agency (BRIN)","correspondingAuthor":true,"prefix":"","firstName":"Aerma","middleName":"","lastName":"Hastuty","suffix":""},{"id":314138180,"identity":"1aca8521-7fa7-4aa6-a099-6de934f84289","order_by":1,"name":"Wibowo Mangunwardoyo","email":"","orcid":"","institution":"Indonesian University","correspondingAuthor":false,"prefix":"","firstName":"Wibowo","middleName":"","lastName":"Mangunwardoyo","suffix":""},{"id":314138181,"identity":"e6478de7-b209-469c-baff-7c6d3dad697b","order_by":2,"name":"Maggy Thenawijaya Suhartono","email":"","orcid":"","institution":"Bogor Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Maggy","middleName":"Thenawijaya","lastName":"Suhartono","suffix":""},{"id":314138182,"identity":"cec0357f-9483-4c9f-a0cf-760b51c32e19","order_by":3,"name":"Nanik Rahmani","email":"","orcid":"","institution":"National Research and Innovation Agency (BRIN)","correspondingAuthor":false,"prefix":"","firstName":"Nanik","middleName":"","lastName":"Rahmani","suffix":""},{"id":314138183,"identity":"38976fe5-0ca4-4596-b02a-987748cb5c44","order_by":4,"name":"Pamungkas Rizky Ferdian","email":"","orcid":"","institution":"National Research and Innovation Agency (BRIN)","correspondingAuthor":false,"prefix":"","firstName":"Pamungkas","middleName":"Rizky","lastName":"Ferdian","suffix":""},{"id":314138184,"identity":"cb770990-8079-4d4f-b9e0-3d04b1c65366","order_by":5,"name":"Iman Hidayat","email":"","orcid":"","institution":"National Research and Innovation Agency (BRIN)","correspondingAuthor":false,"prefix":"","firstName":"Iman","middleName":"","lastName":"Hidayat","suffix":""}],"badges":[],"createdAt":"2024-06-12 05:42:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4567703/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4567703/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59405044,"identity":"74d8e998-b930-4b95-8f3e-4f03efe7f8ce","added_by":"auto","created_at":"2024-07-01 11:15:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7295524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Colony of the endophytic bacterium strain InaCC-B1657 on NA medium; \u003cstrong\u003eb\u003c/strong\u003e Qualitative screening of proteolytic activity on SMA medium; \u003cstrong\u003ec\u003c/strong\u003e Qualitative screening of fibrinolytic activity on FPA medium; \u003cstrong\u003ed\u003c/strong\u003e Bayesian phylogenetic tree of the 16S rRNA sequences. PP values are shown in front of the branch node. The scale bar represents the expected changes per site\u003c/p\u003e","description":"","filename":"Fig1ad.png","url":"https://assets-eu.researchsquare.com/files/rs-4567703/v1/9cdb56ba335a9beceacbe617.png"},{"id":59405048,"identity":"cd45d94d-424b-4433-9fd9-4782afbb1270","added_by":"auto","created_at":"2024-07-01 11:15:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1422345,"visible":true,"origin":"","legend":"\u003cp\u003eFibrinolytic enzyme of \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657; \u003cstrong\u003ea\u003c/strong\u003e Total enzyme activity; \u003cstrong\u003eb\u003c/strong\u003e Specific activity of the enzyme; \u003cstrong\u003ec \u003c/strong\u003eProtein precipitation using (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e; \u003cstrong\u003ed\u003c/strong\u003e Chromatogram profile of the enzyme; \u003cstrong\u003ee\u003c/strong\u003e Fractionation of the enzyme; \u003cstrong\u003ef\u003c/strong\u003e SDS-PAGE analysis of purified enzyme after gel filtration (molecular weight is indicated by arrow)\u003c/p\u003e","description":"","filename":"Fig2af.png","url":"https://assets-eu.researchsquare.com/files/rs-4567703/v1/17c6b33f300970680968bb83.png"},{"id":59405047,"identity":"001907c3-34b0-44e7-8a88-d971df2e59b8","added_by":"auto","created_at":"2024-07-01 11:15:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":799285,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of various factors on the fibrinolytic enzyme activity; \u003cstrong\u003ea \u003c/strong\u003eTemperature; \u003cstrong\u003eb\u003c/strong\u003e pH; \u003cstrong\u003ec\u003c/strong\u003e Metal ions; \u003cstrong\u003ed\u003c/strong\u003e Inhibitors\u003c/p\u003e","description":"","filename":"Fig3ad.png","url":"https://assets-eu.researchsquare.com/files/rs-4567703/v1/554086a14bb2e5ec127afdb0.png"},{"id":59405046,"identity":"46079f27-fb3f-4ef6-bc83-2aa2394fdc93","added_by":"auto","created_at":"2024-07-01 11:15:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3669230,"visible":true,"origin":"","legend":"\u003cp\u003eVisualization of the complete genome from the \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 using Proksee (https://proksee.ca) (Vpr gene is indicated by arrow)\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4567703/v1/8351499febca39a4ce73bd3f.png"},{"id":59405672,"identity":"88c9696f-e194-4183-a729-edfeb04629b0","added_by":"auto","created_at":"2024-07-01 11:23:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5308402,"visible":true,"origin":"","legend":"\u003cp\u003eThe 3D visualization of the predicted model protein; \u003cstrong\u003ea\u003c/strong\u003e by SWISS-MODEL; \u003cstrong\u003eb\u003c/strong\u003e by I-TASSER\u003c/p\u003e","description":"","filename":"Fig5ab.png","url":"https://assets-eu.researchsquare.com/files/rs-4567703/v1/edc3bc6df0b994d196c6c7cf.png"},{"id":59406446,"identity":"e3d564cb-9f1b-4790-b669-1345cc06b921","added_by":"auto","created_at":"2024-07-01 11:32:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29880277,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4567703/v1/9d57a07b-80f5-4f1e-a73a-373e3c1704f5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characteristics of Thermo-stable Serine Peptidase Vpr from Endophytic Bacillus cereus strain InaCC-B1657","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFibrinolytic enzymes act as thrombolytic agents by activating plasminogen to form plasmin to degrade fibrin, followed by breaking down thrombin. The accumulation of fibrin in the blood can cause myocardial infarction and other cardiovascular diseases (CVDs) (Hwang et al. 2007). Currently, the trend of heart and blood vessel disease is not only suffered by the elderly population but also affects many young people 75% of deaths are caused by heart and blood vessel disease. Before 2022, data from the World Health Organization (WHO) showed that about 17.9\u0026nbsp;million people die yearly due to CVDs. While advancements in pharmacological therapies for CVDs have been substantial, discovering drugs specifically to their target within the cardiovascular system remains a challenge. Searching for novel and specific metabolites or biologically active compounds for CVDs therapy is necessary to overcome this hurdle.\u003c/p\u003e \u003cp\u003eHence, over the past few decades, exploring novel fibrinolytic enzymes has become a hotspot for researchers in CVDs therapy. A promising application of bacterial-derived fibrinolytic enzymes prevents and treats vascular occlusion due to their advantages in cost-benefit ratio and large-scale production. Several genera of bacteria are known to have the potential to produce fibrinolytic enzymes with specific characteristics, such as \u003cem\u003eStreptococcus\u003c/em\u003e (Buiting et al. 1990), \u003cem\u003eAeromonas\u003c/em\u003e (Cho et al. 2011), Lactic Acid Bacteria (\u003cem\u003eLactococcus\u003c/em\u003e, \u003cem\u003eVagococcus\u003c/em\u003e, \u003cem\u003eWeisella\u003c/em\u003e) (Thokchom and Joshi 2014), \u003cem\u003ePaenibacillus\u003c/em\u003e (Vijayaraghavan and Prakash 2014; Lu et al. 2010), \u003cem\u003ePseudoalteromonas\u003c/em\u003e (Vijayaraghavan et al. 2015), \u003cem\u003eSerratia\u003c/em\u003e (Taneja et al. 2017), \u003cem\u003eStreptomyces\u003c/em\u003e (Verma et al. 2018), \u003cem\u003eStenotrophomonas\u003c/em\u003e (Taneja et al. 2019), \u003cem\u003eBacillus\u003c/em\u003e (Leite et al. 2022), and \u003cem\u003eAcinetobacter\u003c/em\u003e (Umay et al. 2023). These bacteria are typically found in soil, fermented foods, and as endophytes in various plant species. Among these resources, bacterial endophytes have the potential to produce fibrinolytic enzymes with specialized metabolic pathways and desirable properties that may not be present in commonly explored sources. These enzymes may possess unique characteristics, such as high fibrinolytic activity or stability under specific conditions, with unique characteristics.