Beta-N-acetyl hexosaminidase Fmshx (G417A) and application thereof

Abstract

The invention discloses beta-N-acetyl hexosaminidase Fmshx (G417A). The amino acid sequence of the beta-N-acetyl hexosaminidase Fmshx (G417A) is shown as SEQ ID NO. 3. The application of the enzyme in preparation of GlcNAc and the application of the enzyme in multi-enzyme synergetic degradation of chitin are specifically as follows: the enzyme, lysing polysaccharide monooxygenase and chitinase synergistically degrade chitin to obtain GlcNAc. According to the invention, beta-N-acetyl hexosaminidase Fmshift is subjected to mutation modification to obtain the beta-N-acetyl hexosaminidase Fmshift (G417A) with higher hydrolytic activity, and the hydrolytic activity of the beta-N-acetyl hexosaminidase Fmshift (G417A) on pNP-GlcNAc and (GlcNAc) 2 is respectively 8.8 times and 2.3 times that of the beta-N-acetyl hexosaminidase Fmshift (G417A) before mutation. The method disclosed by the invention has very important environmental protection and industrial significance on degradation of crystal chitin and preparation of GlcNAc, and has important industrial production value and economic value.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of β - N -Acetylaminohexosidase Fmshex (G417A) and its application in multi-enzyme synergistic degradation of crystalline chitin, belong to the technical field of functional enzyme. BACKGROUND

[0002] α -Chitin is the second most abundant natural polysaccharide in nature, and is also a rich source of high-value bioactive molecules, but the complex crystal structure leads to low bioavailability. The composition unit of chitin sugar chain, N -Acetyl-D-glucosamine (GlcNAc) has ideal biological activity, is an ideal and potential precursor for the preparation of many high-value compounds, and has higher application value. Therefore, it is necessary to explore efficient and environmentally friendly degradation α -Chitin preparation method of GlcNAc is a research hotspot.

[0003] In the previous study, the team excavated a kind of lytic polysaccharide monooxygenase OsLPMO10A (LPMOs), which is described in Chinese invention patent CN 116790696 A. LPMOs can effectively destroy the chitin crystal structure, and its synergistic effect with chitinase can enhance the destruction of chitin crystal structure. But the product of this hydrolysis system is a mixture of chitobiose (GlcNAc) 2 and GlcNAc, which does not meet the demand of commercial uniformity, and needs to use β - N -Acetylaminohexosidase (EC 3.2.1.52) further hydrolyzes (GlcNAc) 2, thereby improving the purity of GlcNAc. Therefore, it is necessary to excavate β - N -Acetylaminohexosidase, which is of great significance for constructing a high-efficiency multi-enzyme synergistic degradation system of crystalline chitin. SUMMARY

[0004] In view of the above prior art, the present application provides a kind of β - N -Acetylaminohexosidase Fmshex (G417A) and its application.

[0005] The present application is realized by the following technical solutions: A kind of β - N -Acetylaminohexosidase Fmshex (G417A), the amino acid sequence is as shown in SEQ ID NO.3.

[0006] The β -N - the coding gene of acetylhexosaminidase Fmshex (G417A), the nucleotide sequence of which is shown in SEQ ID NO. 4.

[0007] The β - N - the use of acetylhexosaminidase Fmshex (G417A) in the preparation of GlcNAc.

[0008] The β - N - the use of acetylhexosaminidase Fmshex (G417A) in the synergistic degradation of chitin by multiple enzymes, including the following three ways: (1) β - N - acetylhexosaminidase Fmshex (G417A) and chitinase synergistically degrade chitin; (2) chitin is degraded by chitinase and chitinase to obtain chitooligosaccharide, and then β - N - acetylhexosaminidase Fmshex (G417A) hydrolyzes chitooligosaccharide to obtain GlcNAc; (3) β - N - acetylhexosaminidase Fmshex (G417A) and chitinase and chitinase synergistically degrade chitin to obtain GlcNAc.

[0009] Further, the chitin is selected from α - chitin or / and β - chitin.

