Carbohydrate microcapsules tailored and grafted for covalent immobilization of glucose isomerase for pharmaceutical and food industries

Carrageenan is one of the most common carbohydrates utilised in the entrapment industry to immobilise cells and enzymes. However, it lacks functionality. Carrageenan has been grafted to produce fructose by covalently immobilising glucose isomerase (GI). Fructose is one of the most widely used sweeteners in beverages, food production, and the pharmaceutical business. Up to 91.1 U g−1 gel beads are immobilised by the grafted beads. Immobilized GI has a Vmax of 13.8 times that of the free enzyme. pH of immobilized GI was improved from 6.5–7 to 6–7.5 that means more stability in wide pH range. Also, optimum temperature was improved and become 65–75 °C while it was at 70 °C for free enzyme. The immovability and tolerance of the gel beads immobilised with GI over 15 consecutive cycles were demonstrated in a reusability test, with 88 percent of the enzyme's original activity retained, compared to 60 percent by other authors. These findings are encouraging for high-fructose corn syrup producers.


Introduction
Enzymes with excellent catalytic properties have been well studied as catalysts because of their unique physicochemical behavior. Enzymes are remarkably effective in the versatile reactions (Aluísio et al. 2022;Gabrielly et al. 2022), such as hydrolysis, esterification, transesterification, alcoholysis (Carlos et al. 2016), and C-C bond formation. Enzymes have been recently used as a potential biocatalyst (Gledson et al. 2017;Katerineda et al. 2022) in a large number of biotechnological sciences; more specifically, these include dairy products (cheese recovery, flavor enhancement and the production of Enzyme-modified cheese (EMC), detergents, pharmaceuticals (ibuprofen, naproxen), chemicals (Lima et al. 2021), agriculture products (pesticides, insects), and oil (Francisco et al.2021) chemistry (fat and oil hydrolysis, and the synthesis of bio-detergents). Enzymes act as a good catalyst therefore Moreira et al. 2020), its production and utilization may be a better alternative of chemical catalysts (Lima et al. 2021;Fernandez-Lopez et al. 2015;Nathalia et al. 2019).
The immobilisation approach ensures that the enzyme can be used continuously, making the immobilised enzyme more suited and advantageous than the free-enzyme state. This is due to the ease with which the immobilised enzyme may be regenerated, resulting in a low-cost industrial process. Adsorption (Salah et al. 2008), crosslinking (Elnashar and Hassan 2014), entrapment (Betancor et al. 2008;Hassan et al. 2016), or a combination of these approaches (D'Souza 1999) are the most often used enzyme immobilization techniques.
The enzyme immobilization for its economic use is, therefore, essential since can improve the selectivity of the enzyme, repeatability, the range of substrates, and the separation of the enzyme/ cells from the raw materials. Among other advantages of immobilization of enzyme, one can mention the increased enzyme/ cells and enzyme/ cells contact with the raw materials, prevention of the enzyme ejaculation, and improvement in the stability of the enzymes three-dimensional structure (Dong-Mei and Chen 2020; Yale et al. 2021;Sahar et al. 2021).
In the future, according to Street, GI will be the most significant industrial enzyme (Street 1977). For example, GI transforms glucose to fructose, which is utilised as a sucrose alternative in the pharmaceutical industry (Barclay et al. 2012). Because GI has a financial interest in enzyme immobilisation, a variety of methods are applied, including glutaraldehyde crosslinking. This method of immobilization is a widely used method that relies on the formation of strong covalent bond between the support material and enzyme via the functional groups which are found on their surfaces (Tyagi et al. 1999), entrapment in polyacrylamide (Yuan et al. 2016), and adsorption on DEAE-Cellulose (Chen and Anderson 1979). Diethyleaminoethyl cellulose (DEAE-C) (Gul et al. 2009) and alginate beads have been used to immobilise the gastrointestinal enzyme (Bao et al. 2011).
κ-Carrageenan is one of the most common carriers utilised in the entrapment method of immobilising cells and enzymes (Belyaeva et al. 2004). Carrageenans are generally utilised for enzyme immobilisation by the creation of non-covalent bonds (entrapment/ encapsulation) due to their lack of functions, according to Elnashar et al. 2020. Unfortunately, propagation of the biocatalyst from the carrier is a common feature of enzyme entrapment in a hydrogel, especially for enzymes with molecular weights less than 300 KDa. Many researchers have worked in this field, and some fascinating results have been obtained (Daniel et al. 2010;Elnashar et al. 2009;Elnashar 2011;Yuan et al. 2016).
