A method for synthesizing glucosamine by one-step enzymatic method with fructose as substrate

Abstract

The application discloses a method for synthesizing glucosamine by one-step enzymatic method with fructose as a substrate and belongs to the technical field of bioengineering. The application uses fructose as raw material, adopts glucosamine-6-phosphate synthetase (Glucosamine-6-phosphate synthetase, EC 5.3.1.19) and an amino donor to directly catalyze fructose into glucosamine (Fig. 1). The application only needs one enzyme to catalyze and synthesize glucosamine with fructose as raw material, and the conversion rate reaches 77.2-92.6%, which is not only simpler than the existing published enzyme synthesis technical route, but also 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 one-step enzymatic synthesis method for glucosamine using fructose as a substrate, 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 control, enhancement of mitochondrial glutathione antioxidant capacity, and prolonging cell lifespan. Therefore, glucosamine is currently widely used and has important applications in medicine, food, cosmetics, and other fields.

[0004] Currently, there are two main methods for producing glucosamine: (1) Chitin hydrolysis method, including chitin acid hydrolysis and chitinase hydrolysis. Among them, chitin acid hydrolysis is the most commonly used method for producing glucosamine. This method first uses high-concentration hydrochloric acid to hydrolyze chitin to obtain acetylglucosamine, and then deacetylates acetylglucosamine to obtain glucosamine. This production method is highly susceptible to the supply of shrimp and crab chitin raw materials. On the other hand, the acid treatment will generate a large amount of wastewater that needs to be treated. In addition, some people who are allergic to shrimp and crab raw materials will have allergic reactions after consuming glucosamine prepared by this method. Chitinase hydrolysis method also uses chitin as raw material and hydrolyzes it to produce glucosamine under the action of chitinase. Although this method has less environmental pollution, it also has disadvantages such as limited raw material supply and allergic reactions. (2) Microbial fermentation method. This method uses metabolic engineering to modify Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, etc. to obtain metabolic engineering production strains. The strains use glucose, starch, etc. as raw materials to produce glucosamine. While this method has advantages such as not being limited by the source of chitin raw materials and having high production efficiency, it is difficult to genetically modify the microbial metabolic pathways and has disadvantages such as difficulty in controlling the genetic stability of engineered strains and 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, the process is complex, the synthesis efficiency of the glucosamine product is easily affected, and quality control is difficult. Summary of the Invention

[0006] This invention uses fructose as a substrate and utilizes glucosamine-6-phosphate synthetase (EC 5.3.1.19) to catalyze the synthesis of glucosamine from fructose. It requires only one enzyme and has the advantages of easier process control, high production efficiency, and low 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 bio-enzyme catalysis. 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 health.

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

[0009] The first object of the present invention is to provide a recombinant bacterium that expresses glucosamine-6-phosphate synthase as shown in (a) or (b):

[0010] (a) The amino acid sequence is shown in SEQ ID NO.2;

[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 glucosamine-6-phosphate synthase activity.

[0012] In one embodiment, the recombinant bacteria uses Escherichia coli, Bacillus subtilis, or Pichia pastoris as the host.

[0013] In one embodiment, the recombinant bacteria uses Escherichia coli BL21(DE3) as the host.

[0014] In one embodiment, the recombinant bacteria use the pET, pCold, and pPIC series as expression vectors.

[0015] In one embodiment, the recombinant bacteria uses pET28a(+) as an expression vector.

[0016] The second objective of this invention is to provide a method for synthesizing glucosamine, wherein fructose is used as the sole substrate, and glucosamine is synthesized by catalysis using the aforementioned recombinant bacteria, the fermentation broth of the aforementioned recombinant bacteria, and / or glucosamine-6-phosphate synthase and an amino donor.

[0017] In one embodiment, the glucosamine-6-phosphate synthase is a protein as shown in (a) or (b):

[0018] (a) The amino acid sequence is shown in SEQ ID NO.2;

[0019] (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 glucosamine-6-phosphate synthase activity.