[0010] Further, the lytic polysaccharide monooxygenase is selected from lytic polysaccharide monooxygenase OsLPMO10A.

[0011] Further, the chitinase is selected from chitinase ChiB, i.e. chitinase SmChi B described in CN 116790696 A.

[0012] Further, in specific applications, to α - add lytic polysaccharide monooxygenase OsLPMO10A, chitinase ChiB and β-N-acetylhexosaminidase Fmshex (G417A) to the chitin solution, and enzymatically degrade to obtain GlcNAc.

[0013] Still further, the α - the concentration of the chitin solution is 5 g / L; the enzyme amount of the lytic polysaccharide monooxygenase OsLPMO10A and the chitinase ChiB is 3 μM; and theβ The amount of N-acetylhexosaminidase Fmshex (G417A) is 2 μM.

[0014] Further, the enzymolysis condition is that the temperature is 45 ℃ and the time is 24 h.

[0015] Further, the α The chitin solution is prepared by adding chitin into acetate buffer solution, and mixing uniformly to obtain the chitin solution; wherein the concentration of the acetate buffer solution is 10 mM, and the pH is 6.0. α The chitin, and mixing uniformly to obtain the chitin solution; wherein the concentration of the acetate buffer solution is 10 mM, and the pH is 6.0.

[0016] A method for preparing GlcNAc by degrading chitin, comprising the following steps: α The method for preparing GlcNAc by degrading chitin, comprising the following steps: α The method for preparing GlcNAc by degrading chitin, comprising the following steps: β The method for preparing GlcNAc by degrading chitin, comprising the following steps:

[0017] Further, the α The concentration of the chitin solution is 20 g / L; the amount of the lytic polysaccharide monooxygenase OsLPMO10A and the chitinase ChiB is 3 μM; and the amount of the N-acetylhexosaminidase Fmshex (G417A) is 2 μM. β The amount of the N-acetylhexosaminidase Fmshex (G417A) is 2 μM.

[0018] Further, the enzymolysis condition is that the temperature is 45 ℃ and the time is 24 h.

[0019] Further, the α The chitin solution is prepared by adding chitin into acetate buffer solution, and mixing uniformly to obtain the chitin solution; wherein the concentration of the acetate buffer solution is 10 mM, and the pH is 6.0. α The chitin, and mixing uniformly to obtain the chitin solution; wherein the concentration of the acetate buffer solution is 10 mM, and the pH is 6.0.

[0020] The present application is directed to β - N The N-acetylhexosaminidase Fmshex is subjected to mutation and modification, and the N-acetylhexosaminidase Fmshex (G417A) with higher hydrolysis activity is obtained. β - N The N-acetylhexosaminidase Fmshex (G417A) has the hydrolysis activity of 8.8 times and 2.3 times of the N-acetylhexosaminidase Fmshex on NP-GlcNAc and (GlcNAc)2, respectively. p NP-GlcNAc and (GlcNAc)2, respectively. β - N NP-GlcNAc and (GlcNAc)2, respectively. αchitin, lytic polysaccharide monooxygenase OsLPMO10A, chitinase ChiB, β - N The synergistic enzymatic hydrolysis system of acetylhexosaminidase Fmshex (G417A) can efficiently degrade crystalline chitin to prepare GlcNAc. β - N The acetylhexosaminidase Fmshex (G417A) has very important environmental protection and industrial significance for the degradation of crystalline chitin and the preparation of GlcNAc, and has important industrial production value and economic value.

[0021] The various terms and phrases used in the present application have the general meanings known to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 : β - N Schematic diagram of the result of acetylhexosaminidase Fmshex docking with (GlcNAc)2 molecules.

[0023] Figure 2 SDS-PAGE electrophoresis diagram, wherein M1 and M2 are standard proteins; Os is lytic polysaccharide monooxygenase OsLPMO10A; ChiB is chitinase ChiB; G417A is acetylhexosaminidase Fmshex (G417A). β - N Acetylhexosaminidase Fmshex (G417A).

[0024] Figure 3 Effect of temperature on the relative enzyme activity of acetylhexosaminidase Fmshex (G417A). β - N Schematic diagram of the effect of acetylhexosaminidase Fmshex (G417A) on the relative enzyme activity.

