Enzymatic preparation method of glucosamine
阅读说明：本技术 氨基葡萄糖的酶催化制备方法 (Enzymatic preparation method of glucosamine ) 是由 陈延静 詹金明 王松叶 于 2021-10-22 设计创作，主要内容包括：本发明是一种氨基葡萄糖的酶催化制备方法,属于生物工程技术领域。该方法包括：采用葡萄糖异构酶催化,将D-葡萄糖转化为D-果糖；以及采用转氨酶、氨基供体化合物和弱氧化剂化合物将D-果糖转化为氨基葡萄糖；葡萄糖异构酶来源于凝结芽孢杆菌、树枝状黄杆菌、橄榄色链霉菌、凝结芽孢杆菌、树枝状黄杆菌、橄榄色链霉菌；转氨酶来源于枯草芽孢杆菌、贝莱斯芽孢杆菌、短小芽孢杆菌、地衣芽孢杆菌、唾液链球菌、马里乳杆菌；本发明利用生物酶通过体外催化D-葡萄糖实现制备氨基葡萄糖的方法,初始原料为D-葡萄糖或D-果糖所需的葡萄糖异构酶和转氨酶可通过构建大肠杆菌基因工程表达菌,采用“一锅法”催化直接得到终产物氨基葡萄糖,方法具有原料廉价、生产成本低、环境友好等优点。(The invention relates to an enzyme catalysis preparation method of glucosamine, belonging to the technical field of bioengineering. The method comprises the following steps: converting D-glucose into D-fructose by adopting glucose isomerase catalysis; and converting D-fructose to glucosamine using a transaminase, an amino donor compound, and a weak oxidant compound; the glucose isomerase is derived from Bacillus coagulans, Flavobacterium arborescens, Streptomyces olivaceus, Bacillus coagulans, Flavobacterium arborescens, and Streptomyces olivaceus; the transaminase is derived from Bacillus subtilis, Bacillus belgii, Bacillus pumilus, and Bacillus licheniformis 、 Streptococcus salivarius, lactobacillus mali; the invention relates to a method for preparing glucosamine by in vitro catalyzing D-glucose by utilizing biological enzyme, wherein the initial raw material is glucose isomerase and transaminase required by D-glucose or D-fructoseThe final product glucosamine is directly obtained by constructing escherichia coli genetic engineering expression bacteria and adopting one-pot catalysis, and the method has the advantages of cheap raw materials, low production cost, environmental friendliness and the like.)
1. An enzymatic preparation method of glucosamine, comprising: converting D-glucose into D-fructose by adopting glucose isomerase catalysis; and converting D-fructose to glucosamine using a transaminase, an amino donor compound, and a weak oxidant compound;
the glucose isomerase is derived from bacillus coagulans (A), (B) and (C)Bacillus coagulans) GenBank accession number KYC 85466; or from Flavobacterium arborescens: (Flavobacterium arborescens) GenBank accession number OAZ 44030; or from Streptomyces olivorubicus (Streptomyces olivochromogenes) GenBank accession number KUN 44528; or from Bacillus coagulans (Bacillus coagulans) GenBank accession number KYC 85466; or from Flavobacterium arborescens: (Flavobacterium arborescens) GenBank numbering is OAZ 44030; or from Streptomyces olivorubicus (Streptomyces olivochromogenes) GenBank accession number KUN 44528;
the transaminase is derived from bacillus subtilis (A), (B)Bacillus subtilis) GenBank accession No. QHF 59041; or from Bacillus belgii (B.), (B.) (Bacillus velezensis) GenBank accession No. AFZ 92582; or from Bacillus pumilus (Bacillus pumilus) GenBank accession number is SNV 15900; or from Bacillus licheniformis (Bacillus licheniformis) GenBank accession number VEH 77736; or from Streptococcus salivarius (Streptococcus salivarius) GenBank accession number KXU 58123; or from Lactobacillus Marylanicus (A), (B), (C)Lactobacillus mali) GenBank accession number KRN 26726.
2. The process for the enzymatic preparation of glucosamine according to claim 1, wherein: the amino donor compound is one or any mixture of two or more of D-alanine, isopropylamine, tert-butylamine and phenylethylamine; the weak oxidant compound is selected from one or any mixture of two or more of phosphite, organic peroxy acid and copper oxide.
3. The enzymatic production method of glucosamine according to claim 1 or 2, characterized in that: the temperature of the catalytic reaction is 20-70 ℃, preferably 35-40 ℃ or 30-37 ℃; the pH for the catalytic reaction is 4.0-9.0, preferably 6.7-7.5.
4. The enzymatic production method of glucosamine according to claim 1 or 2, characterized in that: when the two catalytic reaction steps are carried out simultaneously, the catalytic reaction time is 1-48h, preferably 20-30 h.
5. The enzymatic production method of glucosamine according to claim 1 or 2, characterized in that: the two catalytic reaction steps are carried out in steps, the catalytic reaction time of each step is independently carried out for 1 to 15 hours, and preferably 5 to 8 hours.
6. The enzymatic production method of glucosamine according to claim 1 or 2, characterized in that: the concentration of substrate glucose in the reaction system is 1-200g/L, preferably 8-20g/L or 8-35 g/L; the concentration of the glucose isomerase in the reaction system is 0.1-20U/mL, preferably 4-8U/mL; the concentration of transaminase is 0.1-20U/mL, preferably 4-8U/mL.
7. The enzymatic production method of glucosamine according to claim 1 or 2, characterized in that: the concentration of the amino donor compound in the reaction system is 0.1 to 3mM, preferably 1 to 2 mM; the concentration of the weak oxidant compound is 0.5-20mM, preferably 8-12 mM.
8. The enzymatic production method of glucosamine according to claim 1 or 2, characterized in that: the reaction system contains the cofactor pyridoxal phosphate in a concentration of 0.1 to 2mM, preferably 0.8 to 1.5 mM.
9. The enzymatic production method of glucosamine according to claim 1 or 2, characterized in that: the reaction system also contains a buffer solution, and the concentration of the buffer solution is 20-300 mM, preferably 80-150 mM; the buffer is preferably HEPES buffer, Tris-HCl buffer, MOPS buffer or citrate buffer.
