Alginate lyase is an enzyme that catalyzes the hydrolysis of alginate into alginate oligoalginates. To enhance enzyme stability and recovery, a facile strategy for alginate lyase immobilization was developed. The novel magnetic chitosan microspheres were synthesized and used as a carrier to immobilize alginate lyase. Immobilization of alginate lyase on magnetic chitosan microspheres was successful as proved by the Fourier transform infrared spectroscopy and X-ray diffraction spectra. The enzyme immobilization exhibited best performance at MCM dosage = 1.5 g/L, adsorption time = 2.0 h, glutaraldehyde concentration = 0.2%, and immobilization time = 2.0 h. The optimal pH of the free alginate lyase was 7.5, and this pH value was shifted to 8.0 after immobilization. No difference was observed in the optimal temperature (45°C) for the immobilized and free enzymes. The immobilized alginate lyase displayed superior thermal stability compared with the free alginate lyase. The Km of the free and immobilized enzymes were 0.05 mol/L and 0.09 mol/L, respectively. The immobilized alginate lyase retained 72% of its original activity after 10 batch reactions. This strategy was a promising method to immobilize alginate lyase.
Research Article
Preparation, structural characterization and enzymatic properties of alginate lyase immobilized on magnetic chitosan microspheres
https://doi.org/10.21203/rs.3.rs-3325686/v1
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Journal Publication
published 30 Dec, 2023
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Alginate lyase
Immobilization
Magnetic chitosan microspheres
Enzyme stability
Enzyme recovery
Alginate lyase is an enzyme catalyzing alginate into alginate oligoalginates. The mechanism of alginate lyases action is that they cleave 1, 4-glycosidic bond of alginate via a beta-elimination mechanism [1]. Numerous alginate lyases have been isolated from Vibrio sp., Pseudomonas aeruginosa, Microbulbifer sp., and so on [2, 3]. Alginate lyases are divided into endolytic and exolytic modes based on the cleavage sites on alginate polysaccharide [4]. Alginate lyases have been used to degrade alginate in Saccharina latissima, Laminaria japonica, and other algae into alginate oligoalginates [5, 6]. They also have been used as pharmaceutical enzymes, and enzymatic tools for biotechnology and molecular biology [7]. The advantages of using alginate lyases to produce alginate oligoalginates from alginate are green technology, a high reaction rate under mild reaction conditions, high specificity, low energy consumption and side reactions [8]. However, the high cost, enzyme instability and difficulty in recovery of enzymes greatly limit their application in industry.
Immobilized enzyme technology can overcome the instability and difficulty in recovery. Enzyme immobilization is a kind of technology which uses a carrier to restrain enzymes in a certain area and still maintains the specific catalytic activity of the enzymes, making them recoverable and reusable [9]. Methods of enzyme immobilization include physical methods (adsorption and entrapment) and chemical methods (cross-linking and covalent binding) [10]. Enzyme activity usually decreases upon immobilization [11]. The carrier material is particularly important for the immobilized efficiency of enzymes and enzyme stability. The carriers used for the immobilization of enzymes include natural organic materials, inorganic materials, organic polymers and magnetic carriers [12]. Recently, the immobilization of alginate lyase draws great attention due to reusing the enzyme, high operational stability and easy recovery of product from the enzymatic hydrolysates [13].
Magnetic nanomaterials are considered as a kind of green material. Unlike filtration and centrifugation, magnetic nanomaterials can be recovered by magnet, which is easy, cost effective and efficient in preventing enzyme loss [14]. Among the magnetic nanomaterials, Fe3O4 nanoparticles have been used for the immobilization of cellulase, lipase, amylase with good immobilized efficiency due to their small particle sizes, large specific surface area and strong magnetic properties [15–17]. Attaching inorganic or polymeric coatings to Fe3O4 nanoparticles can improve the functionality or biocompatibility of these nanostructures which can avoid the phenomenon magnetically induced self-aggregation of magnetic cores [18]. Natural polymer gel carriers (chitosan, calcium alginate, etc.) and organic synthetic polymers (polyacrylamide, polyethyleneimine, etc.) are often used as the base materials for carrier preparation because of the functional groups on their surfaces. Among them, chitosan (β-1,4-2-amino-2-deoxy-D-glucose) is an aminopolysaccharide whose amino (-NH2) and hydroxyl (-OH) groups of its molecular chain can be easily chemically modified as active sites for biological binding [19]. Chitosan is one of the most abundant polysaccharides in nature and with low cost, biodegradability, good biocompatibility and non-toxicity has wide application in water treatment, enzyme immobilization, etc. [20]. There is little information on the immobilization of alginate lyases on magnetic chitosan microspheres.
