Method for preparing chitobiose by double-enzyme catalysis method
Chitobiose is prepared by the dual enzyme catalytic method, using natural raw materials such as shrimp shells, and using recombinantly expressed chitin hydrolase and deacetylase for enzyme reaction, solving the problem of efficient preparation of high-purity chitobiose and achieving high yield and low cost green production.
Patent Information
- Application Number
- CN202510299478.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-13
- Publication Date
- 2025-07-11
AI Technical Summary
It is difficult to efficiently prepare high-purity specific structural chitobiose in the prior art, and chemical synthesis methods have a greater impact on the environment and are costly.
Chitin hydrolase and chitin deacetylase were prepared by recombinant expression using the dual enzyme catalysis method, and natural raw materials such as shrimp shells were used to carry out enzyme reaction in the buffer system to prepare chitobiose.
Significantly improves the yield and purity of chitosaccharide, reduces environmental pollution, is suitable for large-scale production, and is suitable for food and medical fields.
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Figure CN120290665A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of green biosynthesis by multi-enzyme catalysis, and relates to a method for preparing chitobiose by double-enzyme catalysis, and particularly relates to a method for preparing two specific-structured chitobioses by tandem enzyme catalysis from shrimp shells, crab shells or colloidal chitin. Background Art
[0002] Chitin is a natural polysaccharide, mainly existing in the cell walls of fungi, the exoskeletons of crustaceans and insects. It is the second largest polysaccharide in the world after cellulose, and about 10 6 tons of chitin are produced annually in the marine products industry. Chitin is a polysaccharide connected by β-1,4 glycosidic bonds, mainly composed of N-acetylglucosamine (GlcNAc). After deacetylation of chitin, chitosan is obtained, and its basic structural unit is glucosamine (Glucosamine, GlcN). After chitosan is depolymerized by acid hydrolysis, physical hydrolysis or enzymatic hydrolysis, it becomes chitosan oligosaccharides. However, due to the difficulty in controlling the degree and site of deacetylation, the current chitosan oligosaccharide products are mostly mixtures of different structures. Preparing chitosan oligosaccharides with specific structures is beneficial to the development of their functions, the study of action mechanisms, and the application and promotion in different fields such as agriculture, food, and medicine.
[0003] Chitobiose is a chitosan oligosaccharide with a polymerization degree of 2, usually a disaccharide with a specific structure formed by connecting two β-D-glucosamines and / or β-D-N-acetylglucosamines through β-1,4-glycosidic bonds. Its chemical structure can be expressed as GlcNAc-β(1→4)-GlcNAc (abbreviated as AA), GlcN-β(1→4)-GlcNAc (abbreviated as DA), GlcNAc-β(1→4)-GlcN (abbreviated as AD), GlcN-β(1→4)-GlcN (abbreviated as DD). This specific structure endows chitobiose with unique properties and functions. The amino and hydroxyl functional groups on the glucosamine unit endow it with certain reactivity and biological activity. For example, it can be chemically modified such as acetylation and quaternization, thereby changing its physicochemical properties and biological functions. With this specific structure, specific-structured chitobiose can participate in physiological processes such as cell recognition and signal transduction in the biomedical field; in the agricultural field, it can bind to plant cell surface receptors, triggering the immune response and growth regulation mechanism of plants; in the fields of food preservation and healthcare, it also shows important application value due to its antioxidant function.
[0004] The activity of chitosan oligosaccharide is closely related to its structure, such as degree of polymerization, degree of deacetylation, deacetylation site, etc. At present, chitobiose (AA and DD) can be obtained by chemical or enzymatic hydrolysis and separation and purification, while chitobiose with specific structure (AD and DA) can only be obtained through complex chemical synthesis steps and has a greater impact on the environment. AD and DA can be prepared by chemical or enzymatic deacetylation reaction using AA as a substrate. However, the process has the limitation of high cost. In contrast, using natural renewable resources such as shrimp shells as raw materials to directly obtain chitobiose with specific structure has great industrial application value. Summary of the Invention
[0005] In order to solve the above technical problems existing in the background art, the present invention provides a method for preparing chitobiose by double enzyme catalysis, which can significantly improve the yield and purity of chitobiose and effectively reduce environmental pollution.
