Thermostable Collagenase Derived from Streptomyces scabies Demonstrates Selective Antibacterial Activity Against Infections in Diabetic Foot Ulcers

Abstract

Diabetic foot ulcers (DFUs) and other chronic wounds are major global health challenges, often complicated by infections and delayed healing due to excessive collagen accumulation. Microbial collagenases offer an enzymatic alternative to surgical debridement by selectively degrading collagen and potentially limiting microbial colonization. In this study, an isolated and characterized thermostable collagenase from Streptomyces scabies from rhizospheric soil in Al-Lith thermal springs, Saudi Arabia, is investigated. Identification was confirmed via 16S rRNA sequencing, and enzyme production was optimized on gelatin agar. Partial purification was achieved through ammonium sulfate precipitation and dialysis, and molecular weight (~25 kDa) was determined by Sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Activity was assessed under varying temperatures, pH, substrates, and metal ions, while antibacterial potential was tested against Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The collagenase exhibited optimal activity at 80 °C and pH 9, stability under thermophilic and alkaline conditions, activation by Fe²⁺, and notable antibacterial effects at higher concentrations. These results demonstrate that S. scabies collagenase exhibits selective antibacterial activity in vitro, suggesting its potential as an enzymatic tool for further evaluation in diabetic foot debridement and infection control.

1. Introduction

Diabetic foot ulcers (DFUs) are among the most crippling of the many systemic consequences linked to diabetes mellitus (DM), a serious global health concern. The International Working Group on the Diabetic Foot (IWGDF) defines DFUs as open lesions or sores that develop on the foot and often spread from the deep dermal layer into the epidermis. In diabetic patients, poor glycemic management and immunological dysfunction frequently make these ulcers worse. Peripheral neuropathy, ischemia, and opportunistic infections are the usual causes of these ulcers. DFUs have a complicated and frequently multi-microbial etiology. Pathogenesis is largely influenced by both Gram-positive cocci, especially methicillin-resistant Staphylococcus aureus (MRSA), and Gram-negative bacteria, including Proteus species, Klebsiella species, and Escherichia coli [1,2].

Typically, wound dressing, metabolic stabilization, antibiotic medication, and mechanical or debridement surgery of necrotic tissue are all part of standard care for DFUs. Adjunctive techniques that enhance healing while lowering antibiotic reliance are necessary, as therapeutic success is limited by recurrent recurrence, poor vascularization, and the emergence of antimicrobial resistance [3]. Compared to surgical techniques, enzymatic debridement has drawn interest as a possible substitute, especially when using collagenase-based preparations that offer a selective, non-invasive, or preservation of tissue procedure [4]. Skin integrity and wound repair depend heavily on collagen, the main building block of tendons, skin, and connective tissue [5]. Healing is hampered in chronic wounds like DFUs by dysregulated matrix turnover and excessive collagen deposition [6]. A regulated clearance of necrotic tissue, the ejection of bioactive peptides, and the promotion of vasculature and cellular migration are all made possible by collagenase, which is a proteolytic enzyme that precisely hydrolyzes collagen fibers [7,8]. These enzymes have been used in tissue digestion and wound treatment since the initial discovery of microbial collagenase enzymes at the beginning of the 1960s [9]. Under physiological settings, the thermal stability, enzymatic efficiency, and substrate specificity of collagenases produced from bacteria like Clostridium histolyticum and actinomycetes like Streptomyces spp. have been investigated [10]. The significance of these enzymes is further highlighted by the rising antibiotic resistance among DFU infections. An investigation conducted in a Peruvian hospital revealed that E. Coli and Pseudomonas aeruginosa isolates from DFUs had high levels of multidrug resistance, including resistance to β-lactam antibiotics because of the synthesis of extended-spectrum β-lactamase (ESBL) [11]. In addition, the COVID-19 pandemic made this problem worse by increasing resistance to antibiotics levels from 36% in 2019 to 63% in 2020 due to unmonitored use [12]. These results highlight how urgent it is to create substitute, non-antibiotic-based treatment options. Collagenases produced by actinomycetes have shown the most promise among microbiological sources. Streptomyces sp. isolated collagenase showed the characteristics of a “true collagenase,” breaking down both native and denatured collagen. Its molecular weight was about 75 kDa, and its catalytic activity was increased when Ca²⁺ and Mg²⁺ were present [13]. Comparative examination of various Streptomyces isolates showed that the generation of collagenase is outside the cell phase of growth, dependent, and significantly affected by the composition of the medium, especially when collagen is the only source of nitrogen [14]. Because of their quick growth, genetic adaptability, and capacity to flourish in a variety of settings, microbial enzymes provide scalable, economical, and environmentally friendly manufacturing platforms from a biotechnological standpoint. In healthcare, food processing, and pharmaceuticals, microbiological enzymes such as amylases, lipases, and collagenases already have revolutionary functions [15]. The goal of this study is to identify, isolate, and describe a heat-stable collagenase from the Streptomyces scabies strain, considering these diagnostic and industrial implications. To promote potential uses for preventing infection in chronic wounds, its biochemical characteristics, thermal endurance, and antibacterial properties against DFU-related pathogens are examined.

