https://orcid.org/0000-0001-5811-1561
Figures
Abstract
Ninety-one elastase-producing bacterial isolates were recovered from different localities of the Eastern Province of Saudi Arabia. Elastase from the best isolate Priestia megaterium gasm32, from luncheon samples was purified to electrophoretic homogeneity using DEAE-Sepharose CL-6B and Sephadex G-100 chromatographic techniques. The recovery was 17.7%, the purification fold was 11.7x, and the molecular mass was 30 kDa. Enzymatic activity was highly repressed by Ba2+ and almost completely lost by EDTA, but it was greatly stimulated by Cu2+ ions, suggesting a metalloprotease type. The enzyme was stable at 45°C and pH 6.0–10.0 for 2 hours. Ca2+ ions considerably enhanced the stability of the heat-treated enzyme. The Vmax and Km against the synthetic substrate elastin–Congo red were 6.03 mg/mL, and 8.82 U/mg, respectively. Interestingly, the enzyme showed potent antibacterial activity against many bacterial pathogens. Under SEM, most bacterial cells showed loss of integrity, damage, and perforation. SEM micrographs also showed a time-dependent gradual breakdown of elastin fibers exposed to elastase. After 3 hours, intact elastin fibers disappeared, leaving irregular pieces. Given these good features, this elastase may be a promising candidate for treating damaged skin fibers with the inhibition of contaminating bacteria.
Citation: AlShaikh-Mubarak GA, Kotb E, Alabdalall AH, Aldayel MF (2023) A survey of elastase-producing bacteria and characteristics of the most potent producer, Priestia megaterium gasm32. PLoS ONE 18(3): e0282963. https://doi.org/10.1371/journal.pone.0282963
Editor: Fuad Ameen, King Saud University, SAUDI ARABIA
Received: November 8, 2022; Accepted: February 27, 2023; Published: March 13, 2023
Copyright: © 2023 AlShaikh-Mubarak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: EK received a fund from the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia through a project number IF-2020-028-BASRC. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: BSA, Bovine serum albumin; EDTA, Ethylene diamine tetraacetic acid; Kcat, Catalytic rate constant; Km, Michaelis–Menten constant; LB broth, Luria-Bertani broth; LB plot, Lineweaver–Burk plot; LM, Light microscope; MDR, Multidrug resistance; MHB, Muller Hinton Broth; SEM, Scanning electron microscopy; TCA, Trichloroacetic acid; Vmax, Maximum velocity
Introduction
Elastin is a basic matrix component responsible for tissue flexibility. It is found in the skin, arteries, lungs, and other tissues. Depending on the tissue type, elastin is present in variable amounts, forming fibers of extensively cross-linked protein. It is insoluble and has a high degree of hydrophobicity. These characteristics are relatively stable and durable in tissues unless one or more proteinases degrades the elastin fibers [1].
Elastases are special types of proteolytic enzymes that can degrade elastin as a specific substrate. They are subdivided into different groups of proteases, particularly serine proteases, aspartic proteases, thiol proteases, and metalloenzymes [2]. They are different in their catalysis and substrate specificity [1]. They were initially discovered in the viscera of animals. Nonetheless, this source is insufficient to meet the needs of industry and daily use. Microorganisms can produce high levels of elastases [3].
For several years, bacterial elastases were classified as metalloproteinases, which require Zn2+ ions as cofactors for their activity. Streptomyces fradiae and Bacillus thermoproteolyticus elastases are among the most potent elastolytic proteinases discovered to date because they are 4-8-fold more effective than pancreatic elastases [4].
The requirement for unique eco-friendly bacterial-derived elastases that can be used in various industrial and pharmaceutical applications has increased. They have widespread usage in food processing, medical treatment, and the chemical industry [5]. Additionally, elastases immobilized on a bandage are used to treat various ailments, including carbuncles, damaged skin, furuncles, and burns [6].
The majority of microbial elastases are produced by Pseudomonas aeruginosa [7], Staphylococcus epidermidis [8], Bacillus licheniformis [5], Bacillus subtilis [9], and Chryseobacterium indologenes [3]. Unfortunately, most of them have low stability under environmental conditions.
Antimicrobial resistance is wreaking havoc on people’s health. As a result, ten million people have died worldwide [10]. In this research, we aimed to produce a highly stable elastase that can hydrolyze elastin fibers efficiently while also having antibacterial activity. Therefore, it can be applied in the treatment of damaged skin and the targeting of contaminating bacteria.
Materials and methods
Materials
Elastin–Congo red, azocasein, and EDTA were purchased from Sigma‒Aldrich (USA). Sephadex G-100 FF, DEAE-Sepharose CL-6B, and SDS‒-PAGE reagents were procured from Pharmacia Biotech (Sweden). The other materials were sourced from regional providers.