Carrageenans have poor thermal stability and mechanical qualities, while being biocompatible and affordable. Some study was done to improve their mechanical and thermal properties, and it was discovered that adding 3,6-anhydro-D-galactose 2-sulphate and gum boosted their mechanical strength (Wahba and Hassan 2017). To alleviate the problem of low thermal stability, the carrageenan gel was treated with polyamine compounds to generate a polyelectrolyte complex. Polyamine groups can improve the heat stability of carrageenan gels (Chao et al. 1986). To increase the thermal stability of carrageenan gel, natural polyamine, chitosan, and polyethylenimine were utilised (Elnashar 2010). GI (EC 5.3.1.5) catalyses the reversible isomerization of D-glucose to D-fructose, making it one of the most significant enzymes in industry. This conversion is critical, particularly in the production of high-fructose corn syrup (HFCS) (Gill et al. 2006). Because fructose is sweeter than all other sugars/carbohydrates, there is a financial incentive to produce it. In addition, because fructose has a higher solubility than sucrose, it is less prone to clump in a variety of foods. Despite the fact that fructose is the sweetest of the naturally occuring caloric sweeteners found in fruits, honey, and some vegetables, it should be ingested in moderation because calories are still present.
Immobilized GI can thus offer numerous benefits in biotechnological and industrial applications. Pure fructose (not HFCS), reusability, ease of product separation from the enzyme, and increased enzyme stability at various temperatures and pH levels without affecting enzyme characteristics are just a few of the benefits. In addition to the kinetic constants of immobilised and free GI, several factors have been investigated. Fourier Transform infrared (FTIR) and scanning electron microscopy are used to monitor the steps involved in preparing the hydrogels and immobilising the enzyme (SEM).
The gastrointestinal enzyme (GI) is a high-cost intracellular enzyme (Tumturk et al. 2008). The catalytic process requires large doses of the enzyme to obtain maximal effectiveness, therefore GI has a high Km value, which makes it more expensive. Furthermore, the free GI's Vmax is relatively low. As a result, it's critical to keep GI immobilised on a costeffective carrier with a low Km/Vmax ratio. Unfortunately, only a few carriers are regarded cost-effective for industrial usage among the several immobilised GI such as Kamal et al. 2014 who immobilized glucose isomerase (GI) by entrapment into Poly(acrylic acid) P(AA) and Poly(acrylic acid-co-2-Acrylamido 2-methyl Propane sulfonic acid) P(AA-co-AMPS) polymer networks, the enzyme carriers were prepared by radiation induced copolymerization in the presence of (Methylene-bisacrylamide) (MBAA) as a crosslinking agent. And Neifar et al. 2020 who immobilized glucose isomerase GICA from Caldicoprobacteralgeriensis by ionic adsorption on polymethacrylate carriers (Sepabeads EC-EA and EC-HA) or covalent attachment to glyoxal agarose.
Many researchers and governments have aimed to immobilise GI onto carrageenan to generate high fructose syrup with high yields. Carrageenan, on the other hand, lacks functions for covalent interactions, therefore this was restricted to physical interaction. The first is to provide additional amine groups to the carrageenan surface, which enable glutaraldehyde reaction while simultaneously functioning as a spacer arm (ligand) to increase the distance between the enzyme and the support, so reducing steric effects and enhancing enzyme immobilisation yield (Bonazza et al. 2018;Elnashar et al. 2013a, b). The length of the spacer arm could play a crucial role in enzyme immobilization. In a previous study by Elnashar and Hassan (2014), they studied the spacer arm's length, and their findings were further optimized his approach was further studied using three different polymers based on alginate, carrageenan and chitosan Elnashar et al. (2014). The second is the development of multi-ionic linkages (a network) between the protonated amino groups (cationic polyethylenimine) and the sulphate groups, which hardens the gel beads (anionic carrageenan). This type of ionic network has been shown to be an alternative to covalently crosslinked hydrogels containing sulphate groups in the gel (Elnashar et al. 2008). Given that glutaraldehyde is one of the most widely used activation materials, it is possible to improve enzyme stability by forming a complex with it. Because glutaraldehyde is one of the most widely used activation materials, it can help to improve enzyme stability by forming strong covalent connections between the enzyme and the support, as illustrated in scheme 1. (Barbosa et al. 2014;Elnashar 2010;Elnashar et al. 2013a, b;Elnashar and Yassin 2009a, b). A schematic picture depicts the grafted formulation of the carrageenan beads immobilising GI covalently, and the chemical modification is demonstrated using FTIR and SEM techniques (scheme 1). The enzyme loading capacity, on the other hand, is optimised using a short-chain active ligand (to avoid steric hindrance) and varied enzyme doses. The immobilised and free enzymes' optimal pH and temperature are determined, and the Michaelis constants are investigated using a Hans-Woolf plot. Finally, the immobilized enzyme is checked for its reusability and durability overtime for 15 cycles. • Balance -Precisa -XT 220 A -made in Switzerland. • pH-meter (Jenway 370). • Shaker "roller mixer", Stuart.

