Immobilization of Mutant Phosphotriesterase on Fuller’s Earth Enhanced the Stability of the Enzyme

: Immobilization is a method for making an enzyme more robust in the environment, especially in terms of its stability and reusability. A mutant phosphotriesterase (YT PTE) isolated from Pseudomonas dimunita has been reported to have high proﬁciency in hydrolyzing the S p and R p -enantiomers of organophosphate chromophoric analogs and therefore has great potential as a decontamination agent and biosensor. This work aims to investigate the feasibility of using Fuller’s earth (FE) as a YT PTE immobilization support and characterize its biochemical features after immobilization. The immobilized YT PTE was found to show improvement in thermal stability with a half-life of 24 h compared to that of the free enzyme, which was only 8 h. The stability of the immobilized YT PTE allowed storage for up to 4 months and reuse for up to 6 times. The immobilized YT PTE showed high tolerance against all tested metal ions, Tween 40 and 80 surfactants and inorganic solvents. These ﬁndings showed that the immobilized YT PTE became more robust for use especially with regards to its stability and reusability. These features would enhance the future applicability of this enzyme as a decontamination agent and its use in other suitable


Introduction
The application of enzymes in various processes, especially in the food, pharmaceutical, detergent and biosensing industries has gained much interest in recent times [1]. However, the stability of these enzymes in the environments that they are used in or when used under harsh usage conditions continues to be a matter of concern [2]. Phospotriesterase (PTE) is an enzyme that is known to be able to hydrolyze toxic organophosphate esters (OP) into non-toxic compounds [3,4]. This toxic functionality is widely present in various industries such as in agriculture, petroleum, plasticizer production, textile manufacturing and they have also been used as chemical warfare agents [5,6]. Thus, the utilization of PTE as a means to inactivate OPs is an application that could address concerns about their use and subsequent inactivation after use in these industries. The immobilization of this enzyme onto a support could provide a means for its wider use, a need that has become a matter of great interest due to PTE's wide potential application and in view of the current high costs of cleaning up environmental contamination from the residues of OP use. Previously, PTE has been immobilized onto certain support materials such as agarose, graphene, polyvinylidenefluoride and polyethersulphone [7][8][9]. However, most of this work was for the purpose of detecting the presence of OPs and was only in

Immobilization Process of Recombinant YT PTE
Recombinant YT PTE was obtained from overproduction in E. coli BL21(De3) and subsequently extracted and purified using anion exchange chromatography. The purified enzymes were then immobilized onto FE. When conducting the immobilization, certain process parameters were varied in order to identify the best means of immobilization, encompassing immobilization duration, enzyme loading and pH.
The immobilization of purified YT PTE on FE was studied by varying the immobilization duration in order to identify the duration that results in the highest protein yield. Figure 1 shows the protein yield percentage of the immobilized enzyme. YT PTE showed its highest adsorption onto FE after 2 h incubation, culminating in a 78.9% yield. This high adsorption of FE was likely due to its small particle size, being 100 mesh/149 um, which thus presents a large surface area that could be further expanded (in the presence of water) and also because of its intercalation properties [16,17,26]. Since FE is a part of the smectite group of materials, it has been reported to possess high cation-exchange capacity within the interlayer of its crystalline structure [21,27]. The lowest yield, however, was seen at 30 min incubation, which indicates a probable insufficient adsorption time. In contrast, with PTE that was immobilized onto trityl agarose, the immobilization process took just 10 min to complete [28]. However, this was probably due to the different immobilization method used, namely the entrapment method as opposed to the less complicated physical adsorption technique utilized in this study.

