A method for directly catalyzing the synthesis of glucosamine from fructose by using a biological enzyme

Abstract

The application discloses a method for directly catalyzing fructose to synthesize glucosamine by using biological enzymes, and belongs to the technical field of biology. In the application, the fourth isoleucine of wild-type glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) of Bacillus subtilis is mutated into threonine, the thirteenth alanine is mutated into valine, the 480th leucine is mutated into valine, and the 593rd leucine is mutated into serine. In the application, fructose is used as raw material, and the fructose is directly catalyzed to synthesize glucosamine by using the mutant enzyme GlmS and an amino donor (Fig. 1). In the application, only one enzyme is needed to catalyze and synthesize glucosamine by using fructose as raw material, and the technical route is simple compared with the existing published enzyme synthesis technology, and the application has the advantages of easy process control, high production efficiency and low industrial application cost, and is suitable for industrial popularization and application.

Description

Technical Field

[0001] This invention relates to a method for directly catalyzing the synthesis of glucosamine from fructose using biological enzymes, belonging to the field of bioengineering technology. Background Technology

[0002] Glucosamine is a compound formed by replacing the second hydroxyl group in a glucose molecule with an amino group. It is readily soluble in water and hydrophilic solvents and is an important functional monosaccharide. It is an essential component of human mucosal secretions, connective tissue, skin, tendons, ligaments, and cartilage. Glucosamine is found in almost all organisms, including bacteria, fungi, and plants and animals. It is a major component of biological glycoproteins and proteoglycans, and also a major component of chitin.

[0003] Studies have shown that glucosamine possesses several unique physiological activities in the human body, including cartilage protection and repair, anti-inflammatory and analgesic effects, treatment of rheumatoid arthritis, improvement of immune regulation and enhancement of immune function, antibacterial infection, enhancement of mitochondrial glutathione antioxidant capacity, and prolonging cell lifespan. Therefore, glucosamine is currently widely used in medicine, food, and cosmetics. For example, in the pharmaceutical industry, glucosamine can stimulate the biosynthesis of chondroitin proteoglycans, thus serving as a raw material for treating rheumatoid arthritis and osteoarthritis. In the food industry, glucosamine, due to its various physiological functions such as absorbing free radicals, anti-aging, promoting weight loss, antibacterial activity, and regulating the endocrine system, is used in the production of food additives and health foods. In the cosmetics industry, acetylglucosamine is one of the monomers for synthesizing hyaluronic acid (also known as hyaluronic acid), an indispensable substance in high-end cosmetics, primarily used as a high-molecular-weight water-absorbing and moisturizing agent.

[0004] Currently, there are two main methods for producing glucosamine: chitin hydrolysis and microbial fermentation. Chitin hydrolysis is further divided into chitin acid hydrolysis and chitinase hydrolysis. A common drawback is that it is limited by raw material supply, and some people allergic to shrimp and crab feed may experience allergic reactions when using glucosamine prepared by this method. Chitin acid hydrolysis generates a large amount of wastewater that requires treatment, potentially causing environmental pollution. Chitinase hydrolysis, also using chitin as a raw material, hydrolyzes it to produce glucosamine under the action of chitinase. While this method has less environmental pollution, it also suffers from limitations in raw material supply and the risk of allergic reactions. Microbial fermentation primarily uses metabolic engineering techniques to modify bacteria such as *Escherichia coli*, *Bacillus subtilis*, and *Corynebacterium glutamicum* to obtain metabolically engineered production strains. These strains use glucose and starch as raw materials to produce glucosamine. Although this method has advantages such as not being limited by chitin sources and having high production efficiency, the genetic modification of microbial metabolic pathways is difficult, and there are drawbacks such as difficulty in controlling the genetic stability of engineered strains and the easy generation of metabolic byproducts, which affect industrial applications and increase production costs.