[0006] In order to achieve the above object, the present invention adopts the following technical scheme:
[0007] A method for preparing chitobiose by double enzyme catalysis, characterized in that: the method for preparing chitobiose by double enzyme catalysis includes the following steps:
[0008] 1) Obtain a substrate containing chitin;
[0009] 2) Prepare chitinase and chitin deacetylase by recombinant expression;
[0010] 3) Carry out an enzymatic reaction on the substrate containing chitin obtained in step 1) with the chitinase and chitin deacetylase obtained in step 2) in a buffer system to obtain chitobiose.
[0011] The substrate containing chitin in the above step 1) is shrimp shell, crab shell and / or colloidal chitin.
[0012] The chitinase in the above step 2) is chitinase MBP-BliCHI; the chitin deacetylase is MBP-NodB or MBP-VcCDA.
[0013] The preparation method of the above chitinase MBP-BliCHI is:
[0014] a.1) Construct a recombinant plasmid pMAL-p5x-BliCHI;
[0015] a.2) Transform the recombinant plasmid pMAL-p5x-BliCHI constructed in step a.1) into the Escherichia coli expression strain BL21(DE3), pick monoclonal colonies, expand the culture, induce expression with IPTG, centrifuge to collect the bacteria, ultrasonically disrupt, and centrifuge to collect the supernatant;
[0016] a.3) Analyze the product obtained in step a.2) by SDS-PAGE and purify it through a Ni-NTA affinity chromatography purification column;
[0017] a.4) Dialyze the product obtained in step a.3) overnight in Tris-NaCl buffer to obtain chitinase MBP-BliCHI.
[0018] The preparation method of the above chitin deacetylase MBP-NodB is as follows:
[0019] b.1) Construct the recombinant plasmid pMAL-p5x-NodB;
[0020] b.2) Transform the recombinant plasmid pMAL-p5x-NodB constructed in step b.1) into Escherichia coli BL21(DE3), pick a single clone, and perform large-scale culture; induce expression with IPTG, collect the bacterial cells by centrifugation, disrupt them by sonication, and collect the supernatant by centrifugation;
[0021] b.3) Analyze the product obtained in step b.2) by SDS-PAGE and purify it through a Ni-NTA affinity chromatography purification column;
[0022] b.4) Dialyze the product obtained in step b.3) overnight in Tris-NaCl buffer to obtain chitin deacetylase MBP-NodB.
[0023] The preparation method of the above chitin deacetylase MBP-VcCDA is as follows:
[0024] c.1) Construct the recombinant plasmid pMAL-p5x-VcCDA;
[0025] c.2) Transform the recombinant plasmid pMAL-p5x-VcCDA constructed in step c.1) into Escherichia coli BL21(DE3), pick a single clone, and perform large-scale culture; induce expression with IPTG, collect the bacterial cells by centrifugation, disrupt them by sonication, and collect the supernatant by centrifugation;
[0026] c.3) Analyze the product obtained in step c.2) by SDS-PAGE and purify it through a Ni-NTA affinity chromatography purification column;
[0027] c.4) Dialyze the product obtained in step c.3) overnight in Tris-NaCl buffer to obtain chitin deacetylase MBP-VcCDA.
[0028] When the above chitin deacetylase is MBP-NodB, the specific implementation method of step 3) is:
[0029] Mix chitinase MBP-BliCHI, chitin deacetylase MBP-NodB and a chitin-containing substrate in proportion to obtain a mixture. Place the mixture in a buffer for an enzymatic reaction, followed by desalting and lyophilization to finally obtain chitobiose DA with a purity of not less than 90%.
[0030] The above chitinase MBP-BliCHI, chitin deacetylase MBP-NodB and the chitin-containing substrate are mixed in a mass ratio of 1:1:100 - 1000; the buffer is 50 mM Tris-HCl or 50 mM ammonium bicarbonate; the pH of the buffer is 8.0; the time of the enzymatic reaction is 0.5 h - 72 h; the reaction temperature of the enzymatic reaction is 30 - 40 °C.
