Antibacterial, Antioxidant and Anticancer Activities of the Streptomyces PML5 strain isolated from Carbonate rocksin the Amazon

Antibacterial, Antioxidant and Anticancer Activities of the Streptomyces PML5 strain isolated from Carbonate rocksin the Amazon | 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 Antibacterial, Antioxidant and Anticancer Activities of the Streptomyces PML5 strain isolated from Carbonate rocksin the Amazon Karine Rodrigues do Nascimento Chaves, Maria Lucila Texeira de Andrade França, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4069286/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 5 You are reading this latest preprint version Abstract The aim of this study was to investigate the antimicrobial and cytotoxic potential of Streptomyces sp. isolated from carbonate rock in the Amazon. The antimicrobial activity was verified by the diffusion technique in agar against pathogens such as Micrococcus lutteus ATCC 7468, Streptococcus pneumoniae ATCC700699; Proteus vulgaris ATCC 13315, Escherichia coli ATCC 10536, Acinetobacter baumanii ATCC 19606.Cytotoxic activity was investigated ​​by the inhibition of cell growth (%) of samples tested in human colorectal carcinoma cell line (HCT116), at a concentration of 10 µg/mL (mean ± SEM; n = 2). The isolate showed a broad spectrum of antimicrobial activity against test microorganisms. This isolate showed maximum antibacterial activity against Escherichia coli ATCC 10536 (13 ± 0.0 mm) and Proteus vulgaris ATCC 13315 (12.8 ± 0.29 mm) and the most resistant microorganism against antibacterial activity of this isolate was Acinetobacter baumanii ATCC 19606 (11.5 ± 1.15 mm). This metabolites was extracted by solid : liquid method with ethyl acetate organic solvent. ​​The percentage of inhibition of cell growth of the crude extract tested in a human colorectal carcinoma line (HCT116) was 96,55%. The determination of antioxidant activity by capturing the free radical ABTS had an inhibition of 35.60 ± 2.01%, while the antioxidant potential by capturing the free radical DPPH had an inhibition of 10.65 ± 1.95%. The LC-MS analysis showed the presence of four peaks, where it was identified that the absorbance of substances 5,791 and 13,556 were similar to 9,921 and 17,077, respectively; this suggests the possibility of PML 5 having a metabolic pathway synthesizing substances of the same class. The CG-MS of the extract showed the presence of 10 volatile compounds. The main compound was Cyclododecane (83.75%) compared to other bioactive compounds. These results reinforce the potential of Amazonian biodiversity, revealing that actinobacteria produce antimicrobial and cytotoxic bioactive substances of interest to the pharmaceutical industry. Streptomyces. Bioinnovation. Biotechnological potential. Cytotoxic Potential. Antioxidant action Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION The Amazon biome boasts the largest expanse of rainforest on the planet, and is widely recognized for its abundant biological richness. However, it is important to note that, despite the essential contribution of microorganisms to ecosystems and their critical role in ecological and biogeochemical processes, research and exploration of microbiodiversity remain relatively scarce when compared to investigations involving plants and animals ( 1 ). Thus, the bioprospecting of actinobacteria offers an innovative perspective in the search for antimicrobial resources, with emphasis on little-explored environments, such as sedimentary formations, such as Amazonian carbonate rocks ( 2 ). The Itaituba Formation, located in the western region of Pará in the Brazilian Amazon, is a remarkable geological deposit dating back approximately 300 million years. Mostly composed of carbonate rocks, this sedimentary formation offers a rich source of information about the environmental and geological conditions that prevailed during the Permian Period ( 3 ). Inserted in the Amazon Basin, this formation composes one of the largest and oldest sedimentary basins in Brazil, preserving crucial geological events that influenced the deposition of sediments and shaped the local landscape over time, being mainly made up of minerals such as calcite and dolomite, a composition that reflects the intense biogenic activity that occurred during the Permian Period. The characteristics of these rocks surviving time indicate the presence of biogeochemical processes of degradation of organic matter, nutrient cycling, and release of chemical substances into the environment, which may result from the associated antimicrobial activity ( 3 ). In this scenario, the genus Streptomyces plays an important role, due to the diversity of substances with pharmacological properties and diversified chemical structure, such as antibiotics, antiparasitics, anticancer, as well as enzymes of industrial application. This genus is composed of Gram-positive, filamentous bacteria with a high content of guanine and cytosine ( 4 ). The objective of this research was to evaluate the antimicrobial, antioxidantand anticanceractivity of volatile compounds produced by Streptomyces associated with Amazonian carbonate rocks. MATERIALS AND METHODS Isolation of Streptomyces from Amazonian carbonate rock One sample of 10g carbonate rock was collected on Carbonate Rocks from the Itaituba Formation (04º16'34" S and 55º59'01" W) in Itaituba, Pará, Brazil. The rock sample were obtained from about 10 cm depth, numbered and stored in sterile zip-lock bags. The sample were taken to the laboratory and kept at 4°C for further study. The sample was mixed with 90 ml of sterile distilled water to make a uniform suspension and serially diluted up to 10 − 5 . About 100 µl suspension from this sample was evenly spread on the surface of Arginine Yeast Agar Medium (AYA) – (Compositiom: L-arginine 0.3 g, Glucose 1 g, Glycerol 1 g, K 2 HPO 4 0.3 g, MgSO 4 *7H 2 O 0.2 g, KCl 0.3 g, Yeast extract 1 g, Agar 20 g, pH: 7.4) ( 5 ). The media was supplemented with 64 µg/ml of antifungal (Nistatin) to prevent the growth of fungi. The plates were then kept at 30°C temperature for three weeks. Distinct colonies with morphological features such as dry, wrinkled, leathery, and powdery appearance were isolated and purified using the streak method on yeast-malt extract agar medium (ISP Medium No. 2, ISP2) and also maintained at − 80°C in glycerol suspension (20%, v/v) ( 6 ). Characterizations of Potent Isolate Morphological and Biochemical Characterization Characterization of the strain was carried out according to Goodfellow and Cross (1984). The substrate and aerial mycelia color, colony shape, and pigmentation of isolate were recorded. The scanning electron microscope (SEM) analysis of the isolate was done to observe spore chain and surface morphology with the help of an SEM instrument (LEO 5410LV). The biochemical characterization was performed to study the nutritional and metabolic capabilities of the isolate, and the tests were done by actinomycetes protocols ( 6 ). Prospecção de clusters gênicos de síntese de PSK e NRPS A series of primers was used to detect genes related to the antibiotic biosynthesis pathway, such as the so-called polykettide synthase (PKS-II), and non-ribosomal peptides (NRPS): PKSII: KS𝛼 (5’-TSGCSTGCTTGGAYGCSATC-3’) e KS𝛽 (5’- TGGAANCCGCCGAABCCTCT-3’); NRPS: A3F (5’-GCS TAC SYS ATS TAC ACS TCS GG-3’) e A7R (5’-SAS GTC VCC SGT SCG GTA S-3’) ( 7 ). A PCR (50µL) consisted of 100 ng genomic DNA (1µL), 5x Trans start Fast PfU buffer (10µL), primer 10µM (1 µL), 2,5 mM DNTP´s (4µL), Trans Start Fast Pfu polymerase (1µL), PCR stimulant (5x) (5µL), MgSO 4 50mM (2µL) e ddH2O 25 µL). O ciclo de PCR ocorrerá segundoas condições: 98 º C por 4’; 30 ciclos de 94 º C (1’), 57,5 º C (30’’), 72 º C (1’/ kb); extensão de 72 º C (10’). All PCR products will be separated by 1.2% agarose gel electrophoresis and bands between 1,200-1,400, 600, 1,400 and 700–800 bp will be classified as products of the PKSII, and NRPS genes ( 7 ). Sequencing reactions will be done using the BigDye Terminator v3.1 Cycle Sequencing Kit (código 4337456). he runs will be made in 36cm capillaries using the POP7 polymer and the generated contigs will be analyzed by the Sequencing Analysis 7.0 software using the Base Caller KB. The acquired sequences will be manually aligned using the GenBank database with BLAST. Sequences with 98–100% homology will be considered for molecular taxonomic analysis. Multiple sequence alignment will be performed for each generated sequence and the sequences from the GeneBank database with the CLUSTAL X program. The phylogenetic tree will be constructed using the neighbor-joining and maximum parsimony methods in the Molecular Evolutionary Genetic Analysis software (MEGA version 11.0.13) based on bootstrap values of 1,000 replications ( 7 ). Primary Screening for Antagonistic Activity The primary screening of isolates was performed by agar block method Ichikawa; Date; Ishikura; Ozaki, (1971) against S treptococcus pneumoniae ATCC700699; Micrococcus lutteus ATCC7468; Staphylococcus aureus ATCC14458; Staphylococcus epidermidis ATCC12228; Aeromonas hydrophyla INCQS00318; Klebsiella pneumoniae OXA48; Escherichia coli ATCC10536; Acinetobacter baumannii ATCC19606; Proteus vulgaris ATCC13315; Escherichia coli ATCC25922 e Salmonella enteritidis INCQS00268. The antagonistic activity of actinomycete isolates was recorded to select potential isolate with bioactivity ( 8 ). Evaluation of Anticancer Activity by MTT Assay Human tumor cell lines HCT-116 (colorectal carcinoma) were obtained from the American Type Culture Collection (ATCC). Cells were maintained in RPMI 1640 (HCT-116) media supplemented with 10% fetal bovine serum, supplemented with 1% antibiotics (penicillin + streptomycin) at 37°C with 5% CO2 and 95% relative humidity. To maintain cultures in exponential growth, cells were split periodically. Cytotoxic activity was evaluated by the MTT method (Mosmann 1983). An aliquot of 2 x 10³ cells was inserted per well into 96-well plates (10 4 cells/well in 200 µL/well) and left to adhere for 24 h. Each sample, dissolved in DMSO, was added to their respective wells, making up to final concentration 10 µg/mL, with each concentration tested in duplicate, and