Strain isolation
Throughout this inspection, twenty samples of seawater and soil were collected from Tarout Island (26° 33’ 59.99" N, 50° 03’ 60.00" E), Al Khobar city (26° 13’ 1.8876’’ N, 50° 11’ 49.6968’’ E), and Al Ahsa city (25° 25’ 27.59" N, 49° 37’ 11.39" E) in the Eastern Province of Saudi Arabia from August to November 2019. Additionally, we collected five samples of crude camel milk, five shrimp shell waste samples, five crude cheese samples, and five processed meat samples from the Al Ahsa public market. Protease production was detected by growing isolates on 25% (v/v) skimmed-milk agar containing (gr/L) agar (15), NaCl (5), beef extract (1) peptone (5), and yeast extract (2). The medium pH was adjusted to 7.0, and incubation was performed at 37°C for 24 hours to visualize the positive hydrolytic colonies. For further experiments, pure cultures were stored at -80°C in 20% glycerol.
Characterization of the most potent isolates at the species level
Extraction of DNA from the bacterial cells was performed according to Dellaporta et al. [11] as follows: 20 mg of freshly harvested cells was broken up with 0.5 mL of Dellaporta buffer (100 mM Tris-HCl pH 8, 500 mM NaCl, 50 mM ethylenediaminetetraacetate (EDTA), 10 mM β-mercaptoethanol). Specifically, 33 μL of 20% sodium dodecyl sulfate (SDS, w/v) was added to the cells and they were vortexed and incubated at 65°C for 10 min. Then, 160 μL of 5 M potassium acetate was added, and the mixture was vortexed, followed by centrifugation for 10 min at 10,000 rpm. The supernatant was collected, and 450 μL of it was combined with the same volume of phenol, chloroform, and isoamyl-alcohol mixture at a ratio of 25:24:1. The mixture was vortexed for 5 min prior to centrifugation at 10,000 rpm for 5 min. Then, 400 μL of the upper phase was removed, mixed with a half volume of isopropanol, vortexed, and centrifuged at 14,000 rpm for 10 min. The nucleic acid precipitate was cleaned with 70% ethanol and centrifuged at 10,000 rpm for 5 min. The precipitate was finally resuspended in 100 μL of distilled water.
Amplification of the 16S rRNA gene was performed using the universal primers 1492R 5′-TACGGYTACCTTGTTACGACTT and 27F 5′-AGAGTT TGATCMTGGCTCAG-3′ [12]. The main steps of polymerase chain reaction (PCR) were as follows: 94°C for 5 min, denaturation at 94°C for 45 sec, annealing at 55°C for 60 sec, and extension at 72°C for 60 sec. After thirty cycles, the last extension was performed at 72°C for 10 min (Applied Biosystems™ Veriti™ Thermal Cycler, 96-Well, USA). The QIAquick® PCR Purification Kit was employed to purify the PCR products according to the manufacturer’s guidelines. The DNA sequences in both directions were then determined at Macrogen Inc. (Korea) and uploaded to Blastn of GenBank and a similarity check was run. For phylogenetic inference analysis, the closely related sequences were used alongside the sequences retrieved for all bacteria to establish phylogenetic trees using MEGA11 software. Information on the phylogenetic tree is available in S3 Fig.
Enzyme production and protein measurement
The crude enzyme was produced in a minimal medium composed of (%, w/v) peptone (0.5), fructose (0.5), KH2PO4 (0.08), NaCl (0.3), yeast extract (0.04), MgSO4.7H2O (0.05), K2HPO4 (0.02), CaCl2 (0.01), MnSO4 (0.01), FeSO4 (0.002) and ZnSO4 (0.002). The medium pH was adjusted to 7.0 and then dispensed at 20% (v/v) in 250 mL Erlenmeyer flasks. A2% (v/v) inoculum of 12-hour-old cultures (1.0×108 cfu/mL) in LB broth was used. LB consisted of (%, w/v) tryptone (1.0), NaCl (0.5), and yeast extract (0.5) at pH 7.0. Fermentation was allowed for 48 hours at 37°C with shaking at 200 rpm. Centrifugation was performed at 5,000 rpm for 20 min using a refrigerated centrifuge (Hermle Labortechnik GmbH, Z 326 K, Germany) at 4°C to separate the bacterial cells. Supernatants were used for the determination of elastase productivity and protein concentration as described by Kotb et al. [13] with slight modifications.
The amount of protein was quantified by measuring the absorbance of the culture filtrate at 280 nm. The optical densities were converted to weights from the standard curve of varying concentrations of bovine serum albumin (BSA).
Enzyme assays
Dual plate assays: A preliminary protease assay was performed on Petri dishes containing 2% (w/v) skimmed milk and 1.5% (w/v) agar [14]. In parallel, an elastase assay was performed in nutrient agar plates supplemented with elastin–Congo red as a substrate [15]. Isolates were checked for enzyme productivity using spotting and well plate techniques. After creating wells with a diameter of 7 mm on Petri dishes, 0.1 mL of crude enzymes was introduced to every well. Petri dishes were then incubated at 37°C for 24 hours. Zones of substrate hydrolysis were used as a measure of enzyme productivity.