[0025] Figure 4 Effect of pH on the relative enzyme activity of acetylhexosaminidase Fmshex (G417A). β - N Schematic diagram of the effect of acetylhexosaminidase Fmshex (G417A) on the relative enzyme activity.

[0026] Figure 5 Effect of incubation at 40℃ for different time on the relative enzyme activity of acetylhexosaminidase Fmshex (G417A). β - N Schematic diagram of the effect of acetylhexosaminidase Fmshex (G417A) on the relative enzyme activity.

[0027] Figure 6 Effect of incubation at different pH conditions for different time on the relative enzyme activity of acetylhexosaminidase Fmshex (G417A). β - N Schematic diagram of the effect of acetylhexosaminidase Fmshex (G417A) on the relative enzyme activity.

[0028] Figure 7 The effects of different insulation times at 4℃ on β - N Schematic diagram of the effect of acetaminohexosidase Fmshex (G417A) on relative enzyme activity.

[0029] Figure 8 Schematic diagram of the effect of enzymatic hydrolysis temperature on the relative yield of chitosan oligosaccharides.

[0030] Figure 9 Schematic diagram of the effect of enzymatic hydrolysis pH on the relative yield of chitosan oligosaccharides.

[0031] Figure 10 : β - N Schematic diagram showing the effect of the amount of acetylaminohexosidase Fmshex (G417A) added on the relative yield of chitosan oligosaccharides.

[0032] Figure 11 Schematic diagram showing the effect of substrate concentration on chitin conversion rate and GlcNAc concentration. Detailed Implementation

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

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

[0035] The invention adopts p NP-GlcNAc, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0036] The invention adopts α - Chitosan, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0037] The chitosan oligosaccharides used in this invention, namely chitobiose (GlcNAc)2, chitotriose (GlcNAc)3, chitotetraose (GlcNAc)4, chitopentose (GlcNAc)5, and chitohexaose (GlcNAc)6, were all purchased from Qingdao Bozhi Huili Biotechnology Co., Ltd.

[0038] Example 1 β - N- Modification of acetylaminohexosidase Fmshex β - N - Acetaminohexosidase Fmshex was screened from the NCBI website from *Pseudomonas karyotes* (…). Fulvivirga maritima The protein fragment (GenBank: WP_233776848.1) belongs to the GH20 family. After removing the signal peptide, its amino acid sequence is shown in SEQ ID NO.1.

[0039] β - N The amino acid sequence of acetaminohexosidase Fmhex is shown in SEQ ID NO.1, as follows: .

[0040] β - NThe nucleotide sequence of the gene encoding acetylhexosaminidase Fmhex is shown as SEQ ID NO. 2, as follows (direction 5'-3'):

[0041] Studies have shown that, β - N - Acetylhexosaminidase Fmshex has the ability to degrade chitinous oligosaccharides, and the enzyme activity of hydrolyzing (GlcNAc)2 is 15.68±1.28 (U / mg), and the enzyme hydrolysis efficiency is low. Therefore, the present application is mutated to obtain β - N - acetylhexosaminidase with higher hydrolysis activity.

[0042] The structure model of acetylhexosaminidase Fmshex is constructed by using the SWISS MODEL protein modeling server. β - N - Acetylhexosaminidase Fmshex. In order to enhance the action efficiency of the enzyme and the substrate (GlcNAc)2, the structure model of β - N - Acetylhexosaminidase Fmshex is docked with (GlcNAc)2. In order to find as many active mutants as possible, the present application selects all amino acid residues within a range of 4 angstroms around the ligand (GlcNAc)2, a total of 11 sites (as shown in Figure 1 ). According to whether the amino acid is a component of the active pocket, the evolutionary conservation of the amino acid, and the nature of the amino acid, etc., the glycine at position 417 is mutated to alanine, and the specific method is: the codon corresponding to the amino acid at position 417 on the acetylhexosaminidase Fmshex coding gene is changed from the glycine codon "GGC" to the alanine codon "GCA", obtaining the mutant: Fmshex(G417A), which is heterologously expressed and its activity is studied. β - N - Acetylhexosaminidase Fmshex. In order to enhance the action efficiency of the enzyme and the substrate (GlcNAc)2, the structure model of