incubated for 72 h. Doxorubicin was used as a positive control, while negative control groups received the sample dilution vehicle, DMSO. At the end of the incubation period, the supernatant in each well was replaced with fresh media added with 0.5 mg/mL of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)). After 3h, the supernatant was removed, the precipitate product was dissolved in 150 µL DMSO and the absorbance was measured using a multiplate reader at 570 nm. Absorbance values for each treatment were transformed to percentage of cell growth inhibition based on positive (100% inhibition) and negative (0% inhibition) control parameters, for analysis of qualitative data. Inhibitory concentration values were calculated by nonlinear regression using GraphPad Prism 8 ( 9 ). Evaluation of Antioxidant Activity The determination of the antioxidant activity occurred only with the most promising strain in cytotoxic activity, PML5, which will have its extract tested by the capture of the ABTS free radical performed in a spectrophotometer. The ABTS radical was prepared from the reaction of 5 mL of ABTS stock solution (7 mM) with 88 µL of potassium persulfate solution (140 mM) and kept at room temperature in the dark for 16 hours. After the necessary time, 1 mL of the mixture was diluted in ethyl alcohol until an absorbance of 0.7 nm ± 0.05 nm at 734 nm was obtained in a Shimadzu UV 1800 spectrometer (Shimadzu® 1800, Kyoto, Japan). Then, a standard trolox curve (2,000 µM) was constructed with concentrations ranging from 100 µM to 2,000 µM. In a dark environment, an aliquot of 30 µL of each trolox solution (100 µM, 500 µM, 1000 µM, 1500 µM and 2,000 µM) was transferred to test tubes and 3.0 mL of the ABTS radical solution was mixed. Then, it was homogenized in a vortex and after 6 min of mixing, the readings were performed at 734nm. Ethyl alcohol was used as white to calibrate the spectrophotometer. To determine the antioxidant activity in the sample, the same procedure as above was repeated with the dilution of the oil ( 10 ). The percentage of inhibition was calculated according to Eq. 1 and the final concentration was expressed in µM Trolox /g. The analysis was performed in triplicate. Equation 1: % INHIBITION = (Control Abs - Sample Abs)/Control Abs x100 Antioxidant activity by DPPH free radical capture was performed in a UV 1800 spectrophotometer (Shimadzu®, Kyoto, Japan) at a wavelength of 515 nm. The DPPH radical solution was prepared from 24 mg of DPPH in 100 mL of ethanol. Then, 10 mL of the solution was removed and transferred to a 100 mL volumetric flask and the volume was completed with ethanol to obtain the working solution. In a dark environment, an aliquot of 150 µL of the sample was transferred to test tubes, mixed with 5,085 mL of the DPPH radical of the working solution and homogenized in a vortex. After 30 minutes of reaction, the reading was performed in a spectrophotometer. Ethyl alcohol was used as white to calibrate the spectrophotometer. Antioxidant activity was calculated based on a standard Trolox curve (50µM-1000 µM). The percentage of inhibition was calculated according to Eq. 1 and the final concentration was expressed in µM Trolox /g( 11 ). LC-MS Analysis of the Promising Insulator The analyses by high performance liquid chromatography coupled to an ultraviolet spectrum scanning photodiode detector (HPLC-UV-DAD) were developed on an Agilent 1260 chromatograph equipped with a 60 mm flow cell with detection speed at 80 Hz and detector by array of ultraviolet and visible spectrum scanning photodiodes (range between 200 to 600 nm). For the analytical-scale analyses, a reversed-phase Zorbax Eclipse plus C 18 column (150 x 4.6 mm) and 3.5 µm particle diameter maintained at 45 o C was used as a stationary phase, with a mobile phase flow of 1.0 mL.min-1 and 3µL of sample injection volume, prepared at a concentration of 1 mg.mL−1 . The wavelengths used to record the chromatograms were 254, 280, 325 and 352 nm. The gradient elution system consisted of acidified water with 0.1% acetic acid (eluent A; Synth) and acetonitrile (eluent B; Merck) on the following schedule: 0-10min.: 10–25%B; 10-30min.: 25–50%B; 30-50min.: 50–100%B; 50-60min.: 100%B. GC-MS analysis of the promising isolate The extracts were analyzed by gas chromatography (GC) in an Agilent CG 6850 equipment coupled to an Agilent 5975C mass spectrometer (EM) containing a 30m x 0.25mm HP-5MS column and 0.25µm film. The sample injection volume was 1µL in splitless mode, with Helium as the carrier gas at a rate of 1mL.min-1. The method used has an initial oven temperature of 100°C, maintained for 5 minutes, and a heating rate of 5° C.min−1 until reaching a temperature of 320°C, which was maintained at the end of heating for another 8 minutes. The injector, quadrupole and ion source temperatures were, respectively, 300°C, 180°C and 280°C. Mass spectra detections were performed by electron impact ionization (EI) at 70 eV, in full-scan acquisition mode in the m/z range 50–800 m/z at 2.66 scan.s-1. Metabolite identifications were performed by comparison of pattern retention time and by comparison with the NIST spectral library (v2.0, 2008) using Match and R-Match comparison values above 900. RESULTS AND DISCUSSION The PML5 isolate showed poorly developed white airy mycelium and yellow vegetative mycelium, a relevant taxonomic criterion for the identification of Streptomyces ( 12 , 13 ) (Fig. 2). The development of aerial mycelium occurred after seven days of cultivation in ISP2 medium in a greenhouse at 30ºC, something common to actinobacteria, since its growth is slow and can take from 7 to 15 days for complete development at this temperature ( 14 , 15 ). Regarding the arrangement of the spores, the presence of flexuous filaments in branched chains, short closed filaments was identified, all these characteristics converging with the typical morphological profile of the genus Streptomyces , cited by Azuma (2012) ( 16 ). The microscopic characteristics observed in the isolates showed organization and shape compatible with the genus Streptomyces , as described in the literature by Alam et.al. (2022). ( 17 ). The biochemical and physiological characteristics of the isolate made it possible to identify the biochemical profile of the isolate with growth in lipase (3,58) and esterase (3,58), biocatalyst enzymes that are used biotechnologically in the synthesis of surfactants, textile, cosmetic and pharmaceutical industries, in addition to participating in the production of biofuels, acting without the need for cofactors, with high stability in organic solvents and wide specificity of substrates ( 18 ). The PML5 isolate was positive for the enzyme Catalase, which in the study by Fang et.al. (2021) was described as a substance directly involved in the biosynthesis of secondary metabolites in 2,634 Streptomyces strains with catalytic properties capable of being exploited in different ways ( 19 ). When exposed to nitrogen sources, PML5 was positive for L-histidine, serine, valine and L-asparigine. Each of the amino acids—L-histidine, serine, valine, and L-asparagine—plays key roles as sources of nitrogen in the metabolic processes of organisms. During the metabolism of L-histidine, the nitrogen present can be released and utilized in the synthesis of other nitrogen-containing molecules, such as nucleotides and neurotransmitters ( 20 ). Similarly, Serine and L-asparagine, also containing amino groups, contribute as sources of nitrogen in metabolic pathways. When metabolized, these amino acids release nitrogen, which is then used in the synthesis of a variety of molecules vital for cellular function ( 21 ). Valine can be incorporated into other nitrogen-containing molecules. These processes are essential for balancing the nitrogenous metabolism of organisms, ensuring adequate nitrogen availability for various biological functions, including protein and nucleic acid synthesis ( 20 , 21 ). The growth of PML5 in these nitrogen sources suggests that these microorganisms may be present in the environment where these compounds are found and may be involved in the production of bioactive compounds. PML5 grew in media with carbon source D-xylose, Sucrose, Maltose, D-mannose, D-lactose and Glucose. D-xylose is a monosaccharide, commonly used in industry as a carbon source in microbiological culture media. On the other hand, sucrose is a disaccharide composed of glucose and fructose, widely found in food and used in the food industry and in microbiological culture media. Maltose, another disaccharide composed of two glucose molecules, is often employed as a carbon source in the food industry and microbiology ( 22 ). D-mannose, a monosaccharide isomer of glucose, is commercially synthesized and used in dietary supplements due to its beneficial health properties. D-lactose, a disaccharide, is an important source of carbon and energy for infants and is used in the food industry in dairy production. Finally, glucose, a monosaccharide, is essential for the metabolism of organisms and is used in various industrial sectors, from food and beverages to biofuels and pharmaceutical production. These carbon sources play crucial roles in the nutrition and metabolism of various organisms and have significant applications in industry and scientific research ( 23 ). The growth of PML5 strains in these media indicates the ability of actinobacteria to use these carbohydrates as a carbon source, which can influence the degradation and cycling of nutrients in the carbonate rock environment. The Antibiotic Susceptibility Test showed that the PML5 strain is sensitive to Bacitracin (23mm) and Azithromycin (35mm) and resistant to Nalidixic Acid, Cefepime, Tetracycline, Cethazidine, Rhinoampiline, Oxacillin, Penicillin, Ketalhotin, Cephalitriaxone, Amphotericin B and Amoxicillin. Resistance to drug classes may be related to the production of secondary metabolites of PML5 similar to the antibiotics tested. Quinolone, Penicillin, and 1st-, 3rd-, and 4th-generation cephalosporins are classes of broad-spectrum antibiotics with different modes of action. The simultaneous production of these classes may suggest that PML 5 has the ability to synthesize compounds that act