[0020] 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.

[0021] In one embodiment, the fermentation reaction broth is prepared by ultrasonically crushing the fermentation broth obtained from the fermentation of the recombinant bacteria.

[0022] Those skilled in the art will understand that fructose can also be prepared from glucose via glucose isomerase.

[0023] In one embodiment, the reaction system of the catalytic synthesis also contains inorganic salts and Triton X-100.

[0024] In one embodiment, the temperature of the catalytic reaction is 20-60°C.

[0025] In one embodiment, the pH of the catalytic reaction is 5.0-9.0.

[0026] In one embodiment, the catalytic reaction time is 0.5-48 h.

[0027] In one embodiment, the concentration of the substrate fructose in the reaction system is 1-160 g / L.

[0028] In one embodiment, the amount of recombinant bacteria added to the reaction system is 1-70 g / L.

[0029] In one embodiment, the concentration of glucosamine-6-phosphate synthase in the reaction system is 1-100 U / mL.

[0030] In one embodiment, the concentration of the inorganic ammonium salt in the reaction system is 50-1000 mM.

[0031] In one embodiment, the inorganic salt is KCl or MgCl2.

[0032] In one embodiment, the concentration of KCl or MgCl2 in the reaction system is 10-100 mM.

[0033] 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.

[0034] In one embodiment, the concentration of the buffer solution in the reaction system is 20-300 mM.

[0035] The present invention also provides the application of the above-mentioned recombinant bacteria, the above-mentioned recombinant bacterial fermentation reaction broth and / or glucosamine-6-phosphate synthase in the fields of medicine, food or cosmetics.

[0036] In one embodiment, the fermentation reaction broth is prepared by ultrasonically crushing the fermentation broth obtained from the fermentation of the recombinant bacteria.

[0037] The present invention also provides the application of the above-described method for resynthesizing glucosamine in the preparation of products containing glucosamine in the fields of medicine, food or cosmetics.

[0038] The present invention also provides the use of the above-mentioned recombinant bacteria, the above-mentioned recombinant bacterial fermentation broth and / or glucosamine-6-phosphate synthase in the preparation of products containing glucosamine.

[0039] In one embodiment, the fermentation reaction broth is prepared by ultrasonically crushing the fermentation broth obtained from the fermentation of the recombinant bacteria.

[0040] The present invention also provides the application of the above-described method for resynthesizing glucosamine in the preparation of products containing glucosamine.

[0041] Beneficial effects:

[0042] This invention provides a recombinant bacterium expressing glucosamine-6-phosphate synthase. The host of the recombinant bacterium can be engineered Escherichia coli, Bacillus subtilis, or Pichia pastoris. According to conventional techniques in the art, glucosamine-6-phosphate synthase can be expressed in different host cells through codon optimization, etc.

[0043] This invention utilizes recombinant bacteria overexpressing glucosamine-6-phosphate synthase, crude enzyme solution obtained from fermentation of the recombinant bacteria, and / or purified enzyme solution, with fructose as the sole substrate and an appropriate amount of amino donor added, to ferment and obtain glucosamine with a conversion rate of 77-92%. This invention is simpler than existing disclosed enzymatic synthesis technologies, and the process is easier to control, with higher production efficiency and lower application costs, making it suitable for industrial promotion and application. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the technology for synthesizing glucosamine using fructose as a raw material and catalyzing glucosamine-6-phosphate synthase.

[0045] Figure 2 This is a high-performance liquid chromatography (HPLC) chromatogram of the whole-cell synthesis of glucosamine using fructose as a raw material and ammonium chloride as an amino donor, catalyzed by recombinant Escherichia coli expressing glucosamine-6-phosphate synthase.

[0046] Figure 3 This is a high-performance liquid chromatography (HPLC) chromatogram of the synthesis of glucosamine from Escherichia coli cells lysed with fructose as a raw material and ammonium chloride as an amino donor, using recombinant glucosamine-6-phosphate synthase as a catalyst.