Experimental techniques
Unless otherwise mentioned, all tests were performed in triplicate, and data are presented as means SD (n = 3) unless otherwise noted. The Student's t-test was used to establish the significance level (p-value) of each concentration effect.

Preparation of κ-carrageenan beads
κ-carrageenan powder (2.5 g) was dissolved in warmed 100 mL distilled water (at 70 °C), yielding 2.5% (w/v) carrageenan gel beads. The Innotech Encapsulator, model IE-50, was used to manufacture uniform gel beads of 300 µm in size, as illustrated in Fig. 1 (Elnashar et al. 2020). The gel bead diameter was in the 300 µm range when using a nozzle. Prior to treatment, the beads were hardened by soaking them in 0.3 M potassium chloride (KCl) for 3 h.

Activation of gel beads
For 3 h at room temperature, gel beads of κ-carrageenan were immersed in 4% (v/v) Polyethyleneimine (PEI), pH 9.5. Using distilled water, the aminated beads were rinsed to eliminate any unreacted PEI. The GA solution was then used to react with the aminated beads (Carr-PEI) (2.5% for 3 h). The beads were then thoroughly rinsed in distilled water to remove any unreacted GA. Finally, as illustrated in scheme (1), the activated beads were ready for the next phases of immobilisation (Elnashar, 2005).

Enzyme immobilization
To determine the highest enzyme immobilisation efficiency, one gram of treated gel beads was soaked in solutions of GI solutions comprising 97-1455 units (U) for 1h at room temperature and pH 7. The -C=Nbond was formed by the interaction of the amino group (-NH 2 ) in the enzyme with the free -C=O group on glutaraldehyde (scheme (1)). .

Immobilization efficiency (I.E.) using our locally prepared GI
One gram of gel beads was soaked for one hour at room temperature in 15 mL GI solutions of 97-1455 U. (RT). From Eq. 1, the immobilisation efficiency (I.E.) was obtained as follows:

Determination of GI activity
The GI activity was measured according to assay conditions. The amount of GI enzyme required to produce 1 unit of GI activity was defined asthe amount of GI enzyme needed to produce 1 μmol of fructose under assay conditions.

pH profile
The effect of varying pHs on free and immobilised enzyme activity was investigated. Five units of immobilised and free GI were incubated for one hour at 70 °C in a 10 mL 1 M glucose solution at pH 5-9. (Torres & Batista-Viera 2017). The activity at each pH has been given as a percentage of the activity at the optimal pH, which is 100% of the activity.