\u003c/p\u003e \u003cp\u003eResearch on fibrinolytic enzymes derived from bacteria, especially \u003cem\u003eBacillus\u003c/em\u003e, has been interesting for several decades (Bode et al. 1996; Leite et al. 2022). Several species of \u003cem\u003eBacillus\u003c/em\u003e, such as \u003cem\u003eB. licheniformis\u003c/em\u003e (Buiting et al. 1990), \u003cem\u003eB. cereus\u003c/em\u003e (Ghosh et al. 2009), \u003cem\u003eB. subtilis\u003c/em\u003e (Choi et al. 2010), \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e (Heo et al. 2013), \u003cem\u003eB. vallismortis\u003c/em\u003e (Cheng et al. 2015), \u003cem\u003eB. circulans\u003c/em\u003e (Yogesh and Halami 2015), \u003cem\u003eB. tequilensis\u003c/em\u003e (Xin et al. 2018), \u003cem\u003eB. velezensis\u003c/em\u003e (Yang et al. 2020), and \u003cem\u003eB. flexus\u003c/em\u003e (Al Farraj et al. 2020) have been reported producing fibrinolytic enzyme with high fibrin specificity. The fibrinolytic enzymes from \u003cem\u003eBacillus\u003c/em\u003e also have the potential to be developed as alternatives to indirect mechanisms of tissue plasminogen activator (tPA), streptokinase, and urokinase (Weng et al. 2017; Chen et al. 2018) and the direct mechanisms of thrombus lysis (plasmin use and nattokinase) (Ali and Bavisetty 2020). This is because t-PA, streptokinase, urokinase, plasmin use, and nattokinase have adverse side effects, such as allergic reactions and fatal complications that cause intracerebral bleeding in patients (Frias et al. 2021). In addition, these thrombolytic agents are also high-costs and low-specificity for fibrin. Therefore, the discovery of more effective fibrinolytic enzymes that do not cause side effects and are low-cost has become increasingly necessary in the last few decades, along with the increase in CVDs cases. In this study, screening, isolation, and optimization of fibrinolytic enzymes from bacterial endophytes isolated from papaya leaves (\u003cem\u003eC. papaya\u003c/em\u003e) were carried out to discover fibrinolytic enzymes with specific characteristics. The strain showing fibrinolytic activity was identified using molecular phylogenetic analysis based on the 16S rRNA sequence.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eBacterial Endophyte Isolation\u003c/h2\u003e\n \u003cp\u003eMature and healthy leaves of \u003cem\u003eC. papaya\u003c/em\u003e were collected from the Cibinong Science and Technology Campus of the National Research and Innovation Agency of Indonesia (BRIN), West Java (Latitude: approximately 6\u0026deg; 28\u0026rsquo; 54.01\u0026quot; S; Longitude: approximately 106\u0026deg; 51\u0026rsquo; 15.01\u0026quot; E; Elevation: 130 m.). Isolation of bacteria was conducted on a Nutrient Agar (NA) medium using the surface sterilization method described by (Shukla and Wahla 2019).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eProteolytic and Fibrinolytic Activity Screening\u003c/h2\u003e\n \u003cp\u003eScreening of proteolytic activity was carried out using the agar diffusion method on Skim Milk Agar (SMA), and the fibrinolytic screening on fibrin plate agar (FPA) (Taneja et al. 2017). Fibrin and protein hydrolysis index (FHI and PHI) values were calculated as follow:\u003c/p\u003e\n \u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e$$\\text{H}\\text{y}\\text{d}\\text{r}\\text{o}\\text{l}\\text{y}\\text{s}\\text{i}\\text{s} \\text{I}\\text{n}\\text{d}\\text{e}\\text{x}= \\frac{\\text{C}\\text{l}\\text{e}\\text{a}\\text{r} \\text{Z}\\text{o}\\text{n}\\text{e} \\text{D}\\text{i}\\text{a}\\text{m}\\text{e}\\text{t}\\text{e}\\text{r}}{\\text{C}\\text{o}\\text{l}\\text{o}\\text{n}\\text{y} \\text{o}\\text{f} \\text{W}\\text{e}\\text{l}\\text{l} \\text{D}\\text{i}\\text{a}\\text{m}\\text{e}\\text{t}\\text{e}\\text{r}}$$\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eThe highest positive protease and fibrinolytic activity isolates were deposited at the Indonesian Culture Collection (InaCC).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eBacterial Endophyte Identification\u003c/h2\u003e\n \u003cp\u003eA 48-h bacterial colony on NA medium was used for the DNA extraction using the Geneaid Presto\u0026trade; Mini gDNA Bacteria Kit. The primer pairs used in the amplification of the 16S rRNA gene region were 27F (Ludwig et al. 1993), 1492R (Wilson et al. 1990), 357F (Kim et al. 2011), and 907R (Muyzer et al. 1995). PCR amplification was carried out using the following conditions: pre-denaturation at 94\u0026deg;C for 2 min, followed by 30 cycles (94\u0026deg;C for 2 min of denaturation, 55\u0026deg;C for 1 min of annealing, 72\u0026deg;C for 1 min of extension, and final extension at 72\u0026deg;C for 10 min). The quality of the PCR product was determined by electrophoresis using 1.5% agarose gel. The PCR products were sent to FirstBASE (Malaysia) for sequencing.\u003c/p\u003e\n \u003cp\u003eA newly nucleotide sequences were edited in ChromasPro v2.6.6 and aligned with the homologous sequences obtained from the NCBI GenBank through BLASTN program. Multiple sequence alignment was conducted with MEGA v11 software (Tamura et al. 2021). The Bayesian phylogeny analysis was reconstructed in BEAST v2.7.6 using default parameters (Douglas et al. 2022). The chain length was set to 1,000,000, the tree was sampled every 1,000 steps, the trace log was set to 100, and the screening was adjusted to 1,000. The results were analyzed with Tracer v1.7 (Rambaut et al. 2018). The burn-in percentage was set to 10, the posterior probability (PP) was limited to 0.75 in TreeAnnotator v1.10, and the PP support for the specified branches was displayed in the DensiTree v2 (Bouckaert and Heled 2014).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eFibrinolytic Activity Assay\u003c/h2\u003e\n \u003cp\u003eA total of 2 mL of bacterial inoculant (OD value 1.0) was added to 100 mL of fermentation medium (peptone 0.5 g, dextrin 2 g, yeast extract 0.15 g, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 0.4 g, Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 0.04 g, CaCO\u003csub\u003e3\u003c/sub\u003e 0.3 g, distilled water 100 mL), then incubated in a shaker incubator for 48 h, 37\u0026deg;C, at 120 rpm. The fermentation product was centrifuged at 10,000 rpm and 4\u0026deg;C, for 10 min. The centrifuged supernatant was used as a crude enzyme. A total of 0.4 mL of 0.5% (w/v) fibrinogen solution was added to 1.4 mL of 50 mM Tris-HCl buffer, pH 7.8, then incubated at 37\u0026deg;C for 5 min. A 0.1 mL of thrombin (20 U/mL) was further added and incubated at 37\u0026deg;C for 10 min, then 0.1 mL of enzyme solution was added and incubated again at 37\u0026deg;C for 60 min. Inactivation of the enzyme was conducted by adding 1 mL of 0.2 M TCA (trichloroacetic acid) and centrifuged at 10,000 rpm for 5 min at 4\u0026deg;C, then the fibrinolytic enzyme activity was measured using a spectrophotometer at 275 nm. The fibrinolytic activity was calculated as follows (Cupp-Enyard 2008):\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\" width=\"524\" height=\"92\"\u003e\u003c/p\u003e\n \u003cp\u003ewhere:\u003c/p\u003e\n \u003cp\u003eA: Total volume (mL) of assay\u003c/p\u003e\n \u003cp\u003eB: Time of assay (min) as per Unit definition\u003c/p\u003e\n \u003cp\u003eC: Volume of enzyme (mL) of enzyme used\u003c/p\u003e\n \u003cp\u003eD: Volume (mL) used in colorimetric determination\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eCrude Fibrinolytic Enzyme Production\u003c/h2\u003e\n \u003cp\u003eProduction of fibrinolytic enzymes was carried out using the following composition: peptone 5 g, dextrin 20 g, yeast extract 1.5 g, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 4 g, Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 0.4 g, CaCO\u003csub\u003e3\u003c/sub\u003e 3 g, and 1000 mL of distilled water. A 2% bacterial inoculum with an OD (optical density) value of 1.0 was added into 50 mL of medium, then incubated at 30\u0026deg;C for 48 h. After the incubation, the inoculum in the 50 mL medium was added into 2 L of production medium and incubated at 30\u0026deg;C, 150 rpm for 96 h. Bacterial inoculum was separated from the enzyme by centrifugation at 13,000 rpm, 4\u0026deg;C, and 10 min. The final supernatant was filtered using a cellulose acetate membrane with 0.22 \u0026micro;m pores and stored at 4\u0026deg;C for purification.