Bifunctional fusion enzyme as well as construction method and application thereof

Abstract

The invention relates to a bifunctional fusion enzyme as well as a construction method and application thereof. The construction method of the bifunctional fusion enzyme comprises the following steps: carrying out PCR (Polymerase Chain Reaction) amplification on a lytic polysaccharide monooxygenase gene BcLPMO08 and a chitinase gene BcChi49 of bacillus cereus CGMCC (China General Microbiological Culture Collection Center) No.4348; the method comprises the following steps: constructing a bifunctional fusion enzyme BcLPMO-linker-BcChi recombinant expression vector mediated by different linkers by using a seamless cloning technology; a recombinant vector containing the bifunctional fusion enzyme BcLPMO-linker-BcChi gene is expressed in a host cell, so that the bifunctional fusion enzyme BcLPMO-linker-BcChi gene is obtained. The fusion enzyme has two catalytic structural domains with completely different functions, namely oxidation and hydrolysis cleavage glycosidic bond activity. A substrate channel is formed under the synergistic effect of proximity induction between the bacillus amyloliquefaciens and the bacillus amyloliquefaciens, chitin substances can be effectively degraded to produce the chitosan oligosaccharide, the production cost of the enzyme is reduced, and the bacillus amyloliquefaciens has potential industrial application prospects in sustainable conversion of biomass.

Description

Technical Field

[0001] The present invention relates to the technical fields of genetic engineering and bioengineering, and particularly relates to a bifunctional fusion enzyme, a construction method thereof, and an application thereof. Background Art

[0002] Chitin is a linear polymer composed of β-(1→4) glycosidic bonds connecting N-acetyl-D-glucosamine (GlcNAc), and has unique application values in aspects such as food, medicine, agriculture, and materials. However, chitin is insoluble in water and most organic solvents, and its application is greatly restricted. The hydrolysis product of chitin, N-acetyl chitooligosaccharide, also known as chitooligosaccharide (CTOS), has relatively good solubility and significant physiological activities, such as antibacterial, antioxidant, antitumor, improving metabolic disorders, and enhancing immunity. In addition, CTOS can also promote plant growth, induce plant immune responses, and protect soil colony structures. However, a major challenge in chitin enzymatic conversion is that most chitinases have difficulty accessing the chitin chains stacked into lattices. In recent years, it has been found that lytic polysaccharide monooxygenases (LPMOs) can bypass this geometric and thermodynamic barrier. It can oxidatively cleave β-(1→4) glycosidic bonds, disrupt the surface topology, and form chain ends on insoluble substrates so that chitinases can further biotransform them.

[0003] The complete enzymatic conversion of chitin based on LPMOs and chitinases is a promising candidate for chemical or physical pretreatment strategies. Chinese invention patent CN202210868807.1 discloses a lytic polysaccharide monooxygenase BsLPMO10A from Bacillus subtilis. Its synergistic effects with a series of glycoside hydrolases indicate that BsLPMO10A significantly accelerates the saccharification of dextran, xylan, cellulose, and chitin. Chinese invention patent CN202210092989.8 discloses a lytic polysaccharide monooxygenase PILPMOCB3. Its synergistic effects with commercial cellulase, xylanase, or chitinase can improve the degradation efficiency of cellulose, lignocellulose, xylan, and chitin. However, the post-expression mixed application of single enzymes makes the entire process relatively complex and is not conducive to industrial production. To avoid this problem, Chinese invention patent CN202211297855.6 discloses a method using Streptomyces sp. SCUT-1 as the starting strain to co-express the lytic polysaccharide monooxygenase LPMO and chitinase Chi19 using the efficient promoter scutP1. Nevertheless, there are still mass transfer problems in the free multi-enzyme catalytic system, and the catalytic efficiency needs to be further improved.

[0004] Attaching the LPMO catalytic domain to a glycoside hydrolase (GH) module to shorten the spatial distance between LPMO and GH, forming an unprecedented bifunctional enzyme with the ability to hydrolyze and oxidatively cleave glycosidic bonds, seems to be a feasible solution. And how to obtain a bifunctional fusion enzyme that can be efficiently applied has become a research hotspot and difficulty. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide a bifunctional fusion enzyme, its construction method and application in view of the deficiencies of the prior art.