Immobilization Process of Recombinant YT PTE
Recombinant YT PTE was obtained from overproduction in E. coli BL21(De3) and subsequently extracted and purified using anion exchange chromatography. The purified enzymes were then immobilized onto FE. When conducting the immobilization, certain process parameters were varied in order to identify the best means of immobilization, encompassing immobilization duration, enzyme loading and pH.
The immobilization of purified YT PTE on FE was studied by varying the immobilization duration in order to identify the duration that results in the highest protein yield. Figure 1 shows the protein yield percentage of the immobilized enzyme. YT PTE showed its highest adsorption onto FE after 2 h incubation, culminating in a 78.9% yield. This high adsorption of FE was likely due to its small particle size, being 100 mesh/149 um, which thus presents a large surface area that could be further expanded (in the presence of water) and also because of its intercalation properties [16,17,26]. Since FE is a part of the smectite group of materials, it has been reported to possess high cation-exchange capacity within the interlayer of its crystalline structure [21,27]. The lowest yield, however, was seen at 30 min incubation, which indicates a probable insufficient adsorption time. In contrast, with PTE that was immobilized onto trityl agarose, the immobilization process took just 10 min to complete [28]. However, this was probably due to the different immobilization method used, namely the entrapment method as opposed to the less complicated physical adsorption technique utilized in this study.  Figure 1 shows that after 2 h of incubation, the adsorptivity of FE towards the enzyme started to decrease. This may have been due to the sensitivity of the enzyme towards mechanical forces [2,29]. During the immobilization process, prolonged stirring may induce cumulative shear stress forces that could impact on an enzyme's structure, causing it to eventually become denatured or alternatively may even cause the enzyme to become detached mechanically from the support [30]. This reversible immobilization process occurred because of non-ionic bonding between the enzyme and the support and would result in the surplus enzyme being left in the supernatant. Thus, it was surmised that the best immobilization process duration for YT PTE onto FE was 2 h at room temperature. Figure 2 shows the effect of sample loading for the YT PTE immobilization process in terms of the percentage of protein yield. At a quantity ratio of 2:1 (protein: support), the immobilization yield was found to be maximal with a value of 68% of the enzyme loaded onto the support. The gradual decline in protein yield might be due to the enzyme having become fully coated and thus being unable to continue adsorption or reaching its  Figure 1 shows that after 2 h of incubation, the adsorptivity of FE towards the enzyme started to decrease. This may have been due to the sensitivity of the enzyme towards mechanical forces [2,29]. During the immobilization process, prolonged stirring may induce cumulative shear stress forces that could impact on an enzyme's structure, causing it to eventually become denatured or alternatively may even cause the enzyme to become detached mechanically from the support [30]. This reversible immobilization process occurred because of non-ionic bonding between the enzyme and the support and would result in the surplus enzyme being left in the supernatant. Thus, it was surmised that the best immobilization process duration for YT PTE onto FE was 2 h at room temperature. Figure 2 shows the effect of sample loading for the YT PTE immobilization process in terms of the percentage of protein yield. At a quantity ratio of 2:1 (protein: support), the immobilization yield was found to be maximal with a value of 68% of the enzyme loaded onto the support. The gradual decline in protein yield might be due to the enzyme having become fully coated and thus being unable to continue adsorption or reaching its saturation limit for the available surface area of FE. Each solid support molecule has a limited adsorption area available that is dependent on the size of the protein (enzyme) being adsorbed, the available surface area for adsorption and the number of sites available for the protein to be adsorbed onto [20,31]. This finding was in agreement with findings in other studies of immobilized enzymes on FE or montmorillonite (MMT) involving enzymes such α-amylase, laccase and tyrosinase. In these studies, it was found that overloading the enzyme decreased the adsorption yield [16,21,32]. Overloading enzymes during adsorption may cause inhibition of the mobility and flexible stretching of the enzyme and this, in turn, may lead to its subsequent inactivation [33][34][35][36]. This effect can be seen by observing the protein yield of immobilized YT PTE where the enzyme yield begins to decrease after the 2:1 concentration, namely becoming 60%, 58% and 58% yields for the 3:1, 4:1 and 5:1 ratios, respectively. Similarly, in another study, it was found that the activity of immobilized lipase obtained from Pseudomonas fluorescens was decreased when it was overloaded during the immobilization process using octyl-agarose beads [35].
Catalysts 2021, 11, x FOR PEER REVIEW 4 of 18 saturation limit for the available surface area of FE. Each solid support molecule has a limited adsorption area available that is dependent on the size of the protein (enzyme) being adsorbed, the available surface area for adsorption and the number of sites available for the protein to be adsorbed onto [20,31]. This finding was in agreement with findings in other studies of immobilized enzymes on FE or montmorillonite (MMT) involving enzymes such α-amylase, laccase and tyrosinase. In these studies, it was found that overloading the enzyme decreased the adsorption yield [16,21,32]. Overloading enzymes during adsorption may cause inhibition of the mobility and flexible stretching of the enzyme and this, in turn, may lead to its subsequent inactivation [33][34][35][36]. This effect can be seen by observing the protein yield of immobilized YT PTE where the enzyme yield begins to decrease after the 2:1 concentration, namely becoming 60%, 58% and 58% yields for the 3:1, 4:1 and 5:1 ratios, respectively. Similarly, in another study, it was found that the activity of immobilized lipase obtained from Pseudomonas fluorescens was decreased when it was overloaded during the immobilization process using octyl-agarose beads [35].  Figure 3 shows the effect of pH during the immobilization process. The highest adsorption of YT PTE onto FE was found at pH 9 in Tris HCl, which demonstrated a 77% protein yield. pH is known to affect protein adsorption by distributing the total net charge of the enzyme onto a surface area, which contains charged amino acids leading to the inactivation of the enzyme and enhanced protein support interactions [16,37]. Furthermore, alkaline conditions are also known to promote nucleophilic activity, especially at enzyme active sites, which contain positively charged amino acids, thereby resulting in high enzyme/support activity [21,38].  Figure 3 shows the effect of pH during the immobilization process. The highest adsorption of YT PTE onto FE was found at pH 9 in Tris HCl, which demonstrated a 77% protein yield. pH is known to affect protein adsorption by distributing the total net charge of the enzyme onto a surface area, which contains charged amino acids leading to the inactivation of the enzyme and enhanced protein support interactions [16,37]. Furthermore, alkaline conditions are also known to promote nucleophilic activity, especially at enzyme active sites, which contain positively charged amino acids, thereby resulting in high enzyme/support activity [21,38].  In this study, immobilization under acidic conditions was not attempted due to the known instability of YT PTE (protein aggregation would occur) at neutral and acidic pH conditions (unpublished data). At the pI value, positive and negative charges are balanced leading to a net zero charge, and thus electrostatic charges predominate, which can cause protein aggregation and eventually their precipitation out of solution. However, if the pH  In this study, immobilization under acidic conditions was not attempted due to the known instability of YT PTE (protein aggregation would occur) at neutral and acidic pH conditions (unpublished data). At the pI value, positive and negative charges are balanced leading to a net zero charge, and thus electrostatic charges predominate, which can cause protein aggregation and eventually their precipitation out of solution. However, if the pH is too far removed from the pI value, it can also lead to protein aggregation due to the dominance of positive or negative charges at the surface area. Thus, the best equilibrium state for stronger enzyme binding to FE would be close to the pI value (6.9). This finding was similar to those found in other studies conducted on horseradish peroxidase where it was found that they achieved their highest yields near their pI values [29]. Furthermore, a trend towards low yields can be seen when the enzyme is immobilized at an almost neutral pH. This is because when the pH is nearing the pI value of the enzyme, the net charge approaches zero and thus proteins may precipitate out of solution and become less available. Moreover, FE has been reported to have exchangeable cation tunnels and contain a large number of cation compounds such as Mg 2+ , Ca 2+ , Si 2+ and Na 2+ [16,21,22]. Thus, FE would benefit from binding with an enzyme with a predominantly negative charge to allow stronger binding of proteins over the top of the surface area. On the other hand, buffer conditions can also affect enzyme equilibrium conditions and thereby impact the support by changing its ionic condition and pore size. Thus, it is crucial to find the best conditions for enzyme and support binding, which would then lead to higher hydrolysis activity.

