Tyrosinase Enzyme Purification and Immobilization from Pseudomonas sp. EG22 Using Cellulose Coated Magnetic Nanoparticles: Characterization of Bioactivity in Melanin Product

doi:10.21203/rs.3.rs-3100351/v1

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Tyrosinase Enzyme Purification and Immobilization from Pseudomonas sp. EG22 Using Cellulose Coated Magnetic Nanoparticles: Characterization of Bioactivity in Melanin Product

https://doi.org/10.21203/rs.3.rs-3100351/v1

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published 10 Nov, 2023

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Melanin is a brown-black pigment produced by a variety of organisms and has significant roles in various biological processes such as insect cuticle sclerotization, wound healing, and fruit ripening. The tyrosinase enzyme catalyzes the conversion of tyrosine to melanin. Research on this enzyme and its derivatives has revealed promising uses in the pharmaceutical and biotechnology sectors. The aim of this research is to purify and immobilize the tyrosinase enzyme from Pseudomonas sp. EG22 using cellulose-coated magnetic nanoparticles. Various techniques, such as UV-visible spectroscopy, transmission electron microscopy (TEM), Zeta Sizer Nano ZS, and FTIR, were utilized to examine the synthesized nanoparticles. According to the findings, the nanoparticles exhibited a spherical shape with an average diameter of 12 nm. Furthermore, they possessed a negative surface charge, as evidenced by a polydispersity index (PDI) of 0.260 and a surface potential of -55.7 mV.

The antibacterial and anticancer bioactivity of the enzyme's melanin product is also investigated. Results of the study indicated optimum tyrosinase activity at pH 6 and 35°C and increased with increasing tyrosine concentration. The results indicate that by immobilizing the tyrosinase enzyme on cellulose coated magnetic nanoparticles, its stability can be improved, enabling longer usage. Moreover, this method could prove beneficial in increasing the production of melanin. Produced melanin showed potential antibacterial activity against multi-drug resistant strain of Citrobacter freundii. The potential of melanin pigment to decrease cell survival and induce apoptosis in initiation cells was demonstrated. When treated with the IC₅₀ concentration, HepG2 cells showed reduced resistance to melanin pigment.

apoptosis

immobilization

magnetic nanoparticles

melanin

tyrosinase

Nanoparticles, due to their small size and large surface area, have had a significant impact on many aspects of human life. This has led to rapid growth in the field of nanotechnology as a prominent area of research (Marathe and Doshi 2015).

Scientists have expressed interest in nanoparticles, which are particles that are smaller than 100 nm in size (Wu et al. 2008). Nanoparticles are whether organic or inorganic (Liu et al. 2020). Nanoparticles are considered complex molecules because of their three-layer structure. The nanoparticle's core layer is the central part, while the surface layer can be modified with various molecules or metal ions. Additionally, the outermost layer has different chemical properties compared to the core (Shin et al. 2016).

The structure of these substances determines whether they exist in zero dimensions (0D), one dimension (1D), two dimensions (2D), or three dimensions (3D). Nanoparticles have been used to deliver drugs to specific tissues and improve their ability to withstand enzyme degradation. The classification of nanoparticles will involve various criteria, such as their size, shape, and material properties (Khan et al. 2019).

Polysaccharides derived from cellulose, which is obtained from various plants including cotton, corn cobs, wood, bagasse, and stalks, are the planet's most abundant renewable resources (Jabareen et al. 2021). This polymer component presents a high biotechnological potential. Cellulose has become a popular raw material of late. The growing interest in scientific research can be attributed to the exceptional mechanical properties of nanocellulose over the past decade (Jin et al. 2017). Due to its elongated chain structure, cellulose has been discovered to have utility in the creation of a multitude of nanoparticle varieties and adsorbents.

Additionally, the use of cellulose has been demonstrated to provide effective stabilization of nanoparticles. These findings are supported by studies such as those conducted byXu et al. (2019).

The capping of magnetic nanoparticles is crucial for their various applications, such as catalysis, drug delivery, and environmental cleanup, as it allows for precise manipulation of micro-sized objects at the microscopic scale (Lu et al. 2007; Faraji et al. 2010).

Tyrosinase is an enzyme containing copper that can catalyze the o-hydroxylation of monophenols to catechols, which is also known as monophenolase or cresolase activity. Its substrate, tyrosine, can be converted into an o-diphenol called 3,4-dihydroxyphenylalanine (DOPA) by tyrosinase. The same enzyme can then further oxidise DOPA to produce an o-quinone known as dopaquinone (Zaidi et al. 2014; Biundo et al. 2020).

The production of melanin pigments relies on a complex series of enzymatic and non-enzymatic reactions that transform the o-quinone intermediate into melanin pigments (Slominski and Paus 1993; Ito and Wakamatsu 2003; Hearing 2005).

Tyrosinases are enzymes that can be found in various organisms, including plants, fungi, actinomycetes, and bacteria, both eukaryotic and prokaryotic. The tyr2 tyrosinase gene was identified by analyzing the genome of Trichoderma reesei. The gene encodes a protein that possesses a signal sequence and can be overexpressed in its native host with the aid of a potent promoter (Selinheimo et al. 2006). For instance, Streptomyces tyrosinases are small proteins that are not modified and are released into the nearby environment, playing a role in the production of melanin outside the cells (Halaouli et al. 2006).