10. The process for the enzymatic production of glucosamine by a biological enzyme according to claim 1 or 2, wherein: converting the D-fructose to glucosamine, including pure glucosamine, glucosamine salts and acetylglucosamine; wherein the glucosamine salt is selected from glucosamine hydrochloride, glucosamine sulfate, glucosamine phosphate, glucosamine sulfate sodium chloride complex salt, glucosamine sulfate potassium chloride complex salt, or glucosamine sulfate calcium chloride complex salt.
Technical Field
The invention relates to an enzyme catalysis preparation method of glucosamine, in particular to a method for preparing the glucosamine in vitro by adopting biological enzyme catalysis, belonging to the technical field of biological engineering.
Background
Glucosamine is a compound obtained by substituting hydroxyl at 2-position in a D-glucose molecule with amino, has a chemical name of 2-amino-2-deoxy-D-glucose, is easily soluble in water and hydrophilic solvents, and is an important functional monosaccharide. Glucosamine is present in almost all organisms including bacteria, fungi, plants and animals, and is a major constituent of glycoproteins and proteoglycans, as well as chitosan and chitin.
Glucosamine and derivatives thereof have wide application and important application in the fields of medicine, food, cosmetics and the like. In the pharmaceutical industry, glucosamine sulfate can be used as a raw material drug for treating rheumatoid arthritis by stimulating the biosynthesis of cartilage proteoglycan; in the food industry, glucosamine has various physiological functions of absorbing free radicals in vivo, resisting aging, promoting weight loss, inhibiting bacteria, regulating endocrine of human body and the like, and is used in the production of food additives and health-care food; in the cosmetic industry, acetylglucosamine is a monomer of hyaluronic acid, and is an indispensable substance in high-grade cosmetics.
Currently, there are mainly two methods for producing glucosamine:
(1) the chitin hydrolysis method comprises chitin acid hydrolysis method and chitin enzyme hydrolysis method. Among them, the acid hydrolysis method of chitin is the most commonly used method for producing glucosamine, which comprises first hydrolyzing chitin with high-concentration hydrochloric acid to obtain acetylglucosamine, and then deacetylating the acetylglucosamine to obtain glucosamine. However, the production method is easily affected by the supply of raw materials, the wastewater generated by acid treatment can cause pollution to the environment, and in addition, some people with prawn and crab allergy can generate allergic reaction after eating the glucosamine prepared by the method. The chitin enzyme hydrolysis method takes chitin as a raw material, and generates a glucosamine monomer through hydrolysis reaction under the action of the chitin enzyme, and the method has less environmental pollution but is also limited by factors such as raw material supply, production efficiency, anaphylactic reaction and the like.
(2) A microbial fermentation method for preparing aminoglucose from glucose and starch includes such steps as modifying the genetically engineered bacterial strains of colibacillus and Bacillus subtilis, and preparing aminoglucose from glucose and starch. Although the method has the advantages of no limitation of raw material sources, higher production efficiency, less environmental pollution and the like, the method also has the defects of higher modification difficulty of microbial metabolic pathways, difficult control of genetic stability of engineering bacteria, easy generation of metabolic byproducts and the like. Therefore, there is an urgent need to develop a new method for producing glucosamine with low cost, low pollution, and high efficiency.
Disclosure of Invention
The invention aims to provide a novel method for producing and preparing glucosamine by in vitro catalysis by using biological enzymes, aiming at the defects of the existing method for producing and preparing the glucosamine. The method has the advantages of cheap raw materials, low production cost, high production efficiency, environmental friendliness, safety to human body and the like.
The invention is realized by the following technical scheme:
the invention provides a method for preparing glucosamine by taking D-glucose as a raw material and utilizing biological enzyme through in vitro catalysis, which comprises the following steps: converting D-glucose into D-fructose by adopting Glucose Isomerase (GI) or Xylosidase (XI) as a catalyst; and converting D-fructose into glucosamine using Transaminase (TA), an amino donor compound, and a weak oxidant compound.
The synthetic route of the invention is as follows:
according to the invention, the process comprises a reaction step for converting D-glucose into D-fructose, which is catalyzed by glucose isomerase, or xylose isomerase, which is capable of isomerizing aldoses such as D-glucose, D-xylose and D-ribose into the corresponding ketose.
According to the invention, the process comprises a reaction step for converting D-fructose into glucosamine, which is catalyzed by a transaminase, an amino donor compound, which is one, any mixture of two or more, of D-alanine, isopropylamine, tert-butylamine and phenethylamine, and a weak oxidant compound, which is one, any mixture of two or more, of phosphite, organic peroxyacid and copper oxide, with pyridoxal phosphate (PLP) as a cofactor.
The glucose isomerase of the present invention is derived from Bacillus coagulans (A), (B) and (C)Bacillus coagulans) GenBank accession number KYC 85466; or from Flavobacterium arborescens: (Flavobacterium arborescens) GenBank accession number OAZ 44030; or from Streptomyces olivorubicus (Streptomyces olivochromogenes) GenBank accession number KUN 44528; or from Bacillus coagulans (Bacillus coagulans) GenBank accession number KYC 85466; or from Flavobacterium arborescens: (Flavobacterium arborescens) GenBank numbering is OAZ 44030; or from Streptomyces olivorubicus (Streptomyces olivochromogenes) GenBank accession number KUN 44528;
the transaminase is derived from bacillus subtilis (A), (B)Bacillus subtilis) GenBank accession No. QHF 59041; or from Bacillus belgii (B.), (B.) (Bacillus velezensis) GenBank accession No. AFZ 92582; or from Bacillus pumilus (Bacillus pumilus) GenBank accession number is SNV 15900; or from Bacillus licheniformis (Bacillus licheniformis) GenBank accession number VEH 77736; or from Streptococcus salivarius (Streptococcus salivarius) GenBank accession number KXU 58123; or from Lactobacillus Marylanicus (A), (B), (C)Lactobacillus mali) GenBank accession number KRN 26726.
According to the invention, the catalytic reaction is carried out without addition of NAD (H) and ATP.
It will be appreciated by those skilled in the art that the above steps included in the method of the invention may be carried out simultaneously, for example in a single bioreactor or reaction vessel.
It will be appreciated by those skilled in the art that the above steps included in the method of the invention may also be carried out in steps, for example in one bioreactor or reaction vessel or in a plurality of bioreactors or reaction vessels arranged in series.
According to the invention, the temperature of the catalytic reaction is 20 to 70 ℃, preferably 35 to 40 ℃ or 30 to 37 ℃.
According to the invention, the pH of the catalytic reaction is between 4.0 and 9.0, preferably between 6.7 and 7.5.
