Immobilized Cold-Active Enzymes onto Magnetic Chitosan Microparticles as a Highly Stable and Reusable Carrier for p-xylene Biodegradation in Soil

doi:10.21203/rs.3.rs-1740243/v1

Stability and reusability properties are the two most important factors that determine an enzyme's application in industry. To this end, cold-active crude enzymes from a psychrophile were immobilized on magnetic chitosan microparticles using glutaraldehyde as a linker. Immobilization was optimized based on four variables, such as magnetic particle, chitosan, glutaraldehyde, and enzyme concentrations. The immobilized enzymes were characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscope (SEM). The immobilized enzymes showed improved pH tolerance ranging from 4.0 to 9.0, better temperature stability ranging from 5 to 50, higher storage stability (∼70% activity after 30 days of storage), and more importantly, reusability (∼40% activity after 10 repetitive cycles of usage) compared to their free form. Also, the immobilization of enzymes increased the effectiveness of the enzymatic treatment of p-xylene (10,000 mg/kg) in soil. As a result of the superior catalytic properties of immobilized XMO and C1,2D, they offer great potential for in situ or ex situ bioremediation of pollutants in soil or water.

Cold active

Immobilization

Magnetic

chitosan

Composite

Living systems rely upon enzymes to catalyze a variety of chemical reactions. Due to enzymes' specific properties, such as their chemo-, stereo-, and regioselectivity, they are generally preferred over chemicals as catalysts. Hence, using enzymes has recently received increasing attention for pollutant removal as an environment-friendly technology. However, enzymes are not widely used for remediation purposes due to their high production costs. A useful tool for reducing costs is the immobilization of enzymes as it enables efficient recovery, reuse, and recycling, as well as increased stability under harsh operational conditions such as high/low pH and temperature. An important challenge of using immobilized enzymes is the identification of new matrix materials with appropriate structural and morphological characteristics as well as understanding enzyme-matrix interactions for improving catalysis (Kumari et al. 2018; Agrawal et al. 2020; Wang et al. 2022).

Many different carriers can be used to immobilize enzymes (Xie et al. 2009). Among all of them, chitosan (known as poly-β (1 → 4)-2-amino-2-deoxy-d-glucose) is a linear polysaccharide that has been often used for immobilization of enzymes (Krajewska 2004). Chitosan polymer possesses a number of significant properties, such as its biocompatibility, hydrophilicity, mechanical stability, renewability, and the ease with which it can be prepared in multiple geometric configurations suitable for biodegradation. Chitosan has the additional advantage of being inexpensive, allowing it to be used to prepare low-cost carriers for large-scale applications. Enzymes can be immobilized by cross-linking chitosan and activation by glutaraldehyde (Pospiskova and Safarik 2013). Moreover, magnetic carriers may also be used to separate immobilized enzymes by simple magnet. Magnetic chitosan carriers have already been used for immobilizing several enzymes as depicted in Table 1. In many cases, magnetic chitosan particles are used for immobilization of mesophilic enzymes for various applications (Table 1), but not used for enzymatic biodegradation of petroleum pollutants.

Table 1

Magnetic chitosan-based carriers for enzyme immobilization, their applications and reusability
Enzyme	Carrier	Application	Reusability	Reference
Lipase	Magnetic chitosan beads	Synthesis of flavor esters	5 cycles with more than 70% of its initial activity	(Bayramoglu et al. 2022)
Glucose oxidase and catalase	Magnetic chitosan microspheres	Production of Sodium gluconate from glucose	10 with more than 60% of their remaining activity	(Liu et al. 2022)
Lipase	β-cyclodextrin grafted and aminopropyl-functionalized chitosan/Fe₃O₄ magnetic nanocomposites	Synthesis of fruity flavor esters	15 cycles with a slight decrease (15%) in the relative activity	(Zhao et al. 2022)
Laccase	Magnetic chitosan nanoparticles modified with amino-functionalized ionic liquid containing ABTS	Removal of 2,4-dichlorophenol, bisphenol A, indole and anthracene	6 cycles with a removal efficiency of 93.2% for 2,4-dichlorophenol.	(Qiu et al. 2021)
Lactase (β-d-galactosidase)	Magnetic chitosan microsphere	Biomedical applications	10 recycles with more than 65% of its initial activity	(Ke et al. 2020)
α-amylase	Chitosan-magnetite composite	Food, fermentation, detergent applications, textile and paper industry	10 cycles with 45–55% of its initial activity	(Bindu and Mohanan 2020)
Porcine pancreatic lipase	Magnetic chitosan nanoparticles modified with imidazole-based functional ionic liquid	ND	10 recycles with more than 84.6% of its initial activity	(Suo et al. 2019)
Laccase	Chitosan beads magnetized with Fe₃O₄ NPs	Removal of Evans blue, Direct blue 15, Reactive black 5 and Acid red 37 azo dyes	10 cycles with 47% of remaining activity	(Nadaroglu et al. 2019)
Xylanase	Magnetic chitosan	Production of xylooligosaccharides with food and pharmaceutical applications	3 cycles retaining 50% of its initial activity	(Gracida et al. 2019)
Cellulase	Magnetic Chitosan Nanoparticles	Glucose production	10 cycles with more than 80% of its initial activity	(Zang et al. 2014)
Glucoamylase	Magnetic chitosan microspheres	Production of glucose from starch	10 cycles with 45–68.4% of its initial activity	(Wang et al. 2013)
Lipase and β-galactosidase	Magnetic chitosan microparticles	ND	8 cycles with more than 90% of its initial activity	(Pospiskova and Safarik 2013)
Pullulanase	Magnetic chitosan beads	Improves the saccharification of starch to produce glucose	10 cycles retaining 64.8% residual activity	(Zhang et al. 2009)