[0047] 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 glucosamine-6-phosphate synthase obtained through isolation and purification. Detailed Implementation

[0048] 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.

[0049] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0050] The plasmids and strains involved in the following examples:

[0051] pET28a(+) plasmid: EMD Biosciences (Novagen), CA;

[0052] Top 10 clones of E. coli: Invitrogen, Carlsbad, CA;

[0053] Escherichia coli expression strain E. coli BL21(DE3): Invitrogen, Carlsbad, CA.

[0054] The experimental methods involved in the following embodiments are as follows:

[0055] Methods for determining enzyme activity:

[0056] The method for determining the enzyme activity of glucosamine-6-phosphate synthase 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 is determined 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 measured at 530 nm absorbance.

[0057] The enzyme activity unit of glucosamine-6-phosphate synthase is defined as the amount of enzyme required to catalyze the synthesis of 1 μmol of glucosamine per minute at 37°C. The enzyme activity unit is U / mL.

[0058] The culture media involved in the following examples:

[0059] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0.

[0060] The glucosamine standards used in the following examples were purchased from Sigma-Aldrich, Inc., USA.

[0061] Example 1: Construction of a recombinant E. coli genetically engineered strain expressing glucosamine-6-phosphate synthase

[0062] The complete gene of the glucosamine-6-phosphate synthase gene of E. coli (GenBank number NZ_LVNI01000093.1:358712-360541, sequence as shown in SEQ ID NO.1) was synthesized. The recombinant plasmid was ligated to the pET28a(+) plasmid, which had also undergone double enzyme digestion (restriction sites: Nde I and BamHI), and transformed into competent cells of E. coli TOP10 strain. The plasmid was then plated on LB plates containing kanamycin (30 μg / ml) and cultured at 37°C for 12 h. Positive transformants were then picked, identified, and sequenced.

[0063] The positive monoclonal samples verified by sequencing were inoculated into 5 mL of LB liquid medium containing kanamycin (30 μg / mL) and cultured overnight at 37°C and 180–220 rpm. The plasmid pET28a(+)-GS was extracted and transformed into the expression host E. coli BL21(DE3) to obtain the recombinant expression strain E. coli BL21(DE3) / pET28a(+)-GS.

[0064] SDS-PAGE analysis of total bacterial protein showed that the recombinant expression strain exhibited a clear expression band of glucosamine-6-phosphate synthase 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(+)-GS was preserved by slant culture and then by glycerol storage at -80℃.

[0065] Example 2: Whole-cell catalysis of fructose to glucosamine using recombinant strain E. coli BL21(DE3) / pET28a(+)-GS

[0066] (1) Shake-flask fermentation

[0067] The recombinant strain E. coli BL21(DE3) / pET28a(+)-GS 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. 600 The 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. The cells were induced to express and ferment for 10h at 180-220rpm. 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 10-30g / L wet cells.

[0068] (2) Whole-cell catalytic synthesis of glucosamine

[0069] 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 (0.1% v / v) (50 mM) and Triton X-100 (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.

[0070] (3) Detection of products

[0071] 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.

[0072] The results are as follows Figure 2 As shown, the retention time of both the product and the glucosamine standard sample was approximately 4.5 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 81.3 g / L, and the conversion rate was 81.3% (conversion rate = concentration of glucosamine produced / initial fructose concentration × 100%). Glucosamine can be further separated and purified from the reaction solution.

[0073] Example 3: Whole-cell catalysis of fructose to glucosamine by recombinant strain E. coli BL21(DE3) / pET28a(+)-GS

[0074] (1) Shake-flask fermentation

[0075] The recombinant strain E. coli BL21(DE3) / pET28a(+)-GS 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 culture temperature reached OD. 600 The concentration was set at 0.4-0.6, and then the mixture was transferred to 30℃ for incubation. IPTG was added to a final concentration of 0.2mM, and fermentation was carried out at 180-220rpm for 10h to obtain the fermentation broth. The supernatant was removed by centrifugation and the precipitate was resuspended in Tris-HCl buffer (pH 7.4) to obtain 10-30g / L of wet cells.

