CN113046401A - Method for preparing glucosamine by biological enzyme catalysis - Google Patents
Method for preparing glucosamine by biological enzyme catalysis Download PDFInfo
- Publication number
- CN113046401A CN113046401A CN202110282228.4A CN202110282228A CN113046401A CN 113046401 A CN113046401 A CN 113046401A CN 202110282228 A CN202110282228 A CN 202110282228A CN 113046401 A CN113046401 A CN 113046401A
- Authority
- CN
- China
- Prior art keywords
- glucosamine
- concentration
- glucose
- reaction
- genbank accession
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
Abstract
The invention discloses 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. The method comprises the following steps: converting D-glucose into D-fructose by adopting glucose isomerase or xylose isomerase as an enzyme; and converting D-fructose to glucosamine using a transaminase, an amino donor, and a weak oxidant. Glucose isomerase and transaminase were derived from 4 sources, respectively. The invention relates to a 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 preparation is relatively easy to obtain and low in price. The method adopts a one-pot method for catalysis, the substrate and the enzyme start to react after being added, and the final product glucosamine is directly obtained after the reaction is finished, so that the method has the advantages of low production cost, environmental friendliness and safety.
Description
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 overcome the defects of the existing method for producing and preparing the glucosamine, and provides a novel method for producing and preparing the glucosamine by in vitro catalysis by utilizing biological enzymes. 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 purpose of the invention is realized by the following technical scheme:
the invention provides a method for preparing glucosamine by biological enzyme catalysis, which takes D-glucose as a raw material and realizes the preparation of the glucosamine by in vitro catalysis by using biological enzyme, and 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.
According to the invention, the process comprises a reaction step for converting D-glucose into D-fructose, which is catalyzed by a glucose isomerase, or xylose isomerase, capable of isomerizing aldoses such as D-glucose, D-xylose and D-ribose into the corresponding ketose, which may be derived from Streptomyces olivaceus (S.)Streptomyces olivochromogenesGenBank accession No. KUN 44528), Bacillus coagulans (B.coagulans)Bacillus coagulansGenBank accession number KYC 85466), Flavobacterium arborescens (Flavobacterium arborescensGenBank accession number OAZ 44030), Streptomyces olivaceus (S.olivaceus) ((S.olivaceus)Streptomyces olivochromogenesGenBank accession number KUN 44528).
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 and a weak oxidant compound for transaminationThe enzyme may be derived from Bacillus pumilus (B.) (Bacillus pumilusGenBank accession number SNV 15900), Bacillus licheniformis (Bacillus licheniformis)Bacillus licheniformisGenBank accession number VEH 77736), Streptococcus salivarius (S.salivarius: (S.salivarius)Streptococcus salivariusGenBank accession No. KXU 58123), Lactobacillus Marylanica (Lactobacillus maliGenBank No. KRN 26726), pyridoxal phosphate (PLP) may be used as a cofactor, the amino donor compound is one, or any mixture of two or more of D-alanine, isopropylamine, tert-butylamine and phenethylamine, and the weak oxidant compound is one, or any mixture of two or more of phosphite, organic peroxy acid and copper oxide.
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 ℃ and preferably 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 24 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 6 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 35 g/L.
According to the invention, the concentration of the glucose isomerase in the reaction system is 0.1-20U/mL, preferably 3-8U/mL;
according to the invention, the concentration of transaminase in the reaction system is from 0.1 to 20U/mL, preferably from 3 to 8U/mL.
According to the present invention, the concentration of pyridoxal phosphate (PLP), a cofactor, in the reaction system is 0.1-2mM, preferably 0.8-1.2 mM;
according to the present invention, the concentration of the amino donor in the reaction system is 0.1 to 3mM, preferably 1.0 to 2.5 mM;
according to the present invention, the concentration of the weak oxidizing agent in the reaction system is 0.5 to 20mM, preferably 8 to 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 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 relates to a 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 preparation 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 of the preferred embodiments
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 Streptomyces olivorubidus (Streptomyces olivochromogenesGenBank accession number KUN 44528), or Bacillus coagulans (B.coagulans)Bacillus coagulansGenBank accession number KYC 85466), or Flavobacterium arborescens (Flavobacterium arborescensGenBank accession number OAZ 44030), 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. 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. coli BL21(DE3)/pColdII-AT, after verification 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 enzymatic preparation of glucosamine Using glucose isomerase and transaminase
The Streptomyces olivaceus strain prepared in example 2 was taken (Streptomyces olivochromogenesGenBank accession number KUN 44528) and recombinant glucose isomerase originating from a short shootBacillus (A), (B), (C)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 4 enzymatic preparation of glucosamine Using glucose isomerase and transaminase
The Bacillus coagulans-derived material prepared in example 2 is taken (Bacillus coagulansGenBank accession number KYC 85466) 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 3 to obtain glucosamine having a concentration of 7.0g/L and a conversion of 72%.