There are two key enzymes involved in the oxidation of aromatic rings - Xylene monooxygenase (XMO) and catechol 1,2-dioxygenase (C1,2D). These enzymes can be found in most of the aerobic biodegradation of mono-aromatic petroleum hydrocarbons. For instance, Pseudomonas stutzeri degraded benzene and toluene by expressing toluene/xylene monooxygenase (Wang et al. 2021). Wongbunmak et al., (2020) reported that XMO played critical roles in toluene and m-xylene biodegradation by Bacillus amyloliquefaciens subsp. plantarum strain W1 (Wongbunmak et al. 2020). Moreover, XMO from P. putida mt-2, could catalyze only m-xylene and p-xylene (Panke et al. 1999). Our previous research also showed that cold-active Pseudomonas strains produced cold-active XMO and catechol 2,3 dioxygenase that can degrade p-xylene under low temperatures (< 15ºC). Cold-active enzymes have the potential to provide greater value in terms of sustainable immobilization and production and may be preferred as a result of their higher catalytic activity in comparison to their mesophilic counterparts for biodegradation of contaminants in cold environments. Our previous finding also showed that the inherent instability of these enzymes is one of the important challenges in the industrial and commercial application of these enzymes (Miri et al. 2021b, 2022). In our previous results, the reusability of immobilized enzymes on solid carriers for soil matrices such as biochar was not feasible (Miri et al. 2021b). This leads us to study the immobilization of enzymes on magnetic carriers for ease of separation in the soil.

The current study hence focused on evaluating the stability and reusability of immobilized XMO and C1,2D for biodegradation of p-xylene in highly contaminated soil. Immobilization of cold-active enzymes was optimized for maximum immobilization yield. Then, kinetic parameters of enzymes were determined before and after immobilization. To the best of our knowledge, no reports have systematically evaluated the use of magnetic chitosan carriers for immobilization of cold-active XMO and C1,2D and how these immobilized enzymes can be applied to the degradation of petroleum hydrocarbons in soil.

Bacterial strains and enzyme preparation

Identification and characterization of psychrophilic Pseudomonas S2TR-14 were carried out as mentioned in our previous study (Miri et al., 2021). Briefly, the selected strain was cultured in tryptic soy broth (TSB) at 15±1^∘C and 150 rpm. Optical density at 600 nm (OD600) and the number of viable bacteria by counting colony-forming units (CFU) showed a correlation. Each OD600 value corresponds to 1.2×10⁸ of viable Pseudomonas S2TR-14. Enzyme production was carried out according to our previous study (Miri et al. 2022) with some modifications. 18 g/L of crustacean waste, 2 g/L of yeast extract, and 200 mg/L of p-xylene were used as the source of calcium, proteins, and carbon respectively. After 24h of incubation, the culture was centrifuged at 3810 x g for 20 min. Then, the pellet was collected and washed with phosphate buffer (pH 7.2) and then re-suspended in the same buffer then ultrasonicated at 22-30 kHz frequencies for 10 min on ice (Branson Ultrasonics Corporation, Danbury, CT, USA). It is noticeable that 15 ± 1 ^∘C was considered the optimum temperature for these cold-active enzymes and all tests were carried out at this temperature.

Magnetic particle preparation

There are several methods for the synthesis of Fe₃O₄ depending on the particle size to be obtained. In this study, magnetic microparticles were synthesized by the method of (Singh et al., 2016). Briefly, the magnetic microparticles (MPs) were obtained by reduction of FeCl₂.7H₂O and FeCl₃.6H₂O chlorides in an aqueous ammonia solution under vigorous stirring. 5.4 g of FeCl₃. 6H₂O followed by 2.0 g of FeCl₂. 4H₂O was dissolved in 25 mL of 0.4 M HCl solution. The solution was added dropwise to 250 mL of a 1.5 M NaOH solution under continuous stirring. A black precipitate was formed immediately. The synthesized magnetic microparticles were washed several times using deoxygenated water and separated using a permanent magnet and dried using a vacuum until further use.

Optimization of the immobilization parameters by RSM

Immobilization of enzymes onto magnetic chitosan microparticles was carried out according to Kumari et al. with modification (Kumari et al. 2018). The optimum levels of parameters of enzyme immobilization on magnetic particles for the maximum immobilization yield were determined statistically using the Box-Behnken design of experiment (BBD) in the Response Surface Methodology (RSM). Table S1 shows the defined factors including magnetic particle, chitosan, glutaraldehyde, and enzyme concentrations, along with their related levels. Levels for the factors were selected based on preliminary experiments. Experimental design, model calculation, graph drawing, and statistical analyses were performed using Minitab^® software (Version 8.0.6, Stat-Ease Inc., Minneapolis, USA). The response model was assessed using ANOVA to evaluate its adequacy and significance. A selected concentration of the magnetic microparticles (3 mg/mL stock solution) was taken in a microcentrifuge tube based on the best results of the one at a time method. The microparticles were settled using a magnetic bar. Under shaking conditions, each gram of magnetic microparticles and chitosan was mixed with acetic acid (2% v/v) for 24 hours. These magnetic particles were washed extensively with deoxygenated water and activated using glutaraldehyde through overnight incubation. Later, crude enzyme solution was added and shaked for 2 h. To remove excess glutaraldehyde and free enzymes, these particles were washed five times with 50 mM phosphate buffer (pH=7.2). The obtained particles was kept overnight at 4ºC under the vacuum condition for drying.

