CN115216500A - Method for synthesizing 2' -fucosyllactose - Google Patents
Method for synthesizing 2' -fucosyllactose Download PDFInfo
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- 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
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- 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
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- 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 provides a method for synthesizing 2' -fucosyllactose, belonging to the technical field of enzyme engineering. The invention utilizes glucose as a carbon source to synthesize mannose-6-phosphate, and synthesizes 2'-fucosyllactose through alpha-1, 2-fucosyltransferase and the like, thereby not only reducing the production cost, but also having the characteristics of short reaction time, easily controlled conditions, purer product and higher yield, providing a feasible way for industrial production of 2' -fucosyllactose, and having stronger research value and social and economic benefits. Experimental results show that the 2' -fucosyllactose synthesized by the method has the production amount of 14.56g/L and the yield of 96.75%.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a method for synthesizing 2' -fucosyllactose.
Background
Human Milk Oligosaccharides (HMOs) are the third most nutritive substance in human milk next to lactose and fat, and are present in milk at about 15%. Current research shows that breast milk oligosaccharides have a variety of beneficial effects on newborns. Breast milk oligosaccharides can be used as a novel food additive in infant formula. 2' -fucosyllactose (2 ' -FL) has better function than other beneficial groups, therefore, industrial synthesis of 2' -FL and increasing yield and reducing production cost have become the current research focus. In early scientific research, 2' -FL was extracted from breast milk, cow milk and sheep milk to obtain a small amount of 2' -fucosyllactose, but this is far from meeting the production requirement, so realizing industrial production of 2' -FL is an inevitable requirement for the development of infant formula.
At present, the synthesis methods of 2' -FL are widely divided into two methods: chemical synthesis method and enzymatic synthesis method. In the prior art 1 (ago K, hederos M J, bajza I, et al, kilogram scale chemical synthesis of 2' -fucosylase [ J ] Carbohydrate Research, 2019, 476 71-77.) 2' -FL is synthesized by a chemical synthesis method, although the method is easy to amplify production and has high product purity, the operation steps are complex and difficult to understand, the conditions required by the reaction process are high, and the yield of the high-purity 2' -FL is only 19.8% -27.3%. The enzymatic synthesis method is still in the exploration stage, and the prior art adopts GDP-fucose and lactose to generate 2'-FL by group replacement catalyzed by FUT2 or synthesizes 2' -FL by using alpha-L-fucosidase. The former uses fucose with higher price, which causes higher production cost and lack of practical value, resulting in difficulty in realizing 2' -FL production on a large scale, and also with lower yield, as in prior art 2 (Albermann C, piepersberg W, wehmeier U F. Synthesis of the milk oligosaccharide 2' -hydrolytic using recombinant bacterial enzymes [ J ]. Carbohydrate Research, 2001, 334 (2): 97-103.) the yield of 2' -FL is 65%. Although the latter method can solve the problem of raw material cost by taking pNP-fucose as a glycosyl donor, the transglycosylation efficiency of the enzyme is low, the method cannot be applied to large-scale synthesis of 2' -FL, and the pNP in a reaction product cannot be applied to the field of food. Therefore, the development of safe and inexpensive fucosyl substrates and transglycosidases with high transglycosidation efficiency will be the research direction for realizing the industrial synthesis of 2' -fucosyllactose by alpha-L-fucosidase in the future.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for synthesizing 2' -fucosyllactose, which has the advantages of low cost, short preparation time and high yield.
In order to achieve the above purpose, the invention provides the following technical scheme:
a method of synthesizing 2' -fucosyllactose, the method comprising: (1) Mixing glucose, ATP, 6-phosphoglucokinase, phosphoglucose isomerase and phosphomannose isomerase, and reacting to obtain mannose-6-phosphate; (2) Mannose-6-phosphate, lactose, NADPH, GTP, phosphomannomutase, mannose-1-phosphoguanyltransferase, GDP-mannose-6-dehydrogenase, GDP-fucose synthetase and alpha-1, 2-fucosyltransferase are mixed and reacted to obtain 2' -fucosyllactose.
Preferably, the glucose concentration in the step (1) is 8.00-12.00g/L, and the ATP concentration is 25.00-30.00g/L; the concentration of 6-phosphoglucokinase is 3-5mg/L; the concentration of the phosphoglucose isomerase is 3-8mg/L; the concentration of phosphomannose isomerase is 3-8mg/L.