[0006] To solve the above technical problems, the present invention discloses a bifunctional fusion enzyme, its construction method and application. This bifunctional fusion enzyme combines the catalytic domains of lytic polysaccharide monooxygenase and chitinase, and can improve the efficiency of enzymatic preparation of chito-oligosaccharides. The specific technical solutions are as follows:

[0007] A bifunctional fusion enzyme, comprising the catalytic domain of lytic polysaccharide monooxygenase (BcLPMO08) and the catalytic domain of chitinase (BcChi49), and the catalytic domain of lytic polysaccharide monooxygenase and the catalytic domain of chitinase are fused through a linker; wherein, the amino acid sequence of the lytic polysaccharide monooxygenase is as shown in SEQ ID No.1; the amino acid sequence of the chitinase is as shown in SEQ ID No.2; the amino acid sequence of the linker is any one of SEQ ID No.3 to 8, preferably SEQ ID No.7. The bifunctional fusion enzyme has the activities of oxidizing and hydrolytically cleaving glycosidic bonds. Preferably, in the fusion, the C-terminus of the catalytic domain of lytic polysaccharide monooxygenase is connected to the N-terminus of the catalytic domain of chitinase.

[0008] Preferably, the amino acid sequence of the bifunctional fusion enzyme is selected from any one of SEQ ID No.9 to 14. More preferably, it is SEQ ID No.13.

[0009] More preferably, the construction method of the bifunctional fusion enzyme comprises the following steps:

[0010] S1. Perform PCR amplification on the lytic polysaccharide monooxygenase gene BcLPMO08 and the chitinase gene BcChi49 derived from Bacillus cereus CGMCC No.4348;

[0011] S2. Construct a prokaryotic expression vector of the bifunctional fusion enzyme BcLPMO08-linker-Chi49 mediated by different linkers by seamless cloning technology;

[0012] S3. Express the prokaryotic expression vector containing the bifunctional fusion enzyme BcLPMO-linker-BcChi gene in a host bacterium to obtain a recombinant bacterium;

[0013] S4. Ferment and culture the recombinant bacterium to induce the expression of the bifunctional fusion enzyme;

[0014] S5. Collect the cells obtained by fermentation in S4, ultrasonically disrupt the cells, collect the supernatant, and purify the expressed bifunctional fusion enzyme by Ni + -NTA affinity chromatography.

[0015] Further preferably, in S2, the lytic polysaccharide monooxygenase BcLPMO08 gene and the chitinase BcChi49 gene are obtained by PCR amplification using Bacillus cereus CGMCC No. 4348 as a template, and the linearized vector is obtained by PCR amplification using a basic vector as a template. The basic vector is any one of pET-22b(+), pET-25b(+), pET-26b(+), or pRSFDuet-1; the linearized vector is ligated with the BcLPMO08 gene and the BcChi49 gene by seamless cloning to obtain a prokaryotic expression vector;

[0016] In a second aspect, the present invention provides a gene encoding the bifunctional fusion enzyme described in the first aspect; preferably, its nucleotide sequence is selected from any one of SEQ ID No. 15-20.

[0017] In a third aspect, the present invention provides an expression cassette or a recombinant expression vector containing the gene described in the second aspect.

[0018] Among them, for the recombinant expression vector, its basic vector is any one of pET-22b(+), pET-25b(+), pET-26b(+), or pRSFDuet-1. Preferably, it is pET-22b(+).

[0019] In a fourth aspect, the present invention provides a recombinant bacterium containing the expression cassette or the recombinant expression vector described in the third aspect. Preferably, the host bacterium of the recombinant bacterium is Escherichia coli.

[0020] In a fifth aspect, the present invention provides the application of the bifunctional fusion enzyme described in the first aspect, or the expression cassette or the recombinant expression vector described in the third aspect, or the recombinant bacterium described in the fourth aspect in degrading chitinous substances to prepare chitooligosaccharides.

[0021] Among them, the chitinous substances include any one of α-chitin, β-chitin, or shrimp shell powder. Preferably, the chitinous substances further include colloidal chitin.

[0022] Among them, chitooligosaccharides are prepared by degrading chitinous substances using the bifunctional fusion enzyme described above. Among them, the reaction system includes 0.1 - 3 μM bifunctional fusion enzyme, 10 - 50 g / L chitinous substances, and 0.1 - 2 mM ascorbic acid; the reaction time is 12 - 36 h, and the reaction temperature is 20 - 55 °C. Preferably, the reaction system includes 1 μM bifunctional fusion enzyme, 10 g / L chitinous substances, and 1 mM ascorbic acid, and reacts at 50 °C for 24 h.