Effect of Temperature on Immobilized YT PTE Activity and Stability
The effect of temperature on immobilized and free YT PTE activity is depicted in Figure 4. In this study, it was observed that immobilized YT PTE's optimum activity temperature was 40 • C, which was similar to that of the free enzyme. This is consistent with the findings from earlier studies that involved other enzymes such as α-amylase, catalase and laccase, which were immobilized onto FE and MMT, where it was also found that there was no shift in their optimum temperature [16,28,32]. In contrast to this, it has been found that for tyrosinase isolated from Agaricus bisporus, its optimum activity temperature shifted 5 • C lower than that of the free enzyme [21]. This enzyme was immobilized onto a mixture of FE and gelatin using both direct adsorption and entrapment technique. The changes in the immobilized enzyme's (physical and chemical) properties were probably contributed to by hydrophobic and ionic forces in addition to other secondary interactions. Furthermore, the immobilized YT PTE in this study showed higher hydrolysis activity than the free enzyme at temperatures ranging 20-30 • C.  It was found that the thermal stability of immobilized YT PTE exhibited more stable behavior compared to the free enzyme as is seen in Figure 5. The immobilized YT PTE showed a 10-15% decrease in activity for every 1 h incubation compared to the free enzyme, which instead showed a 20-25% decrease in activity. The calculated half-life of the immobilized YT PTE and the free enzyme was 24 h and 8 h, respectively. These results It was found that the thermal stability of immobilized YT PTE exhibited more stable behavior compared to the free enzyme as is seen in Figure 5. The immobilized YT PTE showed a 10-15% decrease in activity for every 1 h incubation compared to the free enzyme, which instead showed a 20-25% decrease in activity. The calculated half-life of the immobilized YT PTE and the free enzyme was 24 h and 8 h, respectively. These results showed that the enzyme was protected from denaturing and deactivation by the FE through the preservation of its structure [21,32]. This finding is in agreement with that of Babavatan and his coworkers where they found that immobilized lipase was protected from denaturing by the support [39]. In addition to this, FE is also known to be a fire-retardant material, and is able to displace heat, which may also be a reason for its stability [18]. Therefore, the improved thermostability of immobilized YT PTE is an important observation and is likely to have an impact on practical applications of this enzyme. It was found that the thermal stability of immobilized YT PTE exhibited more stable behavior compared to the free enzyme as is seen in Figure 5. The immobilized YT PTE showed a 10-15% decrease in activity for every 1 h incubation compared to the free enzyme, which instead showed a 20-25% decrease in activity. The calculated half-life of the immobilized YT PTE and the free enzyme was 24 h and 8 h, respectively. These results showed that the enzyme was protected from denaturing and deactivation by the FE through the preservation of its structure [21,32]. This finding is in agreement with that of Babavatan and his coworkers where they found that immobilized lipase was protected from denaturing by the support [39]. In addition to this, FE is also known to be a fireretardant material, and is able to displace heat, which may also be a reason for its stability [18]. Therefore, the improved thermostability of immobilized YT PTE is an important observation and is likely to have an impact on practical applications of this enzyme.

Effect of pH on Immobilized YT PTE Activity
The effect of pH on the activity of immobilized and free YT PTE is shown in Figure  6. Immobilized YT PTE showed only slight changes in optimum pH, where its highest activity was detected at pH 8. However, the overall hydrolysis trend remained almost the same under alkaline conditions. Since FE is a polycationic support that contains various metal ions, it can adsorb OH − and H + ions in the vicinity of the enzyme. [16]. Changes in

Effect of pH on Immobilized YT PTE Activity
The effect of pH on the activity of immobilized and free YT PTE is shown in Figure 6. Immobilized YT PTE showed only slight changes in optimum pH, where its highest activity was detected at pH 8. However, the overall hydrolysis trend remained almost the same under alkaline conditions. Since FE is a polycationic support that contains various metal ions, it can adsorb OH − and H + ions in the vicinity of the enzyme. [16]. Changes in pH value are known to affect the conformation and the degree of dissociation of a substrate and thus lead to either high or low catalysis conditions [40]. In Figure 6, the immobilized YT PTE hydrolysis activity was found to decrease at pH 6 and below as well as at pH 10 and above. These effects when pH changes from acidic to basic have also been observed in other enzymes immobilized onto FE and MMT [16,21,39], thereby indicating that immobilized YT PTE shares a similar profile to these other enzymes, which also require a lower pH for their maximum activity as compared to their free enzyme state. Previously, in a study on catalase immobilized onto clay, it was seen that its optimum activity shifted to occur under more alkaline conditions because the immobilization resulted in the addition availability of larger, inorganic hydroxyl cations [28]. However, it must also be noted that some immobilized enzymes maintain a similar pH profile for optimal activity with their free form [32]. quire a lower pH for their maximum activity as compared to their free enzyme state. Previously, in a study on catalase immobilized onto clay, it was seen that its optimum activity shifted to occur under more alkaline conditions because the immobilization resulted in the addition availability of larger, inorganic hydroxyl cations [28]. However, it must also be noted that some immobilized enzymes maintain a similar pH profile for optimal activity with their free form [32]