According to the invention, when the above steps are carried out simultaneously, the catalytic reaction time is 1 to 48 hours, preferably 20 to 30 hours.
According to the invention, when the above steps are carried out stepwise, the catalytic reaction times of the individual steps are carried out independently of one another for a period of from 1 to 15 hours, preferably from 5 to 8 hours.
According to the invention, the concentration of the substrate glucose in the reaction system is 1 to 200g/L, preferably 8 to 20g/L or 8 to 35 g/L.
According to the invention, the concentration of the glucose isomerase in the reaction system is 0.1-20U/mL, preferably 4-8U/mL; the concentration of transaminase is 0.1-20U/mL, preferably 4-8U/mL.
According to the present invention, the concentration of pyridoxal phosphate (PLP), a cofactor, in the reaction system is 0.1-2mM, preferably 0.8-1.5 mM; the concentration of the amino donor is 0.1-3mM, preferably 1-2 mM; the concentration of the weak oxidizing agent is 0.5-20mM, preferably 8-12 mM.
According to the present invention, the reaction system further comprises a buffer. It will be appreciated by those skilled in the art that various buffers can be used in the present invention, such as HEPES buffer, Tris-HCl buffer, MOPS buffer, citrate buffer, and the like. The concentration of the buffer solution in the reaction system is 20-300 mM, preferably 80-150 mM.
According to the invention, D-fructose is converted into glucosamine, including pure glucosamine, glucosamine salts and acetylglucosamine; wherein the glucosamine salt is selected from glucosamine hydrochloride, glucosamine sulfate, glucosamine phosphate, glucosamine sulfate sodium chloride complex salt, glucosamine sulfate potassium chloride complex salt, or glucosamine sulfate calcium chloride complex salt.
According to the present invention, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention, based on the common knowledge in the art.
Compared with the prior art, the invention has the positive improvement effects that:
the invention provides a brand-new method for preparing glucosamine by catalyzing D-glucose in vitro by using biological enzyme, wherein the initial raw material is D-glucose or D-fructose, the price is low, the required glucose isomerase and transaminase can be obtained by constructing escherichia coli genetic engineering expression bacteria and then preparing a large amount by fermentation, and the method is relatively easy to obtain and low in price.
The method provided by the invention is a one-pot method for catalysis, the substrate and the enzyme are added and then start to react, and the final product glucosamine is directly obtained after the reaction is finished. Compared with other existing production and preparation methods, the preparation method of the glucosamine biological enzyme has the advantages of cheap raw materials, low production cost, environmental friendliness, safety to human bodies and the like, and is suitable for popularization.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Some material information used in the examples of the present invention are as follows:
pColdII plasmid (Takara, Dalian, China);
escherichia coli cloned strainE. coli DH5α（Invitrogen，Carlsbad，CA）；
Escherichia coli expression strainE. coli BL21(DE3)（Invitrogen，Carlsbad，CA）；
Example 1 construction of engineered expression Strain containing glucose isomerase Gene and transaminase Gene
According to Bacillus coagulans (A), (B)B. coagulans) The glucose isomerase of (2) is also called xylose isomerase gene nucleotide sequence (GenBank accession number KYC85466) and Bacillus subtilisB. subtilis) The transaminase gene nucleotide sequences (GenBank accession number QHF 59041) are respectively subjected to whole gene synthesis, are connected with pColdII plasmids through enzyme digestion and ligation reaction, are transformed into escherichia coli DH5 alpha strain competent cells, are coated with LB plates containing aminobenzyl antibiotics (30 mu g/ml), are cultured at 37 ℃ for 12 hours, and then positive transformants are picked, identified and sequenced. Inoculating the positive monoclonal antibody into 5mL LB liquid culture medium containing 30 ug/mL ampicillin, culturing at 37 deg.C overnight, respectively extracting two recombinant plasmids, and transforming into expression hostE. coliBL21(DE3) to obtain recombinant strainsE. coliBL21(DE3)/pColdII-GI and recombinant strainsE. coliBL21(DE3)/pColdII-AT, after the confirmation of shake flask pilot fermentation, the recombinant strains were subjected to slant storage and glycerol storage AT-80 ℃. The LB medium comprises the following components: 10g/L Tryptone (Tryptone), 5g/L Yeast extract (Yeast extract), 10g/L sodium chloride (NaCl), pH 7.4. Definition of enzyme activity unit: the amount of enzyme required to oxidize 1. mu. mol of substrate per minute is one enzyme activity unit U.
Example 2 recombinant expression preparation of glucose isomerase and transaminase
1) Seed culture: recombinant strain preserved on slantE. coliBL21(DE3)/pColdII-GI and recombinant strainsE. coliBL21(DE3)/pColdII-AT are respectively inoculated to an LB liquid culture medium containing 30 mu g/ml ampicillin, and cultured for 8-10 h AT 37 ℃ to obtain seed liquid;
2) fermentation culture: the seed solution was inoculated into LB liquid medium containing 30. mu.g/ml ampicillin in an inoculum size of 1% by volume, and cultured at 37 ℃ to OD600The value is 0.5, then the strain is transferred to 15 ℃ for culture, isopropyl-beta-D-galactoside (IPTG) with the final concentration of 0.1mM is added, the rotation speed is 160 r/min, the induction expression is carried out for 24h, thalli are collected, the total protein of the whole strain is analyzed by utilizing polyacrylamide gel electrophoresis (SDS-PAGE), the obvious glucose isomerase and transaminase recombinant expression protein bands are shown after the gene engineering strain is induced, and the molecular weight of the bands is consistent with the expected molecular weight. The LB liquid culture medium has the following final concentration composition: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and deionized water as solvent, pH 7.4, 30. mu.g/ml ampicillin was added before use. Under these conditions, the expression level of the recombinant protein is about 50% of the total amount of the bacterial cells, and most of the recombinant protein is in a soluble state. After induction expression, the cells were disrupted by sonication and Ni was used+Purifying by column affinity chromatography to obtain soluble recombinant glucose isomerase and transaminase, wherein the imidazole elution concentration in the affinity chromatography is 400 mM.
Example 3, a process for the enzymatic preparation of glucosamine,
the method comprises the following steps: d-glucose is converted into D-fructose by adopting the glucose isomerase prepared in the example 2 for catalysis; and converting D-fructose into glucosamine using the transaminase, the amino donor compound, and the weak oxidant compound prepared in example 2.