[0076] (2) Whole-cell catalytic synthesis of glucosamine

[0077] The wet bacterial cells (10 g / L) obtained in step (1) were used as catalysts, D-fructose (30 g / L) was used as substrates, ammonium chloride (NH4Cl) (300 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, and Tris-HCl buffer (pH 7.4) was used as buffer. The whole-cell catalytic transformation reaction was carried out at 37 °C for 8 h to obtain the reaction solution.

[0078] (3) Detection of products

[0079] 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.

[0080] The results showed that the retention time of both the product and the glucosamine standard was approximately 4.5 min. The concentration of glucosamine was directly proportional to the response intensity of the HPLC characteristic peak of glucosamine. After 8 hours of reaction, the concentration of glucosamine was 27.8 g / L, with a conversion rate of 92.6%. Glucosamine can be further separated and purified from the reaction solution.

[0081] Example 4: Utilizing crude enzyme solution containing glucosamine-6-phosphate synthase to catalyze the synthesis of glucosamine from fructose.

[0082] (1) Shake-flask fermentation

[0083] The recombinant strain E. coli BL21(DE3) / pET28a(+)-GS 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. 600 The concentration was set at 0.4-0.6, and then the mixture was transferred to 30℃ for incubation. IPTG was added to a final concentration of 0.2mM, and fermentation was carried out at 180-220rpm for 10h to obtain the fermentation broth.

[0084] (2) Crude enzyme solution catalyzes the synthesis of glucosamine

[0085] The fermentation broth obtained in step (1) was centrifuged to obtain bacterial cells, and the bacterial cells were resuspended in Tris-HCl buffer (pH 7.4). The bacterial 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 a crude enzyme solution containing glucosamine-6-phosphate synthase. The enzyme activity was measured to be 17.3 U / mL.

[0086] Using crude enzyme solution (10 g / L) as catalyst, 100 g / L D-fructose as substrate, and 500 mM ammonium chloride (NH4Cl) as 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 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.

[0087] (3) Detection of products

[0088] 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.

[0089] The results are as follows Figure 3 As shown, the retention time of the product with the glucosamine standard sample is approximately 4.5 min. The concentration of glucosamine is directly proportional to the response intensity of the HPLC characteristic peak of glucosamine. After 6 hours of reaction, the concentration of glucosamine is 77.2 g / L, and the conversion rate is 77.2%. Glucosamine can be further separated and purified from the reaction solution.

[0090] Example 5: Utilizing a crude enzyme solution containing glucosamine-6-phosphate synthase to catalyze the synthesis of glucosamine from fructose.

[0091] (1) Shake-flask fermentation

[0092] The recombinant strain E. coli BL21(DE3) / pET28a(+)-GS 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. 600 The concentration was set at 0.4-0.6, and then the mixture was transferred to 30℃ for incubation. IPTG was added to a final concentration of 0.2mM, and fermentation was carried out at 180-220rpm for 12h to obtain the fermentation broth.

[0093] (2) Crude enzyme solution catalyzes the synthesis of glucosamine

[0094] The fermentation broth obtained in step (1) was centrifuged to obtain bacterial cells, and the bacterial cells were resuspended in Tris-HCl buffer (pH 7.4). The cells were then sonicated (the sonication conditions were: ice bath, 300W, 12S disruption, 10S interval) to obtain a crude enzyme solution containing glucosamine-6-phosphate synthase. The enzyme activity was measured to be 19.1 U / mL.

[0095] Using the crude enzyme solution (5 g / L) as a catalyst, 30 g / L D-fructose as a substrate, and 300 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.

[0096] (3) Detection of products

[0097] 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.