Protease assay: Cultures of the ninety-one bacterial isolates were shake-incubated at 37°C for 48 hours in minimal medium to allow enzyme productivity. The cell-free supernatants (CFS) were obtained after centrifugation at 5,000 rpm for 20 min using a cooling centrifuge at 4°C. The reactants were 0.5 mL of 0.08% (w/v) azocasein solution as a substrate and 0.5 mL of CFS. The azocasein was dissolved in 0.2 M potassium phosphate buffer at pH 7.0. Then, the mixture was incubated at 37°C for 30 min, and 1 mL of 10% (w/v) trichloroacetic acid (TCA) was added. After incubation in a crushed ice bath for 1 hour, the unreacted azocasein was precipitated by centrifugation. An equal volume of supernatant and NaOH (1 N) were mixed, and the absorbances were measured at 440 nm [16, 17]. A blank was prepared by the same procedure, replacing the enzyme with distilled water. Under usual test conditions, one unit (U) of enzyme activity was defined as the quantity of enzyme that produced an increase in 0.01 absorbance at 440 nm per minute. The tests were performed in triplicate, and the mean values were recorded as units of protease activity.
Elastase assay: Every 1 mL of elastase was mixed with 20 mg of the substrate Congo red elastin suspended in 2 mL of 0.2 mol/l acid buffer (pH 7.4) with shake incubation at 37°C for 20 min. The reaction was ended by adding 100 μL of 0.12 M EDTA (pH 8.0). Following centrifugation, the absorbance at 495 nm was calculated with a spectrophotometer calibrated to a control elastin–Congo red sample incubated without the enzyme [18]. One unit (U) of elastase was equivalent to the quantity of enzyme causing a rise in A495 by 0.01 after 1 hour of incubation under the standard assay conditions.
Enzyme purification
The elastolytic enzyme from the most potent isolate was purified through 3 main stages: fractional precipitation with (NH4)2SO4, DEAE-Sepharose CL-6B anion-exchange, and Sephadex G100 FF gel permeation chromatography. Bacteria separated from the fermentation medium by centrifugation at 5000 rpm for 15 min. (NH4)2SO4 crystals were added slowly to the crude enzyme preparation until 70% saturation was reached. Agglomerated enzyme molecules were collected by centrifugation (7,000 × g for 10 min, 4°C) after overnight incubation at 4°C. The pellet was mixed with the least volume of 100 mM phosphate buffer (pH 7.4, buffer A) necessary for resuspension. Dialysis was then performed against buffer A to remove ammonium sulfate and other undesirable salts. The concentrated dialysate was then applied onto a DEAE-Sepharose CL-6B column (0.5×5 cm2) equilibrated with buffer A. A linear rise of 0–1 M NaCl in 0.1 M borate buffer (pH 9.4, buffer B) was used as an eluent. Fractions showing elastase activity were collected and concentrated for application through a second Sephadex G100 FF column (2.0 × 45 cm2). The elution rate was adjusted to 1 mL/3 min. The elastase active fractions were merged and lyophilized for characterization studies. To examine the homogeneity and molecular mass of the target elastase, SDS‒PAGE analysis was performed with a 5% (w/v) stacking gel and a 15% (w/v) separating gel [19].
Effect of pH on enzymatic activity and stability
Enzyme activity was tested at pH values of 4 to 12 in a suitable buffer by incubating crude enzyme substrate under standard assay conditions. Experiments were carried out utilizing the typical enzyme assay with the substrate azocasein (0.8%, w/v) with incubation at 45°C for 30 min based on the result of the optimal temperature for maximal enzyme activity, which was 45°C. As previously stated, relative activity was determined. For the pH stability test, buffered enzyme fractions were preincubated at pH 4–12 for 2 hours at 45°C. The residual activity against the substrate was then assayed at pH 8.0 and 45°C.
Effect of temperature on the enzymatic activity and stability
The optimal temperature for elastase activity was determined by mixing elastase with its substrate at a ratio of 1:1 and incubating at 20–50°C for 30 min. The relative elastase activities were determined as the percentage of the maximum activity (100%) under the standard assay conditions.
Relative activity = [Total activity (U/mL) × 100] ÷ Maximum activity (U/mL) [20]
A thermal stability assay was designed to determine the temperature range within which the tested elastase maintained its activity. The enzyme preparation was incubated alone in a water bath for 15, 30, 45, and 60 min at 50, 60, 70, and 80°C. The residual elastase activity was assessed against the substrate at 45°C for 30 min as previously mentioned.
The impact of calcium ions on thermostability was determined by preincubating the elastase preparation at 60°C in the presence of CaCl2. Calcium chloride was tested at concentrations of 2, 5, 10, and 15 mM. The remaining activity was then determined after 60 min of heat exposure. The nonheated enzyme was considered a control and expressed as 100% activity.
Effect of metallic ions and elastase inhibitors
The effect of various cations, such as Co2+, Ba2+, Mg2+, Ca2+, Hg2+, Fe3+, Na+, Mn2+, Fe2+, Mg2+, Cu2+, and Zn2+on elastase activity was examined. The enzyme was incubated at 35°C for 2 hours with metallic ions at a concentration of 5 mM.
The mixture was then incubated with 5.0 mL of 0.08% (w/v) azocasein as a substrate in 200 mM potassium phosphate buffer (pH 7.0) at 45°C for another 2 hours.
For the inhibition studies, the enzyme was preincubated for 2 hours with two concentrations of EDTA (5 and 10 mM) at room temperature, and then the residual activity was measured under the optimum assay conditions. The activity of the enzyme in the absence of metal ions and inhibitors defined as 100% activity.