[0005] Therefore, there is an urgent need to develop new methods for producing glucosamine that are low-cost, highly efficient, and highly stable, such as conducting research on enzymatic synthesis technology of glucosamine. On January 21, 2020, Chinese invention patent CN110714042A disclosed an enzymatic method for preparing glucosamine. The core strategy of this method is to first synthesize fructose 6-phosphate from glucose and phosphate using phosphorylase and isomerase respectively, then use glucosamine-6-phosphate deaminase to catalyze the conversion of fructose 6-phosphate into glucosamine 6-phosphate, and finally use an enzyme that dephosphates the group to catalyze the removal of the phosphate group from glucosamine 6-phosphate to finally produce glucosamine. This method requires the participation of at least four different enzymes, the steps are cumbersome, and the process is difficult to control, affecting the synthesis efficiency of the glucosamine product. Summary of the Invention

[0006] This invention uses fructose as a raw material and utilizes a mutant of glucosamine-6-phosphate synthase (EC2.6.1.16, abbreviated as GlmS) to directly catalyze the synthesis of glucosamine from fructose. Only this one enzyme is required, which is simpler than the existing publicly available glucosamine enzymatic synthesis technology route. It has the advantages of easier process control, higher production efficiency, and lower industrial application cost.

[0007] The purpose of this invention is to address the shortcomings of existing methods for producing glucosamine by providing a novel and simpler method for synthesizing glucosamine using a mutant enzyme. Compared with existing methods, this method has the advantages of simple technical route, easy process control, high production efficiency, low production cost, environmental friendliness, and product safety for human use.

[0008] This invention is achieved through the following technical solution:

[0009] The first object of the present invention is to provide an glucosamine-6-phosphate synthase mutant, as described below (a) or (b):

[0010] (a) Based on the amino acid sequence of SEQ ID NO.2, isoleucine (Ile) at position 4 is mutated to threonine (Thr), alanine (Ala) at position 13 is mutated to valine (Val), leucine (Leu) at position 480 is mutated to valine (Val), and leucine (Leu) at position 593 is mutated to serine (Ser).

[0011] (b) A protein derived from (a) whose amino acid sequence has been substituted, deleted, or added with one or more amino acids and which has GlmS activity.

[0012] A second object of the present invention is to provide a gene encoding the above-mentioned mutant enzyme, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0013] A third objective of this invention is to provide a vector carrying the aforementioned genes.

[0014] A fourth objective of this invention is to provide microbial cells that express the aforementioned genes or vectors.

[0015] In one embodiment, the microbial cells include, but are not limited to, Pichia pastoris, Escherichia coli, and Bacillus subtilis.

[0016] The fourth objective of this invention is to provide a method for preparing glucosamine, using fructose as a substrate, and catalyzing the synthesis of glucosamine by utilizing the aforementioned microbial cells, the fermentation broth of the aforementioned microbial cells, and / or the aforementioned gene-encoded glucosamine-6-phosphate synthase mutant and amino donor.

[0017] In one embodiment, the amino donor is an inorganic ammonium salt or glutamine, wherein the inorganic ammonium salt includes, but is not limited to, one or more of ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium carbonate.

[0018] In one embodiment, the fermentation reaction broth is prepared by ultrasonically disrupting the fermentation broth obtained from the fermentation of the microbial cells.

[0019] Those skilled in the art will understand that fructose can also be prepared from glucose via glucose isomerase.

[0020] In one embodiment, the reaction system of the catalytic synthesis also contains inorganic salts and Triton X-100.

[0021] In one embodiment, the temperature of the catalytic reaction is 20-60°C.

[0022] In one embodiment, the pH of the catalytic reaction is 5.0-9.0.

[0023] In one embodiment, the catalytic reaction time is 0.5-48 h.

[0024] In one embodiment, the concentration of the substrate fructose in the reaction system is 1-160 g / L.

[0025] In one embodiment, the amount of microbial cells added to the reaction system is 1-70 g / L.

[0026] In one embodiment, the concentration of the glucosamine-6-phosphate synthase mutant in the reaction system is 1-100 U / mL.

[0027] In one embodiment, the concentration of the inorganic ammonium salt in the reaction system is 50-1000 mM.

[0028] In one embodiment, the inorganic salt is KCl or MgCl2.

[0029] In one embodiment, the concentration of KCl or MgCl2 in the reaction system is 10-100 mM.

[0030] In one embodiment, the reaction system further contains a buffer solution, which includes, but is not limited to, Tris-HCl buffer, HEPES buffer, phosphate buffer, and citrate buffer.