The amino donor compound is selected from D-alanine, isopropylamine, tert-butylamine and phenylethylamine; the weak oxidant compound is selected from one of phosphite, organic peroxy acid and copper oxide. The temperature of the catalytic reaction is 35 ℃; the pH of the catalyzed reaction was 6.7. When the two catalytic reaction steps are carried out simultaneously, the catalytic reaction time is 20 h. The concentration of substrate glucose in the reaction system is 8 g/L; the concentration of glucose isomerase in the reaction system is 4U/mL; the transaminase concentration was 4U/mL.
The concentration of the amino donor compound in the reaction system was 1 mM; the concentration of the weak oxidant compound was 8 mM. The reaction system contained pyridoxal phosphate as a cofactor at a concentration of 0.8 mM. The reaction system also contained a buffer solution, the concentration of which was 80 mM. The buffer is HEPES buffer.
Example 4, an enzymatic preparation of glucosamine,
the method comprises the following steps: d-glucose is converted into D-fructose by adopting the glucose isomerase prepared in the example 2 for catalysis; and converting D-fructose into glucosamine using the transaminase, the amino donor compound, and the weak oxidant compound prepared in example 2.
The amino donor compound adopts D-alanine and isopropylamine 1: 1, preparing a mixture; the weak oxidant compound is phosphite and organic peroxy acid 1: 2, and (3) preparing a mixture. The temperature of the catalytic reaction is 0 ℃; the pH of the catalyzed reaction was pH 7.5. When the two catalytic reaction steps are carried out step by step, the catalytic reaction time of each step is independent of that of each other for 5 hours. The concentration of substrate glucose in the reaction system is 20 g/L; the concentration of glucose isomerase in the reaction system is 8U/mL; the transaminase concentration was 8U/mL.
The concentration of the amino donor compound in the reaction system was 2 mM; the concentration of the weak oxidant compound was 12 mM. The reaction system contained pyridoxal phosphate as a cofactor at a concentration of 1.5 mM. The reaction system also contained a buffer solution, the concentration of which was 150 mM. The buffer solution is Tris-HCl buffer solution or MOPS buffer solution.
Example 5, an enzymatic preparation of glucosamine,
the method comprises the following steps: d-glucose is converted into D-fructose by adopting the glucose isomerase prepared in the example 2 for catalysis; and converting D-fructose into glucosamine using the transaminase, the amino donor compound, and the weak oxidant compound prepared in example 2.
The amino donor compound is selected from tert-butylamine and phenethylamine according to the ratio of 2: 1; the weak oxidant compound is organic peroxy acid and copper oxide, wherein the weight ratio of the organic peroxy acid to the copper oxide is 3: 1, in a mixture of the components. The temperature of the catalytic reaction is 28 ℃; the pH of the catalyzed reaction was 7. When the two catalytic reaction steps are carried out simultaneously, the catalytic reaction time is 24 hours; the concentration of substrate glucose in the reaction system is 10 g/L; the concentration of glucose isomerase in the reaction system is 6U/mL; the transaminase concentration was 7U/mL.
The concentration of the amino donor compound in the reaction system was 1.5 mM; the concentration of the weak oxidant compound was 10 mM. The reaction system contained pyridoxal phosphate as a cofactor at a concentration of 1.0 mM. The reaction system also contains a buffer solution, and the concentration of the buffer solution is 120 mM. The buffer solution is MOPS buffer solution or citrate buffer solution.
Example 6, an enzymatic preparation of glucosamine,
the method comprises the following steps: d-glucose is converted into D-fructose by adopting the glucose isomerase prepared in the example 2 for catalysis; and converting D-fructose into glucosamine using the transaminase, the amino donor compound, and the weak oxidant compound prepared in example 2.
The amino donor compounds are isopropylamine and tert-butylamine which are mixed according to a ratio of 1: 1; the weak oxidant compound is selected from phosphite, organic peroxy acid and copper oxide according to the weight ratio of 1: 1: 1 to form a mixture. The temperature of the catalytic reaction is 30 ℃; the pH of the catalyzed reaction was 6.5. When the two catalytic reaction steps are carried out simultaneously, the concentration of the substrate glucose in the reaction system is 100g/L within 24 h; the concentration of glucose isomerase in the reaction system is 10U/mL; the transaminase concentration was 6U/mL.
The concentration of the amino donor compound in the reaction system was 3 mM; the concentration of the weak oxidizer compound was 15. The reaction system contained pyridoxal phosphate as a cofactor at a concentration of 1.8 mM. The reaction system also contained a buffer solution, the concentration of which was 200 mM. The buffer is selected from HEPES buffer, Tris-HCl buffer, MOPS buffer or citrate buffer.
Example 7, experiment for the preparation of glucosamine using glucose isomerase and transaminase catalysis:
recombinant glucose isomerase and transaminase were prepared as in example 2. Glucosamine was quantitatively analyzed by High Performance Liquid Chromatography (HPLC). The chromatographic column is amino column, the mobile phase is 80% acetonitrile water solution, the flow rate is 1mL/min, the column temperature is 40 ℃, and the detector is a Waters2414 refractive index detector. The glucosamine standard sample retention time is about 10.3 min. Glucosamine concentration is proportional to the response intensity of the HPLC characteristic peak of glucosamine. 1mL of a reaction mixture containing 10g/L D-glucose, 2mM isopropylamine or D-alanine, 100mM HEPES buffer (pH 7.0), 1mM pyridoxal phosphate, 10mM phosphite or organic peroxyacid, 5U/mL glucose isomerase, 5U/mL transaminase was reacted at 37 ℃ for 24 hours. After the reaction is finished, adding acetonitrile with the same volume to the reaction system to stop the reaction, centrifuging at 12000 r/min for 10min, and taking supernate to measure the concentration of glucosamine in the reaction solution through high performance liquid chromatography. When the reaction was carried out for 24 hours, the glucosamine concentration was 7.3g/L and the conversion was 73%.