Temperature profile
The effect of temperature on the catalytic activity of immobilised and free GI was investigated by incubating 5 U of both free and immobilised GI in 10 mL of 1 M glucose solution at pH 7 for 1 h at various temperatures ranging from 40-90 °C. (Santos et al. 2015a, b;Seyhan and Dilek, 2008). The ideal temperature was discovered, and enzymatic activity at this temperature is expressed as 100% activity, with activity at other temperatures expressed as a percentage of that activity.

Km and Vmax of immobilized and free GI
KM and Vmax were used to determine the affinity of the immobilised and free enzymes for their substrates. 5U of GI was incubated for 1 h at 70 °C and pH 7 in various concentrations of glucose solutions ranging from 200 to 2000 mM. The reaction conditions were inspired by Seyhan and Dilek 2008, who employed glucose concentrations ranging from 100-1500 mM (Galvão et al. 2018).

Operational stability
The operational stability of immobilised GI was studied. The GI was used for 15 cycles of 60 min each to convert glucose to fructose, which was created from prepared whey using β-galactosidase. One-gram beads carrying 8 units of immobilised GI were mixed with 10 mL of β-galactosidase-pretreated whey. The reaction was kept at 70 °C for 60 min. The amount of fructose was evaluated after the immobilised GI was withdrawn from the reaction mixture. The beads were thoroughly rinsed in buffer solution before being placed in a new substrate solution. This method was done numerous times, with the first activity being 100% and the subsequent activities being expressed as a percentage of the first.

Scanning electron microscope (SEM)
Four separate sets of beads (κ-carrageenan (A), aminated κ-carrageenan (B), activated κ-carrageenan (C), and enzyme immobilised on κ-carrageenan (D)) were produced and activated as described in the preceding section for comparison reasons. After that, the activated gel beads were either lyophilized immediately or soaked for 18 h in 0.05 M citrate-phosphate buffer. Scanning Electron Microscopy (SEM; S-590, Hitachi) was used to evaluate the surface morphology of the lyophilized beads in order to investigate the porosity of the gel beads and prove that changes happened on the surface after each reaction.

Results and discussion
The Innotech Encapsulator was utilised to manufacture homogenous gel beads of κ-carrageenan with a size of 300 µm, as illustrated in Fig. 1. Figure 2 shows that utilising 1445 U enzyme solutions (crude enzyme, molecular weight ranges from 52,000 to 191,000 according to Chen 1980) with a 6.3 percent immobilisation effectiveness, a maximum enzyme loading capacity of 91.1 U g-1 gel beads was achieved. However, we chose not to utilise the maximum loaded enzyme in future experiments since it had a low I.E., and it would be more cost-effective to use formulations like those that produced 72 U g-1 gel beads with an I.E. of 37.3 percent. This formula does not indicate the greatest I.E., but it does show the most likely economic value. To achieve the maximum enzyme loading capacity, we took into consideration several factors such as the ligand's chain length (Elnashar and Hassan 2014), as the long chain ligand would enable multilayer/multipoint attachment of the immobilized enzyme. Such steric hindrance might result in a loss of the enzyme's activity due to a change in its 3D structure.

Determination of optimum pH profile
Because the interaction of protein groups with the support is affected by the pH, changing the immobilisation pH can change the orientation of enzyme molecules on the support. The last nucleophile blocking of the support allowed for the elimination of the support's chemical reactivity, preventing unwanted enzyme-support covalent connections and serving as a helpful reaction end point (dos Santos et al. 2015a, b). During its isoelectric point, the enzyme does not work well. Because it denatures near this value, the GI isoelectric point is 3. GI is most stable and crystalline in the pH range of 6 to 8, and denatures fast below pH 5 (Carrell et al. 1989).
As shown in Fig. 3, the optimum pHs for the free and immobilized GI were 6.5-7 and 6-7.5, respectively. Statistical analysis (one paired-ample t-test) showed a significant differentiation between the mean results of free and immobilized formulae of enzyme, with P* = 0.052 (P ≤ 0.05 is significant). The data showed that the immobilization process has increased the enzyme stability (wider pH) compared to the free enzyme. This could be attributed to the fixed configuration of the immobilized GI inside or on the surface of the beads, which increases the stability and tolerability of the enzyme towards the surrounding pH. Our results reveal a better enzyme stability when compared to the results obtained by other authors; for example, Arun and Srevastava (2016) found it at pH 6.5 (Arun and Srivastava 2016), while Hayrettin et al. (2008) and Kamal et al. (2014) found it at pH 7.5, and Pawar and Deshmukh (1994) found it at pH 7.5.

