CHENG Yimeng, LI Zhaoyue, SUN Huihui, *, ZHAO Ling, LIU Zhen, CAO Rong, 3), LIU Qi, and MAO Xiangzhao, 3), *

Immobilization of Chitosanase on Magnetic Nanoparticles:Preparation, Characterization and Properties

CHENG Yimeng1), 2), LI Zhaoyue2), SUN Huihui2), *, ZHAO Ling2), LIU Zhen1), CAO Rong2), 3), LIU Qi2), and MAO Xiangzhao1), 3), *

1) College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China 2) Department of Food Engineering and Nutrition,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China 3) Laboratory for Marine Drugs and Bioproducts of Qingdao, National Laboratory for Marine Science and Technology, Qingdao 266237, China

Chitosanase could cleave β-1,4-linkage of chitosan to produce chitooligosaccharides (COS) with diverse biological activities. However, there are many limitations on the use of free chitosanase in industrial production. Enzyme immobilization is generally considered a valuable strategy in industrial-scale applications. In this study, the chitosanase Csn-BAC fromsp. MD-5 was immobilized on Fe3O4-SiO2magnetic nanoparticles (MNPs) to enhance its properties, which could be recovered easily from reaction media using magnetic separation. The activities of Csn-BAC immobilized with MNPs (MNPs@Csn-BAC) were determined with temperature and pH, and the thermal- and pH-stabilities, respectively. The reusability of the MNPs@Csn-BAC was determined in repeated reaction cycles. Immobilization enhanced the thermal and pH stability of Csn-BAC compared with the free enzyme. After eight reaction cycles using MNPs@Csn-BAC, the residual enzyme activity was 72.15%. Finally, the amount of COS released by MNPs@Csn-BAC was 1.86 times higher than that of the free Csn-BAC in the catalytic performance experiment. The immobilized Csn-BAC exhibits broad application prospects in the production of COS.

chitosanase; immobilization; magnetic nanoparticles; chitooligosaccharides

1 Introduction

Chitooligosaccharides (COS), the only natural basic amino oligosaccharide, are hydrolyzed products of partially deacetylated chitin (chitosan). They are composed of D-glucosamine and-acetyl-D-glucosamine linked by β-1,4-glycosidic bonds (Muanprasat and Chatsudthipong, 2017). COS have various physiological activities, including antitumor, anti-inflammatory, antibacterial, and hypo- lipidemic properties (Sun, 2020). Chitosanase (EC3.2.1.132) catalyzes the hydrolysis of β-1,4-glycosidic bonds of chitosan to produce COS (Thadathil and Velappan, 2014). Compared with chemical and physical methods, which usually need harsh conditions such as acidic environment, high temperature and high pressure, use of chitosanase is environmentally friendly. Thus, the production of COS using chitosanase has received extensive attention.

There are many limitations on the use of free enzymes in industrial production, such as instability, poor resistance to acid/alkali, and difficulty in separation and purification of products. Enzyme immobilization is generally considered a valuable strategy in industrial-scale applications, and is an effective method to ameliorate the above problems (Sheldon and van Pelt, 2013). Enzymes can be attached to carriers through physical adsorption, ionic and covalent bond interactions. The adsorption is based on the physical or ion interaction between the surface of immobilized carriers and enzymes (Bayat., 2015), while the covalent binding represents that enzymes are covalently linked to the immobilized material functional group (Bouabidi., 2018). Although immobilized enzymes show significant advantages, their application in biocatalytic processes has been limited because of the high technical requirements of operation and cost limitations (Hart- mann and Kostrov, 2013). Having high surface-to-volume ratio and admirable mass-transfer performance, nanomaterials are considered excellent enzyme immobilization materials. Magnetic nanoparticles are effective for enzyme immobilization because of their small core size, low toxicity, and high paramagnetism. The immobilized enzymes can be recovered easily by employing an external magnetic field, and then reused (Aggarwal, 2021). Different kinds of magnetic supports have already been exploited for the enzyme immobilization. (Xie and Zang, 2017) prepared hydroxyapatite-encapsulated γ-Fe2O3na- noparticles and used it to immobilize lipasecovalent linkages, which exhibited a strong magnetic responsiveness and high activities. They also immobilized lipase on the magnetic imidazole-based ionic liquids-functionalized composites, which could be used to produce trans-free plastic fats efficiently and environmentally (Xie and Zang, 2018). Besides lipase (Xia., 2021), magnetic nano- particles have been reported as an immobilized material for other enzymes, such as fibrinolytic protease (Khankari, 2021) and co-immobilization of α-neo-agarose hydrolase and β-agarase (Wang, 2018). All these constructs showed excellent performance, which demonstrated the potential of enzyme immobilization on magnetic nanoparticles. However, similar applications in chitosanase are relatively rare. In our previous study, we immobilized chitosanase onto magnetic nanoparticles and improved its thermal and pH stabilities (Wang, 2018). However, the amount of enzyme immobilized was low (25.1mg chitosanase per g magnetic nanoparticles), and the residual activity of immobilized chitosanase decreased to 67.5% after five cycles of use. These issues restricted the industrial applications of immobilized chitosanase.