[0031] When the above chitin deacetylase is chitin deacetylase MBP-VcCDA, the specific implementation method of step 3) is: Mix chitinase MBP-BliCHI, chitin deacetylase MBP-VcCDA and a chitin-containing substrate in proportion to obtain a mixture. Place the mixture in a buffer for an enzymatic reaction, followed by desalting and lyophilization to finally obtain chitobiose AD with a purity of not less than 90%.
[0032] The above chitinase MBP-BliCHI, chitin deacetylase MBP-VcCDA and the chitin-containing substrate are mixed in a mass ratio of 1:1:100 - 1000; the buffer is 50 mM PBS or 50 mM ammonium bicarbonate; the pH of the buffer is 8.0; the time of the enzymatic reaction is 0.5 h - 72 h; the reaction temperature of the enzymatic reaction is 30 - 40 °C.
[0033] The advantages of the present invention are:
[0034] The present invention provides a method for preparing chitobiose by double enzyme catalysis, which includes: 1) obtaining a substrate containing chitin; 2) preparing chitin hydrolase and chitin deacetylase by recombinant expression; 3) performing an enzymatic reaction on the chitin-containing substrate obtained in step 1) with the chitin hydrolase and chitin deacetylase obtained in step 2) in a buffer system to obtain chitobiose. The two specific structural chitobioses produced by the present invention can be directly obtained by combining chitin hydrolase MBP-BliCHI with chitin deacetylase MBP-NodB or chitin deacetylase MBP-VcCDA, using shrimp shell powder, crab shell powder or colloidal chitin as the substrate. That is, by using the abundant and inexpensive raw materials of shrimp shells and crab shells in nature, in a way of enzymatic tandem catalysis, two specific structural chitobioses GlcN-β(1→4)-GlcNAc and GlcNAc-β(1→4)-GlcN with high value and purity up to over 90% can be directly obtained in one step, significantly improving the yield and purity of chitobiose, and reducing environmental pollution at the same time. The method of the present invention has the advantages of high efficiency, environmental protection and low cost, and the product purity is up to over 90%. It is suitable for large-scale production and has broad application prospects. Because of its good safety and biocompatibility, it can also be extended to the food and medical fields. Description of the Drawings
[0035] Figure 1 It is a diagram of the protein expression and purification results of chitin hydrolase MBP-BliCHI;
[0036] Figure 2 It is a diagram of the protein expression and purification results of chitin deacetylase MBP-NodB;
[0037] Figure 3 It is a diagram of the protein expression and purification results of chitin deacetylase MBP-VcCDA;
[0038] Figure 4 It is a high-resolution mass spectrum diagram of the product after the tandem catalytic reaction of chitin by chitin hydrolase MBP-BliCHI and chitin deacetylase MBP-NodB, and the product is DA;
[0039] Figure 5 It is a high-resolution mass spectrum diagram of the product after the tandem catalytic reaction of chitin by the combination of chitin hydrolase MBP-BliCHI and chitin deacetylase MBP-NodB, and the product is AD;
[0040] Figure 6 It is a TLC diagram of the products A and AA of the single catalytic reaction of chitin hydrolase MBP-BliCHI;
[0041] Figure 7It is a TLC comparison chart of the products AD and DA of the tandem catalytic reaction of the chitin hydrolase MBP-BliCHI and the chitin deacetylase MBP-NodB or chitin deacetylase MBP-VcCDA;
[0042] Figure 8 The diagram is a schematic diagram of the method for preparing chitobiose by the double enzyme catalysis method provided by the present invention. DETAILED DESCRIPTION
[0043] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.
[0044] Example 1: Expression and purification of chitin hydrolase MBP-BliCHI
[0045] First, the pMAL-p5x-BliCHI plasmid was constructed (pMAL-p5x has its own MBP tag that can promote soluble protein expression), and the Bacillus licheniformis DSM13 strain BliCHI gene was obtained according to the GenBanK database, with the NCBI number ACW83016.1, and sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis (1710 bp). Then, it was connected with pMAL-p5x through the BamHI and EcoRI restriction enzyme sites to obtain the pMAL-p5x-MBP-BliCHI recombinant vector.
