EB01 biofilm and application thereof in preparation of immobilized enzyme

Abstract

The invention discloses an EB01 biofilm and application thereof in preparation of immobilized enzyme, and belongs to the technical field of immobilized enzyme. The EB01 biofilm is prepared by the following method: constructing a recombinant expression vector CsgB-CsgA (5 * Lys), and introducing the recombinant expression vector into escherichia coli to obtain engineered escherichia coli; and culturing the engineered escherichia coli to obtain the biofilm. The invention also discloses application of the EB01 biofilm in preparation of immobilized enzyme. The invention also discloses an immobilized double enzyme, wherein the immobilized double enzyme is prepared by loading the beta-galactosidase BgaD and the L-arabinose isomerase CaLAI on an EB01 biofilm. According to the invention, enzyme immobilization can be realized without additional gene modification of the enzyme, and the immobilization has universality. Through rational design of Curli fibers, the enzyme immobilization process is simplified, efficient enzyme immobilization is achieved, and a universal solution is provided for the field of enzyme immobilization.

Description

Technical Field

[0001] This invention relates to an EB01 biofilm and its application in the preparation of immobilized enzymes, belonging to the field of immobilized enzyme technology. Background Technology

[0002] Bacterial biofilms (BF) have demonstrated their potential as novel immobilization carriers due to their high stability, high loading capacity, and biocompatibility. Bacterial biofilms refer to large aggregates of bacteria that adhere to a contact surface, secreting polysaccharide matrix, fibrin, lipid proteins, etc., to encapsulate themselves within it. Curli, a typical functional amyloid fibrous material composed of CsgA and CsgB subunits, is an important component of the extracellular matrix during biofilm formation, playing crucial roles in regulating cell surface adsorption capacity, intercellular interactions, stabilizing three-dimensional structures, and promoting the formation of mature biofilms.

[0003] Engineered *E. coli* biofilms have shown great potential in the field of enzyme immobilization due to their programmability, dynamic self-healing ability, and high specific surface area. Their unique nanoporous structure provides ample interfaces for enzyme loading and reactions, offering a large three-dimensional surface area, deep pores, and channels. However, existing strategies for enzyme immobilization based on engineered biofilms, such as protein-protein pairing and affinity peptide binding, have significant limitations. Protein-protein pairing requires fusing specific binding domains of the target enzyme and CsgA protein, respectively, to achieve enzyme immobilization on the engineered biofilm through specific recognition between these domains. This process necessitates modification of the binding domains, which can easily lead to decreased or even lost enzyme activity; furthermore, the additional gene modification makes the process cumbersome. Affinity peptide binding requires specific peptide screening for specific enzymes to obtain highly specific affinity peptides. This process is cumbersome and lacks universality; immobilization of a particular enzyme requires re-screening for specific affinity peptides. Both of the above methods rely on additional gene modification or screening steps for enzymes, resulting in high technical barriers, complex operations, and long development cycles, making it difficult to meet the needs of industrial production for efficient and universal platforms. Currently, there are no reports of universally applicable and simple biofilm immobilization platforms. Summary of the Invention

[0004] To address the limitations of existing technologies, this invention provides an EB01 biofilm and its application in the preparation of immobilized enzymes. This invention achieves enzyme immobilization without requiring additional gene modification, and the immobilization method is universal and applicable to the rapid immobilization of various enzymes. Through the rational design of Curli fibers, this invention simplifies the enzyme immobilization process, avoiding cumbersome gene fusion or affinity peptide screening steps, thus achieving highly efficient immobilization. The novel immobilization platform based on engineered E. coli biofilms constructed in this invention combines high loading capacity, strong stability, and ease of operation, providing a universal solution for the field of immobilized enzymes.

[0005] This invention is achieved through the following technical solution: A method for preparing an EB01 biofilm is as follows: constructing a recombinant expression vector CsgB-CsgA (5×Lys), introducing it into Escherichia coli to obtain engineered Escherichia coli; culturing the engineered Escherichia coli to obtain a biofilm; the nucleotide sequence of CsgB-CsgA (5×Lys) is shown in SEQ ID NO.5.

[0006] Furthermore, the specific method for culturing engineered Escherichia coli is as follows: The engineered Escherichia coli is activated in LB liquid medium containing 100 μg / mL Amp to obtain a seed culture; the seed culture is inoculated at a rate of 2% into 100 mL of M63 medium containing 100 μg / mL Amp, and cultured at 37°C in a shaker until the bacterial culture reaches OD. 600 The concentration of IPTG was 0.5, and a final concentration of 0.3 mM was added. The mixture was then cultured in a constant temperature shaker at 30 °C for 48 h to induce the production of the target curli fibers. After induction, the mixture was left to stand at room temperature for 12 h. The curli fibers then self-assembled to form the EB01 biofilm, which was then obtained.

[0007] The EB01 biofilm prepared using the above method is used in the preparation of immobilized enzymes and as a carrier for immobilized enzymes.

[0008] An immobilized dual enzyme is formed by loading β-galactosidase BgaD and L-arabinose isomerase CaLAI onto an EB01 biofilm.

[0009] The method for preparing the immobilized dual enzyme is as follows: Take 10 mg of EB01 biofilm, add 200 μL of a dual enzyme mixture containing β-galactosidase BgaD and L-arabinose isomerase CaLAI, add glutaraldehyde solution to make the concentration 0.4% (volume ratio), and incubate at 18℃ and 130 r / min for 1.5 h to obtain the enzyme; wherein, in the dual enzyme mixture, the total concentration of the two enzymes is 2 mg / mL, and the mass ratio of β-galactosidase BgaD to L-arabinose isomerase CaLAI is 20:1.

[0010] Application of the immobilized dual enzymes in the preparation of new agarotriose and D-tagatose.

[0011] Furthermore, in specific applications, with α- A mixture of agarose oligosaccharides prepared by agarase AgaA33 degradation of agarose was used as a substrate and reacted at 50°C for 6–12 h.