2. Materials and Methods

All reagents used in this study were of analytical grade to ensure experimental accuracy and reproducibility. The chemicals included Type I and Type III hydrolyzed bovine collagen (Doctor’s Best, Tustin, CA, USA) used as substrates for enzyme assays, and Tris Base (Sigma-Aldrich, Burlington, MA, USA) for Tris-HCl buffer with pH 7.5, that maintaining optimal reaction conditions, Ninhydrin reagent (Sigma-Aldrich, Burlington, MA, USA) for colorimetric detection of amino groups, and gelatin from bovine skin (Sigma, Burlington, MA, USA) employed as a substrate for enzyme induction and activity evaluation.

2.1. Microorganisms

The actinomycete strain Streptomyces scabies was isolated from rhizospheric soil collected near the Al-Lith hot spring, Saudi Arabia. Soil samples were collected in sterile tubes, air-dried, and serially diluted before being spread onto starch nitrate agar medium. Plates were incubated at 30 °C for 7 days. Morphologically distinct colonies were purified and maintained on slant agar at 4 °C for further analysis. The isolate was identified and taxonomically verified through sequence submission and confirmation in the National Collection of Industrial, Marine and Food Bacteria (NCIMB) in United Kingdom. Phylogenetic placement of the strain was illustrated based on its registered accession data. Clinical bacterial isolates associated with diabetic foot infections—Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus—were obtained under approved ethical criteria from the Clinical Microbiology Laboratory, King Abdulaziz University Hospital (Jeddah, Saudi Arabia).

2.2. Primary Screening for Collagenolytic Activity

Primary screening for collagenase production was carried out using a modified collagen agar medium that contained (g/L): 0.5 g K₂HPO₄, 0.2 g MgSO₄·7H₂O, 0.5 g NaCl, 5 g gelatin, and 15 g agar, adjusted to pH 7.0 before autoclaving. A loopful of S. scabies colony was spot inoculated onto the plates and incubated at 30 °C for 5 days. Clear hydrolysis zones surrounding colonies were visualized after flooding the plates with acidic mercuric chloride solution (1.5% w/v) to indicate collagen degradation [16,17].

2.3. Collagenase Assay

Collagenase activity was quantitatively determined using the modified ninhydrin method. The reaction mixture contained 0.2 mL of enzyme extract, 10 mg of soluble collagen (Type I or III, Doctor’s Best, Tustin, CA, USA) dissolved in 0.8 mL of 50 mM Tris-HCl containing 4 mM CaCl₂, pH 7.5, and incubated at 37 °C for 30 min. The reaction was stopped by adding 1 mL of 0.1 M acetic acid (TCA), followed by centrifugation at 8000 rpm for 10 min. Supernatants were mixed with ninhydrin reagent (Sigma-Aldrich, Saint Louis, MO, USA) and heated in boiling water for 15 min. After cooling, the absorbance was measured at 570 nm. One unit (U) of enzyme activity was defined as the amount of enzyme releasing 1 µmol of L-leucine equivalents per minute under the assay conditions [18,19].

