Chitosanase CsnQB-E206A-elgii and its application in preparing chitosan oligosaccharide functional beverage

Abstract

The present invention discloses a chitosanase CsnQB‑E206A‑elgii and its application in the preparation of chitosan oligosaccharide functional beverages, belonging to the technical field of chitosanase. The amino acid sequence of the chitosanase CsnQB‑E206A‑elgii is shown in SEQ ID NO.5. The application of the chitosanase CsnQB‑E206A‑elgii in the degradation of chitosan. The application of the chitosanase CsnQB‑E206A‑elgii in the preparation of chitosan oligosaccharide functional beverages. The chitosanase CsnQB‑E206A‑elgii of the present invention is obtained by mutation modification of the chitosanase CsnQB, has higher enzyme activity on colloid chitosan and powdered chitosan, can be used to hydrolyze chitosan in different forms, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to chitosanase CsnQB-E206A-elgii and application thereof in preparing chitosan oligosaccharide functional beverage, belonging to the technical field of chitosanase. Background Art

[0002] Chitosanase (EC.3.2.1.132) is a glycoside hydrolase that catalyzes the degradation of chitosan. It produces chitooligosaccharides by specifically hydrolyzing the β-1,4 glycosidic bonds in chitosan. Since chitosanase has almost no degradation activity on powdered chitosan, and chitosan is insoluble in water and can only be dissolved in dilute acid solutions, chitooligosaccharides are usually prepared by degrading acid-soluble chitosan using chitosanase. However, chitosan has the inherent characteristics of a high molecular weight polymer, and even a low concentration of chitosan solution has a significant viscosity, which leads to a significant inhibition of the catalytic activity and efficiency of chitosanase, greatly hindering the large-scale application of enzymatic degradation of chitosan.

[0003] Chitosan oligosaccharides have the advantages of high solubility, low molecular weight and good biocompatibility. They have biological activities such as antioxidant, anti-inflammatory and antibacterial, anti-tumor, immunomodulatory, and promotion of intestinal microbial growth, and are widely used.

[0004] Changing the substrate binding of chitosanase by reasonable design and enhancing its ability to degrade chitosan to achieve efficient preparation of chitosan oligosaccharides is a theoretically feasible method. The team of the inventor of the present invention has discovered chitosanase CsnQB in previous studies, and the relevant content is recorded in Chinese invention patent CN 118240805 A, and has been appropriately modified to obtain chitosanase CsnQB-D35E and chitosanase CsnQB-E203A. The enzyme activity of the modified chitosanase has been greatly improved, and the relevant content of chitosanase CsnQB-E203A is recorded in Chinese invention patent CN 118406672 A. Although these modifications have improved the activity of chitosanase on acid-soluble chitosan, there is still room for improvement in the improvement, and the activity of chitosanase on powdered chitosan has not been improved. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides a chitosanase CsnQB-E206A-elgii and application thereof in the preparation of chitosan oligosaccharide functional beverages, belonging to the technical field of chitosanase.

[0006] The present invention is achieved through the following technical solutions:

[0007] A chitosanase CsnQB-E206A-elgii, whose amino acid sequence is shown in SEQ ID NO.5.

[0008] The chitosanase CsnQB-E206A-elgii is used in degrading chitosan.

[0009] Furthermore, the chitosan is selected from colloid chitosan or powdered chitosan.

[0010] The chitosanase CsnQB-E206A-elgii is used in the preparation of chitosan oligosaccharide functional beverages. The chitosan oligosaccharide functional beverages refer to beverage products added with chitosan oligosaccharides and having health functions, and the health functions specifically refer to: enhancing immune regulation, promoting intestinal health, assisting in lowering blood sugar and blood lipids, and anti-oxidation effects. The chitosan oligosaccharide functional beverage contains chitosan oligosaccharides, and the chitosan oligosaccharides are prepared by degrading chitosan using chitosanase CsnQB-E206A-elgii.

