WO2020011237A1 - 氨基葡萄糖的酶法制备 - Google Patents

氨基葡萄糖的酶法制备 Download PDF

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WO2020011237A1
WO2020011237A1 PCT/CN2019/095638 CN2019095638W WO2020011237A1 WO 2020011237 A1 WO2020011237 A1 WO 2020011237A1 CN 2019095638 W CN2019095638 W CN 2019095638W WO 2020011237 A1 WO2020011237 A1 WO 2020011237A1
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phosphate
glucosamine
reaction
concentration
enzyme
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PCT/CN2019/095638
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French (fr)
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游淳
孟冬冬
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中国科学院天津工业生物技术研究所
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Priority to EP19833631.5A priority Critical patent/EP3859003A4/en
Priority to KR1020217000075A priority patent/KR20210031678A/ko
Priority to US17/260,168 priority patent/US20210277437A1/en
Priority to JP2021500954A priority patent/JP2021531758A/ja
Publication of WO2020011237A1 publication Critical patent/WO2020011237A1/zh

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    • C12Y305/99—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in other compounds (3.5.99)
    • C12Y305/99006—Glucosamine-6-phosphate deaminase (3.5.99.6)
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    • C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
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    • C12Y504/02002—Phosphoglucomutase (5.4.2.2)

Definitions

  • the invention relates to a method for preparing glucosamine, in particular to a method for preparing glucosamine by in vitro enzyme catalysis, and belongs to the field of enzyme-catalyzed preparation of glucosamine.
  • Glucosamine is a compound in which the 2-position hydroxyl group in the D-glucose molecule is replaced by an amino group, and is an important functional monosaccharide. Glucosamine is found in almost all organisms, including bacteria, yeast, filamentous fungi, plants, and animals. It is the main component of glycoproteins and proteoglycans, as well as the main component of chitosan and chitin.
  • Glucosamine and its derivatives are widely used and have important applications in the fields of medicine, food, cosmetics and so on.
  • glucosamine sulfate can be used as a raw material for the treatment of rheumatoid arthritis by stimulating the biosynthesis of cartilage proteoglycans.
  • glucosamine has the ability to absorb free radicals produced in the body, anti-aging, Promotes weight loss, bacteriostasis, and regulates human endocrine and other physiological functions. It is used in the production of food additives and health foods.
  • acetylglucosamine (GlcNAc) is a monomer of hyaluronic acid and is currently a high-end An indispensable substance in cosmetics.
  • the chitin hydrolysis method includes a chitin acid hydrolysis method and a chitinase hydrolysis method.
  • the chitosan acid hydrolysis method is the most commonly used glucosamine production method. This method first hydrolyzes chitin with high concentration hydrochloric acid to obtain acetylglucosamine, and then deacetylates acetylglucosamine to obtain glucosamine.
  • the production method will be affected by the supply of raw materials, and the wastewater generated by the acid treatment will seriously pollute the environment.
  • some people allergic to shrimp and crab raw materials will have an allergic reaction after eating the glucosamine prepared by this method.
  • the chitinase hydrolysis method uses chitin as a raw material and hydrolyzes under the action of chitinase to generate monomeric glucosamine. Although this method has less environmental pollution, it is also limited by factors such as raw material supply, low production intensity, and allergic reactions.
  • Microbial fermentation method mainly uses engineering bacteria such as E. coli, Bacillus subtilis and other raw materials to metabolize glucose to produce glucosamine. This method has the advantages of not being limited by the source of raw materials, short fermentation time, low environmental pollution, and safe for the human body. However, it has the disadvantages of difficulty in metabolic transformation of microbial engineering bacteria and easy production of metabolic by-products.
  • the invention relates to an in vitro enzymatic method for preparing glucosamine.
  • the method catalyzes the production of glucosamine through an in vitro enzymatic reaction system.
  • the method has the advantages of cheap raw materials, rich sources, low production costs, environmental friendliness, and safety to the human body.
  • the invention provides a method for preparing glucosamine by using an in vitro enzyme-catalyzed reaction, which comprises: catalyzing glucosamine-6-phosphate deaminase (EC 3.5.99.6, GlmD) to catalyze fructose 6-phosphate (F6P) and ammonium salts are converted to glucosamine 6-phosphate (GlcN6P); and an enzyme capable of dephosphorylation group is used to catalyze the dephosphorylation group of GlcN6P to produce glucosamine (GlcN).
  • glucosamine-6-phosphate deaminase EC 3.5.99.6, GlmD
  • F6P fructose 6-phosphate
  • GlcN6P ammonium salts are converted to glucosamine 6-phosphate
  • an enzyme capable of dephosphorylation group is used to catalyze the dephosphorylation group of GlcN6P to produce glucosamine (Glc
  • ammonium salts can be used in the present invention.
  • the ammonium salt is selected from one, two or more of any mixture of ammonium sulfate, ammonium chloride, ammonium bisulfate, ammonium nitrate, ammonium carbonate, and ammonium bicarbonate.
  • the method further comprises a reaction step of converting glucose 6-phosphate (G6P) to F6P, which step is catalyzed by phosphorglucose isomerase (EC5.3.1.9, PGI).
  • the method further comprises a reaction step of converting glucose 1-phosphate (G1P) to G6P, which step is catalyzed by glucose phosphoglucomutase (EC 5.4.2.2, PGM).
  • G1P glucose 1-phosphate
  • PGM glucose phosphoglucomutase
  • the method further comprises a reaction step of converting a substrate and a phosphate to G1P, the substrate is a disaccharide, a polysaccharide or any mixture thereof comprising a D-glucose unit, and this step adopts Enzyme catalysis of substrate and phosphate conversion to G1P.
  • the phosphate is selected from any one, two or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate. More preferably, the phosphate is derived from potassium dihydrogen phosphate and / or dipotassium hydrogen phosphate.
  • the disaccharide containing a D-glucose unit is sucrose
  • sucrose phosphorylase EC 2.4.1.7, SP
  • the polysaccharide containing D-glucose unit is selected from the group consisting of starch, starch derivatives or any mixture thereof, and is catalyzed by starch phosphorylase ( ⁇ -glucan phosphorylase (EC 2.4.1.1, ⁇ GP)) Phosphate is converted to G1P.
  • starch phosphorylase ⁇ -glucan phosphorylase (EC 2.4.1.1, ⁇ GP)
  • Phosphate is converted to G1P.
  • the starch or starch derivative is selected from soluble starch, soluble amylose, soluble amylopectin, starch dextrin, maltodextrin, and maltopolysaccharide.
  • the starch is soluble starch.
  • the D-glucose unit-containing polysaccharide is further selected from cellulose, cellulose derivatives, or any mixture thereof.
  • the cellulose derivative is selected from the group consisting of cellulosic polysaccharide, cellobiose, or any mixture thereof.
  • the cellulose derivative is a product of cellulose after acid or enzyme pretreatment, and the product is cellulosic polysaccharide, cellobiose, or any mixture thereof.
  • the cellulose derivative is a cellulosic polysaccharide.
  • D-glucose unit-containing polysaccharide contains cellulose and / or cellulosic polysaccharides, such as cellulose and / or cellulosic polysaccharides
  • cellodextrin phosphorylase EC 2.4.1.49, CDP
  • cellobiose phosphorylase EC 2.4.1.20, CBP
  • CBP cellobiose phosphorylase
  • the polysaccharide containing the D-glucose unit contains cellobiose
  • cellobiose for example, cellobiose, cellobiose phosphorylase (EC 2.4.1.20, CBP) is used to catalyze the conversion of the cellobiose and phosphate to G1P .
  • CBP cellobiose phosphorylase
  • the above steps included in the method of the present invention can also be performed simultaneously, for example, they can be performed in a bioreactor or a reaction vessel. Preferably, the above steps included in the method of the present invention are performed simultaneously.
  • the present invention provides a method for preparing glucosamine by using an in vitro enzyme-catalyzed reaction, comprising: taking a disaccharide, a polysaccharide or any mixture thereof containing a D-glucose unit as a substrate, adding a phosphate, an ammonium salt, and D-glucose unit-containing disaccharides, polysaccharides, or any mixture thereof, and an enzyme that converts phosphate to G1P, glucose phosphomutase, phosphoglucoisomerase, glucosamine 6-phosphate deaminase, and capable of dephosphorylation Catalyzed reaction to obtain glucosamine.
  • glucosamine 6-phosphate deaminase from various sources can be used in the present invention.
  • the glucosamine 6-phosphate deaminase can be derived from E. coli (UniProt number P0A759), Bacillus subtilis (UniProt number O35000), Giardia blue (UniProt number V6TL01), or Thermococcus kokokarkarensis (UniProt number Q5JDU3) Wait.
  • the enzyme capable of dephosphorylation is a substrate-specific 6-phosphate glucosamine phosphatase (GlmP).
  • the 6-phosphate glucosamine phosphatase can be derived from In E. coli (UniProt No. P77475, P27848, P0AE22, etc.), Bacteroides thetaiotaomicron (UniProt No. Q8A759), and the like.
  • thermophilic enzyme UniProt number Q5JJ45 from Thermococcus kodakarensis is annotated as a predicted phosphatase (HAD superfamily). Its nucleotide sequence is shown in SEQ ID ID NOs: 1 and its amino acid sequence is shown in SEQ ID ID NOs: 2 . In the present invention, the substrate specificity of Q5JJ45 is identified. The results show that Q5JJ45 is a heat-resistant enzyme capable of catalyzing GlcN6P for a dephosphorylation reaction, and has the function of catalyzing GlcN6P to produce glucosamine.
  • the enzyme capable of dephosphorylation group is encoded by a nucleotide comprising at least 60%, at least 70%, at least 80%, at least 90% with SEQ ID NOs: 1 %, At least 95%, at least 99%, or 100% sequence identity; preferably, the nucleotides are at least 60%, at least 70%, at least 80%, at least A nucleotide sequence that is 90%, at least 95%, at least 99%, or 100% sequence identical.
  • the enzyme capable of dephosphorylation comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% sequence identity with SEQ ID NOs: 2 Amino acid sequence; preferably, the enzyme capable of dephosphorylation has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% sequence with SEQ ID NOs: 2 Identity amino acid sequence.
  • the enzyme capable of dephosphorylation is a sugar phosphatase (UniProt No. Q5JJ45) derived from Thermococcus kodakarensis.
  • the starch phosphorylase can be derived from E.
  • sucrose phosphorylase Can be derived from Bifidobacterium adolescentis (UniProt number A0ZZH6), Thermoanaerobacterium thermosaccharolyticum (UniProt number D9TT09), etc .
  • Fibrous polysaccharide phosphorylase can be derived from Clostridium thermothermum (UniProt number A3DJQ6), Clostridium Unistertorcorium No.
  • cellobiose phosphorylase can be derived from Clostridium thermocellum (UniProt No. A3DC35), Thermotoga neapolitana (UniProt No. B9K7M6), and the like.
  • Glucose phosphomutase can be derived from Clostridium thermocellum (Uniprot number A3DEW8), Thermococcus kodakarensis (UniProt number Q68BJ6), etc .; phosphoglucose isomerase can be derived from Clostridium thermocellum (Uniprot number A3DBX9) ), Thermothermophilus (Thermus thermophilus, Uniprot number Q5SLL6) and the like.
