Preparation method of microalgae carrier and application of microalgae carrier in immobilized enzyme

Abstract

The invention discloses a preparation method of a microalgae carrier and application of the microalgae carrier in immobilized enzyme. According to the method, chlorella, nannochloropsis and schizochytrium are taken as objects, and microalgae residues are modified to be prepared into a microalgae carrier by utilizing pretreatment of a deep eutectic solvent in which the ratio of n (ChCl) to n (glycerol) is 1: 2 in combination with an ultrasonic-assisted technology, and the microalgae carrier is used for immobilizing lipase. According to the method, the utilization rate of microalgae can be increased, resource waste and environmental pollution are reduced, a systematic technical path is provided for full-component high-value utilization of microalgae grease and residues, and application and expansion of green enzyme engineering in the fields of food processing and biological manufacturing are expected to be promoted.

Description

Technical Field

[0001] This invention belongs to the field of novel carrier development and immobilized enzyme technology, specifically relating to a method for preparing a microalgae carrier and its application in immobilized enzymes. Background Technology

[0002] Microalgae are widely considered an ideal biological resource for sustainable oil production due to their rapid growth rate, high photosynthetic efficiency, ability to be cultivated on a large scale in non-arable land environments, and high lipid accumulation. Their oils are rich in ω-3 polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have significant nutritional and pharmacological effects in cardiovascular health regulation, nervous system development, and inflammatory response inhibition, demonstrating enormous market potential in the functional food, pharmaceutical, and health product industries.

[0003] However, microalgal cell walls are generally composed of multi-layered structures containing cellulose, glycoproteins, silica, or other recalcitrant components. Their dense structure and chemical stability present challenges for traditional oil release methods such as solvent extraction and mechanical crushing during industrialization, including high energy consumption, high toxicity of chemical solvents, limited oil yield, and easy oxidation and degradation of PUFAs. Furthermore, the large amount of algal residue generated after oil extraction is often directly discarded or used for low-value purposes, not only wasting high-value components but also increasing waste treatment and environmental management costs. This "single-product-oriented" processing model is significantly at odds with the current green development concept of biorefining and high-value utilization of all components.

[0004] In recent years, deep eutectic solvents (DES) have become a research hotspot for microalgal cell disruption and functional component extraction due to their low toxicity, biodegradability, recyclability, and highly efficient ability to disrupt biomass cell walls. DES can significantly improve lipid release efficiency by disrupting hydrogen bond networks and hydrophobic interactions, thereby reducing chemical pollution while maintaining the bioactivity of PUFAs. Furthermore, combining DES pretreatment with ultrasound-assisted technology can further enhance mass transfer rates and cell disruption efficiency, achieving a gentler and more efficient lipid extraction process.

[0005] Notably, after DES and ultrasonic treatment, the cell wall and cytoplasmic structure of algal residue undergoes significant reconstruction, manifested as increased porosity, larger specific surface area, and exposure of active groups such as hydroxyl and amino groups. These structural and chemical changes provide unique advantages for its secondary utilization as a bio-based carrier. In recent years, enzyme immobilization technology has rapidly developed in food processing, bioenergy, and environmental remediation. The pore structure, surface chemical properties, and biocompatibility of the carrier have a decisive influence on the catalytic performance and stability of immobilized enzymes. Compared with traditional inorganic carriers (such as silica and zeolite), microalgal cellulose is a renewable, flexible, and hydrophilic source with good functionalization potential. It can achieve efficient enzyme-carrier interface interactions without introducing toxic components, thereby enhancing enzyme activity and stability.

[0006] Lipases, as important biocatalysts, possess strong substrate selectivity and the ability to catalyze esterification / hydrolysis / transesterification reactions under mild conditions, and have been widely used in food flavor regulation, oil modification, biodiesel synthesis, and the preparation of chiral compounds. However, free lipases often face problems such as thermal inactivation, sensitivity to pH fluctuations, and difficulty in recycling and reusing in industrial applications, significantly limiting their economic feasibility. Immobilization technology can not only improve the operational stability and reusability of enzymes, but also achieve an "activation" effect by altering the enzyme molecular conformation and microenvironment, that is, increasing the catalytic efficiency per unit enzyme protein. Summary of the Invention

[0007] Based on the defects and shortcomings of the existing technology, the present invention aims to provide a method for preparing microalgae carriers and their application in immobilized enzymes.

[0008] This invention utilizes microalgal residues to immobilize lipases and evaluates their feasibility as immobilized enzyme carriers. Results show that microalgal carriers exhibit excellent activity enhancement effects for lipase immobilization. Microalgal carriers treated with a eutectic solvent (Chlorella vulgaris, Micrococcus pseudomicrophylla, and Schizochytrium) at a molar ratio of n(ChCl):n(glycerol) = 1:2 showed the strongest immobilized GTL2 enzyme activity, reaching 288.14 U / mg, 323.76 U / mg, and 206.48 U / mg, respectively, which are 3.00, 3.37, and 2.15 times that of the free enzyme. Through single-factor analysis and response surface methodology, optimization was performed on ultrasonic power, ultrasonic time, and GTL2 addition amount. The highest immobilized enzyme activities obtained with the three microalgal carriers were 433.41 U / mg, 383.93 U / mg, and 230.46 U / mg, respectively, representing increases of 1.50, 1.19, and 1.12 times compared to the unoptimized conditions. The microalgal carrier exhibits a spherical shape, and its particle size and potential experiments indicate that it is a nanoscale material with higher dispersion stability. SEM and FT-IR results show that the enzyme is loaded onto the carrier surface. Catalytic performance analysis of the immobilized lipase demonstrates that the immobilization of GTL2 on the microalgal carrier does not affect its optimal reaction conditions, but it enhances the stability in organic reagents, provides some protection under high-temperature conditions, significantly improves the hydrolytic ability of specific substrates, and markedly enhances storage stability.

[0009] The first objective of this invention is to provide a method for preparing a microalgae carrier, comprising the following steps: S1. Microalgae and eutectic solvent are mixed at a mass ratio of 1:15, shaken at 70°C and 100 rpm for 2 h, and then ultrasonically treated at 37°C and 70% ultrasonic power for 1 h to obtain a microalgae-eutectic solvent mixture; wherein the eutectic solvent is a eutectic solvent containing choline chloride and glycerol in a molar ratio of 1:2. S2. Mix the microalgae-eutectic solvent mixture and anhydrous ethanol at a ratio of 1 g: 15 mL, place at 70℃ and 100 rpm for 2 h and shake, then centrifuge to separate the microalgae residue, wash with pure water until the pH value is neutral, freeze dry to obtain the microalgae carrier.

[0010] Preferably, the microalgae are Chlorella, Micrococcus pseudomicrococcus, or Schizochytrium; the freeze-drying is performed by pre-freezing at -20°C for more than 4 hours, and then freeze-drying at a vacuum of 0°C and -80°C for 24 hours.

[0011] A second objective of this invention is to provide a microalgae carrier prepared by the method described above.

[0012] A third objective of this invention is to provide a method for preparing microalgae-immobilized enzymes using the aforementioned microalgae carrier, comprising the following steps: S1. Mix the microalgae carrier with 0.05 mol / L, pH 8.0 Tris-HCl buffer at a ratio of 0.1 g: 10 mL, and place it under ultrasonic power of 30-50% for 1-10 min to obtain the dispersed microalgae carrier; S2. Add lipase to the dispersed microalgae carrier, stir at 100 r / min for 2-3 h in an ice bath, centrifuge at 5000 r / min for 5 min to collect and wash the precipitate to obtain the microalgae carrier immobilized enzyme; the amount of lipase added is 0.020%-0.080% of the mass of the microalgae carrier.

[0013] Preferably, the microalgae carrier is a Chlorella carrier, and the method includes the following steps: S1. Mix the Chlorella carrier with 0.05 mol / L, pH 8.0 Tris-HCl buffer at a ratio of 0.1 g: 10 mL, and place it under ultrasonic power of 40-44% for 3.7-5 min to obtain dispersed Chlorella carrier; S2. Lipase was added to the dispersed Chlorella carrier, and stirred at 100 r / min for 3 h in an ice bath. The mixture was then centrifuged at 5000 r / min for 5 min to collect and wash the precipitate, thus obtaining the Chlorella carrier immobilized enzyme as a microalgal carrier immobilized enzyme. The amount of lipase added was 0.040%-0.045% of the mass of the Chlorella carrier.

