CN111909973B - Method for synthesizing D- (+) -glucuronic acid through biocatalysis and application of D- (+) -glucuronic acid - Google Patents
Method for synthesizing D- (+) -glucuronic acid through biocatalysis and application of D- (+) -glucuronic acid Download PDFInfo
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Abstract
The invention discloses a method for synthesizing D- (+) -glucuronic acid by biological catalysis and application thereof. The method comprises the steps of taking sucrose as a substrate, adding enzyme for catalytic reaction to obtain D- (+) -glucuronic acid; wherein the enzyme comprises sucrose phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase, inositol monophosphatase and inositol oxidase, the enzyme catalysis system can obtain high conversion rate, and in addition, the conversion rate of D- (+) -glucuronic acid can be further improved by adding phosphate, phosphofructokinase and phosphoglucose isomerase into the reaction system. The method has the advantages of high utilization rate of raw materials, high conversion rate of D- (+) -glucuronic acid, simple steps, low production cost, little pollution and little influence on the environment, and can realize the large-scale production of the D- (+) -glucuronic acid.
Description
Technical Field
The invention belongs to the field of enzyme catalysis production of D- (+) -glucuronic acid, and particularly relates to a method for synthesizing D- (+) -glucuronic acid through biocatalysis and application thereof.
Background
D- (+) -glucuronic acid (C)6H10O7D-glucuronic acid), which is a compound formed by oxidizing a primary alcohol hydroxyl group of glucose to form a carboxyl group, is unstable in aqueous solution and is easily converted to form 3,6 gluconolactone, and the two are in a tautomeric equilibrium state in aqueous solution. Glucuronic acid in the presence of a strong acid under heatingSusceptible to decarboxylation to CO2Furan, and the like.
Glucuronic acid is widely present in animals and plants and has important biological functions. It can combine with toxic substance containing hydroxyl, amino, carboxyl, sulfhydryl and other groups, enhance the aqueous solution of toxic substance, and discharge it out of kidney quickly, thus playing the role of detoxification. Meanwhile, the glucuronic acid can be combined with an active group-phenolic hydroxyl group on the molecule of the combined substance, so that the biological effects of related hormones and medicines are reduced. The derivative glucuronolactone is also a health care medicine, which is beneficial to the detoxification of liver, can prevent and treat epidemic hepatitis, liver cirrhosis, food and drug poisoning, and is also an additive of functional beverage and health care food. With the increasing health concept and the increasing demand for biomass, the demand for glucuronic acid is increasing.
The current production methods of D- (+) -glucuronic acid mainly comprise: chemical oxidation, polysaccharide hydrolysis and biological fermentation.
The chemical oxidation method mainly adopts inorganic oxidation or catalytic oxidation, and the main method for industrially producing glucuronic acid and lactone in China at present is the inorganic oxidation method: the inorganic oxidation method has poor selectivity, the oxidation degree can not be effectively controlled, the product is difficult to separate and recycle, and the environmental pollution is serious; the main components of the catalytic oxidation are homogeneous catalytic oxidation and heterogeneous catalytic oxidation.
The polysaccharide hydrolysis method is to hydrolyze polyuronic acid or polysaccharide containing uronic acid existing in nature by acid or alkali, and extract and separate to obtain uronic acid. However, since the glycosidic bond between glucuronic acids is very stable, strong reaction conditions such as addition of strong acid or strong base are required, but under such conditions, glucuronic acid is broken and the recovery rate is low.
The biological fermentation method is a method for producing glucuronic acid by fermenting a specific substance capable of forming or presupposing glucuronic acid with a specific strain or a biological enzyme. At present, relatively few researches on the production of glucuronic acid by a biological fermentation method are carried out, and the microbial fermentation method has the defects of long production period, difficult separation and purification and the like; the enzymatic synthesis has the advantages of short production period, simple separation and purification, small environmental pollution, high specificity and the like, and the enzymatic synthesis has a great development prospect in producing the glucuronic acid.
Therefore, it is highly desirable to develop a new method for producing D- (+) -glucuronic acid with low cost, low pollution and high yield.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for synthesizing D- (+) -glucuronic acid by biocatalysis, the method is used for producing the D- (+) -glucuronic acid by catalyzing sucrose with low cost by using in vitro multi-enzyme, and the method has the advantages of high yield of the D- (+) -glucuronic acid, high conversion rate of raw materials, low production cost, no pollution and the like.