[0012] This invention modifies the CsgA gene expressing curli amyloid protein using genetic engineering techniques, and then transfers it into the gene to induce lysine residues. E. coli The EB01 engineered biomembrane was constructed by expressing and assembling amino-rich cells in BL21::ΔCsgA competent cells. This invention systematically characterized the EB01 biomembrane using CR, CV staining, TEM, and SEM, obtaining an ideal material for enzyme immobilization. This invention successfully co-immobilized BgaD and CaLAI enzymes onto the EB01 biomembrane using glutaraldehyde-mediated amino cross-linking technology. Unlike traditional methods for immobilizing enzymes on engineered biomembranes, this method is universal and not limited to BgaD or CaLAI enzymes. Through optimization of immobilization conditions, the optimal immobilization conditions were determined: glutaraldehyde concentration 0.4%, immobilization time 1.5 h, enzyme ratio (CaLAI:BgaD) 20:1, and enzyme dosage 2 mg / mL. Compared with other traditional aminated materials, the EB01 biofilm exhibits a higher enzyme loading (39.88 mg / g) and enzyme activity recovery rate (37.27%), which is mainly attributed to its three-dimensional nanoporous structure, large specific surface area, stable chemical environment and good biocompatibility.

[0013] This invention investigated and compared the enzymatic properties of immobilized dual enzymes. The results showed that the immobilized enzyme exhibited superior characteristics compared to the free dual enzyme in terms of temperature, pH, operational stability, and storage stability. Under high temperature and extreme pH conditions, the immobilized enzyme maintained high activity, demonstrating broader tolerance and stability. Furthermore, the immobilized enzyme exhibited good reusability in the agarose cascade reaction pathway, maintaining high catalytic efficiency even after five cycles, thus reducing production costs. When this immobilized enzyme was applied to the agarose cascade reaction using A4 as a substrate, the yield of NA3 reached 0.25 g / L and the yield of d-tagatose reached 0.10 g / L at equilibrium. The EB01 co-immobilized enzyme of this invention provides an efficient and stable enzyme catalytic platform for the targeted preparation of NA3 and d-tagatose, and has broad prospects for industrial application.

[0014] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0015] Figure 1 : Plasmid diagram of recombinant plasmid pET21a-CsgB-CsgA(5×Lys).

[0016] Figure 2 : Positive clone verification results.

[0017] Figure 3 Photographs of Congo red and crystal violet staining, with the two tubes on the left stained with Congo red and the two tubes on the right stained with crystal violet.

[0018] Figure 4 The result of relative binding amount.

[0019] Figure 5 TEM image of a biofilm, with the upper side being... E. coli The biofilm of BL21::ΔCsgA is shown below the biofilm of strain EB01.

[0020] Figure 6 SEM image of biofilm, where the upper side is... E. coli The biofilm of BL21::ΔCsgA is shown below the biofilm of strain EB01.

[0021] Figure 7 Effect of glutaraldehyde concentration on the activity of immobilized dual enzymes.

[0022] Figure 8 Effect of immobilization time on the activity of immobilized dual enzymes.

[0023] Figure 9 Effect of the ratio of two enzymes on the activity of immobilized two enzymes.

[0024] Figure 10 Effect of enzyme dosage on the activity of immobilized dual enzymes.

[0025] Figure 11 Effect of reaction temperature on the catalytic activity of free and immobilized dual enzymes.

[0026] Figure 12 The effect of reaction pH on the activity of free and immobilized dual enzymes.

[0027] Figure 13 Results of temperature stability determination for free and immobilized dual enzymes.

[0028] Figure 14 Results of pH stability determination for free and immobilized dual enzymes.

[0029] Figure 15 Results of the determination of the operational stability of the immobilized dual enzymes.

[0030] Figure 16 Results of the determination of storage stability of free and immobilized dual enzymes.

[0031] Figure 17 Operational stability of co-immobilized enzymes.

[0032] Figure 18 : Reaction time curve of co-immobilized enzyme. Detailed Implementation

[0033] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0034] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0035] Experiment 1: Preparation and Characterization of EB01 Biofilm 1.1 Main Experimental Materials Used for cloning and expression of biofilm genes in EB(BL21)-01-XXH (EB01 for short). E. coli The BL21::ΔCsgA strain is preserved in our laboratory. It was knocked out using the λ-Red recombination system. E. coli The CsgA gene in the BL21(DE3) strain genome was used to obtain this mutant strain. Specific procedures were performed according to the following literature: Binding Peptide-Guided Immobilization of Lipases with Significantly Improved Catalytic Performance Using Escherichia coli BL21(DE3) Biofilms as a Platform.

[0036] The empty expression vector pET21a(+) is a conventional plasmid in our laboratory.

[0037] The plasmid pET21a(+)-CsgB-CsgA is a plasmid constructed using conventional methods and preserved in our laboratory. The nucleotide sequence of the CsgB-CsgA gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it expresses is shown in SEQ ID NO.2.

[0038] The nucleotide sequences of the CsgB-CsgA genes are shown below (direction 5'-3') (Note: the underlined "TAA" is the stop codon, which divides the expressed protein into two segments, namely CsgB protein and CsgA protein): AAAAACAAATTGTTATTTATGATGTTAACAATACTGGGTGCGCCTGGGATTGCAGCCGCAGCAGGTTATGATTTAGCTAATTCAGAATATAACTTCGCGGTAAATGAATTGAGTAAGTCTTCATTTAATCAGGCAGCCATAATTGGTCAAGCTGGGACTAATAATAGTGCTCAGTTACGGCAGGGAGGCTCAAAACTTTTGGCGGTTGTTGCGCAAGAAGGTAGT AGCAACCGGGCAAAGATTGACCAGACAGGAGATTATAACCTTGCATATATTGATCAGGCGGGCAGTGCCAACGATGCCAGTATTTCGCAAGGTGCTTATGGTAATACTGCGATGATTATCCAGAAAGGTTCTGGTAATAAAGCAAATATTACACAGTATGGTACTCAAAAAACGGCAATTGTAGTGCAGAGACAGTCGCAAATGGCTATTCGCGTGACACAACGT TAAGGATCCAAGGAGATAGTACATATGAAACTTTTAAAAGTAGCAGCAATTGCAGCAATCGTATTCTCCGGTAGCGCTCTGGCAGGTGTTGTTCCTCAGTACGGCGGCGGCGGTAACCACGGTGGTGGCGGTAATAATAGCGGCCCAAATTCTGAGCTGAACATTTACCAGTACGGTGGCGGTAACTCTGCACTTGCTCTGCAAACTGATGCCCGTAACTCTGACTTGACTATTACCCAGCATGGCGGCGGTAATGGTGCAGATGTTGGTCAGGGCTCAGATGACAGCTCAATCGATCTGACCCAACGTGGCTTCGGTAACAGCGCTACTCTTGATCAGTGGAACGGCAAAAATTCTGAAATGACGGTTAAACAGTTCGGTGGTGGCAACGGTGCTGCAGTTGACCAGACTGCATCTAACTCCTCCGTCAACGTGACTCAGGTTGGCTTTGGTAACAACGCGACCGCTCATCAGTAC。