Kinetic parameters
The dynamics of elastase were tested against the substrates azocasein and elastin–Congo red. The reaction rate was assessed at 45°C and pH 8.0 thereafter, and the enzymatic activity was determined as described previously. The Michaelis–Menten constant (Km) and the maximum reaction velocity (Vmax) were then calculated from the linear regression equation created from the Lineweaver–Burk plot (LB plot). The turnover number (Kcat) was determined by dividing the value of Vmax by the adopted elastase concentration.
Antibacterial activity of elastase
The antibacterial potential of the tested elastase was evaluated against several strains of bacterial pathogens, including Escherichia coli, Salmonella spp., S. aureus, Shigella boydii ATCC 9207, and P. aeruginosa ATTC 27853. The agar well diffusion method was adopted as illustrated by Khan et al. [21]. The agar layers were swabbed with bacteria at 1.0×108 colony forming units/mL of physiological saline. Then, 6-mm diameter holes were pierced off aseptically with a sterilized cork borer. Each well received a certain concentration of the purified enzyme (100 U, 50 U, and 25 U) in a total volume of 100 μl, and then incubation was performed at 37°C for 24 hours. The inhibition areas around the wells were taken as direct measurements of the antibacterial activity.
The exact MIC values against the most affected bacteria S. boydii ATCC 9207 and S. aureus subsp. aureus Rosenbach ATCC 25923 were measured by the broth dilution method with few modifications [22]. An initial stock preparation of 4 mL for the tested elastase was adjusted at 100 U/mL in Muller Hinton Broth (MHB). Then, two-fold dilutions were made in 2 mL MHB tubes till reaching 6.25 U/mL concentration of the enzyme. Exactly, 200 μL from a bacterial culture suspension prepared in physiological saline at 1.0×108 colony forming units/mL was inoculated into each broth culture. A broth culture with no bacteria served as the negative control (0% growth) while, a broth tube not involving the tested hydrolytic enzyme served as the positive control (100% growth). Incubation was done for 24 hours at 37°C. MIC was defined as the mean of the lowest concentration inhibiting bacteria and the highest concentration allowing the growth of bacteria.
Scanning electron microscopy (SEM) examination
TESCAN VEGA3 SEM was used to observe the staphylolytic activity and shigellalytic activity of the target elastase on the exterior features of cells. S. aureus subsp. aureus Rosenbach ATCC 25923 and S. boydii ATCC 9207 were cultivated in LB broth for 24 hours at 37°C. Bacterial cells were harvested, and a fraction was suspended in an enzyme preparation for 20 hours at 37°C. A second fraction served as a blank control. Cells were collected at 5000 rpm for 20 min, rinsed two times in 0.2 M sodium phosphate buffer, pH 7.5, and loaded on adhesive SEM stubs for sample processing. Dehydration was performed with 30%-95% ethanol, and then the cells were incubated at 35°C for 2 hours to allow them to dry completely. Sputter coating with chromium for 15 min was performed to enhance the electron conductivity on the sample surfaces. Finally, TESCAN VEGA3 SEM was used to inspect the exterior shape of both sets at a low voltage of 20 keV.
In vitro application of elastase in the degradation of elastin fibers
Exactly, 0.5 mg of elastin fibers were mixed with one mL of elastase preparation in 50 mM Tris-HCl (pH 8.0) buffer and incubated for 3 hours at 37°C. The elastin fibers were separated by natural settling in an ice bath for 1 hour. This procedure was performed twice in physiological saline to clean the elastin fibers thoroughly. Elastin fibers in 50 mM Tris-HCl (pH 8.0) without elastase were utilized as a blank control. The fiber status was observed each hour [23] under LM and SEM. For SEM analysis, the fiber fractions were lyophilized before being placed on the SEM metal stub, coated with a 5 nm thick chromium layer and investigated with a Tescan VEGA3 SEM at 5.0 kV [24].
Statistical analysis
SPSS Statistics Ver. 20.1 (IBM® SPSS® Statistics, Armonk, NY, USA) was employed to analyze the raw results. P values of less than 0.05 were judged statistically significant. Unless otherwise indicated, all data are expressed as the mean ± standard deviation (SD) of three repeats.
Results
Strain isolation
In the present study, ninety-one proteolytic isolates were recovered from different localities of the Eastern Province of Saudi Arabia. Clear zones around growing colonies were considered a positive indication of enzyme productivity. Isolates 25, 27, 28, 32, 34, 37, 43, 71, 82, and 91 were the highest producers of proteases and elastases on skimmed milk agar and elastin–Congo red agar. Isolate gasm32 displayed the highest proteolytic and elastolytic activity compared to the others (S1 Fig). Therefore, it was chosen for further research.