Determination of optimum temperature profile
The thermal stability of covalently immobilized GI onto chemically activated κ-carrageenan beads is one of the most important application criteria for industrial applications (Manoel et al. 2015a, b;Palomo et al. 2002). The optimum temperatures for the free and immobilized enzyme were 70 & 65-75 °C respectively, as shown in Fig. 4. Statistical analysis (one paired -sample t-test) showed a significant differentiation between the mean results of free and immobilized formulae of enzyme, with P* = 0.0380 (P ≤ 0.05 is significant). The results show that the immobilization process made the enzyme more stable at a wider temperature range than it did the free enzyme.
The interesting observation results from a close inspection of the Figure, is the existence of wide range of temperature starting from 60 °C to 82.5 °C, about 22.5 °C, in which the immobilized enzyme kept from 90 to 100% of its relative activity compared to 10 °C only for the free counterpart starting from 62.5 °C to 72.5 °C. It is worthy to mention here that the response of the immobilized enzyme to variation of environment temperature is less sensitive than the free one. This behavior can be related to the formation of multivalent linkage with the enzyme. This leads to "restrict" of enzyme structure conformational changes as a result of environmental changes. The immobilized enzyme maintained a greater rigidity and was more resistant to unfolding at higher temperatures than its free form (Sanjay G, Sugunan S (2006)). Therefore, the immobilized enzyme could work in harsh environmental conditions with less activity loss compared to its free counterpart (Le-Tien et al. 2004). On the other hand, the decline rate of activity of the immobilized enzyme is seems less than the free counterpart. This behavior may be referred to the part of the enzyme immobilized via entrapment into the polyelectrolyte polymer matrix which stabilized by the formation by a kind of "polyelectrolyte" results from the interaction between the functional groups charges over the enzyme molecules surface and the negative and positive charges of the carrageenan and polyethyleneimine parts of the polymer matrix. This part is freer and more flexible to affect by the surrounding environment temperature than the covalently immobilized part. The activation of the polyethyleneimine with gultaraldehyde could also participates in a kind of chemical crosslinking resulted in reduction of the polymer matrix pores size, which also contributes to improving of the efficiency of the entrapment process of the enzyme and preventing its leakage. In conclusion, the immobilization strategy used, which is a mix between entrapment and covalent immobilization, is the responsible about the obtained behavior. Tor et al. 1989 supports this finding, as he suggested that the immobilization process forms a cage surrounding the enzyme. That cage protects/isolates the immobilized enzyme from the bulk temperature and reduces the temperature of the environment surrounding the enzyme. High temperature is also favored, to avoid microbial contamination. Our results were comparable to those found in literature, where the optimum temperatures for the immobilized enzyme were 70 °C (Buchholz 1992); 60 °C (Fagir and Abu-reesh 1998;Sorenseon and Emborg 1989), 65 °C (Kamal et al. 2014) and 75 °C (Pawar and Deshmukh 1994). The optimum temperature was also found to be 70 °C for the free enzyme, and 85 °C for the immobilized enzyme (Ge et al. 1998). Recently, Sanchez et al. 2016 studied the impact of Bovine trypsin immobilization on glyoxyl-agarose using two different preparations on the stability and inactivation behavior of the immobilized enzyme. The first one was reduced just after immobilization (less covalent attachment points), while the other was left to continue the enzyme support reaction (higher number of covalent attachment points).
This strategy is a guarantee of the identical orientation of the enzyme regarding the support surface and the identical physical properties of the support. Then, the second preparations were submitted to inactivation under different conditions: thermal and solvent inactivation under different pH values. The results confirm that the structures of the different preparations were very different, suggesting that the inactivation ways were different for each enzyme preparation and dependent on the inactivation conditions. This information is very relevant for the design of strategies for enzyme stabilization, as shown by the fact that the inactivation may follow different conformational changes depending on the degree of enzyme rigidification and inactivation conditions.