  • the temperature of the catalytic reaction is 30-70 ° C, more preferably 30-50 ° C, and most preferably 37 ° C.
  • the pH of the catalytic reaction is 5.0-8.0, more preferably 6.0-7.5, and most preferably 7.0.
  • the time for the catalytic reaction is 1-48 hours, more preferably 8-36 hours, more preferably 10-24 hours, and most preferably 20 hours.
  • the time for each step to catalyze the reaction is independently from 0.5 to 10 hours, more preferably from 1 to 3 hours, and most preferably from 2 hours.
  • the substrate concentration in the reaction system is 1-200 g / L, more preferably 5-50 g / L, more preferably 8-20 g / L, and most preferably 10 g / L.
  • the concentration of the enzyme that converts the substrate into G1P in the reaction system is 0.1-10 U / mL, more preferably 0.2-5 U / mL, more preferably 1-3 U / mL, and most preferably 2 U / mL.
  • the concentration of glucose phosphomutase is 0.1-10U / mL, more preferably 0.2-5U / mL, more preferably 1-3U / mL, and most preferably 2U / mL; preferably, glucosamine phosphate
  • the concentration of the isomerase is 0.1-10U / mL, more preferably 1-5U / mL, and most preferably 3U / mL; preferably, the concentration of glucosamine 6-phosphate deaminase is 0.1-10U / mL, more preferably 0.2-5U / mL, more preferably 1-3U / mL, most preferably 2U / mL; preferably, the concentration of the enzyme capable of dephosphorylation is 0.1-10U / mL, and still more preferably 0.2-5U / mL , More preferably 1-3 U / mL, and most preferably 2 U / mL.
  • the concentration of the ammonium salt in the reaction system is 50-500 mM, more preferably 100-300 mM, and most preferably 200 mM.
  • the concentration of the phosphate in the reaction system is 1-150 mM, further preferably 2-50 mM, more preferably 10-30 mM, and most preferably 20 mM.
  • the phosphate generated in the process of the GlcN6P dephosphorylation group to generate glucosamine (GlcN) can be used as the phosphorus in the step of converting the disaccharide, polysaccharide or any mixture thereof containing D-glucose unit to G1P source.
  • the above reaction system further contains a magnesium salt.
  • a magnesium salt such as magnesium chloride, magnesium sulfate, and the like.
  • the magnesium salt is magnesium chloride.
  • the concentration of the magnesium salt in the reaction system is 1-20 mM, more preferably 2-15 mM, and most preferably 10 mM.
  • the reaction system further contains a buffer solution.
  • buffer solution e.g., HEPES buffer, Tris-HCl buffer, MOPS buffer, citrate buffer such as sodium citrate buffer, and the like.
  • the buffer is a HEPES buffer.
  • concentration of the buffer in the reaction system is 20-300 mM, preferably 50-200 mM, and most preferably 100 mM.
  • the catalytic reaction is performed under conditions without ATP and without NAD (H).
  • the starch, starch derivative, or any mixture thereof contains an alpha-1,6 glycosidic bond (e.g., soluble starch, soluble amylopectin, amylodextrin, maltodextrin, maltopolysaccharide)
  • the method of the present invention further includes a reaction step of isoamylase (EC 3.2.1.68, IA) hydrolysis of ⁇ -1,6-glycosidic bonds in the substrate.
  • isoamylases from various sources can be used in the present invention.
  • the isoamylase can be derived from Sulfolobus tokodaii (UniProt No. Q973H3), Flavobacterium sp. (UniProt No. O32611), and the like.
  • the concentration of the isoamylase in the reaction system is 0.1-10 U / mL, more preferably 0.5-2 U / mL, and most preferably 1 U / mL.
  • reaction step of hydrolyzing ⁇ -1,6-glucosidic bond in the substrate by using isoamylase and the above reaction step can be performed in steps, for example, it can be performed in a bioreactor or reaction vessel or in series
  • the multiple bioreactors or reaction vessels arranged may be performed simultaneously with the above reaction steps, for example, they may be performed in one bioreactor or reaction vessel.
  • the step of hydrolyzing the ⁇ -1,6-glucosidic bond in the substrate using an isoamylase is performed before the reaction step of converting the substrate and the phosphate to G1P, that is, the ⁇ - After the 1,6-glycosidic bond is obtained after the substrate treated with isoamylase is obtained, other reaction steps are performed. At this time, the other reaction steps may be performed stepwise or simultaneously.
  • the substrate concentration in the reaction system is 1-300 g / L, more preferably 10-200 g / L, more preferably 50-150 g / L, and most preferably 100 g / L; preferably, isostarch
  • the concentration of the enzyme is 0.1-10 U / mL, more preferably 0.5-2 U / mL, and most preferably 1 U / mL; preferably, the pH of the catalytic reaction is 4-8, more preferably 4.5-6.5, and most preferably 5.5;
  • the reaction is performed at 10-99 ° C for 0.5-72 hours, further preferably at 30-95 ° C for 1-48 hours, more preferably at 50-90 ° C for 6-24 hours, and most preferably at 85 ° C for 12 hours.
  • the reaction system further contains a magnesium salt and a buffer solution.
  • magnesium salts can be used in the present invention, such as magnesium chloride, magnesium sulfate, and the like.
  • the magnesium salt is magnesium chloride.
  • the concentration of the magnesium salt in the reaction system is 0.01-10 mM, further preferably 0.1-5 mM, more preferably 0.2-1 mM, and most preferably 0.5 mM.
  • various buffers can be used in the present invention, such as sodium acetate buffer, HEPES buffer, citrate buffer such as sodium citrate buffer, etc.
  • the buffer is sodium acetate buffer .
  • the concentration of the buffer in the reaction system is 1-50 mM, further preferably 2-20 mM, more preferably 3-10 mM, and most preferably 5 mM.
  • the present invention when the substrate is starch, a starch derivative, or any mixture thereof (e.g., soluble starch, soluble amylose, soluble amylopectin, starch dextrin, maltodextrin, maltopolysaccharide), the present invention
  • the method further comprises a reaction step catalyzed by 4- ⁇ -glucanotransferase (EC 2.4.1.25, 4GT).
  • 4- ⁇ -transglucosidase from various sources can be used in the present invention.
  • the 4- ⁇ -transglucosidase can be derived from Thermococcus littoralis (UniProt number O32462), Bacillus subtilis (UniProt number L8AG91), Clostridium butyricum, UniProt number Q59266 )Wait.
  • the concentration of 4- ⁇ -transglucosidase in the reaction system is 0.1-10U / mL, more preferably 0.2-5U / mL, more preferably 0.5-2U / mL, and most preferably 1U / mL.
  • reaction step catalyzed by 4- ⁇ -transglucosidase and the above steps can be performed step by step, for example, it can be performed in one bioreactor or reaction vessel or in multiple biological reactions arranged in series. It can be carried out in a reactor or a reaction vessel, and can be performed simultaneously with the above steps, for example, it can be carried out in a bioreactor or a reaction vessel.
  • the step of catalyzing the reaction using 4- ⁇ -transglucosidase is performed after the reaction of converting the substrate and the phosphate to G1P is performed for a period of time.
  • the reaction of converting the substrate and the phosphate to G1P is performed for 0.5-30 hours, preferably 5-20 hours, and most preferably 10 hours after adding 4- ⁇ -transglucosidase to the reaction system.
  • Starch is a mixture of amylose and amylopectin with different chain lengths. Amylose glucose units are connected by ⁇ -1,4 glycosidic bonds. Amylopectin is connected to the starch main chain by ⁇ -1,6 glycosidic bonds. Starch phosphorylase cannot hydrolyze ⁇ -1,6 glycosidic bonds. The addition of isoamylase, a debranching enzyme capable of hydrolyzing ⁇ -1,6 glycosidic bonds in starch, can increase the yield of G1P in the reaction system. In addition, starch phosphorylase hydrolyzes starch, starch derivatives, or any mixture thereof, and releases G1P. The final products are maltose and maltotriose.
  • 4- ⁇ -transglucosidase can be added to the reaction system, which can polymerize short-chain oligosaccharides into long-chain oligosaccharides, and the Long-chain oligosaccharides can be reused by starch phosphorylase to improve starch utilization.
  • the enzyme-catalyzed reaction of the present invention adopts the following method:
  • soluble starch treated with isoamylase is used as a substrate, and magnesium chloride, phosphate, ammonium salt, HEPES buffer (pH 7.0), starch phosphorylase, glucose phosphate mutase, and phosphoglucose are added.
  • Isomerase, 6-phosphate glucosamine deaminase, and an enzyme capable of dephosphorylation are catalyzed to obtain glucosamine.
  • 4- ⁇ -transglucosidase is added to the reaction system after the reaction has been performed for a period of time.
  • 10 g / L of soluble starch treated with isoamylase is used as a substrate, and 10 mM magnesium chloride, 20 mM potassium dihydrogen phosphate, 200 mM ammonium chloride, 100 mM HEPES buffer solution ( pH 7.0), 2U / mL starch phosphorylase, 2U / mL glucose phosphomutase, 3U / mL glucoglucose isomerase, 2U / mL 6-phosphate glucosamine deaminase, 2U / mL capable of dephosphorylation Enzyme (preferably 6-phosphate glucosamine phosphatase), the reaction mixture is catalyzed at 37 ° C for 30 hours to obtain glucosamine.
  • 1 U / mL 4- ⁇ -transglucosidase is also added to the reaction system when the reaction is carried out for 10 h.
  • the invention also provides the use of 6-phosphate glucosamine deaminase and an enzyme capable of dephosphorylation group (preferably 6-phosphate glucosamine phosphatase) in the preparation of glucosamine, preferably the application of catalysis of F6P to glucosamine.
  • an enzyme capable of dephosphorylation group preferably 6-phosphate glucosamine phosphatase
  • the present invention also provides an enzyme comprising a nucleotide-encoding enzyme having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NOs: 1
  • a nucleotide-encoding enzyme having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NOs: 1
  • the nucleotides preferably in catalysis of glucosamine 6-phosphate (GlcN6P) to glucosamine (GlcN); preferably, the nucleotides have at least 60% and at least 70% of SEQ ID NOs: 1
  • a nucleotide sequence that has a sequence identity of%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.
  • the enzyme comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NOs: 2; preferably, The enzyme has an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NOs: 2.
  • the enzyme is a sugar phosphatase (UniProt number Q5JJ45) derived from Thermococcus kodakarensis.
  • the present invention also provides glucosamine prepared by the above method.
  • glucosamine is prepared for the first time by using an in vitro enzyme-catalyzed reaction, in particular, for the first time, glucosamine 6-phosphate deaminase and an enzyme capable of dephosphorylation group are used to catalyze F6P to produce glucosamine.
  • the method of the invention can not only obtain the target product with a good conversion rate, but also the obtained product has the advantages of being safe to the human body and the like.
  • the method of the present invention can use a variety of raw materials, for example, disaccharides, polysaccharides, or any mixtures thereof containing D-glucose units can be used.
  • the method of the present invention has the advantages of cheap raw materials, abundant sources, low production costs, environmental friendliness, and safety to the human body, and is suitable for popularization.