According to Eq. (1), immobilization yields were calculated and used to evaluate the response of independent variables (Box et al., 1978).

Enzymes kinetic parameter analysis

To describe the kinetic behavior, v_max, K_m, and catalytic efficiency (K_cat/K_m) were determined by the Michaelis-Menten model as described by (Kwean et al. 2018)with some modifications. The Michaelis-Menten model, as given in Eq 2. was used to describe the kinetic behavior of enzymes.

Where v and v_max correspond to the rate of substrate utilization and its maximum rate, C_Scorresponds to the initial substrate concentration and K_m corresponds to the half-substrate constant (for enzyme-substrate affinity indication). Different substrate concentrations (0.1–1.0 mM) were used to determine the initial rate of production of the compounds (p-cresol for XMO and C1,2D for muconic acid). Following that, the parameters were calculated using GraphPad Prism version 9.3.1.

Enzyme leaching test

As a leaching test, 0.1 g of immobilized enzymes were incubated in 1 mL of sodium phosphate buffer (pH 7.0) for 1 h, 6 h, 12 h, 24 h, and 48 h while continuously stirring. The particles were separated using a magnet and then the supernatant was used for enzyme activity. The leaching (%) was calculated using Eq. (3):

Effect of pH and temperature

For pH stability, 500 μL of free enzymes (40 U/mg C1,2D and 20 U/mg XMO) and 0.1 mg of immobilized enzymes (initial activity 400 U/g of C1,2D per gram of support and 200 U/g of XMO per gram of support) were prepared in vials containing 2 mL of different buffers (pH of 2 to 10). The residual enzyme activity of free and immobilized samples was determined. The thermostability test was carried out by incubating free and immobilized enzymes at different temperatures (5-50 °C) for 8 hours and then residual activity was assessed in the same way explained for pH stability.

Biodegradation and reusability tests

Bioremediation experiments were carried out in 150 mL sealed serum bottles containing 25 mL of contaminated groundwater or 5 g of soil sample and 0.2 g of immobilized enzyme. The groundwater sample was characterized as hard water due to the presence of carbonates. The soil and groundwater were originally contaminated with p-xylene (10,000 mg/Kg in soil and 200 mg/L in groundwater). Due to the volatile nature of p-xylene, the samples have been artificially contaminated until reaching the concentration at the contaminated site. Before the beginning of incubation, soil moisture was adjusted to 70% of water holding capacity using groundwater. Three sets of controls were designed: 1) control treatment using sterilized soil/groundwater by autoclave (121 °C, 30 min) that simulated abiotic losses 2) reference controls without any treatment that simulated natural attenuation 3) control treated with magnetic chitosan microparticles without enzymes that determined the adsorption of p-xylene to the carrier. To study the reusability of the immobilized XMO and C1,2D, the immobilized enzymes were removed using a magnet and washed with methanol after the first cycle of enzymatic reaction. It was later suspended in a fresh reaction mixture to evaluate the enzyme activity. The initial activity of the enzymes was considered as 100% and activity in the cycle was reported as a relative percentage of the initial activity.

Analytical methods

Gas Chromatography/Mass Spectrometry (GC/MS)

For sample preparation, 0.05 g of soil was mixed with 5 mL of methanol as the extracting solvent in 10 mL headspace vials containing. Then, samples were vortexed for 5 minutes and 50 µL of sub-sample was removed. A gas-tight syringe was used to remove the sub-samples (50 µL) from the liquid phase and prepare them in 40 mL standard GC-headspace vials. Each vial was containing 5 mL of ultrapure water, and 10 μL of fluorobenzene-D5 (100 mg/L) as an internal standard. p-xylene concentration was measured using a 7890A Gas Chromatograph coupled to a Saturn Mass Detector (GC/MS) equipped with a 40-trap automated headspace sampler as described in detail in our previous study (Miri et al. 2022). The concentration of the substrates and compounds was determined by measuring the areas of their peaks and a calibration curve.

Fourier transform infrared (FT-IR) spectroscopy

FTIR spectra of each sample were measured using a FTIR spectrophotometer (Nicole IS50) equipped with an attenuated total reflectance (ATR). Each spectrum was repeated with 16 scans per sample in the spectral range from 500 to 4000 cm^-1 at room temperature. The average of spectra for each sample was used for plotting.

Scanning electron microscope (SEM)

The morphological changes of MPs before and after coating with chitosan and enzyme immobilization were evaluated by a scanning electron microscope (Thermofisher Quanta 3D). The acceleration voltage and working distance for each image were 30 kV and 1–5 mm respectively. Before observation, the samples were coated with a gold-conductive layer.

Liquid Chromatography with tandem mass spectrometry (LC-MS/MS)

For the identification of proteins, the enzyme mixture was prepared using the S-Trap mini spin column (Protifi, USA) according to the manufacturer's instructions. The peptides were analyzed using a mass spectrometer coupled to an EASY-nLC 1000 system (Thermo Fisher Scientific, USA) and using Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, USA) to determine their mass. Proteome Discoverer (version 2.2, Thermo Fisher Scientific, USA) was used to process the raw data. The identification of a protein was established with greater than 99.0% probability and contained at least two distinct peptides. The Protein Prophet algorithm assigned probabilities to proteins. Then the data was aligned with the protein database of the Universal Protein Resource at UniProt (www.uniprot.org) and BLASTP (https://blast.ncbi.nlm.nih.gov).