2.4. Optimization of Collagenase Production

Optimization experiments were conducted to determine the effect of different factors on enzyme yield. Each parameter varied independently while keeping others constant. Inoculum size (2%, 4%, 6%, 8%, and 10% v/v) was tested using 24 h-old S. scabies cultures in 50 mL of production medium within 250 mL Erlenmeyer flasks, incubated at 30 °C and 121 rpm for 5 days. pH optimization was performed within a range of 3.0–9.0 using appropriate buffer systems for acidic and alkaline conditions. Temperature optimization ranged from 25 °C to 80 °C. The influence of carbon (glucose, sucrose, fructose, Lactose; 1% w/v) and nitrogen sources (yeast extract, peptone, and tryptone; 0.5% w/v) was also examined to study whether they can significantly influence microbial growth and collagenase yield in some microorganisms. In addition, the day of incubation was evaluated to determine its effect on enzyme production, and each condition was conducted in triplicate.

2.5. Enzyme Purification

The crude enzyme extract was obtained by centrifugation of the culture broth at 8000 rpm for 15 min at 4 °C. The supernatant was subjected to ammonium sulfate precipitation at 30% saturation under constant stirring at 4 °C for 1 h. The precipitate was collected by centrifugation and re-dissolved in 0.05 M Tris-HCl buffer (pH 7.5). Desalting was achieved through overnight dialysis against the same buffer at 4 °C. The partially purified enzyme was used for subsequent analyses.

2.6. Molecular Weight Determination

The molecular weight and purity of the enzyme were determined using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli [20]. A 12.5% resolving gel and 5% stacking gel were used. Samples (20 µg of protein per lane) were mixed with loading buffer containing β-mercaptoethanol, heated at 95 °C for 5 min, and loaded into the gel. After electrophoresis, protein bands were stained with Coomassie Brilliant Blue R-250 and compared with pre-stained molecular weight markers (Thermo Fisher Scientific, Waltham, MA, USA).

2.7. Antibacterial Activity and Statistical Analysis

The antibacterial potential of the purified collagenase was assessed using the agar well diffusion method on Mueller–Hinton agar (MHA) plates. Bacterial cultures of P. aeruginosa, K. pneumoniae, and S. aureus were adjusted to 0.5 McFarland standard (~1.5 × 10⁸ CFU/mL) and spread evenly on MHA plates. Wells (6 mm in diameter) were made using a sterile cork borer, and 50 µL or 100 µL of enzyme solution were added. Plates were incubated at 37 °C for 48 h, and inhibition zones were measured in millimeters. All experiments were conducted in triplicate, and the results were expressed as mean ± standard deviation (SD). Statistical analyses were performed using IBM SPSS Statistics software (version 26). Differences among experimental groups were evaluated using one-way ANOVA followed by Tukey’s post hoc test (for optimization parameters) and unpaired t-tests (for antibacterial assays). A p-value < 0.05 was considered statistically significant.

3. Results

3.1. Morphological and Molecular Identification

The taxonomic identity of the strain was verified through sequence submission in NCIMB and phylogenetic placement within the S. scabies, showing 99.6% sequence identity with Streptomyces scabies (Figure 1a). On starch-nitrate agar medium, the isolate exhibited distinctive white aerial and substrate mycelia (Figure 1b). Extracellular collagenase production was confirmed by preliminary screening on mineral gelatin agar, where a clear hydrolytic zone surrounding the S. scabies colonies indicated enzymatic activity (Figure 1c.)