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003ePartial Purification and SDS-PAGE Analysis\u003c/h2\u003e\n \u003cp\u003eThe supernatant was first precipitated using 70% of (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (ammonium sulphate) at 4\u0026deg;C, then left overnight in a slow stirrer. It was then centrifuged at 13,000 rpm, 10 min, and at 4\u0026deg;C. The Precipitate was further dissolved using phosphate buffer (50 mM, pH 7.0), and continued with dialysis using a 10 kDa cut-off cellulose membrane. The resulting enzyme was diluted 100 times. A total of 5 mL of dialysate was applied in a Hi-Trap Q FF ion exchange column connected to an FPLC system - AKTA Pure, equilibrated using phosphate buffer (50 mM, pH 7.0). A protein was eluted using 1 M NaCl solution in phosphate buffer (50 mM, pH 7.0) at a 2 mL/min flow rate. The protein content was measured at 595 nm, and the fibrinolytic enzyme activity at 275 nm. Fractions with activity were concentrated using a centric Amicon\u0026reg; Ultra Centrifugal Filter 10 kDa MWCO. Samples were stored at -20\u0026deg;C. The molecular weight and homogeneity of the pure enzyme were determined using SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) (Gallagher and Wiley 2008).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of pH, Temperature, Metal Ions, and Inhibitors\u003c/h2\u003e\n \u003cp\u003eAn assay on the effect of pH and temperature on the fibrinolytic enzyme activity refers to the method published by Lee et al. (2005). A total of 0.1 mL of enzyme solution was added and incubated for 30 minutes with temperature variations of 30, 40, 50, 60, 70, and 80\u0026deg;C. The effects of metal ions and inhibitors on the enzyme activity were conducted using the method described by Liu et al. (2017). Several monovalent and divalent metal ions such as CaCl\u003csub\u003e2\u003c/sub\u003e, CoCl\u003csub\u003e2\u003c/sub\u003e, CuSO\u003csub\u003e4\u003c/sub\u003e, FeCl\u003csub\u003e2\u003c/sub\u003e, HgCl\u003csub\u003e2\u003c/sub\u003e, KCl, MgSO\u003csub\u003e4\u003c/sub\u003e, MnSO\u003csub\u003e4\u003c/sub\u003e, and ZnSO\u003csub\u003e4\u003c/sub\u003e were used in this study. In addition, ethylene diamine tetra-acetic acid (EDTA), ethylene glycol-bis-(\u0026beta;-aminoethyl ether)-N, N, N\u0026prime;, N\u0026prime;-tetra-acetic acid (EGTA), phenyl methyl sulfonyl fluoride (PMSF), and N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) [25] were used in the inhibition assay.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eWhole Genome Sequencing\u003c/h2\u003e\n \u003cp\u003eGenomic DNA was extracted from the 48-h isolates using the Quick-DNA MagBead Kit (Zymo Research). DNA quality and concentrations were estimated by NanoDrop Spectrophotometer and Qubit Fluorometer. Amplicons were prepared for nanopore sequencing using the ONT Native Barcoding Expansion Kits. Library preparations were conducted using Kits from Oxford Nanopore Technology. The libraries were multiplexed on FLO-MIN106 flow cells and run on the GridION X5. GridION sequencing was operated by MinKNOW V23.04.5. Base calling was performed using Guppy V6.5.7 with high accuration mode (Wick et al. 2019). The quality of reads was visualized using NanoPlot (De Coster et al. 2018). Filtering was performed by Filtlong and De novo Assembly was performed using Flye V2.8.3. Mapping and Genome polishing were done four times with Minimap2 (Li 2018), Racon (v.1.5.0), and three times polishing with Medaka V1.7.2. The closely related species of the assembled sequence and genome completeness were determined using dfast-qc V0.4.2 and BUSCO V5.4.4 (Sim\u0026atilde;o et al. 2015). The quality of the assembled sequence was determined using QUAST V5.0.2 (Gurevich et al. 2013). Genome visualization was conducted using Circos software (Krzywinski et al. 2019).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eIdentification of Gene Encoding Fibrinolytic, Prediction of Functional Sites, Physicochemical Properties, and Subcellular Localization\u003c/h2\u003e\n \u003cp\u003eGenome annotation and the full-length gene encoding fibrinolytic identification were performed using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) service system (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bv-brc.org/\u003c/span\u003e\u003c/span\u003e). Genomic mapping of fibrinolytic proteins was performed using Proksee (CG-View) software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cgview.ca/\u003c/span\u003e\u003c/span\u003e). Homologous sequences of the amino acids fibrinolytic gene protein were obtained from the BLASTP program. In addition, a similarity searching of the fibrinolytic gene protein to the type amino acids sequence was also conducted using the BLAST tool for sequence similarity searching of the Universal Protein Resource (UniProt) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/blast\u003c/span\u003e\u003c/span\u003e). A prediction about the protein family, catalytic domain, and active sites related to the gene responsible for producing the fibrinolytic enzyme was conducted using ScanProsite (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://prosite.expasy.org/scanprosite/\u003c/span\u003e\u003c/span\u003e) (Sigrist et al. 2016). In addition, the physical and chemical properties of the gene-encoded fibrinolytic enzyme were evaluated using the ProtParam assessment tool available on the ExPASy server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://web.expasy.org/protparam/\u003c/span\u003e\u003c/span\u003e). The subcellular localization of the gene-encoded fibrinolytic enzyme was determined using PSORTb v.3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.psort.org/psortb/\u003c/span\u003e\u003c/span\u003e) (Yu et al. 2010).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eIn-Silico Structural Modeling\u003c/h2\u003e\n \u003cp\u003eThe protein structure of the minor extracellular protease Vpr gene in this study was predicted using two methods, including SWISS-MODEL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003c/span\u003e) (Waterhouse et al. 2018) and I-TASSER (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://zhanggroup.org/I-TASSER/\u003c/span\u003e\u003c/span\u003e) (Yang et al. 2014; Yang and Zhang 2015). Both methods use homology modeling techniques to create a protein structure model based on the sequence of amino acid residues. The SWISS-MODEL automatically builds the predicted protein model by searching for protein model templates using BLAST (Camacho et al. 2009), HHblits (Steinegger et al. 2019), and AlphaFold DB (V\u0026aacute;radi et al. 2022). The best template is chosen based on sequence coverage, sequence similarity, and GMQE (Global Model Quality Estimate) score. This template is then used to predict the protein model. In addition, I-TASSER suggests the protein model result along with the predicted secondary structure, predicted solvent accessibility, predicted normalized B-factor, C-score, TM-score, and other parameters. The Ramachandran Plot (RC Plot) was utilized to validate the protein structures.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eGenBank Submission\u003c/h2\u003e\n \u003cp\u003eThe 16S rRNA and alkaline protease gene sequences were submitted to GenBank under the accession numbers LC804381 and PP530334 (protein_id=\u0026quot;WXU37559\u0026quot;), respectively. The whole genome data was submitted to NCBI GenBank (Accession Number: JBCEZT000000000) with the BioProject ID PRJNA1090300.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eScreening of Proteolytic and Fibrinolytic Activity\u003c/h2\u003e\n \u003cp\u003eOut of 25 different endophytic bacterial strains tested, bacterial endophyte strain BFP1 (Culture collection accession number: InaCC-B1657) exhibited a high fibrin hydrolysis index (FHI) (fibrinolytic activity) of 2.60 and a protein hydrolysis index (PHI) (proteolytic activity) of 1.73, suggesting its ability to break down both fibrin and protein. This was demonstrated by the clear zones around the colony of the bacterial strain (Fig. 1a-d).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eIdentification of Bacterial Endophyte\u003c/h2\u003e\n \u003cp\u003eIn the phylogenetic analysis of strain InaCC-B1657, a total of 19 homologous \u003cem\u003eBacillus\u003c/em\u003e sequences were included from the BLAST results. \u003cem\u003eBacillus megaterium\u003c/em\u003e strain IAM 13418 (GenBank Accession number: KJ569088.1) was selected as an outgroup (Fig. 1d). The Bayesian inference analysis showed that all homologous \u003cem\u003eB. cereus\u003c/em\u003e group (s.lat.) sequences nested in the same clade with 100% PP. Sequence of strain InaCC-B1657 was also nested in the same clade with \u003cem\u003eB. cereus\u003c/em\u003e strain ATCC 14579 (NR074540.1) to form a monophyletic clade with 88% PP. This indicates that strain InaCC-B1657 belongs to \u003cem\u003eB. cereus\u003c/em\u003e s.lat. The finding of this study represents the first documentation of \u003cem\u003eB. cereus\u003c/em\u003e s.lat., an endophytic bacterium isolated from \u003cem\u003eC. papaya\u003c/em\u003e, as a potential producer of fibrinolytic enzymes from Indonesia.