on different bacterial targets and with different mechanisms of action. Resistance to rifamine and antifungal polyene may indicate the ability of the isolate to synthesize compounds with antituberculosis and antifungal properties, respectively( 24 ). The PML5 strain showed a positive response to PKSII and NRPS, indicating its potential for the production of polyquetides and also non-ribosomal peptides. The presence of PKSII and NRPS is reported by Paulus et.al. (2022) and Alonso et.al. (2021) as part of genome mining for identification of biosynthetic genes from natural products. The discovery of these clusters is necessary for the search for new bioactive compounds, in order to then create the necessary conditions for the clusters to produce them, for which the PML5 strains were provided with carbon and nitrogen sources necessary for their growth and metabolism ( 23 , 25 ) The PML5 isolate showed antagonistic activity against gram-positive and gram-negative pathogens, as shown in Chart 1 Table 1 Halos of pathogen growth inhibition against isolates Pathogens PML5 Streptococcus pneumoniae ATCC700699 12 ± 0,58 Staphylococcus aureus ATCC14458 12 ± 1,0 Staphylococcus epidermidis ATCC12228 12 ± 0,56 Micrococcus lutteus ATCC7468 12 ± 1,00 Escherichia coli ATCC25922 15 ± 0,00 Escherichia coli ATCC10536 13 ± 0,00 Klebsiella pneumoniae OXA48 12 ± 0,59 Acinetobacter baumannii ATCC19606 13 ± 1,15 Salmonella enteritidis INCQS00268 16 ± 0,00 Proteus vulgaris ATCC13315 13 ± 0,29 Aeromonas hydrophyla INCQS00318 10 ± 0,58 The antagonistic activity revealed a broad antimicrobial spectrum against Gram-positive and Gram-negative bactéria of medical interest in humans, with emphasis on Salmonella enteritidis INCQS00268, exhibiting na averafe inhibition halo of 16 ± 0,0 mm. The most resistant pathogen was Aeromonas hydrophyla INCQS00318with na inhbition halo of 10 ± 1,15 mm. In the research by Bhat e Nayaka (2023), the cave isolate YC69 had a greater halo of inhibition against Staphylococcus aureus and Escherichia coli when compared to the other 69 isolates. Similarly, Jaroszewicz et.al. (2021) tested isolates from limestone rocks, where the one with the highest spectrum against Staphylococcus aureus, Salmonella enterica, Enterococcus sp., Escherichia coli , and Pseudomonas aeruginosa for anticâncer testing ( 26 , 27 ). The acetatethyl extract of the PML5 isolate showed greater cytotoxic action (96.5%) against the growth of human colorectal cancer cells (HCT-116). The cytotoxic action of extracts of PML11 and PML15 isolates against HCT-116 cells was 33.40% and 58.55%, respectively. Researchers Bhat and Nayaka (2023) obtained a 41.98% inhibition of cell proliferation in vitro by subjecting the YC69 isolate against human cervical cancer cells (HeLa) to cytotoxic testing. For Jaroszewicz et.al. (2021), the isolates of Streptomyces sp. selected for cytotoxic testing reduced the viability of the breast cancer cell line (T47D). ( 26 ) The use of the cytotoxic MTT assay facilitates the determination of the cytotoxicity of a sample, although it does not clarify its mechanism of action, the analysis of cytotoxicity represents a crucial stage in the process of creating new drugs for clinical use ( 28 ). Researcher Huang et.al. (2023) tested the cytotoxic action of mangrove sediment isolate 4503, with morphological and chemical properties converging to the genus Streptomyces. The isolate showed cytotoxic activity against nasopharyngeal carcinoma (NPC) cell lines with antiproliferation, antimetastasis, induction of cell cycle arrest and apoptosis, in addition to increasing the production of reactive oxygen species, which led to the need to also explore the antioxidant potential of the isolate ( 29 ). The antioxidant action detected by the ABTS method showed an action of 35.60 + 2.01%, while the antioxidant potential by the capture of the free radical DPPH had an inhibition of 10.65 + 1.95%. The antioxidant activity revealed by the ABTS and DPPH method suggests a significant capacity of actinobacteria isolated from carbonate rocks in the Amazon to neutralize free radicals, reflecting a promising biological potential for health and technology applications. This capacity is evidenced by the outstanding performance of the actinobacterium Streptomyces sp., isolated from the Caatinga, whose cultural filtrates demonstrated high antioxidant activity through the DPPH and ABTS methods, in addition to having high phenolic and flavonoid contents ( 30 ). For Rammali et.al. (2022) a response percentage greater than 30% demonstrates the high capacity of secondary metabolites of actinobacteria to capture the ABTS free radical. For Weslati et.al. (2023) A DPPH eliminator response above 1.3 µg is highly active. By relating the research with the result of 35.60% of activity in the capture of ABTS and 138.56 µg of PML5, a greater potential of the carbonate rock isolate is evidenced ( 31 ). In addition, previous studies highlight the importance of ABTS and DPPH methods as reliable tools for evaluating the antioxidant capacity of plant extracts and isolated compounds. Weslati, Simões, Texeira, Parpot et.al. (2023) detailed the ability of several antioxidants to react with the DPPH radical, providing a solid basis for future comparisons of antioxidant activity. These methods are widely recognized for their efficacy in quantifying antioxidant capacity, which is fundamental for studies such as the present one, which seeks to explore the potential of new sources of antioxidants ( 32 ). The metabolic pathway of PML5 was developed to synthesize four different substances, as shown in Fig. 5, where well-defined peaks were identified. In the mass spectrum, it was identified that the absorbance of substances 5,791 and 13,556 were similar to 9,921 and 17,077, respectively. This indicates that they have the same chromophores with conjugated double bonds, i.e., the same skeleton, suggesting the possibility that they are of the same class (Fig. 4 ). GC-MS analysis of the extract showed strong antimicrobial and antitumor activity (Table 6). We identified 10 different volatile compounds from the active metabolite of the isolate (between 14.81min and 37.53 min) and were recognized for their antimicrobial and antitumor activities. Cyclododecane (83.75%) was the main compound identified in the extract. Table 2 Identification of chemical compounds of the PML5 isolate using GC-MS analysis. Time (min) Area (%) M.W (g/mol) Molecular Formula Compound Name Bioactivity 14.816 0.76 206.32 C 14 H 22 O 2,4-Di-tert-butylphenol Antitumor activity ( 33 ) 19.029 83.75 168,32 C 12 H 24​ ​ ​ Cyclododecane Antimicrobial and antitumor activity ( 34 ) 21.255 1.11 316.5 C 18 H 36 O 4 Tetradecanoic acid, 12-methyl-, methyl ester, (S)- Antioxidant and antitumor activity ( 35 ) 23.144 2.76 270.5 C 17 H 34 O 2 Pentadecanoic acid, 14-methyl-, methyl ester Antioxidant and antitumor activity ( 36 ) 23.551 1.32 196,2 C 10 H 16 N 2 O 2 Cyclo(L-prolyl-L-valine) Antimicrobial activity ( 37 ) 23.902 2.96 210.27 C 11 H 18 N 2 O 2 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- Antimicrobial activity ( 38 ) 25.262 0.62 316.5 C 18 H 36 O 4 Tetradecanoic acid, 12-methyl-, methyl ester, (S)- Antioxidant and anti-rumoral activity ( 35 ) 25.537 1.08 168.32 C 12 H 24 Cyclododecane Antimicrobial and antitumor activity ( 34 ) 32.289 1.36 172.18 C 8 H 12 O 4 4-Octene, (Z) Antimicrobial Activity ( 39 ) 37.536 4.27 166.13 C 8 H 6 O 4 1,4-Benzenedicarboxylic acid Antimicrobial Activity ( 40 ) The components determined by GC-MS showed antimicrobial, antioxidant and antitumor activities. 2,4-Di-tert-butylphenol has anticancer properties against Mycobacterium tuberculosis, antioxidant activity of 2,4-DTB, and response of 88% and 89% against colon cancer and uterine cancer, respectively, in Kaari's research; Joseph; Manikkam; Kalyanasundaram et al., (2023). The compound Cyclododecane, which appeared in two moments (19.02min and 25.53min) during chromatography. In the literature, it is described as a substance present in medications for bronchitis, hemomatirua, epilepsy, asthma, leprosy, eczema, debre and jaundice, in addition to having antitumor and antioxidant capacity. The Tetradecanoic acid, 12-methyl-, methyl ester, (S)- also appeared twice during the analysis (21.25min and 25.26min) and its action has antioxidant and antitumor activity as well as the Pentadecanoic acid, 14-methyl-, methyl ester (KEMUNG; TAN; CHAN; SER et al., 2020). Other antimicrobial substances were Cyclo(L-prolyl-L-valine), Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-, 4-Octene, (Z), 1,4-Benzenedicarboxylic acid, which have antimicrobial potential against gram-positive and negative bacteria, some even resistant to antimethicillin resistant Staphylococcus aureus (MRSA)( 40 ). CONCLUSIONS The carbonate rocks of the Itaituba Formation are a rich source of underexplored environmental and geological information for new bioactive microorganisms. The study investigated the isolation, characterization and application of actinobacteria isolated from carbonate sediments, called PML5. The morphological and molecular profile showed convergent patterns with the genus Streptomyces sp . The antimicrobial activity was more sensitive for Escherichia coli ATCC 10536 (13 ± 0.0 mm) and Proteus vulgaris ATCC 13315 (12.8 ± 0.29 mm) and more resistant for Acinetobacter baumanii ATCC 19606 (11.5 ± 1.15 mm). The cytotoxic test inhibited the colorectal cancer cell line by 96.55% and the antioxidant action protected by the ABTS method showed an action of 35.60 + 2.01%, while the antioxidant potential by capturing the DPPH free radical had a limitation of 10.65 + 1.95%. The LC-MS analysis showed the presence of four peaks, where it was identified that the absorbance of substances 5,791 and 13,556 were similar to 9,921 and 17,077, respectively; This suggests the possibility that PML 5 has a metabolic route synthesizing substances of the same class. The GC-MS of the extract showed the presence of 10 volatile compounds. The main compound was Cyclododecane (83.75%) compared to other bioactive compounds. These results reinforce the potential of Amazonian biodiversity, revealing that actinobacteria produce antimicrobial and cytotoxic bioactive substances