Molecular characterization and phylogenetic inference of the most potent isolates at the species level
All DNA samples extracted with the QIAGEN Chromosomal kit were subjected to PCR (S2 Fig). The amplified PCR products were electrophoresed on a 1% (w/v) agarose gel. The extracted DNA appeared as single bands on the agarose gel after electrophoresis, confirming its purity. The partial 16S rRNA sequences of all elastase-producing bacteria were compared with those of other bacterial strains deposited in GenBank using the similarity matrix, which was determined by the number of base differences (Table 1). The neighbor-joining phylogenetic tree constructed with MEGA11 software revealed that the gasm32 isolate recovered from the luncheon sample was a strain of Priestia megaterium, as it was clustered with P. megaterium strains (S3 Fig).
Enzyme purification
Gasm32 elastase was separated from the broth of Priestia megaterium gasm32 using several procedures described in Table 2. The final specific activity of elastase reached 11.7-fold with a recovery of 17.7% compared with the initial concentration of elastase in the CFS of P. megaterium gasm32. SDS‒PAGE of the final elastase-active fractions revealed one band at 30 kDa (Fig 1). The previous version of the SDS‒PAGE gel is presented in S4 Fig.
Effect of pH value on enzymatic reactivity and stability
The results in Fig 2 indicated that the maximum reaction activity for elastase was at pH 8.0. In addition, the enzyme was stable at pH 6–10 for 2 hours. Above or below this particular pH range, the elastase stability drastically decreased.
Effect of temperature on elastase activity and stability
The optimal temperature for maximal elastase activity was observed at 45°C (Fig 3A). The results in Fig 3B revealed that the enzyme was heat-stable up to 60°C for 15 min. The half- lives (t1/2) were 14.22 min, 12.45 min, 11.01 min, and 8.36 min at 50°C, 60°C, 70°C, and 80°C, respectively (Fig 3C). As shown in Fig 3D, the enzyme stability was substantially improved by addition of Ca2+ ions compared to the control test (without CaCl2).
Relation of reaction temperatures to elastase activity (A) and stability (B). The calculated half-lives (t1/2) are presented in Panel C and the effect of calcium ions on elastase thermostability is shown in Panel D.
Effect of metallic ions and enzyme inhibitors
The results in Table 3 show the effect of Co2+, Ba+2, Mg2+, Ca2+, Hg2+, Fe3+, Na+, Mn2+, Fe2+, Zn2+, and Cu2+ ions in addition to EDTA on the activity of elastase. Except for Cu2+ and Mn2+, none of the metal ions tested stimulated the enzyme activity of the elastase. Enzyme activity was inhibited by Ba2+, Hg2+, Fe3+ and Na+ but was enhanced by the addition of Cu2+ and Mn2+ ions. However, EDTA almost wholly repressed the enzymatic activity of the elastase at the tested concentrations.
Determination of kinetic parameters of elastase
The results of the enzyme kinetic study using azocasein and elastin–Congo red as substrates in terms of Km and Vmax were calculated from the LB plots (Fig 4). On azocasein, elastase had Km and Vmax values of 4.20 mg/mL and 188.67 U/mg, respectively. The Kcat was 0.232 s-1, and the catalytic efficiency (Kcat/Km) was 0.055 M-1 s-1. On elastin–Congo red, elastase had Km and Vmax values of 6.03 mg/mL and 8.818 U/mg, respectively. The Kcat was 0.27 s-1, and the catalytic efficiency was 0.046 M-1 s-1.
Lineweaver–Burk plot showing the enzyme catalysis against azocasein (Panel A) and elastin–Congo red (Panel B).
Antibacterial activity of elastase
The results of the well diffusion method showed that the tested elastase exhibited antibacterial activity against most of the tested clinical isolates, including S. aureus subsp. aureus ATCC BAA-1026 (24 mm), S. aureus subsp. aureus Rosenbach ATCC 25923 (26 mm), S. aureus subsp. aureus ATCC 43300 (23 mm), S. aureus subsp. aureus ATCC 29213 (24 mm), S. aureus ATCC-BAA-976 (19 mm), Salmonella enterica subsp. Enterica serovar Enteritidis ATCC 13076 (14 mm), Salmonella spp. (15 mm), and Shigella boydii ATCC 9207 (29 mm). However, the elastase showed no antibacterial activity against E. coli ATCC 8739, E. coli ATCC 25922, P. aeruginosa ATTC 27853, and E. coli ATCC 35218. The MICs of elastase determined by broth dilution method against S. boydii ATCC 9207 and S. aureus subsp. aureus Rosenbach ATCC 25923 were 37.5 U and 18.75 U, respectively (S5 Fig). The antibacterial activity of elastase against S. boydii ATCC 9207 and S. aureus subsp. aureus Rosenbach ATCC 25923 is presented in S6 Fig.
The SEM study on the most affected harmful bacteria showed that most cells presented damage and perforation. Some cells were observed to have reduced size, and the majority appeared to be fused and melted. These images show the loss of shape and integrity, which led to cell death (Fig 5).
In vitro application of elastase in the degradation of elastin fibers
LM images show multiple longitudinal and transverse crevices on the elastin fiber surface after 1 hour of treatment with the tested elastase (Fig 6B). The size and number of crevices expanded with time (Fig 6C). After 3 hours of enzymatic treatment, fibers were fractured lengthwise by gradual longitudinal fractures and broken into pieces by progressive transverse cracks (Fig 6D).
