Enhanced High-Fructose Corn Syrup Production: Immobilizing Serratia marcescens Glucose Isomerase on MOF (Co)-525 Reduces Co2+ Dependency in Glucose Isomerization to Fructose

The escalating demand for processed foods has led to the widespread industrial use of glucose isomerase (GI) for high-fructose corn syrup (HFCS) production. This reliance on GIs necessitates continual Co2+ supplementation to sustain high catalytic activity across multiple reaction cycles. In this study, Serratia marcescens GI (SmGI) was immobilized onto surfaces of the metal-organic framework (MOF) material MOF (Co)-525 to generate MOF (Co)-525-GI for use in catalyzing glucose isomerization to generate fructose. Examination of MOF (Co)-525-GI structural features using scanning electron microscopy-energy dispersive spectroscopy, Fourier-transform infrared spectroscopy, and ultraviolet spectroscopy revealed no structural changes after SmGI immobilization and the addition of Co2+. Notably, MOF (Co)-525-GI exhibited optimal catalytic activity at pH 7.5 and 70 °C, with a maximum reaction rate (Vmax) of 37.24 ± 1.91 μM/min and Km value of 46.25 ± 3.03 mM observed. Remarkably, immobilized SmGI exhibited sustained high catalytic activity over multiple cycles without continuous Co2+ infusion, retaining its molecular structure and 96.38% of its initial activity after six reaction cycles. These results underscore the potential of MOF (Co)-525-GI to serve as a safer and more efficient immobilized enzyme technology compared to traditional GI-based food-processing technologies.


Introduction
The global demand for high-fructose corn syrup (HFCS), a widely used sweetener, has surged in recent decades, securing its place as the world's second most consumed sugar source [1,2].Remarkably, HFCS, with double the sweetness of sucrose at equivalent caloric levels, holds a significant role in the food, beverage, and baking industries due to its non-crystallizing properties at high concentrations [3,4].Additionally, the use of HFCS in food processing contributes to reduced tooth decay.The production of HFCS hinges on glucose isomerase (GI), a catalyst converting D-glucose to D-fructose by isomerizing aldose to ketose [5,6].In the enzymatic hydrolysis of starch for HFCS, the rate-limiting step is the glucose isomerization [7].The isomerization reaction operates within a thermodynamic equilibrium, and elevated reaction temperatures enhance the conversion rate, underscoring the value of heat-resistant GIs for boosting HFCS yield [8,9].
Currently, two primary methods have been employed to enhance the temperature tolerance of glucose isomerase (GI).One approach involves screening genes to identify GI naturally tolerant to high temperatures.Another method focuses on immobilizing native Foods 2024, 13, 527 3 of 13 While technological advancements have been made in the field of GI immobilization onto MOF carriers, research in this field is still predominantly focused on isomerization reactions requiring supplementation with metal ion activators, which must be removed from the final reaction product, which is an expensive and difficult task.However, the subsequent removal of these ions poses a significant challenge.Overcoming this obstacle may entail conducting these reactions without the initial addition of metal ions in future research.Yet, within the realm of MOF-based enzyme immobilization, no studies have yet emerged aiming to diminish or eliminate the necessity for added metal ion activators while supporting isomerization reactions.This includes investigations into reaction systems employing MOF-immobilized GIs.
In this study, MOF-525 coordination with Co 2+ was harnessed to generate MOF (Co)-525 that served as a carrier for the immobilization of Serratia marcescens glucose isomerase (SmGI) used to synthesize MOF (Co)-525-GI.Subsequently, MOF (Co)-525-GI was subjected to UV-adsorption, SEM-EDS, FTIR spectral analyses, and zeta potential analysis.The enzymatic activity and kinetic analyses of free SmGI and immobilized SmGI under various pH and temperature conditions with or without added Co 2+ were performed.Additionally, MOF (Co)-525-GI storage stability and reusability were assessed to reveal its industrial potential.Remarkably, MOF (Co)-525-GI was able to effectively catalyze glucose-to-fructose isomerization without continuous supplementation with Co 2+ , highlighting its promise as an immobilized GI for use in industrial HFCS production.

