Isolation, Molecular Characterization, And Biotechnological Evaluation Of Streptomyces Isolates From Najaf Soil For Enzyme Production, Antimicrobial, And Anticancer Activities

Actinomycetes are a diverse group of Gram-positive filamentous bacteria widely distributed in terrestrial and aquatic environments. These microorganisms are characterized by branching mycelial structures similar to fungal hyphae and possess a high guanine–cytosine (G+C) content greater than 55%, which places them within the phylum Actinobacteria (Farda et al., 2020). Actinomycetes play an important ecological role in soil ecosystems by participating in the decomposition of complex organic materials and nutrient cycling, thereby contributing significantly to soil fertility and environmental sustainability. Among the various actinomycete genera, Streptomyces represents the most abundant and widely studied group in soil environments. Members of this genus are well known for their ability to produce a wide range of secondary metabolites, antibiotics, enzymes, and other biologically active compounds with important applications in medicine and biotechnology (Chiani et al., 2010; Dai et al., 2021). These microorganisms are particularly recognized as one of the most important natural sources of antibiotics used in modern medicine.

Several species of Streptomyces have been identified as major producers of industrial enzymes and therapeutic compounds. Important species include Streptomyces griseus, which produces the antibiotic streptomycin; Streptomyces rimosus, a producer of oxytetracycline; Streptomyces aureofaciens, known for producing chlortetracycline; Streptomyces venezuelae, which produces chloramphenicol; and Streptomyces avermitilis, which produces avermectins, widely used as antiparasitic agents (Barka et al., 2016; Xie et al., 2022). In addition, species such as Streptomyces coelicolor and Streptomyces lividans are frequently used as model organisms in molecular biology and genetics due to their well-characterized genomes and metabolic pathways (Goodfellow et al., 2012).

Actinomycetes produce numerous extracellular enzymes that enable them to degrade complex organic compounds in soil and sediment ecosystems. These enzymes include amylases, cellulases, proteases, gelatinases, lectinases, and ureases, which contribute significantly to the breakdown of organic matter and microbial nutrient utilization (Gluve & Deshmukh, 2012). Because of their enzymatic versatility, actinomycetes have attracted considerable interest for applications in industrial biotechnology, pharmaceutical production, environmental bioremediation, and agricultural processes. Among these enzymes, amylases are widely used in industrial processes, particularly in starch processing industries. Amylases catalyze the hydrolysis of starch into simpler sugars such as oligosaccharides and monosaccharides, which are used in the production of syrups and various food products. In textile industries, amylase enzymes are also used in the desizing process of fabrics, where they remove starch coatings from textile fibers (Tonkova, 2006). Several actinomycete strains have been reported to produce amylase enzymes with significant industrial importance (Kar & Ray, 2008).

Another important enzyme group produced by actinomycetes is cellulases, which are extracellular enzymes responsible for the degradation of cellulose. Cellulose is one of the most abundant organic polymers in nature and represents a major component of plant biomass. Cellulolytic microorganisms therefore play an essential role in the bioconversion of cellulose into fermentable sugars, which can be used for the production of bioethanol and other bio-based products (Balamurugan et al., 2011). Due to their efficiency and versatility, cellulases have gained significant attention for applications in pulp and paper industries, textile processing, biofuel production, brewing, agriculture, and food industries (Kuhad et al., 2011). The enzymatic hydrolysis of cellulose into commercially valuable products such as biofuels and animal feed has therefore become an important area of research in biotechnology (Barros et al., 2010).

In addition to hydrolytic enzymes, actinomycetes are capable of producing enzymes with therapeutic applications, such as L-asparaginase, which has been widely used in cancer chemotherapy. L-asparaginase is an important enzyme used in the treatment of acute lymphoblastic leukemia and lymphosarcoma, particularly in pediatric patients (Khamna et al., 2009; Kumar & Selvam, 2011). The search for new microbial sources of L-asparaginase continues to be an important research focus because different microbial strains may produce enzymes with improved therapeutic efficiency and reduced side effects (Savitri & Azmi, 2003). Proteases represent another major class of enzymes produced by actinomycetes and are among the most commercially valuable enzymes in biotechnology. These enzymes catalyze the hydrolysis of peptide bonds in proteins and have extensive applications in detergent formulations, food processing, pharmaceuticals, leather treatment, and waste management (Deng et al., 2010; Ribitsch et al., 2010).