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eCrude Fibrinolytic Enzyme Activity and Partial Purification\u003c/h2\u003e\n \u003cp\u003eBased on initial observations on the crude enzyme of fibrinolytic activity, the \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 exhibited the highest activity, reaching 36.61 U/mL (Fig. 2a) with a specific activity of 0.807 U/\u0026micro;g protein at 24 h (Fig. 2b). Enzyme purification was further performed on crude enzyme obtained from \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657. The process involved enzyme protein precipitation using (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e at varying concentrations, ranging from 40\u0026ndash;100%. The study demonstrated that the highest enzyme activity was achieved at 70% (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e saturation, yielding a fibrinolytic enzyme activity of 7.47 U/mL (Fig. 2c). In this study, only one peak of fibrinolytic activity was observed in the eluate of the primary fraction with fibrinolytic activity applied to the Hi-Trap Q FF column. The fractions 18 to 25 showed fibrinolytic activity, with the 20th fraction exhibiting the highest level (3.12 U/mL). This fraction had a protein content of 0.00133 mg/mL (Fig. 2d-e). This final stage of purification showed impressive results, with a purity level 2.89 times higher and an activity recovery of 8.52%, with a specific activity of 2,345.86 U/mg protein (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). These results suggest that the \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 is a promising candidate for producing fibrinolytic enzyme protein owing to its highly specific activity. Using SDS-PAGE, the purified fibrinolytic enzyme from \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 was found to be 98.53096 kDa (Fig. 2f). This is in close accordance with the molecular weight of the amino acids calculated from the fibrinolytic gene (GenBank Accession Number: PP530334, 98.502 kDa) (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSummary of purification results for fibrinolytic enzyme from \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSteps\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal Protein (mg/mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal Activity\u003c/p\u003e\n \u003cp\u003e(U/mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecific Activity\u003c/p\u003e\n \u003cp\u003e(U/mg prot.)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRecovery (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFold Purification\u003c/p\u003e\n \u003cp\u003e(x fold)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrude extract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.04519\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e810.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70% Ammonium sulphate precipitation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2,134.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHi-Trap Q FF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.00133\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2,345.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eActivity of Fibrinolytic Enzyme at Different pH and Temperature\u003c/h2\u003e\n \u003cp\u003eEnzymes have specific temperature and pH requirements for optimal function. This study found that the fibrinolytic enzyme was active at all temperatures tested (30\u0026ndash;90\u0026deg;C), with its highest activity occurring at 50\u0026deg;C (Fig. 3a). The fibrinolytic enzyme also demonstrated optimal activity at pH 7.0 and remained stable within the pH range of 6.0\u0026ndash;10.0 (Fig. 3b). These results suggest that the fibrinolytic enzyme produced is most active at a temperature of 50\u0026deg;C and a pH of 7.0, with activity measured at 28.68 U/mL (Fig. 3a) and 32.10 U/mL (Fig. 3b), respectively. Additionally, after being incubated for 24 h, the enzyme retained 89.6, 88, 85.6, and 75.3% of its relative activity at 60, 70, 80, and 90\u0026deg;C, respectively, of that measured at 50\u0026deg;C (Fig. 3a). Similarly, after being incubated for 24 h at different pH conditions, the enzyme retained 74.4% of its activity at pH 6, 90.7% of its activity at pH 8, 85% of its activity at pH 9, and 75.1% of its activity at pH 10 (Fig. 3b). Therefore, this study shows that the fibrinolytic enzyme produced by \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 is thermostable and functions optimally under neutral to basic pH conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of Ion Metals and Inhibitors\u003c/h2\u003e\n \u003cp\u003eThis study found that adding Cu\u003csup\u003e2+\u003c/sup\u003e metal ion (10 mM) increased the activity of fibrinolytic enzymes to 53.11 U/mL, while the presence of Fe\u003csup\u003e2+\u003c/sup\u003e ion (10 mM) reduced the activity to 3.10 U/mL (Fig. 3c). Inhibitor analysis showed that PMSF (5 mM) and TPCK (5 mM) also inhibited the enzymes activity while adding EDTA (5 mM) and EGTA (5 mM) increased the activity (Fig. 3d). The addition of EDTA and EGTA increased the activity of enzymes to 30.03 U/mL (9.6-fold) and 32.67 U/mL (10.5-fold), respectively, from 3.12 U/mL.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eThe Genome of\u003c/strong\u003e \u003cstrong\u003eBacillus\u003c/strong\u003e \u003cstrong\u003ecereus strain InaCC-B1657\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eA current study identified a gene in the \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 that produces a fibrinolytic enzyme called the Vpr gene (Fig. 4). The \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 genome (GB Accession Number: JBCEZT000000000) comprises a circular chromosome containing 5,257,484 base pairs (bp). The N50 of the assembled genome was found to be 5,183,747 bp. The genome has an average G\u0026thinsp;+\u0026thinsp;C content of 35.33% and 5,364 genes (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Among these genes, 5,211 were predicted to be coding sequences (CDSs), consisting of 5,085 CDSs with protein and 126 CDSs without protein. Additionally, 107 tRNAs, 42 rRNAs, and five ncRNAs were detected. Through a BLASTp of the NCBI GenBank, it has been discovered that there is a close relationship between the current fibrinolytic-encode gene and S8 serine peptidase of \u003cem\u003eB. cereus\u003c/em\u003e and \u003cem\u003eB. thuringiensis\u003c/em\u003e isolates. The BLASTp results of the Vpr gene of \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 (915 amino acids) showed a 100% similarity with S8 family serine peptidase of \u003cem\u003eB. cereus\u003c/em\u003e (Accession number: WP_048538152.1) (max score: 1864, total score: 1864, query coverage: 100%). This result agrees with the current biochemical analysis, which has revealed a high level of inhibition with serine protease inhibitors (PMSF and TPCK), confirming that the Vpr gene of \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 is a serine protease. A BLAST result from the UniProt protein sequence database showed 97.8% similarity to the minor extracellular protease Vpr gene of type species of \u003cem\u003eB. cereus\u003c/em\u003e strain ATCC 14579, 93.1% to minor extracellular protease Vpr gene of the \u003cem\u003eB. anthracis\u003c/em\u003e strain ATCC 14578, and 76.6% to the peptidase S8 gene of \u003cem\u003eB. manliponensis\u003c/em\u003e strain JCM 15802.