[0043] Example 2 β - N - Preparation of acetylhexosaminidase The expression vector is constructed by a conventional method, and the acetylhexosaminidase Fmshex is prepared by heterologous expression. β - N - Acetylhexosaminidase Fmshex, and the mutant Fmshex(G417A) designed in Example 1, the steps are as follows: (1) Constructing a recombinant expression vector: artificially synthesizing the gene fragment shown in SEQ ID NO. 2, and the modified gene fragment (the codon at position 417 is replaced by the alanine codon in Example 1), and connecting it to the pET-28a(+) expression vector to construct a recombinant plasmid.

[0044] (2) Constructing a recombinant engineering bacterium: transforming the recombinant plasmid constructed above into Escherichia coli BL21(DE3) competent cells were screened for positive transformants using LB agar plates containing kanamycin sulfate. Clones were validated by colony PCR using T7 universal primers.

[0045] (3) Cultivation of recombinant engineered bacteria: Positive clones were selected and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate, and cultured at 37℃ and 220 rpm for 12 h. 1% of the culture was inoculated into 50 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate, and cultured at 37℃ and 220 rpm until the OD value reached 0.6; 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and expression was induced at 18℃ for 16 h.

[0046] (3) Extraction: Take the culture medium, centrifuge at 8000 rpm for 5 min at 4℃, collect the bacterial cells, resuspend them in Tirs-HCl buffer (10 mM, pH 7.5), sonicate for 30 min, centrifuge at 12000 rpm for 30 min, and the supernatant is the crude enzyme solution.

[0047] (4) Purification: The crude enzyme solution was purified by affinity chromatography using a Ni-NTA nickel column. The column was equilibrated with 10 mM imidazole solution (10 mM imidazole, 150 mM NaCl, 50 mM Tris-HCl), and then weakly binding proteins were eluted with 20 mM imidazole solution (20 mM imidazole, 150 mM NaCl, 50 mM Tris-HCl). The target protein was eluted with 200 mM imidazole solution (200 mM imidazole, 150 mM NaCl, 50 mM Tris-HCl). The eluent was collected and concentrated using an ultrafiltration tube with a molecular weight cutoff of 30 kDa to obtain a pure enzyme solution containing the mutant Fmshex (G417A). SDS-PAGE analysis was performed, and the results are shown below. Figure 2 As shown, the purified proteins all exhibited a single band with a molecular weight of approximately 75 kDa, consistent with the prediction.

[0048] Meanwhile, pure enzyme solutions containing the cleaving polysaccharide monooxygenase OsLPMO10A and the chitinase ChiB were prepared using conventional methods. Their SDS-PAGE electrophoresis images are shown below. Figure 2 As shown.

[0049] Example 3 β - N -Assay of acetylaminohexosidase activity use p The NP colorimetric method was used to determine enzyme activity. The reaction system consisted of 90 μL of 1 mM [a specific enzyme / reagent]. pNP-GlcNAc solution, 10 μL pure enzyme solution (prepared in Example 2), 100 μL phosphate buffer with a concentration of 50 mM. Reaction at 40°C for 10 min. After the reaction, 200 μL sodium carbonate solution with a concentration of 200 mM was added to terminate the reaction, and the absorbance value at 410 nm was detected. Detection was performed three times, and the average value was taken.

[0050] One unit (U) of β - N - Acetylhexosaminidase activity is defined as: 1 μmol of NP required for the enzyme per minute p NP.

[0051] It was determined that, β - N - The activity of acetylhexosaminidase Fmshex was 1291 ± 43.69 U / mg; the enzyme activity of mutant Fmshex (G417A) was 11384 ± 106 U / mg, which was β - N - 8.8 times that of acetylhexosaminidase Fmshex.