Extracting the tyrosinase enzyme involves its acquisition from the culture supernatant. Although obtaining tyrosinase from certain sources can be challenging due to limited availability and purity, isolating bacterial tyrosinase is comparatively simple (Faccio et al. 2012; Mayowa and Marilize 2022). Variations in the relative activities, optimal pH, and molecular weight of tyrosinases are observed among different sources. Microbial tyrosinase can be produced through submerged culture or solid-state fermentation (Zaidi et al. 2014).

Quantitative assays in submerged cultures were employed to enhance the production of intracellular tyrosinase in different strains of Pycnoporus cinnabarinus and Pycnoporus sanguineus. The tyrosinase gene in Escherichia coli was induced using a bacteriophage T7 promoter, leading to the production of melanin pigments in agar plates and liquid cultures upon the addition of copper and tyrosine (Zaidi et al. 2014).

The objective of this research is to utilize magnetic nanoparticles for the isolation and purification of the tyrosinase enzyme, as well as to analyze the production of melanin pigment. Additionally, the study aims to explore the possible anticancer properties of synthesized melanin.

Chemicals

The microbiological media utilized in this study were commercially available and prepared by following the manufacturer's instructions prior to each experiment. Dehydrated forms of the media were used and reconstituted as per the provided guidelines. Tryptone soy medium (TSB and TSA), Muller-Hinton agar (MHA), and Kings medium A base were procured from HiMedia Laboratories Pvt. Ltd (India). To ensure high quality, analytical grade chemicals and solvents were employed, primarily obtained from Sigma-Aldrich or other specified sources. The liquid compounds used for cell culture were obtained from a local supplier (El-Ezaby Pharmacy Inc, Egypt) and stored appropriately at 4°C. For in vitro cell growth and proliferation, it was imperative to use a nutrient-rich medium containing balanced salts and essential compounds. One commonly employed medium for this purpose is Roswell Park Memorial Institute (RPMI-1640) medium.

Cell Line and Culture Conditions

The HepG2 ATCC® HB-8065™ human cancer cell line was acquired from the Tissue Culture Department at VACSERA in Dokki, Egypt. The cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% antibiotic-gentamicin solution, and 1% sodium pyruvate. The cells were maintained at a temperature of 37°C, with 5% carbon dioxide and 95% humidity. They exhibited an epithelial morphology and adhered to the culture flask. The cells were grown until they reached 80% confluence in 75 cm² flasks. Subsequently, the cells were harvested, counted, and washed with phosphate-buffered saline (PBS). All materials and equipment used in the study were obtained from Lonza, Belgium.

Bacterial Isolate

The Bacteriology lab at the Faculty of Science, Helwan University generously provided a bacterial isolate that displayed a noticeable green dispersed pigment. This isolate was picked up as a pure colony in another study, symbolled as EG22 and maintained on TSA slants for further investigation. It was sub-cultured monthly and kept at a temperature of 4^oC to sustain its growth.

Sequencing and phylogenetic analysis of 16S rRNA.

DNA extraction and PCR

The bacterial strain's genomic DNA was isolated using the NucleoSpin® DNA RapidLyse Kit (Macherey-Nagel) as per the manufacturer's guidelines. The 16S rRNA gene was amplified through Polymerase Chain Reaction (PCR) on a Bio-Rad T100™ Thermal Cycler, using two broad-range universal primers: an upstream primer (5-AGA GTT TGA TCC TGG CTC AG-3) and a downstream primer (5-ACG GCT ACC TTG TTA CGA CTT-3). The PCR reaction mixtures were prepared according to the provided instructions. Each mixture consisted of 1µl of genomic DNA and 1µl of each specified primer. To this, 12.5µl of KAPA HiFi HotStart ReadyMix (2x) from KAPA Biosystems was added, along with 9.5µl of RNase-free water, resulting in a total volume of 25µl. The amplification protocol for the 16S rRNA gene proceeded as follows: an initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 20 seconds, annealing at 57°C for 20 seconds, and extension at 72°C for 20 seconds. Finally, a final extension step was performed at 72°C for 5 min. The PCR product was purified using the QIAquick PCR Purification Kit (Qiagen) and subsequently subjected to unidirectional sequencing using the high-throughput sequencer NovaSeq 6000 (Illumina, San Diego, CA, USA) at BGI Genomics in China.

The Geneious Prime 2021 was employed to analyze the 16S rRNA gene and generate a phylogenetic tree. This analysis facilitated the identification of the bacterial strain and the mapping of its evolutionary lineage.

Preparation of magnetic nanoparticles

Carboxy methyl cellulose (CMC) was used as a reducing and stabilizing agent to synthesize magnetic Fe3O4 nanoparticles. A solution of Fe (III) chloride and Fe (II) sulphate was prepared, and the Fe (II) solution was rapidly combined with the Fe (III) solution and stirred for 30 min. To achieve a black color, 30% ammonium hydroxide was added to the solution. Subsequently, a mixture of 50 ml of 0.1% carboxymethylcellulose solution was added. The magnetic nanoparticles obtained were rinsed several times with alcohol and distilled water until a pH value of 7.0 was reached. In a solution containing 100 ml ethanol, 5% tetra ethyl ortho silicate (TEOS) was combined with 1 gm of cellulose coated magnetic nanoparticles (Si-CMC-MNPs) in 50 ml of purified water.

The nanoparticles were dispersed by sonicating them for 30 min. The hybrid nanoparticles obtained were collected using a magnet, washed with ethanol and purified water, and then dried at room temperature under reduced pressure.