Purification and Culture of SmGI
Serratia marcescens was activated and inoculated in 1 L of LB medium and cultured at 28 • C with 170 r/min for 24 h.The bacteria in the medium were centrifuged at 5000× g for 30 min.The precipitate was washed three times with Tris-HCl (50 mM pH 7.2) buffer.one gram of precipitate was collected and dissolved in 10 mL Tris-HCl (50 mM pH 7.2) buffer.Then, the precipitate solution was broken by an ultrasonic crusher with an ice bath.Then, the solution was centrifuged at 8000× g for 10 min to collect the supernatant.The supernatant was heated at 70 • C for 30 min, and centrifuged at 8000× g for 20 min with 4 • C. The supernatant was centrifuged at 1500× g for 30 min by a 10 kD ultrafiltration tube and the SmGI solution was collected.The SmGI size was determined by SDS-Page, and the protein concentration was determined using an Enhanced BCA Protein Assay Kit (Shanghai Beyotime P0010, Shanghai, China).The final concentration of SmGI was adjusted to 5.28 mg/mL.

Preparation of MOF (Co)-525
Synthesis of MOF-525 was performed using the method reported by Chang et al. [37].ZrOCl 2 • 8H 2 O (105 mg, 40.73 mmol) and benzoic acid (1.35 g, 1.38 mol) were ultrasonically dissolved in 8 mL of DMF, and then the resulting transparent solution was incubated at 80 • C for 2 h.After the solution was allowed to cool to room temperature, TCPP (47 mg, 7.43 mmol) was added to the mixture, and then it was ultrasonicated for 20 min.Thereafter, it was heated at 80 • C for 24 h and then allowed to cool to room temperature, during which a precipitate formed that was subsequently collected by centrifugation at 10,000× g at 4 • C for 10 min.The resulting pellet was washed three times with DMF and then dried under a vacuum at 120 • C for 12 h to generate the final MOF-525 preparation.
For MOF (Co)-525 preparation, 60 mg of MOF-525 was dissolved in 300 mL of DMF, and then CoCl 2 •6H 2 O (300 mg, 4.20 mmol) was dissolved in the abovementioned solution.After reacting at 120 • C for 18 h, the solution was centrifuged at 10,000× g, and then the resulting MOF (Co)-525 precipitate was washed with DMF three times and dried under a vacuum at 120 • C for 12 h to generate the final MOF (Co)-525 preparation.-525-GI were obtained using a JSM-6010LA SEM system (CITACHI, Tokyo, Japan).X-ray diffraction (XRD) was carried out using Cu-Ka radiation (α = 1.5418Å) over a 2θ range of 5-25 at a rate of 1/min using an Empyrean diffractometer (Malvern PANalytical B.V., Almelo, The Netherlands).Fourier-transform infrared (FTIR) spectra of samples prepared using the KBr pellet method were obtained using an FTIR spectrophotometer (Bruker, Ettlingen, Germany), and then data spanning the wavelength range of 4000 cm −1 to 500 cm −1 were collected and analyzed.UV-Vis absorption spectra were obtained using a SHIMADZU UV-2700 spectrophotometer (UV-2700 Shimadzu, Tokyo, Japan).Zeta potential analysis was performed using a particle size analyzer (Malvern MS 3000, Bristol, UK).Co 2+ concentrations in reaction solutions were measured using an ICE 3500 atomic absorption spectrometer (Thermo Scientific, Waltham, MA, USA).