[0031] In one embodiment, the concentration of the buffer solution in the reaction system is 20-300 mM.

[0032] The present invention also provides the application of the above-mentioned microbial cells, the above-mentioned microbial cell fermentation reaction broth and / or the above-mentioned gene-encoded glucosamine-6-phosphate synthase mutant in the fields of medicine, food or cosmetics.

[0033] In one embodiment, the fermentation reaction broth is prepared by ultrasonically disrupting the fermentation broth obtained from the fermentation of the microbial cells.

[0034] The present invention also provides the application of the above method in the preparation of glucosamine-containing products in the fields of medicine, food or cosmetics.

[0035] The present invention also provides the use of the above-mentioned microbial cells, the above-mentioned microbial cell fermentation reaction broth and / or the above-mentioned gene-encoded glucosamine-6-phosphate synthase mutant in the preparation of products containing glucosamine.

[0036] In one embodiment, the fermentation reaction broth is prepared by ultrasonically disrupting the fermentation broth obtained from the fermentation of the microbial cells.

[0037] The present invention also provides the application of the above method in the preparation of products containing glucosamine.

[0038] The positive and progressive effects of this invention are as follows:

[0039] This invention provides a novel method for synthesizing glucosamine using a GlmS mutant enzyme. The raw material is fructose, and the catalytic synthesis of glucosamine requires only one enzyme—GlmS. By constructing an engineered strain that recombinantly expresses the GlmS mutant enzyme, the GlmS mutant enzyme catalyst is prepared in large quantities through fermentation using the engineered strain. This method is inexpensive and readily available. After a certain reaction time, the yield of glucosamine reaches 84.4–87.5 g / L. This invention is simpler than existing enzymatic synthesis techniques, with easier process control, higher production efficiency, and lower application costs, making it suitable for industrial-scale application. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the technology for synthesizing glucosamine using fructose as a raw material and catalyzed by the GlmS mutant enzyme.

[0041] Figure 2 This is a high-performance liquid chromatography (HPLC) chromatogram of the whole-cell synthesis of glucosamine from Escherichia coli using fructose as a raw material and ammonium chloride as an amino donor, catalyzed by recombinant expression of the mutant enzyme GlmSmu.

[0042] Figure 3 This is a high-performance liquid chromatography (HPLC) chromatogram of the synthesis of glucosamine from E. coli cells lysed using fructose as a raw material and ammonium chloride as an amino donor, catalyzed by crude enzyme solution of recombinant expression mutant enzyme GlmSmu.

[0043] Figure 4 This is a high-performance liquid chromatography (HPLC) chromatogram of the synthesis of glucosamine using fructose as a raw material and ammonium chloride as an amino donor, catalyzed by the isolated and purified mutant enzyme GlmSmu. Detailed Implementation

[0044] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0045] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0046] The plasmids and strains involved in the following examples:

[0047] pET28a(+) plasmid: EMD Biosciences (Novagen), CA;

[0048] Top 10 clones of E. coli: Invitrogen, Carlsbad, CA;

[0049] Escherichia coli expression strain E. coli BL21(DE3): Invitrogen, Carlsbad, CA.

[0050] The experimental methods involved in the following embodiments are as follows:

[0051] (1) Method for determining GlmS enzyme activity

[0052] The enzyme activity assay method is as follows: The reaction system consists of 1 mL of 100 mM Tris-HCl buffer (pH 7.4), including 30 mM glutamine, 10 g / L fructose, 50 mM KCl, and 0.1 mL of enzyme solution. The reaction is carried out at 37°C for 60 min, then 0.5 mL of acetonitrile is added to terminate the reaction. The supernatant is centrifuged and the glucosamine content is determined. The glucosamine content detection method is as follows: 0.5 mL of the reacted sample is added to 1 mL of acetylacetone reagent and reacted at 90°C for 1 h. After cooling to room temperature, 10 mL of 96% (v / v) ethanol is slowly added, and finally 1 mL of DMAB reagent is added. After mixing, the mixture is allowed to stand at room temperature for 1 h, and the glucosamine content is detected at 530 nm absorbance.