Tyr-Si-CMC-MNPs were synthesized by mixing 1 g of prepared Si-CMC-MNPs with an aqueous solution containing 0.4 g/l of tyrosine. The pH of the magnetic nanoparticle suspension was adjusted to 9.0 by adding a 1 M NaOH solution. The mixture was heated to 90°C and stirred for 6 hours. After cooling to room temperature, the suspension was washed twice with alcohol and six times with distilled water. The final volume of the suspension was adjusted to 100 ml by adding distilled water.

Characterization of biosynthesized magnetic nanoparticles.

UV-vis spectroscopy was conducted using the UVS-260D spectrophotometer to investigate absorption maxima in the range of 190–800 nm. The Nanotechnology Centre at Helwan University, Egypt, utilized the Zeta Sizer Nano ZS instrument to determine the size and zeta potential of MNPs.

To evaluate the surface charge of the particles, a 100 µl sample of the nanoparticles was combined with 1 ml of deionized water and subjected to a 150-mV electric field at ambient temperature. The size and morphology of the nanoparticles were examined at the Nano Technology Centre in the Egyptian Petroleum Research Institute using JEOL electron microscopy. The examination was conducted at room temperature using a copper grid coated with amorphous carbon. A drop of the solution was placed on the grid and allowed to dry before further investigation. The transmission electron microscope (TEM) was set to 80 kV to observe the grid. The PerkinElmer ATR Sample Base Plate DIAMOND FTIR spectrometer, which is situated at the Central Laboratory of the Faculty of Science in Helwan University, Egypt, was utilized to directly examine the functional groups of the magnetic nanoparticles (MNPs) within the spectral range of 4000 − 450 cm^-1 (Sterner et al. 2008).

Purification of tyrosinase enzymes

200 µl/ml of Tyr-Si-CMC-MNPs were added to a solution containing a lysate of Pseudomonas sp. EG22 after being disrupted using an ultrasonic homogenizer for 2 min at 10% amplitude (Model 150VT Ultrasonic Homogenizer, Biologics, USA), then, tyrosinase enzyme bind to the surface of the Tyr-Si-CMC-MNPs through the interaction between the tyrosine ligand of the nanoparticles and the enzyme. Following a gentle shaking period of 2 hours at 25°C, an external magnetic field is utilized to separate the magnetic nanoparticles with the attached tyrosinase enzyme from the remaining proteins in the solution. Phosphate buffer solution (0.1 M, pH 7.0) is used to wash the magnetic nanoparticles in order to remove any proteins that are bound nonspecifically. Finally, the tyrosinase enzyme is extracted by using an elution buffer containing L-tyrosinamide, which competes with L-tyrosine residues on the surface of the Tyr-Si-CMC-MNPs to release the enzyme that is attached to the nanoparticles. The elution buffer consists of 50 mM Tris-HCl at pH 7.5, 300 mM NaCl, and 250 mM L-tyrosinamide in a final volume of 10 ml.

SDS-Polyacrylamide Gels (SDS-PAGE)

The protein samples were prepared by adding a loading buffer containing Tris-HCl (pH 6.8), glycerol, SDS, 2-mercaptoethanol, and bromophenol blue. The resulting mixture was diluted to a concentration of 15 µg per lane and cooled. The proteins were separated using SDS-PAGE, with a separating gel and stacking gels, in an electrophoresis unit at 120 V for 2.5 hours. After electrophoresis, the gel was stained with Coomassie brilliant blue G-250 for 1 hours and then de-stained with distilled water (Lawrence and Besir 2009).

Immobilization of tyrosinase with magnetic nanoparticles

The method for introducing amino groups to CMC magnetic nanoparticles (CMC-MNPs) involves using a bifunctional crosslinker named 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) along with an amino-containing compound known as ethylenediamine (EDA). The procedure begins with the dispersion of 10 mg of CMC-MNPs in 10 ml of a buffer solution, such as phosphate-buffered saline (PBS), at a pH of around 4.5. Subsequently, 10 mg of EDC and 5 mM of a coupling agent hydroxybenzotriazole (HOBt) are added to the solution.

Tyrosinase immobilization was performed using 250 mg of NH2-CMC-MNPs in a 250 ml Erlenmeyer flask containing a 2.0% glutaraldehyde solution in phosphate buffer (0.1 M, pH 7.0). The NH2-CMC-MNPs were activated and washed with 0.1 M acetic acid solution and 0.1 M phosphate buffer. The activated aminated CMC magnetic nanoparticles (NH2-CMC-MNPs) were then used to immobilize tyrosinase on their surface (Ozalp et al. 2015).

Following purification, the NH2-CMC-MNPs were mixed with phosphate-buffered saline to achieve a concentration of 10 mg/ml. The resulting diluted MNPs were combined with 200 µl of the tyrosinase enzyme and incubated at 25°C with gentle agitation for a duration of 3 hours (Zhou et al. 2013, 2014). Once immobilized, the tyrosinase product (Tyr@ase-NH₂-CMC-MNPs) was purified using dialysis and concentrated through magnetic separation.

Melanin production and estimation

Melanin production was achieved by adding either purified or immobilized tyrosinase enzyme (100 mg) to a solution containing 0.4g/l of aqueous tyrosine. A 10^− 3 M copper sulphate solution (100 µl) was added and incubated at room temperature for 30 min. The estimation of melanin production was conducted by Osuna et al. (2023) through the utilization of a method that entailed the measurement of the absorbance of melanin at 475 nm (Osuna et al. 2023).