[0039] The amino acid sequence of CsgB-CsgA protein is shown below (Note: The connection between CsgB protein and CsgA protein is indicated by "*"): KNKLLFMMLTILGAPGIAAAAGYDLANSEYNFAVNELSKSSFNQAAIIGQAGTNNSAQLRQGGSKLLAVVAQEGSSNRAKIDQTGDYNLAYIDQAGSANDASISQGAYGNTAMIIQKGSGNKANITQYGTQKTAIVVQRQSQMAIRVTQR*GSKEIVHMKLLKVAAIAAIVFSGSALAGVVPQYGGGGNHGGGGNNSGPNSELNIYQYGGGNSALALQTDARNSDLTITQHGGGNGADVGQGSDDSSIDLTQRGFGNSATLDQWNGKNSEMTVKQFGGGNGAAVDQTASNSSVNVTQVGFGNNATAHQY。

[0040] M63 medium (1 L): Dissolve 15.6 g of M63 medium substrate (purchased from Qingdao Sangon Biotech Co., Ltd.) in 900 mL of ultrapure water. Adjust the pH to 7.0 with potassium hydroxide solution, and then bring the volume to 1 L with pure water. Sterilize at 121℃ for 15 min. Before use, add 10 mL of sterile 20% glycerol and 1 mL of sterile 1 M MgSO4 to each liter of medium.

[0041] 1.2 Experimental Methods (1) Construction of EB01 engineered bacteria Based on the gene sequence of pET21a(+)-CsgB-CsgA and the lysine (Lys) sequence, specific primers EB01-F and EB01-R were designed and synthesized by Qingdao Sangon Biotech Co., Ltd. The nucleotide sequences are shown below (direction 5'-3'): EB01-F: gtttaactttaagaaggagatataCATATGAAAAACAAATTGTTATTTATGATG, as shown in SEQ ID NO.3; EB01-R: GTGGTGGTGCTCGAGTTACTTCTTCTTCTTCTTGCTGCCACCGCCACCGCTACCG-3', as shown in SEQ ID NO.4.

[0042] Using plasmid pET21a(+)-CsgB-CsgA as an amplification template, specific primers were used to amplify the plasmid pET21a-CsgB-CsgA (5×Lys) with 5 lysine modifications. The nucleotide sequence of the CsgB-CsgA (5×Lys) gene fragment is shown in SEQ ID NO.5, and the amino acid sequence of the protein it expresses is shown in SEQ ID NO.6.

[0043] The nucleotide sequence of the CsgB-CsgA(5×Lys) gene fragment is shown below (direction 5'-3'): AAAAACAAATTGTTATTTATGATGTTAACAATACTGGGTGCGCCTGGGATTGCAGCCGCAGCAGGTTATGATTTAGCTAATTCAGAATATAACTTCGCGGTAAATGAATTGAGTAAGTCTTCATTTAATCAGGCAGCCATAATTGGTCAAGCTGGGACTAATAATAGTGCTCAGTTACGGCAGGGAGGCTCAAAACTTTTGGCGGTTGTTGCGCAAGAAGGTAGTAGCAACCGGGCAAAGATTGACCAGACAGGAGATTATAACCTTGCATATATTGATCAGGCGGGCAGTGCCAACGATGCCAGTATTTCGCAAGGTGCTTATGGTAATACTGCGATGATTATCCAGAAAGGTTCTGGTAATAAAGCAAATATTACACAGTATGGTACTCAAAAAACGGCAATTGTAGTGCAGAGACAGTCGCAAATGGCTATTCGCGTGACACAACGTTAAGGATCCAAGGAGATAGTACATATGAAACTTTTAAAAGTAGCAGCAATTGCAGCAATCGTATTCTCCGGTAGCGCTCTGGCAGGTGTTGTTCCTCAGTACGGCGGCGGCGGTAACCACGGTGGTGGCGGTAATAATAGCGGCCCAAATTCTGAGCTGAACATTTACCAGTACGGTGGCGGTAACTCTGCACTTGCTCTGCAAACTGATGCCCGTAACTCTGACTTGACTATTACCCAGCATGGCGGCGGTAATGGTGCAGATGTTGGTCAGGGCTCAGATGACAGCTCAATCGATCTGACCCAACGTGGCTTCGGTAACAGCGCTACTCTTGATCAGTGGAACGGCAAAAATTCTGAAATGACGGTTAAACAGTTCGGTGGTGGCAACGGTGCTGCAGTTGACCAGACTGCATCTAACTCCTCCGTCAACGTGACTCAGGTTGGCTTTGGTAACAACGCGACCGCTCATCAGTACGGCGGTGGCGGTAGCGGTGGCGGTGGCAGCAAGAAGAAGAAGAAG。

[0044] The amino acid sequence of the CsgB-CsgA(5×Lys) protein is shown below (direction 5'-3'): KNKLLFMMLTILGAPGIAAAAGYDLANSEYNFAVNELSKSSFNQAAIIGQAGTNNSAQLRQGGSKLLAVVAQEGSSNRAKIDQTGDYNLAYIDQAGSANDASISQGAYGNTAMIIQKGSGNKANITQYGTQKTAIVVQRQSQMAIRVTQR*GSKEIVHMKLL KVAAIAAIVFSGSALAGVVPQYGGGGNHGGGGNNSGPNSELNIYQYGGGNSALALQTDARNSDLTITQHGGGNGADVGQGSDDSSIDLTQRGFGNSATLDQWNGKNSEMTVKQFGGGNGAAVDQTASNSSVNVTQVGFGNNATAHQYGGGGSGGGGSKKKKK.

[0045] The constructed recombinant plasmid pET21a-CsgB-CsgA(5×Lys) was transformed into [the target cell] via thermal shock. E. coli In BL21::ΔCsgA competent cells, the strain whose PCR product was verified to be positive and whose sequencing was correct was the engineered Escherichia coli that produced the target curli fiber. This recombinant strain was named EB01 engineered strain.