Preferably, the reaction temperature of the step (1) is 20-30 ℃, the pH value is 6-8, and the time is 20-30h.
Preferably, the pH value in the step (1) is adjusted by a phosphate buffer solution, and the concentration of the phosphate buffer solution is 2.00-5.00g/L.
Preferably, the concentration of the mannose-6-phosphate in the step (2) is 6.00 to 10.00g/L; the lactose concentration is 15-20g/L; NADPH concentration is 20.00-25.00g/L; the GTP concentration is 13.00-18.00g/L; the concentration of the phosphomannose mutase is 1.00-3.00g/L; the concentration of mannose-1-phosphate guanylyltransferase is 1.00-3.00g/L; the concentration of GDP-mannose-6-dehydrogenase is 1.00-3.00g/L; the concentration of the GDP-fucose synthetase is 1.00-3.00g/L; the concentration of alpha-1, 2-fucosyltransferase is 1.00-3.00g/L.
Preferably, the reaction temperature of the step (2) is 20-30 ℃, the pH value is 7-9, and the time is 20-30h.
Preferably, the pH value in the step (2) is adjusted by a phosphate buffer solution, and the concentration of the phosphate buffer solution is 3.00-8.00g/L.
Preferably, the alpha-1, 2-fucosyltransferase, phosphomannomutase, mannose-1-phosphoguanyltransferase, GDP-mannose-6-dehydrogenase and GDP-fucose synthetase are obtained by expression and purification of recombinant engineering bacteria.
Compared with the prior art, the invention has the following beneficial effects:
the invention utilizes glucose as a carbon source to synthesize mannose-6-phosphate, and synthesizes 2'-fucosyllactose through alpha-1, 2-fucosyltransferase and the like, the reaction process is easy to control, the product is purer, the yield is higher, a feasible way is provided for the industrial production of the 2' -fucosyllactose, the production method reduces the production cost, and has stronger basic theoretical research value and social and economic benefits. Experimental results show that the method for synthesizing the 2'-fucosyllactose has the advantages that the yield of the 2' -fucosyllactose is 14.56g/L, the purity is 95.26 percent, and the yield is as high as 96.75 percent.
Drawings
FIG. 1 is a diagram of the reaction path of the in vitro multi-enzyme cascade catalytic synthesis of the present invention;
FIG. 2 shows the electrophoretic patterns of marker, manB, manC, gmd, wcAG proteins; marker, manB, manC, gmd and wcAG are arranged from left to right in sequence;
FIG. 3 is an electrophoretogram of the futC protein;
FIG. 4 is a graph showing the progress of mannose-6-phosphate;
FIG. 5 is a progress curve of 2' -fucosyllactose.
Detailed Description
The invention provides a method for synthesizing 2' -fucosyllactose, which comprises the following steps: (1) Mixing glucose, ATP, 6-phosphoglucokinase, phosphoglucose isomerase and phosphomannose isomerase, and reacting to obtain mannose-6-phosphate; (2) Mannose-6-phosphate, lactose, NADPH, GTP, phosphomannomutase, mannose-1-phosphoguanyltransferase, GDP-mannose-6-dehydrogenase, GDP-fucose synthetase and alpha-1, 2-fucosyltransferase are mixed and reacted to obtain 2' -fucosyllactose.
The mechanism of the method for synthesizing 2'-fucosyllactose is shown in figure 1, and the 2' -fucosyllactose is obtained by reacting in-vitro multi-enzyme cascade catalytic synthesis.
In the present invention, the glucose concentration in step (1) is preferably 8.00 to 12.00g/L, more preferably 10g/L; the ATP concentration is preferably 25.00-30.00g/L, more preferably 28.50g/L; the concentration of 6-phosphoglucokinase is preferably 3-5mg/L, more preferably 4mg/L; the concentration of the phosphoglucose isomerase is preferably 3-8mg/L, and more preferably 5mg/L; the concentration of the phosphomannose isomerase is preferably 3-8mg/L, and more preferably 6mg/L; the reaction temperature is preferably 20-30 ℃, more preferably 25 ℃, the pH value is preferably 6-8, more preferably 7, and the time is preferably 20-30h, more preferably 24h; the pH value is preferably adjusted by means of a phosphate buffer, the concentration of which is preferably from 2.00 to 5.00g/L, more preferably 4.00g/L.