Another object of the present invention is to provide the use of said method for the biocatalytic synthesis of D- (+) -glucuronic acid.
The purpose of the invention is realized by the following technical scheme:
a method for synthesizing D- (+) -glucuronic acid by biological catalysis comprises the steps of taking sucrose as a substrate, adding enzyme for catalytic reaction to obtain D- (+) -glucuronic acid; wherein the content of the first and second substances,
the enzymes include Sucrose phosphorylase (SP, EC:2.4.1.7), Phosphoglucomutase (PGM, EC:5.4.2.2), Inositol-1-phosphate synthase (Inositol-1-phosphate synthase, MIPS, EC:5.5.1.4), Inositol monophosphatase (IMP, EC:3.1.3.25) and Inositol oxidase (Myo-Inositol oxydagenase, MIOX, EC: 1.13.99.1).
The dosage of the sucrose is calculated according to the addition of the sucrose in the reaction system with the final concentration of 10-60 mmol/L; preferably, the final concentration of the catalyst in the reaction system is 30-50 mmol/L.
The dosage of the sucrose phosphorylase is calculated according to the addition of the sucrose phosphorylase in the final concentration of 0-10U/mL of the reaction system (the dosage of the sucrose phosphorylase is not zero); preferably, the addition is calculated according to the final concentration of the compound in the reaction system being 1-10U/mL; more preferably, it is calculated by adding it at a final concentration of 1U/mL in the reaction system.
The dosage of the phosphoglucomutase is calculated according to the addition of the phosphoglucomutase in the final concentration of 0-10U/mL of the reaction system (the dosage of the phosphoglucomutase is not zero); preferably, the addition is calculated according to the final concentration of the compound in the reaction system being 1-10U/mL; more preferably, it is calculated by adding it at a final concentration of 1U/mL in the reaction system.
The dosage of the inositol-1-phosphate synthetase is calculated according to the addition of the inositol-1-phosphate synthetase in the final concentration of 0-10U/mL of the reaction system (the dosage of the enzyme is not zero); preferably, the addition is calculated according to the final concentration of the compound in the reaction system of 2-10U/mL; more preferably, it is calculated by adding it at a final concentration of 2U/mL in the reaction system.
The dosage of the inositol monophosphatase is calculated according to the addition of the inositol monophosphatase in the final concentration of 0-10U/mL in the reaction system (the dosage of the inositol monophosphatase is not zero); preferably, the addition is calculated according to the final concentration of the compound in the reaction system being 1-10U/mL; more preferably, the concentration of the catalyst is calculated according to the addition of the catalyst in the reaction system, wherein the final concentration of the catalyst is 1-2U/mL.
The dosage of the inositol oxidase is calculated according to the addition of the inositol oxidase in the final concentration of 0-10U/mL in the reaction system (the dosage of the enzyme is not zero); preferably, the final concentration of the catalyst in the reaction system is 5-10U/mL.
The enzyme also includes glucose isomerase and phosphoglucokinase.
The dosage of the glucose isomerase is calculated according to the addition of the glucose isomerase in the reaction system with the final concentration of 0-10U/mL (the dosage of the enzyme is not zero); preferably, the addition is calculated according to the final concentration of the compound in the reaction system being 1-10U/mL; more preferably, it is calculated by adding it at a final concentration of 1U/mL in the reaction system.
The dosage of the phosphoglucokinase is calculated according to the addition of the phosphoglucokinase in the final concentration of 0-10U/mL of the reaction system (the dosage of the enzyme is not zero); preferably, the addition is calculated according to the final concentration of the compound in the reaction system being 1-10U/mL; more preferably, it is calculated by adding it at a final concentration of 1U/mL in the reaction system.
The catalytic reaction is carried out in a buffer solution system.
The buffer solution is one of Tris-HCl buffer solution, PBS buffer solution and HEPES buffer solution; preferably 50-350 mmol/L Tris-HCl buffer solution; more preferably 250mmol/L Tris-HCl buffer solution.
The system for catalyzing the reaction also contains phosphate (inorganic phosphate ion PO)4 3+) Metal ions and cysteine.
The phosphate is disodium hydrogen phosphate (NaH)2PO4)。
The concentration of the disodium hydrogen phosphate in the catalytic reaction system is 10-50 mmol/L; preferably 20 mmol/L.