(Panel A: control treatment, Panel B: enzyme treatment for 1 hour, Panel C: enzyme treatment for 2 hours, and Panel D: enzyme treatment for 3 hours).
SEM micrographs also show the time-dependent progressive breakdown of elastin fibers exposed to elastase. Fig 7 shows the dramatic strategy by which the fibers were degraded. Cavities were observed on the surface of elastic fibers after 1 hour of treatment (Fig 7B), which expanded gradually in depth and width as the exposure time increased (Fig 7C and 7D). After 3 hours, intact elastin fibers disappeared, leaving irregular pieces in the field of view (Fig 7E and 7F).
SEM micrographs of elastin degradation by the target elastase (Panel A represents the control, Panel B represents 1 hour of elastase treatment, Panels C and D represent 2 hours of elastase treatment, and Panels E and F represent 3 hours of elastase treatment).
Discussion
Bacterial elastases are mainly grouped under the metalloproteinase family [21]. Indeed, screening for bacteria with elastolytic activity is a direct method for obtaining elastases with desired characteristics. In this study, we screened for bacteria-producing elastases in different localities of Saudi Arabia. The broader sample size and the several sampling locations led to more positive isolation of enzyme producers. However, there are some difficulties in the isolation of bacteria adapted to grow under extreme environments of pressure, pH, temperature, and/or in environments with high concentrations of metals.
Tsai et al. [25] isolated alkaline elastase extracellularly produced by the alkalophilic Bacillus strain Ya-B from soil collected around the Tokyo area. Clark et al. [26] isolated a unique alkaline elastase from Micrococcus luteus from human skin in the late exponential and stationary growth phases. He et al. [27] obtained elastase from Bacillus sp. EL31410 from the soil at a meat-processing factory in Hangzhou, China. Qihe et al. [28] isolated an elastase from Bacillus sp. EL31410, which is a good meat tenderizer. This elastase could be a promising substitute for papain as a good meat tenderizer. Malanicheva et al. [29] isolated Bacillus megaterium from leached chernozem soil (Kras-nodar krai). Kotb [30] isolated Bacillus megaterium KSK-07 from kishk, a traditional Egyptian fermented food. Uttatree et al. [31] isolated Bacillus megaterium from marine sediment in the Gulf of Thailand at a depth of 24 meters. Lei et al. [3] isolated a novel elastase from Chryseobacterium indologenes from the mud of a meat market in Ya’an city. Elastase is expected to have potential application in meat tenderization because of its specific hydrolysis toward elastin. Kotb et al. [13] reported that among a total of 120 clinical specimens that were collected from the hospitals of Zagazig University (Zagazig, Egypt) from different sources, including pus, urine, wounds, sputum, eyes, blood, and ear infections, only 54 isolates of Pseudomonas aeruginosa ZuhP13 were positive for cytotoxic serine-elastase productivity. Zupetic et al. [32] isolated Pseudomonas aeruginosa from 238 unique ICU patients belonging to two tertiary-care centers inside the University of Pittsburgh Medical Center health system, and 75% of the isolates produced elastase activity.
The most potent isolate was gasm32, and it was identified molecularly as P. megaterium. It showed excellent hydrolytic activity against both casein and elastin (S1 Fig). The selected isolate had an elastase productivity of 771.3 U/mL (7.713 A495) (Table 1), which is better than the values found by Zins et al. [33] for P. aeruginosa PAO1 (0.39 A495), P. aeruginosa VMS1120 (0.20 A495), P. aeruginosa VMS7931 (0,109 A495), and C. violaceum 98–9187 (1.09 A495), and the values found by Olson and Ohman [34] for P. aeruginosa PAO1 (0.06 A495), P. aeruginosa FRD2 (0.13 A495), and P. aeruginosa DG1 (0.14 A495).
The secreted elastase from P. megaterium gasm32 was purified to electrophoretic homogeneity and produced a prominent band on SDS‒PAGE indicating a 30 kDa molecular mass (Fig 1). Among other investigators, a 34 kDa molecular mass was reported for an elastase from P. aeruginosa [35], a 26 kDa molecular mass was reported for an elastase from C. indologenes [3], and a 23.7 kDa molecular mass was reported for an elastase from the alkalophilic Bacillus strain Ya-B [25].
The characteristic properties of the elastases were investigated to achieve the highest activity and determine to which family they belonged. The best reaction pH for gasm32 elastase was determined to be 8.0 (Fig 2), which is the same as that of P. aeruginosa [36] and the B. megaterium-TK1 strain isolated from saltwater [37]. This optimal pH was different from those obtained for other elastases in other studies [38], such as the alkaline elastase from Micrococcus luteus (pH 9.3, [26]), B. megaterium (pH 7.5, [29]), P. aeruginosa ZuhP13 (pH 7.5, [13]), and the alkalophilic Bacillus strain Ya-B (pH 11.75, [25]).
The pH stability of gasm32 elastase was observed at pH 6.0 to 10.0 for 2 hours (Fig 2). A wide pH stability spectrum was also observed for the elastase of C. indologenes (pH 5.0–10.5, [3]), the elastase of Flavobacterium odoratum (pH 5.0–10.0, [39]), and the serine-elastase of P. aeruginosa ZuhP13 (pH range of 6.0–9.0, [13]). The high alkaline character of gasm32 elastase revealed by its activity and stability over a wide pH range also suggests its suitability for use under alkaline conditions such as detergent additives.