  • the inventors also found that when starch, starch derivatives, or any mixture thereof is used as a substrate to catalyze the production of glucosamine, the addition of isoamylase and 4- ⁇ -transglucosidase can greatly improve the yield of the target product.
  • FIG. 1 is a schematic diagram of an in vitro enzymatic pathway for preparing glucosamine using starch as a substrate.
  • ⁇ GP starch phosphorylase
  • PGM glucose phosphomutase
  • PGI glucoglucose isomerase
  • GlmD 6-phosphate glucosamine deaminase
  • GlmP 6-phosphate glucosamine phosphatase.
  • FIG. 2 shows the change in Gibbs energy between the intermediates of the in vitro enzymatic pathway for preparing glucosamine using starch as a substrate.
  • Figure 3 shows SDS-PAGE detection of key enzymes for the production of glucosamine using starch as a substrate.
  • M Marker.
  • Figure 4 shows the analysis of glucosamine by HPLC.
  • 4A is an HPLC peak chart of a glucosamine standard;
  • 4B is a quantitative analysis of the concentration of glucosamine by HPLC. The intensity of the glucosamine peak can be used to quantify the concentration of the obtained glucosamine.
  • FIG. 5 is an HPLC analysis of glucosamine 6-phosphate deaminase involved in the in vitro enzyme system catalyzed by soluble starch to synthesize glucosamine.
  • 5A is the result of HPLC analysis of in vitro enzyme-catalyzed soluble starch to produce glucosamine
  • 5B is the reaction progress curve of in vitro enzyme-catalyzed soluble starch to synthesize glucosamine.
  • FIG. 6 is a reaction progress curve of in vitro enzyme-catalyzed synthesis of glucosamine by IA-treated soluble starch.
  • Fig. 7 is a curve of the reaction process of IA-treated soluble starch to synthesize glucosamine in vitro after optimizing the enzyme concentration.
  • FIG. 8 Curve of the reaction process of in vitro enzyme-catalyzed conversion of sucrose to glucosamine.
  • FIG. 9 is a reaction progress curve of in vitro enzyme-catalyzed conversion of fibrin to glucosamine.
  • Soluble starch ACROS product, product number: 424490020;
  • pET20b vector Novagen, Madison, WI;
  • Escherichia coli BL21 (DE3), Invitrogen, Carlsbad, CA;
  • Fig. 1 The catalytic pathway for the conversion of starch into glucosamine through an in vitro enzyme-catalyzed system is shown in Fig. 1.
  • the enzymes capable of dephosphorizing groups are 6-phosphate glucosamine phosphatase as an example.
  • Figure 2 shows the Gibbs energy change in the reaction between intermediates of the enzyme-catalyzed pathway that converts starch to glucosamine.
  • the starch phosphorylase is derived from E.
  • coli (Uniprot number A0A0A0HB49); (2) the glucose phosphomutase is derived from Clostridium thermocellum (Uniprot number A3DEW8); (3) phosphate Glucose isomerase is derived from Clostridium thermocellum (Uniprot number A3DBX9); (4) 6-phosphate glucosamine deaminase is derived from Bacillus subtilis (Uniprot number O35000); (5) can dephosphorize The enzyme is derived from the 6-phosphate glucosamine phosphatase (Uniprot No. Q8A759) of Bacteroides thetaiotaomicron.
  • the above genomic DNA can be obtained from the ATCC official website (www.atcc.org). Through Simple Cloning (You C, Zhang XZ, Zhang Y-HP. 2012. Simple Cloning via direct transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis.Appl.Environ.Microbiol.78 (5): 1593-5 .) Method, the above genes were cloned into pET20b vector (Novagen, Madison, WI), and the corresponding expression vectors pET20b-Ec ⁇ GP, pET20b-CtPGM, pET20b-CtPGI, pET20b-BsGlmD, pET20b-BtGlmP were obtained. The recombinant protein was expressed in E. coli BL21 (DE3) and purified, and the results of the protein purification are shown in FIG. 3.
  • the enzyme activity measurement of glucose phosphomutase derived from Clostridium thermocellum was performed in 100 mM HEPES buffer (pH 7.0) containing 10 mM magnesium chloride. Using 10 mM glucose 1-phosphate as a substrate, the reaction was performed at 37 ° C for 10 minutes, and the amount of 6-phosphate glucose (G6P) produced was measured.
  • the method for detecting the amount of G6P is as follows: take 40 ⁇ l of the sample solution containing G6P, add 200 ⁇ l of 100 mM HEPES buffer containing 2 mM magnesium chloride, 0.15 mM NAD + , 0.5 U / mL glucose 6-phosphate dehydrogenase (G6PDH) Solution (pH 7.0), reacted at 37 ° C for 30 minutes, measured the absorbance at 340 nm, and calculated the amount of NADH produced.
  • G6PDH glucose 6-phosphate dehydrogenase
  • Enzyme activity measurement of starch phosphorylase derived from E. coli was performed in 100 mM HEPES buffer (pH 7.0) containing 10 mM magnesium chloride and 1 U / mL glucose phosphomutase. Using 5 g / L soluble starch as a substrate, the reaction was performed at 37 ° C for 10 minutes, and the amount of glucose 6-phosphate produced was measured. The experimental results showed that the specific activity of the E. coli-derived starch phosphorylase at 37 ° C was 5.6 U / mg.
  • the enzyme activity measurement of phosphoglucose isomerase derived from Clostridium thermocellum was performed in a 100 mM HEPES buffer (pH 7.0) containing 10 mM magnesium chloride. Using 10 mM fructose 6-phosphate as a substrate, the reaction was performed at 37 ° C for 10 minutes, and the amount of 6-phosphate glucose produced was measured. The experimental results show that the specific enzyme activity of phosphoglucose isomerase derived from Clostridium thermocellum at 37 ° C is 396 U / mg.
  • the enzymatic activity measurement of glucosamine 6-phosphate deaminase derived from B. subtilis was performed in a 100 mM HEPES buffer (pH 7.0) containing 10 mM magnesium chloride. Using 10 mM fructose 6-phosphate and 100 mM ammonium chloride as substrates, the reaction was performed at 37 ° C for 10 minutes, and the amount of 6-phosphate glucosamine (GlcN6P) produced was measured.
  • the method for detecting the amount of GlcN6P is as follows: Take 50 ⁇ l of a sample containing GlcN6P and add 100 ⁇ l acetylacetone reagent (take 1.5mL acetylacetone, dissolved in 50mL 1.25mol / L sodium carbonate solution), boil for 20 minutes, and cool to room temperature Slowly add 1 mL of 96% (v / v) ethanol, and then add 100 ⁇ l of p-dimethylaminobenzaldehyde (DMAB) reagent (weigh 1.6g of DMAB dissolved in 30mL of concentrated hydrochloric acid and 30mL of 96% ethanol) and mix Uniform, left at room temperature for 30 minutes, measured the absorbance at 530nm, and calculated the GlcN6P content according to the standard curve.
  • the experimental results show that the specific enzyme activity of glucosamine 6-phosphate deaminase derived from Bacillus subtilis at 37 ° C is 10 U / mg
  • an enzyme capable of dephosphorylation group having specific dephosphorization activity to GlcN6P is one of the key points of the present invention.
  • 100 mM HEPES buffer (pH 7.0) containing 10 mM magnesium chloride we measured the dephosphorization activity of the enzymes capable of dephosphorylation on G1P, G6P, F6P, and GlcN6P.
  • the experimental results are shown in Table 1.
  • the 6-phosphate glucosamine phosphatase from Bacteroides thetaiotaomicron has a higher specific enzyme activity for GlcN6P and exhibits specific GlcN6P dephosphorization activity.
  • Thermococcus kodakarensis-derived sugar phosphatase (predicted sugar phosphatase (HAD) superfamily) has a dephosphorization activity of GlcN6P substrate of 0.011 U / mg at 70 ° C.
  • This embodiment uses enzymes to catalyze the synthesis of glucosamine from soluble starch in vitro.
  • five enzymes were recombinantly expressed: ⁇ GP derived from E. coli, PGM derived from Clostridium thermocellum, PGI derived from Clostridium thermocellum, GlmD derived from Bacillus subtilis, and GlmP derived from B. polymorpha ( Table 2).
  • Quantitative analysis of glucosamine was performed using high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the chromatographic column used was an amino column, the mobile phase was an 80% acetonitrile aqueous solution, the flow rate was 1 mL / min, and the column temperature was 40 ° C.
  • the detector used was a refractive index detector.
  • the standard sample detection is shown in Figure 4A.
  • the glucosamine retention time is about 9.6 minutes.
  • the concentration of glucosamine is directly proportional to the response intensity of the HPLC characteristic peak of glucosamine.
  • the standard curve is shown in Figure 4B.
  • Starch is a mixed polysaccharide of ⁇ -1,4 and ⁇ -1,6, which cannot be completely hydrolyzed by starch phosphorylase.
  • Isoamylase IA, EC 3.2.1.68 can hydrolyze ⁇ -1,6 glycosidic bonds in starch, thereby helping starch phosphorylase to hydrolyze substrates and improve the yield of glucosamine.
  • the isoamylase is derived from Sulfolobus tokodaii (UniProt No. Q973H3). Introduce the expression vector pET20b-St3 reported in the literature (Cheng, K.et. For protein expression and purification.
  • a 5 mM sodium acetate buffer (pH 5.5) containing 100 g / L of soluble starch was added with 0.5 mM magnesium chloride and 1 U / mL isoamylase, and treated at 85 ° C for 12 hours.
  • Example 4 Enzyme-catalyzed IA-treated soluble starch to synthesize glucosamine after optimizing enzyme concentration
  • Starch phosphorylase phosphorylates the soluble amylase-treated soluble starch.
  • the remaining substrates are maltotriose and maltose.
  • 4- ⁇ -transglucosidase (4GT, EC 2.4.1.25) can extend the short-chain malto-oligosaccharide sugar chain, which is further utilized by starch phosphorylase, and then converted to glucosamine to improve the product yield.
  • the 4- ⁇ -transglucosidase is derived from Thermococcus littoralis, and its UniProt number is O32462.
  • primer F2 TGTTTAACTTTAAGAAGGAGATATA ATGGAAAGAATAAACTTCATATTTG
  • R2 CAGTGGTGGTGGTGGTGGTGC TCGAGTCAAAGCTCCCTGAACCTTACCGTG
  • pET20b-St4GT Protein expression and purification were performed by E. coli BL21 (DE3).