[0053] Enzyme activity unit definition: The amount of enzyme required to catalyze the synthesis of 1 μmol of glucosamine per minute at 37℃ is defined as 1 enzyme activity unit, and the enzyme activity unit is U / mL.

[0054] The culture media involved in the following examples:

[0055] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0.

[0056] The glucosamine standards used in the following examples were purchased from Sigma-Aldrich, Inc., USA.

[0057] Example 1: Construction of a recombinant E. coli strain expressing the GlmS mutant enzyme

[0058] The GlmS mutant gene fragment with the nucleotide sequence shown in SEQ ID NO.1 was synthesized from the whole gene. The gene fragment and pET28a(+) plasmid were digested with NdeI and BamHI, and the digested gene fragment and plasmid fragment were ligated by ligation reaction. The resulting recombinant plasmid was transformed into competent cells of E. coli TOP10 strain and plated on LB plates containing kanamycin (Kan, 30 μg / ml). After incubation at 37°C for 12 h, positive transformants were picked, identified, and sequenced.

[0059] The positive monoclonal strains that were successfully sequenced were inoculated into 5 mL of LB liquid medium containing 30 μg / mL kanamycin and cultured overnight at 37°C and 180–220 rpm. The recombinant plasmid was extracted and transformed into the expression host E. coli BL21(DE3) to obtain the recombinant expression strain E. coli BL21(DE3) / pET28a(+)-GlmSmu.

[0060] SDS-PAGE analysis of total bacterial protein showed that the recombinant expression strain exhibited a clear expression band of GlmS enzyme after induction with IPTG (Isopropylβ-D-1-thiogalactopyranoside), and the molecular weight of the band was consistent with the expected molecular weight. The recombinant strain E. coli BL21(DE3) / pET28a(+)-GlmSmu was preserved by slant culture and glycerol storage at -80℃.

[0061] The recombinant strain was inoculated into a 250 mL shake flask containing 25-50 mL of LB medium and incubated at 37°C and 180-220 rpm until the bacterial culture reached OD500. 600 The concentration was 0.4-0.6, and then the culture was transferred to 30℃ and IPTG was added to a final concentration of 0.2mM for induction. Expression was induced at 180-220rpm for 10h to obtain fermentation broth. The enzyme activity of the mutant enzyme GlmSmu in the fermentation broth was measured. The recombinant strain was preserved as a slant and stored in glycerol at -80℃.

[0062] Example 2: Whole-cell catalysis of fructose to glucosamine using recombinant strain E. coli BL21(DE3) / pET28a(+)-GlmSmu

[0063] (1) Shake-flask fermentation

[0064] The recombinant strain E. coli BL21(DE3) / pET28a(+)-GlmSmu obtained in Example 1 was inoculated into a 250 mL shake flask containing 25–50 mL of LB medium and cultured at 37 °C and 180–220 rpm until the bacterial culture reached OD. 600The concentration was 0.4-0.6, and then the cells were transferred to 30℃ for induction with IPTG at a final concentration of 0.2mM. Expression was induced at 180-220rpm for 10h. The fermentation broth was obtained, the supernatant was removed by centrifugation, and the precipitate was resuspended in Tris-HCl buffer (pH 7.4) to obtain 10-40g / L wet cells.

[0065] (2) Whole-cell catalytic synthesis of glucosamine

[0066] The wet bacterial cells (30 g / L) obtained in step (1) were used as catalysts, D-fructose (100 g / L) was used as substrates, and ammonium chloride (NH4Cl) (500 mM) was used as amino donors. KCl (50 mM) and Triton X-100 (0.1% v / v) (0.1% v / v) were added to the reaction system. The buffer was Tris-HCl buffer (pH 7.4). The whole-cell catalytic transformation reaction was carried out at 37 °C for 12 h to obtain the reaction solution.

[0067] (3) Product testing

[0068] The reaction solution obtained in step (2) was centrifuged at 12000 r / min for 5 min, and the supernatant was taken to determine the concentration of glucosamine in the reaction solution by high performance liquid chromatography (HPLC). The chromatographic column used was an amino column, the mobile phase was 80% acetonitrile aqueous solution, the flow rate was 1 mL / min, the column temperature was 40℃, and the detector used was an ultraviolet detector with a detection wavelength of 195 nm.