[0014] Preferably, the microalgae carrier is a *Micrococcus pseudocarrier*, and the method includes the following steps: S1. Mix the pseudomicrococcus carrier with 0.05 mol / L, pH 8.0 Tris-HCl buffer at a ratio of 0.1 g: 10 mL, and place it under ultrasonic power of 38-40% for 2.5-2.7 min to obtain dispersed pseudomicrococcus carrier; S2. Lipase was added to the dispersed *Chlorella pseudocarrier*, stirred for 2 h at 100 r / min in an ice bath, centrifuged at 5000 r / min for 5 min to collect and wash the precipitate, and the immobilized enzyme of *Chlorella pseudocarrier* was obtained as microalgae carrier immobilized enzyme; the amount of lipase added was 0.049%-0.060% of the mass of *Chlorella pseudocarrier*.

[0015] Preferably, the microalgae carrier is a fissile chytrid carrier, and the method includes the following steps: S1. Mix the Schizochytrium carrier with 0.05 mol / L, pH 8.0 Tris-HCl buffer at a ratio of 0.1 g: 10 mL, and place it under ultrasonic power of 40-44% for 2.5-2.7 min to obtain dispersed Schizochytrium carrier. S2. Lipase was added to the dispersed Schizochytrium carrier, stirred for 2 h at 100 r / min in an ice bath, centrifuged at 5000 r / min for 5 min to collect and wash the precipitate, and the immobilized enzyme of Schizochytrium carrier was obtained as microalgal carrier immobilized enzyme; the amount of lipase added was 0.040% of the mass of Schizochytrium carrier.

[0016] Preferably, the precipitate is washed three times with a 0.05 mol / L Tris-HCl buffer solution at pH 8.0, and the lipase is GTL2.

[0017] The fourth objective of this invention is to provide a microalgae carrier-immobilized enzyme prepared using the aforementioned method.

[0018] A fifth objective of this invention is to provide the application of the microalgae carrier-immobilized enzyme in the hydrolysis of esters.

[0019] The beneficial effects of this invention are: Microalgae are considered an important source of sustainable oil resources due to their rapid growth, high lipid accumulation, and lack of arable land occupation. However, the large amount of microalgae residue generated after oil extraction is often disposed of as waste, leading to resource waste and environmental pollution. This invention proposes a "dual-cycle high-value utilization" strategy: using Chlorella (… Chlorella ), Micrococcus pseudocarpa ( Nannochloropsis ) and fissicular chytrids ( Schizochytrium Using microalgae as a representative example, this invention achieves efficient oil extraction through DES pretreatment combined with ultrasound-assisted technology. The extracted algal residue is then chemically / physically modified to prepare a porous microalgal cellulose carrier for lipase immobilization. This invention not only provides a systematic technical path for the high-value utilization of all components of microalgal oils and residues but also has the potential to promote the application of green enzyme engineering in food processing and biomanufacturing. Furthermore, the green eutectic solvent used in this invention is characterized by low toxicity, recyclability, and efficient disruption of biomass structure, making it a hot topic for microalgal cell wall disruption. Attached Figure Description

[0020] Figure 1These are scanning electron microscope images of microalgal carriers and their immobilized enzymes; (a) is Chlorella raw material, (b) is Chlorella carrier after pretreatment, (c) is Chlorella immobilized enzyme, (d) is Microcholezosus raw material, (e) is Microcholezosus carrier after pretreatment, (f) is Microcholezosus immobilized enzyme, (g) is Schizochytridium raw material, (h) is Schizochytridium carrier after pretreatment, and (i) is Schizochytridium immobilized enzyme.

[0021] Figure 2 (a) is the FTIR spectrum of Chlorella raw material and its carrier; (b) is the FTIR spectrum of Micrococcus pseudomicrococcus raw material and its carrier; and (c) is the FTIR spectrum of Schizochytrium raw material and its carrier.

[0022] Figure 3 The immobilization time affects enzyme activity; (a), (b), and (c) show the effects of immobilization time on the enzyme activity immobilized on Chlorella, Micrococcus pseudocarrier, and Schizochytrium carriers, respectively.

[0023] Figure 4 The effects of lipase addition on enzyme activity are shown in (a), (b), and (c), which respectively show the effects of lipase addition on the enzyme activity immobilized on Chlorella carrier, Micrococcus pseudomicrococcus carrier, and Schizochytrium carrier.

[0024] Figure 5 The effect of ultrasound time on enzyme activity is shown in (a), (b), and (c), respectively. The effect of ultrasound time on the enzyme activity immobilized on Chlorella carrier, Micrococcus pseudomicrococcus carrier, and Schizochytrium carrier is shown in (a), (b), and (c), respectively.

[0025] Figure 6 The effects of ultrasonic power on enzyme activity are shown in (a), (b), and (c), which respectively show the effects of ultrasonic power on the enzyme activity immobilized on Chlorella carriers, Micrococcus pseudomicrococcus carriers, and Schizochytrium carriers.

[0026] Figure 7 These are response surface plots and contour plots of enzyme activity immobilized on Chlorella vectors.

[0027] Figure 8 These are response surface plots and contour plots of enzyme activity immobilized on the *Micrococcus pluvialis* carrier.

[0028] Figure 9 These are response surface plots and contour plots of enzyme activity immobilized on the Schizochytrium carrier.

[0029] Figure 10 The effects of different temperatures and pH on the activity of immobilized enzymes are shown in (a), (b), and (c), respectively. The effects of different temperatures and pH on the enzyme activity of Chlorella carriers, Micrococcus pseudocarriers, and Schizochytrium carriers are shown in (a), (b), and (c), respectively.

[0030] Figure 11The effects of different incubation temperatures on the stability of immobilized enzymes are shown in (a), (b), and (c), respectively. These are the effects of different incubation temperatures on the enzyme stability of Chlorella, Micrococcus pseudocarrier, and Schizochytrium carrier.

[0031] Figure 12 This is a determination of the substrate specificity of immobilized enzymes; (a), (b), (c), and (d) represent the hydrolytic capacity of Chlorella residue carrier, Micrococcus pseudomicrococcus residue carrier, Schizochytrium residue carrier, and free enzymes for different substrates, respectively.

[0032] Figure 13 This is a determination of the tolerance of immobilized enzymes to organic reagents; (a), (b), (c), and (d) are the tolerances of enzymes immobilized on Chlorella residue, Micrococcus pseudomicrococcus residue, Schizochytrium residue, and free enzymes to organic reagents, respectively.

[0033] Figure 14 This is a determination of the storage stability of immobilized enzymes; (a), (b), (c), and (d) are the tolerance of enzymes immobilized on Chlorella residue, Micrococcus pseudomicrococcus residue, Schizochytrium residue, and free enzymes to organic reagents, respectively.

[0034] Figure 15 This is a determination of the reusability of immobilized enzymes; (a), (b), and (c) are the reusability of enzymes immobilized on Chlorella residue, Microsporum pseudomicrosporum residue, and Schizochytrium residue, respectively. Detailed Implementation

[0035] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0036] Example 1 1. Test Methods 1.1 Determination of total fat content in microalgae The total lipid content in *Chlorella vulgaris*, *Schizochytrium*, and *Microchondria* was determined according to the Soxhlet extraction method in GB5009.6—2016. The specific method was as follows: 5 g of thoroughly mixed microalgae sample (m0) was weighed and placed in an evaporating dish. The sample was evaporated to dryness in a boiling water bath (100℃), then dried in an oven at 100℃±5℃ for 30 min. After drying, the sample was finely ground and transferred entirely into a filter paper cassette. The evaporating dish and the glass rod with the sample adhering to it were cleaned with absorbent cotton soaked in ether, and the absorbent cotton was placed inside the filter paper cassette. Place the filter paper tube into a Soxhlet extractor and connect it to a receiving flask (m2) that has been dried to constant weight. Add 660 mL of petroleum ether and heat in a 45°C water bath, continuously refluxing the petroleum ether for 10 h. Weigh and remove the receiving flask, recover the petroleum ether, and evaporate the solvent in the receiving flask to dryness in a 45°C water bath when 1-2 mL of solvent remains. Then dry at 100°C ± 5°C for 1 h, cool in a desiccator for 0.5 h, and weigh. Repeat the drying, cooling, and weighing steps until constant weight is achieved, until the difference between two weighings does not exceed 2 mg. Finally, record m1. Design three parallel experiments and take the average value. Calculate the total fat content in the microalgae according to formula (1).