[0046] (2) Preparation of biofilm The constructed EB01 engineered bacteria were activated in LB liquid medium containing 100 μg / mL Amp. The seed culture was inoculated at a rate of 2% into 100 mL of M63 medium containing 100 μg / mL Amp and cultured at 37°C in a shaker until the bacterial culture reached OD500. 600 The concentration of IPTG was increased to 0.5, and the mixture was incubated in a shaker at 30°C for 48 h to induce the production of the target curli fibers. After induction, the mixture was allowed to stand at room temperature for 12 h, during which the curli fibers self-assembled to form an EB01 biofilm. The resulting biofilm solution was centrifuged at 8000 r / min for 2 min, the supernatant was discarded, and the membrane was washed with PBS buffer to obtain the biofilm.

[0047] 1.3 Characterization of EB01 biofilm (1) Characterization of Congo red and crystal violet Take 1 mL of biofilm solution and incubate it with 30 μL of crystal violet (0.1%) solution at 25 °C for 15 min. The amount of bound crystal violet is calculated by measuring the decrease in absorbance at 550 nm; this value represents the amount of biofilm biomass. Separately, take 1 mL of biofilm solution and incubate it with 10 μL of Congo red (25 mg / mL) solution at 25 °C for 15 min. The amount of bound Congo red is calculated by measuring the decrease in absorbance at 495 nm; this value represents the amount of curli fiber generated. For EB01 and... E. coli The binding amounts of BL21::ΔCsgA and BL21::ΔCsgA were compared to determine the formation of engineered biofilms.

[0048] (2) TEM and FE-SEM characterization The morphology and structure of biofilms were evaluated using TEM and FE-SEM. Biofilm solution was centrifuged at 8000 r / min for 2 min, the supernatant was discarded, and the sample was washed with PBS buffer and fixed overnight in 2.5% glutaraldehyde solution. The fixed sample was washed three times with PBS and dehydrated with anhydrous ethanol. The treated sample was then dropped onto a copper grid and observed after the anhydrous ethanol had completely evaporated. E. coli The biofilm formed by BL21::ΔCsgA was used as a control group to compare the morphological differences between the two.

[0049] 1.4 Results and Discussion (1) Construction of recombinant strains The plasmid map of recombinant plasmid pET21a-CsgB-CsgA(5×Lys) is shown below. Figure 1 As shown, five consecutive Lys codons were introduced at the C-terminus of the CsgA gene to provide more amino groups.

[0050] Using the pET21a-CsgB-CsgA plasmid previously constructed in the laboratory as a template, specific primers containing the 5×Lys mutation site were designed, and site-directed mutagenesis was performed using whole-plasmid PCR. Dpn After digestion of the template with enzyme I, the pET21a-CsgB-CsgA(5×Lys) plasmid was obtained. The plasmid was then transformed into... E. coli BL21::ΔCsgA competent cells were plated on LB solid medium containing 100 μg / mL Amp and incubated upside down at 37°C for 12 h. Single colonies were picked for positive clone verification, and the results are as follows. Figure 2 As shown, the band size is consistent with the theoretical value of 1136 bp, proving the successful construction of EB01. To further confirm the accuracy of the recombinant plasmid, the strains with correct verification results were sequenced. Strains with sequencing results completely identical to the map sequence were stored at -20℃ for future use in subsequent biofilm assembly and functional studies.

[0051] (2) Detection of curli fiber formation by Congo red and crystal violet staining Congo red (CR) binds to amyloid protein and is commonly used to determine the content of curli fiber; while crystal violet (CV) is used to determine the total amount of biomass. Both are used to assess EB01 biofilm and E. coli Images of BL21::ΔCsgA biofilm stained with Congo red and crystal violet are shown below. Figure 3 As shown, the EB01 biofilm exhibits stronger binding affinity for CR, resulting in a more pronounced staining effect. Further analysis using absorbance calculations revealed the relative binding amounts of the biofilm to both CR and CR. The results of the relative binding amounts are shown below. Figure 4 As shown, the results indicated no significant difference in CV binding amounts between the two biofilms, suggesting that their total biomass was similar, thus ruling out the interference of biomass on CR staining results. Meanwhile, the CV binding amount of the EB01 biofilm was significantly higher than that of the CR biofilm. E. coli BL21::ΔCsgA biofilm, indicating E. coli BL21::ΔCsgA gene knockout resulted in the absence of curli fibers, while pET21a(+)-CsgB-CsgA(5×Lys) was successfully secreted and assembled into functional curli fibers, which can be used for subsequent enzyme immobilization.

[0052] (3) TEM and SEM observation of biofilm morphology TEM and SEM E. coli The biofilm structures of strains BL21::ΔCsgA and EB01 were characterized, and TEM images of the biofilms are shown below. Figure 5 As shown, the SEM image of the biofilm is as follows. Figure 6 As shown. TEM analysis shows that, E. coli The biofilm structure of strain BL21::ΔCsgA is relatively loose, with a lack of obvious extracellular matrix connections between cells. In contrast, the biofilm of strain EB01 has abundant extracellular matrix, forming obvious fibrous material.

[0053] SEM characterization results further confirmed this difference. E. coli The surface structure of BL21::ΔCsgA is relatively sparse, with relatively independent cell structures and a lack of obvious extracellular material aggregation. In contrast, the biofilm of strain EB01 exhibits a dense fibrous mesh structure, and obvious extracellular material aggregation can be observed.

[0054] The above experimental results show that the significantly enriched curli nanofiber structure of the EB01 strain provides stronger structural stability and higher extracellular matrix content for its biofilm. This dense fiber mesh structure not only enhances the mechanical strength of the biofilm, but also provides ideal carrier support for the efficient assembly of enzymes, thereby significantly increasing the enzyme immobilization load and providing an important structural basis for enzyme immobilization using the EB01 strain.

[0055] Experiment 2 Preparation and Characterization of Immobilized Enzymes 2.1 Two-enzyme immobilization (1) Immobilization of β-galactosidase BgaD and L-arabinose isomerase CaLAI on EB01 biofilm was achieved through amino cross-linking with glutaraldehyde. The immobilization steps were as follows: 20 mg of EB01 biofilm was gently mixed with 200 μL of pure BgaD and CaLAI enzyme solution (total concentration of the two enzymes was 2 mg / mL, and the mass ratio of BgaD to CaLAI was 20:1), and glutaraldehyde solution was added to a final concentration of 0.4%. The mixture was then incubated at 18℃ and 150 r / min for 1.5 h for cross-linking. The immobilized enzymes were recovered by centrifugation, and the immobilized enzymes were washed with PBS buffer until no protein was detected in the supernatant.