[0069] The results are as follows Figure 2 As shown, the retention time of the glucosamine standard sample was approximately 4.1 min. The glucosamine concentration was directly proportional to the response intensity of the HPLC characteristic peak of glucosamine. After 12 h of reaction, the concentration of glucosamine was 87.5 g / L, and the conversion rate was 87.5% (conversion rate = concentration of glucosamine produced / initial fructose concentration × 100%), which was 61.1% higher than that obtained using wild-type GlmS (which produced a glucosamine concentration of 26.4 g / L under the same conditions). Glucosamine could be further separated and purified from the reaction solution.

[0070] Example 3: Utilizing crude enzyme solution containing the mutant enzyme GlmSmu to catalyze the synthesis of glucosamine from fructose.

[0071] (1) Shake-flask fermentation

[0072] The recombinant strain E. coli BL21(DE3) / pET28a(+)-GlmSmu obtained in Example 1 was inoculated into a 250 mL shake flask containing 25-50 mL of LB medium and cultured at 37°C and 180-220 rpm until the bacterial culture reached OD. 600The concentration was 0.4-0.6, and then the mixture was transferred to 30℃ for induction with IPTG at a final concentration of 0.2mM. Expression was induced at 180-220rpm for 10h to obtain the fermentation broth.

[0073] (2) Crude enzyme solution catalyzes the synthesis of glucosamine

[0074] The fermentation broth obtained in step (1) was centrifuged to obtain cell precipitate, and the cells were resuspended in Tris-HCl buffer (pH 7.4). The cells were then disrupted by sonication (the conditions for sonication were: ice bath, 300W, disruption for 10 seconds, and interval for 10 seconds) to obtain crude enzyme solution containing GlmS mutant enzyme. The enzyme activity was measured to be 21.1 U / mL.

[0075] Using the crude enzyme solution (10 g / L) as a catalyst, 100 g / L D-fructose as a substrate, and 500 mM ammonium chloride (NH4Cl) as an amino donor, a final concentration of 50 mM KCl was added to the reaction system. The buffer solution was Tris-HCl buffer (pH 7.4). The conversion reaction was carried out at 37 °C for 6 h. After the reaction was completed, 25% volume of acetonitrile was added to the reaction system to terminate the reaction and obtain the reaction solution.

[0076] (3) Detection of products

[0077] The reaction solution obtained in step (2) was centrifuged at 12000 r / min for 5 min, and the supernatant was taken to determine the concentration of glucosamine in the reaction solution by high performance liquid chromatography (HPLC). The chromatographic column used was an amino column, the mobile phase was 80% acetonitrile aqueous solution, the flow rate was 1 mL / min, the column temperature was 40℃, and the detector used was an ultraviolet detector with a detection wavelength of 195 nm.

[0078] The results are as follows Figure 3 As shown, the retention time of the glucosamine standard sample was approximately 4.1 min. The concentration of glucosamine was directly proportional to the response intensity of the HPLC characteristic peak of glucosamine. After 6 hours of reaction, the concentration of glucosamine was 84.4 g / L, and the conversion rate was 84.4%. Glucosamine can be further separated and purified from the reaction solution.

[0079] Example 4: Utilizing the mutant enzyme GlmSmu purified enzyme to catalyze the synthesis of glucosamine from fructose.

[0080] (1) Shake-flask fermentation

[0081] The recombinant strain E. coli BL21(DE3) / pET28a(+)-GlmSmu obtained in Example 1 was inoculated into a 250 mL shake flask containing 25-50 mL of LB medium and cultured at 37°C and 180-220 rpm until the bacterial culture reached OD. 600The concentration was 0.4-0.6, and then the mixture was transferred to 30℃ for induction with IPTG at a final concentration of 0.2mM. Expression was induced at 180-220rpm for 10h to obtain the fermentation broth.

[0082] (2) Pure enzyme-catalyzed synthesis of glucosamine

[0083] The fermentation precipitate obtained in step (1) was centrifuged to obtain cell precipitate, and the cells were resuspended in Tris-HCl buffer (pH 7.4). The cells were then sonicated (sonication conditions: ice bath, 300W, 10s disruption, 10s interval) to obtain a crude enzyme solution containing GlumS. The crude enzyme solution was further processed with Ni... + The GlmS mutant enzyme was isolated and purified by column affinity chromatography, and the enzyme activity was measured to be 23.7 U / mL.