[0023] Beneficial effects:

[0024] (1) In the present invention, a bifunctional fusion enzyme BcLPMO-linker-BcChi containing the catalytic domains of lytic polysaccharide monooxygenase and chitinase is constructed by genetic engineering technology. This fusion enzyme has both the ability to hydrolyze and oxidatively cleave glycosidic bonds.

[0025] (2) In the present invention, BcLPMO-linker-BcChi is obtained, and the "proximity-induced" synergistic effect of its two catalytic domains is stronger and more stable. The formation of the substrate channel can adjust the reaction rate in real time according to the activity state of each other, ensuring that the entire catalytic process is coherent and efficient, and the catalytic activity is significantly improved compared with single catalytic domain enzymes and free multi-enzyme complex systems.

[0026] (3) The bifunctional fusion enzyme BcLPMO-linker-BcChi obtained in the present invention can efficiently degrade chitinous substances to produce chitooligosaccharides, avoiding the post-expression and mixed application of single enzymes and simplifying the production process.

[0027] (4) The chitooligosaccharides obtained by the method of the present invention can be used as high-quality raw materials for products such as animal and plant nutritional additives. Description of the drawings

[0028] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0029] Figure 1 SDS-PAGE diagram of bifunctional fusion enzymes connected by different linker peptides.

[0030] Figure 2 BcLPMO enzyme activity after expression of bifunctional fusion enzymes connected by different linker peptides.

[0031] Figure 3 BcChi enzyme activity after expression of bifunctional fusion enzymes connected by different linker peptides.

[0032] Figure 4 Yield of chitooligosaccharides produced by bifunctional fusion enzymes degrading chitinous substances.

[0033] Figure 5 HPLC chromatogram of the catalytic product of the bifunctional fusion enzyme, where 1-6 are GlcNAc, (GlcNAc) 2 , (GlcNAc) 3 , (GlcNAc) 4 , (GlcNAc) 5 and (GlcNAc) 6 respectively, where GlcNAc represents N-acetylglucosamine. Detailed implementation manners

[0034] The present invention will be further described in detail below in conjunction with examples and the accompanying drawings, but the implementation manners of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0035] Detection method for the enzyme activity of lytic polysaccharide monooxygenase BcLPMO: The reaction system (1 mL) contains 20 mM Tris-HCl buffer (pH = 7.0), 1 mM 2,6-dimethoxyphenol, 100 μM H 2 O 2 , and 0.5 μM BcLPMO. After reacting at 30 °C for 5 min, the absorbance is measured at 469 nm. The defined unit of BcLPMO enzyme activity is the amount of enzyme required to produce 1 μmol of xylopyronequinone (ε 469 = 53200 M -1 ·cm -1 ) per minute.

[0036] Detection method for the enzyme activity of chitinase BcChi: The reaction system (1 mL) contains 50 mM PBS buffer (pH = 6.0), 2.5 g / L colloidal chitin, and 50 mg / L BcChi. After reacting at 55 °C for 30 min, 1 mL of DNS reagent is added, and it is heated in a boiling water bath for 5 min, cooled to room temperature, and after centrifugation, the supernatant is taken to measure the absorbance at 540 nm. The defined unit of BcChi enzyme activity is the amount of enzyme required to produce 1 μmol of reducing sugar GlcNAc per minute.

[0037] Example 1 Construction of bifunctional fusion enzyme BcLPMO-linker-BcChi

[0038] The BcLPMO and BcChi described in the present invention are derived from Bacillus cereus CGMCC No. 4348. The detailed information of this strain can be referred to Chinese Patent CN201210056365.7. Using the genomic DNA of Bacillus cereus CGMCC No. 4348 as a template, the coding gene fragments of lytic polysaccharide monooxygenase BcLPMO08 (whose amino acid sequence is shown in SEQ ID No. 1) and chitinase BcChi49 (whose amino acid sequence is shown in SEQ ID No. 2) were amplified by PCR respectively, and the catalytic domains of these two enzymes were fused and expressed using different linkers.