The aim of this work was to synthesize a novel magnetic chitosan microspheres (MCM). Furthermore, the alginate lyase was immobilized by using MCM and glutaraldehyde as the carrier and crosslinking agent, respectively. In addition, the structural characterization and enzymatic properties of the immobilized alginate lyase were investigated.
Materials
Fe3O4 nanoparticles, alginate lyase, 3, 5-dinitrosalicylic acid and other reagents were provided by Merck Chemicals Co., Ltd. (Shanghai City, China). The activity of alginate lyase was 20, 000 U/g.
Preparation of MCM
Chitosan of 1.0 g was put into a 250-mL glass flask. Then 100 mL acetic acid was added. Chitosan and acetic acid were mixed evenly. Fe3O4 nanoparticles of 1.0 g were put into the above solution. The mixtures were dispersed ultrasonically (400W ultrasonic power, 6 s interval, 30 s ultrasonic and 30 min total working time). The dispersed mixtures were subsequently added to the Span-80 / liquid paraffin system (160 mL of liquid paraffin and 10 mL of Span-80) under stirring (120 rpm, 30 min). Afterward, 16 mL of 5% (w/v) glutaraldehyde was added. It was kept in a water bath at 40°C for 1.0 h. Its pH was regulated to 9.0 with 1 mol/L NaOH / HCl. After that, the mixtures were kept at 70°C for 2.0 h. The products were collected with a magnet. Next, the collected products were washed thoroughly with 100 mL petroleum ether, 100 mL acetone, and 200 mL distilled water successively. Finally, the washed products were dried at 60°C for 3.0 h and the dried products were MCM.
Immobilization of alginate lyase
For MCM dosage experiment, 0.05 g (0.5 g/L MCM dosage), 0.10 g (1.0 g/L MCM dosage), 0.15 g (1.5 g/L MCM dosage), 0.20 g (2.0 g/L MCM dosage), 0.25 g (2.5 g/L MCM dosage), and 0.30 g (3.0 g/L MCM dosage) MCM were added into 100 mL disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (DPB) (2.0 mol/L, pH 7.2) for 12.0 h, respectively. The MCM was separated by the magnet and then dissolved in 50 mL of 0.6 g/L alginate lyase solution. The mixtures were agitated at 130 rpm in an oscillator for 1.0 h.
For adsorption time experiment, 0.15 g MCM (1.5 g/L MCM dosage) was added into 100 mL DPB for 12.0 h. The MCM was separated by the magnet and then dissolved in 50 mL of 0.6 g/L alginate lyase solution. The mixtures were agitated at 130 rpm for 0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h, respectively.
For glutaraldehyde concentration experiment, 0.15 g MCM (1.5 g/L MCM dosage) was added into 100 mL DPB for 12.0 h. The MCM was separated by the magnet and then dissolved in 50 mL of 0.6 g/L alginate lyase solution. The mixtures were agitated at 130 rpm for 2.0 h. After that, 0.0% (0.00 mL glutaraldehyde), 0.1% (0.05 mL glutaraldehyde), 0.2% (0.10 mL glutaraldehyde), 0.3% (0.15 mL glutaraldehyde), 0.4% (0.20 mL glutaraldehyde), and 0.5% (0.25 mL glutaraldehyde) glutaraldehyde concentrations were carried out and the MCM was cross-linked for 2.0 h.
For immobilization time experiment, 0.15 g MCM (1.5 g/L MCM dosage) was added into 100 mL DPB for 12.0 h. The MCM was separated by the magnet and then dissolved in 50 mL of 0.6 g/L alginate lyase solution. The mixtures were agitated at 130 rpm for 2.0 h. After that, 0.2% glutaraldehyde concentration was used and the MCM was cross-linked for 1.0 h, 2.0 h, 3.0 h, 4.0 h, 5.0 h, and 6.0 h, respectively.
In all experiments, the alginate lyase-bonded MCM was collected by the magnet after cross-link and washed with DPB for 5 times. Thereafter, the immobilized alginate lyase was lyophilized in a freeze drier and stored at 4°C before use.