Example 8 construction of engineered expression Strain containing glucose isomerase Gene and transaminase Gene
According to Streptomyces olivorubidus (Streptomyces olivochromogenesGenBank accession number KUN 44528), or Bacillus coagulans (B.coagulans)Bacillus coagulansGenBank accession number KYC85466), or Flavobacterium arborescens (Flavobacterium arborescensGenBank accession number OAZ44030), or Streptomyces olivaceus (S.olivaceusStreptomyces olivochromogenesGenBank accession number KUN 44528) of glucose isomerase, also known as xylose isomerase, and Bacillus pumilus (Bacillus pumilus: (B.pumilus) ((B.pumilus))Bacillus pumilusGenBank accession number SNV 15900), or Bacillus licheniformis (Bacillus licheniformisGenBank accession number VEH 77736), or Streptococcus salivarius (S.salivarius)Streptococcus salivariusGenBank accession number KXU 58123), or Lactobacillus Marylanicum (A)Lactobacillus maliGenBank number KRN 26726), through enzyme digestion and ligation reaction, to be connected with pColdII plasmid, transformed into Escherichia coli DH5 alpha strain competent cells, coated with LB plate containing aminobenzyl antibiotic (30. mu.g/ml), cultured at 37 ℃ for 12h, picked, identified positive transformant and sequenced. Will be examinedInoculating the positive single clone to 5mL LB liquid culture medium containing 30 ug/mL ampicillin, culturing at 37 deg.C overnight, respectively extracting two recombinant plasmids, and transforming into expression hostE. coliBL21(DE3) to obtain recombinant strainsE. coliBL21(DE3)/pColdII-GI and recombinant strainsE. coliBL21(DE3)/pColdII-AT, after the confirmation of shake flask pilot fermentation, the recombinant strains were subjected to slant storage and glycerol storage AT-80 ℃. The LB medium comprises the following components: 10g/L Tryptone (Tryptone), 5g/L Yeast extract (Yeast extract), 10g/L sodium chloride (NaCl), pH 7.4. Definition of enzyme activity unit: the amount of enzyme required to oxidize 1. mu. mol of substrate per minute is one enzyme activity unit U.
Example 9 recombinant expression preparation of glucose isomerase and transaminase
1) Seed culture: recombinant strain preserved on slantE. coliBL21(DE3)/pColdII-GI and recombinant strainsE. coliBL21(DE3)/pColdII-AT are respectively inoculated to an LB liquid culture medium containing 30 mu g/ml ampicillin, and cultured for 8-10 h AT 37 ℃ to obtain seed liquid;
2) fermentation culture: the seed solution was inoculated into LB liquid medium containing 30. mu.g/ml ampicillin in an inoculum size of 1% by volume, and cultured at 37 ℃ to OD600The value is 0.5, then the strain is transferred to 15 ℃ for culture, isopropyl-beta-D-galactoside (IPTG) with the final concentration of 0.1mM is added, the rotation speed is 160 r/min, the induction expression is carried out for 24h, thalli are collected, the total protein of the whole strain is analyzed by utilizing polyacrylamide gel electrophoresis (SDS-PAGE), the obvious glucose isomerase and transaminase recombinant expression protein bands are shown after the gene engineering strain is induced, and the molecular weight of the bands is consistent with the expected molecular weight. The LB liquid culture medium has the following final concentration composition: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and deionized water as solvent, pH 7.4, 30. mu.g/ml ampicillin was added before use. Under these conditions, the expression level of the recombinant protein is about 50% of the total amount of the bacterial cells, and most of the recombinant protein is in a soluble state. After induction expression, the cells were disrupted by sonication and Ni was used+Purifying by column affinity chromatography to obtain soluble recombinant glucose isomerase and transaminase, wherein the imidazole elution concentration in the affinity chromatography is 400 mM.
Example 10 enzymatic preparation of glucosamine Using glucose isomerase and transaminase
The Streptomyces olivaceus strain prepared in example 9 was taken (Streptomyces olivochromogenesGenBank accession number KUN 44528) and a recombinant glucose isomerase derived from Bacillus pumilus (Bacillus pumilus: (II)Bacillus pumilusGenBank accession number SNV 15900). Glucosamine was quantitatively analyzed by High Performance Liquid Chromatography (HPLC). The chromatographic column is amino column, the mobile phase is 80% acetonitrile water solution, the flow rate is 1mL/min, the column temperature is 40 ℃, and the detector is a Waters2414 refractive index detector. The glucosamine standard sample retention time is about 10.3 min. Glucosamine concentration is proportional to the response intensity of the HPLC characteristic peak of glucosamine. 1mL of a reaction mixture containing 10g/L D-glucose, 2mM isopropylamine or D-alanine, 100mM HEPES buffer (pH 7.0), 1mM pyridoxal phosphate, 10mM phosphite or organic peroxyacid, 5U/mL glucose isomerase, 5U/mL transaminase was reacted at 37 ℃ for 24 hours. After the reaction is finished, adding acetonitrile with the same volume to the reaction system to stop the reaction, centrifuging at 12000 r/min for 10min, and taking supernate to measure the concentration of glucosamine in the reaction solution through high performance liquid chromatography. The reaction was carried out for 24 h. The glucosamine concentration was 7.5g/L and the conversion was 69%.
Example 11 enzymatic preparation of glucosamine Using glucose isomerase and transaminase
The Bacillus coagulans-derived material prepared in example 9 is taken (Bacillus coagulansGenBank accession number KYC85466) and recombinant glucose isomerase derived from Bacillus licheniformis (Bacillus licheniformis: (II)Bacillus licheniformisGenBank accession No. VEH 77736). The reaction was carried out for 36 hours in the same manner as in example 10 to obtain a glucosamine concentration of 7.0g/L and a conversion of 72%.
Example 12 enzymatic preparation of glucosamine Using glucose isomerase and transaminase
Taking Flavobacterium arborescens (F) as the source prepared in example 9Flavobacterium arborescensGenBank accession OAZ44030) and from Streptococcus salivarius (A. salivarius)Streptococcus salivarius，GenBank No. KXU 58123). The reaction was carried out for 48 hours in the same manner as in example 10. The glucosamine concentration was 8.0g/L and the conversion was 80%.
Example 13 enzymatic preparation of glucosamine Using glucose isomerase and transaminase
The Streptomyces olivaceus strain prepared in example 9 was taken (Streptomyces olivochromogenesGenBank accession number KUN 44528) and a recombinant glucose isomerase derived from Lactobacillus Marylanica (L.))Lactobacillus maliGenBank accession No. KRN 26726). The reaction was carried out for 36 hours in the same manner as in example 10, and the reaction was carried out for 24 hours. The glucosamine concentration was 7.8g/L and the conversion was 78%.