Effect of Metal Ions on Immobilized YT PTE Activity
Metal ions generally enhance hydrolysis activity, but certain metal ions instead decrease it. This is because metal ions generally are able to act as electrophiles that seek out possible sharing of electron pairs with other atoms. This experiment aimed to test the ability of FE to protect the enzyme from denaturation [41]. Figure 7 shows the effect of metal ions on the activity of immobilized and free YT PTE. The immobilized YT PTE showed a stimulatory effect when treated with 1 mM of metal ions in comparison to the free enzyme. The highest relative activity of immobilized YT PTE was detected in the presence of Ni 2+ , which showed 127.8% activity compared to the free enzyme. Other metal ions such as Cu 2+ , Co 2+ , Fe 2+ and Zn 2+ were also able to activate the hydrolysis activity of immobilized YT PTE. In contrast, the free enzyme showed a 35% decrease in hydrolysis activity compared to the control. This is probably because the FE protected the enzyme from direct exposure to the metal ions by stabilizing its microenvironment [23,42].

Effect of Metal Ions on Immobilized YT PTE Activity
Metal ions generally enhance hydrolysis activity, but certain metal ions instead decrease it. This is because metal ions generally are able to act as electrophiles that seek out possible sharing of electron pairs with other atoms. This experiment aimed to test the ability of FE to protect the enzyme from denaturation [41]. Figure 7 shows the effect of metal ions on the activity of immobilized and free YT PTE. The immobilized YT PTE showed a stimulatory effect when treated with 1 mM of metal ions in comparison to the free enzyme. The highest relative activity of immobilized YT PTE was detected in the presence of Ni 2+ , which showed 127.8% activity compared to the free enzyme. Other metal ions such as Cu 2+ , Co 2+ , Fe 2+ and Zn 2+ were also able to activate the hydrolysis activity of immobilized YT PTE. In contrast, the free enzyme showed a 35% decrease in hydrolysis activity compared to the control. This is probably because the FE protected the enzyme from direct exposure to the metal ions by stabilizing its microenvironment [23,42]. Immobilized YT PTE showed a slight decline in hydrolysis activity (98.9%) when tested with 1 mM of Mn 2+ . Although Mn 2+ is known to be a strong binder of carboxylic groups in the enzyme and is able to prevent the interaction between negatively and positively charged groups [43], it did not show any dramatic effects when the enzyme, already immobilized onto FE, was compared to the free enzyme. This finding is also in congruence with the findings from a study on acid phosphatase that was immobilized onto clay. The immobilized enzyme showed no significant changes in its hydrolysis activity when treated with Mn 2+ . The clay was thought to preserve the enzyme from any negative effects caused by Mn 2+ [44]. Immobilized YT PTE showed a slight decline in hydrolysis activity (98.9%) when tested with 1 mM of Mn 2+ . Although Mn 2+ is known to be a strong binder of carboxylic groups in the enzyme and is able to prevent the interaction between negatively and positively charged groups [43], it did not show any dramatic effects when the enzyme, already immobilized onto FE, was compared to the free enzyme. This finding is also in congruence with the findings from a study on acid phosphatase that was immobilized onto clay. The immobilized enzyme showed no significant changes in its hydrolysis activity when treated with Mn 2+ . The clay was thought to preserve the enzyme from any negative effects caused by Mn 2+ [44].

Effect of Surfactant on the Activity of Immobilized YT PTE
Two types of commercial surfactants are commonly found in the cleaning industry, namely anionic surfactants and non-ionic surfactants [45]. The effects of these surfactants on the stability of immobilized YT PTE and free enzyme were tested. The results obtained are shown in Figure 8. The immobilized YT PTE showed a stimulatory effect when exposed to Tween 40 and Tween 80, measuring 111.3% and 103.8% activity, respectively, compared to the free enzyme. The surfactants did not seem to have a discernable effect on the hydrolysis activity of the enzyme itself. This can be seen when the treated free enzyme was found to have slightly decreased hydrolysis activity, measuring 98.1% and 90.7%, respectively, after the surfactant was applied. This effect was similar to that seen with immobilized lipase isolated from Pseudozyma hubeiensis Strain HB85A where it was found that there was enhanced catalytic activity when treated with 1% Tween 80 [46]. Moreover, Tween is a non-ionic surfactant that does not typically break protein-protein interactions that would lead to the denaturation of enzymes [47]. In addition, non-ionic surfactants cause a slight expansion of the interlayer space, leading to improved surface and substrate adsorption as seen with bentonite [48]. In contrast, anionic surfactants, namely sodium dodecyl sulfate (SDS), caused the immobilized YT PTE to show an immediate decline in activity when treated with a 1% solution of SDS i.e., a decline of 15.7%. The SDS not only decreased the activity of the immobilized enzyme, but it also deactivated the free enzyme. Thus, this finding shows the ability of FE to protect some of the enzyme from denaturation by buffering its microenvironment. The anionic surfactant was able to disrupt non-covalent bonds in the protein structure and this then led to the loss of enzyme conformation [49]. However, it did not affect the support (FE), neither in terms of its adsorption nor surface area. There have been reports that cationic surfactants can significantly enhance the adsorption of similar smectite groups such as those found in FE [48]. Hence, the use of immobilized YT PTE in the cleaning industry has potential because of these findings.