Km and Vmax of free and immobilized GI
The Km and Vmax of the immobilized and free GI were calculated by using the Hanes-Woolf plot method as in Fig. 5. Table 1 shows the calculated values. The km for the immobilized enzyme was 492 mM which was higher than that of the free one, 29.3 mM. This indicates that the immobilized Optimum temperature for free and covalently immobilized GI enzyme needs more substrate concentrations than does the free one (Hassan et al. 2019a, b). The results of the free Km is in agreement with published data by Tumturket al.2008, as he reported that the Km value of free GI was 17.9 mM. The increase in Km value of the immobilized form might be because of diffusional limitations, steric effects and changes in enzyme structure which influence the ability of the substrate to react with the enzyme active sites (Awad et al. 2016). Mohy Eldin et al. 2011, successes in overcoming the diffusion limitation problem of the substrate through developing of an affinity immobilization technique by combining entrapment and multicovalent immobilizations strategies to immobilize glucoamylase enzyme into ρ-benzoquinone-activated alginate beads. They found that the K m values of free and entrapped glucoamylase were found to be almost identical. The key point in their strategy was keeping the active site protected through affinity towards the polymeric matrix used for the immobilization process, which subsequently followed by formation of multicovalent bonds with the enzyme structure leading to "Fix" the enzyme structure in the best conformational one. In the other hand, reducing of the microcapsules size to the nano-scale could be very helpful in overcoming of the substrate diffusion problem and getting K m value close to the free counterpart. The maximum reaction velocity (Vmax) value for the immobilized enzyme, 1117 μmol min −1 , was higher than the Vmax of the free enzyme, 81 μmolmin −1 . The increase of the enzyme's Vmax after immobilization is highly favored in industry, as more products are produced per min compared to the free enzyme. This could be explained as follows: for the enzyme to be efficient, the loss of entropy, coming from the binding between enzyme and substrate to form the enzyme-substrate complex, must be paid by the released binding energy from the favoring interaction between substrate and enzyme (Seyhan and Dilek 2008).Although the Km has increased after the immobilization process, however, the Vmax of the immobilized enzyme has increased too. Using the same amount of free and immobilized enzyme, by comparing the ratios of the free enzyme's Vmax (81 μmol min −1 ) to its Km (29.3 mM),with that of the immobilized one(Km: 492 mM and Vmax: 1117 μmol min −1 ), the result is 2.76 and 2.27, respectively. The catalytic efficiency (Vmax/Km) of the immobilized enzyme was 82.2% of that of the free one, and this could be attributed to product inhibition or slight enzyme mis-conformation after immobilization (Ahmed et al. 2019).Overall, the result is positive, as the immobilization process has many benefits including operational stability that the free enzyme doesn't afford.

Operational stability
The most important elements of the industrial applicability of immobilised enzymes are their reuse, and the simplicity of separation. Immobilized enzymes may be more beneficial than free enzymes if their stability is improved. The reusability of enzyme particles is very important while considering enzymatic reactions. Enzyme reusability was accounted for continuous application of the enzyme. The immobilised GI was employed 15 times for 60 min per cycle, as shown in Fig. 6, and the residual activity was roughly 88 percent of the starting activity (Awad et al. 2016). After being encapsulated in poly (acrylic acid-co-2acrylamido 2-methyl propane sulfonic acid) P (AAco-AMPS), it preserved 81% of its initial activity after being utilised 15 times (Kamal et al. 2014). When adsorbed on Indion resin, however, it showed  inferior stability with repeated use, losing around 40% of its initial activity after five cycles and 61% after the seventh cycle. More than one reason could be given in explanation of the loss of durability and stability of the immobilized GI enzyme. The first reason is the incomplete removal of the product (Fructose) from the microcapsules leading to partially masking of the active sites and causing of a kind of substrate-product "traffic jam". The second possible reason is the loss of part of the physically entrapped enzymes with successive use and washing due to decline of the matrix mechanical stability. The last reason is the irreversible conformational changes of the enzyme structure towards denaturized form. A proposed solution os such issue could be suggested through washing of the immobilized GI microcapsules after each use cycle by potassium chloride solution and adding of a slight portion of free enzyme every fixed number of reuse cycles.