[0098] The results showed that the retention time of the product with the glucosamine standard was approximately 4.5 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 25.7 g / L, and the conversion rate was 85.6%. Glucosamine can be further separated and purified from the reaction solution.

[0099] Example 6: Utilizing purified glucosamine-6-phosphate synthase to catalyze the synthesis of glucosamine from fructose.

[0100] 1) Shake flask fermentation

[0101] The recombinant strain E. coli BL21(DE3) / pET28a(+)-GS 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. 600 The concentration was set at 0.4-0.6, and then the mixture was transferred to 30℃ for incubation. IPTG was added to a final concentration of 0.2mM, and fermentation was carried out at 180-220rpm for 10h to obtain the fermentation broth.

[0102] (2) Pure enzyme-catalyzed synthesis of glucosamine

[0103] The fermentation broth obtained in step (1) was centrifuged to obtain bacterial cells, which were then resuspended in Tris-HCl buffer (pH 7.4) and sonicated (the sonication conditions were: ice bath, 300W, 10s disruption, 10s interval) to obtain a crude enzyme solution containing glucosamine-6-phosphate synthase. The crude enzyme solution was further processed using Ni... + The synthase was purified by column affinity chromatography, and the enzyme activity was measured to be 22.9 U / mL.

[0104] Using pure enzyme solution (5 g / L) as catalyst, 100 g / L D-fructose as substrate, and 500 mM ammonium chloride (NH4Cl) as 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.

[0105] (3) Detection of products

[0106] 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.

[0107] The results are as follows Figure 4 As shown, the retention time of the product with the glucosamine standard sample is approximately 4.5 min. The glucosamine concentration is directly proportional to the response intensity of the HPLC characteristic peak of glucosamine. After 3 hours of reaction, the concentration of glucosamine is 78.4 g / L, and the conversion rate is 78.4%. Glucosamine can be further separated and purified from the reaction solution.