[0046] The recombinant plasmid pMAL-p5x-BliCHI with correct sequencing was transformed into Escherichia coli BL21 (DE3), and a single clone was picked and inoculated into 10 mL LB liquid medium (containing 100 μg / mL ampicillin), and cultured overnight at 200 r / min and 37°C. The next day, the culture was expanded according to the inoculum size of 2%-5% (exemplarily, the inoculum size of 4% was used in the present invention), and cultured in a shaking incubator at 37°C and 200 r / min until the OD 600 When the OD is 0.6 to 0.8 (for example, the OD finally achieved in this embodiment 6000.72), add IPTG to a final concentration of 0.2mM, and induce for 16h at 16℃ and 200r / min. Centrifuge at 4℃ and 4000r / min for 10min, discard the supernatant, and collect the bacteria. Add an appropriate amount of PBS to resuspend the bacteria, freeze and thaw repeatedly in liquid nitrogen and 37℃ for 3 times, and ultrasonically disrupt for 30min. Centrifuge at 4℃ and 10000r / min for 30min, collect the supernatant for SDS-PAGE analysis and pass through Ni-NTA affinity chromatography purification column. The fusion protein MBP-BliCHI bound to the Ni-NTA affinity chromatography purification column was eluted with different concentrations of imidazole (10mM, 25mM, 50mM, 100mM, 500mM), dialyzed overnight in Tris-NaCl buffer, and the concentration was determined by BCA protein concentration determination kit, 10% glycerol was added and stored at -80℃ for later use. The Tris-NaCl buffer solution includes HCl with a final concentration of 100 mM Tris and NaCl with a final concentration of 200 mM, and the pH of the Tris-NaCl buffer solution is 7.5. The purification results are shown in Figure 1 As shown, MBP-BliCHI contains an MBP tag, BI represents protein before induction, AI represents protein after induction, SP represents soluble protein, and PP represents purified protein.
[0047] Example 2: Expression and purification of chitin deacetylase MBP-NodB
[0048] First, the pMAL-p5x-NodB plasmid was constructed (pMAL-p5x has its own MBP tag that can promote soluble protein expression), and the NodB gene of Rhizobium sp.GRH2 strain was obtained according to the GenBank database, with the NCBI number AJW76244.1, and sent to Shanghai Biotechnology Co., Ltd. for synthesis (666 bp). Then, it was cloned into the pMAL-p5x plasmid through BamHI and EcoRI to obtain the pMAL-p5x-NodB recombinant vector.
[0049] The recombinant plasmid pMAL-p5x-NodB with correct sequencing was transformed into Escherichia coli BL21 (DE3), and a single clone was picked and inoculated into 10 mL LB liquid medium (containing 100 μg / mL ampicillin), and cultured overnight at 200 r / min and 37°C. The next day, the culture was expanded according to the inoculation amount of 2%-5% (exemplarily, the inoculation amount in this example is 3%), and cultured in a shaking incubator at 37°C and 200 r / min until the OD 600 When OD is 0.6 to 0.8 (for example, OD 6000.67), add IPTG to a final concentration of 0.2mM, and induce for 16h at 16℃ and 200r / min. Subsequently, centrifuge for 10min at 4℃ and 4000r / min, discard the supernatant, and collect the bacteria. Add an appropriate amount of PBS to resuspend the bacteria, freeze and thaw repeatedly in liquid nitrogen and 37℃ for 3 times, and ultrasonically disrupt for 30min. Centrifuge for 30min at 4℃ and 10000r / min, collect the supernatant for SDS-PAGE analysis and pass through Ni-NTA affinity chromatography purification column. The fusion protein MBP-NodB bound to the Ni-NTA affinity chromatography purification column was eluted with different concentrations of imidazole (10mM, 25mM, 50mM, 100mM, 500mM), dialyzed overnight in Tris-NaCl buffer, and the concentration was determined by BCA protein concentration determination kit, 10% glycerol was added and stored at -80℃ for use. The Tris-NaCl buffer solution includes HCl with a final concentration of 100 mM Tris and NaCl with a final concentration of 200 mM, and the pH of the Tris-NaCl buffer solution is 7.5. The purification results are shown in Figure 2 As shown, MBP-NodB contains an MBP tag and PP indicates the purified protein.