[0039] Two genes, BcLPMO08 and BcChi49, were used to construct a bifunctional fusion protein in the mode of BcLPMO-linker-BcChi and ligated to the basic vector pET-22b(+). Six linkers were selected, namely 3 flexible linkers: F1: GGGGSGGGGSGGGGS (SEQ ID No. 3), F2: EGKSSGSGSESKST (SEQ ID No. 4), F3: GSAGSAAGSGEF (SEQ ID No. 5) and 3 rigid linkers: R1: EAAAKEAAAKEAAAK (SEQ ID No. 6), R2: APAPAPAPAPAPAP (SEQ ID No. 7), R3: VDEAAAKSGR (SEQ ID No. 8). Six bifunctional fusion enzymes were obtained by ligating through the above 6 linkers respectively, and their amino acid sequences are shown in SEQ ID Nos. 9-14 respectively, and are encoded by the nucleotide sequences shown in SEQ ID Nos. 15-20 respectively. The specific steps are as follows:

[0040] 1) Inoculate Bacillus cereus CGMCC No. 4348 into LB medium and shake culture at 37°C and 200 rpm for 12-16 h to obtain the seed liquid of Bacillus cereus CGMCC No. 4348; take 4 mL of the seed liquid of Bacillus cereus CGMCC No. 4348 and extract genomic DNA using a rapid DNA extraction and detection kit.

[0041] 2) Using primers pET-22b(+)-F and pET-22b(+)-R, and taking the plasmid vector pET-22b(+) as a template, prepare the linearized vector pET-22b(+) by inverse PCR amplification.

[0042] 3) Using primers BcLPMO-F, BcLPMO-R and BcChi49-F1-F, BcChi49-R, with the genomic DNA of Bacillus cereus CGMCC No. 4348 as a template, the gene fragments of BcLPMO08 and BcChi49 were amplified by PCR respectively. The linearized vector pET-22b(+) was ligated with the BcLPMO08 and BcChi49 fragments by seamless cloning to obtain the recombinant vector pET-22b(+)-BcLPMO-F1-BcChi.

[0043] 4) Using primers BcLPMO-F, BcLPMO-R and BcChi49-F2-F, BcChi49-R, with the genomic DNA of Bacillus cereus CGMCC No. 4348 as a template, the gene fragments of BcLPMO08 and BcChi49 were amplified by PCR respectively. The linearized vector pET-22b(+) was ligated with the BcLPMO08 and BcChi49 fragments by seamless cloning to obtain the recombinant vector pET-22b(+)-BcLPMO-F2-BcChi.

[0044] 5) Using primers BcLPMO-F, BcLPMO-R and BcChi49-F3-F, BcChi49-R, with the genomic DNA of Bacillus cereus CGMCC No. 4348 as a template, the gene fragments of BcLPMO08 and BcChi49 were amplified by PCR respectively. The linearized vector pET-22b(+) was ligated with the BcLPMO08 and BcChi49 fragments by seamless cloning to obtain the recombinant vector pET-22b(+)-BcLPMO-F3-BcChi.

[0045] 6) Using primers BcLPMO-F, BcLPMO-R and BcChi49-R1-F, BcChi49-R, with the genomic DNA of Bacillus cereus CGMCC No. 4348 as a template, the gene fragments of BcLPMO08 and BcChi49 were amplified by PCR respectively. The linearized vector pET-22b(+) was ligated with the BcLPMO08 and BcChi49 fragments by seamless cloning to obtain the recombinant vector pET-22b(+)-BcLPMO-R1-BcChi.

[0046] 7) Using primers BcLPMO-F, BcLPMO-R and BcChi49-R2-F, BcChi49-R, with the genomic DNA of Bacillus cereus CGMCC No. 4348 as a template, the gene fragments of BcLPMO08 and BcChi49 were amplified by PCR respectively. The linearized vector pET-22b(+) was ligated with the BcLPMO08 and BcChi49 fragments by seamless cloning to obtain the recombinant vector pET-22b(+)-BcLPMO-R2-BcChi.

[0047] 8) Using primers BcLPMO-F, BcLPMO-R and BcChi49-R3-F, BcChi49-R, with the genomic DNA of Bacillus cereus CGMCC No. 4348 as a template, the gene fragments of BcLPMO08 and BcChi49 were amplified by PCR respectively. The linearized vector pET-22b(+) was ligated with the BcLPMO08 and BcChi49 fragments by seamless cloning to obtain the recombinant vector pET-22b(+)-BcLPMO-R3-BcChi.

[0048] 9) PCR amplification system: ddH 2 O 20 μL, 2*Phanta Max Master Mix (Dye Plus) 25 μL, primer F 2 μL, primer R 2 μL, template DNA 1 μL.

[0049] Plasmid inverse PCR amplification conditions: Pre-denaturation at 95 °C for 30 s; Denaturation at 95 °C for 15 s; Annealing at 60 °C for 15 s; Extension at 72 °C (the extension rate of the enzyme is 30 - 60 s / kb, and the specific time is set according to the length of the amplified fragment); Cycle 30 times; Final extension at 72 °C for 5 min; Incubate at 12 °C.