The metal ion is Mg2+，Ca2+，Fe2+，Co2+，Cu2+And Mn2+At least one of; preferably Mg2+And Fe2 +At least one of; more preferably Mg2+。
The concentration of metal ions in the catalytic reaction system is 2-5 mmol/L; preferably 2 mmol/L.
The cysteine is preferably L-cysteine.
The concentration of cysteine in the catalytic reaction system is 1-2 mmol/L; preferably 2 mmol/L.
The temperature of the catalytic reaction is 20-50 ℃; preferably 35 deg.c.
The time of the catalytic reaction is 20-80 hours; preferably 60 hours.
The pH value of the catalytic reaction is 5.5-8.5; preferably 7.5.
The catalytic reaction system is a 2mL catalytic reaction system and comprises the following components:
50-350 mmol/L Tris-HCl buffer solution, 10-50 mmol/L NaH2PO42-5 mmol/L metal ion, 1-2 mmol/L L-cysteine, 0-10U/mL Sucrose Phosphorylase (SP), 0-10U/mL Phosphoglucomutase (PGM), 0-10U/mL inositol-1-phosphate synthase (MIPS), 0-10U/mL Inositol Monophosphatase (IMP) and 0-10U/ML Inositol Oxidase (MIOX), 10-60 mmol/L sucrose, and pH 7.5; wherein the dosage of the enzyme is not zero.
The metal ion is Mg2+，Ca2+，Fe2+，Co2+，Cu2+And Mn2+At least one of; preferably Mg2+And Fe2 +At least one of; more preferably Mg2+。
The system of the catalytic reaction is a 2mL catalytic reaction system, and the composition is preferably as follows:
50-350 mmol/L Tris-HCl buffer solution, 20mmol/L NaH2PO42mmol/L metal ion, 2mmol/L L-cysteine, 1U/mL Sucrose Phosphorylase (SP), 1U/mL Phosphoglucomutase (PGM), 2U/mL inositol-1-phosphate synthase (MIPS), 1-2U/mL Inositol Monophosphatase (IMP) and 5-10U/ML Inositol Oxidase (MIOX), 30-50 mmol/L sucrose, and pH 7.5.
The metal ion is Mg2+，Ca2+，Fe2+，Co2+，Cu2+And Mn2+At least one of; preferably Mg2+And Fe2 +At least one of; more preferably Mg2+。
The system of the catalytic reaction is a 2mL catalytic reaction system, and the composition is further preferably as follows:
50-350 mmol/L Tris-HCl buffer solution, 10-50 mmol/L NaH2PO42-5 mmol/L metal ion, 1-2 mmol/L L-cysteine, 0-10U/mL Sucrose Phosphorylase (SP), 0-10U/mL Phosphoglucomutase (PGM), 0-10U/mL inositol-1-phosphate synthase (MIPS), 0-10U/mL Inositol Monophosphatase (IMP) and 0-10U/ML Inositol Oxidase (MIOX), 0-10U/mL Glucose Isomerase (GI), 0-10U/mL phosphoglucokinase (PPGK), 10-60 mmol/L sucrose, and pH 7.5; wherein the dosage of the enzyme is not zero.
The metal ion is Mg2+，Ca2+，Fe2+，Co2+，Cu2+And Mn2+At least one of; preferably Mg2+And Fe2 +At least one of; more preferably Mg2+。
The system of the catalytic reaction is a 2mL catalytic reaction system, and the composition is further preferably as follows:
50-350 mmol/L Tris-HCl buffer solution, 20mmol/L NaH2PO42mmol/L metal ion, 2mmol/L L-cysteine, 1U/mL Sucrose Phosphorylase (SP), 1U/mL Phosphoglucomutase (PGM), 2U/mL inositol-1-phosphate synthase (MIPS), 1-2U/mL Inositol Monophosphatase (IMP), 5-10U/ML Inositol Oxidase (MIOX), 1U/mL Glucose Isomerase (GI), 1U/mL phosphoglucokinase (PPGK), 30-50 mmol/L sucrose, and pH 7.5.
The metal ion is Mg2+，Ca2+，Fe2+，Co2+，Cu2+And Mn2+At least one of; preferably Mg2+And Fe2 +At least one of; more preferably Mg2+。
The application of the method for synthesizing D- (+) -glucuronic acid by biological catalysis in the production of D- (+) -glucuronic acid.