[0052] The above determination results show that the mutant Fmshex (G417A) has high hydrolysis activity and great application potential, and the present application formally names it as β - N - Acetylhexosaminidase Fmshex (G417A), the amino acid sequence of which is shown in SEQ ID NO. 3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 4.

[0053] β - N - The amino acid sequence of acetylhexosaminidase Fmshex (G417A) is shown in SEQ ID NO. 3, as follows: MSCSTSVPKEEEYNSPISIIPEPVSLEQGKGALIINENTKLLFAKDDEEAQKIAESFADQFSAASGISLAVEESAEGKVEGGILFSVNENLDVPEEGYELKVDDAGVSIVGKDHAGLFYGMQTLKQLLPPAIESTEKVEGEKWLVPLVSIQDYPRYKWRGLHLDVSRHFSTVDFVKKYIDNMAMHKLNTFHWHLTDDQGWRIEIKKYPKLTEIGAYRDETLVGHAGSTEFDGKRYGGFYTQEEIKEVVAYAQERHITVVPEIELPGHATAAVASYPELGVTGNRPKVVTEWGVFLDIYGVQDETFEFLQDVLTEVMELFPSKYIHIGGDEAWKDQWKASAEVQEKIKELGLKDEHELQSWFITRIEQFVNSKGRQIIGWDEILEGGLAPNAAVMSWRGEEGGIAAAKEKHNVVMTPAGYVYFDHYQGDPQFEPLQISGYTTLERVYSYDPTPEVLSPEEQKYILGAQANVWSEYLPTSEMVEYVVFPRLAALSEVLWTPLENKDWEAFKEKIPNQLKRYDYRGINYSKSIYYVTYDVEDKAGTEKLVVSLKNQFGLSKMYYTTDGSEPTKESSLYEGPLYLDEGTTLKAVAIQDGEPMSKVTEITVEKPKEEEK.

[0054] β - N The nucleotide sequence of the gene encoding acetylhexosaminidase Fmshex (G417A) is shown as SEQ ID NO. 4, as follows (direction 5'-3'):

[0055] Example 4 β - N - Determination of the enzymatic properties of acetylhexosaminidase Fmshex (G417A) Determination of the optimum temperature: The enzyme activity at different temperatures was determined according to the determination method of Example 3, with the highest enzyme activity being taken as 100%, and the relative enzyme activity at different temperatures was calculated, with the results shown in Table 1, and the optimum reaction temperature was 40°C. Figure 3 Determination of the optimum temperature: The enzyme activity at different temperatures was determined according to the determination method of Example 3, with the highest enzyme activity being taken as 100%, and the relative enzyme activity at different temperatures was calculated, with the results shown in Table 1, and the optimum reaction temperature was 40°C.

[0056] Determination of the optimum pH: The enzyme activity was determined according to the determination method of Example 3 at pH 4.0-10.0 (the buffers used were citrate buffer at pH 4.0-6.0, phosphate buffer at pH 6.0-8.0, Tris-HCl buffer at pH 7.0-9.0, and Gly-NaOH buffer at pH 9.0-10.0), with the highest enzyme activity being taken as 100%, and the relative enzyme activity at different pH conditions was calculated, with the results shown in Table 2, and the optimum pH was 6.0. Figure 4 Determination of the optimum pH: The enzyme activity was determined according to the determination method of Example 3 at pH 4.0-10.0 (the buffers used were citrate buffer at pH 4.0-6.0, phosphate buffer at pH 6.0-8.0, Tris-HCl buffer at pH 7.0-9.0, and Gly-NaOH buffer at pH 9.0-10.0), with the highest enzyme activity being taken as 100%, and the relative enzyme activity at different pH conditions was calculated, with the results shown in Table 2, and the optimum pH was 6.0.

[0057] Determination of the temperature stability: The pure enzyme solution was incubated at 40°C for different time (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, with the highest enzyme activity being taken as 100%, and the relative enzyme activity after incubation at 40°C for different time was calculated, with the results shown in Table 3. As can be seen from the figure, Figure 5 Determination of the temperature stability: The pure enzyme solution was incubated at 40°C for different time (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, with the highest enzyme activity being taken as 100%, and the relative enzyme activity after incubation at 40°C for different time was calculated, with the results shown in Table 3. As can be seen from the figure, β - N - Acetylhexosaminidase Fmshex (G417A) incubated at 40°C for 12 h could maintain about 60% activity, indicating that the enzyme had good temperature stability.