Effect of Different Solvents on the Activity of Immobilized YT PTE
The organic solvent tolerance stability of immobilized YT PTE and the free enzyme arise shown in Figure 9. This study aimed to explore the immobilized enzyme's robustness for potential applications, such as in environmental decontamination where it is known that there are organic solvents present. The immobilized YT PTE showed tolerance to 25% v/v of hexane and xylene as evidenced by its hydrolysis activity being measured at 138.5% and 106%, respectively, compared to the control. A similar effect was also seen with the free enzyme where its hydrolysis activity was enhanced to 122.7% and 106.6%. respectively. Thus, it can be seen that the hydrolysis activity was enhanced even more In contrast, anionic surfactants, namely sodium dodecyl sulfate (SDS), caused the immobilized YT PTE to show an immediate decline in activity when treated with a 1% solution of SDS i.e., a decline of 15.7%. The SDS not only decreased the activity of the immobilized enzyme, but it also deactivated the free enzyme. Thus, this finding shows the ability of FE to protect some of the enzyme from denaturation by buffering its microenvironment. The anionic surfactant was able to disrupt non-covalent bonds in the protein structure and this then led to the loss of enzyme conformation [49]. However, it did not affect the support (FE), neither in terms of its adsorption nor surface area. There have been reports that cationic surfactants can significantly enhance the adsorption of similar smectite groups such as those found in FE [48]. Hence, the use of immobilized YT PTE in the cleaning industry has potential because of these findings.

Effect of Different Solvents on the Activity of Immobilized YT PTE
The organic solvent tolerance stability of immobilized YT PTE and the free enzyme arise shown in Figure 9. This study aimed to explore the immobilized enzyme's robustness for potential applications, such as in environmental decontamination where it is known that there are organic solvents present. The immobilized YT PTE showed tolerance to 25% v/v of hexane and xylene as evidenced by its hydrolysis activity being measured at 138.5% and 106%, respectively, compared to the control. A similar effect was also seen with the free enzyme where its hydrolysis activity was enhanced to 122.7% and 106.6%. respectively. Thus, it can be seen that the hydrolysis activity was enhanced even more when using FE as a support. The stability of the enzyme towards exposure to organic solvents was likely the result of a number of factors such as its ability to maintain its secondary structure, the presence of intramolecular disulfide linkages and the presence of certain amino acids that prevented the penetration of the organic solvent into the internal structure of the protein [49][50][51]. In contrast to when the organic solvent was below log p = 2.0, the immobilized YT PTE and free enzyme showed a dramatic decline in their hydrolysis activity when compared to control. This might be due to the loss of water molecules that maintain protein conformation mobility by interrupting the hydrogen bonds in the enzyme [51,52]. Organic solvents below log p = 2.0 are known as non-polar organic solvents, where they have the tendency to strip water molecules from enzymes [53]. However, some of the immobilized YT PTE was able to survive denaturation because of the protection provided by the support, which enabled the balancing of the environmental changes that occurred. This finding is in concurrence with the findings obtained from a study of an enzyme obtained from Candida antarctica, which was immobilized onto MMT, where its hydrolysis activity was found to be enhanced in the presence of solvents [39]. Thus, organic solvent tolerant enzymes would likely have applications where they can be used in poor quality aquatic mediums where enzyme catalysis is needed because of their enhanced stability and hydrolysis activity in these conditions.

Storage Stability and Reusability
A key factor for immobilization is the ability of the immobilized enzyme to retain its hydrolysis activity after storage and its ability to be reused repeatedly [2,31]. The stability of immobilized YT PTE is depicted in Figure 10. The experimental data from the immobilized YT PTE showed stability of up to 79.7% when it was stored at 4 °C over a period of 4 months as is seen in Figure 10. In other studies on immobilized phosphotriesterase, it was found that they were able to retain up to 90% of its activity when stored at 4 °C [9]. In contrast, free YT PTE hydrolysis activity has been found to show a sharp decline to 60% activity after 2 months of storage at 4 °C. Similarly, a study using a commercial free phosphotriesterase preparation (SsoPox W263F) showed a decrease of 90% of its activity after 1 month of storage at 4 °C [54]. This decline in activity may be due to the environmental changes in storage such as irregular environmental temperature (prolonged opening of storage device doors), humidity and ambient luminance during storage that may have led to enzyme inactivation. These findings support the claim that the immobilized YT PTE does display better storage stability compared to the free enzyme. In contrast to when the organic solvent was below log p = 2.0, the immobilized YT PTE and free enzyme showed a dramatic decline in their hydrolysis activity when compared to control. This might be due to the loss of water molecules that maintain protein conformation mobility by interrupting the hydrogen bonds in the enzyme [51,52]. Organic solvents below log p = 2.0 are known as non-polar organic solvents, where they have the tendency to strip water molecules from enzymes [53]. However, some of the immobilized YT PTE was able to survive denaturation because of the protection provided by the support, which enabled the balancing of the environmental changes that occurred. This finding is in concurrence with the findings obtained from a study of an enzyme obtained from Candida antarctica, which was immobilized onto MMT, where its hydrolysis activity was found to be enhanced in the presence of solvents [39]. Thus, organic solvent tolerant enzymes would likely have applications where they can be used in poor quality aquatic mediums where enzyme catalysis is needed because of their enhanced stability and hydrolysis activity in these conditions.