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Abstract

提供了一种氨基葡萄糖的制备方法，尤其涉及通过体外酶催化制备氨基葡萄糖的方法，属于氨基葡萄糖的酶催化制备领域。所述方法包括：采用6-磷酸氨基葡萄糖脱氨酶(glucosamine-6-phosphate deaminase，EC 3.5.99.6，GlmD)催化，将6-磷酸果糖(F6P)和铵盐转化为6-磷酸氨基葡萄糖(GlcN6P)；和采用能够脱磷酸基团的酶催化GlcN6P脱磷酸基团生成氨基葡萄糖(GlcN)。

Description

氨基葡萄糖的酶法制备
本申请要求2018年7月13日向中国国家知识产权局提交的，专利申请号为201810772487.3，发明名称为“氨基葡萄糖的酶法制备”在先申请的优先权。该申请的全文通过引用的方式结合于本申请中。
技术领域
本发明涉及氨基葡萄糖的制备方法，尤其涉及通过体外酶催化制备氨基葡萄糖的方法，属于氨基葡萄糖的酶催化制备领域。
背景技术
氨基葡萄糖是D-葡萄糖分子中2位羟基被氨基取代后的化合物，是一种重要的功能性单糖。氨基葡萄糖几乎存在于所有有机体中，包括细菌、酵母、丝状真菌、植物以及动物体，是糖蛋白和蛋白聚糖的主要组成成分，同时也是壳聚糖和甲壳素的主要组成成分。
氨基葡萄糖及其衍生物应用十分广泛，在医药、食品、化妆品等领域均具有重要用途。在医药工业方面，氨基葡萄糖硫酸盐能通过刺激软骨蛋白聚糖的生物合成从而能作为治疗抗风湿性关节炎的原料药；在食品工业方面，氨基葡萄糖具有吸收体内产生的自由基、抗衰老、促进减肥、抑菌以及调节人体内分泌等多种生理功能，被用于食品添加剂和保健食品的生产中；在化妆品工业方面，乙酰氨基葡萄糖(GlcNAc)是透明质酸的单体，是目前高档的化妆品中不可缺少的一种物质。
目前，主要有两种生产氨基葡萄糖的方法。(1)甲壳素水解法，包括甲壳素酸水解法和甲壳素酶水解法。其中，甲壳素酸水解法是最常用的氨基葡萄糖生产方法，该方法首先用高浓度盐酸水解几丁质得到乙酰氨基葡萄糖，然后将乙酰氨基葡萄糖脱乙酰基得到氨基葡萄糖。该生产方法会受到原料供应的影响，因酸处理而产生的废水会严重污染环境，另外，一些对虾蟹原料过敏的人群在食用由该方法制备得到的氨基葡萄糖后会出现过敏反应。甲壳素酶水解法以甲壳素为原料，在甲壳素酶的作用下水解反应生成单体氨基葡萄糖。该方法虽然对环境污染较小，但是同样受到原料供应、生产强度低、导致过敏反应等因素的限制。(2)微生物发酵法，主要利用工程菌如大肠杆菌、枯草芽孢杆菌等代谢葡萄糖等原料生产氨基葡萄糖。该方法具有不受原料来源限制、发酵时间短、环境污染低、对人体安全等优点，但是 存在微生物工程菌代谢改造困难、易产生代谢副产物等缺点。
因此，亟待开发一种低成本、低污染的生产氨基葡萄糖的新方法。
发明内容
本发明涉及一种体外酶法制备氨基葡萄糖的方法，该方法通过体外酶反应体系催化生产氨基葡萄糖，该方法具有原料廉价、来源丰富、生产成本低、环境友好、对人体安全等优点。
本发明通过如下技术方案来实现：
本发明提供了一种利用体外酶催化反应制备氨基葡萄糖的方法，包括：采用6-磷酸氨基葡萄糖脱氨酶(glucosamine-6-phosphate deaminase，EC 3.5.99.6，GlmD)催化，将6-磷酸果糖(F6P)和铵盐转化为6-磷酸氨基葡萄糖(GlcN6P)；和采用能够脱磷酸基团的酶催化GlcN6P脱磷酸基团生成氨基葡萄糖(GlcN)。
根据本发明，各种铵盐均可用于本发明。优选地，所述铵盐选自硫酸铵、氯化铵、硫酸氢铵、硝酸铵、碳酸铵、碳酸氢铵中的一种、两种或者更多种的任意混合物。
根据本发明，优选地，所述方法还包括将6-磷酸葡萄糖(G6P)转化为F6P的反应步骤，该步骤采用磷酸葡糖异构酶(phosphoglucose isomerase，EC 5.3.1.9，PGI)催化。
根据本发明，优选地，所述方法还包括将1-磷酸葡萄糖(G1P)转化为G6P的反应步骤，该步骤采用葡萄糖磷酸变位酶(phosphoglucomutase，EC 5.4.2.2，PGM)催化。
根据本发明，优选地，所述方法还包括将底物和磷酸盐转化为G1P的反应步骤，所述底物为包含D-葡萄糖单元的二糖、多糖或其任意混合物，该步骤采用能将底物和磷酸盐转化为G1P的酶催化。
根据本发明，各种磷酸盐均可用于本发明。优选地，所述磷酸盐选自磷酸二氢钾、磷酸氢二钾、磷酸二氢钠、磷酸氢二钠中的一种、两种或者更多种的任意混合物。更优选地，磷酸盐来自于磷酸二氢钾和/或磷酸氢二钾。
根据本发明，优选地，所述包含D-葡萄糖单元的二糖为蔗糖，采用蔗糖磷酸化酶(sucrose phosphorylase,EC 2.4.1.7，SP)催化将其和磷酸盐转化为G1P。
根据本发明，优选地，所述包含D-葡萄糖单元的多糖选自淀粉、淀粉衍生物或其任意混合物，采用淀粉磷酸化酶(α-glucan phosphorylase，EC 2.4.1.1，αGP)催化将其和磷酸盐转化为G1P。优选地，所述淀粉或淀粉衍生物选自可溶性淀粉、可溶性直链淀粉、可溶性支链淀粉、淀粉糊精、麦芽糊精、麦芽多糖。优选地，所述淀粉为可溶性淀粉。
根据本发明，优选地，所述包含D-葡萄糖单元的多糖还选自纤维素、纤维素衍生物或其任意混合物。优选地，所述纤维素衍生物选自纤维多糖、纤维二糖或其任意混合物。优选地，所述纤维素衍生物为纤维素经过酸或酶预处理后的产物，该产物为纤维多糖、纤维二糖或其任意混合物。优选地，所述纤维素衍生物为纤维多糖。当所述包含D-葡萄糖单元的多糖含有纤维素和/或纤维多糖时，例如为纤维素和/或纤维多糖时，采用纤维多糖磷酸化酶(cellodextrin phosphorylase，EC 2.4.1.49，CDP)催化将其和磷酸盐转化为G1P；优选地，进一步采用纤维二糖磷酸化酶(cellobiose phosphorylase，EC 2.4.1.20，CBP)催化将纤维素和/或纤维多糖降解产生的纤维二糖和磷酸盐转化为G1P。当所述包含D-葡萄糖单元的多糖含有纤维二糖时，例如为纤维二糖时，采用纤维二糖磷酸化酶(cellobiose phosphorylase，EC 2.4.1.20，CBP)催化将其和磷酸盐转化为G1P。
本领域技术人员可以理解，本发明的方法所包括的上述步骤可以分步进行，例如可以在一个生物反应器或反应容器中进行或在串联布置的多个生物反应器或反应容器中进行。
本领域技术人员可以理解，本发明的方法所包括的上述步骤也可以同时进行，例如可以在一个生物反应器或反应容器中进行。优选地，本发明的方法所包括的上述步骤同时进行。
优选地，本发明提供一种利用体外酶催化反应制备氨基葡萄糖的方法，包括：以包含D-葡萄糖单元的二糖、多糖或其任意混合物为底物，加入磷酸盐、铵盐、和能将包含D-葡萄糖单元的二糖、多糖或其任意混合物和磷酸盐转化为G1P的酶、葡萄糖磷酸变位酶、磷酸葡糖异构酶、6-磷酸氨基葡萄糖脱氨酶和能够脱磷酸基团的酶，进行催化反应得到氨基葡萄糖。
本领域技术人员可以理解，各种来源的6-磷酸氨基葡萄糖脱氨酶均可用于本发明。优选地，6-磷酸氨基葡萄糖脱氨酶可以来源于大肠杆菌(UniProt编号P0A759)、枯草芽孢杆菌(UniProt编号O35000)、蓝氏贾第鞭毛虫(UniProt编号V6TL01)或Thermococcus kodakarensis(UniProt编号Q5JDU3)等。
本领域技术人员可以理解，各种来源的能够脱磷酸基团的酶都可用于本发明。根据本发明的一个方面，能够脱磷酸基团的酶为具有底物特异性的6-磷酸氨基葡萄糖磷酸酶(6-phosphate glucosamine phosphatase，GlmP)，优选地，6-磷酸氨基葡萄糖磷酸酶可以来源于大肠杆菌(UniProt编号P77475、P27848、P0AE22等)、多形拟杆菌(Bacteroides thetaiotaomicron，UniProt编号Q8A759)等。
来源于Thermococcus kodakarensis的嗜热酶UniProt编号Q5JJ45被注释为糖磷酸酶(predicted sugar phosphatase，HAD superfamily)，其核苷酸序列如SEQ ID NOs：1所示， 氨基酸序列如SEQ ID NOs：2所示。在本发明中，Q5JJ45的底物特异性被鉴定，结果表明，Q5JJ45是能够催化GlcN6P进行脱磷酸基团反应的耐热酶，具有催化GlcN6P生产氨基葡萄糖的作用。根据本发明的另一个方面，所述能够脱磷酸基团的酶由核苷酸编码，所述核苷酸包含与SEQ ID NOs：1具有至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％序列同一性的核苷酸序列；优选地，所述核苷酸为与SEQ ID NOs：1具有至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％序列同一性的核苷酸序列。优选地，所述能够脱磷酸基团的酶包含与SEQ ID NOs：2具有至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％序列同一性的氨基酸序列；优选地，所述能够脱磷酸基团的酶具有与SEQ ID NOs：2具有至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％序列同一性的氨基酸序列。优选地，所述能够脱磷酸基团的酶为来源于Thermococcus kodakarensis的糖磷酸酶(UniProt编号Q5JJ45)。
本领域技术人员可以理解，各种来源的能将底物转化为G1P的酶、葡萄糖磷酸变位酶、磷酸葡糖异构酶均可用于本发明。优选地，淀粉磷酸化酶可以来源于大肠杆菌(Uniprot编号A0A0A0HB49)、海栖热袍菌(Thermotoga maritima，Uniprot编号G4FEH8)、热纤维梭菌(Clostridium thermocellum，Uniprot编号A3DCB6)等；蔗糖磷酸化酶可以来源于青春双歧杆菌(Bifidobacterium adolescentis，UniProt编号A0ZZH6)、Thermoanaerobacterium thermosaccharolyticum(UniProt编号D9TT09)等；纤维多糖磷酸化酶可以来源于热纤梭菌(Clostridium thermocellum，UniProt编号A3DJQ6)、Clostridium stercorarium(UniProt编号P77846)等；纤维二糖磷酸化酶可以来源于热纤梭菌(Clostridium thermocellum，UniProt编号A3DC35)、Thermotoga neapolitana(UniProt编号B9K7M6)等。葡萄糖磷酸变位酶可以来源于热纤梭菌(Clostridium thermocellum，Uniprot编号A3DEW8)、Thermococcus kodakarensis(UniProt编号Q68BJ6)等；磷酸葡糖异构酶可以来源于热纤梭菌(Clostridium thermocellum，Uniprot编号A3DBX9)、嗜热栖热菌(Thermus thermophilus，Uniprot编号Q5SLL6)等。
根据本发明，优选地，催化反应的温度为30-70℃，更优选为30-50℃，最优选为37℃。
根据本发明，优选地，催化反应的pH为5.0-8.0，更优选为6.0-7.5，最优选为7.0。