Apart from enzyme production, actinomycetes are also recognized as the most prolific producers of antibiotics and other bioactive secondary metabolites. It is estimated that 70–80% of known antibiotics originate from actinomycetes, particularly from species belonging to the genus Streptomyces (Rajivgandhi et al., 2021; Xie et al., 2022). These microorganisms produce a wide range of antimicrobial compounds including anthracyclines, aminoglycosides, carbapenems, cephalosporins, quinolones, and other pharmacologically important molecules (Raja & Prabakarana, 2011). In addition to antimicrobial properties, many actinomycete-derived compounds exhibit antitumor, antiviral, antifungal, immunomodulatory, and anti-inflammatory activities, making them valuable resources for drug discovery (Rani et al., 2021; He et al., 2022).

In recent decades, the emergence of antibiotic-resistant pathogens has become a major global health concern, significantly reducing the effectiveness of many existing antimicrobial drugs. Multidrug-resistant bacteria are increasingly reported worldwide, creating an urgent need for the discovery of novel antimicrobial compounds and alternative therapeutic agents. In this context, actinomycetes remain one of the most promising sources for discovering new antibiotics and bioactive metabolites (Cimermanova et al., 2021; AbdElgawad et al., 2021).

Recent advances in molecular biology and microbial taxonomy, particularly through the use of polymerase chain reaction (PCR) and 16S rRNA gene sequencing, have significantly improved the classification and identification of actinomycetes (Devanshi et al., 2021; Rathore et al., 2021). These molecular techniques allow accurate determination of phylogenetic relationships and facilitate the discovery of previously unrecognized actinomycete species (Jagannathan et al., 2021). In addition to molecular methods, morphological characteristics such as vegetative mycelium formation, aerial hyphae, and spore formation are also important criteria used in actinomycete classification (Mishra et al., 2021; Bhattacharyya et al., 2022).

Considering the remarkable metabolic diversity and industrial importance of actinomycetes, continued exploration of soil microorganisms remains essential for discovering novel enzymes and bioactive compounds. Therefore, the present study aims to isolate and characterize actinomycetes from soil samples collected in Najaf Province, Iraq, and to evaluate their potential for enzyme production, antimicrobial activity, and anticancer applications using biochemical and molecular approaches.

2.0 MATERIALS AND METHODS

2.1 Study Area And Soil Sample Collection

Soil samples were collected from various agricultural and environmental locations in Najaf Province, Iraq, between March and May 2023 to isolate actinomycetes capable of producing biologically active biomolecules. The region is characterized by sedimentary plain soils that support agricultural activities such as wheat, barley, maize, cucumbers, and tomatoes, particularly in areas surrounding palm groves and irrigation systems near the Ghazi River.

A total of seventeen soil samples were collected from seven transects representing different land-use patterns across the study area. Soil samples were obtained using a sterile hand auger at a depth of approximately 20 cm, transferred into sterile polyethylene bags, and transported to the laboratory for further processing. The samples were air-dried at room temperature, crushed using a porcelain mortar and pestle, and passed through a 2-mm sieve to remove debris and large particles prior to analysis (McCauley, 2005).

2.1.1 Soil Texture

Soil texture was determined and spatial distribution maps were generated using Geographic Information System (GIS) techniques (Brown, 2003; Salwan et al., 2011).

2.1.2 Soil Physicochemical Properties

The physicochemical properties of the soil samples were also analysed. Soil pH and electrical conductivity were measured using a 1:1 soil–water extract, where electrical conductivity was measured using an EC meter and pH using a digital pH meter (Richards, 1954; Rhoades et al., 2012). Soil water holding capacity and permeability were determined following standard soil analysis procedures (Walworth, 2012).

2.1.3 Isolation Of Actinomycetes

Isolation of actinomycetes was carried out using the serial dilution plating technique. Soil samples were pre-treated with calcium carbonate and incubated for 24 h to enhance the growth of actinomycetes while suppressing other microorganisms (Hayakawa & Nonomura, 2003). One gram of soil sample was taken and serially diluted up to 10^{− 2} using distilled water as diluent. The mixture was shaken vigorously using a vortex;0.1 ml of each dilution was placed on starch casein agar (soluble starch: 10 g, K₂HPO₄ – 2.0 g, KNO₃ – 2.0 g, Casein – 0.3 g, MgSO₄·7H₂O – 0.05 g, CaCO₃ – 0.02 g, FeSO₄·7H₂O – 0.01 g, Agar – 15.0 g, distilled water – 1000 mL, pH – 7.0 ± 0.1.), and the inoculum was spread properly using a sterile glass spreader. The inoculated plates were allowed to stand at room temperature for 5–10 minutes to allow the liquid to be absorbed and were incubated at 28°C for 7 days.