[0011] The chitosanase CsnQB-E206A-elgii of the present invention is obtained by mutation modification of chitosanase CsnQB. The modification process is: first, mutation modification of chitosanase CsnQB is performed to obtain chitosanase CsnQB-E206A, and the catalytic effect of chitosan is improved after modification; then, a carbohydrate binding module (CBM) is fused to the C-terminus of chitosanase CsnQB-E206A to obtain chitosanase CsnQB-E206A-elgii, and the catalytic effect of chitosan is more significantly enhanced after fusion. The chitosanase CsnQB-E206A-elgii of the present invention has higher enzyme activity on colloid chitosan and powdered chitosan, can be used for efficient hydrolysis of chitosan in different forms, and has broad application prospects. The invention rationally transforms the chitosanase CsnQB, improves its catalytic ability to chitosan in different forms, and has important significance for reducing the production cost of industrial preparation of chitosan oligosaccharides by chitosanase.

[0012] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 :Chitosanase CsnQB-E206A and (GlcN) 5 Schematic diagram of molecular docking simulation.

[0014] Figure 2 : Figure 1 An enlarged schematic diagram of the portion shown in the dashed box.

[0015] Figure 3 : Schematic diagram of the effect of temperature on the relative enzyme activity of chitosanase CsnQB-E206A.

[0016] Figure 4:Schematic diagram of the effect of temperature on the relative enzyme activity of chitosanase CsnQB-E206A-elgii.

[0017] Figure 5 : Schematic diagram of the effect of pH on the relative enzyme activity of chitosanase CsnQB-E206A.

[0018] Figure 6 : Schematic diagram of the effect of pH on the relative enzyme activity of chitosanase CsnQB-E206A-elgii.

[0019] Figure 7 : HPLC chromatograms of the degradation products of powdered chitosan degraded by various chitosanases, wherein DP2, DP3, DP4, DP5, and DP6 refer to standard chitosan oligosaccharides with a degree of polymerization of 2, 3, 4, 5, and 6, respectively, namely, chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose.

[0020] Figure 8 : HPLC chromatograms of the degradation products of colloidal chitosan by various chitosanases.

[0021] Fig. 9 : SEM image of powdered chitosan (5000×).

[0022] Fig.10 :SEM image of powdered chitosan treated with CBM-elgii protein (5000×)

[0023] Fig.11 : SEM image of powdered chitosan (10000×).

[0024] Fig.12 : SEM image of powdered chitosan treated with CBM-elgii protein (10000×). DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0026] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods available in the prior art.

[0027] The colloidal chitosan used in the present invention was purchased from MacLean Biochemical Co., Ltd. (Shanghai, China), and the deacetylation degree was ≥95%.

[0028] The powdered chitosan used in the present invention was purchased from MacLean Biochemical Co., Ltd. (Shanghai, China), with a deacetylation degree of ≥95% and a viscosity of 100-200 mpa.s.

[0029] Example 1 Modification of chitosanase CsnQB

[0030] The amino acid sequence of chitosanase CsnQB is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2.

[0031] The amino acid sequence of chitosanase CsnQB is shown in SEQ ID NO.1, as shown below:

[0032] AGLNKDQKRRAEQLTSIFENGTTEIQYGYVERLDDGRGYTCGRAGFTTATGDALEVVEVYTKAVPNNKLKKYLPELRRLAKEESDDTSNLKGFASAWKSLANDKEFRAAQDKVNDHLYYQPA MKRSDNAGLKTALARAVMYDTVIQHGDGDDPDSFYALIKRTNKKAGGSPKDGIDEKKWLNKFLDVRYDDLMNPANHDTRDEWRESVARVDVLRSIAKENNYNLNGPIHVRSNEYGNFVIKLE.

[0033] The nucleotide sequence of the gene encoding chitosanase CsnQB is shown in SEQ ID NO.2, as shown below (direction 5'-3'):

[0034] .

[0035] Previous studies have shown that the enzyme activity of chitosanase CsnQB is 246.26 U / mg, and the amount of reducing sugar produced after degrading powdered chitosan for 3 hours is 2.35 μmol / ml. Although chitosanase CsnQB has a certain catalytic ability for powdered chitosan, its activity is relatively low. Therefore, the present invention attempts to mutate and modify it in order to obtain a chitosanase with higher activity.