Analysis of Enzymatic Activity of MOF (Co)-525-GI 2.3.1. Determination of SmGI Loading Rate
For the determination of the MOF (Co)-525-GI loading rate, 30 mg of dried MOF (Co)-525 was dissolved in 4 mL of Tris-HCl (50 mM pH 7.2) buffer solution.After sonication for 30 min, 1 mL of 5.28 mg/mL SmGI extracted from S. marcescens was added to the MOF (Co)-525 solution to generate a 5 mL reaction system that was stirred for 2 h at 4 • C.During stirring, a precipitate formed that was collected via centrifugation at 5000× g for 15 min.After centrifugation, the SmGI concentration in the supernatant was determined (X 1 ).The precipitate was washed with Tris-HCl(50 mM pH 7.2) buffer three times, yielding SmGI concentrations in subsequent washes designated as X 2 , X 3 , and X 4 , while the SmGI concentration before its addition to the MOF (Co)-525 solution was designated as X.SmGI concentrations were determined using an Enhanced BCA Protein Assay Kit (Shanghai Beyotime P0010, Shanghai, China).The SmGI load rate (Y) was calculated using the following formula: Effects of pH and Temperature on Immobilized and Free SmGI Enzymatic Activities The effect of pH on the enzymatic activity of free SmGI was measured after 0.3 mL of a solution of free SmGI was added to a 2.7 mL volume of 50% glucose solutions of pH 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, or 9.5.The effect of pH on enzymatic activities of either MOF-525-GI or MOF (Co)-525-GI in a 3 mL reaction system was assessed after 20 mg of these reaction systems were added to 3 mL of 50% glucose solutions of pH 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, or 9.5.All reactions were allowed to proceed at 60 • C for 1 h, and then the immobilized enzyme was removed from reaction supernatants by passing them through 0.22-µm membrane filters.Supernatant fructose concentrations were measured using a Fructose Assay Kit (Nanjing Jiancheng A085-1-1, Nanjing, China).
To assess the temperature optimum for free SmGI, MOF-525-GI, and MOF (Co)-525-GI, 0.3 mL of free SmGI was added to 2.7 mL of a 50% glucose solution at pH 7.5, while 20 mg of MOF (Co)-525-GI was added to 3 mL of a 50% glucose solution at pH 7.5.After the three reaction systems were incubated for 1 h at 30, 40, 50, 60, 70, 80, or 90 • C, the immobilized Foods 2024, 13, 527 5 of 13 enzyme was removed from reaction supernatants by passing it through 0.22 µm membrane filters, and then fructose concentrations in filtrates were measured as described above.

Determination of Biochemical and Operation Properties
The enzymatic activities of free and immobilized SmGI were assessed in 3 mL reaction volumes based on enzyme catalytic activity for the conversion of glucose to fructose.For the assessment of free or immobilized SmGI catalytic activity, 0.3 mL of a solution containing 5.28 mg/mL free SmGI was added to 2.7 mL of deoxygenated solutions containing various concentrations of glucose substrate (2.5, 5, 10, 20, 40, 60, 80, 100, 200, and 400 mM) to generate 3 mL reaction systems, while 20 mg of MOF (Co)-525-GI was added to 3 mL volumes of the same glucose solutions.After incubating each reaction at 60 • C for 1 h, immobilized SmGI was collected using centrifugation for later use.Fructose concentrations of all supernatants were immediately measured after passing supernatants through 0.22 µm membrane filters.K m and V max values of reactions containing free and immobilized SmGI were calculated using the Lineweaver-Burk equation: where V 0 is the reaction rate (µM min −1 ), V max is the maximum reaction rate (µM min −1 ), [S] is the glucose concentration (mM mL −1 ), and K m is the Michaelis-Menten constant.

Assessments of Reusability and Storage Stability of MOF (Co)-525-GI
To assess MOF (Co)-525-GI reusability and storage stability, 20 mg of MOF (Co)-525-GI was added to 3 mL of a 50% (w/v) glucose solution at pH 7.5.After the reaction was allowed to proceed at 70 • C for 1 h, the immobilized enzyme was collected using centrifugation for later use.The fructose concentration in the supernatant was measured after supernatants were passed through 0.22 µm membrane filters, as described in Section 2.3.3.The relative enzymatic activities of free SmGI and MOF (Co)-525-GI stored at 4 • C were determined every 15 days during storage and compared to respective free SmGI and MOF (Co)-525-GI catalytic activities prior to storage (which were set to 100%).

Statistical Analysis
Statistical analyses were performed using GraphPad Prism 10.1 software (GraphPad Software Inc., San Diego, CA, USA) based on calculated mean and standard error values obtained for three experimental replicates.Graphs were analyzed using Origin 2022 (Origin 2022, OriginLab, Northampton, MA, USA).