The optimum reaction temperature for the target elastase was found to be 45°C (Fig 3A). In reviewing the findings of other investigators, 37°C was the optimal reaction temperature for the elastase of C. indologenes [3], 40°C was the optimal reaction temperature for the serine-elastase of P. aeruginosa ZuhP13 [13], the temperature range of 57–59°C was optimal for the elastase of Micrococcus luteus [26], and 60°C was the optimal reaction temperature for Bacillus Ya-B elastase [25].
Our findings showed that gasm32 elastase was thermally stable for 45 min at 50°C (Fig 3B). Thus, it is more heat stable than the elastase of Flavobacterium odoratum (70% inhibition at 50°C for 4 hours, [39]) and the elastase of C. indologenes (50% inhibition at 55°C, [3]). Our elastase was more heat-sensitive than the serine-elastase of P. aeruginosa ZuhP13, which was inhibited 50% at 60°C after 30 min of exposure [13]. These differences may be due to the source of the isolate and the type of bacteria.
Similar to other proteases, our elastase was heat-stabilized in the presence of calcium ions [34]. When 15 mM CaCl2 was added, 90% elastase activity was sustained for 15 min at 60°C (Fig 3D), while there was a 25% decrease in elastase activity in the absence of Ca2+ ions. Moreover, it maintained 51% of its original activity after 60 min in the presence of Ca2+ while in the absence of Ca2+ it retained 3% of its initial activity. The presence of Ca2+ ions also increases the thermal stability of a protease from B. subtilis FBL-1 [40] and the neutral proteases of B. megaterium AU02 [16]. The increased enzyme stability in the presence of calcium ions may be due to the ability of calcium ions to act as an ionic bridge between carboxylic groups of amino acids forming the enzyme molecules, thereby retaining the three dimensions of the enzyme [41]. Additionally, it could be explained by enhancing protein‒protein interactions and associating Ca2+ with autolysis sites to avoid autolysis and thermal unfolding [42].
The impact of various cations on the activity of P. megaterium gasm32 elastase varied significantly (Table 3). Cu2+ and Mn2+ ions stimulated enzyme activity, whereas Mg2+, Zn2+, Fe2+, Co2+, Ca2+, Hg2+, Fe3+, Ba2+, and Na+ ions decreased it. Comparable results were reported by Manavalan et al. [37], who found that the enzyme activity of B. megaterium-TK1 increased in the presence of Mn2+ ions, while it decreased in the presence of Zn2+ and Hg2+ ions. On the other hand, Abd El-Aziz and Hassan [9] found that the elastase from B. subtilis was completely inhibited by Zn2+ and Mn2+. However, Kotb et al. [13] found that the activity of P. aeruginosa serine elastase was not affected in the presence of Hg2+, Cu2+, Zn2+, Co2+, Ca2+, Mn2+, Mg2+, Ba2+, and Fe3+ ions.
The gasm32 enzyme was completely inhibited by EDTA, indicating that it is most likely a metalloprotease. Similar findings were found by Zins et al. [33] and Abd El-Aziz and Hassan [9]. EDTA did not affect enzymatic activity in the study of Kotb et al. [13], as the enzyme they studied was a serine elastase. Metals, especially divalent cations, are essential for some enzymes’ catalytic activity and/or stability because they may act as cofactors and frequently serve as salt or ion bridges among nearby amino acid residues. EDTA is a metal–ion chelating agent that binds divalent metal ions. Therefore, it can inactivate metal ion-requiring enzymes (metalloproteases) [43]. In general, elastases of bacteria such as B. amyloliquefaciens, B. subtilis, Pseudomonas sp., E. coli, and Lysobacter enzymogenes are either serine proteases or metalloproteases, while aspartic proteases and cysteine proteases are mostly found in higher organisms such as animals and plants [44].
The data from LB plots (Fig 4) revealed that the elastase of P. megaterium gasm32 has a catalytic efficiency against azocasein of 0.055 M-1 s-1, a Km of 4.20 mg/mL, and a Kcat of 0.232 s-1. In addition, its catalytic efficiency against elastin–Congo red was 0.046 M-1 s-1, its Km was 6.03 mg/mL, and its Kcat was 0.27 s-1. Given previous research, the catalytic efficiency of the serine-elastase of P. aeruginosa ZuhP13 was 4.62 M-1 s-1, its Km was 1.32 mg/mL, and its Kcat was 1.27 s-1 [13]. The Km of the protease from B. Megaterium AU02 using azocasein was 0.722 mg/mL, and the Vmax was 0.018 U/mg [16].
Antibacterial agents could benefit from the proteolytic potential of bacterial elastases to inhibit MDR pathogenic bacteria. This research provided beneficial results as a step toward developing bioactive peptide compounds. The tested elastase exerted excellent antibacterial activity against well-known antibiotic-resistant bacteria, especially S. boydii ATCC 9207 (Fig 5 and S6 Fig). This bacterium is a Gram-negative type that causes diarrhea (often bloody), fever, and stomach cramps. Antibiotic resistance among Shigella spp. is increasing globally (Center for Diseases Control—CDC, Atlanta, USA).