[0058] Determination of the pH stability: 10 μL of pure enzyme solution was mixed with 100 μL of buffer at different pH (the buffer used was phosphate buffer at pH 6.0-7.0), and the mixture was incubated at 40°C for different time (0 h, 12 h, 24 h, 48 h, 60 h). Then 90 μL of 1 mM p NP-GlcNAc was added, and the enzyme activity was determined according to the determination method of Example 3, with the highest enzyme activity being taken as 100%, and the stability at different pH conditions was calculated, with the results shown in Table 4. As can be seen from the figure, Figure 6 Determination of the pH stability: 10 μL of pure enzyme solution was mixed with 100 μL of buffer at different pH (the buffer used was phosphate buffer at pH 6.0-7.0), and the mixture was incubated at 40°C for different time (0 h, 12 h, 24 h, 48 h, 60 h). Then 90 μL of 1 mM β - N - Acetylhexosaminidase Fmshex (G417A) showed high stability at pH 7.0, with about 70% residual enzyme activity after incubation for 36 h, and had good pH stability.

[0059] Storage stability assay: The pure enzyme solution was placed at 4°C for 60 h, and the enzyme activity was measured every 12 h according to the assay method of Example 3. The relative enzyme activity after storage for different time was calculated with the highest enzyme activity as 100%, and the results are shown in Table 2. Figure 7 As can be seen from Table 2, β - N - The acetylhexosaminidase Fmshex (G417A) can maintain about 60% activity after incubation at 4°C for 60 h, indicating that the enzyme has good storage stability.

[0060] Example 5 β - N Assay of substrate specificity of acetylhexosaminidase Fmshex (G417A) The enzyme activity was detected under standard enzyme activity assay conditions using NP-GlcNAc and chitooligosaccharide as substrates, respectively. p The results are shown in Table 1. β - N The substrate specificity of acetylhexosaminidase Fmshex (G417A) was detected using β - N - acetylhexosaminidase Fmshex as a control. The results are shown in Table 1.

[0061]

[0062] Note: The enzyme activity assay method for chitooligosaccharide was as follows: 3 μM enzyme solution was added to 2 mg / mL chitooligosaccharide and 40 μL pH 6.0 phosphate buffer, and the reaction was carried out at 40°C for 10 min. Then the amount of released β - N acetylglucosamine was determined by high performance liquid chromatography (HPLC). The HPLC assay conditions were as follows: HPLC-RID detection system, the temperature of the chromatographic column (Sugar Pak I) was 75°C, the mobile phase was calcium disodium ethylenediaminetetraacetate (50 mg / L) solution, the flow rate was 0.5 mL / min, and the time was 20 min. β - N The activity unit of acetylhexosaminidase was defined as follows: the amount of enzyme required to generate 1 μmol of β - N acetylglucosamine per minute under the above reaction conditions was one enzyme activity unit (1 U).

[0063] Results: β - N- Acetylhexosaminidase Fmshex(G417A) has more than 30 U / mg activity on (GlcNAc)2, (GlcNAc)4, (GlcNAc)5, and about 20 U / mg activity on (GlcNAc)3, (GlcNAc)6. β - N - The hydrolytic activity of acetylhexosaminidase Fmshex(G417A) on (GlcNAc)2 is 2.3 times that of Fmshex, with a large increase. β - N - The hydrolytic activity of acetylhexosaminidase Fmshex(G417A) on (GlcNAc)2 is 2.3 times that of Fmshex, with a large increase. β - N - Acetylhexosaminidase Fmshex(G417A) has the high activity required for efficient degradation of (GlcNAc)2 to prepare GlcNAc.