[0037] Equation (1): Y (%) = (m1-m2) / m0 × 100%. In Equation (1): Y is the proportion of total fat mass in microalgae to the total mass of microalgae, m1 is the fat content (g) of the receiving bottle after constant weight; m2 is the mass of the receiving bottle, and m0 is the mass of the microalgae sample (g).

[0038] 1.2 Combining ultrasound and pretreatment with eutectic solvent (DES) to extract lipids from microalgae Weigh 1.0 g of dried microalgae sample (m0) and add it to a pre-weighed centrifuge tube (m2). Add different eutectic solvents at a material-to-liquid ratio of 1:15 (g / g) (Table 1). Pre-treat in a constant temperature water bath shaking incubator (70℃, 100 rpm) for 2 h, then sonicate in an ultrasonic cleaner (37℃, 70% ultrasonic power) for 1 h to obtain a microalgae-eutectic solvent mixture. Add petroleum ether reagent to the microalgae-eutectic solvent mixture at a material-to-liquid ratio of 1:15 (g / mL), and extract in a constant temperature water bath shaking incubator (70℃, 100 rpm) for 2 h. After extraction, centrifuge to separate the microalgae residue and organic phase. Set aside the microalgae residue. Recover the petroleum ether from the obtained organic phase using a rotary evaporator. When the organic phase is concentrated to 1 mL-2 mL, evaporate to dryness in a 100℃ water bath, then dry at 100℃ ± 5℃ for 1 h, and cool in a desiccator for 0.5 hours. After h, weigh the sample, repeat the drying, cooling, and weighing steps until constant weight is achieved, until the difference between two weighings does not exceed 2 mg, and finally record m1. Design three parallel experiments and take the average value. Calculate the microalgae lipid extraction rate according to formula (2).

[0039] Equation (2): X (%) = (m1-m2) / (m0×Y)×100%. In Equation (2): X is the lipid extraction rate of microalgae, Y is the proportion of total fat mass in microalgae to the total mass of microalgae, m1 is the content of fat in centrifuge tube after constant weight (g), m2 is the mass of centrifuge tube, and m0 is the mass of microalgae sample (g).

[0040] Table 1 Types of Eutectic Solvents Note: The ratio of hydrogen donor to hydrogen acceptor in the eutectic solvent is a molar ratio; the hydrogen acceptor is choline chloride (ChCl), and the hydrogen donor is glycerol, urea, malic acid, or citric acid.

[0041] 1.3 Preparation of microalgal carriers Weigh 1.0 g of dried microalgae sample and add it to a 50 mL centrifuge tube. Add different proportions of eutectic solvent at a material-to-liquid ratio of 1:15 (g / g) (Table 1). Pre-treat in a constant temperature water bath shaking incubator (70℃, 100 rpm) for 2 h. Then, sonicate in an ultrasonic cleaner (37℃, ultrasonic power 70%) for 1 h to obtain a microalgae-eutectic solvent mixture. Add anhydrous ethanol reagent to the microalgae-eutectic solvent mixture at a material-to-liquid ratio of 1:15 (g / mL). Extract in a constant temperature water bath shaking incubator for 2 h. After extraction, centrifuge to separate the microalgae residue. Wash with pure water until the pH value is neutral, and then freeze-dry (specifically, pre-freeze at -20℃ for more than 4 h, and freeze-dry at vacuum conditions of 0 and -80℃ for 24 h) to obtain the microalgae carrier.

[0042] It is worth noting that in the early experiments, we tried to prepare microalgae carriers using organic reagents such as petroleum ether and methanol, but the results were not good. The immobilization efficiency and enzyme activity of the immobilized enzymes were particularly low, which could not meet the needs of the later stages. Only ethanol prepared the carriers and the subsequent immobilization effect was good. Therefore, anhydrous ethanol was used for the preparation of microalgae carriers in the future.

[0043] 1.4 Immobilized lipase on microalgae carriers Weigh 0.1 g of the microalgal carrier sample and disperse it evenly in 1 mL of Tris-HCl buffer (0.05 mol / L, pH 8.0). Place the sample in an ultrasonic cell disruptor (ultrasonic power: 60%) and disrupt the cell walls for 1 min to ensure uniform dispersion. Add 100 μg of lipase (GTL2, Geobacillus (Bacillus) thermocatenulatus lipase 2, which has been published in Jun Zhang, Miao Tian, ​​Pengmei Lv et al., High Efficiency expression of the thermophiliclipase from Geobacillus thermocatenulatus in Escherichia coli and its application in the enzymatic hydrolysis of rapeseed oil. 3 Biotech (2020) 10:523. https: / / doi.org / 10.1007 / s13205-020-02517-6), after mixing and stirring at 100 r / min for 3 h under ice bath conditions, centrifuged at 5000 r / min for 5 min to separate, the precipitate was washed three times with Tris-HCl buffer (0.05 mol / L, pH 8.0), and the resulting precipitate was the immobilized enzyme. All supernatants were collected for later use, and the volume of supernatant V1 was recorded.

[0044] 1.5 Enzyme Activity Analysis Lipase hydrolyzes 4-nitrophenyl ester (ρNP-P) to produce ρNP (4-nitrophenyl), which is yellow under alkaline conditions. The absorbance at 410 nm was measured, and the concentration of ρNP and enzyme activity were determined using a standard curve method. Enzyme activity recovery and relative enzyme activity were calculated according to equations (4) and (5), respectively. The specific enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of ρNP per minute through hydrolysis of ρNP-P. Specifically, 4-nitrophenyl palmitate was used to determine enzyme activity.

[0045] Formula for calculating enzyme activity (Equation 3): U (U / mg) = [C ρNP ×540 / 400×1500 / 1000×1015 / C p =0.0002025×C ρNP / C p In equation (3), C ρNP C represents the concentration of ρNP after the reaction. p It is the concentration of lipase in the reaction system.

[0046] Equation (4) is as follows: Enzyme activity recovery rate (%) = (immobilized enzyme activity / free enzyme activity) × 100%.

[0047] Equation (5) is as follows: Relative enzyme activity (%) = (Activity of immobilized enzyme to be tested / Optimal immobilized enzyme activity under the same conditions) × 100%.

[0048] 1.6 Evaluation of Lipase Immobilization Effect Lipase GTL2 was immobilized according to the method described in section 1.4 for immobilizing lipase on a microalgae carrier. All supernatants were collected for later use, and the volume of supernatant V1 was recorded.

[0049] Lipase GTL2 solutions with concentrations of 0, 4, 8, 16, 24, 32, 40, and 48 μg / mL were prepared. The p-nitrophenyl palmitate was hydrolyzed using different concentrations of lipase GTL2 solutions and the collected supernatant to produce p-nitrophenol. The absorbance at 410 nm was measured under alkaline conditions (0.5 mol / L NaOH) and a standard curve of lipase concentration versus absorbance at 410 nm was plotted as shown in Equation (6). The concentration of lipase in the supernatant was calculated, and the immobilization efficiency was calculated using Equation (7).

[0050] Equation (6): y = 0.0106x + 0.1508, R² = 0.9904. Where x is the lipase concentration (μg / mL) and y is the absorbance at 410 nm.

[0051] Equation (7): Immobilization efficiency (%) = (m0 - C1V1) / m0 × 100%. Where m0 is the total protein content, C1 is the lipase concentration in the supernatant, and V1 is the volume of the supernatant.