[0036] In order to change the catalytic ability of the enzyme to chitosan and improve the enzymatic properties, based on the rational design of the previous molecular docking results, structural calculation and amino acid sequence consistency analysis, the present invention believes that the glutamic acid at position 206 may be a key site that affects the enzyme's action on chitosan, so a site-directed mutagenesis is performed on this site to change the glutamic acid at position 206 to alanine, specifically by changing the codon corresponding to the glutamic acid at position 206 on the coding gene of chitosanase CsnQB from "GAA" to the codon of alanine "GCG". The chitosanase after site-directed mutagenesis is named chitosanase CsnQB-E206A, and its amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the coding gene of chitosanase CsnQB-E206A is shown in SEQ ID NO.4.

[0037] The structural model of chitosanase CsnQB-E206A was constructed using the SWISSMODEL protein modeling server, and the structural model of chitosanase CsnQB-E206A was compared with (GlcN) 5 Molecular docking for molecular simulation.

[0038] Chitosanase CsnQB-E206A and (GlcN) 5 Schematic diagram of molecular docking simulation Figure 1 As shown, Figure 1 The enlarged schematic diagram of the part shown in the dotted box is shown in Figure 2 As shown. In the wild-type chitosanase CsnQB, there is no hydrogen bond between the end of the polysaccharide molecule and the glutamic acid at position 206. However, when the glutamic acid at position 206 is mutated to alanine, the cyclic structure of the alanine side chain causes the conformation of the polysaccharide molecule in the active pocket to change, thereby forming a hydrogen bond between the amino acid at position 206, as shown in FIG. Figure 2 This conformational change may enhance the catalytic effect of the enzyme on chitosan.

[0039] It should be noted that although the transformation of the chitosanase CsnQB-E206A of the present invention and the chitosanase CsnQB-E203A recorded in the Chinese invention patent CN118406672 A is to mutate glutamic acid to alanine, the three-dimensional structure of the protein after mutation has undergone a completely different change due to the different sites. During the folding process of the protein, the spatial position of each amino acid, the interaction of the side chain and the binding mode with the substrate will be different. In the chitosanase CsnQB-E206A mutation, there was no hydrogen bond between the end of the polysaccharide molecule and glutamic acid No. 206. After the mutation to alanine, a new hydrogen bond was formed, which indicates that the mutation has a positive effect on the stability of the local structure or the substrate binding mode. E203A mutation In chitosanase CsnQB-E203A, the end of the polysaccharide molecule originally had a hydrogen bond with glutamic acid No. 203. After mutation, the hydrogen bond disappeared, but new hydrogen bonds were formed with other surrounding amino acids, indicating that the effect of this site on the overall structure after mutation is different from that of E206A. Moreover, the catalytic center of chitosanase usually includes multiple sites, which work together to stabilize the substrate and promote the catalytic reaction. Although E203 and E206 are both located in the active region, their specific positions in the substrate binding pocket are different, resulting in significant differences in the effects of mutation on substrate affinity, substrate orientation or catalytic activity. E206 is located in the stable region of the binding pocket, and the new hydrogen bonds formed after mutation may enhance the binding of the substrate, thereby improving the catalytic efficiency. E203 is in the key region of substrate release, and the disappearance or redistribution of hydrogen bonds after mutation may affect the dissociation process of the substrate, resulting in different changes in enzymatic properties.

[0040] The amino acid sequence of chitosanase CsnQB-E206A is shown in SEQ ID NO.3, as shown below:

[0041] AGLNKDQKRRAEQLTSIFENGTTEIQYGYVERLDDGRGYTCGRAGFTTATGDALEVVEVYTKAVPNNKLKKYLPELRRLAKEESDDTSNLKGFASAWKSLANDKEFRAAQDKVNDHLYYQPA MKRSDNAGLKTALARAVMYDTVIQHGDGDDPDSFYALIKRTNKKAGGSPKDGIDEKKWLNKFLDVRYDDLMNPANHDTRDEWRASVARVDVLRSIAKENNYNLNGPIHVRSNEYGNFVIKLE.