2.2 Morphological Identification

Colonies exhibiting characteristic actinomycete morphology such as filamentous growth, powdery appearance, and aerial mycelium formation were selected and repeatedly sub-cultured to obtain pure isolates. These isolates were preserved for further biochemical and molecular analyses (Barka et al., 2016).

2.3 Identification

Identification of the actinomycetes was done on the basis of macroscopic and microscopic examination and physiological tests as suggested by Bergey’s Manual of Systematic Bacteriology, 2 ^nd Edition, Vol 5, the Actinobacteria, Part A.

2.4 Macroscopic Characterization.

The isolated actinomycetes were observed for aerial mycelium, submerged mycelium, colors, and diffusible pigments Sapkota et al., 2020.

2.5 Microscopic Observation.

Microscopic examination was performed by cover slip and Gram-staining method. Sterile cover slip was inserted into the solidified starch casein agar medium plate at an inclination of 45° with the agar surface. Actinomycete isolate was inoculated along the surface of the medium that meets the surface of the buried cover slip. It was incubated at 28° C for four days. The cover slip was removed using sterile forceps and placed on an individual clean glass slide, which were then observed at oil immersion objective Sapkota et al., 2020.

2.6 Biochemical Tests.

For all the isolates, a loop full of colony of pure culture of about 7 days of incubation was placed in starch casein broth and incubated at 28° C for 4 days. After the appearance of turbidity, the culture suspension was used for different sugar utilization tests, protein utilization test, and catalase and oxidase tests. Different hydrolysis tests, namely, starch hydrolysis, casein hydrolysis, lipid hydrolysis, and gelatin hydrolysis tests, were performed Sapkota et al., 2020.

2.7 Screening For Enzyme Production

The isolated actinomycete strains were screened for the production of several industrially important enzymes including amylase, cellulase, protease, gelatinase, lipase, and L-asparaginase using standard microbiological methods.

2.7.1 Amylase Production

Amylase activity was determined using starch agar medium, where plates were incubated at 28°C for five days and subsequently flooded with iodine solution. The formation of clear halos around colonies indicated starch hydrolysis and positive amylase production (Gupta et al., 2003; Sharma et al., 2017).

2.7.2 Cellulase Production

Cellulase production was evaluated using carboxymethyl cellulose (CMC) agar medium. After incubation, plates were flooded with iodine solution, and the presence of clear zones around colonies indicated cellulose degradation (Singhania et al., 2013).

2.7.3 Protease Production

Protease activity was detected using skim milk agar medium, where the formation of clear zones surrounding colonies indicated casein hydrolysis and positive protease activity (Rao et al., 2009).

2.7.4 Gelatinase Production

Gelatinase production was evaluated using gelatin agar medium. Plates were incubated for 4–5 days at 28°C, and gelatin hydrolysis was detected by the appearance of clear zones around colonies after the addition of ammonium sulfate solution (Gupta et al., 2002).

2.7.5 Lipase Production

Lipase activity was determined using tributyrin agar or Tween-80 agar medium. Clear halos surrounding colonies after incubation indicated lipid hydrolysis and positive lipase production (Jaeger & Eggert, 2002; Sharma et al., 2017).

2.7.6 L-Asparaginase Enzyme Production

The production of L-asparaginase enzyme was detected using asparagine-glucose agar medium containing phenol red indicator. A color change from yellow to pink indicated ammonia production resulting from L-asparagine hydrolysis, confirming enzyme activity (Verma et al., 2007; Batool et al., 2016).

2.8 Antimicrobial Activity Assay

The antimicrobial activity of the isolated actinomycete strains was evaluated against pathogenic microorganisms including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Morganella sp., Klebsiella pneumoniae, Candida albicans, and Candida glabrata. The assay was performed using the perpendicular streak method on Mueller–Hinton agar medium. In this method, the actinomycete isolate was first streaked across the center of the agar plate and incubated at 28°C for 5 days to allow the production of secondary metabolites. Following incubation, the test pathogens were streaked perpendicularly to the actinomycete growth and the plates were incubated at 37°C for 24 hours. Antimicrobial activity was determined by observing the inhibition zones formed between the actinomycete growth and the test microorganisms, indicating the production of antimicrobial compounds (Balouiri et al., 2016).