SDS-Page of SmGI and SEM-EDS Analysis of MOF (Co)-525-GI
The protein bands of SmGI were observed in 10% acrylamide gel.SDS-PAGE results of SmGI used in the experiment are shown in Figure 1a.The main bind at approximately 60 kD was detected, which is consistent with published reports [38].Also, the purity of the protein bind showed that the protein was suitable for further experiments.The immobilization rate of MOF (Co)-525 for SmGI was 43.2% following the formula in Section 2.3.1, which was higher than previously reported [39].Since the immobilization rate was 43.2%, with the description in Section 2. EDS analysis was conducted to determine the elemental composition of MOF (Co)-525 (Figure 1f).EDS results demonstrated the presence of C, O, S, Co, and Zr in MOF (Co)-525-GI at mass ratios of 46.63%, 10.29%, 20.98%, 0.07%, 0.17%, and 20.93%, respectively.With excess Co 2+ removed from the reaction of MOF-525, the Co 2+ signal showed that Co 2+ successfully coordinated within MOF (Co)-525 (Figure 1f).Sulfur, an element found in proteins but not in MOF (Co)-525, was detected in the EDS spectrum, thus indicating that SmGI was successfully immobilized onto the MOF (Co)-525 substrate.EDS analysis was conducted to determine the elemental composition of MOF (Co)-525 (Figure 1f).EDS results demonstrated the presence of C, O, S, Co, and Zr in MOF (Co)-525-GI at mass ratios of 46.63%, 10.29%, 20.98%, 0.07%, 0.17%, and 20.93%, respectively.With excess Co 2+ removed from the reaction of MOF-525, the Co 2+ signal showed that Co 2+ successfully coordinated within MOF (Co)-525 (Figure 1f).Sulfur, an element found in proteins but not in MOF (Co)-525, was detected in the EDS spectrum, thus indicating that SmGI was successfully immobilized onto the MOF (Co)-525 substrate.
FTIR results obtained for MOF (Co)-525 and MOF (Co)-525-GI within the wavelength range of 4000-500 cm −1 were consistent with spectra reported by Chang et al., thus confirming the successful synthesis of MOF-525 [37].Comparisons of FTIR spectra obtained for MOF-525 and MOF (Co)-525 revealed that added Co 2+ coordinated with MOF-525, as indicated by the appearance of a new peak at 996 cm −1 [41].
After SmGI was immobilized on MOF-525 or MOF (Co)-525, new FTIR spectral peaks corresponding to amide A, amide I, and amide II bonds appeared (Figure 2b).According to published results, the amide A band is mainly attributed to an N-H stretching vibration in resonance with amide II, while the amide I absorption band corresponds primarily to the amide group C=O stretching vibration, and the amide II absorption band corresponds to N-H bending and C-N stretching vibrations [42].Notably, an analysis of SmGI spectral bands within amide A, amide I, and amide II regions of free SmGI and MOF (Co)-525-GI revealed that the structure of SmGI remained intact after immobilization.
Changes in porphyrin UV absorption spectral Soret and Q bands are important indicators of the metallization status of the porphyrin center.As shown in Figure 2c, characteristic UV absorption peak results revealed a maximum MOF-525 absorption peak near 411 nm within the Soret band region and three additional peaks at 528, 566, and 655 nm.The peak at 411 nm was shifted to 431 nm in MOF (Co)-525 due to the coordination of Co with MOF-525, as consistent with published results indicating successful synthesis of Co-TCPP [41,43].Moreover, peaks at 528 nm, 566, and 655 were replaced by peaks at 551 nm and 590 nm, while SmGI UV absorbance spectra did not change after SmGI immobilization.
551 nm and 590 nm, while SmGI UV absorbance spectra did not change after SmGI immobilization.
Zeta potential is an important indicator of colloidal dispersion system stability [44,45].Here, zeta potential measurements revealed the greatest MOF (Co)-525 stability at pH = 3 or pH = 9.At a pH of >5, both MOF-525 and MOF (Co)-525 in the buffer carry negative charges (Figure 2d).In contrast, zeta potentials of MOF-525(Co)-GI obtained under different pH conditions clearly indicate that the material in solution is extremely unstable within the pH range of 4-6 and relatively stable within the pH range of 6-8.Within the latter pH range, good dispersion of MOF (Co)-525-GI enhances its ability to effectively interact with the substrate, thereby increasing its catalytic activity.Zeta potential is an important indicator of colloidal dispersion system stability [44,45].Here, zeta potential measurements revealed the greatest MOF (Co)-525 stability at pH = 3 or pH = 9.At a pH of >5, both MOF-525 and MOF (Co)-525 in the buffer carry negative charges (Figure 2d).In contrast, zeta potentials of MOF-525(Co)-GI obtained under different pH conditions clearly indicate that the material in solution is extremely unstable within the pH range of 4-6 and relatively stable within the pH range of 6-8.Within the latter pH range, good dispersion of MOF (Co)-525-GI enhances its ability to effectively interact with the substrate, thereby increasing its catalytic activity.