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eGenome information of \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFeature\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eObservation\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSize (total length) (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5,257,484\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG\u0026thinsp;+\u0026thinsp;C content (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.33%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of contigs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5,364\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etRNAs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e107\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003erRNAs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003encRNAs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCDSs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5,211\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCDSs (with protein)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5,085\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCDSs (without protein)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e126\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal pseudo genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e126\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHypothetical Protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2,355\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(N50 (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5,183,747\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of N\u0026rsquo;s per 100 kbp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003ePrediction of Functional Sites, Physicochemical Properties, and Subcellular Localization\u003c/h2\u003e\n \u003cp\u003eIn Vpr gene of \u003cem\u003eB. cereus\u003c/em\u003e InaCC-B1657, the Subtilase domain (peptidase S8) was predicted to be located at amino acid position 182\u0026ndash;591 (compared to PROSITE entry PS51892). This prediction included the catalytic triad subtilase, which is composed of Subtilase_Asp204, Subtilase_His237, and Subtilase_Ser531, predicted with a score of 44.621. The physicochemical characterization of the \u003cem\u003eB. cereus\u003c/em\u003e InaCC-B1657 Vpr gene with the sequences of five serine proteases from different \u003cem\u003eBacillus\u003c/em\u003e species is shown in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The molecular weight ranged from 98.5\u0026ndash;99.2 kDa, with the Vpr gene of \u003cem\u003eB. cereus\u003c/em\u003e InaCC-B1657 weighting 98.5 kDa, which was consistent with the weight detected in vitro by SDS-PAGE. The pI scores, which determine the pH at which a molecule or surface carries no net electrical charge, ranged from 5.33 to 6.15, with the Vpr gene of \u003cem\u003eB. cereus\u003c/em\u003e InaCC-B1657 scoring 6.15. The predicted instability index was \u0026lt;\u0026thinsp;40 (29.18\u0026ndash;31.03), indicating that the protein was stable. The aliphatic index of the Vpr gene of \u003cem\u003eB. cereus\u003c/em\u003e InaCC-B1657 was 85.05, indicating its enhanced thermo-stability over a broad range of temperatures. These findings agreed with the biochemical characterization, which showed that the fibrinolytic enzyme of \u003cem\u003eB. cereus\u003c/em\u003e InaCC-B1657 was active at all temperatures tested (30\u0026ndash;90\u0026deg;C), with its highest activity at 50\u0026deg;C. The number of negatively charged amino acids (Asp\u0026thinsp;+\u0026thinsp;Glu) was higher than the positively charged amino acids (Arg\u0026thinsp;+\u0026thinsp;Lys) for all sequences, with the Vpr gene of \u003cem\u003eB. cereus\u003c/em\u003e InaCC-B1657 having a ratio of 95/87, indicating its alkalo-halo stability. Furthermore, the GRAVY (Grand Average of Hydropathy) index ranged from \u0026minus;\u0026thinsp;0.370 to -0.343, with the Vpr gene of \u003cem\u003eB. cereus\u003c/em\u003e InaCC-B1657 having an index of -0.353, which suggests that it is a hydrophilic serine protease enzyme that exhibits strong interaction with water. A subcellular localization analysis showed that the Vpr gene of \u003cem\u003eB. cereus\u003c/em\u003e InaCC-B1657 is situated beyond the confines of the cell. The enzyme is known to be responsible for the breakdown of fibrin in blood clots, and its localization outside the cell suggests that it can act on external substrates. The PSORTb score of 9.98 further supports the extracellular localization of the enzyme (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePhysicochemical properties of the Vpr gene from \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 compared to similar serine proteases from \u003cem\u003eBacillus\u003c/em\u003e species obtained from NCBI GenBank database and UniProt\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReference Number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOrganism\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN Amino Acids\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMW\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAsp\u0026thinsp;+\u0026thinsp;Glu /Arg\u0026thinsp;+\u0026thinsp;Lys\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTheoritical pI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGRAVY Index\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInstability Index\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAliphatic Index\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e915\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98502.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95/87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.353\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e85.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWP_048538152.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e915\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98502.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95/87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.353\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e85.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQ818B5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e strain ATCC 14579\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e917\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99047.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97/88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.361\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e85.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWP_086407785.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus thuringiensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e915\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98687.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96/88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.358\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e85.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA0A6H3AKS4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eB. anthracis\u003c/em\u003e strain ATCC 14578\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e917\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98751.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98/87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.343\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e86.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA0A073JYX1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eB. manliponensis\u003c/em\u003e strain JCM 15802\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e915\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99266.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e102/79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.370\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e83.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSubcellular localization of the gene-encoded fibrinolytic enzyme of \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 using PSORTb\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLocalization\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePSORTb Score\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCytoplasmic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCytoplasmic Membrane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCell Wall\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eExtracellular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFinal Prediction: Extracellular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eIn-Silico Structural Modeling\u003c/h2\u003e\n \u003cp\u003eA BLAST result from UniProtKB database shows protein with accession number A0A0D0PQG3.1 as the most homolog sequence. Therefore, this sequence was used as a template. Because the status of the A0A0D0PQG3.1 protein is obsolete in the database, we selected an identical amino acid sequence from the database (Accession number: A0A9X0JT16) as a template protein. According to SWISS-MODEL\u0026apos;s evaluation, the A0A9X0JT16 was then chosen for modeling due to its superior sequence identity and GMQE ratings. Using the AlphaFold DB technique, the alignment revealed a 98.8% match with the template in a span of 917 residues. Furthermore, the model generated with the A0A9X0JT16 template showed a GMQE score of 0.89 in the SWISS-MODEL analysis, signifying a high-quality estimate within the 0 to 1 range. Based on the template protein on the SWISS-MODEL analysis, the active sites were predicted at Asp204, His237, and Ser531 (Fig.