Example 14, glucosamine preparation of glucosamine hydrochloride:
to the aqueous glucosamine solution obtained in example 10, hydrochloric acid was added in an equimolar or molar excess amount to conduct a salt-forming reaction for 1 hour. And then freeze-drying or concentrating under reduced pressure to remove water, cooling, adding a precipitator for crystallization, carrying out solid-liquid separation after crystallization is finished, and drying the solid to obtain the glucosamine hydrochloride.
Example 15, glucosamine preparation of glucosamine sulfate:
to the aqueous glucosamine solution obtained in example 11, sulfuric acid was added in an equimolar or molar excess amount to conduct a salt-forming reaction for 1 hour. And then freeze-drying or concentrating under reduced pressure to remove water, cooling, adding a precipitator for crystallization, carrying out solid-liquid separation after crystallization is finished, and drying the solid to obtain glucosamine sulfate.
Example 16 preparation of glucosamine sulfate Potassium chloride Complex salt with glucosamine:
to the aqueous glucosamine solution obtained in example 12, sulfuric acid was added in an equimolar or molar excess amount to conduct a salt-forming reaction for 1 hour. After the reaction is finished, adding equal mol of potassium chloride, and carrying out complex reaction for 1 hour under the condition of 35-45 ℃. And then freeze-drying or concentrating under reduced pressure to remove water, cooling, adding a precipitator for crystallization, carrying out solid-liquid separation after crystallization is finished, and drying the solid to obtain the glucosamine sulfate potassium chloride composite salt.
Example 17 preparation of glucosamine sulfate sodium chloride complex salt from glucosamine:
to the aqueous glucosamine solution obtained in example 13, sulfuric acid was added in an equimolar or molar excess amount to conduct a salt-forming reaction for 1 hour. After the reaction is finished, adding equimolar sodium chloride, and carrying out complex reaction for 1 hour under the condition of 35-45 ℃. And then freeze-drying or concentrating under reduced pressure to remove water, cooling, adding a precipitator for crystallization, carrying out solid-liquid separation after crystallization is finished, and drying the solid to obtain the glucosamine sulfate sodium chloride composite salt.
Example 18 preparation of glucosamine sulfate calcium chloride complex salt with glucosamine:
to the aqueous glucosamine solution obtained in example 10, sulfuric acid was added in an equimolar or molar excess amount to conduct a salt-forming reaction for 1 hour. After the reaction is finished, adding calcium chloride with the same mole, and carrying out complex reaction for 1 hour under the condition of 35-45 ℃. And then freeze-drying or concentrating under reduced pressure to remove water, cooling, adding a precipitator for crystallization, carrying out solid-liquid separation after crystallization is finished, and drying the solid to obtain the glucosamine sulfate calcium chloride composite salt.
Example 19 preparation of glucosamine phosphate glucosamine:
to the aqueous glucosamine solution obtained in example 11, phosphoric acid was added in an equimolar or molar excess amount to conduct a salt-forming reaction for 1 hour. And then freeze-drying or concentrating under reduced pressure to remove water, cooling, adding a precipitator for crystallization, carrying out solid-liquid separation after crystallization is finished, and drying the solid to obtain the glucosamine phosphate.
Example 20 preparation of acetylglucosamine from glucosamine:
to the aqueous glucosamine solution or mixed solvent obtained in example 12, an appropriate amount of a catalyst was added, and then acetic anhydride was added in an equimolar or molar excess amount to carry out an acylation reaction, after completion of the reaction. Concentrating to remove part of the solvent, crystallizing, separating after crystallization, washing, and drying to obtain the acetylglucosamine.
Example 21 preparation of acetylglucosamine from glucosamine:
to the aqueous glucosamine solution or mixed solvent obtained in example 13, an appropriate amount of a catalyst was added, and then acetyl chloride was reacted in an equimolar or molar excess amount for 4 to 6 hours to complete the acylation reaction. Concentrating to remove part of the solvent, crystallizing, separating after crystallization, washing, and drying to obtain the acetylglucosamine.
Example 22, a method for the enzymatic preparation of glucosamine by a biological enzyme:
glucose isomerase and transaminase were prepared as described in example 9.
The method for preparing glucosamine comprises the following steps: is prepared from streptomyces olivaceus (A)Streptomyces olivochromogenes) D-glucose is converted into D-fructose by the catalysis of glucose isomerase with the GenBank number of KUN 44528; and the extract is derived from Lactobacillus Marylanicum (II)Lactobacillus mali) A transaminase, amino donor compound and weak oxidant compound numbered as KRN26726 in GenBank converts D-fructose to glucosamine;
the method comprises the following steps:
the amino donor compound is D-alanine; the weak oxidant compound is phosphite. The temperature of the catalytic reaction is 30 ℃; the pH of the catalyzed reaction was 6.7. The two catalytic steps are carried out simultaneously, and the catalytic reaction time is 20 hours; the concentration of substrate glucose in the reaction system is 8 g/L; the concentration of glucose isomerase in the reaction system is 3U/mL; the transaminase concentration in the reaction system was 3U/mL. The reaction system also contained the cofactor pyridoxal phosphate at a concentration of 0.8 mM. The concentration of the amino donor compound was 1.0 mM; the concentration of the weak oxidant compound was 8 mM. The reaction system also contains a buffer solution, wherein the buffer solution is HEPES buffer solution, and the concentration of the buffer solution is 80 mM. After completion of the reaction, the glucosamine concentration was 7.3g/L and the conversion was 76%.
Example 23, a method for the enzymatic preparation of glucosamine by a biological enzyme:
glucose isomerase and transaminase were prepared as described in example 9.
The method for preparing glucosamine comprises the following steps: derived from streptomyces olivaceus (A)Streptomyces olivochromogenes) D-glucose is converted into D-fructose by the catalysis of glucose isomerase with the GenBank number of KUN 44528; and adoptDerived from streptococcus salivarius (Streptococcus salivarius) A transaminase, amino donor compound and weak oxidant compound of GenBank accession No. KXU58123 converts D-fructose to glucosamine;
the method comprises the following steps:
the amino donor compound is isopropylamine; the weak oxidant compound is organic peroxy acid. The temperature of the catalytic reaction is-37 ℃; the pH of the catalyzed reaction was 7.5. When the two catalysis steps are carried out simultaneously, the catalytic reaction time is 24 hours; the concentration of substrate glucose in the reaction system is 35 g/L; the concentration of glucose isomerase in the reaction system is 8U/mL; the transaminase concentration in the reaction system was 8U/mL. The reaction system also contained the cofactor pyridoxal phosphate at a concentration of 1.2 mM. The concentration of the amino donor compound was 2.5 mM; the concentration of the weak oxidant compound was 12 mM. The reaction system also contains a buffer solution, wherein the buffer solution is Tris-HCl buffer solution, and the concentration of the buffer solution is 150 mM. After completion of the reaction, the glucosamine concentration was 7.5g/L and the conversion was 77%.