[0050] Example 3: Expression and purification of chitin deacetylase MBP-VcCDA
[0051] First, the pMAL-p5x-VcCDA plasmid was constructed (pMAL-p5x has its own MBP tag that can promote soluble protein expression), and the VcCDA gene of Vibrio cholerae strain was obtained according to the GenBanK database, with the NCBI number AAF94439.1, and sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis (1293 bp). Then, it was cloned into the pMAL-p5x plasmid through BamHI and EcoRI to obtain the pMAL-p5x-VcCDA recombinant vector.
[0052] The correctly sequenced recombinant plasmid pMAL-p5x-VcCDA was transformed into Escherichia coli BL21 (DE3), and a single clone was picked and inoculated into 10 mL LB liquid medium (containing 100 μg / mL ampicillin), and cultured at 200 r / min and 37°C overnight. The next day, the culture was expanded according to the inoculum size of 2%-5% (exemplarily, the inoculum size in this example is 4%), and cultured in a shaking incubator at 37°C and 200 r / min until the OD 600 When OD is 0.6 to 0.8 (for example, OD 600(with an OD600 of 0.76), IPTG was added to a final concentration of 0.2 mM, and induction was carried out at 16 °C and 200 r / min for 16 h. Subsequently, centrifugation was performed at 4 °C and 4000 r / min for 10 min. The supernatant was discarded, and the bacterial cells were collected. An appropriate amount of PBS was added to resuspend the bacterial cells, and they were repeatedly frozen and thawed three times in liquid nitrogen and at 37 °C, followed by sonication for 30 min. Centrifugation was carried out at 4 °C and 10,000 r / min for 30 min, and the supernatant was collected for SDS-PAGE analysis and purification through a Ni-NTA affinity chromatography column. The protein MBP-VcCDA bound to the Ni-NTA affinity chromatography column was eluted with different concentrations of imidazole (10 mM, 25 mM, 50 mM, 100 mM, 500 mM), dialyzed overnight in Tris-NaCl buffer, and its concentration was determined using a BCA protein concentration assay kit. 10% glycerol was added and it was stored at -80 °C for later use. Among them, Tris-NaCl buffer includes HCl with a final concentration of 100 mM Tris and NaCl with a final concentration of 200 mM, and the pH of Tris-NaCl buffer is 7.5. The purification results are shown in Figure 3 , where MBP-VcCDA contains an MBP tag, and PP represents the purified protein.
[0053] Example 4: Preparation of colloidal chitin
[0054] Colloidal chitin was prepared as follows: 10 g of crab shell chitin was dissolved in 150 mL of 6 M hydrochloric acid and continuously stirred until the chitin was completely dissolved. Then, the solution was mixed with 150 mL of pre-cooled ethanol and 500 mL of distilled water. The precipitated colloidal chitin was washed with distilled water until the pH reached 7.0 and stored at 4 °C for later use.
[0055] Example 5: Chitin enzymatic reaction
[0056] (1) Reaction of chitin hydrolase MBP-BliCHI
[0057] Shrimp shell powder, crab shell powder, or colloidal chitin were all subjected to hydrolysis reactions with chitin hydrolase MBP-BliCHI at the same concentration. The reaction conditions were in 50 mM ammonium bicarbonate, with a pH of 8.0, a reaction temperature of 40 °C, and a reaction time of 0.5 h - 72 h. The mass ratio of chitin hydrolase MBP-BliCHI to the substrate was 1:100 - 1000. Exemplarily, in this example, the mass ratio used was 1:100. See Figure 6 , as the experiment progressed, high-purity AA disaccharide could be obtained at low concentrations and short times, and GlcNAc (A) monosaccharide would gradually be produced as time increased. Through TLC determination, it was found that the longer the time, the more A monosaccharide was finally hydrolyzed.