\u0026nbsp;5a). Furthermore, the predicted protein model demonstrates structural stability, with over 95% of its amino acid residues situated within the favoured region and over 99% within the allowed region of the Ramachandran plot, suggesting an accurate representation of the natural conformation of the protein. In the protein model prediction using I-TASSER analysis, the I-TASSER predicted model tends to have residues with normalized B-factor values below zero, indicating a relatively stable model. The 3D structural visualization of this model has a C-score of -2.38, which falls within the acceptable confidence range of (-5, 2), where higher values denote more reliable models. Similar to results from the SWISS-MODEL analysis, the I-TASSER analysis indicates that the model aligns with the serine-type peptidase classification and retains proteolytic activity (Fig.\u0026nbsp;5b). In addition, I-TASSER analysis has also forecasted binding sites allowing COFACTOR and COACH, identifying residues with a probability score equal to or more than 75%, notably His237 and Ser531.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study unveils the discovery of \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657, an endophytic bacterium found in \u003cem\u003eC. papaya\u003c/em\u003e, as a promising source of fibrinolytic enzymes from Indonesia. Notably, previous research has reported on the production of these enzymes by several strains of \u003cem\u003eB. cereus\u003c/em\u003e from various sources such as soil, blood, and cattle dung (Deepak et al. 2010; Bajaj et al. 2013; Majumdar et al. 2015; Narasimhan et al. 2015, 2018; Biji et al. 2016; D\u0026rsquo;Souza et al. 2020; Sharma et al. 2020). \u003cem\u003eBacillus\u003c/em\u003e cereus is a member of the \u003cem\u003eB. cereus\u003c/em\u003e group, which includes \u003cem\u003eB. anthracis\u003c/em\u003e, \u003cem\u003eB. thuringiensis\u003c/em\u003e, and \u003cem\u003eB. mycoides\u003c/em\u003e. These bacteria have been extensively studied for their pathogenic potential, particularly concerning foodborne illnesses and food safety. In the genome of \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657, there are several extracellular proteases with significant virulence factors found, including Immune inhibitor A (InhA) metalloprotease, M6 family metalloprotease immune inhibitor InhA1, and M6 family metalloprotease immune inhibitor InhA2 (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This raises concerns about the safety of \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 as a fibrinolytic enzyme producer. For further development, comprehensive risk assessments, including toxicity studies, are essential to determine if \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 can be considered GRAS (Generally Recognized as Safe). The GRAS status is necessary because it provides a solid foundation for utilizing these bacteria in producing fibrinolytic enzymes for various applications, including thrombosis treatment.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenes encoding proteases and their predicted compartmentalization in \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.psort.org/psortb/results.pl\u003c/span\u003e\u003cspan address=\"https://www.psort.org/psortb/results.pl\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene Start\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene End\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStrand\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProtease\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLocalization Score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSubcellular Localization\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e86632\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e84731\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell division-associated, ATP-dependent zinc metalloprotease FtsH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCytoplasmic Membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e182557\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e183732\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerine protease, DegP/HtrA, do-like (EC 3.4.21.-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e271138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e271812\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUncharacterized membrane zinc metalloprotease YwhC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCytoplasmic Membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e598232\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e599902\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExtracellular neutral protease B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eExtracellular\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e637997\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e638710\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCPBP family intramembrane metalloprotease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCytoplasmic Membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e756569\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e752346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS8 family serine peptidase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eExtracellular\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e757208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e756603\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell envelope-bound metalloprotease, Camelysin (EC 3.4.24.-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1235777\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1233030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eminor extracellular protease VpR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eExtracellular\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1941462\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1942703\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSerine protease, DegP/HtrA, do-like (EC 3.4.21.-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2433589\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2435976\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eImmune inhibitor A metalloprotease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eExtracellular\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2540600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2542987\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eImmune inhibitor A metalloprotease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eExtracellular\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3176660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3177853\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS8 family peptidase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eExtracellular\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3326332\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3324821\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePutative neutral metalloprotease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3488700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3487753\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIntracellular serine protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3961206\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3960109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMembrane metalloprotease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCytoplasmic Membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4107404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4105014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM6 family metalloprotease immune inhibitor InhA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eExtracellular\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4250342\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4249074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUncharacterized metalloprotease YhfN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCytoplasmic Membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4663850\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4661451\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM6 family metalloprotease immune inhibitor InhA2 (EC 3.4.24.