Enzyme activity and protein concentration assays

Xylene monooxygenase activity was assayed using the spectrophotometric method as described by (Arenghi et al. 2001) with some modifications. Briefly, 4% xylene was prepared in N, N-dimethylformamide as the substrate. 35 µL of 4% p-xylene was added to the cell lysates and incubated at 15 ± 1 ^∘C. The sub-samples (1 mL) were collected at 3 minutes intervals and mixed with 100 μL of ammonium hydroxide (1 M), 100 μL of 4-amino antipyrine (2%), and 100 μL of 8% potassium ferricyanide. The mixture was briefly centrifuged (14,000 × g) and the production of p-cresol (product of the enzymatic reaction) was determined at 500 nm.

Catechol 1, 2-dioxygenase activity was determined by the measurement of muconic acid production using a spectrophotometric method according to (Li et al. 2014). 600 μL of 10 mM catechol (as the substrate) was added to 2.0 mL phosphate buffer (pH 7.0 ± 0.2), and 400 μL of cell lysate. The absorbance of the mixture was measured at 500 nm every 3 minutes.

To measure the specific activity of target enzymes, the total protein of cell lysates was determined by a bicinchoninic acid (BCA) protein assay kit (Pierce™ Protein Assay Kit, Thermo Scientific, US). One unit of enzyme activity was defined as the amount of enzyme that produces 1 μmol of compounds (p-cresol or muconic acid) per minute per mg of total protein.

Statistical analysis

An analysis of variance (ANOVA) was conducted using SPSS software (Version 23) on a completely randomized design. Analytical and biological tests were performed in triplicate, and the calculated means of the replicates were used along with their standard deviations. The level of significance was set at a p-value of 0.05.

Enzyme mixture characterization

Pseudomonas S2TR-14 was used to produce the cold-active enzyme mixture. Enzyme activity assays showed that the enzyme mixture contained 20 U/mg for XMO and 41 U/mg for C1,2D. This result corroborated with previous studies that showed the highest enzyme production was obtained in the media supplemented with crustacean waste, yeast extract, and 200 mg/L of p-xylene (Miri et al. 2021b, 2022). Yeast extract and crustacean waste offer effective sources of calcium, nitrogen, and proteins that can facilitate bacterial growth. p-xylene can induce the production of XMO and C1,2D as crucial enzymes in the p-xylene biodegradation pathway. p-xylene is usually degraded by aerobic biodegradation through the mono-oxidation of its alkyl groups (by XMO) resulting in several intermediates such as p-toluic acid, p-cresol, etc. The next step of de-aromatization is meta-cleavage by catechol 1, 2 dioxygenases (C1,2D) which is crucial for the detoxification process (Arenghi et al. 2001; Miri et al. 2021b).

In addition, results from LC-MS/MS spectra showed that more than 50 different proteins were detected in the enzyme mixture (Table S2). FDR (False discovery rate) represents the reliability of identified differentially expressed proteins and for the identified proteins, FDR ≥ 1% was considered. Two enzyme sequences were detected in the mixture in the presence of other proteins (Table S2). According to Table S2, XMO and C1,2D showed the highest number of total peptide hits, affirming their identification, and indicating their high abundance in the crude enzyme mixture. Recent research suggests that xylene monooxygenase is capable of directly oxidizing one or two of the methyl groups of the aromatic ring. In addition to catalyzing multi-step oxidation, this enzyme can also produce catecholic or non-catecholic derivatives (Choi et al. 2013).

The main reason for the presence of other proteins is that a crude enzyme mixture was used in this study. The process of enzyme production involves several steps of purification and downstream processing, which must be completed for minimal impurities and high specificity. Each step increases the cost of production, which also limits the application of enzymes (Agrawal et al. 2020). Therefore, using crude enzyme mixtures can significantly reduce the cost of production and result in affordably remediating option for contaminated soil.

Optimization of the immobilization compounds

In the present study, the effect of four parameters, such as magnetic particles, chitosan, glutaraldehyde, and enzyme concentrations on immobilization yield was studied with the help of response surface methodology (RSM) (Table S3). The enzyme activity of C1,2D was considered for the calculation of immobilization yield as this enzyme was the dominant enzyme in the mixture with a higher v_max and ease of testing compared to XMO (as described in the following section). The interaction model between immobilization yield (Y) and the four tested variables is given as Eq 4:

Where, X₁, X₂, X₃, and X₄represent chitosan, glutaraldehyde, magnetic microparticles, and enzymes respectively.

The accuracy and significance of the factors were accomplished by calculating F and P-values. Statistical significance is defined as P > 0.05, whereas nonsignificant means P > 0.1. The ANOVA results showed that the amount of chitosan and introduced enzyme had significant linear effects on immobilization yield (Table 2). In addition, the direct interaction between chitosan and magnetic particles, X₁X₃, (P=0.014) showed a significant influence, while the two-level interaction of other compounds was nonsignificant. The quadratic effects of chitosan (P=0.023), glutaraldehyde (P=0.044), and enzyme (P=0.013) were also significant on immobilization yield.