根据本发明，上述步骤同时进行时，优选地，催化反应的时间为1-48小时，进一步优选为8-36小时，更优选为10-24小时，最优选为20小时。
根据本发明，上述步骤分步进行时，优选地，各步骤催化反应的时间彼此独立地为0.5- 10小时，进一步优选为1-3小时，最优选为2小时。
根据本发明，优选地，反应体系中底物的浓度为1-200g/L，进一步优选为5-50g/L，更优选为8-20g/L，最优选为10g/L。
根据本发明，优选地，反应体系中将底物转化为G1P的酶的浓度为0.1-10U/mL，进一步优选为0.2-5U/mL，更优选为1-3U/mL，最优选为2U/mL；优选地，葡萄糖磷酸变位酶的浓度为0.1-10U/mL，进一步优选为0.2-5U/mL，更优选为1-3U/mL，最优选为2U/mL；优选地，磷酸葡糖异构酶的浓度为0.1-10U/mL，更优选为1-5U/mL，最优选为3U/mL；优选地，6-磷酸氨基葡萄糖脱氨酶的浓度为0.1-10U/mL，进一步优选为0.2-5U/mL，更优选为1-3U/mL，最优选为2U/mL；优选地，能够脱磷酸基团的酶的浓度为0.1-10U/mL，进一步优选为0.2-5U/mL，更优选为1-3U/mL，最优选为2U/mL。
根据本发明，优选地，反应体系中铵盐的浓度为50-500mM，更优选为100-300mM，最优选为200mM。
根据本发明，优选地，反应体系中磷酸盐的浓度为1-150mM，进一步优选为2-50mM，更优选为10-30mM，最优选为20mM。本领域技术人员可以理解，GlcN6P脱磷酸基团生成氨基葡萄糖(GlcN)的过程中产生的磷酸盐可以作为将包含D-葡萄糖单元的二糖、多糖或其任意混合物转化为G1P的步骤中的磷源。
根据本发明，优选地，上述反应体系中还含有镁盐。本领域技术人员可以理解，各种镁盐均可用于本发明，例如氯化镁、硫酸镁等，优选地，镁盐为氯化镁。优选地，反应体系中镁盐的浓度为1-20mM，进一步优选为2-15mM，最优选为10mM。
根据本发明，优选地，上述反应体系中还含有缓冲液。本领域技术人员可以理解，各种缓冲液均可用于本发明，例如HEPES缓冲液、Tris-HCl缓冲液、MOPS缓冲液、柠檬酸盐缓冲液例如柠檬酸钠缓冲液等。优选地，缓冲液为HEPES缓冲液。优选地，反应体系中缓冲液的浓度为20～300mM，优选为50～200mM，最优选为100mM。
根据本发明，所述催化反应在无ATP、无NAD(H)的条件下进行。
在优选的实施方案中，当淀粉、淀粉衍生物或其任意混合物中含有α-1,6糖苷键(例如，可溶性淀粉、可溶性支链淀粉、淀粉糊精、麦芽糊精、麦芽多糖)时，本发明的方法还包括采用异淀粉酶(isoamylase,EC 3.2.1.68，IA)水解底物中α-1,6-糖苷键的反应步骤。
本领域技术人员可以理解，各种来源的异淀粉酶均可用于本发明。优选地，异淀粉酶可以来源于硫化叶菌(Sulfolobus tokodaii，UniProt编号Q973H3)、黄杆菌(Flavobacterium sp.，UniProt编号O32611)等。
根据本发明，优选地，反应体系中异淀粉酶的浓度为0.1-10U/mL，更优选为0.5-2U/mL，最优选为1U/mL。
本领域技术人员可以理解，采用异淀粉酶水解底物中α-1,6-糖苷键的反应步骤与上述反应步骤可以分步进行，例如可以在一个生物反应器或反应容器中进行或在串联布置的多个生物反应器或反应容器中进行，也可以与上述反应步骤同时进行，例如可以在一个生物反应器或反应容器中进行。
优选地，采用异淀粉酶水解底物中α-1,6-糖苷键的反应步骤在将底物和磷酸盐转化为G1P的反应步骤之前进行，即先采用异淀粉酶水解底物中α-1,6-糖苷键，在得到经异淀粉酶处理过的底物后，再进行其他反应步骤，此时其他反应步骤可以分步进行也可以同时进行。此时，优选地，反应体系中底物的浓度为1-300g/L，进一步优选为10-200g/L，更优选为50-150g/L，最优选为100g/L；优选地，异淀粉酶的浓度为0.1-10U/mL，更优选为0.5-2U/mL，最优选为1U/mL；优选地，催化反应的pH为4-8，更优选为4.5-6.5，最优选为5.5；优选地，在10-99℃反应0.5-72小时，进一步优选在30-95℃反应1-48小时，更优选在50-90℃反应6-24小时，最优选在85℃反应12小时。优选地，反应体系中还含有镁盐和缓冲液。本领域技术人员可以理解，各种镁盐均可用于本发明，例如氯化镁、硫酸镁等，优选地，镁盐为氯化镁。优选地，反应体系中镁盐的浓度为0.01-10mM，进一步优选为0.1-5mM，更优选为0.2-1mM，最优选为0.5mM。本领域技术人员可以理解，各种缓冲液均可用于本发明，例如乙酸钠缓冲液、HEPES缓冲液、柠檬酸盐缓冲液例如柠檬酸钠缓冲液等，优选地，缓冲液为乙酸钠缓冲液。优选地，反应体系中缓冲液的浓度为1-50mM，进一步优选为2-20mM，更优选为3-10mM，最优选为5mM。
在优选的实施方案中，当底物为淀粉、淀粉衍生物或其任意混合物(例如可溶性淀粉、可溶性直链淀粉、可溶性支链淀粉、淀粉糊精、麦芽糊精、麦芽多糖)时，本发明的方法还包括采用4-α-转葡糖苷酶(4-α-glucanotransferase，EC 2.4.1.25，4GT)进行催化的反应步骤。
本领域技术人员可以理解，各种来源的4-α-转葡糖苷酶均可用于本发明。优选地，4-α-转葡糖苷酶可以来源于嗜热高温球菌(Thermococcus litoralis，UniProt编号O32462)、枯草芽孢杆菌(Bacillus subtilis，UniProt编号L8AG91)、丁酸梭菌(Clostridium butyricum，UniProt编号Q59266)等。
根据本发明，优选地，反应体系中4-α-转葡糖苷酶的浓度为0.1-10U/mL，进一步优选为0.2-5U/mL，更优选为0.5-2U/mL，最优选为1U/mL。
本领域技术人员可以理解，采用4-α-转葡糖苷酶进行催化的反应步骤与上述步骤可以分步进行，例如可以在一个生物反应器或反应容器中进行或在串联布置的多个生物反应器或反应容器中进行，也可以与上述步骤同时进行，例如可以在一个生物反应器或反应容器中进行。
优选地，采用4-α-转葡糖苷酶催化反应的步骤在将底物和磷酸盐转化为G1P的反应进行一段时间后进行。此时，优选地，在将底物和磷酸盐转化为G1P的反应进行0.5-30小时，优选5-20小时，最优选10小时后在反应体系中添加4-α-转葡糖苷酶。
淀粉是一种由不同链长的直链淀粉和支链淀粉组成的混合物。直链淀粉葡萄糖单元之间以α-1,4糖苷键相连，支链淀粉通过α-1,6糖苷键与淀粉主链相连，淀粉磷酸化酶并不能水解α-1,6糖苷键，在反应体系中加入能够水解淀粉中α-1,6糖苷键的脱支酶—异淀粉酶，能够提高G1P的得率。另外，淀粉磷酸化酶水解淀粉、淀粉衍生物或其任意混合物，释放G1P后，最终产物是麦芽糖和麦芽三糖。为了使淀粉中的葡萄糖单元尽可能多的转化为G1P，可以向反应体系中添加4-α-转葡糖苷酶，它可以将短链的寡聚糖聚合成为长链的寡聚糖，而该长链的寡聚糖又可被淀粉磷酸化酶重新利用，从而提高淀粉的利用率。
作为一个优选实施方案，本发明的酶催化反应采用如下方法：
在一个反应体系中，以经异淀粉酶处理的可溶性淀粉为底物，加入氯化镁、磷酸盐、铵盐、HEPES缓冲液(pH 7.0)、淀粉磷酸化酶、葡萄糖磷酸变位酶、磷酸葡糖异构酶、6-磷酸氨基葡萄糖脱氨酶、能够脱磷酸基团的酶(优选6-磷酸氨基葡萄糖磷酸酶)，进行催化反应，得到氨基葡萄糖。优选地，在反应进行一段时间后向反应体系中加入4-α-转葡糖苷酶。
在进一步优选的实施方案中，在一个反应体系中，以10g/L经异淀粉酶处理的可溶性淀粉为底物，加入10mM氯化镁、20mM磷酸二氢钾、200mM氯化铵、100mM HEPES缓冲液(pH 7.0)、2U/mL淀粉磷酸化酶、2U/mL葡萄糖磷酸变位酶、3U/mL磷酸葡糖异构酶、2U/mL 6-磷酸氨基葡萄糖脱氨酶、2U/mL能够脱磷酸基团的酶(优选6-磷酸氨基葡萄糖磷酸酶)，反应混合物在37℃下进行催化反应30h，得到氨基葡萄糖。优选地，在反应进行10h时向反应体系中还加入1U/mL 4-α-转葡糖苷酶。
本发明还提供6-磷酸氨基葡萄糖脱氨酶和能够脱磷酸基团的酶(优选6-磷酸氨基葡萄糖磷酸酶)在制备氨基葡萄糖中的应用，优选地在催化F6P生成氨基葡萄糖中的应用。
本发明还提供包含与SEQ ID NOs：1具有至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％序列同一性的核苷酸编码的酶在制备氨基葡萄糖中的应用，优选地在催化6-磷酸氨基葡萄糖(GlcN6P)生成氨基葡萄糖(GlcN)中的应用；优选 地，所述核苷酸为与SEQ ID NOs：1具有至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％序列同一性的核苷酸序列。优选地，所述酶包含与SEQ ID NOs：2具有至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％序列同一性的氨基酸序列；优选地，所述酶具有与SEQ ID NOs：2具有至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％序列同一性的氨基酸序列。优选地，所述酶为来源于Thermococcus kodakarensis的糖磷酸酶(UniProt编号Q5JJ45)。
本发明还提供上述方法制备得到的氨基葡萄糖。
本发明首次使用体外酶催化反应制备氨基葡萄糖，特别是首次使用6-磷酸氨基葡萄糖脱氨酶和能够脱磷酸基团的酶催化F6P生成氨基葡萄糖。本发明的方法不仅可以以较好的转化率获得目标产物，而且获得的产物具有对人体安全等优点。此外，本发明的方法可使用多种原料，例如可以使用含D-葡萄糖单元的二糖、多糖或其任意混合物。因此，本发明的方法具有原料廉价、来源丰富、生产成本低、环境友好、对人体安全等优点，适宜推广。此外，发明人还发现，在使用淀粉、淀粉衍生物或其任意混合物为底物催化生成氨基葡萄糖时，加入异淀粉酶和4-α-转葡糖苷酶可以极大地提高目标产物的收率。
附图说明
图1为以淀粉为底物制备氨基葡萄糖的体外酶催化途径的示意图。其中αGP为淀粉磷酸化酶，PGM为葡萄糖磷酸变位酶、PGI为磷酸葡糖异构酶、GlmD为6-磷酸氨基葡萄糖脱氨酶、GlmP为6-磷酸氨基葡萄糖磷酸酶。
图2为以淀粉为底物制备氨基葡萄糖的体外酶催化途径的中间体之间的反应吉布斯能量变化。
图3为SDS-PAGE检测以淀粉为底物制备氨基葡萄糖的关键酶。M：Marker。
图4为利用HPLC分析氨基葡萄糖。其中4A为氨基葡萄糖标准品的HPLC峰图；其中4B为利用HPLC定量分析氨基葡萄糖的浓度，通过氨基葡萄糖峰的强度可以对所得到的氨基葡萄糖的浓度进行定量。
图5为利用HPLC分析6-磷酸氨基葡萄糖脱氨酶参与的体外酶体系催化可溶性淀粉合成氨基葡萄糖。其中5A为体外酶催化可溶性淀粉生成氨基葡萄糖的高效液相色谱分析结果；其中5B为体外酶催化可溶性淀粉合成氨基葡萄糖的反应进程曲线。
图6为体外酶催化IA处理过的可溶性淀粉合成氨基葡萄糖的反应进程曲线。
图7为优化酶浓度后体外酶催化IA处理过的可溶性淀粉合成氨基葡萄糖的反应进程曲 线。
图8体外酶催化将蔗糖转化为氨基葡萄糖的反应进程曲线。
图9体外酶催化将纤维多糖转化为氨基葡萄糖的反应进程曲线。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