Enzyme activity assay
The immobilized alginate lyase of 50 mg or free alginate lyase of 5 mg was dissolved into 20 mL DPB (2.0 mol/L, pH 7.5), respectively. The activities of the immobilized and free alginate lyase were measured by a 3, 5-dinitrosalicylic acid assay [21]. One unit of enzyme activity of alginate lyase (U/mL or U/g) was defined as the amount of alginate lyase used to produce 1 µg of reducing sugar per minute. Proteins in the enzyme solutions were determined by the Bradford’s method [22]. The enzyme activity recovery (EAR, %), immobilized efficiency (IE, %), and adsorption capacity of MCM (AC, mg/g) were calculated as follows:
where A0 (U/mL or U/g), and A1 (U/mL or U/g) are the activity of the free alginate lyase, and observed activity of the immobilized alginate lyase, respectively. C1 (mg/mL) and C0 (mg/mL) are the protein concentrations of the buffer system after immobilization and the initial protein concentrations of the buffer system, respectively. V (mL) is the volume of the immobilized system. M (g) is MCM weight.
Structural characterization of the immobilized alginate lyase
Characterization of the particle morphology of MCM was through scanning electron micrograph (SEM) [18]. The chemical structures of chitosan, MCM and the immobilized alginate lyase were characterized by the Fourier transform infrared spectroscopy (FTIR), respectively [23]. The crystal structures of Fe3O4 nanoparticles, MCM and the immobilized alginate lyase were measured by using X-ray diffractometer (XRD), respectively [24]. The hysteresis loops of MCM and the immobilized alginate lyase were obtained by vibrating sample magnetometer (VSM), respectively [25].
Characterization of the enzymatic properties of the immobilized alginate lyase
Optimal pH
Each sample of 100 mg sodium alginate was dissolved into 20 mL citric acid-sodium citrate buffer (pH 6.0), 20 mL citric acid-sodium citrate buffer (pH 6.5), 20 mL DPB (pH 7.0), 20 mL DPB (pH 7.5), 20 mL Tris-HCl buffer (pH 8.0), and 20 mL Tris-HCl buffer (pH 8.5), respectively. The immobilized alginate lyase of 10 mg or free alginate lyase of 5 mg was dissolved into the sodium alginate solution, respectively. The mixtures were kept at 40°C for 1.0 h. Then the enzyme activity of the immobilized or free alginate lyase was measured. The maximum enzyme activity was set to 100%. The free alginate lyase was used as control.
Optimal temperature
Sodium alginate of 100 mg was dissolved into 20 mL DPB (pH 7.5). The immobilized alginate lyase of 10 mg or free alginate lyase of 5 mg was dissolved into the sodium alginate solution, respectively. The mixtures were kept for 1.0 h at 35°C, 40°C, 45°C, 50°C, and 55°C, respectively. Then the enzyme activity of the immobilized or free alginate lyase was measured. The maximum enzyme activity was set to 100%. The free alginate lyase was used as control.
Thermal stability
Sodium alginate of 100 mg was dissolved into 20 mL DPB (pH 7.5). The immobilized alginate lyase of 10 mg or free alginate lyase of 5 mg was dissolved into the sodium alginate solution, respectively. The mixtures were reacted for 120 min at 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, and 75°C, respectively. After that, the enzyme activity of the immobilized or free alginate lyase was measured every 20 min. The maximum enzyme activity was set to 100%. The free alginate lyase was used as control.
Determination of Km and Vm
The double reciprocal plot method (Lineweaver-Burk plot) was used to calculate Km and Vm of the immobilized and free alginate lyase, respectively. Sodium alginate of 100 mg was dissolved into 20 mL DPB of pH 7.5 and pH 8.0, respectively. Sodium alginate solution (pH 7.5) or (pH 8.0) of 0.00 mL, 1.00 mL, 1.25 mL, 1.67 mL, 2.50 mL, and 5.00 mL were put into 96-hole plate, respectively. DPB (pH 7.5) or (pH 8.0) of 10.00 mL, 9.00 mL, 8.75 mL, 8.33 mL, 7.50 mL, and 5.00 mL were added into the above sodium alginate solution in turn, respectively. The immobilized alginate lyase of 5 mg was put into each hole to dissolve into the sodium alginate solution (pH 8.0). The free alginate lyase of 1 mg was put into each hole to dissolve into the sodium alginate solution (pH 7.5). The mixtures were kept at 45°C for 10 min. Then the mixtures were determined by Microplate reader.
Operational stability
In this experiment, 100 mg sodium alginate was dissolved into 20 mL DPB of pH 8.0 in a reaction vessel. After that, the immobilized alginate lyase of 10 mg was dissolved into. The mixtures were kept at 45°C for 1.0 h. The immobilized alginate lyase was collected by magnet and washed with DPB for 5 times. The immobilized alginate lyase was resuspended in a fresh substrate solution. This procedure was repeated 10 times. The enzyme activity of each batch was determined. The initial enzyme activity of this experiment was set to 100%.