[0056] (2) Determination of the loading capacity and enzyme activity of the immobilized dual enzymes The loading capacity of the two enzymes was determined using the Bradford method, specifically as follows: the absorbance of the supernatant before and after immobilization was measured at 595 nm, and the protein concentration was calculated using BSA as the standard protein, as shown in the following formula: .

[0057] Where c1 and c2 are the concentrations of the two enzymes before and after immobilization (mg / mL), V1 and V2 are the volumes of the two enzyme solutions before and after immobilization (mL), and M is the mass of the EB01 biofilm (g).

[0058] The activity of the immobilized dual enzymes was determined using a reported standard substrate (lactose), and the D-tagatose content of the product was determined using the cysteine-carbazole method. 20 mg of the immobilized dual enzymes were reacted with 2 g / L lactose as a substrate at 50°C for 2 h. The reaction solution was then subjected to colorimetric determination of the d-tagatose content. The enzyme activity recovery rate was calculated using the following formula: .

[0059] (3) Comparison of immobilization effects To compare the enzyme loading and relative activity of EB01 biofilm with other aminated materials, equal amounts of aminated resin and aminated silica microspheres were immobilized and reacted under the same conditions. The methods for determining enzyme loading and activity were the same as above. The experiment was conducted in triplicate, and the results were averaged.

[0060] 2.2 Exploration of Immobilization Conditions 2.2.1 Effect of glutaraldehyde concentration on immobilized dual enzymes 10 mg of biofilm was weighed and added to 200 μL of a dual-enzyme mixture (total concentration of the two enzymes was 2 mg / mL, and the mass ratio of BgaD to CaLAI was 20:1). Glutaraldehyde solution was added at final concentrations of 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1.0%, respectively. The mixture was incubated at 18℃ and 130 r / min for 1.5 h. The material was washed three times with PBS buffer to remove free glutaraldehyde and enzymes. The yield of D-tagatose, the product of the dual-enzyme cascade reaction, was measured. The highest yield was set as 100%, and other concentrations were expressed as relative yields to investigate the effect of glutaraldehyde concentration on immobilization efficiency. Three parallel experiments were performed, and the results were averaged.

[0061] 2.2.2 Effect of immobilization time on immobilized dual enzymes 10 mg of biofilm was weighed and added to 200 μL of a dual-enzyme mixture (total concentration of the two enzymes was 2 mg / mL, mass ratio of BgaD to CaLAI was 20:1), and a 0.4% glutaraldehyde solution. The mixture was incubated at 18℃ and 130 r / min for different times (1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h). The material was washed three times with PBS buffer to remove glutaraldehyde and free enzymes. The yield of D-tagatose, the product of the dual-enzyme cascade reaction, was measured, with the highest yield set as 100%, and other yields expressed as relative yields. Three parallel experiments were performed, and the results were averaged.

[0062] 2.2.3 Effect of the ratio of two enzymes on immobilized two enzymes 10 mg of biofilm was weighed and added to 200 μL of enzyme mixture (total concentration of both enzymes: 2 mg / mL) and 0.4% glutaraldehyde solution. Enzyme ratios of CaLAI:BgaD were set at 5:1, 10:1, 15:1, 20:1, 25:1, and 30:1 (mass ratio), and incubated at 18℃ and 130 r / min for 1.5 h. The material was washed three times with PBS buffer. The yield of D-tagatose, the product of the dual-enzyme cascade reaction, was measured, with the highest yield set as 100%. Other ratios were expressed as relative yields to investigate the effect of the enzyme ratio on immobilization efficiency. Three parallel experiments were performed, and the results were averaged.

[0063] 2.2.4 Effect of enzyme dosage on immobilized dual enzymes Weigh 10 mg of biofilm and add 200 μL of buffer solution containing a certain amount of free dual enzymes (total concentration of the two enzymes: 0.6 mg / mL–3.0 mg / mL; mass ratio of BgaD to CaLAI: 20:1), pH 8. The final concentration of glutaraldehyde in the system is 0.4%. Incubate at 18℃ and 130 r / min for 1.5 h. Wash the material three times with PBS buffer. Detect the yield of D-tagatose, the product of the dual enzyme cascade reaction. The highest yield was set as 100%, and other enzyme additions were expressed as relative yields to investigate the effect of enzyme addition on immobilization efficiency. Three parallel experiments were conducted, and the results were averaged.

[0064] 2.3 Enzymatic properties of immobilized dual enzymes 2.3.1 Optimal Temperature and Temperature Stability Determination of optimal temperature: Using 2 g / L lactose as a substrate, an appropriate amount of immobilized dual enzymes (prepared under the optimized conditions described above, i.e., 10 mg of biofilm, 0.4% glutaraldehyde, and 1.5 h immobilization time; the total concentration of the two enzymes in 200 μL of enzyme solution was 2 mg / mL, and the mass ratio of BgaD to CaLAI was 20:1) were added. Reactions were carried out at 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, and 70℃. The yield of D-tagatose, the product of the dual enzyme cascade reaction, was measured. The highest yield was set as 100%, and the yields at other temperatures were expressed as a percentage of the highest yield. Three parallel experiments were performed, and the average value of the results was taken.

[0065] Temperature stability determination: Immobilized or free dual enzymes were incubated at 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃ for 2 h, respectively. Using 2 g / L lactose as a substrate, the yield of the cascade reaction product D-tagatose was measured at different temperatures. The yield of unincubated products was set as 100%. Three parallel experiments were conducted, and the average value of the results was taken.

[0066] 2.3.2 Optimal reaction pH and pH stability Determination of optimal pH: Using 2 g / L lactose as a substrate, appropriate amounts of immobilized or free dual enzymes were added. Reactions were carried out at pH 5, pH 6, pH 7, pH 8, pH 9, and pH 10, respectively. The yield of D-tagatose, the product of the dual enzyme cascade reaction, was measured. The highest yield was set as 100%, and the yields at other pH values ​​were expressed as a percentage of the highest yield. Three parallel experiments were conducted, and the results were averaged.

[0067] pH stability determination: Immobilized or free EB01 enzymes were incubated at pH 5–10 for 2 h. Using 2 g / L lactose as substrate, the yield of D-tagatose, the cascade reaction product, was measured at different pH values. The yield of unincubated enzymes was set as 100%. Three parallel experiments were conducted, and the average value was taken.