Optimization of tyrosinase activity.

The study examined the impact of pH, temperature, and substrate concentration on the activity of immobilized tyrosinase. where buffer solutions with pH values ranging from 4 to 8 were created using appropriate chemicals and adjusted with NaOH or HCl. The substrate was dissolved in distilled water for preparation. Additionally, stock solutions of tyrosine at concentrations of 0.1M, 0.05M, and 0.01M were prepared. Subsequently, different concentrations of tyrosine were added to separate tubes for each pH and temperature condition. Each tube consisted of 1 ml of buffer solution, 0.1 ml of tyrosine solution, and 0.1 ml of tyrosinase enzyme. The tubes were subjected to different temperatures (30°C, 35°C, and 40°C) and incubated for ten minutes. The spectrophotometer was utilized to assess the absorbance of the reaction mixture at 475 nm, and the enzyme activity was determined by calculating the rate of absorbance change per minute.

Antibacterial activity of melanin

Antibacterial activity of melanin produced by Pseudomonas sp. EG22 was evaluated using agar paper disc method (Balouiri et al. 2016) against resistant strain of Citrobacter fruendii. Briefly, surface of sterilized MHA was spread with 40 µl of adjusted overnight bacterial culture of 5 log CFU/ml. After that, discs of sterilized papers of about 7 mm diameter were prepared and inoculated with 20 µl of partially purified melanin pigment. The plates were incubated at 37 ℃ for 18–24 hours and subsequently examined for bacterial growth and the presence or absence of clear zones. Any clear zones that formed were then measured in millimeters.

Assessment of cell cytotoxicity by MTT assay

The HepG2 cell line was rinsed with PBS and subsequently exposed to trypsin. After a 24-hour incubation period, the cells were cultured in a 96-multiwell plate at a density of 104 cells per well, using a complete RPMI-1640 growth medium. The cells were then treated with a chemotherapeutic drug. Subsequently, a serial dilution technique was employed to introduce distinct cell culture mediums containing varying concentrations of melanin pigment into the wells. This procedure was repeated thrice for each well.

The cell monolayers were subjected to melanin exposure and incubated for 48 hours at a temperature of 37°C in a CO2 incubator. After the incubation period, the melanin-containing medium was removed, and the cells were rinsed twice with PBS. Then, each well was treated with 0.5 mg/ml of MTT, a yellow compound called methyl thiazolyl tetrazolium bromide, and incubated at 37°C for 4 hours. Once the medium was discarded, dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance of the resulting color was measured at 570 nm using an ELISA reader (Boster Immunoleader, USA) to determine the percentage of viable cells.

Cell survival was calculated by dividing the absorbance of the treated wells by that of the controls and expressing it as a percentage. A correlation between the concentration of a specific melanin and cell viability was plotted to determine the survival curve and half-maximal inhibitory concentration (IC50) of the cancer cell line (Buch et al. 2012).

Detection of apoptosis by flow cytometry

The IC50 concentration of melanin was determined using the MTT assay. This value was divided into IC50/2 and IC50/4 concentrations, which were used to treat a cancer cell line in 25 cm² culture flasks with a cell density of 1×106 cells for 24 hours. After the treatment, the cells were trypsinized, collected by centrifugation, and washed with ice-cold PBS buffer. The resulting cell pellets were then resuspended in 100 µl of binding buffer containing Annexin V-FITC and PI staining solution. The cell suspension was incubated in the dark at room temperature for 15 min and then 400 µl of binding buffer was added. The cells were analyzed using a COULTER® EPICS® XL™ flow cytometer (Beckman Coulter Co., France).

16S rRNA Gene Sequence Analysis

The PCR product was sequenced, revealing that Pseudomonas sp. EG22 had a 93.3% similarity with P. putida strain LCR80 and a 92.5% similarity with P. granadensis strain CPRSM1, as confirmed by BLASTN analysis (Fig. 1).

UV- visible spectrophotometer

The UV spectrophotometer analysis of tyrosine-conjugated magnetic nanoparticles coated with CMC and silicate (Tyr-Si-CMC-MNPs) exhibited a peak at 193 nm, indicating successful conjugation of tyrosine to the magnetic nanoparticles. In contrast, magnetic nanoparticles coated with CMC and silicate alone showed no peak (Si-CMC-MNPs), indicating that the conjugation was specific to the presence of tyrosine.

Zeta size and potential

The magnetic nanoparticles displayed a surface charge of -1.94 mv, whereas the magnetic CMC had a potential of -20.4 mv, the magnetic CMC silicate beads had a potential of -36.9 mv, and the magnetic silicate tyrosine conjugate had a potential of -55.2 mv. The average diameter of the CMC silicate tyrosine magnetic nanoparticles was 81.6 nm, with a polydispersity index (PDI) of 0.260. The nanoparticles exhibited a surface potential of -55.7 mV, indicating their exceptional stability. The surface potential of the magnetic nanoparticles was found to be -1.94 mv, while the surface potential for magnetic CMC was − 20.4 mv, magnetic CMC silicate beads were − 36.9 mv, and magnetic silicate tyrosine conjugate was − 55.2 mv (Fig. 3a). The average size of the CMC silicate tyrosine-coated magnetic nanoparticles (Tyr-Si-CMC-MNPs) was determined to be 81.6 nm, with a polydispersity index (PDI) of 0.260 (Fig. 3b).

