Method for synergistically degrading chitin through multiple enzymes

Abstract

The invention discloses a method for synergistically degrading chitin by multiple enzymes, which comprises the following steps: adding lysing polysaccharide monooxygenase OsLPMO10A, chitinase and beta-N-acetylglucosaminidase Sohex into an alpha-chitin solution, and carrying out enzymolysis to prepare GlcNAc; the amino acid sequence of the beta-N-acetylglucosaminidase Sohex is as shown in SEQ ID NO. 1 (sequence identifier number 1). The invention further discloses the beta-N-acetylglucosaminidase Sohex and application of the beta-N-acetylglucosaminidase Sohex in degradation of chitin, preparation of GlcNAc and synthesis of lactose-N-trisaccharide II. The invention also discloses a method for synthesizing the lactose-N-trisaccharide II. The beta-N-acetylglucosaminidase Sohex with high hydrolytic activity and promotion effect is excavated, the beta-N-acetylglucosaminidase Sohex is combined with lysing polysaccharide monooxygenase and chitinase to be applied to degradation of alpha-chitin to prepare GlcNAc, and the beta-N-acetylglucosaminidase Sohex has very important environmental protection and industrial significance and has important industrial application value and economic value.

Description

Technical Field

[0001] The invention relates to a method for multi-enzyme synergistic degradation of chitin, belonging to the technical field of enzyme engineering. Background Art

[0002] As resource shortages become increasingly severe, there are more global initiatives involving turning to the ocean for food and resources. In addition to being developed as food, aquatic products are also an important source of high-value products, especially wastes rich in macromolecules. α-Chitin is the main component of crustacean shells and the second most abundant polysaccharide resource in nature. It is also a rich raw material for high-value bioactive molecules, with biocompatibility, biodegradability and non-toxicity. However, due to its ordered crystalline state, α-chitin is difficult to dissolve and degrade, resulting in low bioavailability. As a constituent unit of α-chitin, N-acetyl-D-glucosamine (GlcNAc) has ideal biological activity and is an ideal precursor for the preparation of many high-value compounds with higher practicality. In order to maximize the use of waste resources and reduce environmental pressure, people have been exploring efficient ways to convert α-chitin to GlcNAc.

[0003] In addition to the traditional, environmentally unfriendly acid method, there are also many reports on the production of GlcNAc by hydrolyzing colloidal chitin with chitinase (EC 3.2.1.14) and β-N-acetylglucosaminidase (EC 3.2.1.52), ball-milled chitin, ionic liquids and other methods of pre-treated α-chitin. Although chitinase and β-N-acetylglucosaminidase have significant synergistic effects and high conversion rates, the pretreatment of α-chitin greatly increases the industrial cost.

[0004] In order to improve the hydrolysis efficiency of α-chitin without pretreatment, several different hydrolysis methods have been explored. Pichyangkura et al. [Pichyangkura et al. Quantitative production of 2-acetamido-2-deoxy-D-glucose from crystalline chitin by bacterial chitinase. Carbohydr Res 2002, 337 (6), 557-559] used chitinase and β-N-acetylglucosaminidase to degrade α-chitin to produce GlcNAc, with a conversion rate of 41%. Suresh et al. [Suresh et al. Enhanced degradation of α-chitinmaterials prepared from shrimp processing byproduct and production of N-acetyl-D-glucosamine by thermoactive chitinases from soil mesophilic fungi. Biodegradation 2012, 23 (4), 597-607] used the same method to prepare GlcNAc, with a conversion rate of 23.7%. Sashiwa et al. [Sashiwa et al. Production of N-acetyl-D-glucosamine from α-chitin by crude enzymes from Aeromonas hydrophila H-2330. Carbohydr Res 2002,337 (8), 761-763] described another method for converting α-chitin using chitinase purified from Aeromonas hydrophila, which has the same disadvantages. Although some chitinases have been found to hydrolyze α-chitin, the main disadvantages of these studies are low yield, low purity and long time consumption due to the compact structure of chitin. Many chitinases must work in synergy with lytic polysaccharide monooxygenases (LPMOs) to further destroy the crystal structure of chitin and further improve the hydrolysis efficiency. The research group of the inventor of the present invention previously discovered a lytic polysaccharide monooxygenase OsLPMO10A (recorded in Chinese invention patent CN 116790696 A) that can effectively destroy the crystal structure of α-chitin. It works synergistically with chitinase and the conversion rate can reach 30.86% within 24 hours; unfortunately, the product obtained is a mixture of chitooligosaccharides with low GlcNAc purity.Therefore, it is urgent to explore an effective strategy that can simultaneously improve the conversion rate of α-chitin and the purity of GlcNAc. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides a method for the synergistic degradation of chitin by multiple enzymes. At the same time, the present invention also provides a β-N-acetylglucosaminidase Sohex, and its application in the degradation of chitin, the preparation of GlcNAc, and the synthesis of lactose-N-trisaccharide II. The present invention also provides a method for synthesizing lactose-N-trisaccharide II.