本发明实施例中使用的部分材料信息如下：
可溶性淀粉，ACROS公司产品，产品编号：424490020；
pET20b载体，Novagen，Madison，WI；
大肠杆菌表达菌BL21(DE3)，Invitrogen，Carlsbad，CA；
实施例1氨基葡萄糖酶法合成途径中的酶活测定
通过体外酶催化体系将淀粉转化为氨基葡萄糖的催化途径见图1，其中能够脱磷酸基团的酶以6-磷酸氨基葡萄糖磷酸酶为例。图2显示了将淀粉转化为氨基葡萄糖的酶催化途径的中间体之间的反应吉布斯能量变化。在本实施例中，(1)淀粉磷酸化酶来源于大肠杆菌(Uniprot编号A0A0A0HB49)；(2)葡萄糖磷酸变位酶来源于热纤梭菌(Clostridium thermocellum，Uniprot编号A3DEW8)；(3)磷酸葡糖异构酶来源于热纤梭菌(Clostridium thermocellum，Uniprot编号A3DBX9)；(4)6-磷酸氨基葡萄糖脱氨酶来源于枯草芽孢杆菌(Uniprot编号O35000)；(5)能够脱磷酸基团的酶来源于多形拟杆菌Bacteroides thetaiotaomicron的6-磷酸氨基葡萄糖磷酸酶(Uniprot编号Q8A759)。上述基因组DNA都可从ATCC的官方网站(www.atcc.org)上获得。通过Simple Cloning(You C,Zhang XZ,Zhang Y-HP.2012.Simple cloning via direct transformation of PCR product(DNA Multimer)to Escherichia coli and Bacillus subtilis.Appl.Environ.Microbiol.78(5):1593-5.)的方法，将上述基因克隆至pET20b载体(Novagen,Madison,WI)中，分别获得相应的表达载体pET20b-EcαGP、pET20b-CtPGM、pET20b-CtPGI、pET20b-BsGlmD、pET20b-BtGlmP。重组蛋白在大肠杆菌BL21(DE3)中表达，并进行了纯化，蛋白质纯化的结果如图3所示。
来源于热纤梭菌(Clostridium thermocellum)的葡萄糖磷酸变位酶的酶活测定在含有10 mM氯化镁的100mM HEPES缓冲液(pH 7.0)中进行。以10mM 1-磷酸葡萄糖为底物，在37℃下反应10min，测定产生的6-磷酸葡萄糖(G6P)的量。G6P量的检测方法如下：取40μl含有G6P的样品溶液，添加200μl含有2mM氯化镁、0.15mM NAD +、0.5U/mL葡萄糖6-磷酸脱氢酶(glucose 6-phosphate dehydrogenase，G6PDH)的100mM HEPES缓冲液(pH 7.0)，37℃反应30分钟，测定340nm处的吸光度，计算产生的NADH的量。实验结果显示，热纤梭菌来源的葡萄糖磷酸变位酶在37℃下的比酶活为20U/mg。
来源于大肠杆菌(E.coli)的淀粉磷酸化酶的酶活测定在含有10mM氯化镁、1U/mL葡萄糖磷酸变位酶的100mM HEPES缓冲液(pH 7.0)中进行。以5g/L可溶性淀粉为底物，在37℃下反应10min，测定产生的6-磷酸葡萄糖的量。实验结果显示，大肠杆菌来源的淀粉磷酸化酶在37℃下的比酶活为5.6U/mg。
来源于热纤梭菌(Clostridium thermocellum)的磷酸葡糖异构酶的酶活测定在含有10mM氯化镁的100mM HEPES缓冲液(pH 7.0)中进行。以10mM 6-磷酸果糖为底物，在37℃下反应10min，测定产生的6-磷酸葡萄糖的量。实验结果显示，热纤梭菌来源的磷酸葡糖异构酶在37℃下的比酶活为396U/mg。
来源于枯草芽孢杆菌(B.subtilis)的6-磷酸氨基葡萄糖脱氨酶的酶活测定在含有10mM氯化镁的100mM HEPES缓冲液(pH 7.0)中进行。以10mM 6-磷酸果糖和100mM氯化铵为底物，在37℃下反应10min，测定产生的6-磷酸氨基葡萄糖(GlcN6P)的量。GlcN6P量的检测方法如下：取含有GlcN6P的样品50μl加入100μl乙酰丙酮试剂(取1.5mL乙酰丙酮，溶于50mL 1.25mol/L碳酸钠溶液中，配制而成)，煮沸20分钟，冷却至室温，缓慢加入96％(v/v)乙醇1mL，然后加入对二甲氨基苯甲醛(DMAB)试剂(称取1.6g DMAB溶于30mL浓盐酸和30mL 96％的乙醇中，配制而成)100μl，混合均匀，室温放置30分钟，测定530nm处的吸光度，根据标准曲线计算GlcN6P含量。实验结果显示，枯草芽孢杆菌来源的6-磷酸氨基葡萄糖脱氨酶在37℃下的比酶活为10U/mg。
在该反应路径中，对GlcN6P具有特异性脱磷活性的能够脱磷酸基团的酶是本发明的关键点之一。在含有10mM氯化镁的100mM HEPES缓冲液(pH 7.0)中，我们测定了上述能够脱磷酸基团的酶对G1P、G6P、F6P和GlcN6P的脱磷活性。实验结果如表1所示。来自于多形拟杆菌(Bacteroides thetaiotaomicron)的6-磷酸氨基葡萄糖磷酸酶对GlcN6P具有较高的比酶活，并展现出特异的GlcN6P脱磷活性。Thermococcus kodakarensis来源的糖磷酸酶(predicted sugar phosphatase，HAD superfamily)在70℃下对GlcN6P底物的脱磷活性为0.011U/mg。
表1两个能够脱磷酸基团的酶对不同底物的脱磷活性
Figure PCTCN2019095638-appb-000001
a:37℃下测定的比酶活；b:70℃下测定的比酶活。
实施例2体外酶催化可溶性淀粉合成氨基葡萄糖
本实施例采用酶在体外催化可溶性淀粉合成氨基葡萄糖。首先重组表达了五种酶：来源于大肠杆菌的αGP、来源于热纤梭菌的PGM、来源于热纤梭菌的PGI、来源于枯草芽孢杆菌的GlmD、来源于多形拟杆菌的GlmP(表2)。
表2体外合成氨基葡萄糖所用到的酶的信息
Figure PCTCN2019095638-appb-000002
采用高效液相色谱法(HPLC)定量分析氨基葡萄糖。所用色谱柱为氨基柱，流动相为80％乙腈水溶液，流速1mL/min，柱温40℃，所用检测器为示差折光检测器。标准样品检测如图4A所示，氨基葡萄糖保留时间约为9.6分钟。氨基葡萄糖浓度与氨基葡萄糖的HPLC特征峰的响应强度成正比，标准曲线如图4B所示。
将含有10g/L可溶性淀粉、10mM氯化镁、20mM磷酸二氢钾、200mM氯化铵、100mM HEPES缓冲液(pH 7.0)、1U/mL αGP、1U/mL PGM、1U/mL PGI、1U/mL GlmD、1U/mL GlmP的0.5mL反应混合物在37℃下反应30h。反应结束后，向反应体系添加等体积的乙腈终止反应，12000转离心10min，取上清通过高效液相色谱法测定反应溶液中的氨基葡萄糖的浓度(图5A)。反应进行20h时，氨基葡萄糖的浓度为2.4g/L，转化率为24％(图5B)。
产物转化率的计算公式如下：
Figure PCTCN2019095638-appb-000003
实施例3体外酶催化IA处理过的可溶性淀粉合成氨基葡萄糖
淀粉为α-1,4和α-1,6混合连键的多糖，不能被淀粉磷酸化酶完全水解。异淀粉酶(IA，EC 3.2.1.68)能够水解淀粉中的α-1,6糖苷键，从而帮助淀粉磷酸化酶磷酸解底物，提高氨基葡萄糖的得率。
在本实施例中，异淀粉酶来源于硫化叶菌(Sulfolobus tokodaii，UniProt编号Q973H3)。将文献(Cheng,K.et al.Doubling Power Output of Starch Biobattery Treated by the Most Thermostable Isoamylase from an Archaeon Sulfolobus tokodaii.Sci.Rep.5:13184)报道的表达载体pET20b-StIA导入大肠杆菌BL21(DE3)，进行蛋白质表达与纯化。
在含有100g/L可溶性淀粉的5mM乙酸钠缓冲液(pH 5.5)中，添加0.5mM氯化镁和1U/mL异淀粉酶，85℃处理12个小时。
将含有10g/L经异淀粉酶处理的可溶性淀粉、10mM氯化镁、20mM磷酸二氢钾、200mM氯化铵、100mM HEPES缓冲液(pH 7.0)、1U/mL αGP、1U/mL PGM、1U/mL PGI、1U/mL GlmD、1U/mL GlmP的0.5mL反应混合物在37℃下孵育20h。不同时间取样，通过添加等体积的乙腈终止反应，12000转离心10min，取上清通过高效液相色谱法测定氨基葡萄糖的浓度。反应进行10h时，氨基葡萄糖的浓度为3.5g/L，转化率为35％(图6)
实施例4优化酶浓度后体外酶催化IA处理过的可溶性淀粉合成氨基葡萄糖
将含有10g/L经异淀粉酶处理的可溶性淀粉、10mM氯化镁、20mM磷酸二氢钾、200mM氯化铵、100mM HEPES缓冲液(pH 7.0)、2U/mL αGP、2U/mL PGM、3U/mL PGI、2U/mL GlmD、2U/mL GlmP的0.5mL反应混合物在37℃下孵育30h。不同时间取样，通过添加等体积的乙腈终止反应，12000转离心10min，取上清通过高效液相色谱法测定氨基葡萄糖的浓度。反应进行20h时，氨基葡萄糖的浓度为7.12g/L，转化率为71.2％(图7，实线所示)。
实施例5通过添加4-α-转葡糖苷酶，提高氨基葡萄糖的得率
淀粉磷酸化酶将异淀粉酶处理的可溶性淀粉磷酸解，最终剩余的底物为麦芽三糖和麦芽糖。4-α-转葡糖苷酶(4GT，EC 2.4.1.25)能够将短链麦芽寡糖糖链进行延长，进一步被淀粉磷酸化酶利用，进而转化为氨基葡萄糖提高产物得率。
在本实施例中，4-α-转葡糖苷酶来源于嗜热高温球菌Thermococcus litoralis，其UniProt编号为O32462。利用引物F2：TGTTTAACTTTAAGAAGGAGATATA ATGGAAAGAATAAACTTCATATTTG，R2：CAGTGGTGGTGGTGGTGGTGC TCGAGTCAAAGCTCCCTGAACCTTACCGTG，通过Simple Cloning的方法克隆至pET20b载体中，获得相应的表达载体pET20b-St4GT。通过大肠杆菌BL21(DE3)，进行蛋白质表达与纯化。
将含有10g/L经异淀粉酶处理的可溶性淀粉、10mM氯化镁、20mM磷酸二氢钾、200mM氯化铵、100mM HEPES缓冲液(pH 7.0)、2U/mL αGP、2U/mL PGM、3U/mL PGI、2U/mL GlmD、2U/mL GlmP的0.5mL反应混合物在37℃下孵育10h。然后加入终浓度1U/mL的4GT，37℃继续反应至30h。不同时间取样，通过添加等体积的乙腈终止反应，12000转离心10min，取上清通过高效液相色谱法测定反应溶液中的氨基葡萄糖的浓度。反应进行20h时，氨基葡萄糖的浓度为7.93g/L，转化率为79.3％(图7，虚线所示)。
实施例6体外酶催化将蔗糖转化为氨基葡萄糖
将含有10g/L蔗糖、10mM氯化镁、20mM磷酸二氢钾、200mM氯化铵、100mM HEPES缓冲液(pH 7.0)、2U/mL SP、2U/mL PGM、3U/mL PGI、2U/mL GlmD、2U/mL GlmP的0.5mL反应混合物在37℃下孵育20h。不同时间取样，通过添加等体积的乙腈终止反应，12000转离心10min，取上清通过高效液相色谱法测定反应溶液中的氨基葡萄糖的浓度。反应进行10h时，氨基葡萄糖的浓度为4.1g/L，转化率为41％(图8)。