[0108] 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 one-step enzymatic synthesis of glucosamine using fructose as a substrate <130> BAA211113A <160> 2 <170> PatentIn version 3.3 <210> 1 <211> 1830 <212> DNA <213> E. coli <400> 1 atgtgcggta tatttggtat tatctcaagc aatccaataa aggtgtctga actgaagtta 60 cttgctgaac acgccagaca acgtggtaga gattctagtg ggctcattgt taataatcag 120 aatggttatg atatttatcg tgccgattat gatattaaaa aattactagc tcgtgtgaaa 180 atatcagaat acaatcttgc tttagggcac agtcgattaa ttactaatgg tctcactgac 240 aaccaacctg tagttaggga tgaagtatgt gttattcata atggtattat tgttaaccat 300 gaattattat ggtcagaatt ggggcttgaa cgtcggttgg agattgatac agaaattatt 360 gctgctatta cagaaaagca tatattagag acaggtaatc tttctggttt agcaaataag 420 gttcttgatt tatgccaggg tgttgtggcc accgcaataa tatctccatc attaggaaag 480 atgattcttt tctcaaataa tggtagttg tatgtaggtg aaaaaaaaga tattttttat 540 ttctcgtctg agaaataccc attaacgatg ctgagttgtg ataatattaa gcaagtttttt 600 aatgatgaag ttttggttga tataccaata agtaaaaata taaatatcac agactatgct 660 ttaagaaaaa ctaaccttat accaaagtttt ataaaggtta actcggaagc aaaacttctt 720 gaatatcctt gtccagtatt gcggcgttgt acaagatgta ttcttcctga aacgatgcct 780 tacatttcat ttgatgacca tggtgtttgc aactattgcc gaaattacaa gacaagaaat 840 aatcccaagc caaaagaaga gctttttgaa ctggttaagc catatcgacg taatggtaaa 900 ctcgattgta ttgttcctttt ttctggtggt cgtgatagtt gttatgctct tcatcttata 960 gtaaaagaat tacagatgaa accggttact tatacttatg attggggaat ggttactgat 1020 ttaggacgaa ggaatataag cagaatgtgt gctgagctgg gtgtcgaaaa tataatcgtt 1080 gccgatgata tttctaagaa aagacgcaat atagctctaa atttgaaagc gtggttaaaa 1140 tctccacatc tgggaatgat aagtatatta actgctggag ataagcattt tttccgacat 1200 gttgaatctg taaaaaaaca gacaggaatt aatcttaatc tttggggggt aaatccgcta 1260 gaggttactc attttaaatc tggtttttta ggtataccgc ctgattttgc tgaagaacga 1320 gtttatagcc atggatggca gaagcaactt acatatcaaa aattaagatt taaagcaatg 1380 ttgcaaagtc tggggtattt taatcgttca ttatgggata cattatcggg agaatattat 1440 cgtagtttta cagagaagaa agattattat catatcttcg attattggcg ctgggatgaa 1500 aaactaattg atgatacttt aattaatgag tataaatggg aaactgctgt tgataccaat 1560 accacatggc gtattggaga tggtacagct gctttttaca actatattta tttgtcgtt 1620 gcaggtttta ctgagcatga tactttccgg agtaatcaaa tccgtgaagg acaaattacc 1680 agagagcttg cattaaaact agtaaaagat gaaaaccaac ccagatatga aaatattcga 1740 tggtatttgg atgctttagg aatggacttt tcagaggtta ttaaagtaat aaataacatc 1800 cctagattat acgaaggttt ccatgagtaa 1830 <210> 2 <211> 609 <212> PRT <213> Escherichia coli <400> 2 Met Cys Gly Ile Phe Gly Ile Ile Ser Ser Asn Pro Ile Lys Val Ser 1 5 10 15 Glu Leu Lys Leu Leu Ala Glu His Ala Arg Gln Arg Gly Arg Asp Ser 20 25 30 Ser Gly Leu Ile Val Asn Asn Gln Asn Gly Tyr Asp Ile Tyr Arg Ala 35 40 45 Asp Tyr Asp Ile Lys Lys Leu Leu Ala Arg Val Lys Ile Ser Glu Tyr 50 55 60 Asn Leu Ala Leu Gly His Ser Arg Leu Ile Thr Asn Gly Leu Thr Asp 65 70 75 80 Asn Gln Pro Val Val Arg Asp Glu Val Cys Val Ile His Asn Gly Ile 85 90 95 Ile Val Asn His Glu Leu Leu Trp Ser Glu Leu Gly Leu Glu Arg Arg 100 105 110 Leu Glu Ile Asp Thr Glu Ile Ile Ala Ala Ile Thr Glu Lys His Ile 115 120 125 Leu Glu Thr Gly Asn Leu Ser Gly Leu Ala Asn Lys Val Leu Asp Leu 130 135 140 Cys Gln Gly Val Val Ala Thr Ala Ile Ile Ser Pro Ser Leu Gly Lys 145 150 155 160 Met Ile Leu Phe Ser Asn Asn Gly Ser