[0042] The nucleotide sequence of the gene encoding chitosanase CsnQB-E206A is shown in SEQ ID NO.4, as shown below (direction 5'-3'):

[0043] GCGGGCCTGAACAAAGATCAGAAACGCCGCGCGGAACAGCTGACCAGCATTTTTGAAAACGGCACCACCGAAATTCAGTATGGCTATGTGGAACGCCTGGATGATGGCCGCGGCTATACCTGCGGTCGCGCGGGTTTTACCACCGCGACCGGTGATGCGTTAGAAGTGGTGGAAGTGTATACCAAAGCGGTGCCGAACAACAAACTGAAAAAGTATCTGCCGGAACTGCGCCGCCTGGCGAAAGAAGAAAGCGATGATACCAGCAACCTGAAAGGCTTTGCGAGCGCGTGGAAAAGCCTGGCGAATGATAAAGAATTTCGCGCGGCGCAGGATAAAGTGAACGATCATCTGTATTATCAGCCGGCGATGAAACGCAGCGATAACGCGGGCTTAAAAACCGCGCTGGCGCGTGCGGTTATGTATGATACCGTTATTCAGCATGGCGATGGCGATGATCCGGATAGCTTTTATGCGCTGATTAAACGCACCAACAAAAAGGCGGGCGGCAGCCCGAAAGATGGCATTGATGAAAAGAAATGGCTGAACAAATTCCTGGACGTGCGCTATGATGATCTGATGAACCCGGCGAACCATGATACCCGCGATGAATGGCGCGCGAGCGTGGCGCGTGTTGATGTGTTACGCAGCATTGCGAAAGAAAACAACTATAACCTGAACGGCCCGATTCATGTGCGCAGCAACGAATATGGCAACTTTGTGATTAAACTCGAG。

[0044] CBM is a type of non-catalytically active protein structure that can specifically bind to polysaccharides (especially insoluble polysaccharides), thereby increasing the affinity between the enzyme and the substrate and promoting the degradation of macromolecular compounds such as cellulose and chitin. However, chitosanase CsnQB contains only one catalytic domain and lacks a CBM domain. Therefore, in order to further enhance the degradation ability of chitosanase to chitosan, the present invention selects a CBM sequence (elgii) and fuses the sequence to the C-terminus of chitosanase CsnQB-E206A through rational design. The fused chitosanase is named chitosanase CsnQB-E206A-elgii, and its amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence of the coding gene of chitosanase CsnQB-E206A-elgii is shown in SEQ ID NO.6.

[0045] The amino acid sequence of chitosanase CsnQB-E206A-elgii is shown in SEQ ID NO.5, as shown below:

[0046] AGLNKDQKRRAEQLTSIFENGTTEIQYGYVERLDDGRGYTCGRAGFTTATGDALEVVEVYTKAVPNNKLKKYLPELRRLAKEESDDTSNLKGFASAWKSLANDKEFRAAQDKVNDHL YYQPAMKRSDNAGLKTALARAVMYDTVIQHGDGDDPDSFYALIKRTNKKAGGSPKDGIDEKKWLNKFLDVRYDDLMNPANHDTRDEWRASVARVDVLRSIAKENNYNLNGPIHVRSN EYGNFVIKLEPTAPANLTATAVSSSQVNLSWTASTDNVGVKEYKIYRGGTEVGTATGTSYSDTGLNPSTTYSYTVKAYDAAGNASANSNTASATTTDGPSTDTNIAKGKTAKASSQE GSGYEASKAFDGNASTRWASKEGSDPQWIYVDLGKTYSVNKVKLNWEAAYGKNYKIQVSNDSGSPANWTDVYTKTNGKGGVEEITFAAQDARYVRMYGTARGTSYGYSLYEFEVYGP.