[0050] Enzyme gene fragment PCR amplification conditions: Pre-denaturation at 95 °C for 3 min; Denaturation at 95 °C for 15 s; Annealing at 55 °C for 15 s; Extension at 72 °C (the extension rate of the enzyme is 30 - 60 s / kb, and the specific time is set according to the length of the amplified fragment); Cycle 30 times; Final extension at 72 °C for 5 min; Incubate at 12 °C.

[0051] Seamless cloning ligation system: Insert fragment 10 - 100 ng, linearized vector 50 - 100 ng, (the molar ratio of each insert fragment to the vector is 3:1, and the specific addition amount is calculated according to the actual DNA concentrations of the two), 2*Seamless Cloning Mix 10 μL, supplemented with Nuclease free Water to 20 μL.

[0052] The primer sequences involved in the above steps 2) to 8) are shown in Table 1.

[0053] Table 1 PCR amplification primers

[0054]

[0055]

[0056] Example 2 Expression and purification of bifunctional fusion enzyme BcLPMO-linker-BcChi

[0057] 1) The recombinant vectors containing the fusion protein gene constructed in Example 1 were respectively transformed into E. coli BL21(DE)3 to obtain the following recombinant strains:

[0058] E. coli-pET-22b(+)-BcLPMO-F1-BcChi, E. coli-pET-22b(+)-BcLPMO-F2-BcChi, E. coli-pET-22b(+)-BcLPMO-F3-BcChi, E. coli-pET-22b(+)-BcLPMO-R1-BcChi, E. coli-pET-22b(+)-BcLPMO-R2-BcChi and E. coli-pET-22b(+)-BcLPMO-R3-BcChi.

[0059] 2) The recombinant strains expressing the fusion protein were inoculated into LB medium containing 100 μg / mL sodium ampicillin for fermentation. After the fermentation was completed, the cells were collected by centrifugation. After ultrasonic disruption of the cells, the supernatant was collected, and the expressed bifunctional fusion enzyme was purified by Ni + -NTA affinity chromatography. The fermentation conditions were: culture at 37 °C and 200 rpm. When the cell concentration (OD 600 ) reached 0.6 - 0.8, 0.5 mM IPTG was added for induction, the induction temperature was 18 °C, and fermentation was continued for 20 h.

[0060] The predicted molecular weights of the 6 bifunctional fusion enzymes were approximately 83 kDa. As analyzed by SDS-PAGE, as Figure 1 shown, clear protein bands were observed in lanes 2 - 7 in the range of 70 - 100 kDa, indicating that the fusion enzymes were all highly expressed.

[0061] The oxidative activity ( Figure 2 ) of BcLPMO and the hydrolytic activity ( Figure 3)Evaluations were carried out separately. After covalently coupling the two catalytic domains with different linkers, there was not much difference in the oxidation activities among the fusion enzymes, while the hydrolysis activities differed greatly, which might be affected by the type and length of the linker. Among them, both the oxidation activity and hydrolysis activity of BcLPMO-R2-BcChi were the highest.

[0062] Example 3 Production of chitooligosaccharides by degrading chitinous substances with bifunctional fusion enzymes

[0063] The reaction system contained 1 μM of the bifunctional fusion enzyme described in Example 2, 10 g / L of chitinous substances (including any one of α-chitin, β-chitin or shrimp shell powder), and 1 mM of ascorbic acid. The reaction was carried out at 50 °C for 24 h, terminated by boiling for 10 min, and after cooling to room temperature, the supernatant was taken by centrifugation for HPLC analysis.

[0064] This example also included two control experiments. Control 1 (BcChi group): The reaction system contained 1 μM of free BcChi, 10 g / L of chitinous substances, and 1 mM of ascorbic acid; Control 2 (BcLPMO + BcChi group): The reaction system contained 1 μM of free BcLPMO, 1 μM of free BcChi, 10 g / L of chitinous substances, and 1 mM of ascorbic acid.

[0065] The specific HPLC analysis conditions were as follows: The chromatographic column was Shodex Asahipak NH 2 P-50 4E column (250×4.6 mm); the mobile phase was acetonitrile: water (70:30, v / v); the column temperature was 40 °C; the flow rate was 1 mL / min; the detector was a UV detector (VWD); the detection wavelength was 210 nm. According to the corresponding standard curve, the contents of chitooligosaccharides with different degrees of polymerization were calculated through the peak areas.