3.2. Optimization of Collagenase Production

Utilizing a two mL inoculum and 4 percent gelatin as the substrate, collagenase synthesis was optimized throughout a 4-day incubation period at 80 °C and pH 9.0. The most efficient carbon and nitrogen sources among the nutritional sources were found to be sucrose and tryptone, respectively (Figure 2). Enzyme activity exhibited a distinct temperature-dependent pattern. Minimal activity was detected at 37 °C (control, 0.800 ± 0.009 U/mL), while the highest level was achieved at 80 °C (1.714 ± 0.022 U/mL; **** p < 0.0001) (Figure 2a) Activities at 5 °C, 28 °C, 30 °C, and 40 °C were also significantly higher than the control (*** p < 0.001–0.0001), confirming the thermostable nature of the enzyme. The enzyme showed marked pH-dependent activity. Compared with pH 7 (control, 0.569 ± 0.003 U/mL), activity significantly increased at pH 3 (1.256 ± 0.018 U/mL; **** p < 0.0001) and pH 9 (1.508 ± 0.016 U/mL; **** p < 0.0001), while a moderate increase was observed at pH 5 (* p < 0.05). These results demonstrate the enzyme’s stability and functionality in both acidic and alkaline environments (Figure 2b). Enzyme yield was influenced by inoculum concentration. The 4% inoculum resulted in the highest activity (1.360 ± 0.123 U/mL; ** p < 0.01) compared to the 2% control, while higher inoculum sizes (6% and 8%) did not improve production (p > 0.05). This indicates that excessive inoculum can deplete nutrients, limiting enzyme synthesis (Figure 2c). Collagenase production progressively increased from Day 1 (0.825 ± 0.004 U/mL) to Day 4 (1.413 ± 0.0075 U/mL; *** p < 0.0001), followed by a decline on Days 6–7 (** p < 0.05–0.01). This trend suggests that maximum secretion occurs during the mid-log growth phase, with a reduction likely due to enzyme degradation or nutrient limitation in the stationary phase (Figure 2d). Different carbon sources produced significant differences in collagenase activity. Sucrose yielded the highest activity (2.154 ± 0.004 U/mL; **** p < 0.0001) compared with glucose (control, 1.890 ± 0.009 U/mL). Fructose and lactose significantly decreased enzyme activity (**** p < 0.0001), suggesting a metabolic preference for disaccharides in supporting collagenase biosynthesis (Figure 2e). Nitrogen sources also influenced enzyme yield. Tryptone (2.238 ± 0.002 U/mL; **** p < 0.0001) and yeast extract (2.033 ± 0.003 U/mL; *** p < 0.001) both enhanced activities compared with peptone control (1.944 ± 0.004 U/mL). These complex nitrogen sources likely provide essential amino acids and cofactors that stimulate enzyme production (Figure 2f).

3.3. Biochemical Properties of Collagenase

The isolated crude collagenase maintained its activity at 20 °C and a broad pH range (3.0–9.0), with pH 5.0 showing the highest activity. Specificity and inhibition of substrates, while activation studies showed that Fe²⁺ significantly reduced activity, Ca²⁺, Hg^2+, and EDTA exhibited mild inhibitory effects. Substrate preference assays, however, verified a high selectivity for gelatin (

Table 1. Biochemical characterization of S. scabies collagenase.

Category	Condition	Mean	SD	Relative Activity (%)
Temperature (°C)	5	0.089	0.02	20.9
	20	0.425	0.001	100
	37	0.265	0.002	62.3
	60	0.22	0.001	51.7
pH	3	0.195	0.003	85.90
	5	0.227	0.02	100
	7	0.055	0.002	24.22
	9	0.221	0.04	97.3
Substrate conc. (M)	0.15 mL	0.051	0.02	85
	0.25 mL	0.048	0.02	80
	0.35 mL	0.06	0.02	100
	0.45 mL	0.042	0.015	70
Enzyme conc. (mg/mL)	0.05 mL	0.474	0.022	99.7
	0.06 mL	0.473	0.021	99.5
	0.07 mL	0.471	0.02	99.1
	0.08 mL	0.475	0.02	100
Effect of Heavy Metals and Chemicals	Zn++	0.073	0.02	25.3
	Fe++	0.288	0.001	100
	Hg++	0.139	0.001	48.2
	EDTA	0.048	0.003	16.6

Mean ± SD and relative activity (%) of S. scabies crude collagenase under different experimental conditions. (++) It means a positive charge ion for metals.