The invention takes sucrose as substrate, adds sucrose phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase, inositol monophosphatase and inositol oxidase, prepares a multi-enzyme reaction system, and the multi-enzyme catalysis approach comprises: converting one D-glucose unit in sucrose into glucose-1-phosphate by sucrose phosphorylase and producing fructose; glucose-1-phosphate is converted to glucose-6-phosphate by phosphoglucomutase, glucose-6-phosphate is converted to inositol-1-phosphate by inositol-1-phosphate synthase, inositol-1-phosphate is converted to inositol by inositol monophosphatase, and inositol is converted to D- (+) -glucuronic acid by inositol oxidase. The whole reaction is exothermic, i.e. thermodynamically feasible, so that the enzyme catalytic system can obtain high conversion rate.
In the experiment of converting sucrose into D- (+) -glucuronic acid by in vitro multi-enzyme catalysis, in a reaction system, sucrose is used as a raw material, system reaction enzyme is added, and the reaction is carried out for 60 hours at 35 ℃, so that the atom conversion rate of the sucrose finally reaches 80%, and the utilization rate of the raw material is obviously improved. In addition, the conversion rate of D- (+) -glucuronic acid can be improved by adding phosphate, phosphofructokinase and phosphoglucose isomerase to the reaction system; more preferably, the invention adds glucose phosphate isomerase, phosphate and phosphofructokinase into the multi-enzyme system to convert fructose into glucose-6-phosphate, and finally converts all sugar units in sucrose into D- (+) -glucuronic acid by inositol-1-phosphate synthase, inositol monophosphatase and inositol oxidase, thereby further improving the yield and conversion rate of D- (+) -glucuronic acid.
The terms and definitions to which this invention relates:
unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term "enzyme-catalyzed reaction" refers to a chemical reaction that is carried out under the action of a biocatalyst-enzyme.
Compared with the prior art, the invention has the following advantages and effects:
(1) the preparation method of D- (+) -glucuronic acid disclosed by the invention takes cane sugar with wide sources as a substrate, and the substrate is converted into the D- (+) -glucuronic acid by virtue of high-efficiency catalysis of in vitro multiple enzymes in a multiple enzyme reaction system.
(2) In the in vitro synthesis route of the D- (+) -glucuronic acid, the specific activity and the action strength of each enzyme are different, the catalytic efficiency is improved by optimizing the optimal proportion of each enzyme, namely, the process is optimized, and the enzyme capable of utilizing sucrose is added, so that an optimized multienzyme system is established, the conversion efficiency of raw materials and the yield of the D- (+) -glucuronic acid can be obviously improved, the yield and the conversion rate are high, and the separation cost of the D- (+) -glucuronic acid is greatly reduced.
(3) The method has the advantages of high utilization rate of raw materials, high conversion rate of D- (+) -glucuronic acid, namely high yield of D- (+) -glucuronic acid, simple steps, low production cost, little pollution and little influence on the environment, and can realize the large-scale production of D- (+) -glucuronic acid.
Drawings
FIG. 1 is a diagram of the conversion of sucrose to D- (+) -glucuronic acidSchematic diagram of in vitro multiple enzyme catalytic pathway of acid (in the figure, SP: sucrose phosphorylase; PGM: phosphoglucomutase; MIPS: inositol-1-phosphate synthase; IMP: inositol monophosphatase; MIOX: inositol oxidase; P)i: a phosphorus molecule).
FIG. 2 is a diagram showing the results of SDS-PAGE (lane M: protein Marker; SP: sucrose phosphorylase; MIOX: inositol oxidase; PGM: phosphoglucomutase; MIPS: inositol-1-phosphate synthase; IMP: inositol monophosphatase).
FIG. 3 is a graph showing the results of HPLC analysis of D- (+) -Glucuronic acid (Glucose-1-phosphate, Glucose-6-phosphate, Fructose, D- (+) -Glucuronic acid and inositol are distinguished by HPLC).
FIG. 4 is a schematic diagram of the process-optimized in vitro multienzyme catalytic pathway for the conversion of sucrose to D- (+) -glucuronic acid (in the figure, SP: sucrose phosphorylase; PGM: phosphoglucomutase; GI: glucose isomerase; PPGK: phosphoglucokinase; MIPS: inositol-1-phosphate synthase; IMP: inositol monophosphatase; MIOX: inositol oxidase; P.sub.P.sub.i: a phosphorus molecule).