of interest to the pharmaceutical industry. Declarations -Ethical Approval (Missing) -Consent to Participate(Missing) -Consent to Publish (Missing) -Authors Contributions All authors contributed to the conception and design of the study. The preparation of the material, collection and analysis of data were carried out by Karine Rodrigues do Nascimento Chaves, Maria Lucila Texeira de Andrade França, Anna Ludmylla Oliveira Mentes, Pablício Pereira Cardoso, Kamila Brielle Pantoja Vasconcelos, Raphael Carlos Ferrer de Santana, Letícia Veras Costa Lotufo, Anderson Conceição Mendes, Lindalva Maria de Meneses Costa Ferreira, Sara Freitas de Sousa Ramos, Isabella Cristhina Gonçalves Costa, Marcelo J. Pena Ferreira and Gabriel Padilla. The first draft of the manuscript was written by Karine Rodrigues do Nascimento Chaves and Silvia Katrine Rabelo da Silva and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. -Funding This research was supported by the Universidade Federal do Oeste do Pará, Universidade Federal do Pará and Universidade de São Paulo. -Competing Interests The authors declare that they have no conflict of interest. -Availability of data and materials (Missing) The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request. Source data are provided with this paper. References de Azevedo, J. H., & Campos, J. E. G. (2021). Flow patterns and aquifer recharge controls under Amazon rainforest influence: The case of the Alter do Chão aquifer system. Journal of South American Earth Sciences , 112 , 103596. Saleem, M., Hassan, A., Li, F., Lu, Q., Ponomareva, L. V., Parkin, S., et al. (2023). 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C. (2022). Antimicrobial compounds of one Streptomyces celluloflavus strain isolated from Can Gio mangrove soil, Vietnam. GSC Biological and Pharmaceutical Sciences , 19 (3), 120–126. Kemung, H. M., Tan, L. T. H., Chan, K-G., Ser, H-L., Law, J. W. F., Lee, L-H. (2020). Antioxidant Activities of Streptomyces sp. strain MUSC 14 from Mangrove Forest Soil in Malaysia. BioMed Research International . Ser, H., Palanisamy, U., Yin, W., Chan, K., Goh, B., & Lee, L. (2016). Streptomyces malaysiense sp. nov.: A novel Malaysian mangrove soil actinobacterium with antioxidative activity and cytotoxic potential against human cancer cell lines. Scientific reports . ;6. Sharma, N., Koul, M., Joshi, N., Dufossé, L., & Mishra, A. (2024). Fungal-Bacterial Combinations in Plant Health under Stress: Physiological and Biochemical Characteristics of the Filamentous Fungus Serendipita indica and the Actinobacterium Zhihengliuella sp. ISTPL4 under In Vitro Arsenic Stress. Microorganisms . ;12(2). Buzon-Durán, E., Sánchez-Hernández, E., Sánchez-Báscones, M., García-González, M., Hernández-Navarro, S., Correa-Guimarães, A. (2023). Um Revestimento Baseado em Compostos Bioativos de Streptomyces spp. e oligômeros de quitosana para controlar Botrytis cinerea preserva a qualidade e melhora a vida útil das uvas de mesa. Baozhen, F., Dandan, C., Ruixue, J., Erqin, L., & Peiqian L. Bioactivities evaluation of an endophytic bacterial strain Bacillus velezensis JRX-YG39 inhabiting wild grape. BMC Microbiol2022. Essam, S., Neveen, P. M., N, A-R-X., & II, B. M. (2020). A. MA. Antimicrobial and antinematicidal metabolites from Streptomyces cuspidosporus strain SA4 against selected pathogenic bacteria, fungi and nematode. Saudi J Biol Sci. Supplementary Files SupplementaryInformationfiles.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Resubmit revised form; Major revisions required 05 May, 2024 Reviewers agreed at journal 16 Apr, 2024 Reviewers invited by journal 21 Mar, 2024 Editor invited by journal 18 Mar, 2024 First submitted to journal 15 Mar, 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-4069286","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":282072722,"identity":"54271098-b1b8-43fc-90d1-2a2654df108f","order_by":0,"name":"Karine Rodrigues do Nascimento Chaves","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYBACCSA+AGIYMININiCfvQHIKIBIEamFB8g/YIBfCxgYgEk2kFACfi2S7WcfHi6ouJdnzs58dMOHMgt5/pmPj0l/MLCRk2xgfvjoBqYWaZ50g8MzzhQXWzazpd2ccU7CcMbttDSJAwZpxtIMbMbGOZha5BjSGA7ztiUkbjjMY3abtw3oqts5ZkAthxPnMfCwSWPTwv8MqOUfVMtfoBb5m2fwa5GWANnSANXCCNRicIMHomU2Di2SM4C28BxLSNwJ8ksP0C8bz6QlW5wB+kWyGbtfJM6nMX/mqUlI3M5/+NiNH2V18nLHDx+8UVFhIydxvPnhYyxa8AFm0pSPglEwCkbBKEAAALzdZSDOpM86AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3800-7825","institution":"Universidade Federal do Oeste do Para - 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Pena","lastName":"Ferreira","suffix":""},{"id":282072734,"identity":"5270ca5e-7dcd-48f4-ba22-51402379a84b","order_by":12,"name":"Gabriel Padilla","email":"","orcid":"","institution":"Universidade de Sao Paulo Campus de Sao Paulo: Universidade de Sao Paulo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"","lastName":"Padilla","suffix":""},{"id":282072735,"identity":"d83c3927-882d-4cc4-872b-6f3ca5fd1003","order_by":13,"name":"Silvia Katrine Rabelo da Silva","email":"","orcid":"","institution":"Universidade Federal do Oeste do Para - Campus Tapajos: Universidade Federal do Oeste do Para","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Silvia","middleName":"Katrine Rabelo da","lastName":"Silva","suffix":""}],"badges":[],"createdAt":"2024-03-11 03:30:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4069286/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4069286/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53401595,"identity":"e859303c-058f-46ea-a653-af3b4a563180","added_by":"auto","created_at":"2024-03-25 14:38:51","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":219037,"visible":true,"origin":"","legend":"\u003cp\u003eA map of the Carbonate rocks (53°0’00′′.N E 2°0’00′′ .S)\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4069286/v1/7059f466741e9ff17898fca9.jpeg"},{"id":53402320,"identity":"a294ec64-88ed-4375-87ed-bc916758de1e","added_by":"auto","created_at":"2024-03-25 14:46:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":301249,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of PML5. (a) Colony morphology on ISP2 agar after incubation at 30◦C for 14 days. (b) Spore chains of PML5 under scanning electron microscopy\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4069286/v1/2f6aaedacfb3bf929873c028.png"},{"id":53401593,"identity":"35eb3f7f-865e-465a-a2c4-1035e5253b5f","added_by":"auto","created_at":"2024-03-25 14:38:51","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":223727,"visible":true,"origin":"","legend":"\u003cp\u003eChemical profile of PML5 metabolic extract\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4069286/v1/34811b9b415de819eed7764e.jpeg"},{"id":53401596,"identity":"5804140c-94bb-4330-8e31-dcc6ad8632e4","added_by":"auto","created_at":"2024-03-25 14:38:51","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":483561,"visible":true,"origin":"","legend":"\u003cp\u003ePML5 Mass Spectrometry\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4069286/v1/8795503d682683e9e948195c.jpeg"},{"id":53402914,"identity":"a1728d4f-2080-48ab-a8b3-1f4c4d586e0c","added_by":"auto","created_at":"2024-03-25 14:54:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1046188,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4069286/v1/5f00e548-6e9d-40bf-a155-a7087341aa16.pdf"},{"id":53401594,"identity":"52de8d04-b6a6-4573-a9a3-3bc17d548e81","added_by":"auto","created_at":"2024-03-25 14:38:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1415864,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationfiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-4069286/v1/1d0503c0acca8bcae039a7d0.docx"}],"financialInterests":"","formattedTitle":"Antibacterial, Antioxidant and Anticancer Activities of the Streptomyces PML5 strain isolated from Carbonate rocksin the Amazon","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe Amazon biome boasts the largest expanse of rainforest on the planet, and is widely recognized for its abundant biological richness. However, it is important to note that, despite the essential contribution of microorganisms to ecosystems and their critical role in ecological and biogeochemical processes, research and exploration of microbiodiversity remain relatively scarce when compared to investigations involving plants and animals (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThus, the bioprospecting of actinobacteria offers an innovative perspective in the search for antimicrobial resources, with emphasis on little-explored environments, such as sedimentary formations, such as Amazonian carbonate rocks (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Itaituba Formation, located in the western region of Par\u0026aacute; in the Brazilian Amazon, is a remarkable geological deposit dating back approximately 300\u0026nbsp;million years. Mostly composed of carbonate rocks, this sedimentary formation offers a rich source of information about the environmental and geological conditions that prevailed during the Permian Period (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInserted in the Amazon Basin, this formation composes one of the largest and oldest sedimentary basins in Brazil, preserving crucial geological events that influenced the deposition of sediments and shaped the local landscape over time, being mainly made up of minerals such as calcite and dolomite, a composition that reflects the intense biogenic activity that occurred during the Permian Period. The characteristics of these rocks surviving time indicate the presence of biogeochemical processes of degradation of organic matter, nutrient cycling, and release of chemical substances into the environment, which may result from the associated antimicrobial activity (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this scenario, the \u003cem\u003egenus Streptomyces\u003c/em\u003e plays an important role, due to the diversity of substances with pharmacological properties and diversified chemical structure, such as antibiotics, antiparasitics, anticancer, as well as enzymes of industrial application. This genus is composed of Gram-positive, filamentous bacteria with a high content of guanine and cytosine (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe objective of this research was to evaluate the antimicrobial, antioxidantand anticanceractivity of volatile compounds produced by \u003cem\u003eStreptomyces\u003c/em\u003e associated with Amazonian carbonate rocks.