Effects of Temperature and pH on SmGI Enzymatic Activity
Environmental factors such as pH and temperature greatly influence enzyme activity, with excellent stability, serving as an important indicator of immobilized enzyme industrial potential [46].Temperature can provide energy for the isomerization SmGI-catalyzed conversion of glucose to fructose.Based on this premise, we investigated the effect of temperature on free and immobilized SmGI catalytic activities.Our experimental results demonstrated good enzymatic activity of immobilized SmGI at temperatures between 50 and 80 • C, while good enzymatic activity of free SmGI was observed only at 60 • C and optimal MOF (Co)-525-GI enzyme activity was observed only at 70 • C, as shown in Figure 3a.
the activity of the immobilized enzyme decreased drastically, due to potential destabilization of the SmGI conformation and possible decomposition of the MOF-525 nanometal-organic framework under alkaline conditions [49,50].Importantly, the pH of the reaction system tended to decrease with the progression of the SmGI catalytic isomerization reaction.Nevertheless, the relatively greater stability of MOF (Co)-525-GI under weakly acidic conditions as compared to that of free SmGI underscores the greater suitability of MOF (Co)-525-GI for industrial HFCS production.As compared to the optimal temperature of free SmGI, MOF (Co)-525-GI exhibited optimal catalytic activity at a temperature approximately 10 • C higher (Figure 3a).During the process of SmGI conversion of glucose to fructose, a thermodynamic equilibrium exists between glucose and fructose conversion reactions after glucose conversion reaches a certain level [47,48].Notably, with increasing temperature, the conversion of glucose to fructose becomes more favorable and therefore would be better supported by (Co)-525-GI than by free SmGI, highlighting the enhanced suitability of MOF (Co)-525-GI for high-temperature industrial food processing.

Kinetic Studies of MOF (Co)-525-GI Activity
We also assessed MOF (Co)-525-GI stability under different pH conditions within the pH range of 3.5-9.5 (Figure 3b) and observed the highest enzyme activity at pH 7.5 and greatest stability within the pH range of pH 5.5-8.5.However, when the pH was >9, the activity of the immobilized enzyme decreased drastically, due to potential destabilization of the SmGI conformation and possible decomposition of the MOF-525 nanometal-organic framework under alkaline conditions [49,50].Importantly, the pH of the reaction system tended to decrease with the progression of the SmGI catalytic isomerization reaction.Nevertheless, the relatively greater stability of MOF (Co)-525-GI under weakly acidic conditions as compared to that of free SmGI underscores the greater suitability of MOF (Co)-525-GI for industrial HFCS production.

Kinetic Studies of MOF (Co)-525-GI Activity
During the MOF (Co)-525-GI-catalyzed reaction, enzyme activity was detectable over the substrate concentration range of 2.5-400 mM (Figure 4a,c) and a good linear relationship was observed between glucose isomerization rate and concentration at low glucose concentrations.However, as the glucose concentration increased, the reaction rate gradually reached a maximum (Figure 4a,c), as indicated by Lineweaver-Burk double reciprocal analysis results (Figure 4b,d) and the maximum reaction speed (V max ) and K m of SmGI (Table 1).A comparison of K m values obtained for SmGI and MOF (Co)-525-GI revealed a greater value for MOF (Co)-525-GI, indicating reduced enzyme-substrate affinity and associated reduced enzymatic activity due to decreased enzyme dispersion postimmobilization [48].Notably, the substrate affinity of MOF (Co)-525-GI in our experiments was greater than that reported in the literature, highlighting its promise for use in industrial food processing operations [12].applications.Moreover, in the absence of added Co 2+ , the catalytic activity of MOF (Co)-525-GI was higher than that of MOF-525-GI, due to SmGI activation by Co 2+ within the MOF (Co)-525-GI porphyrin center [51].Collectively, these results demonstrate that MOF (Co)-525-GI possessed good long-term substrate affinity and stable enzyme activity in the absence of added metal ion activator, providing a theoretical basis for reducing the use of metal ion activators such as CoCl2 in the HFCS production process.Importantly, the addition of Co 2+ increased the enzymatic activity of free SmGI (Table 1), an effect that may have been due to Co 2+ induction of increased SmGI affinity for the substrate.In contrast, the effect of added Co 2+ on the enzymatic activity of MOF (Co)-525-GI was less pronounced than Co 2+ enhancement of free SmGI activity, whereby in the absence of added Co 2+ (low Co 2+ concentration), MOF (Co)-525-GI exhibited optimal enzyme activity that would render it particularly suitable for use in food-processing applications.Moreover, in the absence of added Co 2+ , the catalytic activity of MOF (Co)-525-GI was higher than that of MOF-525-GI, due to SmGI activation by Co 2+ within the MOF (Co)-525-GI porphyrin center [51].Collectively, these results demonstrate that MOF (Co)-525-GI possessed good long-term substrate affinity and stable enzyme activity in the absence of added metal ion activator, providing a theoretical basis for reducing the use of metal ion activators such as CoCl 2 in the HFCS production process.