[0052] 1.7 Structural characterization of microalgal carriers and immobilized enzymes The external morphology of samples after different treatments was analyzed using scanning electron microscopy. Fourier transform infrared spectroscopy was used in the wavenumber range of 4000-500 cm⁻¹. -1 The changes in functional groups in the samples were analyzed. The prepared carrier and immobilized enzyme were ground and mixed with potassium bromide (KBr) solid at a mass ratio of 1:200. After pelleting, the samples were tested, with KBr as a blank. The potential and particle size of the samples after different treatments were analyzed using a Malvern particle size potential analyzer.

[0053] 1.8 Single-factor optimization of enzyme immobilization on microalgae carriers Weigh 0.1 g of the microalgae carrier sample and disperse it evenly in 1 mL of Tris-HCl buffer (0.05 mol / L, pH 8.0). Place the sample in an ultrasonic cell disruptor and sonicate it for different power levels (10%, 20%, 30%, 40%, 50%, 60%) and durations (0.5 min, 1 min, 2.5 min, 5 min, 10 min, 15 min) to ensure uniform dispersion. Add different amounts of lipase (20 μg, 40 μg, 60 μg, 80 μg, 100 μg) and mix at 100 r / min for a certain period of time (1 h, 2 h, 3 h, 4 h, 5 h) under ice bath conditions. Then, centrifuge at 5000 r / min for 5 min to separate the enzyme. Wash the immobilized enzyme three times with Tris-HCl buffer (0.05 mol / L, pH 8.0). The resulting precipitate is the immobilized enzyme. Collect all the supernatant from the washing and record the volume of the supernatant. Calculate the immobilized enzyme activity, enzyme recovery rate, and enzyme immobilization efficiency.

[0054] 1.9 Response Surface Optimization of Microalgae Immobilized Enzymes Response surface methodology (RSM) experiments were designed based on single-factor results. The Box-Behnken Design (BBD) method was used to optimize the immobilized enzyme activity on three microalgae carriers. Enzyme dosage, sonication time, and sonication power were selected as the main factors, with enzyme activity as the response value. A total of 17 experiments with 3 factors and 3 levels were designed using Design-Expert 13 software to optimize the effects on immobilized enzyme activity.

[0055] 1.10 Enzymatic Properties Analysis of Immobilized Enzymes on Microalgae Carriers Optimal temperature and pH determination: The immobilized enzyme was placed in a water bath at 30℃-70℃ (30℃, 40℃, 50℃, 60℃, 70℃) and neutralized in buffer solutions with pH values ​​of 6.0 (PBS), 7.0 (PBS), 8.0 (PBS and Tris-HCl), and 9.0 (Gly-NaOH) to determine enzyme activity according to the 1.5 enzyme activity method.

[0056] Temperature stability analysis: The three microalgal immobilized enzymes were placed in Tris-HCl at pH 8.0 and incubated in water baths at 30℃-70℃ for 30-180 min, respectively. The enzyme activity was determined according to the method in 1.5.

[0057] Substrate specificity analysis: p-nitrophenol esters with carbon chain lengths of 4, 8, 10, 12, 14, 16, and 18 (p-nitrophenylbutyrate, p-nitrophenyloctanoate, p-nitrophenyl decanoate, p-nitrophenyl laurate, p-nitrophenyl myristate, p-nitrophenyl palmitate, and p-nitrophenyl stearate, respectively) were selected as substrates. The catalytic effects of immobilized enzymes on different substrates were compared according to the enzyme activity assay method in section 1.5 to explore the preference of immobilized lipases for different substrates.

[0058] Storage stability analysis: The immobilized enzyme was stored at -20℃, and the enzyme activity was measured every 48 h according to the 1.5 enzyme activity method to investigate the changes in enzyme activity over 14 days, with the enzyme activity at day 0 of storage as 100%.

[0059] Organic reagent tolerance analysis: 5%, 10%, 25%, and 50% (v / v) of methanol, ethanol, glycerol, n-butanol, n-hexane, and furfural were added to the reaction system for determining enzyme activity, respectively. The enzyme activity was determined according to the 1.5 enzyme activity method, with the enzyme activity of the blank group without added organic reagents being 100%.

[0060] Reusability analysis: Following the enzyme activity assay method in 1.5, the immobilized enzyme was washed with Tris-HCl buffer (0.05 mol / L, pH 8.0) and the assay was repeated. The enzyme activity measured for the first time was taken as 100%, and the number of assays and relative enzyme activity were recorded.

[0061] The specific steps for the reusability assay are as follows: The substrate hydrolysis reaction is carried out in a syringe lined with filter paper. After the reaction is complete, the entire reaction system is collected by pressurizing the syringe and the reaction is terminated. Tris-HCl buffer (0.05 mol / L, pH 8.0) is added to the syringe wall, the syringe is shaken to wash away any remaining substrate, and the washing solution is discharged from the syringe orifice. This washing process is repeated three times with the addition of Tris-HCl buffer (0.05 mol / L, pH 8.0), and enzyme activity is measured. After the above steps are completed, Tris-HCl buffer (0.05 mol / L, pH 8.0) is added to the syringe again for the next enzyme activity assay, and so on.

[0062] 2. Experimental Results 2.1 Extraction and content analysis of microalgal lipids Table 2 shows the total fat content of the three microalgae. Among the three microalgae, Schizochytrium has the highest fat content at 31.91%, while Chlorella has a lower fat content of only 14.46%.

[0063] Table 2 Total lipid content of microalgae Since microalgal biomass is stored within a tough and complex cell wall, the extraction of microalgal lipids depends on effective pretreatment. DES pretreatment of marine microalgae is a green method for cell wall disruption. Table 3 shows that acidic DES (ChCl:citric acid = 1:2) pretreatment resulted in the highest lipid extraction rates for the three microalgae, reaching 85.29%, 80.05%, and 79.39%, respectively, representing increases of 25.35%, 34.31%, and 40.74% compared to the untreated control group. The degree of microalgal cell damage largely depends on the acidity of the DES; the stronger the acidity of the DES, the greater the degree of cell disruption and the more thorough the cell destruction. The increased lipid recovery rate in algal biomass after DES pretreatment may be due to the internal molecular hydrogen bonds of macromolecules (such as cellulose and hemicellulose) within the algal cell wall, which break down the cell wall and allow the contents to flow out, thus increasing the lipid extraction rate.

[0064] Table 3. Effects of different DES cell disruption methods on lipid extraction rate from marine microalgae. Note: "—" indicates that water was used as a blank control instead of the eutectic solvent.

[0065] 2.2 Feasibility of immobilizing lipases on microalgae carriers Microalgal residues were used to immobilize lipase GTL2, and its enzyme activity, immobilization efficiency, and enzyme activity recovery rate were measured to analyze the feasibility of enzyme immobilization using microalgal carriers. The results (Table 4) showed that three microalgal carriers significantly improved the GTL2 enzyme activity recovery rate. The microalgal carrier treated with the eutectic solvent ChCl:glycerol = 1:2 exhibited the highest enzyme activity. The enzyme activities immobilized on Chlorella, Microchondria, and Schizochytrium carriers reached 288.14, 323.76, and 206.48 U / mg, respectively, which were 3.00, 3.37, and 2.15 times that of the free enzyme (GTL2, enzyme activity 95.96 U / mg). This indicates that microalgal carriers have a significant effect on enhancing the activity of lipase GTL2.

[0066] Among the four eutectic solvents, the microalgal carrier treated with ChCl:glycerol = 1:2 showed the strongest synergistic effect on lipase activity, followed by the control group. The acidic eutectic solvents with ChCl:DL-malic acid = 1:2 and ChCl:citric acid = 1:2 showed weaker synergistic effects on the GTL2 lipase activity. This may be because acidic DES has a certain degree of destructive effect on the structure of the microalgal carrier, and acids can also degrade carbohydrates, thus the immobilized enzyme exhibited lower enzyme activity than the control group. Therefore, in subsequent experiments characterizing the structure and enzymatic properties of the immobilized enzyme, the microalgal carrier treated with the eutectic solvent ChCl:glycerol = 1:2 was selected for the experiments.

[0067] Table 4. Activity performance of enzymes immobilized on microalgae carriers Note: "—" indicates that water was used as a blank control instead of the eutectic solvent.