Storage Stability and Reusability
A key factor for immobilization is the ability of the immobilized enzyme to retain its hydrolysis activity after storage and its ability to be reused repeatedly [2,31]. The stability of immobilized YT PTE is depicted in Figure 10. The experimental data from the immobilized YT PTE showed stability of up to 79.7% when it was stored at 4 • C over a period of 4 months as is seen in Figure 10. In other studies on immobilized phosphotriesterase, it was found that they were able to retain up to 90% of its activity when stored at 4 • C [9]. In contrast, free YT PTE hydrolysis activity has been found to show a sharp decline to 60% activity after 2 months of storage at 4 • C. Similarly, a study using a commercial free phosphotriesterase preparation (SsoPox W263F) showed a decrease of 90% of its activity after 1 month of storage at 4 • C [54]. This decline in activity may be due to the environmental changes in storage such as irregular environmental temperature (prolonged opening of storage device doors), humidity and ambient luminance during storage that may have led to enzyme inactivation. These findings support the claim that the immobilized YT PTE does display better storage stability compared to the free enzyme. The reusability of immobilized YT PTE is shown in Figure 11. This parameter can be seen as one that would be of importance for industrial applications, since it is an ability that has the potential to lessen production and application costs, especially for biocatalytic processes that require repetitive and continuous actions. It was found that the immobilized YT PTE retained 80% of its hydrolysis activity even after six times of usage (reuse). This might have been due to the characteristics of FE, which has a large surface area and has cation tunnels that can protect the enzyme from the effects of repeated drying. This experiment did not determine the limits of reusability, but six iterations were used to demonstrate that reusability is possible for the immobilized YT PTE. Further study is needed to determine the limits of reusability of this enzyme construct. Immobilized αamylase and tyrosinase, which used FE as a support, have also showed similar trends [16,21]. The hydrolysis activity of α-amylase and tyrosinase were found to remain at 75% and 77% even after five times of usage, respectively. This finding provides a further advantage for the utilization of immobilized YT PTE and would make it more attractive for use in industrial processes.  The reusability of immobilized YT PTE is shown in Figure 11. This parameter can be seen as one that would be of importance for industrial applications, since it is an ability that has the potential to lessen production and application costs, especially for biocatalytic processes that require repetitive and continuous actions. It was found that the immobilized YT PTE retained 80% of its hydrolysis activity even after six times of usage (reuse). This might have been due to the characteristics of FE, which has a large surface area and has cation tunnels that can protect the enzyme from the effects of repeated drying. This experiment did not determine the limits of reusability, but six iterations were used to demonstrate that reusability is possible for the immobilized YT PTE. Further study is needed to determine the limits of reusability of this enzyme construct. Immobilized α-amylase and tyrosinase, which used FE as a support, have also showed similar trends [16,21]. The hydrolysis activity of α-amylase and tyrosinase were found to remain at 75% and 77% even after five times of usage, respectively. This finding provides a further advantage for the utilization of immobilized YT PTE and would make it more attractive for use in industrial processes. The reusability of immobilized YT PTE is shown in Figure 11. This parameter can be seen as one that would be of importance for industrial applications, since it is an ability that has the potential to lessen production and application costs, especially for biocatalytic processes that require repetitive and continuous actions. It was found that the immobilized YT PTE retained 80% of its hydrolysis activity even after six times of usage (reuse). This might have been due to the characteristics of FE, which has a large surface area and has cation tunnels that can protect the enzyme from the effects of repeated drying. This experiment did not determine the limits of reusability, but six iterations were used to demonstrate that reusability is possible for the immobilized YT PTE. Further study is needed to determine the limits of reusability of this enzyme construct. Immobilized αamylase and tyrosinase, which used FE as a support, have also showed similar trends [16,21]. The hydrolysis activity of α-amylase and tyrosinase were found to remain at 75% and 77% even after five times of usage, respectively. This finding provides a further advantage for the utilization of immobilized YT PTE and would make it more attractive for use in industrial processes.

Surface Morphology of FE before and after Immobilization
The surface morphology of FE was investigated using a Field Emission Scanning Electron Microscope (FESEM) visualizing two samples, with each being a sample of FE and YT PTE immobilized onto FE (Figure 12). The changes in the morphology of the samples of immobilized YT PTE and FE were viewed at a magnification of 50,000×. It was observed that FE appeared to be particulate in a rod-shaped fibrillar form (see Figure 12B). In Figure 12A, the blue arrows indicate the attachment of the enzymes onto FE where they appear as aggregated masses on and among the fibrillar particles of FE. In Figure 12B, these aggregations are not seen and the fibrillar particles of FE are clearly visualized. The differences between the two images could be an indication that the YT PTE has been successfully immobilized onto FE.

Surface Morphology of FE before and after Immobilization
The surface morphology of FE was investigated using a Field Emission Scanning Electron Microscope (FESEM) visualizing two samples, with each being a sample of FE and YT PTE immobilized onto FE (Figure 12). The changes in the morphology of the samples of immobilized YT PTE and FE were viewed at a magnification of 50,000×. It was observed that FE appeared to be particulate in a rod-shaped fibrillar form (see Figure 12B). In Figure 12A, the blue arrows indicate the attachment of the enzymes onto FE where they appear as aggregated masses on and among the fibrillar particles of FE. In Figure 12B, these aggregations are not seen and the fibrillar particles of FE are clearly visualized. The differences between the two images could be an indication that the YT PTE has been successfully immobilized onto FE.  Figure 13 shows the FTIR spectra of immobilized YT PTE, which was overlayed onto the support (FE). Immobilized YT PTE is the red line and FE alone is the purple line. The chemical structure of FE, which predominantly consists of aluminum magnesium silicate is confirmed through the presence of peaks at 3614, 1178 and 974 cm −1 (the purple line),  [57]. In addition, the dotted box in the graph shows the presence of the same peak (1178 and 978 cm −1 ) in both lines. It can thus be concluded that the YT PTE was successfully immobilized onto FE. indicating the presence of bonding vibration of Al-O-Al, Si-O-Si and Si-O-Mg, respectively [55,56]. The red line (immobilized YT PTE) clearly shows the presence of the enzyme at the peaks at 3340 and 1030 cm −1 indicating the presence of vibration of N-H and C-O bonding found in the enzyme [57]. In addition, the dotted box in the graph shows the presence of the same peak (1178 and 978 cm −1 ) in both lines. It can thus be concluded that the YT PTE was successfully immobilized onto FE.