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cb\u003eIsolation of\u003c/b\u003e \u003cb\u003eStreptomyces\u003c/b\u003e \u003cb\u003efrom Amazonian carbonate rock\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOne sample of 10g carbonate rock was collected on Carbonate Rocks from the Itaituba Formation (04\u0026ordm;16'34\" S and 55\u0026ordm;59'01\" W) in Itaituba, Par\u0026aacute;, Brazil. The rock sample were obtained from about 10 cm depth, numbered and stored in sterile zip-lock bags. The sample were taken to the laboratory and kept at 4\u0026deg;C for further study. The sample was mixed with 90 ml of sterile distilled water to make a uniform suspension and serially diluted up to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. About 100 \u0026micro;l suspension from this sample was evenly spread on the surface of Arginine Yeast Agar Medium (AYA) \u0026ndash; (Compositiom: L-arginine 0.3 g, Glucose 1 g, Glycerol 1 g, K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 0.3 g, MgSO\u003csub\u003e4\u003c/sub\u003e*7H\u003csub\u003e2\u003c/sub\u003eO 0.2 g, KCl 0.3 g, Yeast extract 1 g, Agar 20 g, pH: 7.4) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The media was supplemented with 64 \u0026micro;g/ml of antifungal (Nistatin) to prevent the growth of fungi. The plates were then kept at 30\u0026deg;C temperature for three weeks. Distinct colonies with morphological features such as dry, wrinkled, leathery, and powdery appearance were isolated and purified using the streak method on yeast-malt extract agar medium (ISP Medium No. 2, ISP2) and also maintained at \u0026minus;\u0026thinsp;80\u0026deg;C in glycerol suspension (20%, v/v) (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCharacterizations of Potent Isolate\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eMorphological and Biochemical Characterization\u003c/h2\u003e \u003cp\u003eCharacterization of the strain was carried out according to Goodfellow and Cross (1984). The substrate and aerial mycelia color, colony shape, and pigmentation of isolate were recorded. The scanning electron microscope (SEM) analysis of the isolate was done to observe spore chain and surface morphology with the help of an SEM instrument (LEO 5410LV). The biochemical characterization was performed to study the nutritional and metabolic capabilities of the isolate, and the tests were done by actinomycetes protocols (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eProspec\u0026ccedil;\u0026atilde;o de\u003c/b\u003e \u003cb\u003eclusters\u003c/b\u003e \u003cb\u003eg\u0026ecirc;nicos de s\u0026iacute;ntese de PSK e NRPS\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA series of primers was used to detect genes related to the antibiotic biosynthesis pathway, such as the so-called polykettide synthase (PKS-II), and non-ribosomal peptides (NRPS): PKSII: KS\u0026#120572; (5\u0026rsquo;-TSGCSTGCTTGGAYGCSATC-3\u0026rsquo;) e KS\u0026#120573; (5\u0026rsquo;- TGGAANCCGCCGAABCCTCT-3\u0026rsquo;); NRPS: A3F (5\u0026rsquo;-GCS TAC SYS ATS TAC ACS TCS GG-3\u0026rsquo;) e A7R (5\u0026rsquo;-SAS GTC VCC SGT SCG GTA S-3\u0026rsquo;) (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA PCR (50\u0026micro;L) consisted of 100 ng genomic DNA (1\u0026micro;L), 5x Trans start Fast PfU buffer (10\u0026micro;L), primer 10\u0026micro;M (1 \u0026micro;L), 2,5 mM DNTP\u0026acute;s (4\u0026micro;L), Trans Start Fast Pfu polymerase (1\u0026micro;L), PCR stimulant (5x) (5\u0026micro;L), MgSO\u003csub\u003e4\u003c/sub\u003e 50mM (2\u0026micro;L) e ddH2O 25 \u0026micro;L). O ciclo de PCR ocorrer\u0026aacute; segundoas condi\u0026ccedil;\u0026otilde;es: 98\u003csup\u003e\u0026ordm;\u003c/sup\u003eC por 4\u0026rsquo;; 30 ciclos de 94\u003csup\u003e\u0026ordm;\u003c/sup\u003eC (1\u0026rsquo;), 57,5\u003csup\u003e\u0026ordm;\u003c/sup\u003eC (30\u0026rsquo;\u0026rsquo;), 72\u003csup\u003e\u0026ordm;\u003c/sup\u003e C (1\u0026rsquo;/ kb); extens\u0026atilde;o de 72\u003csup\u003e\u0026ordm;\u003c/sup\u003eC (10\u0026rsquo;). All PCR products will be separated by 1.2% agarose gel electrophoresis and bands between 1,200-1,400, 600, 1,400 and 700\u0026ndash;800 bp will be classified as products of the PKSII, and NRPS genes (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSequencing reactions will be done using the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eBigDye Terminator v3.1 Cycle Sequencing Kit\u003c/span\u003e (c\u0026oacute;digo 4337456). he runs will be made in 36cm capillaries using the POP7 polymer and the \u003cem\u003egenerated contigs\u003c/em\u003e will be analyzed by \u003cem\u003ethe Sequencing Analysis\u003c/em\u003e 7.0 software using the Base Caller KB. The acquired sequences will be manually aligned using the GenBank database with BLAST. Sequences with 98\u0026ndash;100% homology will be considered for molecular taxonomic analysis. Multiple sequence alignment will be performed for each generated sequence and the sequences from the GeneBank database with the CLUSTAL X program. The phylogenetic tree will be constructed using the neighbor-joining and maximum parsimony methods in the Molecular Evolutionary Genetic Analysis software (MEGA version 11.0.13) based on bootstrap values of 1,000 replications (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePrimary Screening for Antagonistic Activity\u003c/h2\u003e \u003cp\u003eThe primary screening of isolates was performed by agar block method Ichikawa; Date; Ishikura; Ozaki, (1971) against S\u003cem\u003etreptococcus pneumoniae\u003c/em\u003e ATCC700699; \u003cem\u003eMicrococcus lutteus\u003c/em\u003e ATCC7468; \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC14458; \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e ATCC12228; \u003cem\u003eAeromonas hydrophyla\u003c/em\u003e INCQS00318; \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e OXA48; \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC10536; \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e ATCC19606; \u003cem\u003eProteus vulgaris\u003c/em\u003e ATCC13315; \u003cem\u003eEscherichia coli ATCC25922 e Salmonella enteritidis\u003c/em\u003e INCQS00268. The antagonistic activity of actinomycete isolates was recorded to select potential isolate with bioactivity (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of Anticancer Activity by MTT Assay\u003c/h2\u003e \u003cp\u003eHuman tumor cell lines HCT-116 (colorectal carcinoma) were obtained from the American Type Culture Collection (ATCC). Cells were maintained in RPMI 1640 (HCT-116) media supplemented with 10% fetal bovine serum, supplemented with 1% antibiotics (penicillin\u0026thinsp;+\u0026thinsp;streptomycin) at 37\u0026deg;C with 5% CO2 and 95% relative humidity. To maintain cultures in exponential growth, cells were split periodically. Cytotoxic activity was evaluated by the MTT method (Mosmann 1983). An aliquot of 2 x 10\u0026sup3; cells was inserted per well into 96-well plates (10\u003csup\u003e4\u003c/sup\u003e cells/well in 200 \u0026micro;L/well) and left to adhere for 24 h. Each sample, dissolved in DMSO, was added to their respective wells, making up to final concentration 10 \u0026micro;g/mL, with each concentration tested in duplicate, and incubated for 72 h. Doxorubicin was used as a positive control, while negative control groups received the sample dilution vehicle, DMSO. At the end of the incubation period, the supernatant in each well was replaced with fresh media added with 0.5 mg/mL of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)). After 3h, the supernatant was removed, the precipitate product was dissolved in 150 \u0026micro;L DMSO and the absorbance was measured using a multiplate reader at 570 nm. Absorbance values for each treatment were transformed to percentage of cell growth inhibition based on positive (100% inhibition) and negative (0% inhibition) control parameters, for analysis of qualitative data. Inhibitory concentration values were calculated by nonlinear regression using GraphPad Prism 8 (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of Antioxidant Activity\u003c/h2\u003e \u003cp\u003eThe determination of the antioxidant activity occurred only with the most promising strain in cytotoxic activity, PML5, which will have its extract tested by the capture of the ABTS free radical performed in a spectrophotometer. The ABTS radical was prepared from the reaction of 5 mL of ABTS stock solution (7 mM) with 88 \u0026micro;L of potassium persulfate solution (140 mM) and kept at room temperature in the dark for 16 hours. After the necessary time, 1 mL of the mixture was diluted in ethyl alcohol until an absorbance of 0.7 nm\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 nm at 734 nm was obtained in a Shimadzu UV 1800 spectrometer (Shimadzu\u0026reg; 1800, Kyoto, Japan). Then, a standard trolox curve (2,000 \u0026micro;M) was constructed with concentrations ranging from 100 \u0026micro;M to 2,000 \u0026micro;M. In a dark environment, an aliquot of 30 \u0026micro;L of each trolox solution (100 \u0026micro;M, 500 \u0026micro;M, 1000 \u0026micro;M, 1500 \u0026micro;M and 2,000 \u0026micro;M) was transferred to test tubes and 3.0 mL of the ABTS radical solution was mixed. Then, it was homogenized in a vortex and after 6 min of mixing, the readings were performed at 734nm. Ethyl alcohol was used as white to calibrate the spectrophotometer. To determine the antioxidant activity in the sample, the same procedure as above was repeated with the dilution of the oil (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The percentage of inhibition was calculated according to Eq.\u0026nbsp;1 and the final concentration was expressed in \u0026micro;M Trolox /g. The analysis was performed in triplicate.