Transmission electron microscope (TEM)

Based on the TEM images (Fig. 3(A) and (b)), it can be observed that the magnetic silicate tyrosine conjugate had a spherical shape with an average size of 12 nm.

FT-IR Analysis

Figure 4 displays the FT-IR spectra of cellulose, MNPs, tyrosine, and Tyr-Si-CMC-MNPs to compare the functional groups involved in metal ion reduction and nanoparticle stability. The O-H group stretching mode is shown by IR bands ranging from 3650 − 3250 cm^− 1 in all spectra. The absorption bands at around 547 and 551 cm-1 in the MNPs and Tyr-Si-CMC-MNPs spectra correspond to the Fe-O stretching mode (Fig. 5). The hypothesis of the binding of magnetic carboxy methyl cellulose silicate to tyrosine is illustrated in Fig. 6.

Protein gel electrophoresis

It is likely that the purified tyrosinase enzyme was analyzed using protein gel electrophoresis. The method used in this study separates proteins by their size and charge, allowing for the detection of the purified enzyme and any potential impurities. The resulting protein bands would be visualized using staining technique. This analysis would confirm the purity and integrity of the purified tyrosinase enzyme. Based on Fig. 7, the protein gel profile indicates that the purified tyrosinase enzyme using magnetic nanoparticles has a molecular weight of 62.2 KDa.

Enzyme activity

The absorbance at 475 nm was measured to determine the concentration of dopachrome, the product of the immobilized tyrosinase enzyme. The results demonstrated a direct relationship between the concentration of dopachrome and absorption, suggesting a higher level of melanin production.

The comparison between the immobilized magnetic tyrosinase enzyme and the free tyrosinase enzyme revealed that the highest concentration of melanin production was observed at pH 6 for both enzymes. At pH 5 and pH 7, the concentration of produced melanin was also high for both enzymes. In contrast, at pH levels of 4 and 8, both the immobilized and free enzymes exhibited reduced levels of melanin production, as shown in Fig. 8 and Table 1.

Table 1

Comparative melanin production by immobilized and free tyrosinase enzyme across a pH range of 4 to 8
PH	OD 475 of Immobilized enzyme	OD 475 of Free enzymes
4	0.444	0.073
5	0.712	0.547
6	0.757	0.665
7	0.644	0.339
8	0.461	0.236

Based on these findings, it can be inferred that a pH of 6 is the most favorable for melanin production using both the immobilized and free tyrosinase enzymes. This pH provides an optimal environment for efficient enzyme activity and effective synthesis of melanin. Any deviation from the pH range of 4 to 8 may have a detrimental effect on melanin production, possibly due to reduced enzyme activity or instability. The absorbance peak of dopachrome, which is the product of the tyrosinase enzyme, was recorded at 475 nm. As the concentration of dopachrome increased, so did the level of absorption.

Throughout all temperatures tested, the optical density (OD) at 475 nm consistently demonstrated that the immobilized enzyme had higher melanin production in comparison to the free enzyme. At 30°C, the OD of the immobilized enzyme was 0.948, while the free enzyme had an OD of 0.75. This pattern persisted at 35°C and 40°C, with the immobilized enzyme exhibiting greater melanin absorption than the free enzyme. Notably, the immobilized enzyme at 35°C exhibited the highest concentration of melanin, as indicated by the highest OD value, indicating the greatest melanin production. No significant difference in activity was observed between the free and immobilized enzyme at a temperature of 40°C, as shown in Fig. 9 and Table 2.

Table 2

Comparative Melanin Production by immobilized and free enzyme at at 30, 35, and 40 ^oC.
Temperature	OD 475 of Immobilized enzyme	OD 475 of Free enzymes
30	0.948	0.75
35	1.338	0.825
40	1.077	0.095

In this study, the production of melanin was examined by immobilizing the tyrosinase enzyme on magnetic nanoparticles at various concentrations of tyrosine. The production of melanin initially rose with increasing tyrosine concentrations until it reached a maximum point, after which it declined. The highest melanin production was observed at an optical density (OD) of 1.338, when the concentration of tyrosine was 0.1. Table 3 and Fig. 10 present the specific OD values at different tyrosine concentrations. At a tyrosine concentration of 0.01, a lower melanin production was observed with an OD of 0.948. As the tyrosine concentration increased to 0.5, the melanin level also increased, resulting in an OD of 1.168.

Table 3

Investigating Melanin Production by immobilized tyrosinase at varied tyrosine concentrations
Conc. mM	Immobilized enzyme OD 475 nm
0.01	0.948
0.1	1.338
0.5	1.168

The storage time of the tyrosinase enzyme immobilized with magnetic nanoparticles resulted in a decrease in melanin production. The concentration of melanin reached its maximum level (OD: 1.356) during the initial month of storage, but gradually declined over time. The results indicated that the melanin concentration decreased gradually in the second, third, fourth, fifth, and sixth months of storage, as shown in Table 4 and Fig. 11, with concentrations corresponding to OD: 1.345, 1.323, 1.256, 0.994, and 0.893, respectively.

Table 4

Assessing the stability of enzyme activity over extended time intervals (1–6 Months).
Time in month	Immobilized enzyme OD 475 nm
1	1.356
2	1.345
3	1.323
4	1.256
5	0.994
6	0.893

Antibacterial activity

The paper disc method was used to investigate the antibacterial activity of melanin pigment against a multi-drug resistant strain of Citrobacter fruendii. Results represented in Fig. 12 showed that the pathogen was sensitive to our melanin product with an inhibition zone of 18 mm, indicating its antibacterial activity.