[0064] Example 6 β - N - Optimization of conditions for acetylhexosaminidase Fmshex(G417A) for cascade degradation of chitin To α - Add lytic polysaccharide monooxygenase OsLPMO10A, chitinase ChiB, and β - N-acetylhexosaminidase Fmshex(G417A) to the chitin solution, and investigate the synergistic effect of lytic polysaccharide monooxygenase OsLPMO10A, chitinase ChiB, and β - N - N-acetylhexosaminidase Fmshex(G417A) to optimize the conditions for cascade degradation of chitin.

[0065] The initial reaction conditions are as follows: α - The concentration of the chitin solution is 20 g / L, and the enzyme dosage of lytic polysaccharide monooxygenase OsLPMO10A and chitinase ChiB is 3 μM (referring to the concentration of the enzyme in the reaction system after addition), β - N - The enzyme dosage of acetylhexosaminidase Fmshex(G417A) is 2 μM; the enzyme hydrolysis temperature is 45°C, and the enzyme hydrolysis time is 24 h.

[0066] The α - Chitin solution is prepared by the following method: adding α - Chitin is mixed to obtain; wherein the concentration of the acetate buffer solution is 10 mM, and the pH is 6.0.

[0067] The produced GlcNAc is determined by HPLC, and the method is the same as in Example 5.

[0068] The production of chitooligosaccharide was calculated according to the following formula: Chitooligosaccharide production = GlcNAc release (mg) + (GlcNAc)2 release (mg).

[0069] The conversion rate of chitin was calculated according to the following formula: Chitin conversion rate (%) = GlcNAc release (mg) / initial α chitin content (mg) x 100%.

[0070] During the investigation, each single factor condition in the initial condition was adjusted to optimize the enzymatic temperature, initial pH, enzyme dosage and substrate concentration, which were specifically as follows.

[0071] (1) Optimization of enzymatic temperature: within the range of 30-50℃, the production of chitooligosaccharide at different enzymatic temperatures was determined by HPLC method, and the relative production was calculated with the highest value as 100%, and the results were shown in Figure 8 The production of chitooligosaccharide was the highest at 45℃, and was at a relatively high level within the range of 40-50℃.

[0072] (2) Optimization of initial pH: within the range of pH 5.0-10.0 (adjusting the acetate buffer in the initial condition to acetate buffer at pH 5.0, phosphate buffer at pH 6.0-8.0, Tris-HCl buffer at pH 7.0-9.0, and Gly-NaOH buffer at pH 9.0-10.0), the production of chitooligosaccharide at different pH was determined by HPLC method, and the relative production was calculated with the highest value as 100%, and the results were shown in Figure 9 The production of chitooligosaccharide was the highest when using acetate buffer at pH 6.0.

[0073] (3) Optimization of enzyme dosage: the enzyme dosage of both lytic polysaccharide monooxygenase OsLPMO10A and chitinase ChiB was kept unchanged, both being 3 μM, and the addition amount of β N acetylhexosaminidase Fmshex(G417A) was changed (1, 1.5, 2, 2.5, 3 μM, respectively). The production of chitooligosaccharide at different enzyme dosages was determined by HPLC method, and the relative production was calculated with the highest value as 100%, and the results were shown in Figure 10 β N The production of chitooligosaccharide was the highest when the enzyme dosage of acetylhexosaminidase Fmshex(G417A) was 2.0 μM.

[0074] (4) Optimization of substrate concentration: the substrate concentration was changed α ​​​- Chitosan concentrations (5, 10, 20, 30, 40, 50 g / L) were used to determine the chitosan conversion rate and the concentration of the product GlcNAc at different substrate concentrations using HPLC. The results are as follows: Figure 11 As shown. Although the yield of GlcNAc increases with increasing substrate concentration, α - The conversion rate of chitin is highest at a concentration of 5 g / L. Increasing the substrate concentration will lead to a decrease in conversion rate and waste of resources.

[0075] In summary, the optimal reaction conditions are: α The chitin solution concentration was 5 g / L, and the amount of both the polysaccharide-lysing monooxygenase OsLPMO10A and chitinase ChiB added was 3 μM. β - N The amount of acetylaminohexosidase Fmshex (G417A) added was 2 μM; the enzymatic hydrolysis temperature was 45℃, and the hydrolysis was carried out in acetate buffer at pH 6.0 for 24 h. At this time, the chitin conversion rate was 28.77 ± 1.77%.