).

3.4. Enzyme Purification and Molecular Weight

Through dialysis and 30% ammonium sulfate precipitation, the enzyme was purified while maintaining its activity. A clear band at around 25 kDa was seen by SDS-PAGE, which is suggested to be S. scabies collagenases (Figure 3).

3.5. Antibacterial Activity

The purified collagenase demonstrated remarkable antibacterial activity against the tested clinical isolates (Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa). A clear concentration-dependent pattern was observed, as increasing the enzyme volume from 50 µL to 100 µL resulted in larger inhibition zones for all tested bacteria. At 50 µL, inhibition zones measured 50 mm for S. aureus, 35 mm for K. pneumoniae, and 25 mm for P. aeruginosa. When the enzyme concentration was doubled to 100 µL, the inhibition zones increased to 55 mm, 40 mm, and 35 mm, respectively (

Table 2. Inhibition zone diameters (mm) for two collagenase concentrations (50 µL and 100 µL) against selected pathogenic bacteria.

Bacterial Strain	Collagenase (50 µL)	Collagenase (100 µL)	p-Value	Significance
S. aureus	50 ± 1.0	35 ± 1.5	0.001	Significant
K. pneumoniae	35 ± 0.5	40 ± 1.0	0.07	NS
P. aeruginosa	35 ± 0.8	25 ± 1.0	0.003	Significant

Data presented as zone of inhibition ± SD (n = 2); significance assessed by unpaired two-tailed t-test; NS: not significant.

; Figure 4). All antibacterial assays were performed in triplicate, and the results are expressed as mean ± standard deviation (SD). Statistical analysis using unpaired t-tests revealed significant differences between the two enzyme concentrations (p < 0.05) for each bacterial strain, confirming a concentration-dependent enhancement of inhibitory activity. These findings highlight the reproducibility and statistical reliability of the observed antibacterial effects, supporting the enzyme’s potential as a broad-spectrum antimicrobial agent. The overall inhibition trend and comparative activity profiles are further illustrated in (Figure 5).