Data analysis
All experiments were carried out at random. All trials were performed in triplicate. Analysis of variance (ANOVA) of the data was carried out as described in a previous study [26].
Optimization of immobilization conditions
Immobilization conditions affected the EAR highly [27]. To enhance the EAR of alginate lyase, immobilization conditions including MCM dosage, adsorption time, glutaraldehyde concentration and immobilization time were optimized. MCM was used as a carrier for enzyme immobilization. To investigate the effect of the MCM dosage on the EAR of alginate lyase, MCM with six different dosages (0.5 g/L, 1.0 g/L, 1.5 g/L, 2.0 g/L, 2.5 g/L, and 3.0 g/L) at an adsorption time of 1.0 h without glutaraldehyde was applied. The effect of the MCM dosage on the EAR is shown in Fig. 1(a). The optimal dosage of MCM for immobilization was 1.5 g/L with the highest EAR of 35%. The EAR increased significantly (P < 0.05) with increasing MCM dosage from 0.5 g/L to 1.5 g/L and decreased significantly (P < 0.05) above 1.5 g/L. The results showed that low MCM dosage had low EAR. The possible reason was that only a few enzyme molecules were immobilized on the carrier with a low dosage of MCM. On the contrary, excessive dosage of MCM made the carrier not fully used, which also led to the low EAR [28].
Previous studies suggested that a proper time was necessary for better adsorption between the carriers and enzymes [29]. To investigate the effect of adsorption time on the EAR, different adsorption times (0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h) with 1.5 g/L MCM without glutaraldehyde were carried out. The immobilization efficiency reached peak value of 41% at 2.0 h (Fig. 1(b)). The EAR increased significantly (P < 0.05) with increasing adsorption time up to 2.0 h and decreased significantly (P < 0.05) above 2.0 h. The results showed that further prolonging incubation caused the mass loss of EAR. It might be that shaking for a long time would cause the shedding of adsorbed enzyme molecules during the preparation process [30]. In addition, each carrier MCM was absorbed by more than one enzyme molecule on the surface, which affected the contact between the immobilized alginate lyase and the substrate making its enzyme activity not fully expressed [30].
Glutaraldehyde was a cross-linking agent for enzyme immobilization. It could combine with the amino groups on the surface of enzyme molecules to form Schiff bases [31]. The firmness of binding of the enzyme molecule to the carrier depended on glutaraldehyde concentration [32]. However, high concentration of glutaraldehyde could lead to inactivation of the enzyme / protein, which reduced the immobilization greatly [33]. Different glutaraldehyde concentrations (0.0%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%) at an adsorption time of 2.0 h and immobilization time of 2.0 h with 1.5 g/L MCM were carried out. The EAR increased before they decreased along with increasing glutaraldehyde concentration (Fig. 1(c)). The optimal glutaraldehyde concentration was 0.2% with the maximum EAR of 60%. Compared with 0.0% glutaraldehyde (the EAR of 41%), the EAR of 0.2% glutaraldehyde was increased by 46%. It indicated that glutaraldehyde greatly prompted the immobilization.
Immobilization time greatly affected the EAR. As shown in Fig. 1(d), the EAR of alginate lyase increased with prolonging immobilization time from 0.0 h to 2.0 h at an adsorption time of 2.0 h with 1.5 g/L MCM and 0.2% glutaraldehyde. It may be the increase in enzyme loading. The EAR reached peak value of 63% at 2.0 h. This suggested that the monolayer adsorption of alginate lyase reached its saturation point. When the immobilization time was too long, the over-loading of alginate lyase could lead to a multi-layered stacking of enzyme, which would block the substrate molecules from accessing to the internal alginate lyase and thus decreased the enzyme activity [34].
Under the optimized conditions of immobilization (1.5 g/L MCM, 2.0 h adsorption time, 0.2% glutaraldehyde and 2.0 h immobilization time), the AC of MCM was determined. The AC of MCM was 178 mg/g. Alginate lyase showed excellent IE of 72%.
Structural characterization of the immobilized alginate lyase
The photograph and SEM photo of Fig. 1 showed MCM particles. As shown in Fig. 1, the entrapment method allowed obtaining a spheroidal structure of MCM with an average size of 30 ± 20 nm, and there were surface wrinkles on MCM, which were in favor of enzyme immobilization. MCM exhibited an obvious agglomeration phenomenon, which could be mainly caused by the coercive force of magnetic particles or magnetic dipole interactions between particles [18].