[0066] As Figure 4 shown, the yields of chitooligosaccharides produced by degrading 3 chitinous substances (α-chitin, β-chitin and shrimp shell powder) with 6 bifunctional fusion enzymes were all higher than those of the BcChi group and the BcLPMO + BcChi group. Among them, the yield of chitooligosaccharides of the R2 fusion enzyme was the highest. When using α-chitin as the substrate, it was 5.35 times that of the BcChi group and 1.64 times that of the BcLPMO + BcChi group; when using β-chitin as the substrate, it was 4.68 times that of the BcChi group and 1.48 times that of the BcLPMO + BcChi group; when using shrimp shell powder as the substrate, it was 4.13 times that of the BcChi group and 1.58 times that of the BcLPMO + BcChi group. The present invention further analyzed the product of the bifunctional fusion enzyme BcLPMO-R2-BcChi with the best catalytic effect. As Figure 5As shown, BcLPMO-R2-BcChi degrades α-chitin mainly to produce GlcNAc and (GlcNAc) 2 .

[0067] The construction of the bifunctional fusion enzyme further enhances the synergy between LPMO and GH. The formation of the substrate channel shortens the distance between the two catalytic domains, and they promote each other, resulting in a significantly higher catalytic activity compared to the single catalytic domain enzyme and the free multi-enzyme complex system. In addition, the CBM5 domain at the C-terminus of BcLPMO is retained during the construction process, which helps to anchor the two catalytic domains to the substrate. This data is higher than the currently reported literature and the published invention patents. For example, the reports by Zhao et al. (Bioconversion of α-chitin by a lytic polysaccharide monooxygenase OSLPMO10A coupled with chitinases and the synergistic mechanism analysis, J. Agric. Food Chem. 2024, 72, 7256-7265), as well as the public data of Chinese invention patents CN202210868807.1, CN202210092989.8, CN202211297855.6, etc.

[0068] The present invention provides a bifunctional fusion enzyme, its construction method and application, and the specific implementation manners cover but are not limited to the technical paths described in the examples. It should be emphasized that those skilled in the art can make several improvements and modifications on the premise of following the core principle of the present invention, and these improvements and modifications should also be included in the protection scope of the claims of the present invention. Each component not clearly defined in this example can be realized by the prior art.

Claims

1. A bifunctional fusion enzyme, characterized in that: The invention comprises a catalytic domain of a lytic polysaccharide monooxygenase and a catalytic domain of a chitinase, wherein the catalytic domain of the lytic polysaccharide monooxygenase and the catalytic domain of the chitinase are fused via a connecting peptide; The amino acid sequence of the lytic polysaccharide monooxygenase is shown as SEQ ID No.1; the amino acid sequence of the chitinase is shown as SEQ ID No.2; and the amino acid sequence of the connecting peptide is any one of SEQ ID Nos.3 to 8.

2. The bifunctional fusion enzyme according to claim 1, characterized in that: The amino acid sequence of the bifunctional fusion enzyme is selected from any one of SEQ ID No. 9-14.

3. A gene encoding the bifunctional fusion enzyme according to claim 1 or 2.

4. The gene according to claim 3, characterized in that The nucleotide sequence of the gene is selected from any one of SEQ ID No. 15 to 20.

5. An expression cassette or recombinant expression vector containing the gene according to claim 3.

6. The expression cassette or recombinant expression vector according to claim 5, characterized in that: The basic vector of the recombinant expression vector is any one of pET-22b, pET-25b, pET-26b or pRSFDuet-1.

7. A recombinant bacterium containing the expression cassette or recombinant expression vector according to claim 5 or 6.

8. Use of the bifunctional fusion enzyme according to claim 1 or 2, or the expression cassette or recombinant expression vector according to claim 5, or the recombinant bacterium according to claim 7 in degrading chitin-like substances to prepare chito-oligosaccharides.

9. The use according to claim 8, characterized in that: The chitinous substance includes any one of α-chitin, β-chitin or shrimp shell powder.

10. The use according to claim 8, characterized in that: The bifunctional fusion enzyme is used to degrade chitin substances to prepare chitosan oligosaccharides, wherein the reaction system includes 0.1-3 μM bifunctional fusion enzyme, 10-50 g / L chitin substances and 0.1-2 mM ascorbic acid; the reaction time is 12 to 36 hours, and the reaction temperature is 20 to 55°C.

Images