Example 5 enzymatic preparation of glucosamine Using glucose isomerase and transaminase
Taking Flavobacterium arborescens (F) as the source prepared in example 2Flavobacterium arborescensGenBank accession OAZ 44030) and from Streptococcus salivarius (A. salivarius)Streptococcus salivariusGenBank accession No. KXU 58123). The reaction was carried out for 48 hours in the same manner as in example 3. The glucosamine concentration was 8.0g/L and the conversion was 80%.
Example 6 enzymatic preparation of glucosamine Using glucose isomerase and transaminase
The Streptomyces olivaceus strain prepared in example 2 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 3, and the reaction was carried out for 24 hours. The glucosamine concentration was 7.8g/L and the conversion was 78%.
Example 7, glucosamine preparation of glucosamine hydrochloride:
to the aqueous glucosamine solution obtained in example 3, 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 8, glucosamine preparation of glucosamine sulfate:
to the aqueous glucosamine solution obtained in example 4, 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 9 preparation of glucosamine sulfate Potassium chloride Complex salt with glucosamine:
to the aqueous glucosamine solution obtained in example 5, 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 10 preparation of glucosamine sulfate glucosamine sodium chloride complex salt with glucosamine:
to the aqueous glucosamine solution obtained in example 6, 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 11 preparation of glucosamine sulfate calcium chloride Complex salt with glucosamine:
to the aqueous glucosamine solution obtained in example 3, 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 12 glucosamine preparation of glucosamine phosphate:
to the aqueous glucosamine solution obtained in example 4, 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 13 preparation of acetylglucosamine from glucosamine:
to the aqueous glucosamine solution or mixed solvent obtained in example 5, 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 14 preparation of acetylglucosamine from glucosamine:
an appropriate amount of a catalyst was added to the aqueous glucosamine solution or mixed solvent obtained in example 6, and then acetyl chloride was reacted in an equimolar or molar excess amount for 4 to 6 hours after completion of the acylation reaction. Concentrating to remove part of the solvent, crystallizing, separating after crystallization, washing, and drying to obtain the acetylglucosamine.
Example 15, a method for the enzymatic preparation of glucosamine by a biological enzyme:
glucose isomerase and transaminase were prepared as described in example 2.
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 16, a method for the enzymatic preparation of glucosamine by a biological enzyme:
glucose isomerase and transaminase were prepared as described in example 2.
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 the medicine is derived 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 17, a method for the enzymatic preparation of glucosamine by a biological enzyme:
glucose isomerase and transaminase were prepared as described in example 2.
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 14, a method for the enzymatic preparation of glucosamine by a biological enzyme:
glucose isomerase and transaminase were prepared as described in example 2.
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%.
Claims (10)
1. A method for preparing glucosamine by biological enzyme catalysis, which is characterized by comprising 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 streptomyces olivaceus (Streptomyces olivaceus) (III)Streptomyces olivochromogenes) GenBank accession number KUN 44528; or from Bacillus coagulans (Bacillus coagulans) GenBank accession number KYC 85466; orDerived 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 pumilus (B), (B)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 method for preparing glucosamine catalyzed by biological enzymes according to claim 1, wherein: the amino donor compound is selected from 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 process for the enzymatic production of glucosamine by a biological enzyme according to claim 1 or 2, wherein: the temperature of the catalytic reaction is 20-70 ℃, and preferably 30-37 ℃; the pH for the catalytic reaction is 4.0-9.0, preferably 6.7-7.5.
4. The process for the enzymatic production of glucosamine by a biological enzyme according to claim 1 or 2, wherein: when the two catalysis steps are carried out simultaneously, the catalytic reaction time is 1-48h, preferably 20-24 h; when the two catalytic steps are carried out stepwise, the catalytic reaction times of the individual steps are carried out independently of one another over a period of from 1 to 15 hours, preferably from 6 to 8 hours.
5. The process for the enzymatic production of glucosamine by a biological enzyme according to claim 1 or 2, wherein: the concentration of substrate glucose in the reaction system is 1-200g/L, preferably 8-35 g/L; the concentration of the glucose isomerase in the reaction system is 0.1-20U/mL, preferably 3-8U/mL; the concentration of transaminase in the reaction system is 0.1-20U/mL, preferably 3-8U/mL.
6. The process for the enzymatic production of glucosamine by a biological enzyme according to claim 1 or 2, wherein: the reaction system also contains the cofactor pyridoxal phosphate, the concentration of which is 0.1-2mM, preferably 0.8-1.2 mM.
7. The process for the enzymatic production of glucosamine by a biological enzyme according to claim 1 or 2, wherein: the concentration of the amino donor compound is 0.1-3mM, preferably 1.0-2.5 mM; the concentration of the weak oxidant compound is 0.5-20mM, preferably 8-12 mM.
8. The process for the enzymatic production of glucosamine by a biological enzyme according to claim 1 or 2, wherein: the reaction system also contains a buffer solution, wherein the buffer solution is selected from a HEPES buffer solution, a Tris-HCl buffer solution, a MOPS buffer solution or a citrate buffer solution, and the concentration of the buffer solution is 20-300 mM, preferably 80-150 mM.