In this study, we simplified the process of immobilization and omitted the step of nanoparticles surface modification. The Fe3O4-SiO2magnetic nanoparticles (MNPs) were prepared and characterized. Chitosanase Csn-BAC fromsp. MD-5 was immobilized on the MNPs and evaluated with a view to improved use of chitosanase in industrial production. Compared with the free enzyme, the stability and catalytic efficiency of the immobilized Csn-BAC were improved.

2 Materials and Methods

2.1 Materials

Chitosan (viscosity, 100–200mPa.s, ≥95% degree of deacetylation [DDA]) was purchased from Shanghai Macklin BioChem Tech Co., Ltd. (Shanghai, China). Try- ptone and Yeast Extract were obtained from Oxoid, Ltd. (Basingstoke, UK). HCl, NaCl, imidazole, 3,5-dini-trosa- licylic acid (DNS), FeCl3·6H2O, NH3·H2O, FeCl2·4H2O, ethanol, and tetraethyl orthosilicate (TEOS) were obtain- ed from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

2.2 Synthesis of Fe3O4-SiO2 MNPs

Fe3O4nanoparticles were synthesized by a coprecipitation method (Du, 2006). In brief, 0.8g FeCl2·4H2O and 2.7g FeCl3·6H2O were placed in 100mL ultrapure water, and stirred using a magnetic agitator in an N2atmosphere for 5min until fully dissolved. After the addition of 4mL 25% NH3·H2O, the solution was allowed to react for 30min in N2. After the reaction, nanometer-scale ferric oxide was separated using an external magnetic field, washed with ethanoland ultrapure water three times in turn, then dried under vacuum at room temperature.

Fe3O4-SiO2nanoparticles were synthesized by the classical Stöber method (Stöber, 1968). Fe3O4(0.5g) was placed in 150mL anhydrous ethanol at room temperature and treated ultrasonically for 30min in an N2atmosphere to produce a uniform suspension. Then, 10mL 25% NH3·H2O and 6mL TEOS (28%) were added to the suspension and the mixture were stirred continuously for 5h at room temperature. Nanometer Fe3O4coated by si- lica were separated from the reaction system using an external magnetic field, washed with ethanol and ultrapure water three times in turn, and dried under vacuum at room temperature.

2.3 Purification of Csn-BAC

The chitosanase Csn-BAC fromsp. MD-5(Gen- Bank: CP021911.1) was recombinantly expressed with vector pET-28a inBL21 (DE3), as in our previous study (Yang, 2020). The recombinant cells were cultivated, harvested, and purified accor- ding to the method of Yang(2020).

2.4 Immobilization of Csn-BAC

MNPs (5.0mg) were uniformly distributed in 500μL solution containing Csn-BAC and PBS (50mmolL−1, pH 7.0). After the reaction was placed in a constant temperature shaker for a period of time, the Csn-BAC immobilized with MNPs (MNPs@Csn-BAC) were washed with PBS (50mmolL−1, pH7.0) three times. The amount of MNPs@Csn-BAC was determined by the Bradford me- thod.

The effects of time, enzyme dose, temperature, and pH on the activity of the MNPs@Csn-BAC were evaluated using colloidal chitosan (1%, w/v; 95% DDA) as the substrate. The time of immobilization ranged from 0.25 to 4 h, and the enzyme activity was determined in PBS (50mmolL−1, pH 7.0). The influence of the dose of Csn-BAC on the activity of MNPs@Csn-BAC was investigated at pH 7.0 using the optimum immobilization time. The effect of temperature (10℃ to 40℃) on the activity of the MNPs@Csn-BAC was determined in PBS (50mmolL−1, pH 7.0) using the optimal immobilization time and enzyme dose. The effect of immobilization pH on the enzyme activity was determined using the optimal immobilization time, temperature and enzyme dose.