Elucidation of the modified gel beads
Fourier transform infrared spectroscopy Figure 7 shows the FT-IR analysis of gel beads at each step of the immobilisation procedure in the range of 400 to 4000 cm −1 . Hassan et al. 2019a, b, used the snailase enzyme to create the illustration. The same procedure used to activate k-carrageenan with glutaraldehyde was employed to immobilise GI, as shown in Fig. 7. The activated carrier with free aldehyde groups interacts to the enzyme via its amino groups, which are plentiful in all enzymes, therefore changing the enzyme should not make a difference. The characteristic peaks seen in each formula are depicted in this diagram. The spectra of κ-carrageenan gel beads are shown in curve "A," while curve "B" reveals a new broad peak at 3400 cm −1 , indicating the existence of κ-carrageenan gel beads. This signifies that the amine group is added to the gel beads. The spectra indicate two new peaks after activation with glutaraldehyde, one referring to a free aldehyde end group (C=O) at 1730 cm −1 and the other corresponding to an N=C group that has formed between the amine group on the surface of the beads and the aldehyde group found in glutaraldehyde (curve "C"). Curve "D" has a larger peak at 3450 cm −1 , showing that the enzyme's amine groups generate a rise in amine group concentrations. We can deduct from the foregoing that the entire immobilisation process was successful. This result was in line with prior research findings (Karam et al. 2018). Figure 8 displaysthe SEM result for κ-carrageenan, aminated κ-carrageenan, activated κ-carrageenan and immobilized GI onto activated κ-carrageenan ( Fig. 8A-D, respectively). The Figures show the changes that happened on the surface in each step after each treatment. For example, in Fig. 8A (untreated carrageenan), the surface is rough (Wahba and Hassan 2017). In Fig. 8B, there is the appearance of many small beads which are attributed to the amination of carrageenan with PEI. In Fig. 8C, the surface is starting to get smoother with a few pebblestructures on its surface which are attributed to the formation of the C=N and free aldehyde end groups (C=O) on its surface as proven in FTIR (Fig. 7). Finally, in Fig. 8D, the surface is getting almost smoother. And that could be attributed to the immobilization of GI on its surface. From the Figures, we can also see the difference in pore size after each step, and this is consistent with other researchers' findings.

Conclusions
To create fructose from glucose, the GI enzyme was covalently attached onto -carrageenan gel beads. The pharmaceutical and food industries benefit greatly from the industrial production of fructose because fructose serves as a cryprotectant, which is used to make medicines more pleasant, and promotes hydrophobic active-ingredient solubility. The optimum pH and temperature of the immobilised GI have been shifted to a wider range than the free one, according to the results. This improves the enzyme's stability and tolerance to changes in pH and temperature, as well as preventing microbial contamination. Furthermore, after 15 reuses (each cycle lasting 60 min), immobilised GI retains 88% of its action, which is beneficial on an industrial level. These findings support the economic and biotechnological advantages of using GI immobilisation to manufacture pure fructose rather than HFCS (which contains glucose). As a result, the findings of this study are regarded as a good model for producing pure fructose from natural and cost-effective sources. The authors suggest that this method be scaled up on a semi-pilot size in the future. This is not a simple process because it will require the use of a bioreactor and changes to the reaction conditions, such as quantity, thermodynamics, and heat transport. As a result, including a chemical engineer is highly suggested.