\u003c/p\u003e \u003cp\u003eEquation 1: % INHIBITION = (Control Abs - Sample Abs)/Control Abs x100\u003c/p\u003e \u003cp\u003eAntioxidant activity by DPPH free radical capture was performed in a UV 1800 spectrophotometer (Shimadzu\u0026reg;, Kyoto, Japan) at a wavelength of 515 nm. The DPPH radical solution was prepared from 24 mg of DPPH in 100 mL of ethanol. Then, 10 mL of the solution was removed and transferred to a 100 mL volumetric flask and the volume was completed with ethanol to obtain the working solution. In a dark environment, an aliquot of 150 \u0026micro;L of the sample was transferred to test tubes, mixed with 5,085 mL of the DPPH radical of the working solution and homogenized in a vortex. After 30 minutes of reaction, the reading was performed in a spectrophotometer. Ethyl alcohol was used as white to calibrate the spectrophotometer. Antioxidant activity was calculated based on a standard Trolox curve (50\u0026micro;M-1000 \u0026micro;M). The percentage of inhibition was calculated according to Eq.\u0026nbsp;1 and the final concentration was expressed in \u0026micro;M Trolox /g(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS Analysis of the Promising Insulator\u003c/h2\u003e \u003cp\u003eThe analyses by high performance liquid chromatography coupled to an ultraviolet spectrum scanning photodiode detector (HPLC-UV-DAD) were developed on an Agilent 1260 chromatograph equipped with a 60 mm flow cell with detection speed at 80 Hz and detector by array of ultraviolet and visible spectrum scanning photodiodes (range between 200 to 600 nm). For the analytical-scale analyses, a reversed-phase Zorbax Eclipse plus C\u003csub\u003e18\u003c/sub\u003e column (150 x 4.6 mm) and 3.5 \u0026micro;m particle diameter maintained at 45\u003csup\u003eo\u003c/sup\u003eC was used as a stationary phase, with a mobile phase flow of 1.0 mL.min-1 and 3\u0026micro;L of sample injection volume, prepared at a concentration of 1 \u003csup\u003emg.mL\u0026minus;1\u003c/sup\u003e. The wavelengths used to record the chromatograms were 254, 280, 325 and 352 nm. The gradient elution system consisted of acidified water with 0.1% acetic acid (eluent A; Synth) and acetonitrile (eluent B; Merck) on the following schedule: 0-10min.: 10\u0026ndash;25%B; 10-30min.: 25\u0026ndash;50%B; 30-50min.: 50\u0026ndash;100%B; 50-60min.: 100%B.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGC-MS analysis of the promising isolate\u003c/h2\u003e \u003cp\u003eThe extracts were analyzed by gas chromatography (GC) in an Agilent CG 6850 equipment coupled to an Agilent 5975C mass spectrometer (EM) containing a 30m x 0.25mm HP-5MS column and 0.25\u0026micro;m film. The sample injection volume was 1\u0026micro;L in \u003cem\u003esplitless\u003c/em\u003e mode, with Helium as the carrier gas at a rate of 1mL.min-1. The method used has an initial oven temperature of 100\u0026deg;C, maintained for 5 minutes, and a heating rate of 5\u0026deg;\u003csup\u003eC.min\u0026minus;1\u003c/sup\u003e until reaching a temperature of 320\u0026deg;C, which was maintained at the end of heating for another 8 minutes. The injector, quadrupole and ion source temperatures were, respectively, 300\u0026deg;C, 180\u0026deg;C and 280\u0026deg;C. Mass spectra detections were performed by electron impact ionization (EI) at 70 eV, in \u003cem\u003efull-scan\u003c/em\u003e acquisition mode in the m/z range 50\u0026ndash;800 m/z at 2.66 scan.s-1. Metabolite identifications were performed by comparison of pattern retention time and by comparison with the NIST spectral library (v2.0, 2008) using \u003cem\u003eMatch\u003c/em\u003e and \u003cem\u003eR-Match\u003c/em\u003e comparison values above 900.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eThe PML5 isolate showed poorly developed white airy mycelium and yellow vegetative mycelium, a relevant taxonomic criterion for the identification of \u003cem\u003eStreptomyces\u003c/em\u003e (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) (Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eThe development of aerial mycelium occurred after seven days of cultivation in ISP2 medium in a greenhouse at 30\u0026ordm;C, something common to actinobacteria, since its growth is slow and can take from 7 to 15 days for complete development at this temperature (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding the arrangement of the spores, the presence of flexuous filaments in branched chains, short closed filaments was identified, all these characteristics converging with the typical morphological profile of the genus \u003cem\u003eStreptomyces\u003c/em\u003e, cited by Azuma (2012) (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe microscopic characteristics observed in the isolates showed organization and shape compatible with the genus \u003cem\u003eStreptomyces\u003c/em\u003e, as described in the literature by Alam \u003cem\u003eet.al.\u003c/em\u003e (2022). (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe biochemical and physiological characteristics of the isolate made it possible to identify the biochemical profile of the isolate with growth in lipase (3,58) and esterase (3,58), biocatalyst enzymes that are used biotechnologically in the synthesis of surfactants, textile, cosmetic and pharmaceutical industries, in addition to participating in the production of biofuels, acting without the need for cofactors, with high stability in organic solvents and wide specificity of substrates (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe PML5 isolate was positive for the enzyme Catalase, which in the study by Fang \u003cem\u003eet.al.\u003c/em\u003e (2021) was described as a substance directly involved in the biosynthesis of secondary metabolites in 2,634 \u003cem\u003eStreptomyces\u003c/em\u003e strains with catalytic properties capable of being exploited in different ways (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen exposed to nitrogen sources, PML5 was positive for L-histidine, serine, valine and L-asparigine. Each of the amino acids\u0026mdash;L-histidine, serine, valine, and L-asparagine\u0026mdash;plays key roles as sources of nitrogen in the metabolic processes of organisms. During the metabolism of L-histidine, the nitrogen present can be released and utilized in the synthesis of other nitrogen-containing molecules, such as nucleotides and neurotransmitters (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSimilarly, Serine and L-asparagine, also containing amino groups, contribute as sources of nitrogen in metabolic pathways. When metabolized, these amino acids release nitrogen, which is then used in the synthesis of a variety of molecules vital for cellular function (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eValine can be incorporated into other nitrogen-containing molecules. These processes are essential for balancing the nitrogenous metabolism of organisms, ensuring adequate nitrogen availability for various biological functions, including protein and nucleic acid synthesis (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe growth of PML5 in these nitrogen sources suggests that these microorganisms may be present in the environment where these compounds are found and may be involved in the production of bioactive compounds.\u003c/p\u003e \u003cp\u003ePML5 grew in media with carbon source D-xylose, Sucrose, Maltose, D-mannose, D-lactose and Glucose. D-xylose is a monosaccharide, commonly used in industry as a carbon source in microbiological culture media. On the other hand, sucrose is a disaccharide composed of glucose and fructose, widely found in food and used in the food industry and in microbiological culture media. Maltose, another disaccharide composed of two glucose molecules, is often employed as a carbon source in the food industry and microbiology (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eD-mannose, a monosaccharide isomer of glucose, is commercially synthesized and used in dietary supplements due to its beneficial health properties. D-lactose, a disaccharide, is an important source of carbon and energy for infants and is used in the food industry in dairy production. Finally, glucose, a monosaccharide, is essential for the metabolism of organisms and is used in various industrial sectors, from food and beverages to biofuels and pharmaceutical production. These carbon sources play crucial roles in the nutrition and metabolism of various organisms and have significant applications in industry and scientific research (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe growth of PML5 strains in these media indicates the ability of actinobacteria to use these carbohydrates as a carbon source, which can influence the degradation and cycling of nutrients in the carbonate rock environment.\u003c/p\u003e \u003cp\u003eThe Antibiotic Susceptibility Test showed that the PML5 strain is sensitive to Bacitracin (23mm) and Azithromycin (35mm) and resistant to Nalidixic Acid, Cefepime, Tetracycline, Cethazidine, Rhinoampiline, Oxacillin, Penicillin, Ketalhotin, Cephalitriaxone, Amphotericin B and Amoxicillin.\u003c/p\u003e \u003cp\u003eResistance to drug classes may be related to the production of secondary metabolites of PML5 similar to the antibiotics tested. Quinolone, Penicillin, and 1st-, 3rd-, and 4th-generation cephalosporins are classes of broad-spectrum antibiotics with different modes of action. The simultaneous production of these classes may suggest that PML 5 has the ability to synthesize compounds that act on different bacterial targets and with different mechanisms of action. Resistance to rifamine and antifungal polyene may indicate the ability of the isolate to synthesize compounds with antituberculosis and antifungal properties, respectively(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe PML5 strain showed a positive response to PKSII and NRPS, indicating its potential for the production of polyquetides and also non-ribosomal peptides. The presence of PKSII and NRPS is reported by Paulus \u003cem\u003eet.al.\u003c/em\u003e (2022) and Alonso \u003cem\u003eet.al.