Assessments of MOF (Co)-525-GI Reusability and Stability
Immobilized SmGI was assessed for reusability, with the results demonstrating that MOF (Co)-525-GI retained 96.38% of its initial activity after six reaction cycles (Figure 5a).Moreover, the structure of the immobilized enzyme remained intact during cycling, as indicated by unchanging UV spectra during cycling (Figure 5c) and unchanging FTIR spectra indicating complete retention of Co 2+ during cycling (Figure 5d).These results demonstrate that immobilized SmGI retained greater enzyme activity over six reaction cycles than that retained by free SmGI, thereby enhancing the industrial applicability of SmGI for food processing.
Atomic absorption spectrophotometric measurements of Co 2+ concentration in the MOF (Co)-525-GI reaction system over six reaction cycles (Figure 5b) indicated that during cycling, the Co 2+ concentration remained essentially constant and was approximately one-thousandth that of the free SmGI reaction system with continuous Co 2+ supplementation.Therefore, coordination of Co 2+ at the MOF (Co)-525-GI porphyrin center plays an important role in SmGI enzyme activity by reducing added Co 2+ to maintain enzyme activity over repeated reaction cycles.These results clarified that the construction of a reaction system for HFCS production with low Co 2+ addition is possible.
indicated by unchanging UV spectra during cycling (Figure 5c) and unchanging FTIR spectra indicating complete retention of Co 2+ during cycling (Figure 5d).These results demonstrate that immobilized SmGI retained greater enzyme activity over six reaction cycles than that retained by free SmGI, thereby enhancing the industrial applicability of SmGI for food processing.
Atomic absorption spectrophotometric measurements of Co 2+ concentration in the MOF (Co)-525-GI reaction system over six reaction cycles (Figure 5b) indicated that during cycling, the Co 2+ concentration remained essentially constant and was approximately one-thousandth that of the free SmGI reaction system with continuous Co 2+ supplementation.Therefore, coordination of Co 2+ at the MOF (Co)-525-GI porphyrin center plays an important role in SmGI enzyme activity by reducing added Co 2+ to maintain enzyme activity over repeated reaction cycles.These results clarified that the construction of a reaction system for HFCS production with low Co 2+ addition is possible.An analysis of storage stabilities of free SmGI and MOF (Co)-525-GI based on enzymatic activities measured every 15 days revealed that SmGI stored at 4 • C retained 73.79% of its initial enzymatic activity after 15 days, 28.54% of its initial activity after one month, and no activity after 60 days (Figure 6).In contrast, after 30 days of storage, MOF (Co)-525-GI enzymatic activity remained higher (85.92%) than that reported for SmGI immobilized onto the upper critical solution temperature (UCST)-responsive polymers carrier [48].Moreover, on the 15th day of storage, MOF (Co)-525-GI residual activity was significantly higher than that of free SmGI and remained high (47.4%)after 90 days of storage.Therefore, immobilization endowed SmGI with significantly greater long-term storage stability compared to that of free SmGI, further highlighting MOF (Co)-525-GI suitability for food-processing applications.An analysis of storage stabilities of free SmGI and MOF (Co)-525-GI based on enzymatic activities measured every 15 days revealed that SmGI stored at 4 °C retained 73.79% of its initial enzymatic activity after 15 days, 28.54% of its initial activity after one month, and no activity after 60 days (Figure 6).In contrast, after 30 days of storage, MOF (Co)-525-GI enzymatic activity remained higher (85.92%) than that reported for SmGI immobilized onto the upper critical solution temperature (UCST)-responsive polymers carrier [48].Moreover, on the 15th day of storage, MOF (Co)-525-GI residual activity was significantly higher than that of free SmGI and remained high (47.4%)after 90 days of storage.Therefore, immobilization endowed SmGI with significantly greater long-term storage stability compared to that of free SmGI, further highlighting MOF (Co)-525-GI suitability for food-processing applications.