Example 24, a method for the enzymatic preparation of glucosamine by a biological enzyme:
glucose isomerase and transaminase were prepared as described in example 9.
The method for preparing glucosamine comprises the following steps: derived from Bacillus coagulans (Bacillus coagulans) D-glucose is converted into D-fructose by the catalysis of glucose isomerase with GenBank number KYC 85466; and the extract is derived from bacillus licheniformis (B)Bacillus licheniformis) GenBank accession No. VEH77736, the amino donor compound and the weak oxidant compound convert D-fructose to glucosamine;
the amino donor compound is tert-butylamine; the weak oxidant compound is copper oxide. The temperature of the catalytic reaction is 35 ℃; the pH of the catalyzed reaction was 7.2. The two catalytic steps are carried out step by step, and the catalytic reaction time of each step is independently carried out for 6 h. The concentration of substrate glucose in the reaction system is 25 g/L; the concentration of glucose isomerase in the reaction system is 5U/mL; the transaminase concentration in the reaction system was 6U/mL. The reaction system also contained pyridoxal phosphate as a cofactor, at a concentration of 1.0 mM. The concentration of the amino donor compound was 1.5 mM; the concentration of the weak oxidant compound was 10 mM. The reaction system also contains a buffer solution, wherein the buffer solution is MOPS buffer solution, and the concentration of the buffer solution is 120 mM. After completion of the reaction, the glucosamine concentration was 7.8g/L and the conversion was 79%.
Example 25, a method for the enzymatic preparation of glucosamine by a biological enzyme:
glucose isomerase and transaminase were prepared as described in example 9.
The method for preparing glucosamine comprises the following steps: using a strain derived from Flavobacterium arborescens (Flavobacterium arborescens) D-glucose is converted into D-fructose by the catalysis of glucose isomerase with the GenBank number of OAZ 44030; and the extract is derived from Bacillus pumilus (B), (B)Bacillus pumilus) The transaminase, amino donor compound and weak oxidant compound, GenBank accession number SNV15900, converts D-fructose to glucosamine;
the method comprises the following steps:
the amino donor compound is phenethyl ammonia; the weak oxidant compound is phosphite. The temperature of the catalytic reaction is 35 ℃; the pH of the catalyzed reaction was 7.0. The two catalytic steps are carried out step by step, and the catalytic reaction time of each step is independent of each other for 8 hours. The concentration of substrate glucose in the reaction system is 25 g/L; the concentration of glucose isomerase in the reaction system is 6U/mL; the transaminase concentration in the reaction system was 5U/mL. The reaction system also contained the cofactor pyridoxal phosphate at a concentration of 1.2 mM. The concentration of the amino donor compound was 2.0 mM; the concentration of the weak oxidant compound was 11 mM. The reaction system also contains a buffer solution, wherein the buffer solution is a citrate buffer solution, and the concentration of the buffer solution is 110 mM. After the reaction was completed, the glucosamine concentration was 8.0g/L and the conversion was 80%.
Example 26 construction of engineered expression Strain containing glucose isomerase Gene and transaminase Gene
According to Flavobacterium arborescens: (F. arborescens) The nucleotide sequence of the glucose isomerase gene (GenBank accession OAZ44030) also called xylose isomerase and Bacillus belezii (B. velezensis) The transaminase gene nucleotide sequences (GenBank accession number AFZ 92582) are respectively subjected to whole gene synthesis, are connected with pColdII plasmids through enzyme digestion and ligation reaction, are transformed into escherichia coli DH5 alpha strain competent cells, are coated with LB plates containing aminobenzyl antibiotics (30 mu g/ml), are cultured at 37 ℃ for 12 hours, and then positive transformants are picked, identified and sequenced. Inoculating the positive monoclonal antibody into 5mL LB liquid culture medium containing 30 ug/mL ampicillin, culturing at 37 deg.C overnight, respectively extracting two recombinant plasmids, and transforming into expression hostE. coliBL21(DE3) to obtain recombinant strainsE. coliBL21(DE3)/pColdII-GI and recombinant strainsE. coliBL21(DE3)/pColdII-AT, after the confirmation of shake flask pilot fermentation, the recombinant strains were subjected to slant storage and glycerol storage AT-80 ℃. The LB medium comprises the following components: 10g/L Tryptone (Tryptone), 5g/L Yeast extract (Yeast extract), 10g/L sodium chloride (NaCl), pH 7.4. Definition of enzyme activity unit: the amount of enzyme required to oxidize 1. mu. mol of substrate per minute is one enzyme activity unit U.
Example 27 recombinant expression preparation of glucose isomerase and transaminase
1) Seed culture: recombinant strain preserved on slantE. coliBL21(DE3)/pColdII-GI and recombinant strainsE. coliBL21(DE3)/pColdII-AT are respectively inoculated to an LB liquid culture medium containing 30 mu g/ml ampicillin, and cultured for 8-10 h AT 37 ℃ to obtain seed liquid;
2) fermentation culture: the seed solution was inoculated into LB liquid medium containing 30. mu.g/ml ampicillin in an inoculum size of 1% by volume, and cultured at 37 ℃ to OD600The value is 0.5, then the strain is transferred to 15 ℃ for culture, isopropyl-beta-D-galactoside (IPTG) with the final concentration of 0.1mM is added, the rotation speed is 160 r/min, the induction expression is carried out for 24h, thalli are collected, the total protein of the whole strain is analyzed by utilizing polyacrylamide gel electrophoresis (SDS-PAGE), the obvious glucose isomerase and transaminase recombinant expression protein bands are shown after the gene engineering strain is induced, and the molecular weight of the bands is consistent with the expected molecular weight. The LB liquid culture medium has the following final concentration composition: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and deionized water as solvent, pH 7.4, 30 μ g/ml before useAmpicillin. Under these conditions, the expression level of the recombinant protein is about 50% of the total amount of the bacterial cells, and most of the recombinant protein is in a soluble state. After induction expression, the cells were disrupted by sonication and Ni was used+Purifying by column affinity chromatography to obtain soluble recombinant glucose isomerase and transaminase, wherein the imidazole elution concentration in the affinity chromatography is 400 mM.