\u003c/em\u003e (2021) as part of genome mining for identification of biosynthetic genes from natural products. The discovery of these clusters is necessary for the search for new bioactive compounds, in order to then create the necessary conditions for the clusters to produce them, for which the PML5 strains were provided with carbon and nitrogen sources necessary for their growth and metabolism (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe PML5 isolate showed antagonistic activity against gram-positive and gram-negative pathogens, as shown in Chart 1\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHalos of pathogen growth inhibition against isolates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePathogens\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePML5\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\u003eStreptococcus pneumoniae\u003c/em\u003e ATCC700699\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;0,58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC14458\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12 \u0026plusmn; 1,0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e ATCC12228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;0,56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMicrococcus lutteus\u003c/em\u003e ATCC7468\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;1,00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e ATCC25922\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;0,00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e ATCC10536\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;0,00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e OXA48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12 \u0026plusmn; 0,59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAcinetobacter baumannii\u003c/em\u003e ATCC19606\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13 \u0026plusmn; 1,15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSalmonella enteritidis\u003c/em\u003e INCQS00268\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;0,00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eProteus vulgaris\u003c/em\u003e ATCC13315\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;0,29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAeromonas hydrophyla\u003c/em\u003e INCQS00318\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;0,58\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 antagonistic activity revealed a broad antimicrobial spectrum against Gram-positive and Gram-negative bact\u0026eacute;ria of medical interest in humans, with emphasis on \u003cem\u003eSalmonella enteritidis\u003c/em\u003e INCQS00268, exhibiting na averafe inhibition halo of 16\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0 mm. The most resistant pathogen was \u003cem\u003eAeromonas hydrophyla\u003c/em\u003e INCQS00318with na inhbition halo of 10\u0026thinsp;\u0026plusmn;\u0026thinsp;1,15 mm.\u003c/p\u003e \u003cp\u003eIn the research by Bhat e Nayaka (2023), the cave isolate YC69 had a greater halo of inhibition against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e when compared to the other 69 isolates. Similarly, Jaroszewicz et.al. (2021) tested isolates from limestone rocks, where the one with the highest spectrum against \u003cem\u003eStaphylococcus aureus, Salmonella enterica, Enterococcus\u003c/em\u003e sp., \u003cem\u003eEscherichia coli\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e for antic\u0026acirc;ncer testing (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe acetatethyl extract of the PML5 isolate showed greater cytotoxic action (96.5%) against the growth of human colorectal cancer cells (HCT-116). The cytotoxic action of extracts of PML11 and PML15 isolates against HCT-116 cells was 33.40% and 58.55%, respectively.\u003c/p\u003e \u003cp\u003eResearchers Bhat and Nayaka (2023) obtained a 41.98% inhibition of cell proliferation in vitro by subjecting the YC69 isolate against human cervical cancer cells (HeLa) to cytotoxic testing. For Jaroszewicz et.al. (2021), the isolates of Streptomyces sp. selected for cytotoxic testing reduced the viability of the breast cancer cell line (T47D). (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe use of the cytotoxic MTT assay facilitates the determination of the cytotoxicity of a sample, although it does not clarify its mechanism of action, the analysis of cytotoxicity represents a crucial stage in the process of creating new drugs for clinical use (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResearcher Huang et.al. (2023) tested the cytotoxic action of mangrove sediment isolate 4503, with morphological and chemical properties converging to the genus Streptomyces. The isolate showed cytotoxic activity against nasopharyngeal carcinoma (NPC) cell lines with antiproliferation, antimetastasis, induction of cell cycle arrest and apoptosis, in addition to increasing the production of reactive oxygen species, which led to the need to also explore the antioxidant potential of the isolate (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe antioxidant action detected by the ABTS method showed an action of 35.60\u0026thinsp;+\u0026thinsp;2.01%, while the antioxidant potential by the capture of the free radical DPPH had an inhibition of 10.65\u0026thinsp;+\u0026thinsp;1.95%. The antioxidant activity revealed by the ABTS and DPPH method suggests a significant capacity of actinobacteria isolated from carbonate rocks in the Amazon to neutralize free radicals, reflecting a promising biological potential for health and technology applications. This capacity is evidenced by the outstanding performance of the actinobacterium Streptomyces sp., isolated from the Caatinga, whose cultural filtrates demonstrated high antioxidant activity through the DPPH and ABTS methods, in addition to having high phenolic and flavonoid contents (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor Rammali et.al. (2022) a response percentage greater than 30% demonstrates the high capacity of secondary metabolites of actinobacteria to capture the ABTS free radical. For Weslati et.al. (2023) A DPPH eliminator response above 1.3 \u0026micro;g is highly active. By relating the research with the result of 35.60% of activity in the capture of ABTS and 138.56 \u0026micro;g of PML5, a greater potential of the carbonate rock isolate is evidenced (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition, previous studies highlight the importance of ABTS and DPPH methods as reliable tools for evaluating the antioxidant capacity of plant extracts and isolated compounds. Weslati, Sim\u0026otilde;es, Texeira, Parpot et.al. (2023) detailed the ability of several antioxidants to react with the DPPH radical, providing a solid basis for future comparisons of antioxidant activity. These methods are widely recognized for their efficacy in quantifying antioxidant capacity, which is fundamental for studies such as the present one, which seeks to explore the potential of new sources of antioxidants (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe metabolic pathway of PML5 was developed to synthesize four different substances, as shown in Fig.\u0026nbsp;5, where well-defined peaks were identified.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the mass spectrum, it was identified that the absorbance of substances 5,791 and 13,556 were similar to 9,921 and 17,077, respectively. This indicates that they have the same chromophores with conjugated double bonds, i.e., the same skeleton, suggesting the possibility that they are of the same class (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGC-MS analysis of the extract showed strong antimicrobial and antitumor activity (Table\u0026nbsp;6). We identified 10 different volatile compounds from the active metabolite of the isolate (between 14.81min and 37.53 min) and were recognized for their antimicrobial and antitumor activities. Cyclododecane (83.75%) was the main compound identified in the extract.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIdentification of chemical compounds of the PML5 isolate using GC-MS analysis.\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\u003eTime (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArea (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM.W\u003c/p\u003e \u003cp\u003e(g/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMolecular Formula\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCompound Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBioactivity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14.816\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e206.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2,4-Di-tert-butylphenol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAntitumor activity (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e83.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e168,32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e24​\u003c/sub\u003e​\u003csub\u003e​\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCyclododecane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAntimicrobial and antitumor activity (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21.255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e316.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e36\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTetradecanoic acid, 12-methyl-, methyl ester, (S)-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAntioxidant and antitumor activity (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23.144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e270.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePentadecanoic acid, 14-methyl-, methyl ester\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAntioxidant and antitumor activity (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23.551\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e196,2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCyclo(L-prolyl-L-valine)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAntimicrobial activity (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23.902\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e210.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e11\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAntimicrobial activity (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25.262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e316.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e36\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTetradecanoic acid, 12-methyl-, methyl ester, (S)-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAntioxidant and anti-rumoral activity (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25.537\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e168.