2.5 Analysis of MNPs and MNPs@Csn-BAC

The Fe3O4nanoparticles and MNPs were scanned by transmission electron microscopy (TEM, MIC-JEM1200 EX). The FT-IR spectras were recorded by a Fourier tran- sform infrared spectroscopy (Thermo Scientific Nicolet iS10, Waltham, MA USA). A vibrating sample magnetometer (VSM, PPMS-9, Quantum Design, San Diego, CA, USA) was used to analyze the paramagnetism of Fe3O4, MNPs, and MNPs@Csn-BAC in the test range of −20000–20000Oe.

2.6 Assay of Csn-BAC Activity

Enzyme activity was determined by measuring the amount of reducing sugar product using the DNS method with minor modification (Yang, 2020). Standard assays were performed in a reaction mixture containing phosphate buffer (50mmolL−1, pH7), colloidal chitosan (1%, w/v; 95% DDA), and an appropriate amount of free Csn-BAC or MNPs@Csn-BAC. One unit of enzyme activity is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute.

2.7 Characterization of MNPs@Csn-BAC

Determination of optimum temperature for activity and thermal stability: MNPs@Csn-BAC were placed in 500 μL buffer and mixed with 500μL colloidal chitosan (1% w/v, 95% DDA), and the enzyme activity was determined at 30–70℃. MNPs@Csn-BAC were incubated at 30– 70℃ for 1h, then the thermal stability of MNPs@Csn- BAC was determined by measuring the residual enzyme activity under standard conditions.

Determination of optimal pH and pH stability: MNPs@ Csn-BAC were reacted with colloidal chitosan (1%, w/v; 95% DDA) in different pH buffers (citric acid, pH 3.0– 6.0; phosphate, pH 6.0–8.0 Tris-HCl, pH 8.0–9.0), and the optimal pH for activity was determined. After incubating the enzyme in the abovementioned buffers at 30℃ for 1h, the residual activity was measured to determine the pH stability of MNPs@Csn-BAC.

Reusability: MNPs@Csn-BAC were reused eight times, and the enzyme activities in each reaction were determined. After the completion of each reaction, the MNPs @Csn-BAC were separated from the reaction system under the action of an external magnetic field, and washed three times with PBS before re-use.

Catalytic efficiency: The free Csn-BAC and MNPs@ Csn-BAC with the same initial activity were incubated with excess chitosan in PBS (50mmolL−1, pH 7.0), respectively, and the catalytic properties of them were investigated by measuring the hydrolysis of chitosan.

3 Results and Discussion

3.1 Synthesis and Characterization of Fe3O4, MNPs and MNPs@Csn-BAC

The schematic diagram of the immobilization was illustrated in Fig.1A. The size and shape of Fe3O4and Fe3O4-SiO2were observed by TEM and showed in Figs. 1B and 1C. The average diameter of the spherical Fe3O4nanoparticles was 10–20nm, and the distribution was relatively uniform. Nanoparticles with larger diameters were observed in Fig.1C, which was speculated to be the silicon dioxide layer on the surface of the Fe3O4nanoparticles (compared with Fig.1B). The MNPs show a core-shell structure. The diameter of MNPs produced in this study was approximately 50nm. The silicon dioxide layer, produced by the hydrolysis of TEOS, not only protects Fe3O4from oxidation, but also makes the surface of the magnetic beads rich in hydroxyl groups and thus increasing their dispersion in solution.

Fig.1 Synthesis and characterization of Fe3O4, Fe3O4-SiO2 magnetic nanoparticles (MNPs) and MNPs@Csn-BAC. (A) Schematic diagram of the immobilization of chitosanase onto the MNPs. Transmission electron microscopy images of Fe3O4 (B) and Fe3O4-SiO2 (C) nanoparticles. (D) FT-IR spectrum of MNPs, MNPs@Csn-BAC and purified Csn-BAC. (E) Hysteresis loops of Fe3O4, MNPs, and MNPs@Csn-BAC. Photographs of (F) the MNPs dispersed in aqueous solution, and (G) the MNPs attracted by an external magnetic field for 15s.

FT-IR spectras of MNPs, MNPs@Csn-BAC and purified Csn-BAC were presented in Fig.1D. The absorption peaks appeared at 573 and 966cm−1were attributed to the stretching vibration of the Fe-O and Si-O bonds, respectively, which proved the successful preparation of MNPs. In addition, the absorption peak of the peptide bond at 1623 cm−1 in the spectrum of MNPs@Csn-BAC proved that thechitosanase was successfully immobilized on the MNPs (Pan, 2009; Jang and Lim, 2010; Wang, 2018).