4. Discussion

This study identifies Streptomyces scabies as a promising microbial source of extracellular collagenase with dual therapeutic potential—enzymatic debridement and antimicrobial activity. The enzyme exhibited exceptional biochemical properties, including high thermal stability, alkaline pH tolerance, and selective inhibitory effects against Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa, three major pathogens commonly associated with diabetic foot ulcers (DFUs). S. scabies collagenase demonstrated remarkable thermostability, maintaining activity at elevated temperatures with an optimum at 80 °C and pH 9.0. These properties surpass those reported for several other thermostable microbial collagenases, including (60–70 °C) [21,22], Thermoactinomyces (60–65 °C) [23], Nocardiopsis dassonvillei (60 °C) [24], and Bacillus licheniformis (50 °C) [25,26,27]. Furthermore, the alkaline optimum pH of S. scabies collagenase (pH 9.0) aligns with other bacterial collagenases, such as those from Thermoactinomyces [23] and Bacillus spp. [25,26,27], while clearly exceeding the activity ranges of fungal collagenases derived from Aspergillus and Penicillium (pH 6.5–8.3) [28,29]. These features underline its suitability for industrial and biomedical applications that demand stability under harsh processing conditions. Consistent with other microbial collagenases, S. scabies collagenase activity was inhibited by Ca²⁺ and enhanced by heavy metals such as Fe²⁺, supporting its classification within the metalloprotease or serine-protease family. Notably, its relatively small molecular weight (~25 kDa) is lower than most reported collagenases, including those from Thermoactinomyces (50 kDa) [22,23], Nocardiopsis (150 kDa) [24], and Aspergillus (28.7 kDa) [28]. This reduced size may facilitate improved tissue penetration, reduced immunogenicity, and enhanced compatibility with biomedical formulations. Enzymatic debridement is increasingly favored over surgical methods in DFU management due to its precision, reduced invasiveness, and ability to promote wound healing. Although some studies report limited or biased data [30,31], enzymatic approaches have consistently demonstrated superior outcomes in tissue regeneration, epithelialization, and wound closure [32,33,34]. In line with these findings, S. scabies collagenase exhibited robust collagenolytic activity with therapeutic selectivity. Importantly, Collagenase displays direct antimicrobial action inhibition against major DFU pathogens [35,36]. Inhibition zone assays revealed strong activity against S. aureus (50 mm at 50 µL), K. pneumoniae (35–40 mm), and P. aeruginosa (25–35 mm). Interestingly, the reduced sensitivity of S. aureus at higher enzyme volumes suggests possible protein aggregation or inhibitory byproducts, a phenomenon previously observed in other proteolytic systems. The dual-action profile of S. scabies collagenase is particularly valuable. Existing enzymatic formulations, such as clostridial collagenase (Iruxol^®/Iruksan), combine debridement with antimicrobial effects [37]. Similarly, bromelain-based preparations (e.g., NexoBrid^®) have shown tissue selectivity in burn care [38], while clostridial collagenase remains the only FDA-approved agent for chronic wound debridement, with well-documented cost-effectiveness and therapeutic benefits [39,40]. Beyond wound care, collagenases have been applied in Dupuytren’s disease (Xiaflex^®) [41] and shown to promote cell migration and tissue remodeling in alveolar and dermal wound models through the release of bioactive peptides [42,43,44]. Marine-derived peptides, such as those from Aluterus monoceros, further highlight the broad wound-healing potential of collagenolytic systems [45]. The biomedical relevance of S. scabies collagenase extends beyond wound debridement. Its gentle collagenolytic action resembles that of Vibrio alginolyticus collagenase, which has been used for high-viability mesenchymal stem cell (MSC) isolation [46]. Unlike endogenously secreted collagenases from gut flora, which can contribute to tissue pathology such as anastomotic leakage [47], S. scabies collagenase is externally applied, minimizing systemic risks. Pathogenic collagenases, such as those from Peptostreptococcus magnus in DFU isolates [48], illustrate the need to distinguish between virulence factors and controlled therapeutic enzymes. The stability, selective action, and external application of S. scabies collagenase support its clinical safety profile.

Furthermore, collagenase therapy has shown efficacy in neonatal wound care with minimal discomfort [49], suggesting broad clinical tolerability. Diabetic and infected wounds are often characterized by elevated endogenous collagenase activity and exacerbated tissue damage [50]. Controlled application of exogenous collagenase may rebalance the wound environment toward healing. Indeed, clostridial collagenases have been shown to modulate inflammatory responses by reducing TNF-α and IL-6 while enhancing anti-inflammatory mediators [39]. Precision activity, such as selective degradation of fibrotic collagen without affecting collagen VI, is particularly advantageous for chronic wound environments [51]. S. scabies collagenase’s alkaline pH preference aligns with the biochemical milieu of DFUs [52]. Overall, the findings of this study suggest that the collagenase produced by Streptomyces scabies possesses significant biotechnological potential due to its high catalytic efficiency, thermostability, and antibacterial activity under alkaline conditions. This study comprehensively characterized the enzyme’s physicochemical and functional properties, highlighting its suitability as a candidate for enzymatic debridement and infection control in chronic wounds. Nevertheless, the current work was limited to in vitro experiments. Further studies employing biofilm models and in vivo systems are essential to validate its therapeutic potential, biocompatibility, and safety before clinical translation.

5. Conclusions

In this study, a thermostable collagenase from Streptomyces scabies isolated from rhizospheric soil in Al-Lith thermal springs, Saudi Arabia, was successfully characterized. The enzyme was partially purified, and its molecular weight (~25 kDa) was determined by SDS-PAGE. Collagenase activity was optimal at 80 °C and pH 9, remained stable under thermophilic and alkaline conditions, and was enhanced by Fe²⁺ ions. The enzyme also exhibited selective antibacterial activity against Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. These findings highlight the potential of S. scabies collagenase as a promising enzymatic tool for in vitro antibacterial applications and provide a basis for further evaluation in wound management strategies, including diabetic foot infections.