Moreover, the gasm32 elastase strongly inhibited S. aureus subsp. aureus Rosenbach ATCC 25923 (Fig 5 and S6 Fig). This pathogen is a Gram-positive bacterium related to food poisoning, respiratory diseases, and skin infections. S. aureus has obtained numerous strategies to develop resistance to almost all antibiotics. Hence, antibiotic treatment is not always successful [45]. Similarly, elastase showed activity against Salmonella spp., a common Gram-negative human pathogen. The majority of Salmonella strains cause salmonellosis. Other Salmonella strains cause typhoid fever or paratyphoid fever. Salmonella s resistance to essential antibiotics has increased, limiting treatment options for people with severe infections (Center for Diseases Control—CDC, Atlanta, USA).
SEM analysis of the most affected bacteria, S. boydii and S. aureus (Fig 5), revealed that cells became fused and malformed with surface shrinkage. In addition, prominent perforation of cells was very clear in the case of S. aureus (Fig 5C). Ultimately, these effects might lead to cell death. Regarding other investigators, the alkaline protease produced by B. tequilensis ZMS-2 was found to be effective against human pathogens such as S. aureus (27 mm inhibition zone), Klebsiella pneumonia (17 mm), E. coli (15 mm), and B. licheniformis (20 mm) [21].
The in vitro application of gasm32 elastase in the degradation of macroscopic insoluble elastin fibers was confirmed using both LM and SEM. Elastin fiber degradation began on the surface of specific areas. This resulted in larger fissures and cavities, as shown by LM (Fig 6) and SEM (Fig 7). Over time, the fissures and cavities on the elastin fiber surface expanded, causing the fiber to break into small fragments, which were then further broken down into tiny peptides and amino acid residues [46].
Conclusion
In conclusion, an elastase with good characteristics was recovered and purified from the local bacterium P. megaterium gasm32. The productivity was greatly maximized after optimizing the physical and nutritional conditions (unpublished data). Additionally, the factors affecting its activity and stability were studied. Our study confirmed that it is not only able to hydrolyze elastin efficiently but also has antibacterial activity, including activity against skin burn pathogens. Therefore, it may be a candidate for regenerating and healing elastin fibers of damaged skin with antibacterial activity against contaminating bacteria. For future work and perspective, it is recommended to examine its mode of action and safety for industrialization and commercialization before it is used as a commercial pharmaceutical product.
Supporting information
S1 Fig.
Protease activity of isolate gasm32 on skimmed milk agar plates (A-B) and elastase activity on nutrient agar- elastin plates (C-D). Panels a and c represent the spot tests, while Panels b and d represent the well tests after 24 hours of incubation at 37°C. In the well test, 100 μl of crude enzyme was added into each 7 mm diameter well.
https://doi.org/10.1371/journal.pone.0282963.s001
(DOCX)
S2 Fig. Agarose gel electrophoresis showing the 700 bp PCR amplicons for P. megaterium gasm32, B. aryabhattai gasm34, K. pneumoniae gasm37, Serratia marcescens gasm82, Serratia marcescens gasm91, Macrococcus caseolyticus gasm25, Proteus mirabilis gasm43, Proteus sp. gasm71, Enterobacter cloacae gasm27, and Lactococcus lactis gasm28.
The positive control was E. coli BUN001, while the negative control consisted of the master mix with deionized sterile water. DNA was extracted using the QIAGEN kit. L contained a DNA marker.
https://doi.org/10.1371/journal.pone.0282963.s002
(DOCX)
S3 Fig. A neighbor-joining phylogenetic tree for P. megaterium gasm32.
The sequence retrieved is marked. The bootstrap value is indicated by the scale bar.
https://doi.org/10.1371/journal.pone.0282963.s003
(DOCX)
S4 Fig. The previous version of SDS-PAGE of the purified elastase using 5% stacking gel and 15% separating gel.
https://doi.org/10.1371/journal.pone.0282963.s004
(DOCX)
S5 Fig.
The MIC of elastase determined by broth dilution method against: (A) S. aureus subsp. aureus Rosenbach ATCC 25923 was 18.75 U/ml, (B) S. boydii ATCC 9207 was 37.5 U/ml.
https://doi.org/10.1371/journal.pone.0282963.s005
(DOCX)
S6 Fig.
Antibacterial activity of elastase against pathogenic strains: (A) S. boydii ATCC 9207, (B) S. aureus subsp. aureus Rosenbach ATCC 25923.
https://doi.org/10.1371/journal.pone.0282963.s006
(DOCX)
Acknowledgments
The authors would like to acknowledge the authorities of the Basic and Applied Scientific Research Center (BASRC) of Imam Abdulrahman bin Faisal University (IAU) for providing the required equipment and infrastructure to carry out this research. We are also grateful to the advisors of King Faisal University’s Pest & Plant Disease Unit for their assistance in the molecular characterization of bacterial isolates. Furthermore, we thank the authorities of King Fahd University Hospital and Imam Abdulrahman Al Faisal Hospital for providing us with the clinical strains.
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