9. The process for the enzymatic production of glucosamine by a biological enzyme according to claim 1 or 2, wherein: the D-fructose is converted into glucosamine, including pure glucosamine, glucosamine salts and acetylglucosamine.
10. The method for preparing glucosamine catalyzed by biological enzymes according to claim 9, wherein: the glucosamine salt is 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.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110282228.4A CN113046401A (en) | 2021-03-16 | 2021-03-16 | Method for preparing glucosamine by biological enzyme catalysis |
| CN202111235321.6A CN113817788B (en) | 2021-03-10 | 2021-10-22 | Enzymatic preparation method of glucosamine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110282228.4A CN113046401A (en) | 2021-03-16 | 2021-03-16 | Method for preparing glucosamine by biological enzyme catalysis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113046401A true CN113046401A (en) | 2021-06-29 |
Family
ID=76513135
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110282228.4A Pending CN113046401A (en) | 2021-03-10 | 2021-03-16 | Method for preparing glucosamine by biological enzyme catalysis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113046401A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113817788A (en) * | 2021-03-10 | 2021-12-21 | 江苏澳新生物工程有限公司 | Enzymatic preparation method of glucosamine |
| WO2023004772A1 (en) * | 2021-07-30 | 2023-02-02 | 武汉百氨汇基生物技术有限公司 | Fructosamine deglycase vector, transgenic cell line and genetically engineered bacterium expressing fructosamine deglycase, and use of fructosamine deglycase |
-
2021
- 2021-03-16 CN CN202110282228.4A patent/CN113046401A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113817788A (en) * | 2021-03-10 | 2021-12-21 | 江苏澳新生物工程有限公司 | Enzymatic preparation method of glucosamine |
| CN113817788B (en) * | 2021-03-10 | 2024-02-02 | 江苏澳新生物工程有限公司 | Enzymatic preparation method of glucosamine |
| WO2023004772A1 (en) * | 2021-07-30 | 2023-02-02 | 武汉百氨汇基生物技术有限公司 | Fructosamine deglycase vector, transgenic cell line and genetically engineered bacterium expressing fructosamine deglycase, and use of fructosamine deglycase |
| EP4151742A4 (en) * | 2021-07-30 | 2023-03-22 | Wuhan Bai ' Anhuiji Biotechnology Co., Ltd | Fructosamine deglycase vector, transgenic cell line and genetically engineered bacterium expressing fructosamine deglycase, and use of fructosamine deglycase |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113046401A (en) | Method for preparing glucosamine by biological enzyme catalysis | |
| CN109652484B (en) | Method for efficiently catalytically synthesizing L-carnosine by whole cells | |
| CN108342374B (en) | Chitinase and application thereof | |
| KR20210031678A (en) | Enzymatic preparation method of glucosamine | |
| CN109593805B (en) | Method for synthesizing L-carnosine by using L-amino acid ligase one-step method | |
| CN112795605A (en) | Method for preparing glucosamine through enzyme catalysis | |
| CN101633931A (en) | Hyaluronidase expression vector and application thereof | |
| CN109022408B (en) | A kind of novel algin catenase Aly08 and its application | |
| CN114480350A (en) | Application of carrageenase in degrading kappa-carrageenan and furcellaran | |
| CN107384990A (en) | A kind of method that external enzyme law catalysis heparosan prepares heparin | |
| JP2024515083A (en) | Enzyme composition for preparing β-nicotinamide mononucleotide and its application | |
| CN103627691B (en) | A kind of immobilization glutathione synthetase and its preparation and application | |
| CN113817788A (en) | Enzymatic preparation method of glucosamine | |
| CN114410611B (en) | Kunmu polysaccharide degrading enzyme OUC-BsLam26 and application thereof | |
| CN112553272B (en) | Method for improving hyaluronic acid yield | |
| US20210238576A1 (en) | L-aspartate alpha-decarboxylase Mutant and Application thereof | |
| CN109609536B (en) | Method for synthesizing L-carnosine by whole cells in one step | |
| CN113151377A (en) | Enzymatic preparation method for preparing glucosamine from glucose and enzyme application | |
| CN114196655A (en) | Heat-resistant Kunmu polysaccharide degrading enzyme OUC-SaLam66 and application thereof | |
| CN114015675A (en) | Lambda-carrageenase OUC-LuV and application thereof | |
| CN114231511A (en) | Dianhydrofructose hydrolase mutant E160F with improved heat stability | |
| Ojima et al. | Production of value-added materials from alginate using alginate lyases and 4-deoxy-l-erythro-5-hexoseulose uronic acid–metabolic enzymes from alginolytic bacteria and marine gastropods | |
| JPH06261754A (en) | N-acetyl-d-glucosamine deacetylase | |
| JP6171598B2 (en) | Process for producing β-mannoside | |
| CN114891772B (en) | Deacetylase mutant and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20210629 |
|
| WD01 | Invention patent application deemed withdrawn after publication |