Purification of Recombinant YT PTE
Recombinant E. coli BL21 (DE3) harboring the pET51b/YT PTE construct was cultured at 25 °C and expressed using autoinduction media [58] for 32 h. The cells were harvested through centrifugation at 12,000× g for 30 min at 4 °C. The cells were then lysed by sonication and centrifuged again at 12,000× g for 30 min to separate soluble from insoluble proteins. Purification was accomplished using an anion exchange XK16/20 chromatography column (GE Healthcare, Chicago, IL, USA) packed with Q Sepharose resin. The column was equilibrated with a binding buffer (Tris-HCl pH 9) and then loaded with the crude enzyme buffered at pH 9. The column was washed with washing buffer of up to 5 column volumes to wash out any unbound protein. The purified protein was then eluted using 0.5 M NaCl buffered to pH 9. The eluted purified protein from each fraction was then assayed for enzyme activity. The purified enzymes were then stored at 4 °C for use in further experiments.

Immobilization Process of YT PTE
The effect of time on the immobilization of YT PTE was conducted at different time durations. Two milligrams of Fuller's Earth-Merck (the support) were weighed and placed in a vial. The support was washed with purified water 3 times and filtered using either filter paper or a nano filter. Two milliliters of pH 9 buffer, which contained 4 mg of pure enzyme (in liquid form), was then mixed with the support in a beaker and then separate samples were stirred continuously over various durations, namely 30 min, 1, 2 and 3 h at room temperature. The protein concentration of the solution after stirring was determined using the Bradford method as mentioned in Section 3.2 below. A magnetic stirrer was used to stir the solutions and it was set at 200 rpm for the duration of the test time

Purification of Recombinant YT PTE
Recombinant E. coli BL21 (DE3) harboring the pET51b/YT PTE construct was cultured at 25 • C and expressed using autoinduction media [58] for 32 h. The cells were harvested through centrifugation at 12,000× g for 30 min at 4 • C. The cells were then lysed by sonication and centrifuged again at 12,000× g for 30 min to separate soluble from insoluble proteins. Purification was accomplished using an anion exchange XK16/20 chromatography column (GE Healthcare, Chicago, IL, USA) packed with Q Sepharose resin. The column was equilibrated with a binding buffer (Tris-HCl pH 9) and then loaded with the crude enzyme buffered at pH 9. The column was washed with washing buffer of up to 5 column volumes to wash out any unbound protein. The purified protein was then eluted using 0.5 M NaCl buffered to pH 9. The eluted purified protein from each fraction was then assayed for enzyme activity. The purified enzymes were then stored at 4 • C for use in further experiments.

Immobilization Process of YT PTE
The effect of time on the immobilization of YT PTE was conducted at different time durations. Two milligrams of Fuller's Earth-Merck (the support) were weighed and placed in a vial. The support was washed with purified water 3 times and filtered using either filter paper or a nano filter. Two milliliters of pH 9 buffer, which contained 4 mg of pure enzyme (in liquid form), was then mixed with the support in a beaker and then separate samples were stirred continuously over various durations, namely 30 min, 1, 2 and 3 h at room temperature. The protein concentration of the solution after stirring was determined using the Bradford method as mentioned in Section 3.2 below. A magnetic stirrer was used to stir the solutions and it was set at 200 rpm for the duration of the test time span. The mixture was filtered out from the solution using a nano filter and allowed to dry in an oven at a temperature of 37 • C for 12 h. Any unbound enzyme was then washed out until there was no hydrolysis activity detected in the wash. The unbound enzyme was also measured for protein content using the Bradford method [59]. Immobilization adsorption was calculated as the protein yield percentage.
Calculation of protein yield: The percentage of yield = ( total protein − unbound protein ) Total protein × 100 The determination of the most suitable enzyme concentration to load the support was done by using different concentrations of the enzyme. The enzyme concentration was determined using different ratios of enzyme to the support, i.e., 1:1, 2:1, 3:1, 4:1 and 5:1 (mg:mg). The unchanged factor was the support while the variable factor was the enzyme. Each mixture was incubated for 2 h at room temperature and stirred at 200 rpm (the optimum adsorption period determined earlier). The mixture was then filtered and dried at 37 • C for 12 h. Any unbound enzyme was washed out until there was no hydrolysis activity detected. The unbound enzyme was measured for protein content using the Bradford method. Immobilization adsorption was calculated as the protein yield percentage as shown above.
The effect of pH on the adsorption of protein onto the support was next determined. 2 mg of FE was weighed for each of the preparations to be tested. The enzyme was prepared in different types of buffers ranging from pH 7 to pH 12 at a concentration of 6 mg/mL. FE was mixed with the enzyme little by little at room temperature and stirred at 200 rpm for 2 h. The immobilized enzyme was then filtered and washed with buffer until no hydrolysis activity was detected then allowed to dry overnight. The unbound enzyme was measured for protein content using the Bradford method. Immobilization adsorption was calculated in protein yield percentage.