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCyclododecane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAntimicrobial and antitumor activity (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e32.289\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e172.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4-Octene, (Z)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAntimicrobial Activity (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e37.536\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e166.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1,4-Benzenedicarboxylic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAntimicrobial Activity (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e)\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 components determined by GC-MS showed antimicrobial, antioxidant and antitumor activities. 2,4-Di-tert-butylphenol has anticancer properties against Mycobacterium tuberculosis, antioxidant activity of 2,4-DTB, and response of 88% and 89% against colon cancer and uterine cancer, respectively, in Kaari's research; Joseph; Manikkam; Kalyanasundaram et al., (2023). The compound Cyclododecane, which appeared in two moments (19.02min and 25.53min) during chromatography. In the literature, it is described as a substance present in medications for bronchitis, hemomatirua, epilepsy, asthma, leprosy, eczema, debre and jaundice, in addition to having antitumor and antioxidant capacity.\u003c/p\u003e \u003cp\u003eThe Tetradecanoic acid, 12-methyl-, methyl ester, (S)- also appeared twice during the analysis (21.25min and 25.26min) and its action has antioxidant and antitumor activity as well as the Pentadecanoic acid, 14-methyl-, methyl ester (KEMUNG; TAN; CHAN; SER et al., 2020). Other antimicrobial substances were Cyclo(L-prolyl-L-valine), Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-, 4-Octene, (Z), 1,4-Benzenedicarboxylic acid, which have antimicrobial potential against gram-positive and negative bacteria, some even resistant to antimethicillin resistant Staphylococcus aureus (MRSA)(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThe carbonate rocks of the Itaituba Formation are a rich source of underexplored environmental and geological information for new bioactive microorganisms. The study investigated the isolation, characterization and application of actinobacteria isolated from carbonate sediments, called PML5.\u003c/p\u003e \u003cp\u003eThe morphological and molecular profile showed convergent patterns with the genus \u003cem\u003eStreptomyces sp\u003c/em\u003e. The antimicrobial activity was more sensitive for \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 10536 (13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 mm) and \u003cem\u003eProteus vulgaris\u003c/em\u003e ATCC 13315 (12.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 mm) and more resistant for \u003cem\u003eAcinetobacter baumanii\u003c/em\u003e ATCC 19606 (11.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 mm). The cytotoxic test inhibited the colorectal cancer cell line by 96.55% and the antioxidant action protected by the ABTS method showed an action of 35.60\u0026thinsp;+\u0026thinsp;2.01%, while the antioxidant potential by capturing the DPPH free radical had a limitation of 10.65\u0026thinsp;+\u0026thinsp;1.95%.\u003c/p\u003e \u003cp\u003eThe LC-MS analysis showed the presence of four peaks, where it was identified that the absorbance of substances 5,791 and 13,556 were similar to 9,921 and 17,077, respectively; This suggests the possibility that PML 5 has a metabolic route synthesizing substances of the same class. The GC-MS of the extract showed the presence of 10 volatile compounds. The main compound was Cyclododecane (83.75%) compared to other bioactive compounds.\u003c/p\u003e \u003cp\u003eThese results reinforce the potential of Amazonian biodiversity, revealing that actinobacteria produce antimicrobial and cytotoxic bioactive substances of interest to the pharmaceutical industry.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e-Ethical Approval (Missing)\u003cbr\u003e-Consent to Participate(Missing)\u003cbr\u003e-Consent to Publish (Missing)\u003cbr\u003e-Authors Contributions\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the conception and design of the study. The preparation of the material, collection and analysis of data were carried out by Karine Rodrigues do Nascimento Chaves, Maria Lucila Texeira de Andrade Fran\u0026ccedil;a, Anna Ludmylla Oliveira Mentes, Pabl\u0026iacute;cio Pereira Cardoso, Kamila Brielle Pantoja Vasconcelos, Raphael Carlos Ferrer de Santana, Let\u0026iacute;cia Veras Costa Lotufo, Anderson Concei\u0026ccedil;\u0026atilde;o Mendes, Lindalva Maria de Meneses Costa Ferreira, Sara Freitas de Sousa Ramos, Isabella Cristhina Gon\u0026ccedil;alves Costa, Marcelo J. Pena Ferreira and Gabriel Padilla. The first draft of the manuscript was written by Karine Rodrigues do Nascimento Chaves and Silvia Katrine Rabelo da Silva and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e-Funding\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Universidade Federal do Oeste do Par\u0026aacute;, Universidade Federal do Par\u0026aacute; and Universidade de S\u0026atilde;o Paulo.\u003c/p\u003e\n\u003cp\u003e-Competing Interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e-Availability of data and materials (Missing)\u003c/p\u003e\n\u003cp\u003eThe authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request. Source data are provided with this paper. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ede Azevedo, J. H., \u0026amp; Campos, J. E. G. 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Saudi J Biol Sci.\u003c/span\u003e\u003c/li\u003e\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":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Streptomyces. Bioinnovation. Biotechnological potential. Cytotoxic Potential. Antioxidant action","lastPublishedDoi":"10.21203/rs.3.rs-4069286/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4069286/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe aim of this study was to investigate the antimicrobial and cytotoxic potential of \u003cem\u003eStreptomyces\u003c/em\u003e sp. isolated from carbonate rock in the Amazon. The antimicrobial activity was verified by the diffusion technique in agar against pathogens such as \u003cem\u003eMicrococcus lutteus\u003c/em\u003e ATCC 7468, \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e ATCC700699; \u003cem\u003eProteus vulgaris\u003c/em\u003e ATCC 13315, \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 10536, \u003cem\u003eAcinetobacter baumanii\u003c/em\u003e ATCC 19606.Cytotoxic activity was investigated ​​by the inhibition of cell growth (%) of samples tested in human colorectal carcinoma cell line (HCT116), at a concentration of 10 \u0026micro;g/mL (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM; n\u0026thinsp;=\u0026thinsp;2). The isolate showed a broad spectrum of antimicrobial activity against test microorganisms. This isolate showed maximum antibacterial activity against \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 10536 (13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 mm) and \u003cem\u003eProteus vulgaris\u003c/em\u003e ATCC 13315 (12.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 mm) and the most resistant microorganism against antibacterial activity of this isolate was \u003cem\u003eAcinetobacter baumanii\u003c/em\u003e ATCC 19606 (11.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 mm). This metabolites was extracted by \u003cem\u003esolid\u003c/em\u003e:\u003cem\u003eliquid\u003c/em\u003e method with ethyl acetate organic solvent. ​​The percentage of inhibition of cell growth of the crude extract tested in a human colorectal carcinoma line (HCT116) was 96,55%. The determination of antioxidant activity by capturing the free radical ABTS had an inhibition of 35.60\u0026thinsp;\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;2.01%, while the antioxidant potential by capturing the free radical DPPH had an inhibition of 10.65\u0026thinsp;\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;1.95%. The LC-MS analysis showed the presence of four peaks, where it was identified that the absorbance of substances 5,791 and 13,556 were similar to 9,921 and 17,077, respectively; this suggests the possibility of PML 5 having a metabolic pathway synthesizing substances of the same class. The CG-MS of the extract showed the presence of 10 volatile compounds. The main compound was Cyclododecane (83.75%) compared to other bioactive compounds. These results reinforce the potential of Amazonian biodiversity, revealing that actinobacteria produce antimicrobial and cytotoxic bioactive substances of interest to the pharmaceutical industry.\u003c/p\u003e","manuscriptTitle":"Antibacterial, Antioxidant and Anticancer Activities of the Streptomyces PML5 strain isolated from Carbonate rocksin the Amazon","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-25 14:38:46","doi":"10.21203/rs.3.rs-4069286/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Resubmit revised form; Major revisions required","date":"2024-05-05T23:04:07+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-04-16T05:12:46+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-21T04:20:57+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Applied Biochemistry and Biotechnology","date":"2024-03-18T11:35:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Applied Biochemistry and Biotechnology","date":"2024-03-15T12:27:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c872cf81-1f02-45a3-b569-3c1ca7acf814","owner":[],"postedDate":"March 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2024-05-06T03:04:29+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-25 14:38:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4069286","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4069286","identity":"rs-4069286","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}