[0047] The nucleotide sequence of the gene encoding chitosanase CsnQB-E206A-elgii is shown in SEQ ID NO.6, as shown below (direction 5'-3'):

[0048]

[0049] Example 2 Preparation of chitosanase and CBM-elgii protein

[0050] The chitosanase CsnQB, chitosanase CsnQB-E206A, chitosanase CsnQB-E206A-elgii and CBM-elgii proteins were heterologously expressed by conventional methods, and extracted and purified. The steps are as follows:

[0051] (1) Construction of recombinant expression vector

[0052] The coding gene of chitosanase CsnQB was fully synthesized and amplified by PCR to obtain a gene fragment, which was connected with the PET-28a (+) cloning vector using seamless cloning technology to obtain a pET28a plasmid containing the coding gene of chitosanase CsnQB.

[0053] Specific primers were designed for the mutation site, and the pET28a plasmid containing the coding gene of chitosanase CsnQB was used as a template for PCR amplification to obtain the pET28a plasmid containing the coding gene of chitosanase CsnQB-E206A.

[0054] The CBM sequence, the coding gene of elgii, was fully synthesized and amplified by PCR to obtain the elgii gene fragment, which was then connected with the pET-28a (+) cloning vector using seamless cloning technology to obtain the pET28a plasmid containing the coding gene of CBM-elgii.

[0055] Specific primers were designed for the fusion site, and fusion PCR amplification was performed using the pET28a plasmid containing the coding gene of chitosanase CsnQB-E206A and the elgii gene fragment as templates to obtain a fusion plasmid containing the coding gene of chitosanase CsnQB-E206A-elgii.

[0056] The plasmid obtained above was transformed into competent E. coli DH5α cells. Positive transformants were screened using LB plates containing kanamycin sulfate. The clones were verified by PCR and DNA sequencing to obtain recombinant plasmids.

[0057] (2) Construction of recombinant engineered bacteria

[0058] The correctly sequenced recombinant plasmid was extracted and transformed into the host Escherichia coli BL21 competent cells, and the constructed engineered bacteria were grown on kanamycin sulfate resistance plates.

[0059] (3) Expression of chitosanase and CBM protein

[0060] The recombinant engineered bacterial strain grown on the kanamycin sulfate resistance plate was picked up, inoculated into 5 mL of LB liquid culture medium containing 50 μg / mL kanamycin sulfate, and activated at 37°C and 220 rpm for 12 hours; inoculated into 50 mL of LB liquid culture medium containing 50 μg / mL kanamycin sulfate at a 1% inoculum, and cultured at 37°C and 220 rpm until the OD600 value was 0.8; isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0.1 mM to induce the expression of chitosanase and CBM protein for 16 hours.

[0061] (4) Extraction and purification of chitosanase and CBM protein

[0062] After the above-mentioned induction expression, the culture medium was taken, centrifuged at 4°C and 10,000 rpm for 15 minutes, the bacteria were collected, resuspended in Tris-HCl buffer (pH 8.0), ultrasonically disrupted for 15 minutes, and centrifuged at 4°C and 10,000 rpm for 10 minutes to remove cell debris. The supernatant was the crude enzyme solution.

[0063] Crude enzyme solution using Ni - NTA column for affinity chromatography purification: first use 10 mM imidazole solution (10 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl) to balance the column, after loading the sample, use 20 mM imidazole solution (20 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl) to elute the weakly binding impurities, then use 100 mM imidazole solution (100 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl) to elute the target protein, and collect the eluate, which is the pure enzyme solution or protein solution.

[0064] Using the above method, the following were prepared respectively: pure enzyme solution containing chitosanase CsnQB with a protein concentration of 4.552 mg / ml; pure enzyme solution containing chitosanase CsnQB-E206A with a protein concentration of 3.103 mg / ml; pure enzyme solution containing chitosanase CsnQB-E206A-elgii with a protein concentration of 2.560 mg / ml; and protein solution containing CBM-elgii protein with a protein concentration of 5.385 mg / ml.

[0065] Example 3 Determination of enzyme activity

[0066] The activity of chitosanase was determined by dinitrosalicylic acid (DNS) colorimetric method. The reaction system consisted of 10 μL enzyme solution, 90 μL chitosan solution, 100 μL phosphate buffer (pH 8.0), and 40°C for 10 minutes. After the reaction, the mixture was placed in a boiling water bath for 10 minutes, 300 μL of DNS reagent was added, and the mixture was placed in a boiling water bath for 10 minutes for color development. The mixture was centrifuged at 12,000 rpm for 3 minutes, and the absorbance at 540 nm was detected.