2.9 Molecular Characterization

Genomic DNA was extracted from purified actinomycete cultures using standard bacterial DNA extraction protocols. DNA purity and concentration were evaluated using spectrophotometric analysis and agarose gel electrophoresis. Molecular identification of the isolates was performed by PCR amplification of the 16S rRNA gene, which is widely used for bacterial taxonomy and phylogenetic analysis. The amplified PCR products were separated using 1% agarose gel electrophoresis, purified, and sequenced. The obtained sequences were compared with those available in the NCBI GenBank database using BLAST analysis to determine the closest phylogenetic relationships (Clarridge, 2004; Yarza et al., 2014).

2.9 Genomic And Proteomic Analysis

Genomic analysis was conducted to identify gene clusters responsible for secondary metabolite biosynthesis and enzyme production. Gene annotation and pathway prediction were carried out using bioinformatics databases including GenBank, KEGG, and UniProt (Medema et al., 2015). Proteomic analysis was performed using SDS-PAGE, followed by Coomassie Brilliant Blue staining to visualize protein expression profiles. Selected protein bands were further analyzed using mass spectrometry techniques to determine protein identity and biological function (Aebersold & Mann, 2016).

2.10 Bioinformatics Analysis

DNA and protein sequences obtained from the isolates were analyzed using bioinformatics tools to identify genes associated with biomolecule production. Sequence similarity searches were conducted using the BLAST algorithm in the NCBI database. Functional annotation and metabolic pathway analysis were performed using KEGG and UniProt databases, and phylogenetic trees were constructed to evaluate evolutionary relationships among microbial isolates (Kanehisa et al., 2021).

3.0 RESULTS

3.1 Isolation And Morphological Characterization Of Actinomycetes

A total of 37 actinomycete isolates were obtained from soil samples collected from different agricultural sites in Najaf Province, Iraq, using the serial dilution technique. Isolates showed typical morphological characteristics of actinomycetes, under the microscope, filamentous growth, well-developed aerial mycelia, and powdery colony surfaces. Colony pigmentation varied among isolates, with colors ranging from white and gray to pale yellow and cream (Table-01). Based on cultural and morphological characteristics, the isolates were tentatively identified as members of the genus Streptomyces, which is widely known for producing bioactive secondary metabolites and industrially important enzymes (Image-01 & 02).

Image 01: Sampling Sites in Najaf Province, Iraq

Map showing the soil sampling locations in Najaf Province, Iraq, where soil samples were collected for the isolation of actinomycetes. The selected sampling sites represent different agricultural and environmental areas used for microbial isolation and biochemical and morphological investigation.

Image 02 Colony Morphology of Actinomycete Isolates on Agar Plates

Image 1: (A) Representative colony morphology of actinomycete isolates grown, the colonies exhibit variations in color and texture, including white, gray, and pale yellow colonies typical of Streptomyces species (B) Agar medium showing characteristic. (C) Screening the plates for the actinomycetes. (D) Filamentous growth and aerial mycelium formation.

Table 1. Morphological characteristics of selected actinomycete isolate

3.2 ENZYME PRODUCTION SCREENING

All actinomycete isolates were screened for the production of extracellular enzymes including amylase, cellulase, protease, gelatinase, lipase, and L-asparaginase. The results showed that several isolates exhibited strong enzymatic activity, indicating their potential industrial and medical applications. Amylase activity was the most frequently observed enzyme among the isolates, followed by protease and cellulase activities. Gelatinase and lipase activities were detected in fewer isolates, while L-asparaginase activity was observed only in selected isolates, suggesting potential therapeutic importance (Table -2 & Image-03 & Grapgh-01).