FTIR spectroscopy was employed to investigate the structural changes of the immobilized alginate lyase. Figure 3(a) presents the FTIR spectra of the chitosan. The prominent peak at 3445 cm− 1 was associated with the vibrational modes of the -NH2 and -OH functional groups, consistent with typical absorption bands around 3300 cm− 1. The absorption peaks at 2910 cm− 1 and 2850 cm− 1 were assigned to -CH3 and -CH2, respectively. The peaks at 1651 cm− 1 and 1599 cm− 1 were assigned to amide I (C = O stretching vibration) and amide II (N-H in-plane bending vibration), respectively [23]. The peak at 1076 cm− 1 was attributed to the stretching vibration of C-O-C bond in chitosan. The absorption peak at 575 cm− 1 represented the Fe-O bending vibration, which was the characteristic absorption peak of magnetic Fe3O4 (Figs. 3 (b) and (c)). This suggested that the MCM and immobilized alginate lyase remained the structure of Fe3O4 [35]. In Figs. 3 (b) and (c), the absorption peak at 3445 cm− 1 was significantly reduced due to the reaction between -NH2 groups on chitosan and the aldehyde groups of the glutaraldehyde cross-linking agent. The addition of glutaraldehyde led to an increase in the methyl and methylene groups on chitosan. The glutaraldehyde also significantly enhanced the absorption intensity of the range from 2910 cm− 1 to 2850 cm− 1, indicating that magnetic Fe3O4 was successfully cross-linked to chitosan [36]. In Fig. 3(c), the absorption peak intensity at 3445 cm− 1 decreased due to the cross-linking reaction between glutaraldehyde and -NH2 group of alginate lyase. Meanwhile, the absorption peaks at 1655 cm− 1 and 1076 cm− 1 were significantly enhanced due to the amide I and the stretching vibration of C-O-C bond in alginate lyase. The results proved that the alginate lyase was immobilized successfully.
The XRD could reveal the changes of crystal structure of magnetic nanoparticles. Figure 4 showed the XRD of Fe3O4 nanoparticles, MCM and immobilized alginate lyase. The diffraction patterns of Fe3O4 nanoparticles, MCM and immobilized alginate lyase were basically the same. Their diffraction peaks of Fe3O4 crystals all appeared at 2θ of 30.373° (220), 35.742° (311), 43.325° (400), 53.983° (422), 57.338° (511), and 62.911° (440). It indicated that the crystal structure of magnetic Fe3O4 nanoparticles was not destroyed during the preparation of MCM and immobilization [37].
The magnetic properties of magnetic nanoparticles were characterized by the VSM (Fig. 5). Hysteresis curves of both MCM and immobilized alginate lyase are S-shaped without remanence and coercivity. It indicated that both MCM and immobilized alginate lyase had good superparamagnetism [38]. The saturation magnetization strength of MCM and immobilized alginate lyase was 11.34 emu/g and 7.78 emu/g, respectively. The saturation magnetization strength of the immobilized alginate lyase was lower than that of MCM. This was because that MCM was modified by enzymes [39]. The immobilized alginate lyase still showed good superparamagnetism. It was beneficial to recover the immobilized alginate lyase from enzymatic hydrolysates.
Characterization of the enzymatic properties of the immobilized alginate lyase
The optimal pH and temperature
The optimal pH of alginate lyase immobilized on magnetic chitosan microspheres was investigated (Fig. 6). The free enzyme was used as control. The optimal pH values of the free and immobilized enzymes were 7.5 and 8.0, respectively. The optimal pH of the immobilized alginate lyase was higher than that of the free one. It indicated that immobilization contributed significantly to the improvement of pH tolerance. The shift of the enzymatic optimal pH after immobilization was because that the amino groups in the immobilized alginate lyase would adsorb H+ in the solution resulting in the increasing of acidity of the internal microenvironment [40]. In order to offset the impact of its internal microenvironment, the optimal pH of the immobilized enzyme would shift towards alkaline.
The optimal temperature of alginate lyase immobilized on magnetic chitosan microspheres was studied (Fig. 7). The free enzyme was used as control. The optimal temperature of the enzyme did not change after immobilization. The immobilized alginate lyase had no difference in the optimal temperature (45°C) with the free alginate lyase (Fig. 7). However, the immobilized alginate lyase showed higher thermal resistance at high temperature. The relative activity of the immobilized alginate lyase was nearly 40% while the free alginate lyase was less than 10% at 90°C.