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
1. The experimental materials involved in the examples of the present invention are as follows:
sucrose (Sucrose) was purchased from Sigma, product number 424490020; pET28a vector was purchased from Novagen, Madison (Madison), Wis (Wis., USA); coli expression host strain BL21(DE3) was purchased from holo-type gold Biotech, Inc.
2. Sucrose phosphorylase, glucose phosphoglucomutase, inositol-1-phosphate synthase, inositol monophosphatase and inositol oxidase in the catalytic system related to the embodiment of the invention can be obtained by prokaryotic expression according to a genetic engineering method; glucose isomerase (Glucose isomerase, GI, EC 5.3.1.5) was purchased from Sigma, and assigned product number G4166.
3. Enzyme activity (U) in the present invention: the amount of enzyme required to convert 1 micromole of substrate in 1 minute is one activity unit (U).
Example 1 preparation of enzyme
1. Preparation of sucrose phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase, inositol monophosphatase and inositol oxidase:
in the present invention, Sucrose phosphorylase (Sucrose phosphorylase, SP, EC:2.4.1.7) is derived from E.coli BL21(DE3) and has NCBI accession No. NC-012892.2; phosphoglucomutase (PGM, EC:5.4.2.2) is derived from B.methnololus PB1(Bacillus methnololus PB1), and NCBI accession number is NZ _ AFEU01000003.1: 850496-; inositol-1-phosphate synthase (MIPS, EC:5.5.1.4) is derived from S.cerevisiae (Saccharomyces cerevisiae S288C ) with NCBI accession number Chromosome: X; NC _001142.9(134332.. 135933); inositol monophosphatase (Inositol monophophasse, IMP, EC:3.1.3.25) is derived from B.methnoillus PB1 with NCBI accession No. NZ _ AFEU01000003.1:122318 and 123091; inositol oxidase (myo-inositol oxidase, MIOX, EC:1.13.99.1) is derived from a. thaliana (Arabidopsis thaliana), with NCBI accession numbers: NP-194356.2 (myo-inositol oxygenase 4). These genomic DNAs are all available from the official website of NCBI (https:// www.ncbi.nlm.nih.gov /). The 5 genes were obtained from the corresponding genomic DNA by PCR using different primers, and ligated to the vector pET28a by digestion to obtain the corresponding expression vectors pET28a-SP, pET28a-PGM, pET28a-IMP, pET28a-MIPS, pET28 a-MIOX. And the 5 plasmids are transformed into an escherichia coli expression bacterium BL21(DE3) and subjected to protein expression and purification, and the results of protein purification are shown in figure 2. Wherein, the related primer sequences are as follows:
SP-F:5’-GGCGGATCCATGAAACAGAAAATTACGGATTACCT-3’；
SP-R:5’-GGCAAGCTTTTATTTAATCCACATAACCTGCCAGG-3’；
PGM-F:5’-CGGGATCCATGGCTGAAAAGTGGATTGCCGGGGT-3’；
PGM-R:5’-CGCCTCGAGTTACATTTGGTCAATTTTATTCTT-3’；
MIPS-F:5’-GAATTCATGACAGAAGATAATATTGCTC-3’；
MIPS-R:5’-AAGCTTCTACAACAATCTCTCTTCG-3’；
IMP-F:5’-CGGGATCCATGCAATACGAATGGAACCAAATACT-3’；
IMP-R:5’-CGCCTCGAGTCATTCATCAATCATCCCATCCAAT-3’；
MIOX-F:5’-CGGATATCGATGAAAGTTGATGTTGGTCCT-3’；
MIOX-R:5’-CCGCTCGAGTTACCAGGACAGGGTGCCCG-3’。
2. preparation of phosphoglucokinase:
phosphoglucokinase (PPGK), wherein the NCBI (phosphoglucokinase) accession number is NC-007333.1, the source is Thermobifida fuscaYX, the gene is obtained by cloning PCR and is connected to pET28a through an enzyme cutting site to obtain a corresponding expression vector pET28a-TfuPPGK, and the expression vector is transformed into escherichia coli host bacteria BL21(DE3) to carry out protein expression and purification to obtain the protein. Wherein, the related primer sequences are as follows:
PPGK-F:
5’-TTAACTTTAAGAAGGAGATATACATATGGTGAGCAAGGGCGAGGAGGAT-3’；
PPGK-R：
5’-AGTGGTGGTGGTGGTGGTGCTCGAGGGTAAATTTGATATTGTCCAGGCT-3’。
example 2 in vitro Multi-enzyme catalysis of the conversion of sucrose to D- (+) -glucuronic acid
The invention converts sucrose into D- (+) -glucuronic acid by an in vitro multi-enzyme catalytic system (figure 1). These key enzymes include:
1) sucrose Phosphorylase (SP) catalyzing the decomposition of sucrose into glucose-1-phosphate and fructose;
2) phosphoglucomutase (PGM), which catalyzes the conversion of glucose-1-phosphate to glucose-6-phosphate;
3) inositol-1-phosphate synthase (MIPS), which catalyzes the conversion of glucose-6-phosphate to inositol-1-phosphate;
4) inositol Monophosphatase (IMP) which catalyzes the dephosphorylation of inositol-1-phosphate into inositol;
5) inositol Oxidase (MIOX), which catalyzes inositol to D- (+) -glucuronic acid.