Analytical results showed that the optimal ratio for immobilization of enzymes was found to be chitosan 3% (W/V), glutaraldehyde 0.42% (V/V), magnetic microparticles 4.48% (W/V), and enzymes 10.3% (V/V). As expected, the immobilization yield reached up to 98.66%, when the content of chitosan and magnetic particles increased to 3% (w/v) and 4.48% (w/v), the immobilization yield reached the maximum value, and then decreased as the two independent variables increased (Fig. 1A and 1D). Previous studies have demonstrated that chitosan can be used as a cross-linker for enzymes and carrier support. Since chitosan contains amino groups that facilitate the covalent attachment of enzymes (Miri et al. 2021b). Meanwhile, glutaraldehyde as a powerful crosslinker can prevent enzymes leakage from support and chitosan. Reddy & Lee (2013) also mentioned that composites of magnetic chitosan exhibit good sorption properties towards various toxic pollutants in aqueous solutions. In addition to having a fast adsorption rate and high adsorption efficiency, these magnetic composites are also easy to recover and reuse (Reddy and Lee 2013). Although the cross-linking of enzymes adsorbed on aminated supports is possible by glutaraldehyde (Barbosa et al. 2014), the interaction between the amount of glutaraldehyde and other variables on immobilization yield was nonsignificant (Fig. 1b). The leaching test results confirmed that glutaraldehyde prevented the leaching of enzymes from the chitosan-coated magnetic particles.

As described in Fig. 1C and 1D, with increasing dosages of the introduced enzymes (up to 10% V/V), the immobilization yields gradually enhanced and then declined. This downward trend indicated the maximum protein adsorption capacity of the magnetic chitosan carrier. Similarly, Mahdavinia et al., (2018), showed that the adsorption of bovine serum albumin (BSA) by magnetic hydrogel beads continues and then is followed by a decrease in the rate of BSA uptake because of reaching saturation adsorption in the final step (Mahdavinia et al. 2018).

Immobilized and free enzymes Kinetics

The Michaelis-Menten model parameters were detected by measuring the degradation rates of p-xylene and catechol using free and immobilized XMO and C1,2D respectively (Table 3). According to Ma et al. (2013), XMO is specific for toluene as a substrate and catalyzes the oxidation of toluene to catechol as its end product (Ma et al. 2013). Similarly, it has been reported that C1,2D cleaves aromatic rings by showing high specificity for catechol (Kalogeris et al. 2006). The Lineweaver-Burk (LB) graphical method was used to determine the kinetic constants of enzymes in free and immobilized forms in presence of two different substrate concentrations (0.1 and 0.5 mM). Considering that free and immobilized enzymes showed the same the K_m, there is no diffusion limitation for magnetic chitosan microparticles, as shown in Table 3. In addition, it is noteworthy that in contrast to our prior study (Miri et al. 2021b), enzymes are not immobilized on the internal surface of pores in support, thus diffusion limitations of the substrate appear to be negligible in this study because enzymes are immobilized on the surface of the carrier. These results showed that v_max for p-xylene (substrate of XMO) was less than for catechol (substrate of C1,2D). This phenomenon may be attributed to the fact that the initial steps in oxidation require higher activation energy than the latter steps in biodegradation, since the stability of the substrate is decreased in each step of biodegradation (Kwean et al. 2018; Miri et al. 2021a). Table 3 shows that almost no enzyme activity and kinetic parameters were increased or decreased following immobilization. K_cat / K_m of XMO and C1,2D were used to calculate the overall kinetic efficiency (known as the catalytic efficiency). The catalytic efficiency of immobilized enzymes is similar to that of free enzymes. Hence, the loss of enzyme activity due to denaturation and/or strong attachment of enzymes to their support was negligible during immobilization.

Characterization of immobilized enzymes

1. Scanning electron microscope (SEM)

The surface morphology of the supporting material was analyzed to confirm the immobilization of enzymes on magnetic chitosan microparticles. The surface morphology of native MPs and chitosan (Fig. 2I A and 2I B), chitosan-coated MPs (Fig. 2I C), and glutaraldehyde treated MPs (Fig. 2I D), and enzyme immobilized MPs (Fig. 2I E and 2I F) was studied using SEM. Fig. 2E and 2F show that the enzyme has been successfully immobilized onto MPs, as indicated by the white patches in the circle. Although, there is no major effect seen on the surface features of magnetic chitosan microparticles after immobilization of enzymes, clumping and white patches were observed after enzyme immobilization. This observation can be attributed to the changes in the carrier surface after the addition of crude enzymes. Since the molecular weight of xylene monooxygenase and catechol 1,2-dioxygenase are approximately 80 kDa and 35 kDa (Miri et al. 2021a) which corresponds to a particle size lower than 5 nm (Lonappan et al. 2018). Based on the micrographs, a minimum magnification was achieved of 1 μm and further magnification was not possible due to the limitations of the instrument. Similarly, Agrawal et al., (2020) showed changes in the overall morphology of functionalized graphene quantum dots after the immobilization of β-amylase (Agrawal et al. 2020). Lonappan et al., (2018) also observed clumping after immobilization of crude laccase onto micro-biochars (Lonappan et al. 2018).