[0084] Using 5 g / L pure GlmS enzyme solution as a catalyst, 100 g / L D-fructose as a substrate, and 500 mM ammonium chloride (NH4Cl) as an amino donor, the reaction system was supplemented with 50 mM KCl and Tris-HCl buffer (pH 7.4). The conversion reaction was carried out at 37 °C for 3 h. After the reaction was completed, 25% volume of acetonitrile was added to the reaction system to terminate the reaction and obtain the reaction solution.

[0085] (3) Detection of products

[0086] The reaction solution obtained in step (2) was centrifuged at 12000 r / min for 5 min, and the supernatant was taken to determine the concentration of glucosamine in the reaction solution by high performance liquid chromatography (HPLC). The chromatographic column used was an amino column, the mobile phase was 80% acetonitrile aqueous solution, the flow rate was 1 mL / min, the column temperature was 40℃, and the detector used was an ultraviolet detector with a detection wavelength of 195 nm.

[0087] The results are as follows Figure 4 As shown, the retention time of the glucosamine standard sample was approximately 4.1 min. The concentration of glucosamine was directly proportional to the response intensity of the HPLC characteristic peak of glucosamine. After 3 hours of reaction, the concentration of glucosamine was 85.1 g / L, and the conversion rate was 85.1%. Glucosamine can be further separated and purified from the reaction solution.