The thermal stability
The thermal stability of the free and immobilized enzymes was investigated. The free alginate lyase lost almost all its activity at 70°C and 75°C while the immobilized alginate lyase would still retain almost 80% of its activity under 70°C for 120 min and still retained 40% of its activity at 75°C for 60 min (Fig. 8). The results showed that the immobilized alginate lyase exhibited stronger tolerance to high temperatures than that of the free enzyme. It was because of the covalent binding of enzymes on support [40]. The immobilized alginate lyase showed a better thermal stability. The better thermal stability of the immobilized alginate lyase would be a major advantage for its industrial application. The immobilized enzymes can catalyze a reaction under higher temperature conditions.
The Km of the immobilized and free alginate lyase
The Km value of an enzyme can approximately reflect the affinity between substrate and enzyme molecule. The larger the Km value was, the lower affinity between substrate and enzyme molecule was. Km was calculated as 0.05 moL/L and 0.09 moL/L and Vm as 0.50 mmoL/(L·min) and 0.34 mmoL/(L·min) for the free and immobilized form, respectively (Table 1 & Fig. 9). The Km value indicated that the free alginate lyase was more sensitive to the substrate than that of the immobilized alginate lyase. This is because the spatial structure and active site of the enzyme molecules were changed after immobilization [41, 42]. The immobilized alginate lyase formed a certain steric hindrance which hindered the contact between the enzyme molecules and the substrate molecules.
| Enzyme | Km (moL/L) | Vm [mmoL/(L·min)] |
|---|---|---|
| Free alginate lyase | 0.05 | 0.50 |
| Immobilized alginate lyase | 0.09 | 0.34 |
Operational stability
To evaluate the immobilization process, the operational stability was investigated. Retention of more than 70% of alginate lyase activity after 10 reuses was obtained (Table 2). It indicated that the immobilized enzymes performed good operational stability. It could be regarded as the hydrophilic microenvironment that surrounded the enzymes. The enzyme activity decreased slowly during the reuse process. The loss of enzyme activity of the immobilized enzymes might result from leakage of the immobilized alginate lyase and protein denaturation during the catalytic process [43].
| Bach reaction | Relative activity (%) |
|---|---|
| 1 | 98 ± 0 |
| 2 | 97 ± 0 |
| 3 | 94 ± 1 |
| 4 | 92 ± 1 |
| 5 | 89 ± 0 |
| 6 | 88 ± 1 |
| 7 | 84 ± 1 |
| 8 | 81 ± 0 |
| 9 | 75 ± 1 |
| 10 | 72 ± 2 |
A novel method to prepare magnetite chitosan microspheres was developed. Alginate lyase was successfully immobilized on magnetic chitosan microspheres. The enzymatic properties, thermal stability and reusability of the immobilized alginate lyase were enhanced. The results showed that the activity and stability of the immobilized enzymes were enhanced in comparison with the free enzymes. The results suggested that the immobilization of alginate lyase on magnetic chitosan microspheres was a promising strategy. The immobilized alginate lyase had a good potential in industrial application.
Acknowledgments This work was supported in part by grants from the National Natural Science Foundation of China (Grant Nos. 21767017 and 22066016), and Science and Technology Project of Jiangxi Provincial Department of Science and Technology (Grant Nos. 20212BDH81004 and 20192BBH80023). And our sincere thanks to Dr. Paul Chen from University of Minnesota, USA for his language assistance.
Author Contributions Jinmeng Li and Feng Yan: Methodology, Conceptualization, Formal analysis, Investigation, Data analysis, Writing - Original Draft, Visualization. Mengyan Zhang and Binbin Huang: Investigation, Conceptualization, Data analysis, Writing - Review and Editing, Visualization. Xiaodan Wu, Yuhuan Liu, Roger Ruan and Hongli Zheng: Conceptualization, Resources, Writing - review and editing, Visualization, Supervision, Project administration, Funding acquisition.
Funding This work was supported in part by grants from the National Natural Science Foundation of China (Grant Nos. 21767017 and 22066016), and Science and Technology Project of Jiangxi Provincial Department of Science and Technology (Grant Nos. 20212BDH81004 and 20192BBH80023).
Data Availability The data in this research article are fully presented in the manuscript.
Ethical Approval This article does not contain any studies with human participants by any of the authors.
Consent to Participate Not applicable.
Consent for Publication Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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