[0006] The present invention is achieved through the following technical solutions: A method for multi-enzyme synergistic degradation of chitin, comprising the following steps: adding a lytic polysaccharide monooxygenase OsLPMO10A, chitinase and β-N-acetylglucosaminidase Sohex to an α-chitin solution, performing enzymolysis to prepare GlcNAc; The amino acid sequence of the β-N-acetylglucosaminidase Sohex is shown in SEQ ID NO.1; The chitinase is composed of chitinase SmChi A, chitinase SmChi B and chitinase SmChi C, and the molar ratio of the three is 1:1:1.

[0007] Furthermore, the concentration of the α-chitin solution is 5 g / L, the added enzyme amounts of the lytic polysaccharide monooxygenase and chitinase are both 3 μM, and the added enzyme amount of the β-N-acetylglucosaminidase Sohex is 2 μM.

[0008] Furthermore, the enzymatic hydrolysis conditions are: enzymatic hydrolysis temperature 45°C, and enzymatic hydrolysis time 24 h.

[0009] Furthermore, the α-chitoxin solution is prepared by the following method: adding α-chitoxin to a phosphate buffer solution and mixing the mixture; wherein the concentration of the phosphate buffer solution is 10 mM and the pH value is 6.0.

[0010] A β-N-acetylglucosaminidase Sohex, whose amino acid sequence is shown as SEQ ID NO.1.

[0011] The nucleotide sequence of the gene encoding the β-N-acetylglucosaminidase Sohex is shown in SEQ ID NO.2.

[0012] The beta-N-acetylglucosaminidase Sohex is used in degrading chitin or preparing GlcNAc.

[0013] The beta-N-acetylglucosaminidase Sohex is used in synthesizing lactose-N-trisaccharide II.

[0014] A method for synthesizing lactose-N-trisaccharide II is as follows: lactose and β-N-acetylglucosaminidase Sohex are added to a chitobiose solution, and enzymolysis is performed to prepare lactose-N-trisaccharide II.

[0015] Furthermore, the concentration of chitobiose is 25 mM, the concentration of lactose is 250 mM, and the concentration of β-N-acetylglucosaminidase Sohex is 3 μM.

[0016] Furthermore, the enzymolysis conditions are: enzymolysis temperature 50°C, enzymolysis time 2.5 h.

[0017] The present invention expresses β-N-acetylglucosaminidase Sohex for the first time. The specific enzyme activity of the enzyme is 1613 U / mg, the optimum temperature is 50°C, the optimum pH is 7.0, and it has good temperature stability and pH stability. Studies have shown that the enzyme can be used to convert pNP-GlcNAc, (GlcNAc) 2-6 The invention also constructs a synergistic enzymatic hydrolysis system of α-chitin, lytic polysaccharide monooxygenase OsLPMO10A, chitinase, and β-N-acetylglucosaminidase Sohex, which can efficiently prepare GlcNAc.

[0018] The present invention has discovered a new β-N-acetylglucosaminidase with high hydrolysis activity and promotion effect, namely β-N-acetylglucosaminidase Sohex, and combined it with the lytic polysaccharide monooxygenase OsLPMO10A and chitinase to degrade α-chitin to prepare GlcNAc, which has very important environmental and industrial significance, and has important industrial application value and economic value.

[0019] 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

[0020] Figure 1 : SDS-PAGE electrophoresis, wherein M1 and M2 are standard proteins; 1 is the lytic polysaccharide monooxygenase OsLPMO10A; 2 is the chitinase SmChiB; 3 is the β-N-acetylglucosaminidase Sohex; 4 is the chitinase SmChiA; 5 is the chitinase SmChiC.