Wherein, the first step and the second step are reversible, and the subsequent reactions are irreversible, so the enzyme catalysis system can obtain high conversion rate.
The specific experimental steps are as follows:
(1) sucrose phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase, inositol monophosphatase and inositol oxidase were prepared as in example 1.
(2) A2.0 ml reaction was carried out with 50mM Tris-HCl buffer, 20mM NaH2PO4，5mmol/L MgCl22mmol/L L-cysteine (L-cysteine), 1U/mL SP, 1U/mL PGM, 2U/mL MIPS, 2U/mL IMP and 5U/mL MIOX, 50mmol/L sucrose, the pH of the catalytic reaction system is 7.5, the catalytic reaction is carried out at 35 ℃, and the reaction is carried out for 60 hours.
(3) D- (+) -glucuronic acid was analyzed by HPLC (high performance liquid chromatography, column HPX-87H (300 mM. ANG.7.8 mM. ANG.9. mu.m), detector refractivity time difference, mobile phase 5mM dilute sulfuric acid, isocratic elution, flow rate 0.5mL/min, sample amount 10uL, column temperature 65 ℃ C.) according to the reaction time. HPLC can be used to distinguish D- (+) -glucuronic acid, fructose, glucose-1-phosphate or glucose-6-phosphate in the reaction solution, and D- (+) -glucuronic acid can be quantified. The results are shown in FIG. 3: the concentration of D- (+) -glucuronic acid is proportional to the intensity of the characteristic peak of D- (+) -glucuronic acid in HPLC. After the reaction was completed, the final concentration of D- (+) -glucuronic acid was 12.1mM, and the sucrose atom conversion rate was 24.2%.
Example 3 in vitro Multi-enzyme catalysis of the conversion of sucrose to glucuronic acid
(1) Sucrose phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase, inositol monophosphatase and inositol oxidase were prepared as in example 1.
(2) A2.0 ml reaction containing 150mM Tris-HCl buffer, 20mM NaH2PO4，5mmol/L MgCl22mmol/L L-cysteine, 1U/mL SP, 1U/mL PGM, 2U/mL MIPS, 1U/mL IMP and 5U/mL MIOX, 50mmol/L sucrose, the pH of the catalytic reaction system is 7.5, the catalytic reaction is carried out at 35 ℃, and the reaction is carried out for 60 hours.
(3) After the reaction, the final concentration of D- (+) -glucuronic acid was 19.5mM (the quantitative method was the same as in example 2), and the sucrose atom conversion rate reached 39%.
Example 4 catalysis of sucrose conversion to D- (+) -glucuronic acid by in vitro multienzyme by Condition optimization
In order to further improve the conversion rate of the D- (+) -glucuronic acid, the invention optimizes a multienzyme reaction system, and ensures that the raw material sucrose is fully degraded, thereby improving the yield and the conversion rate of the D- (+) -glucuronic acid. The specific experimental steps are as follows:
(1) sucrose phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase, inositol monophosphatase and inositol oxidase were prepared as in example 1.
(2) In a 2.0 ml optimized reaction system containing 200mM Tris-HCl buffer, 20mM NaH2PO4，5mmol/L MgCl22mmol/L L-cysteine, 1U/mL SP, 1U/mL PGM, 2U/mL MIPS, 2U/mL IMP and 5U/mL MIOX, 50mmol/L sucrose, the pH of the catalytic reaction system is 7.5, the catalytic reaction is carried out at 35 ℃, and the reaction is carried out for 60 hours.