[0058] (2) Combined use of chitinase MBP-BliCHI and chitin deacetylase MBP-NodB
[0059] Chitin deacetylase MBP-NodB is an enzyme that acts on the acetyl group at the first position of the non-reducing end and undergoes a combined reaction with chitinase MBP-BliCHI in this experiment. The reaction conditions are in 50 mM ammonium bicarbonate, with a pH value of 8.0, a reaction temperature of 37 °C, and a reaction time of 0.5 h - 72 h (in this example, the reaction time is 36 h). The mass ratio of chitinase MBP-BliCHI, chitin deacetylase MBP-NodB to the substrate containing chitin is 1:1:100 - 1000, and the mass ratio used in this example is 1:1:100. High-purity chitobiose DA can be obtained, and its mass spectrum is as shown in Figure 4 shown. As determined by TLC, the purity is over 90%, as shown in Figure 7 the right figure below. Among them, the structural formula of chitobiose DA is:
[0060]
[0061] (3) Combined use of chitinase MBP-BliCHI and chitin deacetylase MBP-VcCDA
[0062] Chitin deacetylase MBP-VcCDA is an enzyme that acts on the acetyl group at the second position of the non-reducing end and undergoes a combined reaction with chitinase MBP-BliCHI in this experiment. The reaction conditions are in 50 mM ammonium bicarbonate, with a pH value of 8.0, a reaction temperature of 40 °C, and a reaction time of 0.5 h - 72 h (in this example, the reaction time is 36 h). The mass ratio of chitinase MBP-BliCHI, chitin deacetylase MBP-VcCDA to the substrate containing chitin is 1:1:100 - 1000, and the mass ratio used in this example is 1:1:100. High-purity chitobiose AD can be obtained, and its mass spectrum is as shown in Figure 5 shown. As determined by TLC, the purity is over 90%, as shown in Figure 7 the left figure below. Among them, the structural formula of chitobiose AD is:
[0063]
[0064] See Figure 8, is the schematic diagram of the method for preparing chitobiose by the double-enzyme catalysis method provided by the present invention. Among them, Chitin comes from shrimp shells or crab shells. The product of using chitin hydrolase MBP-BliCHI alone is mainly AA and A. The combined use of chitin hydrolase MBP-BliCHI and chitin deacetylase MBP-NodB can obtain high-purity chitobiose DA. The combined use of chitin hydrolase MBP-BliCHI and chitin deacetylase MBP-VcCDA can obtain high-purity chitobiose AD. By combining chitin hydrolase MBP-BliCHI with chitin deacetylase MBP-NodB or chitin deacetylase MBP-VcCDA, using shrimp shell powder, crab shell powder or colloidal chitin as the substrate, that is, utilizing the cheap raw materials shrimp shells and crab shells that exist in large quantities in nature, in the way of enzymatic tandem catalysis, directly obtaining two specific structural chitobioses GlcN-β(1→4)-GlcNAc and GlcNAc-β(1→4)-GlcN with high value and purity up to more than 90% in one step, significantly improving the yield and purity of chitobiose, and at the same time reducing environmental pollution.
Claims
1. A method for preparing chitobiose by double enzyme catalysis, characterized in that: The method for preparing chitobiose by double enzyme catalysis includes the following steps: 1) Obtain a substrate containing chitin; 2) Prepare chitin hydrolase and chitin deacetylase by recombinant expression; 3) Perform an enzymatic reaction on the chitin-containing substrate obtained in step 1) with the chitin hydrolase and chitin deacetylase obtained in step 2) in a buffer system to obtain chitobiose.
2. The method for preparing chitobiose by double enzyme catalysis according to claim 1, wherein: The chitin-containing substrate in step 1) is shrimp shell, crab shell, and / or colloidal chitin.
3. The method for preparing chitobiose by double enzyme catalysis according to claim 1 or 2, characterized in that: The chitin hydrolase in step 2) is chitin hydrolase MBP-BliCHI; the chitin deacetylase is MBP-NodB or MBP-VcCDA.