2. Fourier transform infrared (FTIR) spectroscopy

To verify the immobilization of enzymes onto MPs, FTIR spectra have been compared for the immobilized enzymes (Fig. 2II B) with those of native chitosan (Fig. 2II A), and glutaraldehyde-treated MPs (Fig. 2II C). FTIR spectra for MPs are also presented in Fig. 2II D. FTIR analysis can provide general information regarding the functional groups of the samples and changes after each process (Pourmortazavi et al. 2019). A peak obtained at ~1630 cm⁻¹ (Fig. 2II B) revealed the presence of CONH linkage between MPs composite as organic support and enzyme. Bands at 3264 cm^-1 (Fig. 2A) and ~570 cm⁻¹ (Fig. 2B and 2C) correspond to N–H groups from the chitosan and Fe–O groups from MPs (Pourmortazavi et al. 2019). There was a difference between the vibrational stretching band of Fe–O at 570 to 580 cm^-¹ (Fig. 2B and 2C) and the N–H vibrational stretching band from 1653 to 1633 cm^-¹. A possible explanation for this phenomenon could be that iron ions became bound to NH₂ groups of chitosan and enzymes (Safari and Javadian 2014). The bands at 2918 and 1625 cm^-¹ can be attributed to the CH-stretching and the amide-type 1 vibrational modes of chitosan, respectively.

Based on Beauchamp Spectroscopy Tables, the band at 1056 cm^-1 corresponds to the presence of the C-O group which confirmed the presence of oxygen-containing functional groups on immobilized MPs. Similarly, Agrawal et al., (2020) showed the presence of the C-O group on graphene quantum dots after functionalizing using glutaraldehyde and 3-aminopropyltriethoxysilane (Agrawal et al. 2020).

Enzymes leaching

The results in Fig. S2 showed that the leaching of protein from the magnetic chitosan microparticles with glutaraldehyde was lower than 5% after 48 h under shaking conditions. This is encouraging for the reusability of enzymes immobilized by using both linkers in an aquatic medium. The results confirmed that using one of the linkers led to higher leaching of proteins (around 13% for glutaraldehyde and 21% for chitosan). A few studies suggested that using both chitosan and glutaraldehyde could decrease the amount of protein that is leached from solid supports. For example, Naghdi et al., (2019) showed that only 2% of laccase leached out from functionalized nano-biochar composite after 120 h of incubation (Naghdi et al. 2019). Ariaeenejad et al., (2021) showed that leaching of cellulase, hemicellulase, and cellulase + hemicellulase cocktails from magnetite-cellulose nanocrystals were around 10% after 5h (Ariaeenejad et al. 2021).

Effect of pH and temperature

The effect of pH on both free and immobilized enzymes was determined in this study in the pH range of 2.0 to 10. The maximum activity of free enzymes was present at pH 7, whereas in the case of immobilized enzymes they showed the maximum activity at the pH range of 6.0–9.0. (Fig. 3a). The immobilized enzymes showed greater pH stability (4.0–9.0), indicating that it becomes less sensitive to changes in pH. In the immobilized form of the enzymes, the pH is influenced by the interaction between the charged residues of the amino acids in the enzymes and the functional groups on the matrix. As a result of physicochemical force perturbations at extreme pH, the conformational state of a free enzyme may be altered, which leads to a reduction in its activity. In contrast, immobilized enzyme stability can be attributed to a multipoint attachment of the enzyme to the matrix, which prevents denaturation (Naghdi et al. 2019).

The optimum temperature graph for free and immobilized enzymes revealed that there was no change in optimum temperature at low temperatures after immobilization (Fig. 3b). However, the immobilized XMO and C1,2D showed higher activity than the free enzymes in the temperature range of 25-50°C. Fernandez–Lafuente et al., (2000) reported that immobilized thermophilic catechol 2,3-dioxygenase (C2,3D) from Bacillus stearothermophilus had a much higher optimal temperature (approx. 20°C) than the free enzyme. In that study, C2,3D was immobilized on highly activated glyoxylic agarose beads using multiple covalent links between the enzyme and matrix. It was suggested that covalent links stabilized the quaternary structure of this enzyme and increased the rigidity of the subunit structures (Fernandez–Lafuente et al. 2000). However, to the best of our knowledge, literature showing the effect of immobilization on cold-active C1,2D and XMO is not available. Mukhopadhyay et al., (2015) showed that the activity and stability of cold-active laccase were enhanced after being entrapped in a single-walled nanotube. They also reported that immobilized cold-active laccase exhibited thermostability as well as psychrostability (Mukhopadhyay et al. 2015). Similarly, our results showed immobilized cold-active XMO and C2,3D had high stability at both lower and higher temperatures. A major challenge with cold-active enzymes is their inherent instability, as they are more flexible and unstable near the active site than their mesophilic counterparts (Miri et al. 2019, 2021a). Therefore, immobilization of cold-active enzymes seems a promising method for increasing the structural rigidity of cold-active enzymes and addressing this challenge. The significance of these results is that psychrophilic enzymes can be modified to tolerate drastically high temperatures without changing their primary structure.

Reusability and storage stability

In practical applications, reusability and storage stability are crucial. In industrial and environmental processes, enzyme recyclability has a significant impact on cost reduction. The reusability of the immobilized XMO and C1,2D was conducted in 10 repetitive batch reactions (Fig. 4a). Immobilized XMO and C1,2D could maintain ∼80% of their initial activity after 5 cycles and ∼40% activity after 10 cycles (Fig 4a). These results showed that the immobilized enzymes activity decreased when the recycling number was increased. There are two possible reasons for this observation: 1) enzymes leached out during repeated use and; 2) denaturation and the conformational changes because of repeated use. There is no available literature for the immobilization of cold-active XMO and C1,2D except in our previous study (Miri et al. 2021b). The result of the current study is a significant improvement in the reusability of these enzymes compared to our previous study which showed that XMO and C1,2D retained 15% and 22% of their initial activity after 5 cycles of use (Miri et al. 2021b). One reason for the higher reusability of XMO and C1,2D in current study is the fact of using different immobilization methods and more concentrations of glutaraldehyde and chitosan as enzyme linker agents. In this study, 3% (w/v) chitosan and 0.42% (v/v) glutaraldehyde were used for the preparation of immobilized enzymes. While in our previous study, we used 2% (w/v) chitosan and 0.02% (v/v) glutaraldehyde for immobilization of these enzymes onto nano- and micro-biochar. These results also highlight the importance of optimizing immobilization compounds to maximize immobilization properties. Using glutaraldehyde and chitosan as enzyme linker agent are widely reported in the literature (Barbosa et al. 2014; Verma et al. 2020) however, the immobilization compounds should be optimized for each enzyme.