Determination of Protein Concentration
The Bradford assay method was used to determine protein concentration. It was performed using the commercial Bradford reagent obtained from Fisher Scientific Pte Ltd. [59]. An assay mixture that contained the enzyme solution to be tested plus 0.15 M sodium chloride (100 µL) was prepared in a test tube. One milliliter of the Bradford reagent was then added into the tube and vortexed for 30 s. The mixture was allowed to stand at room temperature for 2 min and absorbance was measured at 595 nm. Bovine serum albumin (BSA) was used as the standard and the concentration was determined between 125-1000 µg/mL.

Phosphotriesterase Assay
The activity of YT PTE was measured using paraoxon in a reaction mixture containing 10 µL of enzyme, 890 µL of 50 mM Hepes pH 7.4 buffer and 100 µL of paraoxon (analytical standard) [60]. The mixture was incubated at 30 • C using a heating block for 10 min. The reaction was then terminated using 100µL of 20% ethanol. Free p-nitrophenol was determined using a colorimetric phosphotriesterase assay method and the absorbance was measured at 412 nm. One unit of hydrolysis activity is defined as the rate of release of one micromole of p-nitrophenol in one minute.

Effect of Temperature
The effect of temperature on the hydrolysis activity of the immobilized enzyme was measured at temperatures ranging from 20 • C to 70 • C at 10 • C intervals over a duration of 10 min. It was then assayed colorimetrically. The thermostability of the immobilized YT PTE was tested by preincubating the immobilized enzyme at the earlier identified optimum temperature (40 • C) for the following durations, i.e., 10 min, 30 min, 1, 4, 8, 16 and 24 h. The preincubation was done using a heating block. The treated immobilized enzyme was then subjected to the phosphotriesterase assay. The control used for this experiment was the untreated enzyme. For comparison, the free enzyme was prepared using the same procedure.

Effect of pH
The effect of pH on the immobilized enzyme was studied by preincubating 2 mg of immobilized YT PTE at different pH values in a range from pH 4 to pH 12 at 40 • C for 10 min. The mixture was then subjected to the phosphotriesterase assay as mentioned in Section 3.3 above. The same method was also applied to the free enzyme for comparison.

Effect of Organic Solvents
A stability study of the immobilized YT PTE towards different organic solvents was then conducted. Two milligrams of the immobilized enzyme was added to a 1 mL solution containing a mixture of pH 9 buffer and 25% v/v organic solvents and then preincubated at the optimum temperature for 10 min. The preincubated enzymes were then assayed for enzyme activity. Each control batch had their own control. The same procedure was applied to the free enzyme. The untreated enzyme was assigned a value of 100% activity.

Effect of Surfactants
The effect of Tween 40, Tween 80, Triton-X and SDS was next tested. Two milligrams of immobilized YT PTE was added to a 1 mL solution containing pH 9 buffer and 1% v/v surfactant at the earlier determined optimum temperature for 10 min. The preincubated immobilized enzymes were then assayed for hydrolysis activity. Each batch (surfactant) had their own control. Relative activity was calculated using the untreated immobilized enzyme as the control.

Storage Stability and Reusability Test
The stability of immobilized YT PTE was determined by measuring the hydrolysis activity after two weeks, one month, two months, three months and four months of storage. The dried immobilized YT PTE was stored at 4 • C in a capped vial. The control used was fresh immobilized YT PTE. In the reusability test, 2 mg of the immobilized YT PTE was weighed, placed in a centrifuge tube and prepared for the hydrolysis test. Then the mixture of immobilized YT PTE, pH 9 buffer and the paraoxon was centrifuged to separate the supernatant from the pellet (immobilized YT PTE). The supernatant was then measured spectrophotometrically at 412 nm. The pellet (immobilized enzyme) was washed with buffer and allowed to dry. This process was repeated six times. The control was calculated as being 100% at the first-time hydrolysis activity was determined.

Field Emission Scanning Electron Microscopy (FESEM)
Topology of immobilized YT PTE was viewed using FESEM. The immobilized enzyme was sent to the Faculty of Engineering, UPNM for visualization. The sample was coated with gold before being analyzed under the FESEM. The FESEM images of the support and the immobilized YT PTE were captured under 50,000× magnification.

Fourier-Transform Infrared Spectroscopy (FTIR)
FTIR measurement was done using a Perkin-Elmer Frontier FT-IR spectroscope and analyzed using Perkin-Elmer Spectrum IR software. The measurement range was carried out at spectrum locations 4000 to 650 cm −1 and over 4 accumulation scans. The spectrometer radiation (IR) was from an ATR crystal. The immobilized enzyme and FE sample were set on top of the instrument's diamond plate. A pressure controller was used to adjust the optimal contact between the sample and the diamond plate, and the measurements were then made and recorded using the instrument's software.

Conclusions
In this study, we successfully immobilized YT PTE onto FE using the direct adsorption method. The immobilized YT PTE demonstrated improvement in its biochemical properties, especially with regards thermostability. In addition, the immobilized enzyme was found to be able to be stored for longer periods. Improved storage time of up to 4 months was also demonstrated compared with the free enzyme. The ability of FE to protect YT PTE from the effects of temperature was demonstrated by the prolongation of its thermal stability from 8 h up to 24 h. Thus, this study demonstrated the potential usefulness of the immobilized YT PTE and provides impetus for further study and exploration of potential future applications.