The paramagnetism of the Fe3O4, MNPs, and MNPs@ Csn-BAC were analyzed using a VSM. Fig.1E showed the hysteresis loops of them. The maximum paramagnetism of Fe3O4was 68.3emug−1, and the maximum paramagnetism of the MNPs was 40.0emug−1. When Csn- BAC was immobilized on MNPs, the magnetization was well preserved, and the maximum paramagnetism was 36.5emug−1. The Fig.1F showed that in the absence of a magnetic field, the MNPs can be uniformly distributed in water. Moreover, the MNPs can be easily separated from the reaction system under the action of an external magnetic field (Fig.1G).

3.2 Optimization of Enzyme Immobilization Conditions

The crude extract of Csn-BAC was purified by Ni-NTA resin. SDS-PAGE results showed that the purified Csn- BAC had a single band and was suitable for immobilization (Supplementary Fig.1).

Various factors that influence enzyme immobilization, including time, enzyme dose, temperature, and pH, were investigated. Fig.2A shows the effect of immobilization time on the quantity and activity of MNPs@Csn-BAC. The enzyme activity of the MNPs@Csn-BAC peaked at an immobilization time of 1h. In contrast, the quantity of MNPs@Csn-BAC reached its maximum at 2h, and then remained stable. This may be attributed to the unstability of enzyme under the immobilization conditions, which caused the activities decreased with the extension of time. On the basis of these results, the optimum immobilization time was considered to be 1h.

Fig.2 Optimization of enzyme immobilization. Effect of reaction time (A), enzyme dose (B), temperature (C), and pH (D) on enzyme loading to the MNPs and relative catalytic activity of the enzyme-loaded MNPs.

The effect of the dose of Csn-BAC (10–200mgg−1MNPs) on the immobilization process is shown in Fig.2B. Immobilized was maximized when the amount of enzyme added was 100mgg−1MNPs. Similarly, the activity of MNPs@Csn-BAC increased with an increasing dose of the enzyme from 10 to 100mgg−1MNPs, then plateaued at higher doses. Thus, we chose 100mgg−1MNPs as the optimum dose of the enzyme.

Fig.2C shows the influence of immobilization tempe- rature on the immobilization process. There was a slight change in the amount of enzyme immobilized in the range 10–30℃, while a remarkable increase was observed at 37℃. However, the same trend was not observed for enzyme activity. Indeed, the highest activity was observed when the immobilization temperature was 20℃. Therefore, 20℃ was chosen as the best immobilization temperature.

The effect of pH on the immobilization process is shown in Fig.2D. The enzyme activity was highest at pH 6.0 in phosphate buffer. Different buffers and pHs had a significant effect on the enzyme activity. The activity was generally low in acidic conditions, and stable around neutral pH. The maximum amount of enzyme immobilization also occurred in PBS at pH 6.0.

On the basis of the above results, the optimum immobilization conditions were as follows: Immobilization time 1h, enzyme amount 100mgg−1MNPs, immobilization temperature 20℃, phosphate buffer, pH 6.0. In these conditions, the amount of Csn-BAC immobilized was 40.02mgg−1MNPs. Compared to our previous study by Wang. (2018) we simplified the process of immobilization, cut down the immobilization time (from 2h to 1h), and improved the amount of enzyme loading by 159% (from 29.74mgg−1to 40.02mgg−1), which exhibited potential for practical applications.

3.3 Effects of Temperature and pH on Activities

The optimum reaction temperatures of free and immobilized Csn-BAC were determined. As shown in Fig.3A, the MNPs@Csn-BAC showed its highest activity at 50℃ and retained >75% of the maximal activity between 30 and 70℃. In contrast, the optimum temperature for activity of the free enzyme was 40℃. The activity decreased markedly at >50℃, and the enzyme was inactivated at 70℃. These results indicated that the carrier can protect the enzyme at high temperature (Li, 2020).

Fig.3 Characterization of free Csn-BAC and MNPs@Csn-BAC. Effects of temperature (A) and pH (B) on the activity of free and MNP@Csn-BAC. Thermal (C) and pH (D) stability of Csn-BAC and MNPs@Csn-BAC.