[0068] 2.3 Structural characterization of microalgal carriers and their immobilized enzymes (1) SEM morphological characterization analysis Morphological analysis of different microalgal raw materials, pretreated microalgal carriers, and their immobilized enzymes was performed using SEM. Figure 1 In (a), (d) and (g), it can be observed that Chlorella, Micrococcus pseudomicrococcus and Schizochytrium are all spheres composed of many small spheres. Micrococcus pseudomicrococcus is more tightly bound, while the gaps between the spheres of Chlorella and Schizochytrium are relatively obvious, the granules are distinct, and the surface is relatively smooth. Figure 1 Images (b), (e), and (h) show the microalgal carriers after pretreatment with the eutectic solvent ChCl:glycerol = 1:2, where the aggregated microspheres of the carriers are dispersed. DES treatment effectively degrades the cell walls of marine microalgae, while ultrasound helps to evenly disperse the degraded microalgal cell wall microspheres. The microalgal carriers obtained from *Schizochytrium* after pretreatment with the eutectic solvent ChCl:glycerol = 1:2 exhibit a relatively regular spherical structure, approaching spherical shape. The microalgal carriers obtained from *Micrococcus pluvialis* after pretreatment with the eutectic solvent ChCl:glycerol = 1:2 show both spherical and ellipsoidal shapes. This may be related to the growth stage of the microalgae; different growth stages result in variations in the size and morphology of the microalgae, thus naturally occurring microalgal carriers cannot achieve uniform size. Figure 1 Images (c), (f), and (i) show SEM images of the enzyme immobilized on the microalgae carrier. It can be observed that the lipase was successfully immobilized on the microalgae residue carrier, with yellow circles and arrows indicating the location of the immobilized enzyme. The carrier maintained structural stability after immobilization without significant damage, indicating that the DES (ChCl:glycerol = 1:2) pretreatment process and enzyme loading process were relatively mild and did not affect the morphology of the carrier.

[0069] (2) Chemical structure infrared spectroscopy analysis (FTIR) Infrared spectroscopy can be used to analyze changes in functional groups within the support and the immobilized enzyme. (3200-3500 cm⁻¹) -1 2927cm -1 1050-1150 cm -1 These peaks correspond to the stretching vibrations of the -OH and CH bonds in cellulose, and the CO stretching vibration of the COC glycosidic bond. These characteristic absorption peaks indicate that the microalgal feedstock contains cellulose. The characteristic absorption peak of glycerol is at approximately 3400 cm⁻¹ (-OH). -1 , Figure 2In comparison to microalgae raw materials, microalgae carriers are typically found at a depth of 3100-3500 cm⁻¹. -1 The increased absorption peak intensity indicates that DES (ChCl:glycerol = 1:2) pretreatment increases the hydroxyl content, suggesting that DES pretreatment can modify the surface of the microalgal carrier to introduce more hydroxyl groups. Lipase at 1650 cm⁻¹... -1 1525 cm -1 1370 cm -1 It exhibits three typical protein absorption peaks: amide I (1600-1700 cm⁻¹), amide II (1500-1580 cm⁻¹), and amide II (1500-1580 cm⁻¹). -1 ) and amide III (1200-1400 cm) -1 Comparing the microalgal carrier (red) and its immobilized enzyme (blue), the immobilized enzyme on the microalgal carrier showed enhanced activity at its characteristic peak, proving that the lipase had been successfully adsorbed and immobilized on the microalgal carrier.

[0070] (3) Analysis of microalgal carrier particle size and zeta potential Table 5 shows that the zeta potential of marine microalgae raw materials and their prepared microalgae carriers is negatively charged. This is because the microalgae raw materials and their prepared carriers contain cellulose, whose surface contains a large number of oxygen-containing groups. These groups can cause the carrier nanoparticles to carry a negative charge through a proton-coupled electron transfer mechanism. The absolute value of the zeta potential of the microalgae carriers increased after DES (ChCl:glycerol = 1:2) pretreatment. This is because after ultrasonic and DES pretreatment, the β-1,4 glycosidic bonds linking glucose in cellulose broke, exposing more hydroxyl groups. The absolute value of the zeta sites of the three microalgae carriers was greater than 30, indicating good dispersion stability and resistance to sedimentation. Observation of particle size changes revealed that the particle size of the raw materials after DES (ChCl:glycerol = 1:2) pretreatment was smaller than that of the raw materials, indicating a decrease in particle size. This suggests that pretreatment effectively disperses the microalgae spheres. The reduced particle size of the microalgae carriers increases the surface area utilization of the microalgae raw materials, increasing the number of sites that can be loaded by lipases.

[0071] Table 5. Particle size and Zeta potential analysis of microalgae raw materials and carriers (4) Single-factor optimization of enzyme immobilization on microalgae carriers The following immobilization process used a Tris-HCl buffer solution with pH=8.0 as the immobilization system, the immobilization temperature was 0℃, and the rotation speed was 125 rpm.

[0072] ①Fixed time The results of the investigation on the effect of immobilization time (1 h–6 h) on enzyme activity are shown in the figure. Figure 3The enzyme addition was 50 μg, the sonication time was 1 min, and the sonication power was 40%. For Chlorella vulgaris and Microchondria fragrans carriers, the immobilization efficiency gradually leveled off with increasing immobilization time. Initially, the carrier surface had sufficient active sites, allowing for rapid enzyme binding. As immobilization time increased, the carrier binding sites became saturated, reducing the available loading sites and decreasing the binding rate until equilibrium was reached. For Schizochytrium fragrans carriers, the immobilization efficiency initially increased and then decreased with increasing immobilization time, but the fluctuations were not significant. This may be because, after reaching equilibrium, weaker lipases might dissociate over time. Observing the changes in the activity of the three immobilized enzymes, the enzyme activity initially increased and then decreased with increasing immobilization time. In the early stages of immobilization, the enzyme activity increased with increasing carrier loading. Subsequently, as the enzyme loading increased, enzyme aggregation between molecules affected diffusion, reducing the accessibility of the active sites to the substrate. Furthermore, prolonged shaking could also lead to some enzyme detachment, resulting in decreased activity. To maximize the utilization of enzyme activity, the optimal immobilization times were selected as 3 h, 2 h, and 2 h for Chlorella vulgaris, Micrococcus pseudocarrier, and Schizochytrium breviscarrier, respectively.

[0073] ② Enzyme addition amount To prevent waste due to incomplete enzyme utilization from excessive enzyme addition, the effect of enzyme addition amount (20 μg-100 μg) on ​​enzyme activity was investigated, which can effectively improve enzyme utilization. The sonication time was 1 min, the sonication power was 40%, and the optimal immobilization time was used. The results of the investigation on the amount of lipase GTL2 added are shown below. Figure 4 As the amount of lipase increased, the immobilization efficiency did not show a significant trend, indicating that the microalgal carrier had a strong enzyme loading effect. With enzyme addition ranging from 20-100 μg, the immobilization efficiency could generally reach over 80%, but the enzyme activity showed a trend of first increasing and then decreasing. The greater the lipase loading, the more enzyme molecules were available, thus increasing enzyme activity. However, further increases in enzyme molecules led to a higher enzyme concentration, eventually reaching saturation, where aggregation between enzyme molecules caused a decrease in enzyme activity. For Chlorella vulgaris, Micrococcus pseudomicrococcus, and Schizochytrium carriers, the enzyme activity reached its highest values ​​at enzyme additions of 40 μg, 60 μg, and 40 μg, respectively. Therefore, response surface methodology was performed using lipase GTL2 addition amounts of 20-60 μg, 40-80 μg, and 20-60 μg, respectively.

[0074] ③ Ultrasound time The preparation of microalgae carriers requires ultrasonic treatment, which effectively disperses the marine microalgae carriers evenly, increases the contact area between the carrier and the enzyme, and facilitates enzyme immobilization. Previous experiments have shown that carbohydrates may have a synergistic effect on lipases, and the release of carbohydrates from the microalgae during ultrasonication may further enhance enzyme activity.