(+ = positive activity, − = no activity)

Table 2. Enzyme production by actinomycete isolates

Image-03 Enzyme Production by Actinomycete Isolates

Graph -01 Enzyme production by actinomycetes isolation

3.3 Antimicrobial Activity Of Actinomycete Isolates

The antimicrobial activity of selected actinomycete isolates was evaluated against several pathogenic microorganisms including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Morganella sp., Klebsiella pneumoniae, Candida albicans, and Candida glabrata. The results demonstrated that several isolates exhibited significant antimicrobial activity, producing inhibition zones ranging from 9 mm to 22 mm. Among the tested pathogens, Staphylococcus aureus showed the highest susceptibility to the actinomycete isolates (Table-03 & Image-04 & Graph-02).

Table 3. Antimicrobial activity of selected actinomycete isolates (zone of inhibition in mm)

Image-04 Antimicrobial Activity of Actinomycete Isolates

Antimicrobial activity of actinomycete isolates against selected pathogenic microorganisms demonstrated by the formation of inhibition zones on agar plates. The clear zones surrounding the colonies indicate the production of antimicrobial metabolites by the isolates.

Graph 02 Antibacterial activity with isolated actinomycetes

3.4 MOLECULAR IDENTIFICATION OF SELECTED ISOLATES

PCR amplification of the 16S rRNA gene from selected isolates produced a DNA fragment of approximately 1500 base pairs, confirming successful amplification. Sequence analysis using the NCBI BLAST database revealed that the isolates showed high similarity with members of the genus Streptomyces. This molecular identification confirmed the taxonomic classification of the isolates as Streptomyces species (Image-05).

Image-05 Agarose Gel Electrophoresis of PCR-Amplified 16S rRNA Gene

Agarose gel electrophoresis showing PCR amplification of the 16S rRNA gene from selected actinomycete isolates. Lane L represents the DNA ladder marker, while the sample lanes show amplified fragments of approximately 1500 bp, confirming successful amplification of the bacterial 16S rRNA gene used for molecular identification.

3.5 Molecular Identification And Phylogenetic Analysis

The molecular identification of the selected actinomycete isolates was carried out through 16S rRNA gene sequencing. Genomic DNA was extracted from four representative isolates (SNA-1, SNA-2, SNA-3, and SNA-4) and the amplified 16S rRNA gene fragments were sequenced. Sequencing was performed at Barcode Biosciences, Bangalore, and the obtained nucleotide sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) available in the NCBI database. The BLAST analysis showed that the sequences obtained from the isolates exhibited 95–98% similarity with members of the genus Streptomyces. These results confirmed that the isolated strains belong to the Actinobacteria group, particularly the genus Streptomyces, which is well known for producing bioactive secondary metabolites. To determine the evolutionary relationships between the isolates and closely related reference strains, the obtained sequences were aligned with sequences retrieved from the NCBI GenBank and EzTaxon databases using Clustal W alignment software. A phylogenetic tree was constructed using the neighbor-joining method to evaluate the phylogenetic relationships among the isolates and related Streptomyces species. The phylogenetic tree analysis demonstrated that the four isolates formed clusters with several known Streptomyces species. The isolate SNA-1 showed a close phylogenetic relationship with Streptomyces fradiae, while SNA-2 clustered with Streptomyces lavendulae. The isolate SNA-3 showed strong similarity with Streptomyces flavofuscus, and SNA-4 clustered with Streptomyces ginsengensis.

These species are widely recognized for their ability to produce antibiotics, enzymes, and othebiologically active secondary metabolite (Phylogenetic tree Figure-01). Bootstrap analysis based on 1000 replicates was performed to assess the reliability of the phylogenetic tree. The resulting dendrogram indicated that the isolates share strong evolutionary relationships with previously reported antibiotic-producing Streptomyces species. Based on molecular identification and phylogenetic clustering, the four isolates were therefore identified as Streptomyces species closely related to Streptomyces fradiae, Streptomyces lavendulae, Streptomyces flavofuscus, and Streptomyces ginsengensis. These results confirm that the isolates SNA-1, SNA-2, SNA-3, and SNA-4 belong to the genus Streptomyces and possess significant potential for the production of bioactive biomolecules with antimicrobial, enzymatic, and anticancer properties. Phylogenetic tree constructed using the neighbor-joining method based on the 16S rRNA gene sequences of four actinomycete isolates (SNA-1, SNA-2, SNA-3, and SNA-4) and closely related Streptomyces species retrieved from the NCBI database. The tree was rooted using Streptomyces himastatinicus ATCC 53653 as an outgroup.

Figure 1. Phylogenetic tree based on 16S rRNA gene sequences of actinomycete isolates.