实施例7体外酶催化将纤维多糖转化为氨基葡萄糖
将含有10g/L纤维多糖(平均聚合度为4.4)、10mM氯化镁、20mM磷酸二氢钾、200mM氯化铵、100mM HEPES缓冲液(pH 7.0)、1U/mL CDP、1U/mL CBP、2U/mL PGM、3U/mL PGI、3U/mL GlmD、2U/mL GlmP的0.5mL反应混合物在37℃下孵育20h。不同时间取样，通过添加等体积的乙腈终止反应，12000转离心10min，取上清通过高效液相色谱法测定反应溶液中的氨基葡萄糖的浓度。反应进行10h时，氨基葡萄糖的浓度为3.44g/L，转化率为34.4％(图9)。
实施例8体外酶催化将6-磷酸果糖转化为氨基葡萄糖
将含有50mM 6-磷酸果糖、10mM氯化镁、200mM氯化铵、100mM HEPES缓冲液(pH 7.0)、1U/mL GlmD、1U/mL GlmP的0.5mL反应混合物在37℃下孵育10h。不同时间取样，通过添加等体积的乙腈终止反应，12000转离心10min，取上清通过高效液相色谱法测定反应溶液中的氨基葡萄糖的浓度。反应进行4h时，氨基葡萄糖的浓度为43.9mM，转化率为87.8％。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种利用体外酶催化反应制备氨基葡萄糖的方法，其特征在于，包括：采用6-磷酸氨基葡萄糖脱氨酶(glucosamine-6-phosphate deaminase，EC 3.5.99.6，GlmD)催化，将6-磷酸果糖(F6P)和铵盐转化为6-磷酸氨基葡萄糖(GlcN6P)；和采用能够脱磷酸基团的酶催化GlcN6P脱磷酸基团生成氨基葡萄糖(GlcN)。
    优选地，所述铵盐选自硫酸铵、氯化铵、硫酸氢铵、硝酸铵、碳酸铵、碳酸氢铵中的一种、两种或者更多种的任意混合物。
  2. 根据权利要求1所述的方法，其特征在于，所述方法还包括将6-磷酸葡萄糖(G6P)转化为F6P的反应步骤，该步骤采用磷酸葡糖异构酶(phosphoglucose isomerase，EC 5.3.1.9，PGI)催化。
    优选地，所述方法还包括将1-磷酸葡萄糖(G1P)转化为G6P的反应步骤，该步骤采用葡萄糖磷酸变位酶(phosphoglucomutase，EC 5.4.2.2，PGM)催化。
    优选地，所述方法还包括将底物和磷酸盐转化为G1P的反应步骤，所述底物为包含D-葡萄糖单元的二糖、多糖或其任意混合物，该步骤采用能将底物和磷酸盐转化为G1P的酶催化。
    优选地，所述磷酸盐选自磷酸二氢钾、磷酸氢二钾、磷酸二氢钠、磷酸氢二钠中的一种、两种或者更多种的任意混合物。
    优选地，所述包含D-葡萄糖单元的二糖为蔗糖，采用蔗糖磷酸化酶(sucrose phosphorylase,EC 2.4.1.7，SP)催化将其和磷酸盐转化为G1P；
    优选地，所述包含D-葡萄糖单元的多糖选自淀粉、淀粉衍生物或其任意混合物，采用淀粉磷酸化酶(α-glucan phosphorylase，EC 2.4.1.1，αGP)催化将其和磷酸盐转化为G1P；
    优选地，所述包含D-葡萄糖单元的多糖还选自纤维素、纤维素衍生物或其任意混合物；优选地，所述纤维素衍生物为纤维素经过酸或酶预处理后的产物；
    优选地，当所述包含D-葡萄糖单元的多糖含有纤维素和/或纤维多糖时，采用纤维多糖磷酸化酶(cellodextrin phosphorylase，EC 2.4.1.49，CDP)催化将其和磷酸盐转化为G1P；优选地，进一步采用纤维二糖磷酸化酶(cellobiose phosphorylase，EC 2.4.1.20，CBP)催化将纤维素和/或纤维多糖降解产生的纤维二糖和磷酸盐转化为G1P；
    优选地，当所述包含D-葡萄糖单元的多糖含有纤维二糖时，采用纤维二糖磷酸化酶(cellobiose phosphorylase，EC 2.4.1.20，CBP)催化将其和磷酸盐转化为G1P。
  3. 根据权利要求1或2所述的方法，其特征在于，所述6-磷酸氨基葡萄糖脱氨酶来源 于大肠杆菌(UniProt编号P0A759)、枯草芽孢杆菌(UniProt编号O35000)、蓝氏贾第鞭毛虫(UniProt编号V6TL01)或Thermococcus kodakarensis(UniProt编号Q5JDU3)；
    优选地，所述能够脱磷酸基团的酶为6-磷酸氨基葡萄糖磷酸酶(6-phosphate glucosamine phosphatase，GlmP)；优选地，所述6-磷酸氨基葡萄糖磷酸酶来源于大肠杆菌(UniProt编号P77475、P27848、P0AE22等)、多形拟杆菌(Bacteroides thetaiotaomicron，UniProt编号Q8A759)；
    优选地，所述能够脱磷酸基团的酶由核苷酸编码，所述核苷酸包含与SEQ ID NOs：1具有至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％序列同一性的核苷酸序列；优选地，所述能够脱磷酸基团的酶包含与SEQ ID NOs：2具有至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％序列同一性的氨基酸序列；优选地，所述能够脱磷酸基团的酶为来源于Thermococcus kodakarensis的糖磷酸酶(UniProt编号Q5JJ45)；
    优选地，所述淀粉磷酸化酶来源于大肠杆菌(Uniprot编号A0A0A0HB49)、海栖热袍菌(Thermotoga maritima，Uniprot编号G4FEH8)、热纤维梭菌(Clostridium thermocellum，Uniprot编号A3DCB6)；
    优选地；所述蔗糖磷酸化酶来源于青春双歧杆菌(Bifidobacterium adolescentis，UniProt编号A0ZZH6)、Thermoanaerobacterium thermosaccharolyticum(UniProt编号D9TT09)；
    优选地，所述纤维多糖磷酸化酶来源于热纤梭菌(Clostridium thermocellum，UniProt编号A3DJQ6)、Clostridium stercorarium(UniProt编号P77846)；
    优选地，所述纤维二糖磷酸化酶来源于热纤梭菌(Clostridium thermocellum，UniProt编号A3DC35)、Thermotoga neapolitana(UniProt编号B9K7M6)；
    优选地，所述葡萄糖磷酸变位酶来源于热纤梭菌(Clostridium thermocellum，Uniprot编号A3DEW8)、Thermococcus kodakarensis(UniProt编号Q68BJ6)；
    优选地，所述磷酸葡糖异构酶来源于热纤梭菌(Clostridium thermocellum，Uniprot编号A3DBX9)、嗜热栖热菌(Thermus thermophilus，Uniprot编号Q5SLL6)。
  4. 根据权利要求1-3中任一项所述的方法，其特征在于，催化反应的温度为30-70℃，更优选为30-50℃，最优选为37℃。
    优选地，催化反应的pH为5.0-8.0，更优选为6.0-7.5，最优选为7.0。
    优选地，各步骤同时进行，催化反应的时间为1-48h，进一步优选为8-36小时，更优选为10-24小时，最优选为20小时。
    优选地，各步骤分步进行，各步骤催化反应的时间彼此独立地为0.5-10小时，进一步优选为1-3小时，最优选为2小时。
    优选地，底物的浓度为1-200g/L，进一步优选为5-50g/L，更优选为8-20g/L，最优选为10g/L。
    优选地，将底物转化为G1P的酶的浓度为0.1-10U/mL，进一步优选为0.2-5U/mL，更优选为1-3U/mL，最优选为2U/mL；
    优选地，葡萄糖磷酸变位酶的浓度为0.1-10U/mL，进一步优选为0.2-5U/mL，更优选为1-3U/mL，最优选为2U/mL；
    优选地，磷酸葡糖异构酶的浓度为0.1-10U/mL，更优选为1-5U/mL，最优选为3U/mL；
    优选地，6-磷酸氨基葡萄糖脱氨酶的浓度为0.1-10U/mL，进一步优选为0.2-5U/mL，更优选为1-3U/mL，最优选为2U/mL；
    优选地，能够脱磷酸基团的酶的浓度为0.1-10U/mL，进一步优选为0.2-5U/mL，更优选为1-3U/mL，最优选为2U/mL。
    优选地，铵盐的浓度为50-500mM，更优选为100-300mM，最优选为200mM。
    优选地，磷酸盐的浓度为1-150mM，进一步优选为2-50mM，更优选为10-30mM，最优选为20mM。
    优选地，反应体系中还含有镁盐；
    优选地，所述镁盐为氯化镁和/或硫酸镁；
    优选地，反应体系中镁盐的浓度为1-20mM，进一步优选为2-15mM，最优选为10mM。
    优选地，反应体系中还含有缓冲液；
    优选地，所述缓冲液选自HEPES缓冲液、Tris-HCl缓冲液、MOPS缓冲液、柠檬酸盐缓冲液；
    优选地，缓冲液为HEPES缓冲液。优选地，缓冲液的浓度为20～300mM，优选为50～200mM，最优选为100mM。
  5. 根据权利要求1-4中任一项所述的方法，其特征在于，当所述淀粉、淀粉衍生物或其任意混合物中含有α-1,6糖苷键时，所述方法还包括采用异淀粉酶(isoamylase,EC 3.2.1.68，IA)水解底物中α-1,6-糖苷键的反应步骤；
    优选地，所述异淀粉酶来源于硫化叶菌(Sulfolobus tokodaii，UniProt编号Q973H3)、 黄杆菌(Flavobacterium sp.，UniProt编号O32611)。
    优选地，反应体系中异淀粉酶的浓度为0.1-10U/mL，更优选为0.5-2U/mL，最优选为1U/mL。
    优选地，采用异淀粉酶水解底物中α-1,6-糖苷键的反应步骤在将底物和磷酸盐转化为G1P的反应步骤之前进行；
    优选地，反应体系中底物的浓度为1-300g/L，进一步优选为10-200g/L，更优选为50-150g/L，最优选为100g/L；
    优选地，异淀粉酶的浓度为0.1-10U/mL，更优选为0.5-2U/mL，最优选为1U/mL；
    优选地，催化反应的pH为4-8，更优选为4.5-6.5，最优选为5.5；
    优选地，在10-99℃反应0.5-72小时，进一步优选在30-95℃反应1-48小时，更优选在50-90℃反应6-24小时，最优选在85℃反应12小时；
    优选地，反应体系中还含有镁盐；优选地，所述镁盐为氯化镁和/或硫酸镁；优选地，反应体系中镁盐的浓度为0.01-10mM，进一步优选为0.1-5mM，更优选为0.2-1mM，最优选为0.5mM。
    优选地，反应体系中还含有缓冲液；优选地，所述缓冲液选自乙酸钠缓冲液、HEPES缓冲液、柠檬酸盐缓冲液；优选地，缓冲液的浓度为1-50mM，进一步优选为2-20mM，更优选为3-10mM，最优选为5mM。