[0075] The effects of different sonication times (0.5 min–15 min) on enzyme activity were investigated under the conditions of 40 μg enzyme addition, optimal immobilization time, and 40% sonication power. The results are as follows: Figure 5 As shown, the immobilization efficiency of the microalgae carrier for lipase GTL2 initially increased and then decreased with increasing sonication time, with the decrease being particularly pronounced for the Chlorella carrier. This may be because, under short-term sonication, the marine microalgae carrier is evenly dispersed in the buffer solution due to ultrasonic cavitation, thus increasing the surface area for enzyme molecule attachment and leading to a higher loading rate. However, as the sonication time continues to extend, the effect of ultrasound becomes too strong, causing structural damage to the marine microalgae carrier and consequently reducing the lipase immobilization efficiency.

[0076] Figure 5 The enzyme activity showed a trend of first increasing and then decreasing with the increase of ultrasound time. On the one hand, as ultrasound improves the aggregation state of marine microalgae carriers and enhances immobilization efficiency, enzyme activity increases. At the same time, sugars may be released from microalgae under ultrasound, which also improves enzyme activity to a certain extent. However, if the ultrasound time is too long, the heat generation may damage the carrier and cause degradation of sugars. Therefore, under prolonged ultrasound treatment, enzyme activity gradually decreases.

[0077] The enzyme activity of Chlorella, Micrococcus pseudomicrococcus, and Schizochytrium vectors reached its maximum at sonication times of 5 min, 2.5 min, and 2.5 min, respectively. Therefore, response surface methodology was optimized at 2.5-10 min, 1-5 min, and 1-5 min, respectively.

[0078] ④ Ultrasonic power The effects of ultrasonic treatment are usually also related to the ultrasonic power. Figure 6The effects of different ultrasonic powers (10%-60%) on the activity of immobilized enzyme GTL2 on microalgal carriers under optimal immobilization time and with an enzyme loading of 40 μg were investigated. With increasing ultrasonic power, the loading rate of the microalgal carrier and enzyme activity initially increased and then decreased. At lower ultrasonic power, the microalgal carriers tended to aggregate, resulting in a relatively small loading area for the lipase and consequently lower enzyme activity. The ultrasonic cavitation effect induced a controllable porous structure in the microalgal cell wall. As the ultrasonic power gradually increased, the uniformity of the microalgal carrier improved, increasing the loading area for the lipase and thus improving the loading efficiency.

[0079] Excessive ultrasonic power can damage the microalgae carrier, impairing its structural integrity and reducing enzyme loading. Furthermore, excessive power can raise local temperatures, potentially promoting carbohydrate degradation and thus reducing the synergistic effect on enzyme activity. Therefore, both loading and enzyme activity tend to decrease when ultrasonic power is too high.

[0080] For Chlorella, Micrococcus pseudomicrococcus, and Schizochytrium carriers, the enzyme activity reached its highest level at an ultrasonic power of 40%. Therefore, an ultrasonic power of 30%-50% was selected for response surface optimization.

[0081] (5) Response surface optimization of GTL2 lipase immobilized on microalgae carrier Based on the results of single-factor experiments, the Box-Behnken Design (BBD) method was used to optimize the activity of enzymes immobilized on microalgae carriers. Enzyme addition amount, sonication time, and sonication power were selected as the investigation factors. Using Design-Expert 13 software, 17 groups of experiments with 3 factors and 3 levels were designed to optimize the factors affecting the immobilized enzyme activity. The design and optimization results of the factors and levels in the response surface methodology are shown in Tables 6 and 7.

[0082] Table 6 Response Surface Design Factor Level Table Table 7 Response Surface Optimization Design and Results ① Optimization of the activity response surface of enzymes immobilized on Chlorella carriers The significance of the quadratic polynomial model was evaluated using analysis of variance (ANOVE). The response surface data results are shown in Table 8. The quadratic regression equation for the immobilized enzyme activity (Y) on the Chlorella residue carrier against timeout (A), ultrasonic power (B), and enzyme addition amount (C) is: Y = -250.36 + 48.02A + 25.18B - 0.09C - 0.49AB - 0.33AC + 0.14BC - 1.47A² - 0.34B² - 0.04C². Comparing the F-values ​​of each factor, the factors affecting the immobilized enzyme activity response value, in order of significance, are: timeout (A) > ultrasonic power (B) > enzyme addition amount (C). Among the influencing factors, the linear terms A and B are highly significant, C is not significant, and the quadratic term A... 2 B 2 C 2 The interaction terms AB, AC, and BC all showed highly significant differences. The model p-value < 0.0001 (significant) and the lack-of-fit terms > 0.05 (insignificant) indicate that the model fits well and there is a high correlation between the actual and predicted values. 2 =0.9860, therefore, this model can well describe the relationship between immobilized enzyme activity and various factors, and can be used to predict the optimal conditions for Chlorella immobilization.

[0083] Table 8. Analysis of variance and significance test of data from the quadratic regression model of enzyme activity immobilized on Chlorella carriers. Note: * indicates a significant difference (P < 0.05); ** indicates an extremely significant difference (P < 0.01).

[0084] In planar contour maps and 3D model diagrams, response surfaces and contour plots can reflect the interactions between factors. The steeper the surface, the denser the distribution of contour lines, and the closer their shape is to an ellipse, the more significant the interaction. From Figure 7 As can be seen from the data, the significance of the interaction effects of the factors is: BC > AC > AB. The contour plots of the pairwise interactions of the three factors are all close to ellipses, indicating that the interaction effects are strong.

[0085] Based on the response surface regression model, the predicted optimal conditions were: sonication time 3.65 min, sonication power 43.95%, and enzyme addition amount 45.22 μg. Under these predicted conditions, the immobilized enzyme activity was 422.18 U / mg. The validation experimental conditions were adjusted to: sonication time 3.7 min, sonication power 44.00%, and enzyme addition amount 45.20 μg. The validation experiment yielded an immobilized enzyme activity of 433.41 U / mg, with a difference of 2.59% from the theoretical value, less than 5.00%, indicating a small discrepancy. Therefore, the optimized conditions for immobilized enzyme activity have high reliability and reference value.

[0086] ② Optimization of enzyme activity response surface of *Microcystis aeruginosa* carrier The response surface methodology results are shown in Table 9. The quadratic regression equation model for the immobilized enzyme activity (Y) on the *Chlorella vulgaris* carrier against ultrasonic time (A), ultrasonic power (B), and enzyme addition amount (C) is: Y = -133.36 - 6.08A + 21.50B + 4.33C + 0.13AB + 0.28AC + 0.06BC - 2.38A² - 0.33B² - 0.07C². Comparing the F-values ​​of each factor, the order of significance of the influence on the immobilized enzyme activity response value is: enzyme addition amount (C) > ultrasonic power (B) > ultrasonic time (A). Among the influencing factors, the linear terms B and C are highly significant, A is not significant, and the quadratic term A... 2 B 2 C 2 The differences were highly significant, with interaction terms AC and BC showing highly significant differences, while AB was not significant. The model p-value < 0.0001 (significant) and the lack-of-fit term > 0.05 (insignificant), indicating that the model fits the actual experiment well and that there is a high correlation between the actual and predicted values. R0 2 =0.9889, therefore, this model can well describe the relationship between immobilized enzyme activity and various factors, and can be used to predict the optimal conditions for immobilized enzymes on the *Chlorella vulgaris* carrier.

[0087] Table 9. Analysis of variance and significance test of data from the quadratic regression model of enzyme activity immobilized on the *Microcystis aeruginosa* carrier. from Figure 8 As can be seen, the significance of the interaction effects of the factors is: AC > AB > BC. The contour plots of the pairwise interactions of the three factors are all close to ellipses, indicating strong interactions. Based on the response surface regression model, the optimal predicted conditions are: sonication time 2.72 min, sonication power 38.35%, and enzyme addition amount 49.35 μg. Under these predicted conditions, the Y value is 371.62 U / mg. The validation experimental conditions were adjusted to: sonication time 2.7 min, sonication power 38.00%, and enzyme addition amount 49.40 μg. The validation experiment showed that the immobilized enzyme activity was 383.93 U / mg, with a difference of 3.21% from the theoretical value, which is less than 5.00%, indicating a small difference. Therefore, the optimized conditions for immobilized enzyme activity have high reliability and reference value.