The effect of pH on enzyme activity was determined in the pH range 3.0–9.0 (Fig.3B). As with free Csn-BAC, the MNPs@Csn-BAC had no activity when the pH was 3.0. The MNPs@Csn-BAC enzyme activity increased gradually with increasing pH, and was maximal in Tris-HCl buffer at pH 8.0, higher than the pH for the greatest activity of the free enzyme (7.0). The free enzyme was inactive at pH 9.0, but the MNPs@Csn-BAC retained 46.83% of its maximal activity at this pH, indicating that the active pH range of the enzyme was improved by immobilization. This may be because of the binding of MNPs with Csn-BAC, which influences the ionization of acidic and basic amino acids around the active site of the enzyme (Cao, 2014).

3.4 Stability Analysis

Thermal stability is important for industrial application of chitosanase. We determined the residual enzyme activity after incubating at 30–70℃ for 1h. As shown in Fig.3C, the MNPs@Csn-BAC retained 75% activity after incubation at 30–60℃. When the incubation temperature was raised to 70℃, the residual activity was <35%. How- ever, the residual enzyme activity of free Csn-BAC was only approximately 45% at 60℃, and the free enzyme was completely inactivated by incubation at 70℃. Immo- bilization can be seen to improve the thermal stability of the enzyme. A possible reason is that immobilization tends to help maintain enzyme conformation (Cao, 2021).

As shown in Fig.3D, similar to free Csn-BAC, incubation at low pH significantly inhibited the activity of the MNPs@Csn-BAC, but at higher pH values, the MNPs@ Csn-BAC displayed better stability than free Csn-BAC. After incubation at pHs 3.0 and 9.0 for 1h, the MNPs@ Csn-BAC retained 10.63% and 72.97% of their initial activity, respectively, while the free Csn-BAC had no activity at either pH. Within a certain range of pH, the carrier may protect the enzyme against the external environment (Fang, 2016). MNPs@Csn-BAC shows acceptable pH stability over a wide range.

3.5 Reusability of MNP@Csn-BAC

Reusability is an important index of immobilized enzymes in industrial production. MNPs can be rapidly se- parated from the reaction system by the action of an external magnetic field, which is one of the main advantages of MNPs (Nguyen, 2019). Fig.4A showed the results of reusability of the MNPs@Csn-BAC. After eight reaction cycles, the MNPs@Csn-BAC retained 72.15% of the initial enzyme activity, which proved the practicality of repeated catalysis. This result was obviously improved compared to our previous study, in which the residual activity of immobilized chitosanase was 67.5% of the initial activity after being recycled 5 times (Wang., 2018; Cao, 2021). The slight decrease of enzyme activity may be because some of the enzyme dissociates from the surface of the MNPs over the process of many reactions (Barbosa, 2013), and/or, in the process of reaction, some of the enzyme molecules were inactivated (Fang, 2016).

Fig.4 Catalytic efficiency of MNPs@Csn-BAC. (A) Reusability of MNPs@Csn-BAC. (B) Time courses of the hydrolysis of chitosan using free and MNPs@Csn-BAC.

3.6 Catalytic Efficiency of Free and Immobilized Chitosanases

Fig.4B shows the time process of the hydrolysis of chi- tosan by free Csn-BAC and MNPs@Csn-BAC. In order to compare the catalytic performance, the initial activities of them were the same. Compared with the free Csn-BAC, the catalytic efficiency of the MNPs@Csn-BAC was higher, which may be related to the improvement of the stability of Csn-BAC by MNPs. After 240min reaction, the amount of COS released by the free Csn-BAC was 314μmol, and that of the MNPs@Csn-BAC was 584 μmol, which was 1.86 times higher than that of the free Csn-BAC. This result was comparable to our previous study by Wang., and the catalytic efficiency was im- proved 1.4 times by immobilization (56.8μmol and 79.4μmol, respectively) (Wang., 2018).

4 Conclusions

Csn-BAC was immobilized on Fe3O4nanoparticles coated with SiO2and without surface modification. Furthermore, the MNPs@Csn-BAC had improved thermal and pH stabilities and high catalytic performance. The immobilized chitosanases retained about 72% of their initial enzyme activity after eight cycles of use. These results demonstrate that MNPs@Csn-BAC has advantages over the free enzyme and is suitable for industrial application to produce COS.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (No. 2019 YFD0901902), the National Natural Science Foundation of China (No. 31801574), the Central Public-interest Sci- entific Institution Basal Research Fund, YSFRI, CAFS (No. 20603022021020), and Qingdao Science and Technology Demonstration and Guidance Project for Benefiting the People (No. 20-3-4-28-nsh).

Supplementary Material

Fig.1 SDS-PAGE analysis of purified Csn-BAC. Lane M, protein markers; Lane 1, crude extract of Csn-BAC; Lane 2, purified Csn-BAC.

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