Leu Tyr Val Gly Glu Lys Lys 165 170 175 Asp Ile Phe Tyr Phe Ser Ser Glu Lys Tyr Pro Leu Thr Met Leu Ser 180 185 190 Cys Asp Asn Ile Lys Gln Val Phe Asn Asp Glu Val Leu Val Asp Ile 195 200 205 Pro Ile Ser Lys Asn Ile Asn Ile Thr Asp Tyr Ala Leu Arg Lys Thr 210 215 220 Asn Leu Ile Pro Lys Phe Ile Lys Val Asn Ser Glu Ala Lys Leu Leu 225 230 235 240 Glu Tyr Pro Cys Pro Val Leu Arg Arg Cys Thr Arg Cys Ile Leu Pro 245 250 255 Glu Thr Met Pro Tyr Ile Ser Phe Asp Asp His Gly Val Cys Asn Tyr 260 265 270 Cys Arg Asn Tyr Lys Thr Arg Asn Asn Pro Lys Pro Lys Glu Glu Leu 275 280 285 Phe Glu Leu Val Lys Pro Tyr Arg Arg Asn Gly Lys Leu Asp Cys Ile 290 295 300 Val Pro Phe Ser Gly Gly Arg Asp Ser Cys Tyr Ala Leu His Leu Ile 305 310 315 320 Val Lys Glu Leu Gln Met Lys Pro Val Thr Tyr Thr Tyr Asp Trp Gly 325 330 335 Met Val Thr Asp Leu Gly Arg Arg Asn Ile Ser Arg Met Cys Ala Glu 340 345 350 Leu Gly Val Glu Asn Ile Ile Val Ala Asp Asp Ile Ser Lys Lys Arg 355 360 365 Arg Asn Ile Ala Leu Asn Leu Lys Ala Trp Leu Lys Ser Pro His Leu 370 375 380 Gly Met Ile Ser Ile Leu Thr Ala Gly Asp Lys His Phe Phe Arg His 385 390 395 400 Val Glu Ser Val Lys Lys Gln Thr Gly Ile Asn Leu Asn Leu Trp Gly 405 410 415 Val Asn Pro Leu Glu Val Thr His Phe Lys Ser Gly Phe Leu Gly Ile 420 425 430 Pro Pro Asp Phe Ala Glu Glu Arg Val Tyr Ser His Gly Trp Gln Lys 435 440 445 Gln Leu Thr Tyr Gln Lys Leu Arg Phe Lys Ala Met Leu Gln Ser Leu 450 455 460 Gly Tyr Phe Asn Arg Ser Leu Trp Asp Thr Leu Ser Gly Glu Tyr Tyr 465 470 475 480 Arg Ser Phe Thr Glu Lys Lys Asp Tyr Tyr His Ile Phe Asp Tyr Trp 485 490 495 Arg Trp Asp Glu Lys Leu Ile Asp Asp Thr Leu Ile Asn Glu Tyr Lys 500 505 510 Trp Glu Thr Ala Val Asp Thr Asn Thr Thr Trp Arg Ile Gly Asp Gly 515 520 525 Thr Ala Ala Phe Tyr Asn Tyr Ile Tyr Tyr Val Val Ala Gly Phe Thr 530 535 540 Glu His Asp Thr Phe Arg Ser Asn Gln Ile Arg Glu Gly Gln Ile Thr 545 550 555 560 Arg Glu Leu Ala Leu Lys Leu Val Lys Asp Glu Asn Gln Pro Arg Tyr 565 570 575 Glu Asn Ile Arg Trp Tyr Leu Asp Ala Leu Gly Met Asp Phe Ser Glu 580 585 590 Val Ile Lys Val Ile Asn Asn Ile Pro Arg Leu Tyr Glu Gly Phe His 595 600 605 Glu

Claims

1. A method for synthesizing glucosamine using fructose as the sole substrate, characterized in that, Glucosamine is synthesized using recombinant bacteria, crude enzyme solution obtained from recombinant bacterial fermentation, or glucosamine-6-phosphate synthase and amino donor synthesized by recombinant bacterial fermentation. The recombinant bacteria is Escherichia coli. E. coli Using BL21(DE3) as the host and pET28a(+) as the expression vector, glucosamine-6-phosphate synthase with the amino acid sequence shown in SEQ ID NO.2 was expressed; the amino donor was ammonium chloride; the catalytic reaction was carried out at a temperature of 37°C and a pH of 7.

4.

2. The method according to claim 1, characterized in that, The crude enzyme solution is prepared by ultrasonically crushing the fermentation broth obtained from the fermentation of the recombinant bacteria.

3. The method according to claim 1, characterized in that, The reaction system synthesized using the recombinant bacteria also contains inorganic salts and Triton X-100.

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