In the present invention, the concentration of mannose-6-phosphate in the step (2) is preferably 6.00 to 10.00g/L, more preferably 8.00g/L; the lactose concentration is 15-20g/L, and more preferably 17g/L; NADPH concentration is 20.00-25.00g/L, more preferably 23.00g/L; the GTP concentration is 13.00-18.00g/L, and more preferably 16g/L; the concentration of the phosphomannose mutase is 1.00-3.00g/L, and more preferably 2.00g/L; the concentration of the mannose-1-phosphate guanyltransferase is 1.00-3.00g/L, more preferably 2.00g/L; the concentration of the GDP-mannose-6-dehydrogenase is 1.00 to 3.00g/L, and the concentration is more preferably 2.00g/L; the concentration of the GDP-fucose synthetase is 1.00-3.00g/L, and more preferably 2.00g/L; the concentration of alpha-1, 2-fucosyltransferase is 1.00-3.00g/L, more preferably 2.00g/L; the reaction temperature is preferably 20-30 ℃, more preferably 25 ℃, the pH value is preferably 7-9, more preferably 8, and the time is preferably 20-30h, more preferably 24h; the pH value is preferably adjusted by means of a phosphate buffer, the concentration of which is preferably from 3.00 to 8.00g/L, more preferably 6.0g/L.
The source of the alpha-1, 2-fucosyltransferase is not particularly limited in the present invention, and any commercially available product that is conventional in the art may be used. The 2'-fucosyllactose synthesized by the alpha-1, 2-fucosyltransferase can not only reduce the dosage of the substrate and the production cost, but also greatly improve the reaction yield, and improve the yield of the 2' -fucosyllactose in the same reaction time to 96.75 percent. As an embodiment, the alpha-1, 2-fucosyltransferase, phosphomannomutase, mannose-1-phosphoguanyltransferase, GDP-mannose-6-dehydrogenase and GDP-fucose synthetase are obtained by expression and purification of recombinant engineered bacteria.
Compared with the prior art, the invention has the following technical advantages: 1) The product has high purity, and is convenient to separate and purify. Compared with in vivo synthesis, the invention does not produce secondary metabolite, and is beneficial to separation and purification of the product. 2) Low cost and high yield. Compared with in vivo synthesis, the engineering bacteria of the invention do not need part of saccharides for growth and propagation, and the yield is improved. 3) The reaction process is easier to control. Compared with the complex in vivo anabolic growth and metabolism network, the method is simple, and has few influencing factors, so that the regulation and control difficulty of the metabolic synthesis process is small, and the production process is easier to control.
The raw materials which are not mentioned in the invention are not specially limited, and the conventional commercial products in the field can be adopted.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the examples futC is alpha-1, 2-fucosyltransferase, manB is phosphomannomutase, manC is mannose-1-phosphoguanyltransferase, gmd is GDP-mannose-6-dehydrogenase, wcaG is GDP-fucose synthetase.
Example 1
Recombinant expression of 2' -fucosyllactose synthesis-related enzymes
1. Construction of plasmids
Coding genes of futC enzyme (GenBank: ABO 61750.1) and futCB enzyme (GenBank: WP-002174293) are subjected to codon optimization and synthesis, nde I and Xho I are respectively inserted into corresponding sites of a plasmid pET-28a vector, and digestion analysis, identification and screening are carried out to obtain corresponding recombinant plasmids.
2. Recombinant bacterium for preparing 2' -fucosyllactose synthesis related enzyme
Introducing recombinant plasmids containing futC and futCB gene sequences into escherichia coli competent cells C43 and JM109 by a chemical method, performing recovery culture, performing coating culture in an LB solid culture medium added with 1 per thousand Kana, performing overnight culture at 37 ℃, selecting colonies with good growth vigor, performing monoclonal culture, and identifying whether the required recombinant bacteria are obtained.