[0021] Figure 2 : Schematic diagram of the effect of temperature change on relative enzyme activity.

[0022] Figure 3: Schematic diagram of the effect of pH change on relative enzyme activity.

[0023] Figure 4 :Schematic diagram of the effect of different incubation times at different temperatures on relative enzyme activity.

[0024] Figure 5 : Schematic diagram of the effect of different incubation times on relative enzyme activity under different pH conditions.

[0025] Figure 6 : Schematic diagram of the detection results of relative enzyme activity under different temperature conditions.

[0026] Figure 7 : Schematic diagram of the detection results of relative enzyme activity under different pH conditions.

[0027] Figure 8 :Schematic diagram of the detection results of relative enzyme activity of β-N-acetylglucosaminidase under different Sohex enzyme addition conditions.

[0028] Fig. 9 : Schematic diagram of the detection results of GlcNAc yield and concentration under different substrate concentration conditions.

[0029] Fig.10 : Liquid phase diagram and primary mass spectrum of cascade degradation of chitin to produce GlcNAc.

[0030] Fig.11 :HPLC and mass spectrum of Sohex transglycosidation product lactose-N-trisaccharide II. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] Example 1 Mining of β-N-acetylglucosaminidase Sohex In order to explore β-N-acetylglucosaminidase with high hydrolysis activity, the present invention screened a β-N-acetylglucosaminidase from Sulfuriroseicoccus oceanibius (GenBank: WP_235203338.1) from the NCBI website. Its amino acid sequence is shown in SEQ ID NO.1, and the present invention names it as β-N-acetylglucosaminidase Sohex. The gene was codon optimized for efficient expression in Escherichia coli, and the nucleotide sequence of the codon-optimized gene is shown in SEQ ID NO.2. The gene fragment was artificially synthesized and connected to the pET-28a (+) expression vector.

[0034] The amino acid sequence of β-N-acetylglucosaminidase Sohex (as shown in SEQ ID NO.1): MLASTPPSIIPKPSTLTLAENASGFTLGADTTIVHSPELKAEAALLADYLKPATGFELKTVGSAAGEGGVIELAMDPSMSDNTGAYSLAAKDGKVTITGTTPAAVFYGIQSLRQILPVEILSDEKVDGVEWSVPAVEVKDEPRFGWRGMHLDVGRHMFEVDDIKKFIDLLALHKLSNFHWHLTEDQGWRVEIKKYPKLTEVGAWRKSTPPYGDRSASDGKRYGGFYTQEQIKEIVAYAAERHITIVPEIDMPGHMAAAIASYPEFGNTDVPNYAPEVCTSWGVHPYVLSPTEETFQFVDDVLGEICELFPSEYIHIGGDEAPKTQWQQSKIAQDVIKREGLKDEHELQSYFIRRVEKMLNKRGRRLIGWDEIREGGLSPSATVMSWRGVGPGIASAKEGNDVVMAPNSHTYFDHYQAPSAQELAKGVEFEAIGGYLPITKLYDFDPVLPGQLTEEENDHVLGVQAQLWTEYMKTWDKVEYMAFPRVAALSEIAWTQPSQKDFKDFAGRLETMMKRYEALGVNAYSDPIPLPPQSLPGTSVDTSLPSHGEYHAVQAHDGKPDTVFWSSRGPQVNDHLTVRFDEPSKGGNIRVFTGGDGKHAGDTLRNGVVEVTSDDSKWVEVAKFDNGMATGEVPAGTLAVRMRATGNQENWLIVKEVVIEEVRRD。

[0035] Nucleotide sequence of the codon-optimized gene (in the 5'-3' direction, as shown in SEQ ID NO.2)

[0036] Example 2 Preparation of β-N-acetylglucosaminidase Sohex Here are the steps: The synthesized plasmid of Example 1 was transferred into competent cells of Escherichia coli BL21 (DE3). Positive transformants were screened using LB plates containing kanamycin sulfate. After the clones were verified by colony PCR using T7 universal primers, positive clones were picked and inoculated in 5 mL of LB liquid culture medium containing 50 μg / mL kanamycin sulfate, and cultured at 37°C and 220 rpm for 12 h. Inoculate 50 mL of LB liquid culture medium containing 50 μg / mL kanamycin sulfate at a 1% inoculum, and culture at 37°C and 220 rpm until the OD value is 0.6; add isopropyl-β-D-thiogalactoside (IPTG) at a concentration of 0.1 mM, and induce expression at 18°C ​​for 16 h.