[0067] One unit (U) of chitosanase activity is defined as the amount of enzyme required to produce 1 μmol of reducing sugar per minute.

[0068] It was determined that the enzyme activity of chitosanase CsnQB was 306.36 U / mg, which was slightly different from the record in Chinese invention patent CN 118240805A (the enzyme activity of chitosanase CsnQB was 246.26 U / mg). The enzyme activity of chitosanase CsnQB prepared in different batches was different in specific values, which was normal and within an acceptable range. The enzyme activity of chitosanase CsnQB-E206A was 728.89 U / mg, and the enzyme activity of chitosanase CsnQB-E206A-elgii was 926.25 U / mg. Compared with chitosanase CsnQB, the enzyme activity of chitosanase CsnQB-E206A increased by 137.91%, and the enzyme activity of chitosanase CsnQB-E206A-elgii increased by 202.34%.

[0069] The chitosan solution is prepared from colloidal chitosan and 10 mg / mL acetic acid solution, and the concentration of chitosan is 20 mg / mL.

[0070] Example 4 Determination of the Optimal Reaction Conditions

[0071] () Determination of optimum temperature: In the range of 30-70°C, the enzyme activities of chitosanase CsnQB-E206A and chitosanase CsnQB-E206A-elgii at different temperatures were determined according to the method of Example 3. The relative enzyme activities at different temperatures were calculated with the highest enzyme activity as 100%.

[0072] Schematic diagram of the effect of temperature on the relative enzyme activity of chitosanase CsnQB-E206A Figure 3 As shown, the optimum reaction temperature is 45°C, the activity is higher in the range of 30-50°C, and the relative activity is higher than 50%, which is basically consistent with chitosanase CsnQB.

[0073] Schematic diagram of the effect of temperature on the relative enzyme activity of chitosanase CsnQB-E206A-elgii Figure 4As shown, the optimum reaction temperature is 45°C, the activity is higher in the range of 30-55°C, and the relative activity is higher than 50%, which is basically consistent with chitosanase CsnQB.

[0074] (2) Determination of the optimum pH: In the range of pH 3.0 to 10.0, the enzymatic activities of chitosanase CsnQB-E206A and chitosanase CsnQB-E206A-elgii at different pH values ​​were determined according to the method of Example 3. The buffers used were: acetate buffer at pH 3.0 to 6.0, phosphate buffer at pH 6.0 to 8.0, Tris-HCl buffer at pH 8.0 to 9.0, and Gly-NaOH buffer at pH 9.0 to 10.0, respectively. "Gly" represents glycine. The relative enzyme activity at different pH values ​​was calculated with the highest enzyme activity being 100%.

[0075] Schematic diagram of the effect of pH on the relative enzyme activity of chitosanase CsnQB-E206A Figure 5 As shown, the optimum pH is 8.0, which is basically consistent with chitosanase CsnQB.

[0076] Schematic diagram of the effect of pH on the relative enzyme activity of chitosanase CsnQB-E206A-elgii Figure 6 As shown, the optimum pH is 8.0, which is basically consistent with chitosanase CsnQB.

[0077] Example 5 Determination of the ability of chitosanase to degrade powdered chitosan

[0078] The degradation ability of chitosanase CsnQB-E206A-elgii on powdered chitosan was determined by the amount of reducing sugar produced. 50 μL of pure enzyme solution containing chitosanase CsnQB-E206A-elgii, 50 μL of ultrapure water, 100 μL of acetate buffer (pH 5.0) and 2 mg of powdered chitosan were mixed and reacted in a water bath shaker at 45°C and 220 rpm for 3 hours; after the reaction, the mixture was heated in a boiling water bath for 10 minutes, 300 μL of DNS reagent was added for reaction, and the mixture was heated in a boiling water bath for 10 minutes for color development; the mixture was centrifuged at 12,000 rpm for 3 minutes, the absorbance at 540 nm was detected, and the amount of reducing sugar produced was calculated by substituting it into the DNS standard curve.