[0088] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims. SEQUENCE LISTING <110> Jiangsu Aoxin Biotechnology Co., Ltd. Jiangnan University <120> A method for directly catalyzing the synthesis of glucosamine from fructose using biological enzymes. <130> BAA211112A <160> 2 <170> PatentIn version 3.3 <210> 1 <211> 1803 <212> DNA <213> Artificial sequence <400> 1 atgtgtggta ctgtaggata tatagggcaa ttggacgtga aggagatctt gcttaagggt 60 ttggagaaac ttgagtaccg cggctatgat agcgcgggta tcgcggttgc taacgaacaa 120 ggcatccacg tttttaaaga gaagggtcgt atcgccgacc tgcgtgaggt ggtcgatgcg 180 aacgtcgagg cgaaggcagg tattggccat acccgttggg cgacccatgg tgaaccgagc 240 tacttaaatg cgcatccgca tcagtccgcg ctgggtcgtt tcacgttggt tcacaacggg 300 gtgattgaga actatgttca actgaagcaa gagtacttac aagatgttga gctgaagtcc 360 gataccgata ccgaagttgt tgtgcaggtt atcgagcagt ttgtcaatgg cggtctcgag 420 accgaggagg cttttcgtaa aaccctgact ttgctcaagg gcagctacgc aattgcgttg 480 ttcgataatg ataaccgtga aaccatcttc gtggccaaga acaaatcgcc gttgctagtt 540 ggcttaggcg atacctttaa tgtcgtggcg tcggacgcca tggcaatgct gcaagttacg 600 aacgaatacg tggagctgat ggataaggaa atggttattg ttaccgacga ccaagtggtg 660 atcaaaaacc tggacggcga tgttatcacc cgcgcatcat acatcgctga actggacgcg 720 tcggatattg agaaaggcac gtacccgcac tatatgttga aggaaactga cgagcaaccg 780 gtggtaatgc gtaaaattat ccagacttat caggacgaaa acggcaaact gtctgtgcct 840 ggtgacatcg cggctgccgt ggctgaagct gaccgcattt atattatcgg ctgcggtacg 900 agctaccatg caggcctggt cggtaaacaa tatatcgaga tgtgggcaaa cgttccggtt 960 gaagtccacg tcgccagcga atttagctac aatatgccac tgttgtccaa gaaaccgctg 1020 ttcatcttcc tgtctcagag cggtgaaacc gcggacagcc gtgcagtgct ggtgcaggtt 1080 aaagcgttgg gtcacaaggc cttgacgata accaacgtgc cgggtagcac cctgtctcgc 1140 gaagcggact acaccctgtt gctgcacgcc ggtccggaaa tcgctgtggc gtccaccaaa 1200 gcgtataccg cacagattgc ggtgctcgcg gtgctggcgt ccgttgcagc tgataaaaat 1260 ggtatcaaca ttggcttcga cctggttaaa gagctgggca tcgctgcgaa cgcgatggaa 1320 gctctgtgtg atcagaaaga tgagatggaa atgattgcgc gcgaatactt gaccgttagc 1380 cgcaatgcat tttttatagg cagaggcctg gactacttcg tttgtgttga aggtgcggtc 1440 aagctgaagg agatcagcta cattcaggct gaaggctttg caggtggtga actgaagcac 1500 ggcacgatcg cgcttatcga gcaaggtact ccggttttcg ccctggctac ccaggagcac 1560 gtgaacctga gtattcgtgg taacgtgaaa gaggttgctg cccgtggtgc gaacacctgc 1620 attatttctc tgaagggtct ggacgacgca gacgaccgct tcgtgctgcc ggaagttaat 1680 ccggcactgg caccgctggt gagcgtggtc ccactgcagc tgattgccta ttatgccgcg 1740 ctgcatcgtg ggtgcgatgt ggataagccg agaaattccg cgaagagcgt taccgtcgag 1800 taa 1803 <210> 2 <211> 600 <212> PRT <213> Artificial Sequence <400> 2 Met Cys Gly Ile Val Gly Tyr Ile Gly Gln Leu Asp Ala Lys Glu Ile 1 5 10 15 Leu Leu Lys Gly Leu Glu Lys Leu Glu Tyr Arg Gly Tyr Asp Ser Ala 20 25 30 Gly Ile Ala Val Ala Asn Glu Gln Gly Ile His Val Phe Lys Glu Lys 35 40 45 Gly Arg Ile Ala Asp Leu Arg Glu Val Val Asp Ala Asn Val Glu Ala 50 55 60 Lys Ala Gly Ile Gly His Thr Arg Trp Ala Thr His Gly Glu Pro Ser 65 70 75 80 Tyr Leu Asn Ala His Pro His Gln Ser Ala Leu Gly Arg Phe Thr Leu 85 90 95 Val His Asn Gly Val Ile Glu Asn Tyr Val Gln Leu Lys Gln Glu Tyr 100 105 110 Leu Gln Asp Val Glu Leu Lys Ser Asp Thr Asp Thr Glu Val Val Val 115 120 125 Gln Val Ile Glu Gln Phe Val Asn Gly Gly Leu Glu Thr Glu Glu Ala 130 135 140 Phe Arg Lys Thr Leu Thr Leu Leu Lys Gly Ser Tyr Ala Ile Ala Leu 145 150 155 160 Phe Asp Asn Asp Asn Arg Glu Thr Ile Phe Val Ala Lys Asn Lys Ser 165 170 175 Pro Leu Leu Val Gly Leu Gly Asp Thr Phe Asn Val Val Ala Ser Asp 180 185 190 Ala Met Ala Met Leu Gln Val Thr Asn Glu Tyr Val Glu Leu Met Asp 195 200 205 Lys Glu Met Val Ile Val Thr Asp Asp Gln Val Val Ile Lys Asn Leu 210 215 220 Asp Gly Asp Val Ile Thr Arg Ala Ser Tyr Ile Ala Glu Leu Asp Ala 225 230 235 240 Ser Asp Ile Glu Lys Gly Thr Tyr Pro His Tyr Met Leu Lys Glu Thr 245 250 255 Asp Glu Gln Pro Val Val Met Arg Lys Ile Ile Gln Thr Tyr Gln Asp 260 265 270 Glu Asn Gly Lys Leu Ser Val Pro Gly Asp Ile Ala Ala Ala Val Ala 275 280 285 Glu Ala Asp Arg Ile Tyr Ile Ile Gly Cys Gly Thr Ser Tyr His Ala 290 295 300 Gly Leu Val Gly Lys Gln Tyr Ile Glu Met Trp Ala Asn Val Pro Val 305 310 315 320 Glu Val His Val Ala Ser Glu Phe Ser Tyr Asn Met Pro Leu Leu Ser 325 330 335 Lys Lys Pro Leu Phe Ile Phe Leu Ser Gln Ser Gly Glu Thr Ala Asp 340 345 350 Ser Arg Ala Val Leu Val Gln Val Lys Ala Leu Gly His Lys Ala Leu 355 360 365 Thr Ile Thr Asn Val Pro Gly Ser Thr Leu Ser Arg Glu Ala Asp Tyr 370 375 380 Thr Leu Leu Leu His Ala Gly Pro Glu Ile Ala Val Ala Ser Thr Lys 385 390 395 400 Ala Tyr Thr Ala Gln Ile Ala Val Leu Ala Val Leu Ala Ser Val Ala 405 410 415 Ala Asp Lys Asn Gly Ile Asn Ile Gly Phe Asp Leu Val Lys Glu Leu 420 425 430 Gly Ile Ala Ala Asn Ala Met Glu Ala Leu Cys Asp Gln Lys Asp Glu 435 440 445 Met Glu Met Ile Ala Arg Glu Tyr Leu Thr Val Ser Arg Asn Ala Phe 450 455 460 Phe Ile Gly Arg Gly Leu Asp Tyr Phe Val Cys Val Glu Gly Ala Leu 465 470 475 480 Lys Leu Lys Glu Ile Ser Tyr Ile Gln Ala Glu Gly Phe Ala Gly Gly 485 490 495 Glu Leu Lys His Gly Thr Ile Ala Leu Ile Glu Gln Gly Thr Pro Val 500 505 510 Phe Ala Leu Ala Thr Gln Glu His Val Asn Leu Ser Ile Arg Gly Asn 515 520 525 Val Lys Glu Val Ala Ala Arg Gly Ala Asn Thr Cys Ile Ile Ser Leu 530 535 540 Lys Gly Leu Asp Asp Ala Asp Asp Arg Phe Val Leu Pro Glu Val Asn 545 550 555 560 Pro Ala Leu Ala Pro Leu Val Ser Val Val Pro Leu Gln Leu Ile Ala 565 570 575 Tyr Tyr Ala Ala Leu His Arg Gly Cys Asp Val Asp Lys Pro Arg Asn 580 585 590 Leu Ala Lys Ser Val Thr Val Glu 595 600