[0037] The culture medium was centrifuged at 8000 rpm for 5 min at 4°C, the bacteria were collected, resuspended in Tirs-HCl buffer (10 mM, pH 7.5), ultrasonically disrupted for 30 min, and centrifuged at 12000 rpm for 30 min. The supernatant was the crude enzyme solution.

[0038] The crude enzyme solution was purified by affinity chromatography using a Ni-NTA nickel column. The column was equilibrated with a 10 mM imidazole solution (10 mM imidazole, 150 mM NaCl, 50 mM Tris-HCl), and then the weakly bound impurities were eluted with a 20 mM imidazole solution (20 mM imidazole, 150 mM NaCl, 50 mM Tris-HCl). The target protein was eluted with a 200 mM imidazole solution (200 mM imidazole, 150 mM NaCl, 50 mMTris-HCl). The eluted components in this part were collected as the purified enzyme solution and tested by SDS-PAGE. The results are shown in Figure 2. Figure 1 As shown, the purified protein showed a single band with a molecular weight of about 75 kDa, which was consistent with the prediction. The ultrafiltration tube with a molecular weight cutoff of 30 kDa was used for concentration to obtain a pure enzyme solution containing β-N-acetylglucosaminidase Sohex.

[0039] Example 3 Determination of β-N-acetylglucosaminidase Sohex enzyme activity The enzyme activity was determined using the pNP colorimetric method. The reaction system consisted of: 90 μL 1 mM p-nitrophenyl-β-N-acetylglucosamine (pNP-GlcNAc), 10 μL pure enzyme solution (prepared in Example 2), and 100 μL 50 mM phosphate buffer. The reaction was carried out at 40°C for 10 min. After the reaction was completed, 200 μL 200 mM sodium carbonate was added to terminate the reaction, and the absorbance at 410 nm was detected.

[0040] One unit (U) of β-N-acetylglucosaminidase activity was defined as the amount of enzyme required to release 1 μmol of pNP per minute.

[0041] The activity of β-N-acetylglucosaminidase Sohex was determined to be 1613 U / mg.

[0042] The pNP-GlcNAc was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0043] Example 4 Determination of the optimal reaction conditions for β-N-acetylglucosaminidase Sohex Determination of optimum temperature: In the range of 30-65°C, the enzyme activity at different temperatures was determined according to the determination method of Example 3. The highest enzyme activity was taken as 100%, and the relative enzyme activity at different temperatures was calculated. The results are as follows: Figure 2 As shown, the optimum reaction temperature is 50°C, and the activity is higher in the range of 45-55°C, with a relative activity higher than 80%.

[0044] Determination of optimum pH: In the range of pH 4.0 to 10.0 (the buffer used is: pH 4.0 to 6.0 citrate buffer, pH 6.0 to 8.0 phosphate buffer, pH 7.0 to 9.0 Tris-HCl buffer, pH 9.0 to 10.0 glycine-sodium hydroxide buffer), the enzyme activity was determined according to the determination method of Example 3, and the highest enzyme activity was taken as 100%, and the relative enzyme activity at different pH was calculated. The results are shown in FIG. Figure 3 As shown, the optimum pH is 7.0.

[0045] Determination of temperature stability: The pure enzyme solution was incubated at different temperatures (45°C, 50°C, 55°C) for different time periods (0 h, 12 h, 24 h, 48 h, 60 h), and then the enzyme activity was determined according to the determination method of Example 3. The highest enzyme activity was taken as 100%, and the relative enzyme activity after incubation at different temperatures for different time periods was calculated. The results are as follows: Figure 4 As shown. Figure 4It can be seen that β-N-acetylglucosaminidase Sohex can still maintain 100% activity after incubation at 45°C for 60 h, indicating that the enzyme has good temperature stability, but is more sensitive to high temperatures above 50°C.