In enzyme immobilization, one of the most critical parameters is storage stability. The stability of both free XMO and immobilized C1,2D was evaluated by storing them at room temperature (25 ± 2 ◦C) in dried form and measuring enzyme activities at certain time intervals. According to Fig. 4b, immobilized enzymes showed activity of more than 70% after 30 days of storage at room temperature. The higher conformational stability of covalently immobilized enzymes may be attributable to the multipoint attachment of enzymes to magnetic chitosan microparticles through glutaraldehyde as a cross-linking agent. Very few reports are available on the immobilization of cold-active enzymes as compared to thermophilic and mesophilic enzymes, but most of the literature reported low thermostability of cold-active enzymes at temperatures higher than 20ºC. This low thermostability is due to molecular flexibility of an active site of cold-active enzymes (Mhetras et al. 2021). The results of current study showed a significant improvement in storage stability of immobilized cold-active enzymes compared to our previous study which reported less than 30% of XMO and 50% of C1,2D remaining activity after 30 days at room temperature (Miri et al. 2021b).

Biodegradation of p-xylene

p-xylene has been identified by the International Tanker Owners Pollution Federation (ITOPF) as one of the top 20 chemicals posing a high level of risk in the category of hazardous and noxious substances (HNS) (Duan et al. 2020). There are numerous applications for p-xylene as a solvent in industry and it is highly mobile under a wide range of environmental conditions in either a liquid, gaseous, or solid form (Mazzeo et al. 2013). p-xylene is considered to be one of the most persistent pollutants due to its high resistance to biodegradation. In aerobic systems, XMO is capable of multistep oxidation and can produce catecholic or non-catecholic derivatives from mono-aromatic hydrocarbons (Choi et al. 2013). Then, as the crucial step in detoxification, para-, meta-, or ortho-cleavage by catechol dioxygenases occurs during the ring de-aromatization of central intermediates (Arenghi et al. 2001). In this study, free and immobilized enzymes (XMO and C1,2D) were used for p-xylene biodegradation (Fig 4). For each test, approximately equal amounts of enzyme mixture in terms of enzyme activity (10 U/mg of XMO and 20 U/mg of C1,2 D) were added either in biodegradation test with free or immobilized form. The changes in the p-xylene concentration versus time are shown in Fig. 5. The degradation results indicated that immobilized enzymes had better biodegradation ability compared to free enzymes (Fig. 5). The possible reason was that when enzymes are immobilized onto adsorbents such as magnetic chitosan, the probability of contact between the substrate molecule and the immobilized enzymes was higher compared to free enzymes, resulting in a high reaction rate.

The decrease in degradation rate observed in the experiment with the free enzymes. This may be explained by their lower activity and stability compared with immobilized enzymes. Qiu et al., (2021) showed that too high substrate concentration (2,4-dichlorophenol) could inhibit the enzymatic reaction in the free form of enzymes (laccase). While immobilized laccase onto modified magnetic chitosan nanoparticles showed a good removal performance even at a high concentration of 2,4-dichlorophenol (Qiu et al. 2021).

Immobilization of cold-adapted enzymes has been the subject of only a few studies, and most have shown that the immobilization method improves the thermal stability of cold-active enzymes (Lee et al. 2017). For example, Rahman et al., (2016) reported that immobilization of cold-active esterase onto Fe₃O₄∼cellulose nanocomposite enhanced catalytic properties such as prolonged half-life, better temperature stability, higher storage stability, improved pH tolerance, and reusability (Rahman et al. 2016).

In practice, the first weeks after enzymatic or nonenzymatic treatments of contaminated soils are important, as they determine the effectiveness of the method. During this time, efforts should be made to enhance the stability of enzymes. Similarly, our results showed that about 20% of p-xylene was degraded after 6 days using free enzymes, while immobilized enzymes degraded 44% of p-xylene after 6 days. These results indicated that immobilization increased the effectiveness of enzymatic treatment of p-xylene contaminated soil.

Two cold-active enzymes (XMO and C1,2D) were immobilized on magnetic chitosan microparticles through cross-linking of glutaraldehyde. Optimization of the amount of each compound involved in immobilization (chitosan, glutaraldehyde, magnetic microparticles, and enzymes) showed that the highest immobilization yield could be achieved up to 98%. Immobilization of these enzymes improved their catalytic characteristics in terms of pH tolerance, thermal stability, reusability, and time of storage stability. Also, immobilized enzymes had a 2-fold higher p-xylene degradation rate in soil compared to the free form. The catalytic improvement makes the immobilized enzymes suitable for the remediation of contaminants such as p-xylene in soil. Generally, the application of enzymes in powder form is significantly easier compared to liquid form in terms of handling, transport, and storage, however, mixing or injection of immobilized enzymes for in-situ or ex-situ application should be studied further.