Cell Viability Assessment

The viability of HepG2 liver cancer cells and the IC50 value of melanin pigment were assessed using the MTT test. Additionally, flow cytometry was employed to identify the various stages of cell death caused by different treatments. These stages included necrotic cells (Annexin-V-/PI+, Q1), late apoptotic cells (Annexin-V+/PI+, Q2), viable cells (Annexin-V-/PI-, Q3), and early apoptotic cells (Annexin-V+/PI-, Q4).

Melanin Response of HepG2 Cells

Figure 13 shows the application of a melanin IC50 concentration (985 µg/ml) to the HepG2 cancer cell line. The graph illustrates the average absorbance, expressed as a percentage of the untreated control cells, plotted against different melanin concentrations.

Detection of Apoptosis in HepG2 Cells

By analyzing Annexin V-FITC/PI, it was determined that the melanin pigment induced apoptosis in HepG2 cells at different stages, as depicted in Fig. 14. The highest percentage of early and late apoptosis (14.75%) was observed when HepG2 cells were exposed to the IC50 concentration of melanin, surpassing the effects of other concentrations.

P. aeruginosa can produce melanin, a black pigment with diverse biological properties such as antimicrobial, anti-inflammatory, and antioxidant effects. Tyrosinase enzymes play a crucial role in the synthesis of melanin by converting tyrosine into dihydroxyphenylalanine (DOPA) and subsequently into melanin. Both free and immobilized enzymes can be utilized for melanin production (Rabaey et al. 2005; Das et al. 2013).

Studies have been carried out to purify and immobilize tyrosinase enzymes for various applications, including the synthesis of melanin (Khan et al. 2005; Chomoucka et al. 2010; Zdarta et al. 2020). Different methods have been employed to purify tyrosinase enzymes, including ammonium sulfate precipitation and chromatography using ion-exchange or affinity with specific ligands. The purified enzymes have been immobilized on different matrices, including magnetic nanoparticles, to enhance their stability and reusability (Al-Abbasy et al. 2021). Enzymes immobilized on chitosan have been shown to have improved operational stability and reusability compared to free enzymes (Sakono et al. 2019; Verma et al. 2020; Fidalgo et al. 2021).

Using silica-coated magnetic nanoparticles and immobilizing tyrosinase on them, we achieved successful melanin production. The immobilized enzyme displayed enhanced catalytic activity and improved reusability compared to the unbound enzyme (Abdollahi et al. 2017, 2019). The UV spectrophotometer analysis confirmed the successful binding of tyrosine to the magnetic nanoparticles, as indicated by the presence of a peak at 193 nm. This absorption behavior of tyrosine is consistent with its known characteristics, as it absorbs UV light within the wavelength range of 190–240 nm, attributed to its aromatic ring structure (Chapman 1963; Steyermark 1976; Wong 2015). Based on the peak range observed in this study, it can be inferred that the tyrosine-linked magnetic nanoparticles are composed of tyrosine molecules. The lack of a peak in the UV spectrophotometer analysis of magnetic nanoparticles coated with CMC and silicate alone suggests that the binding process was targeted solely towards tyrosine. This targeted binding is crucial in ensuring that the magnetic nanoparticles are modified with the desired molecule and can be utilized for specific purposes. The incorporation of tyrosine-coated magnetic nanoparticles has shown promise in drug delivery, as it has been observed that the presence of tyrosine improves the cellular uptake of nanoparticles (Chomoucka et al. 2010). The analysis conducted using TEM revealed that the Tyr-Si-CMC-MNPs possessed a spherical shape with an average diameter of 12 nm and provided information on their size and morphology (Murdock et al. 2008).

The spectrum of Tyr-Si-CMC-MNPs shows 3020.25 cm^− 1 -C-H streching, 1401.73 cm^− 1-C-H bending, the Fe-O-Si stretching vibration at 1025.76 cm^− 1 (Ahangaran et al. 2013; Nalbandian et al. 2015; Lobato et al. 2017; Dawn et al. 2022). The spectrum of tyrosine shows the 3105.72, 3040.60 cm^− 1 =C-H stretching, 2930.03- 2825.20 cm^− 1 -C-H stretching, 2080.06 Cl-H stretching, 1606.16 NH₂ scissoring − 1556, 1584.02 cm^− 1 alkenyl aromatic C = C stretch, 1451 cm^− 1 -CH2 bending, 1329.54 cm^− 1. The combination of stretching of C-C-C bonds and the presence of Phenolic OH groups is observed at a wavenumber of 1112.14 cm-1. Additionally, the presence of C-N bonds is observed at a wavenumber of 1243.04 cm-1, along with the presence of C-O bonds (Anandan et al. 2012). Based on the absorption of IR bands, the cellulose effectively reduced the Fe₃O₄NPs and provided a thorough coating of SiO₂ and tyrosine layers.

The average size of the prepared magnetic nanoparticles, CMC silicate tyrosine (Tyr-Si-CMC-MNPs), was 81.6 nm. These nanoparticles showed a polydispersity index (PDI) of 0.260 and a surface potential of -55.7 mV, indicating excellent stability (Gutierrez and Flores 2018). At 475 nm, the peak absorbance of dopachrome, a byproduct of the tyrosinase reaction, was measured (Osuna et al. 2023). The study examined the production of melanin by both immobilized and free enzymes at various pH levels from 4 to 8. The findings indicated that melanin production reached its peak at pH 6 for both immobilized and free enzymes. However, the immobilized enzyme consistently generated more melanin than the free enzyme across all pH values.