[0046] Determination of pH stability: 10 μL of pure enzyme solution was mixed with 100 μL of buffer solutions of different pH values ​​(the buffer solutions used were: citrate buffer solution of pH 5.0-6.0, phosphate buffer solution of pH 6.0-8.0, and glycine-sodium hydroxide buffer solution of pH 9.0), and the mixture was incubated at 45°C for 72 h. Then 90 μL of 1 mM pNP-GlcNAc was added to the mixture, and the enzyme activity was determined according to the determination method of Example 3. The highest enzyme activity was taken as 100%, and the stability under different pH conditions was calculated. The results are shown in FIG. Figure 5 As shown. Figure 5 It can be seen that β-N-acetylglucosaminidase Sohex showed high stability under pH 9 conditions, with more than 80% residual enzyme activity still remaining after 72 h of incubation, and good pH stability.

[0047] Example 5 Determination of substrate specificity of β-N-acetylglucosaminidase Sohex The enzyme activity was detected under standard enzyme activity assay conditions using colloidal chitin, p-nitrophenyl-β-N-acetylgalactosamine (pNP-GalNAc), pNP-GlcNAc, chitosan, cellobiose, chitooligosaccharide, etc. as substrates to determine the substrate specificity of the enzyme. The results are shown in Table 1.

[0048] Table 1 Substrate specificity of β-N-acetylglucosaminidase Sohex

[0049] Note: The DNS method was used to determine the reducing sugars released from the gelatin chitin. The method for determining the enzyme activity of chitosan oligosaccharides is as follows: take 3 μM enzyme solution, add 2 mg / mL chitosan oligosaccharides, 40 μL pH 6.0 phosphate buffer, and react at 40°C for 10 min. Then the amount of β-N-acetylglucosamine released was determined by high performance liquid chromatography (HPLC). The HPLC determination conditions are: HPLC-RID detection system, chromatographic column (Sugar Pak I) temperature of 75°C, mobile phase of ethylenediaminetetraacetic acid disodium calcium (50 mg / L) solution, flow rate of 0.5 mL / min, and time of 20 min. The activity unit of β-N-acetylglucosamine is defined as: under the above reaction conditions, the amount of enzyme required to generate 1 μmol of β-N-acetylglucosamine per minute is one enzyme activity unit (1U).

[0050] The results showed that β-N-acetylglucosaminidase Sohex had no activity against chitin, chitosan and cellobiose. The highest activity was 1613 U / mg for pNP-GlcNAc. The next was (GlcNAc)2, (GlcNAc)3 and (GlcNAc)3, which had an activity of nearly 60 U / mg. The activity for (GlcNAc)5 and (GlcNAc)6 exceeded 40 U / mg. It can be seen that β-N-acetylglucosaminidase Sohex has the high activity required for the efficient preparation of GlcNAc.

[0051] The colloidal chitosan used in the present invention is prepared by acid-treating α-chitosan, and the preparation method is as follows: adding α-chitosan powder to concentrated hydrochloric acid, adding 10 mg of α-chitosan powder to every 100 ml of concentrated hydrochloric acid, stirring rapidly and uniformly, refrigerating at 4°C for 24 h, then adding 300 mL of 50% ethanol and stirring rapidly to precipitate the colloidal chitosan, repeatedly washing the precipitate with distilled water until it is neutral, and freeze-drying to obtain the colloidal chitosan powder.

[0052] The α-chitin used in the present invention was purchased from Shanghai Aladdin Biotechnology Co., Ltd.

[0053] Example 6 Optimization of conditions for cascade degradation of chitin using β-N-acetylglucosaminidase Sohex The lytic polysaccharide monooxygenase OsLPMO10A, chitinase SmChiA, SmChiB, SmChiC and β-N-acetylglucosaminidase Sohex were added to the α-chitin solution to investigate the synergistic effect of lytic polysaccharide monooxygenase OsLPMO10A, chitinase and β-N-acetylglucosaminidase Sohex, and to optimize the conditions for cascade degradation of chitin.

[0054] The initial reaction conditions were as follows: the concentration of α-chitin solution was 5 g / L, the added enzyme amounts of lytic polysaccharide monooxygenase and chitinase were both 3 μM (referring to the concentration of the enzyme in the reaction system after addition), and the added enzyme amount of β-N-acetylglucosaminidase Sohex was 2 μM; the enzymatic hydrolysis temperature was 45°C, and the enzymatic hydrolysis time was 24 h.