[0068] 2.3.3 Operational stability The operational stability of the immobilized dual enzymes was determined using a repeated batch reaction method. After each reaction, the reaction system was centrifuged at 8000 r / min for 2 min, and the supernatant was collected for product detection. Simultaneously, the immobilized enzymes were collected and washed twice with PBS buffer to remove residual reaction products and unreacted substrate. Fresh substrate solution was added to the washed immobilized dual enzymes, and the reaction was continued for 3 h under the initial conditions. This process was repeated until the D-tagatose yield of the immobilized dual enzymes significantly decreased. Three parallel experiments were conducted, with the product yield of the first reaction considered as 100%, and the average results were taken.

[0069] 2.3.4 Storage stability Take an appropriate amount of immobilized or free dual enzymes and store them in a refrigerator at 4°C. Samples are taken every 4 days, and enzyme activity changes are measured using a standard reaction system. With the initial product yield as 100%, three parallel experiments are performed for each measurement, and the average result is taken. By plotting the product yield change over storage time and comparing the storage stability differences between immobilized and free dual enzymes, the protective effect of immobilization treatment on the conformational stability of enzyme molecules can be further elucidated.

[0070] 2.4 Application of immobilized dual enzymes in agarose cascade reaction pathways Take the immobilized dual enzymes, and... α- A mixture of agarose oligosaccharides prepared by agarase AgaA33 degradation of agarose was used as a substrate and reacted at 50℃. Samples were taken at 2 h, 4 h, 6 h, 9 h, 12 h, and 16 h of reaction time for HPLC analysis to determine the concentrations of NA3 and D-tagatose at different time points. Simultaneously, the operational stability of the immobilized dual enzymes was determined using this mixture as a substrate via a batch reaction method.

[0071] 2.5 Results and Discussion 2.5.1 Enzyme activity assay and comparison of immobilized dual enzymes (1) The effects of two immobilization methods on the immobilization effect were compared: direct immobilization of the biofilm material and immobilization after centrifugation. The results showed that the enzyme loading of direct immobilization was 39.88 mg / g, and the enzyme activity recovery rate was 37.27%. The enzyme loading of immobilization after centrifugation was 27.50 mg / g, and the enzyme activity recovery rate was 26.15%. Direct immobilization was significantly better than immobilization after centrifugation. This result may be closely related to the nanoporous structure characteristics of the biofilm material. Direct immobilization avoids the potential damage to the material structure caused by mechanical force during centrifugation, effectively protecting the integrity of the nanoporous structure, thus providing more binding sites for enzyme molecules and helping to maintain their native conformation and catalytic activity. In contrast, the mechanical force applied during centrifugation may cause the collapse or destruction of the nanoporous structure, ultimately leading to a decrease in enzyme loading. These results provide an important reference for optimizing the enzyme immobilization process. During immobilization, mechanical damage to the carrier material should be minimized to maximize the preservation of its structural integrity and functional properties.

[0072] (2) To systematically evaluate the enzyme immobilization performance of different aminated materials, the enzyme loading and enzyme activity recovery rates of three materials—EB01 biofilm, LX amino resin 1000HA, and aminated silica microspheres—were compared. The experimental results showed that the EB01 biofilm exhibited the best immobilization performance in terms of enzyme loading and enzyme activity recovery rate, with an enzyme loading of 39.88 mg / g, significantly higher than LX amino resin 1000HA (19.82%) and aminated silica microspheres (15.03%). Simultaneously, the enzyme activity recovery rate of 37.27% was also significantly better than LX amino resin 1000HA (15.90%) and aminated silica microspheres (22.36%). The excellent immobilization performance of the EB01 biofilm can be attributed to the following factors: First, its unique nanoporous structure provides a large specific surface area and uniform pore size distribution, which not only significantly improves the enzyme loading capacity but also facilitates mass transfer between substrate and product. Secondly, the stable chemical environment formed by the biofilm effectively buffers changes in the external environment, providing a favorable microenvironment for enzyme molecules and helping to maintain the enzyme's native conformation and catalytic activity. In contrast, LX amino resin 1000HA and aminated silica microspheres may have lower enzyme loading efficiency due to uneven pore size distribution or poor compatibility between surface functional groups and enzyme molecules. These findings indicate that the EB01 biofilm, as a novel enzyme immobilization carrier, has broad application prospects in the field of enzyme engineering.

[0073] 2.5.2 Optimization of Immobilization Conditions 2.5.2.1 Effect of glutaraldehyde concentration on immobilized dual enzymes In enzyme immobilization using glutaraldehyde, optimizing the glutaraldehyde concentration is crucial because it directly affects the catalytic activity and operational stability of the immobilized enzyme. The effect of glutaraldehyde concentration on the activity of immobilized dual enzymes is as follows: Figure 7 As shown, the catalytic activity of the immobilized dual enzymes significantly increased with the glutaraldehyde concentration from 0.05% to 0.4% (v / v). This increased activity is attributed to the enhanced binding strength between the enzyme molecules and the carrier material due to moderate cross-linking, while maintaining the conformational integrity of the enzyme's active site. When the glutaraldehyde concentration exceeded 0.4%, the relative yield of the product decreased. This may be because at higher concentrations of glutaraldehyde, excessive cross-linking occurs between enzyme molecules, disrupting the enzyme's three-dimensional structure and limiting the contact between the enzyme's active site and the substrate, thus reducing the enzyme's catalytic efficiency. Based on the above analysis, a final concentration of 0.4% glutaraldehyde was selected for immobilization.

[0074] 2.5.2.2 Effect of immobilization time on immobilized dual enzymes Immobilization time is one of the key parameters affecting enzyme immobilization efficiency. The effect of immobilization time on the activity of immobilized dual enzymes is as follows: Figure 8 As shown in the figure, the experimental results indicate that the catalytic activity of the immobilized dual enzymes reaches its maximum when the cross-linking time is 1.5 h. However, with further extension of the immobilization time, the yield of D-tagatose gradually decreases. This may be because as the cross-linking time increases, covalent bonds gradually form between the enzyme molecules and the carrier, which helps to improve the immobilization efficiency and stability of the enzyme. When the immobilization time exceeds 1.5 h, the excessively long cross-linking time leads to the formation of too many covalent bonds between the enzyme molecules and the carrier, limiting the enzyme activity. At the same time, glutaraldehyde may cause conformational changes in the enzyme molecules. Excessive conformational changes destroy the active site of the enzyme, leading to a decrease in activity. Therefore, 1.5 h is the optimal immobilization time.