(3) After the reaction, the final concentration of D- (+) -glucuronic acid was 36mM (the quantitative method was the same as in example 2), and the sucrose atom conversion rate reached 72%.
Example 5 conversion of sucrose to D- (+) -glucuronic acid by in vitro multienzyme catalysis by Condition optimization
The process is as in example 3, except that: the initial concentration of Tris-HCl buffer solution in the catalytic reaction system is 100mmol/L, and the catalytic reaction temperature is 20 ℃,30 ℃, 35 ℃,40 ℃, 45 ℃ and 50 ℃.
After the reaction, the final concentration of D- (+) -glucuronic acid was 12mM,14mM,15mM,9mM,2mM,0.5mM (the same quantitative method as in example 2), and the conversion rate of sucrose atoms was 24%, 28%, 30%, 18%, 4%, 1%.
Example 6 catalysis of sucrose conversion to D- (+) -glucuronic acid by in vitro multienzyme by Condition optimization
The process is as in example 3, except that: the initial concentration of Tris-HCl buffer solution in the catalytic reaction system is 100mmol/L, and the pH of the catalytic reaction system is 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5.
After the reaction, the final concentration of D- (+) -glucuronic acid was 12mM,15mM,17mM,19.8mM,21mM,10mM,4mM (the quantitative method was the same as in example 2), and the conversion rate of sucrose atoms was 24%, 30%, 34%, 39.6%, 42%, 20%, 8%.
Example 7 conversion of sucrose to D- (+) -glucuronic acid by in vitro multienzyme catalysis by Condition optimization
The process is as in example 3, except that: the initial concentration of Tris-HCl buffer solution in the catalytic reaction system is 100mmol/L, and 5mmol/L MgCl is added2Respectively replaced by 2mmol/L MgCl2，2mmol/L CaCl2，2mmol/L FeCl2，2mmol/L CoCl2，2mmol/L CuCl2，2mmol/L MnCl。
After the reaction, the final concentration of D- (+) -glucuronic acid was 19mM, 14.8mM, 19.8mM, 11.8mM, 12.8mM, 10.8mM (the quantitative method was the same as in example 2), and the conversion rate of sucrose atoms was 38%, 9.6%, 39.6%, 23.6%, 25.6%, 21.6%.
Example 8 in vitro catalysis of sucrose conversion to D- (+) -glucuronic acid by multienzyme optimization
The process is as in example 3, except that: the initial concentration of Tris-HCl buffer solution in the catalytic reaction system is 100mmol/L, and 50mmol/L of sucrose as a substrate is replaced by 20mmol/L, 30mmol/L, 40mmol/L, 50mmol/L and 60mmol/L respectively.
After the reaction, the final concentrations of D- (+) -glucuronic acid were 10mM, 12mM,15mM, 20mM, and 11mM (the quantitative method was the same as in example 2), and the conversion rates of sucrose atoms were 20%, 24%, 30%, 40%, and 22%.
Example 9 catalysis of the conversion of sucrose to D- (+) -glucuronic acid by in vitro Multi-enzymes by Condition optimization
The process is as in example 3, except that: the buffer solution in the catalytic reaction system is 50mmol/L, 100mmol/L, 150mmol/L, 200mmol/L, 250mmol/L, 300mmol/L and 350mmol/L Tris-HCl buffer solution respectively.
After the reaction, the final concentration of D- (+) -glucuronic acid was 6mM,15mM,19.5mM,28mM,19mM,12mM,8mM (the quantitative method was the same as in example 2), and the conversion rate of sucrose atoms was 12%, 30%, 39%, 56%, 38%, 24%, 16%.
Example 10 conversion of sucrose to D- (+) -glucuronic acid by in vitro multienzyme catalysis by Process optimization
A schematic of the conversion of sucrose to D- (+) -glucuronic acid by a process-optimized in vitro multi-enzyme catalytic system is shown in figure 4.
The specific experimental steps are as follows:
(1) sucrose phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase, inositol monophosphatase, inositol oxidase and phosphoglucokinase were prepared as in example 1.