-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eExtracellular\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4691160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4690561\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell envelope-bound metalloprotease, Camelysin (EC 3.4.24.-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4695656\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4691415\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS8 family serine peptidase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eExtracellular\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4983418\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4982711\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZinc metalloprotease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe biochemical characteristics of the current fibrinolytic enzyme from the \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 are sufficient to support further development (Figs.\u0026nbsp;3a-b). Several studies on the fibrinolytic enzymes from \u003cem\u003eB. cereus\u003c/em\u003e also reported that the optimum activity occurs not only at high-temperature conditions but also at neutral to alkaline pH conditions (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The thermostable nature could facilitate its integration into industrial processes that often involve high-temperature and alkaline pH conditions treatments in the production of bioactive peptides, which are valuable in the pharmaceutical and nutraceutical industries. In order to develop highly efficient fibrinolytic enzymes, several factors that can affect their performance must be evaluated. These include pH, temperature, medium composition, inhibiting and promoting factors. These elements can significantly impact the effectiveness of the enzyme activity and, in several cases, are specifically associated with the activity of the specific enzyme group. This study found that PMSF, TPCK, and ion metal Fe2\u0026thinsp;+\u0026thinsp;inhibited the fibrinolytic enzyme produced by the \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 (Fig.\u0026nbsp;3c-d). These suggest that the enzyme belongs to the serine protease group because the PMSF, TPCK, and metal ion Fe2\u0026thinsp;+\u0026thinsp;were reported as serine metalloprotease or serine protease inhibitors (Hazare et al. 2024). Serine protease (EC 3.4.21) cleaves peptide bonds similarly to other proteases. However, the serine residue in the active site (serine as a nucleophile) can coordinate many other essential functions through protein hydrolysis (Patel 2017). Serine proteases perform various functions, including protein metabolism, digestion, and blood clotting (Grant et al. 2007). Understanding the inhibitory effects of different agents like PMSF, TPCK, and metal ions on these enzymes is crucial for developing strategies to overcome these limitations and develop an efficient enzyme at an industrial level. For instance, metal chelators and other inhibitors, in fact, can help modulate enzyme activity (Akbar and Sharma 2017; Zhao et al. 2019). In addition, several factors also need to be considered to tackle the inhibitors problem, such as understanding their biochemical properties and implementation of gene manipulation methods such as DNA shuffling (Yao et al. 2022) and similar protocols.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOptimum pH and temperature for fibrinolytic enzyme activity of \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 and another \u003cem\u003eB. cereus\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSources\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTemp. (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAuthors\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e NK1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDeepak et al., 2010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e NS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBajaj et al., 2013\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e IND1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVijayaraghavan \u0026amp; Vincent, 2014\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e IND5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiji et al., 2016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e S8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLakshmi et al., 2018\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e SRM-001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNarasimhan et al., 2018\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e RSA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSharma et al., 2020\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e S46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eD\u0026rsquo;Souza et al., 2020\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe genes associated with fibrinolytic activity in \u003cem\u003eBacillus\u003c/em\u003e species are diverse and may encode various fibrinolytic enzymes and proteases. The expression and regulation of proteases/peptidases genes may influence the production, maturation, and activity of fibrinolytic enzymes in various \u003cem\u003eBacillus\u003c/em\u003e species. In this study, the genome sequence of \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 showed 21 proteases/peptidases divided into two groups based on cellular localization, and five proteases are unknown (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Coding sequences of seven extracellular proteases were also found in the genes encoding proteases, including extracellular neutral protease B, minor extracellular protease Vpr, Immune inhibitor A metalloprotease, S8 family peptidase, S8 family serine peptidase, M6 family metalloprotease immune inhibitor InhA1, and M6 family metalloprotease immune inhibitor InhA2. Among these genes, the Vpr and S8 family peptidases are related to the fibrinolytic enzyme activity produced by \u003cem\u003eBacillus\u003c/em\u003e species (Kho et al. 2005; Yao et al. 2020; Syahbanu et al. 2022; Zhou et al. 2022). Both genes belong to serine protease. Proteases are classified into four categories based on the specific catalytic mechanisms and the nature of the active site residues, including serine protease, aspartate protease, cysteine proteases, and metalloproteases (Rao et al. 1998). The Vpr gene belongs to the minor serine protease group and was first discovered in \u003cem\u003eB. subtilis\u003c/em\u003e (Sloma et al. 1991; Huang et al. 2022) and exhibited fibrinolytic enzyme activity (Ghosh et al. 2009; Choi et al. 2010; Yao et al. 2020; Chen et al. 2020; Syahbanu et al. 2022). Ghosh et al. (2009) reported that the Vpr gene has been implicated as a processor protease in the \u003cem\u003eBacillus\u003c/em\u003e system and has been shown to process other secretory pro-proteins and pro-peptides, including its role as a fibrinolytic enzyme (Kho et al. 2005). Yao et al. (2020) also demonstrated that the co-expression of VprSJ4 and aprESJ4 increased the fibrinolytic activity by 117% compared to aprESJ4 single expression in \u003cem\u003eB. subtilis\u003c/em\u003e WB600, indicating the potential involvement of Vpr in fibrinolysis. In addition, a specific catalytic triad of Asp/His/Ser of the Vpr gene of \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 indicates its potential involvement in proteolytic processes, including fibrinolysis.\u003c/p\u003e \u003cp\u003eThe physicochemical properties of the Vpr gene of \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e also showed the overall hydrophobicity or hydrophilicity of a protein or peptide sequence through the GRAVY index measures. A negative GRAVY score of the Vpr gene indicates hydrophilicity of the gene. In addition, the instability index score of 29.74 for the stability of a protein suggests that the protein is predicted to be relatively stable (Gamage et al. 2009), which can be advantageous for its functional properties and potential applications. This condition is also supported by the high aliphatic index score (85.05), which contributes to the structure stability and potential of a protein for functional activity. Therefore, a high aliphatic index value appears to have a beneficial effect on enhancing the thermostability of globular proteins (Ikai 1980). Furthermore, a higher ratio of Asp\u0026thinsp;+\u0026thinsp;Glu residues compared to Arg\u0026thinsp;+\u0026thinsp;Lys residues (95/87) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) exhibited increased stability, while the presence of Arg\u0026thinsp;+\u0026thinsp;Lys residues can impact the folding process and overall stability of the protein (Meuzelaar et al. 2016). A higher ratio of Asp\u0026thinsp;+\u0026thinsp;Glu residues may also influence the electrostatic interactions of protein, potentially impacting its binding to substrates and other molecules (Cheng et al. 2012).