The enhanced production of melanin by the immobilized enzyme can be attributed to several factors. Firstly, the immobilized enzyme displayed superior stability compared to the free enzyme, resulting in reduced degradation. Secondly, the immobilized enzyme had enhanced access to the substrate, resulting in a higher reaction rate. Finally, the immobilized enzyme was less susceptible to inhibition by inhibitors present in the culture medium (Huang et al. 2012; Brena et al. 2013; Stancu 2020).

The results of this study demonstrate that immobilizing enzymes can enhance the efficiency of melanin production compared to non-immobilized enzymes. This improvement is attributed to the increased stability, improved accessibility to substrate, and reduced susceptibility to inhibition of immobilized enzymes. The findings show that magnetic immobilization of the tyrosinase enzyme increases the concentration of melanin produced, particularly at a pH of 6. This may be due to the enhanced stability of the immobilized enzyme at pH 6, allowing for optimal function and higher melanin production. Furthermore, previous studies have shown that the use of magnetic immobilization can enhance the stability, activity, and reuse of enzymes. This is achieved by providing protection against harsh reaction conditions and reducing the leakage of the enzyme (Imarah et al. 2021; Sharma et al. 2021; Csuka et al. 2022).

On the other hand, the highest concentration of melanin produced by the free tyrosinase enzyme was observed at pH 6, but it was still lower compared to the melanin produced by the magnetic immobilized tyrosinase enzyme. This difference can be attributed to the instability of the free enzyme at acidic and basic pH levels, which can lead to enzyme denaturation and a decrease in activity (Johnson et al. 2008; Ren et al. 2011; Yu et al. 2012; Sánchez-Ramírez et al. 2014; Vaghari et al. 2016; Khoshnevisan et al. 2017; Li et al. 2017; Bilal et al. 2018; Darwesh et al. 2020; Fauser et al. 2020). Furthermore, the utilization of unbound enzymes frequently leads to decreased enzyme effectiveness because of their limited reusability and vulnerability to deactivation caused by reaction byproducts and other elements.

The observed increase in melanin production with the immobilized enzyme at all three temperatures can be attributed to several factors. Firstly, the enzyme's stability and activity are enhanced by immobilization, protecting it from denaturation and breakdown and providing an optimal environment for its function. This may explain why the immobilized enzyme produced higher concentrations of melanin compared to the free enzyme at all three temperatures tested. Secondly, the immobilized enzyme's increased accessibility to the substrate could also contribute to the higher melanin production (Khan 2021). The magnetic immobilization technique used in this study may have facilitated contact between the enzyme and substrate, potentially increasing the reaction rate and melanin production. Lastly, the immobilized enzyme was less likely to be inhibited by inhibitors present in the culture medium. This could be because the magnetic immobilization technique allowed for separation of the enzyme from the culture medium, preventing inhibitors from binding to the enzyme and inhibiting its activity (Trindade Ximenes et al. 2021).

The increase in melanin concentration, observed as tyrosine concentration rises, can be attributed to the conversion of tyrosine into melanin facilitated by the tyrosinase enzyme. As the amount of tyrosine increases, there is more substrate available for the enzyme to convert, leading to a higher concentration of melanin. However, once a certain level is reached, the concentration of melanin begins to decline. This decline may be attributed to the inhibition of enzyme activity at high tyrosine concentrations, causing a decrease in melanin production (Wu et al. 2018). The results suggest that the magnetic immobilized tyrosinase enzyme can effectively convert tyrosine to melanin, with the highest concentration obtained at a tyrosine concentration of 0.1. These findings could have potential applications in the development of biosensors or biocatalysts for the detection or degradation of melanin in environmental or medical settings. Further studies could be conducted to optimize the conditions to produce melanin using magnetic immobilized tyrosinase enzyme, as well as to investigate its stability and reusability (Wu et al. 2018; Trindade Ximenes et al. 2021).

The results suggest that the Citrobacter freundii strain used in the study displays resistance to all tested antibiotics, even at the prescribed concentrations. The growing concern over the emergence of resistance to multiple antibiotics in healthcare facilities is due to the challenge it presents in treating infections caused by these bacteria. The melanin disc created an inhibition zone of 18 mm, indicating its ability to inhibit the growth of the tested bacterial strain. Melanin, a black pigment produced by P. aeruginosa, a Gram-negative bacterium known for causing infections in immunocompromised individuals, has been found to have the potential to suppress the growth of C. freundii. Therefore, melanin could be a promising antimicrobial agent.

To assess the anticancer properties of melanin pigment against HepG2 cell lines, the MTT assay was utilized. It is important to note that a 24-hour treatment with chemotherapy agents may not significantly reduce cell viability. Therefore, it may be beneficial to evaluate cell viability after 48 hours (Yu et al. 2015). Evaluating the effectiveness of a drug at an early stage can provide valuable information, as prolonged exposure may lead to misleading results by suggesting similar effects among different substances. When the HepG2 cancer cell line was subjected to different concentrations of melanin pigment, a significant increase in apoptosis induced by the agent was observed, indicating a correlation between lower melanin resistance and increased apoptosis. The influence of melanin pigment treatment on cell viability can be observed by the noticeable expression of the apoptotic marker Annexin V.