(2) In a 2.0 ml optimized reaction system containing 200mM Tris-HCl buffer, 20mM NaH2PO4，5mmol/L MgCl22mmol/L L-cysteine, 1U/mL SP, 1U/mL GI (glucose isomerase), 1U/mL PPGK, 1U/mL PGM, 2U/mL MIPS, 2U/mL IMP and 5U/mL MIOX, 50mmol/L sucrose, the pH of the catalytic reaction system is 7.5, and the catalytic reaction is carried out at 35 ℃ for 60 hours.
(3) After the reaction, the final concentration of D- (+) -glucuronic acid was 38.2mM (the quantitative method was the same as in example 2), and the sucrose atom conversion rate reached 76.4%.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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Claims (3)
1. A method for synthesizing D- (+) -glucuronic acid by biocatalysis is characterized in that: taking sucrose as a substrate, adding enzyme for catalytic reaction to obtain D- (+) -glucuronic acid; wherein the enzymes include sucrose phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase, inositol monophosphatase and inositol oxidase;
the catalytic reaction system is 2 mL and comprises the following components:
50-350 mmol/L Tris-HCl buffer solution, 10-50 mmol/L NaH2PO42-5 mmol/L metal ions, 1-2 mmol/L L cysteine, 1-10U/mL sucrose phosphorylase, 1-10U/mL phosphoglucomutase, 2-10U/mL inositol-1-phosphate synthase, 1-10U/mL inositol monophosphatase, 5-10U/mL inositol oxidase, 30-50 mmol/L sucrose, and the pH is 5.5-8.5;
the metal ion is Mg2+，Ca2+，Fe2+，Co2+ ，Cu2+And Mn2+At least one of;
The temperature of the catalytic reaction is 20-40 ℃;
the time of the catalytic reaction is 20-80 hours.
2. The process for the biocatalytic synthesis of D- (+) -glucuronic acid according to claim 1, characterized in that:
the enzyme also comprises glucose isomerase and phosphoglucokinase;
the catalytic reaction system is 2 mL and comprises the following components:
50-350 mmol/L Tris-HCl buffer solution, 10-50 mmol/L NaH2PO42-5 mmol/L metal ion, 1-2 mmol/L L-cysteine, 1-10U/mL sucrose phosphorylase, 1-10U/mL phosphoglucomutase, 2-10U/mL inositol-1-phosphate synthase, 1-10U/mL inositol monophosphatase, 5-10U/mL inositol oxidase, 1-10U/mL glucose isomerase, 1-10U/mL phosphoglucokinase, 30-50 mmol/L sucrose, and pH 7.5.
3. Use of the method of biocatalytic synthesis of D- (+) -glucuronic acid according to any of claims 1-2 for the production of D- (+) -glucuronic acid.
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| CN105925643A (en) * | 2016-04-15 | 2016-09-07 | 武汉康复得生物科技股份有限公司 | Method for preparing inositol through enzymic catalysis and inositol |
| CN106148292A (en) * | 2016-07-06 | 2016-11-23 | 江南大学 | A kind of inositol oxygenase mutant and application thereof |
| CN108085344A (en) * | 2016-11-23 | 2018-05-29 | 中国科学院天津工业生物技术研究所 | It is a kind of using sucrose as the inositol preparation method of raw material |
| CN110857443A (en) * | 2018-08-24 | 2020-03-03 | 中国科学院天津工业生物技术研究所 | Method for producing inositol by complete phosphorization of cellulose |
| CN110964757A (en) * | 2018-09-30 | 2020-04-07 | 中国科学院天津工业生物技术研究所 | Preparation method of glucaric acid |
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| CN105925643A (en) * | 2016-04-15 | 2016-09-07 | 武汉康复得生物科技股份有限公司 | Method for preparing inositol through enzymic catalysis and inositol |
| CN106148292A (en) * | 2016-07-06 | 2016-11-23 | 江南大学 | A kind of inositol oxygenase mutant and application thereof |
| CN108085344A (en) * | 2016-11-23 | 2018-05-29 | 中国科学院天津工业生物技术研究所 | It is a kind of using sucrose as the inositol preparation method of raw material |
| CN110857443A (en) * | 2018-08-24 | 2020-03-03 | 中国科学院天津工业生物技术研究所 | Method for producing inositol by complete phosphorization of cellulose |
| CN110964757A (en) * | 2018-09-30 | 2020-04-07 | 中国科学院天津工业生物技术研究所 | Preparation method of glucaric acid |
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