[0055] The α-chitoxin solution is prepared by the following method: adding α-chitoxin to a phosphate buffer solution and mixing the mixture; wherein the concentration of the phosphate buffer solution is 10 mM and the pH value is 6.0.

[0056] The lytic polysaccharide monooxygenase OsLPMO10A is prepared by conventional methods (specifically refer to Chinese invention patent CN 116790696 A), and the electrophoresis diagram is as follows Figure 1 shown.

[0057] The chitinase is a chitinase combination (consisting of chitinase SmChi A, chitinase SmChi B and chitinase SmChi C, with a molar ratio of 1:1:1), prepared by conventional methods (specifically refer to Chinese invention patent CN116790696 A), and the electrophoresis diagram is as follows Figure 1 shown.

[0058] The generated GlcNAc was determined by HPLC in the same manner as in Example 5. Considering that 1 mg of chitin can produce a maximum of 1.08 mg of GlcNAc, the yield of GlcNAc was calculated according to the formula: GlcNAc yield = GlcNAc release amount (mg) / "initial α-chitin content (mg) × 1.08" × 100%.

[0059] During the inspection, the conditions of each single factor were adjusted, as shown below for details.

[0060] (1) Determination of the optimum temperature: The activity at different temperatures was determined by HPLC within the range of 30-65°C, with the highest enzyme activity being 100%. The results are as follows: Figure 6 As shown, the optimum reaction temperature is 45°C, and the activity is at a high level in the range of 45-55°C.

[0061] (2) Determination of the optimum pH: Within the range of pH 5.0-10.0 (the phosphate buffer in the initial condition was adjusted to: acetate buffer of pH 5.0-6.0, phosphate buffer of pH 6.0-8.0, Tris-HCl buffer of pH 7.0-9.0, glycine-sodium hydroxide buffer of pH 9.0-10.0), the activity at different pH was determined by HPLC, with the highest enzyme activity being 100%. The results are as follows: Figure 7 As shown, the optimum pH is 6.0, and the yield is at a high level in the pH range of 5.0 to 7.0.

[0062] (3) Determination of the optimal enzyme dosage: The dosage of lytic polysaccharide monooxygenase and chitinase was kept unchanged at 3 μM, and the dosage of β-N-acetylglucosaminidase Sohex was changed (respectively: 0.5, 1, 1.5, 2, 2.5, 3 μM). The products under different enzyme dosages were determined by HPLC method, with the highest enzyme activity as 100%. The results are as follows Figure 8 As shown, the optimal enzyme amount of β-N-acetylglucosaminidase Sohex is 2 μM.

[0063] (4) Determination of the optimal substrate concentration: The addition amounts of lytic polysaccharide monooxygenase, chitinase and β-N-acetylglucosaminidase Sohex were kept at 3 μM, 3 μM and 2 μM respectively, and the concentration of α-chitin was changed (5, 10, 20, 30, 40, 50 and 60 g / L). The yields at different substrate concentrations were determined by HPLC. The results are shown in the figure. Fig. 9 Although the GlcNAc yield increases with the increase of substrate concentration, the conversion rate is the highest when the concentration of α-chitin is 5 g / L. The increase of substrate concentration will lead to a decrease in conversion rate, resulting in a waste of resources.

[0064] In summary, the best reaction conditions are: the concentration of α-chitin solution is 5 g / L, the enzyme dosage of lytic polysaccharide monooxygenase and chitinase is 3 μM, the enzyme dosage of β-N-acetylglucosaminidase Sohex is 2 μM; the enzymatic hydrolysis temperature is 45℃, the enzymatic hydrolysis pH is 6.0, and the enzymatic hydrolysis time is 24 h. At this time, the GlcNAc yield is 42.53%.

[0065] Example 7 Identification of GlcNAc Produced by Cascade Degradation of Chitosan The reaction was carried out according to the optimal reaction conditions of Example 6, and after preliminary identification by HPLC, it was identified again by mass spectrometry. The conditions were: ESI ion source, positive ion mode, primary mass spectrometry. The results are as follows Fig.10 As shown in the mass spectrum, at a mass-to-charge ratio (m / z) of 244 ([M+Na] + ), which is consistent with the molecular weight of the product being 221.