After analyzing the flow cytometry data, it was noticed that HepG2 cells showed higher levels of total apoptotic cell populations (Q2 + Q4) after being exposed to different concentrations of melanin pigment for 24 hours, compared to the control cells. This indicates that HepG2 cells have lower resistance to melanin pigment.

In conclusion, the tyrosinase enzyme from Pseudomonas sp. EG22 was purified and immobilized using cellulose coated magnetic nanoparticles, and the bioactivity of its melanin product was examined. Results indicated that the optimal pH and temperature for tyrosinase activity aligned with prior reports, and that the immobilized magnetic tyrosinase was considerably influenced by pH and temperature. The study revealed that immobilizing the tyrosinase enzyme on cellulose coated magnetic nanoparticles could be an effective approach for increasing melanin production. Particularly at pH 6, the magnetic immobilization of the enzyme enhanced the concentration of produced melanin, highlighting its potential in melanin production. Furthermore, the results showed that the magnetic immobilized tyrosinase enzyme generated more melanin than free tyrosinase enzyme at all three temperatures. The study also demonstrated the enzyme's ability to effectively convert tyrosine to melanin, with the highest concentration observed at a tyrosine concentration of 0.1. Overall, the study underscored the potential of immobilized tyrosinase enzyme for enhancing melanin production. The use of melanin pigment exhibited potential in decreasing cell survival and inducing apoptosis in initiation cells. It was observed that HepG2 cells displayed reduced resistance to cytotoxic melanin pigment when treated with the IC₅₀ concentration, highlighting the fact that the response of cells to anti-cancer drugs can vary depending on the drug concentration, even under normal conditions.

Acknowledgments

Not applicable

Data Availability Statement:

The data supporting the findings of this study, including the raw data files of the flow cytometer (Test1-control.fcs, Test2-quarter IC50.fcs, Test3-half IC50.fcs, and Test4-IC50.fcs), as well as the 16s rRNA sequence and AB1 file for the Pseudomonas sp. EG22 bacterial strain, have been provided as Supplementary material. Additionally, the analytical methods employed in this research, encompassing the purification and immobilization process of the tyrosinase enzyme using cellulose-coated magnetic nanoparticles, along with the evaluation of its bioactivity in melanin production, are available upon request from the corresponding author, Ahmed H. I. Faraag (email: [email protected]). Due to the sensitive nature of the data and the ongoing research in the field, access to the data will be granted after a reasonable request and with the appropriate confidentiality and ethical considerations.

Funding

The authors of this manuscript state that they did not receive any financial assistance, grants, or other forms of support while preparing it. The funding for this research was provided by the Science, Technology & Innovation Funding Authority (STDF) in collaboration with The Egyptian Knowledge Bank (EKB), which offered open access funding.

Competing interests

The authors have no conflicts of interest to declare.

Author Contributions

All authors have made significant contributions to this research and actively participated in the intellectual aspects of the study. Salim Mohamed Abd El-Aziz played a role in conceptualizing the study, developing the experimental design and methodology, supervising the research work, analyzing and interpreting the data, writing the original draft of the manuscript, and revising and editing it. Ahmed Hassan Ibrahim Faraag contributed to the conceptualization of the study, supervised the research work, conducted experimental tasks such as purifying and immobilizing the tyrosinase enzyme and optimizing the cellulose coating process on magnetic nanoparticles, collected and analyzed data, and contributed to manuscript writing, including the methods and results sections. Ayman Meselhi Ibrahim conducted experimental work, specifically characterizing the bioactivity in the melanin product, analyzed and interpreted data from bioactivity assays, provided input for discussing and interpreting the results, and contributed to manuscript writing, particularly the results and discussion sections. Ashraf Albrakati made contributions to the development of the experimental design and methodology, provided guidance and technical support throughout the research work, critically reviewed and revised the manuscript, and ensured the scientific rigor and accuracy of the study. Marwa Reda Bakkar assisted with experimental work, including data collection and analysis, provided support in purifying and immobilizing the tyrosinase enzyme, contributed to manuscript writing, particularly the introduction and discussion sections, and assisted with revising and editing the manuscript to enhance clarity and coherence. All authors have read and approved the final version of the manuscript.

Ethics Approval:

This study did not involve the use of animals.

Consent to Participate

Not applicable.

Consent to publish.

Not applicable.

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Tyrosinase Enzyme Purification and Immobilization from Pseudomonas sp. EG22 Using Cellulose Coated Magnetic Nanoparticles: Characterization of Bioactivity in Melanin Product

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Journal Publication

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Abstract

Figures

Introduction

Materials and methods

Chemicals

Cell Line and Culture Conditions

Bacterial Isolate

DNA extraction and PCR

Preparation of magnetic nanoparticles

Purification of tyrosinase enzymes

SDS-Polyacrylamide Gels (SDS-PAGE)

Immobilization of tyrosinase with magnetic nanoparticles

Melanin production and estimation

Antibacterial activity of melanin

Assessment of cell cytotoxicity by MTT assay

Detection of apoptosis by flow cytometry

Results

16S rRNA Gene Sequence Analysis

UV- visible spectrophotometer

Zeta size and potential

Transmission electron microscope (TEM)

FT-IR Analysis

Protein gel electrophoresis

Enzyme activity

Antibacterial activity

Cell Viability Assessment

Melanin Response of HepG2 Cells

Detection of Apoptosis in HepG2 Cells

Discussion

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1