[0076] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A kind β - N - Acetaminohexosidase Fmshex(G417A), characterized by: The amino acid sequence is shown as SEQ ID NO.

3.

2. The method of claim 1 β - N - a gene encoding acetylhexosaminidase Fmshex (G417A) characterized in that: The nucleotide sequence is shown as SEQ ID NO.

4.

3. The method of claim 1 β - N - Use of acetylhexosaminidase Fmshex (G417A) for the preparation of GlcNAc.

4. The method of claim 1 β - N - Use of acetylhexosaminidase Fmshex (G417A) in the concerted degradation of chitin by multiple enzymes, characterized in that, is one of the following three ways: (1) β - N - Acetylhexosaminidase Fmshex (G417A) synergizes with chitinase in the degradation of chitin; (2) The lytic polysaccharide monooxygenase and chitinase synergistically degrade chitin to obtain chitooligosaccharide, and then β N - The acetylhexosaminidase Fmshex(G417A) hydrolyzes the chitooligosaccharide to obtain GlcNAc;​ (3) β - N - Acetylhexosaminidase Fmshex (G417A) degrades chitin in synergy with lytic polysaccharide monooxygenase and chitinase, resulting in GlcNAc.

5. Use according to claim 4, characterized in that: The chitin is selected from α - chitin or / and β - chitin; the lytic polysaccharide monooxygenase is selected from the lytic polysaccharide monooxygenase OsLPMO10A; the chitinase is selected from the chitinase ChiB.

6. Use according to claim 5, characterized in that: In a specific application, to α - addition of lytic polysaccharide monooxygenase OsLPMO10A, chitinase ChiB and β - N - acetylhexosaminidase Fmshex (G417A), enzymatic hydrolysis, GlcNAc was prepared.

7. Use according to claim 6, characterized in that: The α - The concentration of the chitin solution was 5 g / L; the amount of the cleaving polysaccharide monooxygenase OsLPMO10A and the chitinase ChiB added was 3 μM; β The amount of 2 μM of α-N-acetylglucosidase Fmshex (G417A) was added; the enzymatic hydrolysis conditions were: temperature 45℃, time 24 h. The α The chitin solution is prepared by adding chitin into acetate buffer solution, and mixing to obtain the chitin solution; wherein the concentration of the acetate buffer solution is 10 mM, and the pH is 6.

0. α The chitin solution is prepared by adding chitin into acetate buffer solution, and mixing to obtain the chitin solution; wherein the concentration of the acetate buffer solution is 10 mM, and the pH is 6.

0.

8. A method of degrading α - chitin preparation N - a process for the preparation of acetyl-D-glucosamine, characterized in that: To α - addition of lytic polysaccharide monooxygenase OsLPMO10A, chitinase ChiB and β - N - acetylhexosaminidase Fmshex(G417A), enzymatic hydrolysis, resulting in GlcNAc; the β - N - the amino acid sequence of acetylhexosaminidase Fmshex(G417A) is shown in SEQ ID NO.

3.

9. The degradation of claim 8 α - process for the preparation of GlcNAc from chitin, characterized in that: The α - the concentration of the chitin solution was 20 g / L; the enzyme loading of both the lytic polysaccharide monooxygenase OsLPMO10A and the chitinase ChiB was 3 μΜ; the β - N - the enzyme loading of the acetylhexosaminidase Fmshex(G417A) was 2 μΜ.

10. The degradation of claim 8 α - process for the preparation of GlcNAc from chitin, characterized in that: The enzymolysis conditions are: temperature 45℃, time 24 h; The α The chitin solution is prepared by adding chitin into acetate buffer solution, and mixing to obtain the chitin solution; wherein the concentration of the acetate buffer solution is 10 mM, and the pH is 6.

0. α The chitin solution is prepared by adding chitin into acetate buffer solution, and mixing to obtain the chitin solution; wherein the concentration of the acetate buffer solution is 10 mM, and the pH is 6.0.

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