[0066] Example 8 Use of β-N-acetylglucosaminidase Sohex for the synthesis of lactose-N-triose II First, chitinase SmChiB was used to hydrolyze the colloid chitin to prepare chitobiose. The reaction conditions were 2% colloid chitosan solution, 3 μM / ml of enzyme, 12 h at 45°C, boiling, filtering, freeze-drying and concentration, and then adding water to dissolve into high-concentration chitobiose. The concentration of chitobiose was measured by HPLC, the method was the same as in Example 5.

[0067] Lactose and β-N-acetylglucosaminidase Sohex were added to the chitobiose solution. After addition, the concentrations of chitobiose, lactose and β-N-acetylglucosaminidase Sohex were 25 mM, 250 mM and 3 μM, respectively. The reaction was carried out in a water bath at 50°C for 2.5 h, and the yield of lactose-N-triose II was determined by HPLC, and the conversion rate of lactose-N-triose II was initially achieved to be 11.24%.

[0068] The HPLC method was as follows: HPLC-RID detection system, chromatographic column (XBridge BEH Amide 5 μm), temperature was 45°C, mobile phase was 72% acetonitrile, flow rate was 0.4 mL / min, and the reaction time was 45 min.

[0069] Conversion rate = synthesized lactose-N-triose II concentration (mM) / initial chitobiose concentration (mM) × 100%.

[0070] The lactose was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0071] Example 9 Identification of lactose-N-trisaccharide II synthesized by β-N-acetylglucosaminidase Sohex transglycosylation The reaction of Example 8 was initially identified by HPLC and then identified again by mass spectrometry. The conditions were: ESI ion source, positive ion mode, primary mass spectrometry. The results are as follows Fig.11 As shown in the mass spectrum, the mass-to-charge ratio (m / z) is 546.2 ([M+H] + )568.2([M+Na] + )、584.2([M+K] + ), which is consistent with the molecular weight of the product, 545.

[0072] 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 method for multi-enzyme synergistic degradation of chitosan, characterized in that: The following steps are involved: Adding lytic polysaccharide monooxygenase OsLPMO10A, chitinase and β-N-acetylglucosaminidase Sohex to the α-chitin solution for enzymatic hydrolysis to prepare GlcNAc; The amino acid sequence of the β-N-acetylglucosaminidase Sohex is shown in SEQ ID NO.1; The chitinase is composed of chitinase SmChi A, chitinase SmChi B and chitinase SmChi C, and the molar ratio of the three is 1:1:

1.

2. The method for multi-enzyme synergistic degradation of chitosan according to claim 1, characterized in that: The concentration of the α-chitin solution is 5 g / L, the added enzyme amounts of the lytic polysaccharide monooxygenase and chitinase are both 3 μM, and the added enzyme amount of the β-N-acetylglucosaminidase Sohex is 2 μM.

3. The method for degrading chitosan by multiple enzymes according to claim 1, characterized in that: The enzymolysis conditions are: enzymolysis temperature 45° C., and enzymolysis time 24 hours.

4. The method for degrading chitosan by multiple enzymes according to claim 1, characterized in that: The α-chitoxin solution is prepared by the following method: adding α-chitoxin to a phosphate buffer solution and mixing the solution; wherein the concentration of the phosphate buffer solution is 10 mM and the pH value is 6.

0.

5. A β-N-acetylglucosaminidase Sohex, whose amino acid sequence is shown in SEQ ID NO.

1.

6. Use of the β-N-acetylglucosaminidase Sohex according to claim 5 in degrading chitin or preparing GlcNAc.

7. Use of the β-N-acetylglucosaminidase Sohex described in claim 5 in the synthesis of lactose-N-trisaccharide II.

8. A method for synthesizing lactose-N-trisaccharide II, characterized in that: Lactose and β-N-acetylglucosaminidase Sohex are added to the chitobiose solution for enzymatic hydrolysis to prepare lactose-N-trisaccharide II; the amino acid sequence of the β-N-acetylglucosaminidase Sohex is shown in SEQ ID NO.1;.

9. The method for synthesizing lactose-N-trisaccharide II according to claim 8, characterized in that: The concentration of chitobiose is 25 mM, the concentration of lactose is 250 mM, and the concentration of β-N-acetylglucosaminidase Sohex is 3 μM.

10. The method for synthesizing lactose-N-trisaccharide II according to claim 8, characterized in that: The enzymolysis conditions are: enzymolysis temperature 50° C., and enzymolysis time 2.5 hours.

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