4. The method for preparing chitobiose by double enzyme catalysis according to claim 3, characterized in that: The preparation method of the chitin hydrolase MBP-BliCHI is as follows: a.1) Construct a recombinant plasmid pMAL-p5x-BliCHI; a.2) Transform the recombinant plasmid pMAL-p5x-BliCHI constructed in step a.1) into Escherichia coli BL21(DE3), pick a single clone, and perform an enlarged culture; induce expression with IPTG, centrifuge to collect the cells, ultrasonically disrupt them, and centrifuge to collect the supernatant; a.3) Perform SDS-PAGE analysis and purification through a Ni-NTA affinity chromatography purification column on the product obtained in step a.2); a.4) Dialyze the product obtained in step a.3) overnight in Tris-NaCl buffer to obtain chitin hydrolase MBP-BliCHI.
5. The method for preparing chitobiose by double enzyme catalysis according to claim 3, characterized in that: The preparation method of the chitin deacetylase MBP-NodB is as follows: b.1) Construct a recombinant plasmid pMAL-p5x-NodB; b.2) Transform the recombinant plasmid pMAL-p5x-NodB constructed in step b.1) into Escherichia coli BL21(DE3), pick a single clone, and perform an enlarged culture; induce expression with IPTG, centrifuge to collect the cells, ultrasonically disrupt them, and centrifuge to collect the supernatant; b.3) Perform SDS-PAGE analysis and purification through a Ni-NTA affinity chromatography purification column on the product obtained in step b.2); b.4) Dialyze the product obtained in step b.3) overnight in Tris-NaCl buffer to obtain chitin deacetylase MBP-NodB.
6. The method for preparing chitobiose by double enzyme catalysis according to claim 3, characterized in that: The preparation method of the chitin deacetylase MBP-VcCDA is as follows: c.1) Construct a recombinant plasmid pMAL-p5x-VcCDA; c.2) Transform the recombinant plasmid pMAL-p5x-VcCDA constructed in step c.1) into Escherichia coli BL21(DE3), pick a single clone, and perform an enlarged culture; induce expression with IPTG, centrifuge to collect the cells, ultrasonically disrupt them, and centrifuge to collect the supernatant; c.3) Perform SDS-PAGE analysis and purification through a Ni-NTA affinity chromatography purification column on the product obtained in step c.2); c.4) Dialyze the product obtained in step c.3) overnight in Tris-NaCl buffer to obtain chitin deacetylase MBP-VcCDA.
7. The method for preparing chitobiose by double enzyme catalysis according to claim 3, characterized in that: When the chitin deacetylase is MBP-NodB, the specific implementation method of step 3) is: The chitinase MBP-BliCHI, chitin deacetylase MBP-NodB and a chitin-containing substrate are mixed in a certain proportion to obtain a mixture. The mixture is placed in a buffer solution for an enzymatic reaction, followed by desalting and lyophilization, and finally chitobiose DA with a purity of not less than 90% is obtained.
8. The method for preparing chitobiose by double enzyme catalysis according to claim 7, characterized in that: The mass ratio of the chitinase MBP-BliCHI, chitin deacetylase MBP-NodB to the chitin-containing substrate is 1:1:100 - 1000; the buffer solution is 50 mM Tris-HCl or 50 mM ammonium bicarbonate; the pH of the buffer solution is 8.0; the time of the enzymatic reaction is 0.5 h - 72 h; the reaction temperature of the enzymatic reaction is 30 - 40 °C.
9. The method for preparing chitobiose by double-enzyme catalysis according to claim 3, wherein: When the chitin deacetylase is chitin deacetylase MBP-VcCDA, the specific implementation method of step 3) is: the chitinase MBP-BliCHI, chitin deacetylase MBP-VcCDA and a chitin-containing substrate are mixed in a certain proportion to obtain a mixture. The mixture is placed in a buffer solution for an enzymatic reaction, followed by desalting and lyophilization, and finally chitobiose AD with a purity of not less than 90% is obtained.
10. The method for preparing chitobiose by double enzyme catalysis according to claim 9, wherein: The mass ratio of the chitinase MBP-BliCHI, chitin deacetylase MBP-VcCDA to the chitin-containing substrate is 1:1:100 - 1000; the buffer solution is 50 mM PBS or 50 mM ammonium bicarbonate; the pH of the buffer solution is 8.0; the time of the enzymatic reaction is 0.5 h - 72 h; the reaction temperature of the enzymatic reaction is 30 - 40 °C.