Example 28, a method for the enzymatic preparation of glucosamine:
the method comprises the following steps: d-glucose was converted to D-fructose using the glucose isomerase enzyme prepared in example 27; and converting D-fructose into glucosamine using the transaminase, the amino donor compound, and the weak oxidant compound prepared in example 27.
The amino donor compound is one of D-alanine, isopropylamine, tert-butylamine and phenylethylamine. The weak oxidant compound is one of phosphite, organic peroxy acid and copper oxide. The temperature of the catalytic reaction is 35 ℃; the pH of the catalyzed reaction was 6.7. The two catalytic steps are carried out simultaneously, and the catalytic reaction time is 20 h. The concentration of substrate glucose in the reaction system is 8 g/L; the concentration of glucose isomerase in the reaction system is 2U/mL; the transaminase concentration was 2U/mL. The reaction system also contained the cofactor pyridoxal phosphate at a concentration of 0.8 mM. The concentration of the amino donor compound was 1.5 mM; the concentration of the weak oxidant compound was 6 mM. The reaction system also contains a buffer solution, wherein the buffer solution is selected from HEPES buffer solution, Tris-HCl buffer solution, MOPS buffer solution or citrate buffer solution, and the concentration of the buffer solution is 80 mM.
Example 29, a method for the enzymatic preparation of glucosamine:
the method comprises the following steps: d-glucose was converted to D-fructose using the glucose isomerase enzyme prepared in example 27; and converting D-fructose into glucosamine using the transaminase, the amino donor compound, and the weak oxidant compound prepared in example 27.
The amino donor compounds are D-alanine and isopropylamine in a ratio of 1: 1, in a mixture of the components. The weak oxidant compound is phosphite and organic peroxy acid, and the weight ratio of the phosphite to the organic peroxy acid is 2: 1, in a mixture of the components. The temperature of the catalytic reaction is 40 ℃; the pH of the catalyzed reaction was 7.5. The two catalytic steps are carried out step by step, and the catalytic reaction time of each step is independent of each other for 5 h. The concentration of substrate glucose in the reaction system is 20 g/L; the concentration of glucose isomerase in the reaction system is 8U/mL; the transaminase concentration was 8U/mL. The reaction system also contained the cofactor pyridoxal phosphate at a concentration of 1.5 mM. The concentration of the amino donor compound was 2.5 mM; the concentration of the weak oxidant compound was 15 mM. The reaction system also contains HEPES buffer solution or Tris-HCl buffer solution, and the concentration of the buffer solution is 150 mM.
Example 30, a method for the enzymatic preparation of glucosamine:
the method comprises the following steps: d-glucose was converted to D-fructose using the glucose isomerase enzyme prepared in example 27; and converting D-fructose into glucosamine using the transaminase, the amino donor compound, and the weak oxidant compound prepared in example 27.
The amino donor compound is tert-butylamine and phenethyl ammonia according to the weight ratio of 2: 1, in a mixture of the components. The weak oxidant compound is organic peroxy acid and copper oxide, and the weight ratio of the organic peroxy acid to the copper oxide is 3: 1, in a mixture of the components. The temperature of the catalytic reaction was 38 ℃; the pH of the catalytic reaction is 7.0, the two catalytic steps are carried out simultaneously, and the catalytic reaction time is 28 h. The concentration of substrate glucose in the reaction system is 10 g/L; the concentration of glucose isomerase in the reaction system is 6U/mL; the transaminase concentration was 4U/mL. The reaction system also contained pyridoxal phosphate as a cofactor, at a concentration of 1.0 mM. The concentration of the amino donor compound was 2 mM; the concentration of the weak oxidant compound was 10 mM. The reaction system also contains MOPS buffer solution or citrate buffer solution, and the concentration of the buffer solution is 100 mM.
Example 31, a method for the enzymatic preparation of glucosamine:
the method comprises the following steps: d-glucose was converted to D-fructose using the glucose isomerase enzyme prepared in example 27; and converting D-fructose into glucosamine using the transaminase, the amino donor compound, and the weak oxidant compound prepared in example 27.
The amino donor compound is D-alanine and phenethyl ammonia according to the weight ratio of 1: 2, and (b) a mixture of the components. The weak oxidant compound is phosphite and copper oxide, wherein the weight ratio of phosphite to copper oxide is 4: 1, in a mixture of the components. The temperature of the catalytic reaction is 70 ℃; the pH of the catalyzed reaction was 6. The two catalytic steps are carried out in steps, and the catalytic reaction time of each step is independent of each other for 10 hours. The concentration of substrate glucose in the reaction system is 50 g/L; the concentration of glucose isomerase in the reaction system is 15U/mL; the transaminase concentration was 12U/mL. The reaction system also contains the cofactor pyridoxal phosphate, the concentration of which is 2 mM. The concentration of the amino donor compound was 3mM and the concentration of the weak oxidant compound was 20 mM. The reaction system also contains HEPES buffer solution with the concentration of 200 mM.
Example 32, experiment for the preparation of glucosamine using glucose isomerase and transaminase catalysis:
recombinant glucose isomerase and transaminase were prepared as in example 27. Glucosamine was quantitatively analyzed by High Performance Liquid Chromatography (HPLC). The chromatographic column is amino column, the mobile phase is 80% acetonitrile water solution, the flow rate is 1mL/min, the column temperature is 40 ℃, and the detector is a Waters2414 refractive index detector. The glucosamine standard sample retention time is about 10.3 min. Glucosamine concentration is proportional to the response intensity of the HPLC characteristic peak of glucosamine. 1mL of a reaction mixture containing 10g/L D-glucose, 2mM isopropylamine or D-alanine, 100mM HEPES buffer (pH 7.0), 1mM pyridoxal phosphate, 10mM phosphite or organic peroxyacid, 5U/mL glucose isomerase, 5U/mL transaminase was reacted at 37 ℃ for 24 hours. After the reaction is finished, adding acetonitrile with the same volume to the reaction system to stop the reaction, centrifuging at 12000 r/min for 10min, and taking supernate to measure the concentration of glucosamine in the reaction solution through high performance liquid chromatography. When the reaction was carried out for 24 hours, the glucosamine concentration was 7.0g/L and the conversion was 70%.
The invention is not limited by the specific text described above. The invention can be varied within the scope outlined by the claims and these variations are within the scope of the invention.