\u003c/p\u003e \u003cp\u003eThe predicted protein model of the Vpr gene of \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 shows a stable conformation since its amino acid residues are placed in the favorable region of more than 95% and the allowed region of more than 99% in the Ramachandran plot. The more residues occupying the allowed region correspond to a more stable protein model closely resembling the original experimental structure. This relationship can be attributed to energetically favorable conformations, as indicated by the allowed region, which is more likely to represent native-like protein structures (Laskowski et al. 2013). The 3D visualization of the current predicted protein model by I-TASSER analysis (C-score of -2.38) is still within the recommended range (-5, 2), where the higher value signifies a model with high confidence. The gene ontology of the predicted model by I-TASSER analysis shows that it can be grouped as a serine-type peptidase and has proteolysis activity. This is similar to the result predicted by SWISS-MODEL analysis. The SWISS-MODEL and I-TASSER show conclusive results stating that the protein target was predicted as a serine-type peptidase of the S8 family peptidase. A serine-type peptidase is a group of proteases characterized by the presence of nucleophilic serine residues within the enzyme active site, which play a crucial role in initiating the catalytic activity by forming a bond with substrate protein (Antalis et al. 2010). In this study, the SWISS-MODEL identifies Asp204, His237, and Ser531 as pivotal contributors to catalytic activity, whereas I-TASSER highlights the significance of His237 and Ser531 in catalytic function. These residues comprise a catalytic triad of Asp-His-Ser, which is responsible for peptidase activity and constitutes a characteristic feature of serine-type peptidases. In addition, the secondary structure of Asp204, His237, and Ser531 predicted by the SWISS-MODEL was β-sheet pleated, α-helix, and α-helix, respectively. The secondary structure predicted by the I-TASSER appears to provide a more detailed description, wherein all three residues (Asp204, His237, and Ser531) exhibited coil structures. The predominance of α-helix structure within the catalytic triad area is in accordance with the Cryo-EM visualization results of the Vpr protein reported by Cheng et al. (2023), which indicates the catalytic region of Vpr protein is primarily characterized by α-helix structure.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study determines the \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657, an endophyte from \u003cem\u003eC. papaya\u003c/em\u003e leaves, as a potential candidate in fibrinolytic enzyme production. The optimal activity was found at 50\u0026deg;C and pH 7.0 and remained stable until 80\u0026deg;C and pH 6\u0026ndash;10 for 24 h, indicating its thermo-stability and alkali-halo stability. Fe\u003csup\u003e2+\u003c/sup\u003e, PMSF, and TPCK inhibition to the fibrinolytic enzyme activity suggests that the fibrinolytic enzymes produced by \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 belong to the serine protease group. The genome sequence of \u003cem\u003eB. cereus\u003c/em\u003e strain InaCC-B1657 is 5,257,484 bp and comprises 21 proteases/peptidases, including the coding sequences of seven extracellular proteases. An in-silico study of the protein identifies a catalytic triad (Asp204, His237, and Ser531) as a pivotal contributor to catalytic activity. This triad is responsible for peptidase activity and constitutes a characteristic feature of serine-type peptidases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a grant from the Hibah PUTI Doctoral Programme awarded to Prof. Dr. Wibowo Mangunwardoyo (No. NKB-3295/UN2.RST/HKP.05.00/2020), University of Indonesia. Indriawati and Fina Amreta Laksmi are thanked for laboratory assistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declares that there is no conflict of interest that are relevant to the content of this manuscript. This manuscript has not been published, and it is not under consideration for publication anywhere else.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, methodologies, research experiment, data analysis, writing the draft and final version, AH; Protein purification and genome analysis, NR; In-Silico study, PRF; Research supervision and writing review, WM, MTS, and IH. All authors have approved this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent has been obtained from all participants for both sample collection and data publication in the research study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAkbar SM, Sharma HC (2017) Alkaline serine proteases from \u003cem\u003eHelicoverpa armigera\u003c/em\u003e: potential candidates for industrial applications. 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Microorganisms 10(5):843. https://doi.org/10.3390/microorganisms10050843\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"bacterium, blood clot, Carica papaya, In-silico, protease","lastPublishedDoi":"10.21203/rs.3.rs-4567703/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4567703/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacterial endophytes are a promising source of novel fibrinolytic enzymes with unique metabolic pathways and desirable characteristics that may not be present from conventionally explored sources. This study aimed to characterize fibrinolytic enzymes from selected endophytic bacteria isolated from papaya (\u003cem\u003eCarica papaya\u003c/em\u003e L.) leaves and the genes encoding the enzymes. A strain BFP1 (InaCC-B1657) that showed the highest fibrinolytic activity was identified as \u003cem\u003eBacillus cereus\u003c/em\u003e based on the phylogenetic analysis of the 16S rRNA sequence. The enzyme exhibited optimum activity at 50\u0026deg;C and pH 7.0, and remained stable until 80\u0026deg;C and pH 6\u0026ndash;10 for 24 h. The assay of metal ions and inhibitors on the fibrinolytic enzyme activity found that adding Cu\u003csup\u003e2+\u003c/sup\u003e stimulated, while Fe\u003csup\u003e2+\u003c/sup\u003e reduced the activity. PMSF and TPCK inhibited the enzyme activity, while adding EDTA and EGTA increased the activity. These suggest that the fibrinolytic enzymes belong to the serine protease group. Of the 21 proteases/peptidases determined from the 5,257,484 base pairs (bp) genome, minor extracellular protease Vpr and S8 family peptidase genes were found related to the fibrinolytic enzyme activity. The Vpr gene has a molecular weight of 98.5 kDa. The subtilase domain (peptidase S8 family) and the catalytic triad subtilase active sites (Asp204, His237, and Ser531) were detected. A prediction of physicochemical characteristics of the Vpr gene showed that the enzyme is hydrophilic and exhibited alkali-halo stability and thermo-stability over a broad range of temperatures.\u003c/p\u003e","manuscriptTitle":"Characteristics of Thermo-stable Serine Peptidase Vpr from Endophytic Bacillus cereus strain InaCC-B1657","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-01 11:15:40","doi":"10.21203/rs.3.rs-4567703/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-06-13T16:30:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-13T14:59:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Microbiology","date":"2024-06-12T05:41:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7a005f4c-d66a-4fca-a9fb-a54a1f2e5c05","owner":[],"postedDate":"July 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-07-01T11:15:40+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-01 11:15:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4567703","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4567703","identity":"rs-4567703","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}