[0079] The degradation ability of chitosan powder by chitosanase CsnQB and chitosanase CsnQB-E206A was determined by the same method. The enzyme activities of chitosanase CsnQB and chitosanase CsnQB-E206A in the reaction system were consistent with chitosanase CsnQB-E206A-elgii, and the reaction was carried out under the same conditions.

[0080] The results showed that the reducing sugar production of chitosanase CsnQB was 2.71 μmol / ml, the reducing sugar production of chitosanase CsnQB-E206A was 3.11 μmol / ml, and the reducing sugar production of chitosanase CsnQB-E206A-elgii was 11.19 μmol / ml. Compared with chitosanase CsnQB, the degradation ability of chitosan powder by chitosanase CsnQB-E206A was increased by 14.76%, and the degradation ability of chitosan powder by chitosanase CsnQB-E206A-elgii was increased by 312.92%, with significant improvement.

[0081] Example 6 Identification of degradation products of chitosan in different forms by chitosanase

[0082] Take 50 μL of pure enzyme solution containing chitosanase CsnQB-E206A-elgii, 50 μL of ultrapure water, 100 μL of acetate buffer (pH 5.0) and 2 mg of powdered chitosan, mix them, react in a water bath shaker at 45°C and 220 rpm for 12 hours, and terminate the reaction in a boiling water bath for 20 minutes. At the same time, chitosanase CsnQB and chitosanase CsnQB-E206A were used as controls. The enzyme activities of chitosanase CsnQB and chitosanase CsnQB-E206A in the reaction system were consistent with chitosanase CsnQB-E206A-elgii, and the reaction was carried out under the same conditions.

[0083] 50 μL of pure enzyme solution containing chitosanase CsnQB-E206A-elgii was mixed with 150 μL of chitosan solution (chitosan concentration was 20 mg / mL) and reacted at 40°C for 12 hours; the reaction was terminated by boiling water bath for 20 minutes. At the same time, chitosanase CsnQB and chitosanase CsnQB-E206A were used as controls. The enzyme activities of chitosanase CsnQB and chitosanase CsnQB-E206A in the reaction system were consistent with those of chitosanase CsnQB-E206A-elgii, and the reactions were carried out under the same conditions.

[0084] High-performance liquid chromatography (HPLC) was used for product identification. HPLC is a high-performance size exclusion chromatography method equipped with a refractive index detector (HPSECRID, Agilent 1260, Agilent Technologies, Santa Cruz, CA). The system used a Superdex 30 Increase 10 / 300 GL column (GE Healthcare, Uppsala, Sweden) with 0.2 M ammonium bicarbonate as the mobile phase at a flow rate of 0.4 mL / min. After centrifugation at 10,000 g for 2 minutes, the supernatant was filtered (pore size 0.22 μm, Millipore, Germany), and then 100 μL of the supernatant was injected into the HPLC system. The product components were qualitatively identified based on the corresponding standard curve.

[0085] The HPLC chromatograms of the degradation products of powdered chitosan degraded by various chitosanases are shown in Figure 7 As shown, the HPLC chromatograms of the degradation products of colloid chitosan by various chitosanases are shown in Figure 8 As shown. After chitosanase CsnQB, chitosanase CsnQB-E206A and chitosanase CsnQB-E206A-elgii degraded different forms of chitosan, most of the products were (GlcN) 2 、(GlcN) 3 Since the enzyme activity is set to be consistent, Figure 8 The total abundance of degradation products of degraded chitosan was not significantly different. Figure 7 The results showed that under the same enzyme activity conditions, the total abundance of degradation products of chitosanase CsnQB-E206A after degrading powdered chitosan was slightly higher than that of chitosanase CsnQB, while the total abundance of degradation products of chitosanase CsnQB-E206A-elgii after degrading powdered chitosan was significantly higher than that of chitosanase CsnQB-E206A.