  6. 根据权利要求1-5中任一项所述的方法，其特征在于，当底物为淀粉、淀粉衍生物或其任意混合物时，所述方法还包括采用4-α-转葡糖苷酶(4-α-glucanotransferase，EC 2.4.1.25，4GT)进行催化的反应步骤。
    优选地，4-α-转葡糖苷酶来源于嗜热高温球菌(Thermococcus litoralis，UniProt编号O32462)、枯草芽孢杆菌(Bacillus subtilis，UniProt编号L8AG91)、丁酸梭菌(Clostridium butyricum，UniProt编号Q59266)；
    优选地，反应体系中4-α-转葡糖苷酶的浓度为0.1-10U/mL，进一步优选为0.2-5U/mL，更优选为0.5-2U/mL，最优选为1U/mL；
    优选地，采用4-α-转葡糖苷酶催化反应的步骤在将底物和磷酸盐转化为G1P的反应进行一段时间后进行；优选地，在将底物和磷酸盐转化为G1P的反应进行0.5-30小时，优选5-20小时，最优选10小时后在反应体系中添加4-α-转葡糖苷酶。
  7. 根据权利要求1-6中任一项所述的方法，其特征在于，以10g/L经异淀粉酶处理的可溶性淀粉为底物，加入10mM氯化镁、20mM磷酸二氢钾、200mM氯化铵、100mM  HEPES缓冲液(pH 7.0)、2U/mL淀粉磷酸化酶、2U/mL葡萄糖磷酸变位酶、3U/mL磷酸葡糖异构酶、2U/mL 6-磷酸氨基葡萄糖脱氨酶、2U/mL能够脱磷酸基团的酶，反应混合物在37℃下进行催化反应30h，得到氨基葡萄糖。
    优选地，能够脱磷酸基团的酶为6-磷酸氨基葡萄糖磷酸酶。
    优选地，在反应进行10h时向反应体系中还加入1U/mL 4-α-转葡糖苷酶。
  8. 6-磷酸氨基葡萄糖脱氨酶和能够脱磷酸基团的酶在制备氨基葡萄糖中的应用，优选地在催化F6P生成氨基葡萄糖中的应用；
    优选地，所述能够脱磷酸基团的酶选自6-磷酸氨基葡萄糖磷酸酶。
  9. 包含与SEQ ID NOs：1具有至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％序列同一性的核苷酸编码的酶在制备氨基葡萄糖中的应用，优选地在催化6-磷酸氨基葡萄糖(GlcN6P)生成氨基葡萄糖(GlcN)中的应用；
    优选地，所述核苷酸为与SEQ ID NOs：1具有至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％序列同一性的核苷酸序列。
    优选地，所述酶包含与SEQ ID NOs：2具有至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％序列同一性的氨基酸序列；
    优选地，所述酶具有与SEQ ID NOs：2具有至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％序列同一性的氨基酸序列。
    优选地，所述酶为来源于Thermococcus kodakarensis的糖磷酸酶(UniProt编号Q5JJ45)。
  10. 权利要求1-7中任一项所述的方法制备得到的氨基葡萄糖。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110714042A (zh) * 2018-07-13 2020-01-21 中国科学院天津工业生物技术研究所 氨基葡萄糖的酶法制备

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111518857A (zh) * 2020-06-11 2020-08-11 江苏海飞生物科技有限公司 酶法生产氨基葡萄糖盐及其提纯方法
CN115698313A (zh) * 2021-03-30 2023-02-03 南京农业大学食品科技学院糖组学与糖生物工程研究中心 氨基糖的直接酶法转化工艺；用于该工艺的酶和组合物
CN113201563B (zh) * 2021-04-14 2022-11-29 天津科技大学 一种提高小核菌多糖产量的营养盐及其应用
CN114807098B (zh) * 2022-06-08 2023-06-27 中国科学院青岛生物能源与过程研究所 用于生产胞外纤维素降解酶系的培养方法
CN116162673B (zh) * 2023-04-21 2023-07-18 山东福洋生物科技股份有限公司 一种多种功能糖联产的方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1314951A (zh) * 1998-07-15 2001-09-26 以生物技术资源的名义经营的Dcv公司 生产葡糖胺的方法和原料
JP2005198625A (ja) * 2004-01-19 2005-07-28 Mitsubishi Rayon Co Ltd 微生物の培養方法
CN1685054A (zh) * 2002-09-30 2005-10-19 阿彻-丹尼尔斯-米德兰公司 无细胞生产葡糖胺
EP1923464A1 (en) * 2002-08-30 2008-05-21 Japan Science and Technology Agency Ligase of a hyperthermostable bacterium and chip using the same
JP5424604B2 (ja) * 2008-09-30 2014-02-26 協和発酵バイオ株式会社 グルコサミンの製造法
CN106148425A (zh) * 2015-04-17 2016-11-23 张以恒 肌醇的制备方法
CN107815444A (zh) * 2017-10-31 2018-03-20 中国科学院天津工业生物技术研究所 一种用于atp再生的底盘系统与应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2769992B2 (ja) * 1995-04-25 1998-06-25 工業技術院長 グルコサミン−６−ホスフェートデアミナーゼ
KR101035043B1 (ko) * 2002-07-01 2011-05-19 아르키온 라이프 사이언씨즈 엘엘씨 글루코사민 및 ｎ-아세틸글루코사민의 제조를 위한 물질및 공정
CN110714042A (zh) * 2018-07-13 2020-01-21 中国科学院天津工业生物技术研究所 氨基葡萄糖的酶法制备
CN113122519B (zh) * 2019-12-31 2023-05-26 中国科学院天津工业生物技术研究所 一种耐热6-磷酸氨基葡萄糖磷酸酶突变体及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1314951A (zh) * 1998-07-15 2001-09-26 以生物技术资源的名义经营的Dcv公司 生产葡糖胺的方法和原料
EP1923464A1 (en) * 2002-08-30 2008-05-21 Japan Science and Technology Agency Ligase of a hyperthermostable bacterium and chip using the same
CN1685054A (zh) * 2002-09-30 2005-10-19 阿彻-丹尼尔斯-米德兰公司 无细胞生产葡糖胺
JP2005198625A (ja) * 2004-01-19 2005-07-28 Mitsubishi Rayon Co Ltd 微生物の培養方法
JP5424604B2 (ja) * 2008-09-30 2014-02-26 協和発酵バイオ株式会社 グルコサミンの製造法
CN106148425A (zh) * 2015-04-17 2016-11-23 张以恒 肌醇的制备方法
CN107815444A (zh) * 2017-10-31 2018-03-20 中国科学院天津工业生物技术研究所 一种用于atp再生的底盘系统与应用

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
"Uniprot", Database accession no. A0A0A0HB49
"UniProt", Database accession no. Q973H3
CHENG, K. ET AL.: "Doubling Power Output of Starch Biobattery Treated by the Most Thermostable Isoamylase from an Archaeon Sulfolobus tokodaii", SCI. REP., vol. 5, pages 13184
DATABASE Protein 7 October 2016 (2016-10-07), "predicted sugar phosphatase, HAD superfamily [Thermococcus kodakarensi KOD1]", XP055680957, retrieved from ncbi Database accession no. BAD85923.1 *
LIU DIANLEI, LI PIWU, LI RUIRUI, WANG SHENG, WANG RUIMING: "Research advance of the construction of gene engineering strains for glucosamine", BIOTECHNOLOGY BULLETIN, no. 3, 31 December 2014 (2014-12-31), pages 36 - 41, XP009525425, ISSN: 1002-5464 *
LIU, L.: "Microbial production of glucosamine and N-acetylglucosamine: advances and perspectives", APPL. MICROBIOL. BIOTECHNOL., 11 June 2013 (2013-06-11), pages 6149 - 6158, XP035328673, DOI: 10.1007/s00253-013-4995-6 *
YOU CZHANG XZZHANG Y-HP: "Simple cloning via direct transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis", APPL. ENVIRON. MICROBIOL., vol. 78, no. 5, 2012, pages 1593 - 5, XP055378419, DOI: 10.1128/AEM.07105-11

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110714042A (zh) * 2018-07-13 2020-01-21 中国科学院天津工业生物技术研究所 氨基葡萄糖的酶法制备

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