[0075] 2.5.2.3 Effect of the ratio of two enzymes on immobilized two enzymes Since the ratio and amount of enzymes added affect the efficiency of the cascade reaction and the activity of the immobilized enzyme, the ratio and amount of the two enzymes were optimized. The effect of the ratio of the two enzymes on the activity of the immobilized two enzymes is as follows: Figure 9As shown, with the increase of the relative content of CaLAI, the catalytic activity of the immobilized dual enzymes exhibited a trend of first increasing and then slightly decreasing. The D-tagatose yield reached its maximum when the CaLAI:BgaD ratio reached 20:1. This may be because, with a low proportion of CaLAI, its enzyme activity became the rate-limiting step in the cascade reaction. With the increase of the CaLAI ratio, the catalytic activities of the two enzymes reached a better balance, thereby improving the overall reaction efficiency. However, excessive CaLAI may compete with BgaD for binding sites and create steric hindrance, leading to a decrease in catalytic efficiency. Based on the above analysis, the optimal enzyme ratio CaLAI:BgaD was determined to be 20:1. This ratio ensures the synergistic effect of the two enzymes in the cascade reaction while avoiding competition or inhibition.

[0076] 2.5.2.4 Effect of enzyme dosage on immobilized dual enzymes The amount of enzyme added is another key parameter affecting the performance of immobilized enzymes. The effect of the amount of enzyme added on the activity of immobilized dual enzymes is as follows: Figure 10 As shown, the yield of D-tagatose increased with increasing enzyme concentration, but the catalytic activity decreased with increasing enzyme concentration when the concentration exceeded 2 mg / mL. At lower enzyme concentrations, the binding sites on the carrier surface were not yet saturated, and increasing the enzyme concentration could improve the overall activity of the immobilized dual enzymes. However, studies have shown that excessive enzyme enrichment on the carrier surface can hinder the enzyme's active sites, thereby reducing the enzyme's catalytic efficiency and leading to decreased enzyme activity. Simultaneously, excessively high enzyme concentrations may form a concentration gradient on the carrier surface, potentially hindering substrate diffusion to the enzyme's active site. Therefore, the optimal enzyme concentration was 2 mg / mL, at which point the enzyme loading was 40 mg / g.

[0077] 2.5.3 Study on the enzymatic properties of free and immobilized BgaD & CaLAI 2.5.3.1 Optimal Temperature The effect of reaction temperature on the catalytic activity of free dual enzymes (BgaD & CaLAI) and immobilized dual enzymes is as follows: Figure 11As shown, the catalytic activity of both the free and immobilized dual enzymes increases with increasing temperature. This is because higher temperatures typically increase molecular velocity, thereby increasing the frequency of enzyme-substrate collisions and the reaction rate. The optimal reaction temperature for the immobilized dual enzymes is 50℃, lower than the 55℃ of the free dual enzymes. This may be because the immobilization process alters the enzyme's spatial conformation, increasing its temperature sensitivity. However, when the temperature exceeds 60℃, the activity changes of the free and immobilized dual enzymes show a significant difference. The activity of the free dual enzymes decreases by 55.66%, while the immobilized dual enzymes only decrease by 9.88%, showing a significantly smaller loss of activity. This indicates that immobilization improves the enzyme's thermal stability, reducing its sensitivity to temperature changes at high temperatures and minimizing the possibility of thermal damage to the enzyme structure. This can be attributed to the EB01 biofilm providing a protective microenvironment for the enzyme molecules, reducing the direct damage to the enzyme structure caused by high temperatures. Simultaneously, the covalent bonds formed during immobilization also help maintain the enzyme's conformation. The results show that immobilization can not only regulate the optimal reaction temperature of enzymes, but also significantly enhance their thermal stability, which is of great significance for industrial catalytic reactions carried out under high temperature conditions.

[0078] 2.5.3.2 Optimal pH The effect of reaction pH on the activities of free and immobilized dual enzymes, such as Figure 12 As shown, the optimal pH for both the immobilized and free dual enzymes was 8.0, and this optimal pH remained unchanged after immobilization. However, in an acidic environment of pH 5–6, the immobilized dual enzyme still retained over 60% of its D-tagatose yield, while the free dual enzyme retained only about 30% of its D-tagatose yield. This indicates that the co-immobilized dual enzymes have a wider pH tolerance range. The results suggest that biofilms can provide a more stable microenvironment for enzymes, reducing the impact of external pH changes on enzyme activity.

[0079] 2.5.3.3 Temperature stability Temperature stability is an important indicator for evaluating enzyme performance in practical applications. To investigate the stability of free and immobilized dual enzymes at different temperatures, the remaining activity of both enzymes was measured after incubation at 40℃–70℃ for 2 h. The results of the temperature stability measurements for the free and immobilized dual enzymes are as follows: Figure 13As shown, the residual catalytic activity of both enzymes decreased with increasing temperature. Within the temperature range of 40℃ to 55℃, the difference between the two was small, with the immobilized dual enzyme activity slightly higher than the free dual enzyme. However, at temperatures above 55℃, the yield of the free dual enzyme decreased by 63.7%, while the immobilized dual enzyme only decreased by 19.17%, indicating a significantly lower decrease in catalytic activity for the immobilized dual enzyme. After treatment at 70℃ for 2 hours, the co-immobilized dual enzyme still retained 40% of its yield, while the free dual enzyme was almost completely inactivated. This is because the co-immobilization process forms multi-point covalent bonds between the enzyme and the carrier, enhancing the stability of the enzyme molecule; simultaneously, the E. coli biofilm provides a stable microenvironment for the enzyme, reducing the damage to the enzyme structure caused by heat and giving it better thermal stability.

[0080] 2.5.3.4 pH stability The immobilized and free enzymes were incubated in buffer solutions ranging from pH 5 to 10 for 2 h, and the relative yields of their products at different pH values ​​were measured. The results of the pH stability determination for the free and immobilized enzymes are shown below. Figure 14 As shown, both the free and immobilized dual enzymes were relatively stable at pH 7–8, with minimal changes in D-tagatose production. Within other pH ranges, the immobilized dual enzymes exhibited higher catalytic activity than the free enzymes. At pH 5–6, the immobilized dual enzymes maintained 70.95% and 74.72% of their production, respectively, while the free enzymes only maintained 33.20% and 44.64% of their production. This indicates that immobilization significantly improved the pH stability of the dual enzymes, giving them a wider pH tolerance and demonstrating that the *E. coli* biofilm plays a role in protecting the enzyme's active site under acidic and alkaline conditions.