[0088] ③ Optimization of enzyme activity response surface of *Schizochytrium* carrier immobilized Table 10. Analysis of variance and significance test of the quadratic regression model for enzyme activity immobilized on schistocytic urchin residue carrier. Note: * indicates a significant difference (P < 0.05); ** indicates an extremely significant difference (P < 0.01).

[0089] The response surface methodology results are shown in Table 10. The quadratic regression equation for the immobilized enzyme activity (Y) on the *Schizochytridica* carrier against ultrasound time (A), ultrasound power (B), and enzyme addition amount (C) is: Y = 26.74 - 5.49A + 8.30B + 2.88C + 0.46AB + 0.27AC + 0.01BC - 4.29A² - 0.12B² - 0.04C². The order of significance of the factors affecting the immobilized enzyme activity response value is: ultrasound time (A) > ultrasound power (B) > enzyme addition amount (C). Among the influencing factors, the linear terms A, B, and C are not significant, while the quadratic term A... 2 B 2 C 2 The differences were highly significant, with interaction terms AB and AC showing significant differences, while BC was not significant. The model's p-value < 0.01 (highly significant) and lack-of-fit terms > 0.05 (not significant) indicate that the model fits the actual experiment well, and there is a high correlation between the actual and predicted values. R0 2 =0.9529, therefore, this model can well describe the relationship between immobilized enzyme activity and various factors, and can be used to predict the optimal immobilization conditions of Schizochytrium carriers.

[0090] from Figure 9 The results show that the significance of the interaction effects is AC > AB > BC. The contour plots of the AC and AB interactions are both close to ellipses, indicating strong interactions. Based on the response surface regression model, the optimal predicted conditions are: sonication time 2.66 min, sonication power 37.80%, and enzyme addition 40.13 μg. Under these conditions, the predicted Y value is 234.23 U / mg. The validation experimental conditions were adjusted to: sonication time 2.7 min, sonication power 38.00%, and enzyme addition 40.1 μg. The validation experiment showed an immobilized enzyme activity of 230.46 U / mg, with a difference of 3.21% from the theoretical value, less than 5.00%, indicating a small difference. Therefore, the optimized conditions for immobilized enzyme activity have high reliability and reference value.

[0091] (6) Enzymatic property analysis of lipase immobilized on microalgae carriers ①Optimal temperature and pH The enzyme activity of GTL2 immobilized on three microalgae carriers was measured within the pH range of 6.0-9.0 and the temperature range of 30℃-70℃ to explore the effect of immobilization treatment on the optimal temperature and pH of GTL2. Figure 10As can be seen, the optimal conditions for GTL2 immobilization on the three microalgae carriers are pH=8.0 (Tris-HCl) and temperature=60℃. These are the same as the optimal conditions for the free enzyme, therefore the immobilization technology did not change the optimal reaction conditions for GTL2. Between pH 6.0 and 9.0, enzyme activity initially increased and then decreased with increasing pH, indicating that immobilized GTL2 may undergo denaturation and inactivation in excessively acidic or alkaline systems, thus affecting enzyme activity. Within the temperature range of 30℃ to 60℃, GTL2 enzyme activity showed a slow increase followed by a decrease, reaching its highest level at 60℃. This indicates that excessively low temperatures lead to insufficient activation of the reactive groups in GTL2, resulting in lower activity, while excessively high temperatures cause changes in enzyme structure and inactivation.

[0092] ②Temperature stability For lipases, incubation temperature has a significant impact on enzyme activity. The activity of GTL2 enzyme immobilized on three microalgae carriers was measured within the temperature range of 30℃-70℃ and the incubation time range of 30 min-180 min to explore the protective effect of immobilization on GTL2 at high temperatures. At 30℃, the enzyme activity did not change significantly, indicating that the reactive groups of the enzyme were not fully activated at low temperatures. However, at 70℃, the enzyme activity was almost completely lost, indicating that high temperature caused irreversible denaturation of the enzyme molecules, leading to inactivation.

[0093] Figure 11 In (a), for GTL2 immobilized on Chlorella vectors, enzyme activity decreased with increasing incubation time at 50℃ and 60℃, indicating that prolonged high-temperature incubation leads to reduced enzyme activity. Enzyme activity reached its peak at 60℃ for 30 min, while at 50℃, enzyme activity changed gradually and remained stable within 30 min to 150 min.

[0094] Figure 11 In (b), the enzyme activity of GTL2 immobilized on the *Micrococcus pluvialis* carrier first increased and then decreased with incubation time at 40℃ and 50℃, indicating that the enzyme activity was not fully activated in a short time at low temperatures, but high temperatures may have an impact on enzyme activity as time increases. The enzyme activity reached its highest level at 50℃ and 90 min incubation, while at 40℃, the enzyme activity changed slowly and remained stable within 90 min to 180 min.

[0095] Figure 11 In (c), the enzyme activity of GTL2 immobilized on the Schizochytrium vector at 40℃ first increased and then decreased with incubation time, reaching its peak at 120 min. Although the enzyme activity changed slowly at 30℃ and 50℃, it did not increase significantly, indicating that long-term incubation above 50℃ may lead to changes in the vector or enzyme molecules, resulting in activity loss.

[0096] In summary, incubation at a temperature range of 40℃-50℃ is beneficial for the full expression of enzyme activity, and the enzyme activity can remain stable within a certain incubation period, indicating that the microalgae carrier has a certain protective effect on GTL2.

[0097] ③Substrate specificity pass Figure 12 It was found that, compared with free enzymes, immobilized GTL2 significantly enhanced its hydrolytic ability on various substrates, with the Chlorella-based carrier showing the strongest enhancement. Both immobilized GTL2 and free GTL2 on microalgae carriers tended to hydrolyze C4-chain fatty acids, and their specific enzyme activities decreased with increasing substrate carbon chain length. However, compared with free GTL2, immobilized GTL2 on microalgae carriers also showed improved hydrolytic ability on medium- and long-chain fatty acids (C6-C12). This indicates that microalgae carriers can modulate the substrate specificity of GTL2, a characteristic highly beneficial for biocatalytic processes requiring specific substrate transformation, and can improve the yield of target products.

[0098] ④ Organic reagent tolerance Lipases play an important role in many fields such as organic synthesis, food processing and biofuel manufacturing. In non-aqueous catalytic systems, the enzyme molecule's tolerance to organic solvents is the core parameter that determines its catalytic activity and stability.

[0099] Figure 13 The relative enzyme activities of GTL2 immobilized on different microalgae carriers and free GTL2 in organic reagents (methanol, glycerol, ethanol, n-hexane, furfural, and butanol) at different concentrations (volume fractions of 5%, 10%, 25%, and 50%) were shown.

[0100] Both immobilized and free GTL2 cells were relatively stable in glycerol reagents of varying concentrations. At lower concentrations (5%-25% v / v), the enzyme activity of immobilized GTL2 was enhanced, but decreased at a concentration of 50% v / v. Glycerol at concentrations of 5%-50% v / v exhibited some inhibitory effect on the enzyme activity of free GTL2, although this inhibitory effect was relatively small compared to other organic reagents. These results suggest that low concentrations of glycerol may have a strong promoting effect on immobilized GTL2.

[0101] The activity of free GTL2 was less inhibited in low concentrations of methanol (5%-25% by volume), but showed a relative increase in activity in 50% methanol. Methanol, as a polar organic solvent, may enhance the solubility of hydrophobic substrates (such as lipids) by reducing the water activity of the reaction system, making it easier for the active site of the free enzyme to contact the substrate, thereby improving catalytic efficiency. GTL2 immobilized on *Chlorella vulgaris* and *Schizochytrium* carriers showed a certain synergistic effect in the methanol system; however, enzyme activity gradually decreased with increasing methanol concentration, and GTL2 immobilized on *Chlorella vulgaris* residue was inhibited in the methanol system. This may be because the carrier material or cross-linking agent may hinder the substrate from approaching the enzyme's active site, and the presence of methanol may further exacerbate this steric hindrance, limiting enzyme-substrate contact.

[0102] It is noteworthy that, compared with free enzymes and other carriers, GTL2 immobilized on the *Schizochytrium* carrier showed enhanced enzyme activity in low concentrations of ethanol (5% v / v) and n-butanol (5%-10% v / v). Free GTL2 showed strong inhibitory effects in ethanol and n-butanol. Even though GTL2 immobilized on *Chlorella* and *Microchlorella* carriers did not enhance enzyme activity in ethanol and n-butanol, their relative enzyme activity was higher than that of free enzymes. This indicates that the immobilization of GTL2 on microalgae carriers has a certain protective effect.