Claims

1. A mutant of glucosamine-6-phosphate synthase, characterized in that, Its amino acid sequence is as follows: based on the amino acid of SEQ ID NO.2, isoleucine at position 4 is mutated to threonine, alanine at position 13 is mutated to valine, leucine at position 480 is mutated to valine, and leucine at position 593 is mutated to serine.

2. The gene encoding the glucosamine-6-phosphate synthase mutant of claim 1.

3. A vector carrying the gene of claim 2.

4. Microbial cells expressing the gene of claim 2 or the vector of claim 3.

5. The microbial cell according to claim 4, characterized in that, The microbial cells are Pichia pastoris, Escherichia coli, or Bacillus subtilis.

6. A method for preparing glucosamine, characterized in that, Glucosamine is synthesized using fructose as a substrate and catalyzed by the microbial cells of claim 4 or 5 and / or the glucosamine-6-phosphate synthase mutant and amino donor of claim 1.

7. The method according to claim 6, characterized in that, The fermentation reaction broth of the microbial cells and an amino donor are used to catalyze the synthesis of glucosamine; the fermentation reaction broth is prepared by ultrasonically disrupting the fermentation broth obtained from the fermentation of the microbial cells as described in claim 4 or 5.

8. The method according to claim 6 or 7, characterized in that, The amino donor is an inorganic ammonium salt or glutamine, and the inorganic ammonium salt is one or more of ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium carbonate.

9. The method according to claim 6 or 7, characterized in that, The reaction system for catalytic synthesis also contains inorganic salts and Triton X-100.

10. The method according to claim 6 or 7, characterized in that, The catalytic reaction is carried out at temperatures ranging from 20 to 60°C and at pH values ​​ranging from 5.0 to 9.

0.

11. The use of the microbial cells of claim 4 or 5, the glucosamine-6-phosphate synthase mutant of claim 1, or the method of any one of claims 6 to 10 in the fields of medicine, food, or cosmetics.

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