[0081] 2.5.3.5 Operational stability The reusability of immobilized enzymes is of great significance in industrial production. Because immobilized enzymes can be used multiple times to catalyze reactions, production costs can be significantly reduced; simultaneously, enzyme residue in the product can be minimized, improving production efficiency and product quality. Therefore, to further investigate the performance of the co-immobilized dual enzymes, the operational stability of the EB01 biofilm was studied. The results of the operational stability determination of the immobilized dual enzymes are as follows: Figure 15 As shown, the immobilized dual enzymes maintained over 70% of the initial product yield after seven cycles of catalytic reaction. This is significantly superior to previously reported hybrid crystal co-immobilization. β The galactosidase and l-arabinose isomerase system retains only 50% of the initial yield in the relative conversion of lactose to D-tagatose after the same number of cycles. The good operational stability of the co-immobilized dual enzymes is likely due to the EB01 biofilm's three-dimensional network, which combines good mechanical strength and structural flexibility. This network firmly anchors the enzymes to the surface while reducing enzyme detachment and denaturation under mechanical forces, allowing for repeated use.

[0082] 2.4.3.6 Storage stability Storage stability is an important indicator for evaluating the long-term preservation performance of enzymes in practical applications. To study the storage stability of free and immobilized dual enzymes under low-temperature conditions, both enzymes were stored at 4°C, and their catalytic activity was measured every 4 days. The results of the storage stability determination for the free and immobilized enzymes are as follows: Figure 16 As shown in the figure, the experimental results indicate that the immobilized dual enzymes exhibited significantly better stability than the free dual enzymes throughout the storage period. In the initial storage phase, the catalytic activities of both the free and immobilized dual enzymes remained relatively stable. However, starting from day 12, the activity of the free dual enzymes decreased significantly, while the activity of the immobilized dual enzymes remained relatively stable throughout the storage period. This is mainly because the rigid structure of the curli biofilm provides physical protection for the enzymes, helping to maintain their active conformation and giving the immobilized dual enzymes better storage stability, providing a significant advantage for applications requiring long-term storage.

[0083] 2.5.4 Application of immobilized dual enzymes in agarose cascade reaction pathway Immobilized enzyme technology has significant advantages in industrial catalysis due to its ease of separation and recovery from reaction systems, good operational stability, and wider temperature and pH tolerance range. This invention utilizes... α A4, prepared using agarase AgaA33, was used as a substrate and co-immobilized on the EB01 biofilm. β -Galactosidase BgaD and l-Al CaLAI enabled the efficient preparation of NA3 and D-tagatose. In the preparation reactions of NA3 and D-tagatose, the operational stability of the immobilized dual enzymes was as follows: Figure 17 As shown in the figure. After 5 cycles, the yields of NA3 and D-tagatose remained at 79.58% and 75.72% of their initial yields, respectively, indicating that the co-immobilized dual enzymes have good reusability. This high stability may be attributed to the protective microenvironment provided by the biofilm carrier and the stable covalent bonds formed between the enzyme and the carrier, effectively reducing enzyme inactivation during recycling. Using the prepared A4 as a substrate, the reaction time curve is shown in the figure. Figure 18 As shown, the product yield increased rapidly before 6 hours of reaction, reaching equilibrium at 12 hours. Quantitative analysis using A3 standard showed that the NA3 yield was 0.25 g / L, while the D-tagatose yield was 0.10 g / L. These results indicate that the co-immobilized enzyme can efficiently catalyze the reaction in a short time, effectively achieving the preparation of NA3 and d-tagatose.

[0084] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for preparing an EB01 biofilm, characterized in that: A recombinant expression vector CsgB-CsgA (5×Lys) was constructed and introduced into Escherichia coli to obtain engineered Escherichia coli; the engineered Escherichia coli was cultured to obtain a biofilm; the nucleotide sequence of CsgB-CsgA (5×Lys) is shown in SEQ ID NO.

5.

2. The method for preparing the EB01 biofilm according to claim 1, characterized in that, The specific method for culturing engineered Escherichia coli is as follows: The engineered Escherichia coli is activated in LB liquid medium containing 100 μg / mL Amp to obtain a seed culture; the seed culture is inoculated at a rate of 2% into 100 mL of M63 medium containing 100 μg / mL Amp, and cultured at 37°C in a shaker until the bacterial culture reaches OD. 600 The concentration of IPTG was 0.5, and a final concentration of 0.3 mM was added. The mixture was cultured in a constant temperature shaker at 30℃ for 48 h to induce the production of the target curli fibers. After induction, the mixture was left to stand at room temperature for 12 h. The curli fibers self-assembled to form the EB01 biofilm, which is then obtained.

3. The EB01 biofilm prepared using the preparation method of the EB01 biofilm according to claim 1 or 2.

4. The use of the EB01 biofilm according to claim 3 in the preparation of immobilized enzymes, or in its use as a carrier for immobilized enzymes.

5. An immobilized dual enzyme, characterized in that: It is prepared by loading β-galactosidase BgaD and L-arabinose isomerase CaLAI onto the EB01 biofilm as described in claim 3.

6. The method for preparing the immobilized dual enzymes according to claim 5, characterized in that: Take 10 mg of EB01 biofilm, add 200 μL of a two-enzyme mixture containing β-galactosidase BgaD and L-arabinose isomerase CaLAI, add glutaraldehyde solution to make the concentration 0.4%, and incubate at 18℃ and 130 r / min for 1.5 h to obtain the product; wherein, in the two-enzyme mixture, the total concentration of the two enzymes is 2 mg / mL, and the mass ratio of β-galactosidase BgaD to L-arabinose isomerase CaLAI is 20:

1.

7. The use of the immobilized dual enzymes according to claim 5 in the preparation of neo-agarotriose and D-tagatose.

8. The application according to claim 7, characterized in that: In practical applications, α- A mixture of agarose oligosaccharides prepared by agarase AgaA33 degradation of agarose was used as a substrate and reacted at 50°C for 6–12 h.

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