[0103] Compared with free GTL2, GTL2 immobilized on microalgae carriers showed a certain improvement in relative enzyme activity in methanol, ethanol, glycerol, and n-butanol, indicating that the enzyme immobilized on marine microalgae carriers exhibited superior stability in these organic solvents.

[0104] ⑤ Storage stability GTL2 was immobilized on three different microalgae carriers and its enzyme activity was measured over 14 days. Figure 14 As shown in (a), the enzyme activity of GTL2 immobilized on Chlorella vector remained at 413.93 U / mg ± 12.35 U / mg within 14 days, with a relative enzyme activity of over 92.36%, demonstrating good storage stability. Figure 14 In (b), the enzyme activity of GTL2 immobilized on the Micrococcus pseudocarrier decreased slightly within 14 days, but the enzyme activity remained at 365.33 U / mg ± 10.32 U / mg, retaining 94.35% of the initial enzyme activity, which also showed good storage stability. Figure 14 As shown in (c), the enzyme activity of GTL2 immobilized on the *Schizochytridacna* carrier fluctuated somewhat over 14 days, resulting in a relatively large measurement error. However, the relative enzyme activity remained above 89.40% on day 14. Although the enzyme activity of GTL2 immobilized on the three microalgal carriers decreased slightly during storage, it was still superior to that of free GTL2. In comparison, Figure 14The free enzyme in (d) showed a significant decrease in activity during storage, dropping to 59.48 U / mg on day 14, maintaining only 61.98% of its initial activity, indicating poor storage stability. Therefore, all three microalgal carriers immobilizing GTL2 exhibited superior storage stability compared to free GTL2, with the *Chlorella vulgaris* and *Micrococcus pseudochlorella* carriers showing the best stability. This suggests that immobilizing GTL2 on microalgal carriers can effectively improve its storage stability.

[0105] ⑥ Reuse rate Reusability is one of the key indicators for evaluating the industrial application potential of immobilized lipases. Immobilized lipases with excellent reusability can significantly reduce costs in the production process. Figure 15 The reusability of GTL2 immobilized on three different microalgae carriers was evaluated. The results showed that the relative enzyme activity of GTL2 immobilized on the *Chlorella vulgaris* carrier remained at 44.22% during the first five uses, but gradually decreased with each use, dropping to approximately 20.76% by the tenth use. The relative enzyme activity of GTL2 immobilized on the *Microcystis aeruginosa* carrier remained above 80.00% during the first three uses, then gradually decreased, dropping to approximately 20.86% by the ninth use. The relative enzyme activity of GTL2 immobilized on the *Schizochytrium* carrier remained at 88.65% during the first two uses, but rapidly decreased after the eighth use; after eight uses, the enzyme activity remained at only 23.35% of the initial activity, and by the ninth use, it was almost completely inactivated.

Claims

1. A method of preparing a microalgal carrier, characterized by, The method comprises the following steps: S1. mixing the microalgae and the deep eutectic solvent according to a mass ratio of 1:15, oscillating at 70°C and 100 rpm for 2 h, and then ultrasonic treating at 37°C and an ultrasonic power of 70% for 1 h to obtain a microalgae-deep eutectic solvent mixture; the deep eutectic solvent is a deep eutectic solvent containing choline chloride and glycerol at a molar ratio of 1:2; S2. mixing the microalgae-deep eutectic solvent mixture and anhydrous ethanol according to a ratio of 1 g:15 mL, oscillating at 70°C and 100 rpm for 2 h, and then centrifuging the microalgae residue, washing the microalgae residue with pure water until the pH value is neutral, and freeze-drying to obtain the microalgae carrier.

2. The method of claim 1, wherein, The microalgae are Chlorella vulgaris, Nannochloropsis or Schizochytrium; the freeze-drying is pre-freezing at-20°C for more than 4 h, and then freeze-drying at a vacuum degree of 0 at-80°C for 24 h.

3. A microalgae carrier prepared by the method according to any one of claims 1-2.

4. A method for preparing a microalgal carrier-immobilized enzyme using the microalgal carrier according to claim 3, characterized by, The method comprises the following steps: S1. mixing the microalgae carrier with a Tris-HCl buffer solution with a concentration of 0.05 mol / L and a pH value of 8.0 according to a ratio of 0.1 g:10 mL, and then oscillating at an ultrasonic power of 30-50% for 1-10 min to obtain a dispersed microalgae carrier; S2. adding a lipase to the dispersed microalgae carrier, stirring in an ice bath at 100 r / min for 2-3 h, and then collecting and washing the precipitate by centrifuging at 5000 r / min for 5 min to obtain a microalgae carrier immobilized enzyme; the addition amount of the lipase is 0.020%-0.080% of the mass of the microalgae carrier.

5. The method of claim 4, wherein, The microalgae carrier is a Chlorella vulgaris carrier, which comprises the following steps: S1. mixing the Chlorella vulgaris carrier with a Tris-HCl buffer solution with a concentration of 0.05 mol / L and a pH value of 8.0 according to a ratio of 0.1 g:10 mL, and then oscillating at an ultrasonic power of 40-44% for 3.7-5 min to obtain a dispersed Chlorella vulgaris carrier; S2. adding a lipase to the dispersed Chlorella vulgaris carrier, stirring in an ice bath at 100 r / min for 3 h, and then collecting and washing the precipitate by centrifuging at 5000 r / min for 5 min to obtain a Chlorella vulgaris carrier immobilized enzyme; the addition amount of the lipase is 0.040%-0.045% of the mass of the Chlorella vulgaris carrier.

6. The method of claim 4, wherein, The microalgae carrier is a Nannochloropsis carrier, which comprises the following steps: S1. mixing the Nannochloropsis carrier with a Tris-HCl buffer solution with a concentration of 0.05 mol / L and a pH value of 8.0 according to a ratio of 0.1 g:10 mL, and then oscillating at an ultrasonic power of 38-40% for 2.5-2.7 min to obtain a dispersed Nannochloropsis carrier; S2. adding a lipase to the dispersed Nannochloropsis carrier, stirring in an ice bath at 100 r / min for 3 h, and then collecting and washing the precipitate by centrifuging at 5000 r / min for 5 min to obtain a Nannochloropsis carrier immobilized enzyme; the addition amount of the lipase is 0.040%-0.045% of the mass of the Nannochloropsis carrier. S2. Adding lipase to the dispersed Parachlorella sp. carrier, stirring for 2 h under ice bath and 100 r / min, collecting and washing the precipitate by centrifugation at 5000 r / min for 5 min to obtain the Parachlorella sp. carrier immobilized enzyme as the microalgae carrier immobilized enzyme; the addition amount of the lipase is 0.049%-0.060% of the mass of the Parachlorella sp. carrier.

7. The method of claim 4, wherein, The microalgae carrier is a Schizochytrium sp. carrier, comprising the following steps: S1. Mixing the Schizochytrium sp. carrier with 0.05 mol / L Tris-HCl buffer solution with pH 8.0 at a ratio of 0.1 g:10 mL, and placing under ultrasonic power of 40-44% for 2.5-2.7 min to obtain the dispersed Schizochytrium sp. carrier; S2. Adding lipase to the dispersed Schizochytrium sp. carrier, stirring for 2 h under ice bath and 100 r / min, collecting and washing the precipitate by centrifugation at 5000 r / min for 5 min to obtain the Schizochytrium sp. carrier immobilized enzyme as the microalgae carrier immobilized enzyme; the addition amount of the lipase is 0.040% of the mass of the Schizochytrium sp. carrier.

8. The method of claim 4, wherein, The washing of the precipitate is washing the precipitate three times with 0.05 mol / L Tris-HCl buffer solution with pH 8.0, and the lipase is GTL2.

9. A microalgae carrier immobilized enzyme prepared by the method of any one of claims 4-7.

10. The use of the microalgae carrier immobilized enzyme of claim 9 in hydrolyzing esters.

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