Acknowledgements

The present research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC-Discovery Grant 355254 and Strategic Grant 447075 and Collaborative Research Development Grant 54231) and Techno-Rem Inc. Support from James and Joanne Love Chair in Environmental Engineering at York University is appreciated for the research. We are thankful for Seyyed Mohammadreza Davoodi, Dr. Yi Sheng, Dr. Thomas Robert, Mr. Jean-Marc Lauzon, Mr. Stephane Moise, Magdalena Jaklewicz, and Dr. Joanna Lecka for their kind help and valuable feedback.

Statements and Declarations

There are no conflicts of interest declared by the authors.

Competing Interests and Funding

The authors have no relevant financial or non-financial interests to disclose.

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Table 1. Magnetic chitosan-based carriers for enzyme immobilization, their applications and reusability

Enzyme	Carrier	Application	Reusability	Reference
Lipase	Magnetic chitosan beads	Synthesis of flavor esters	5 cycles with more than 70% of its initial activity	(Bayramoglu et al. 2022)
Glucose oxidase and catalase	Magnetic chitosan microspheres	Production of Sodium gluconate from glucose	10 with more than 60% of their remaining activity	(Liu et al. 2022)
Lipase	β-cyclodextrin grafted and aminopropyl-functionalized chitosan/Fe₃O₄ magnetic nanocomposites	Synthesis of fruity flavor esters	15 cycles with a slight decrease (15%) in the relative activity	(Zhao et al. 2022)
Laccase	Magnetic chitosan nanoparticles modified with amino-functionalized ionic liquid containing ABTS	Removal of 2,4-dichlorophenol, bisphenol A, indole and anthracene	6 cycles with a removal efficiency of 93.2 % for 2,4-dichlorophenol.	(Qiu et al. 2021)
Lactase (β-d-galactosidase)	Magnetic chitosan microsphere	Biomedical applications	10 recycles with more than 65 % of its initial activity	(Ke et al. 2020)
α-amylase	Chitosan-magnetite composite	Food, fermentation, detergent applications, textile and paper industry	10 cycles with 45–55% of its initial activity	(Bindu and Mohanan 2020)
Porcine pancreatic lipase	Magnetic chitosan nanoparticles modified with imidazole-based functional ionic liquid	ND	10 recycles with more than 84.6% of its initial activity	(Suo et al. 2019)
Laccase	Chitosan beads magnetized with Fe₃O₄ NPs	Removal of Evans blue, Direct blue 15, Reactive black 5 and Acid red 37 azo dyes	10 cycles with 47% of remaining activity	(Nadaroglu et al. 2019)
Xylanase	Magnetic chitosan	Production of xylooligosaccharides with food and pharmaceutical applications	3 cycles retaining 50% of its initial activity	(Gracida et al. 2019)
Cellulase	Magnetic Chitosan Nanoparticles	Glucose production	10 cycles with more than 80% of its initial activity	(Zang et al. 2014)
Glucoamylase	Magnetic chitosan microspheres	Production of glucose from starch	10 cycles with 45–68.4% of its initial activity	(Wang et al. 2013)
Lipase and β-galactosidase	Magnetic chitosan microparticles	ND	8 cycles with more than 90% of its initial activity	(Pospiskova and Safarik 2013)
Pullulanase	Magnetic chitosan beads	Improves the saccharification of starch to produce glucose	10 cycles retaining 64.8% residual activity	(Zhang et al. 2009)

Table 2. Regression analysis of four tested variables on immobilization yield from the Box-Behnken experiment.

Source	Coefficient (Coef)	F-value	P-value
Model	95.00	13.15	0.000**
X1-Chitosan	-9.02	6.74	0.017*
X2-Glutaraldehyde	-0.37	0.01	0.906
X3-Magnetic particle	4.75	2.05	0.168
X4-Enzyme	22.78	39.42	0.000**
X1X2	8.64	2.67	0.118
X1X3	16.54	7.31	0.014*
X1X4	5.44	0.71	0.410
X2X3	-6.82	1.44	0.244
X2X4	-0.09	0.00	0.987
X3X4	1.05	0.03	0.868
X1X1	-12.89	6.07	0.023*
X2X2	-10.25	4.62	0.044*
X3X3	-8.24	3.37	0.082
X4X4	-16.43	7.41	0.013*

* Significant (0.01 < P-Value < 0.05). **Very significant (P-value < 0.001).

Table 3. Kinetics parameters for XMO and C1,2D

Enzyme	v_max (mM min^-1)	K_m (mM)	K_cat (min^-1)	Catalytic Efficiency (min^-1 mM^-1)
XMO (Free)	0.45 ± 0.03	1.40 ± 0.12	0.8 ± 0.2	0.57 ± 0.12
XMO (Immobilized)	0.48 ± 0.04	1.39 ± 0.23	0.83 ± 0.25	0.59 ± 0.23
C1,2D (Free)	20.13 ± 0.74	3.71 ± 0.33	1.71 ± 0.34	0.46 ± 0.03
C1,2D (Immobilized)	21.06 ± 0.98	3.69 ± 0.45	1.73 ± 0.13	0.46 ± 0.11

No competing interests reported.

SupplementaryMaterialMarch232022.docx

Immobilized Cold-Active Enzymes onto Magnetic Chitosan Microparticles as a Highly Stable and Reusable Carrier for p-xylene Biodegradation in Soil

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