[0086] The above analysis shows that through site-directed mutation of chitosanase CsnQB, the degradation ability of chitosanase CsnQB-E206A on chitosan has been improved, with higher enzyme activity, and more products are generated by degrading powdered chitosan. Further, by fusing the CBM sequence, the degradation ability of chitosanase CsnQB-E206A-elgii on powdered chitosan has been further significantly enhanced, producing more products, showing its application potential in the industrial efficient degradation of powdered chitosan to produce chitooligosaccharides.

[0087] Example 7 Destructive effect of CBM-elgii protein on powdered chitosan

[0088] Take 4 mg of powdered chitosan and add it to 0.4 ml of protein solution containing CBM-elgii protein (adjust the protein concentration to 0.01 mg / ml), and react in a water bath shaker at 20°C and 180 rpm for 5 hours; after the reaction, centrifuge at 12000 rpm for 10 minutes, discard the supernatant, collect the precipitate, and lyophilize to obtain powdered chitosan treated with CBM-elgii protein.

[0089] The surface morphology of powdered chitosan and powdered chitosan treated with CBM-elgii protein was observed by scanning electron microscopy. The SEM image (5000×) of powdered chitosan is shown in Figure 2. Fig. 9 As shown in the SEM image (10000×) of powdered chitosan Fig.11 The SEM image (5000×) of powdered chitosan treated with CBM-elgii protein is shown in Fig.10 The SEM image (10000×) of powdered chitosan treated with CBM-elgii protein is shown in Fig.12 As shown in the figure, the surface of powdered chitosan without CBM-elgii protein treatment is relatively smooth and compact, making it difficult to be hydrolyzed by enzymes. However, the surface of powdered chitosan treated with CBM-elgii protein becomes rougher, with gaps of varying sizes. These structural changes indicate that the CBM domain destroys the dense structure of powdered chitosan, thereby promoting the subsequent biotransformation process.

[0090] Example 8 Preparation of chitosan oligosaccharide functional beverage

[0091] The components of chitosan oligosaccharide functional beverage are as follows (taking 100 ml as an example): chitosan oligosaccharide 50 mg, concentrated apple juice 15 ml, erythritol 1.2 g, citric acid 0.8 g, vitamin C 30 mg, vitamin B 12 0.01 mg, inulin 1 g, potassium sorbate 0.015 g, and the balance is water.

[0092] The chitosan oligosaccharide is prepared by the following method: taking 5 L of chitosan solution with a concentration of 20 mg / mL, adjusting the pH to 8.0, adding pure enzyme solution containing chitosanase CsnQB-E206A-elgii in an amount of 2 U / ml, reacting at 45°C for 12 hours; spray drying to obtain chitosan oligosaccharide.

[0093] The preparation method of the chitosan oligosaccharide functional beverage is as follows:

[0094] (1) Add concentrated apple juice, erythritol and inulin to 60°C water, stir until fully dissolved, cool to room temperature and set aside;

[0095] (2) Add chitosan oligosaccharide, citric acid, vitamin C and vitamin B to water12 and potassium sorbate, stir until fully dissolved, set aside;

[0096] (3) The solutions prepared in the above (1) and (2) are mixed uniformly to obtain a mixed solution; the mixed solution is mixed with degassed water, sterilized, and cooled to obtain a chitosan oligosaccharide functional beverage.

[0097] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A chitosanase CsnQB-E206A-elgii, characterized in that: The amino acid sequence is shown in SEQ ID NO.

5.

2. Use of the chitosanase CsnQB-E206A-elgii according to claim 1 in degrading chitosan.

3. The use of the chitosanase CsnQB-E206A-elgii according to claim 2 in the degradation of chitosan, characterized in that: The chitosan is selected from colloid chitosan or powdered chitosan.

4. The use of the chitosanase CsnQB-E206A-elgii according to claim 1 in the preparation of chitosan oligosaccharide functional beverages, characterized in that: The chitosan oligosaccharide functional beverage contains chitosan oligosaccharide, and the chitosan oligosaccharide is prepared by degrading chitosan using chitosanase CsnQB-E206A-elgii.

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