Conclusions
In this study, we successfully synthesized MOF (Co)-525-GI by immobilizing SmGI onto the carrier MOF (Co)-525.Our comprehensive analysis of MOF (Co)-525-GI physical and enzymatic characteristics revealed that SmGI retained its crystalline structure after immobilization and exhibited high enzymatic activity even in the absence of added Co 2+ .Notably, the immobilized enzyme displayed an optimal reaction temperature of 70 °C,

Figure 3 .
Figure 3.Effect of temperature and pH for free SmGI and MOF (Co)-525-GI enzyme activity.(a) The effect of temperature on the activity of free SmGI (Red) and MOF (Co)-525-GI (Blue) from 30 °C to 90 °C; (b) the effect of pH on the activity of free SmGI (Red) and MOF (Co)-525-GI (Blue) at pH 3.5-9.5.

Figure 3 .
Figure 3.Effect of temperature and pH for free SmGI and MOF (Co)-525-GI enzyme activity.(a) The effect of temperature on the activity of free SmGI (Red) and MOF (Co)-525-GI (Blue) from 30 • C to 90 • C; (b) the effect of pH on the activity of free SmGI (Red) and MOF (Co)-525-GI (Blue) at pH 3.5-9.5.

Figure 4 .
Figure 4. Influence of substrate concentration on the activity of immobilized enzyme in a reaction system without Co 2+ addition.(a) Activity of free SmGI with substrate concentrations ranging from 2.5-400 mM; (b) Linewever-Burk double reciprocal plot of free SmGI activity at substrate concentrations of 2.5-400 mM; (c) activity of MOF (Co)-525-GI with substrate concentrations ranging from 2.5-400 mM; (d) Linewever-Burk double reciprocal plot of MOF (Co)-525-GI activity at substrate concentrations of 2.5-400 mM.

Figure 4 .
Figure 4. Influence of substrate concentration on the activity of immobilized enzyme in a reaction system without Co 2+ addition.(a) Activity of free SmGI with substrate concentrations ranging from 2.5-400 mM; (b) Linewever-Burk double reciprocal plot of free SmGI activity at substrate concentrations of 2.5-400 mM; (c) activity of MOF (Co)-525-GI with substrate concentrations ranging from 2.5-400 mM; (d) Linewever-Burk double reciprocal plot of MOF (Co)-525-GI activity at substrate concentrations of 2.5-400 mM.

Figure 5 .
Figure 5.The reusability of the MOF (Co)-525-GI.(a) The MOF (Co)-525-GI enzyme activity after six cycles of use.(b) The concentration of Co 2+ in the reaction system after six cycles of use of MOF (Co)-525-GI.(c) The UV spectra of MOF (Co)-525-GI before (Line) and after six cycles using (Dot-line).(d) The FTIR spectra of MOF (Co)-525-GI before (Line) and after six cycles using (Dot-line).

Figure 5 .
Figure 5.The reusability of the MOF (Co)-525-GI.(a) The MOF (Co)-525-GI enzyme activity after six cycles of use.(b) The concentration of Co 2+ in the reaction system after six cycles of use of MOF (Co)-525-GI.(c) The UV spectra of MOF (Co)-525-GI before (Line) and after six cycles using (Dot-line).(d) The FTIR spectra of MOF (Co)-525-GI before (Line) and after six cycles using (Dot-line).

Table 1 .
Kinetic behavior of free GI and immobilized enzymes.