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 vmax 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:
![](https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1655297760.PNG)
Where, X1, X2, X3, and X4 represent 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 Km, 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 vmax 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. Kcat / Km 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.