3. Inducible expression of 2' -fucosyllactose-related synthetase
The successfully transformed recombinant strain is inoculated on 4 mL of liquid culture medium and cultured for 8h (1 per mill kana 4 mu L is required to be added into a test tube) by a constant temperature shaking table at 37 ℃ and 180 rpm. Then, 500. Mu.L of the plasmid was inoculated into 50mL of a medium (containing 50. Mu.L of kana) and shake-cultured at 37 ℃ for 4 hours. Expression was induced by adding 100. Mu.L of IPTG to each flask and culturing overnight at 25 ℃ for 12 h. The cells in the shake flask were centrifuged at 8000 rpm and 4 ℃ for 5 min, the supernatant was discarded, and the cells were collected. The cells were resuspended in 6 mL of Tirs-HCl (pH 8.5) buffer. Then the bacterial cells are broken by ultrasonic waves, the parameters are set to be 15 min, and the power is 30 percent. The supernatant was centrifuged at 13000 rpm for 20 min at 4 ℃. Run time 3s with an interval time of 3s. Crushing and collecting the obtained supernatant, namely the target enzyme solution.
4. Separating and purifying target protein by Ni column affinity chromatography
(1) SDS-PAGE protein electrophoresis verification of induced expression result
1) Preparing a sample: the preparation ratio of the protein electrophoresis sample loading buffer solution to the target enzyme solution is 1;
2) Combining: boiling the sample in water for 10min;
3) Electrophoresis: add marker 10mL and add sample 15 mL to different tracks. The voltage is 120U, the current is 400 mA, and the waiting time is 1 h;
4) Dyeing: dyeing with Coomassie brilliant blue, and heating for 3 min;
5) And (3) decoloring: pouring decolored solution, heating, repeatedly decoloring for 12h, and observing by gel imaging system, wherein the specific results are shown in figures 1-2.
As can be seen from FIGS. 2-3, 2' -fucosyllactose-related enzymes manB, manC, gmd, wcaG, alpha-1, 2-fucosyltransferase futC and futCB which can be synthesized by the strain per se are successfully expressed, and as can be seen from the figure, the molecular weights of crude manB and manC are about 50kDa, the molecular weights of gmd and wcaG are about 34 kDa, and the molecular weights of futC and futCB proteins are about 30 kDa; the expression effect of the futC and futCB enzyme introduced into the competent cell TM109 by the recombinant vector is better than that of the C43 competent strain. Compared with futCB, the expression band of futC is clearest and the expression amount is more, so the experiment selects to purify futC protein and develops the experiment subsequently.
(2) Purification of proteins of alpha-1, 2-fucosyltransferase (futC enzyme), phosphomannomutase (manB), mannose-1-phosphoguanyltransferase (manC), GDP-mannose-6-dehydrogenase (gmd) and GDP-fucose synthetase (wcaG).
Purifying the target enzyme liquid part by utilizing an affinity chromatography method to obtain futC, manB, manC, gmd and wcaG enzyme proteins, wherein the specific process is as follows:
1) Filtering the supernatant with a 0.45 μm diameter filter;
2) Washing with Ni column: washing the resin with ultrapure water of 6-12 times of column volume at a flow rate of 100 cm/h to remove ethanol;
3) Ni column balance: equilibrating the medium with 6-12 times column volume equilibration buffer (20 Mm Tirs-HCl pH 8.5, 400 mM NaCl,20 mM imidazole) at a flow rate of 120 cm/h to ensure that the medium contains solution components and pH consistent with the sample;
4) Sampling: centrifuging and filtering the sample, and slowly loading the sample at the flow speed of 120 cm/h;
5) Impurity washing: eluting nonspecific protein with 12-24 times column volume buffer solution (20 Mm Tirs-HCl pH 8.5, 400 mM NaCl,20 mM imidazole) at flow rate of 120 cm/h, collecting flow-through solution for subsequent analysis;
6) And (3) elution: eluting protein with 6-12 times column volume of elution buffer (20 Mm Tirs-HCl pH 8.5, 200 mM NaCl,20 mM imidazole) at low flow rate, collecting eluate for subsequent analysis;
7) And (3) dialysis: loading the collected eluate into dialysis bag, and placing in dialysate (20 mM Tirs-HCl pH 8.5, 100 mM NaCl,20 mM imidazole);
8) And (3) storage: the dialyzed futC, manB, manC, gmd, wcAG enzyme proteins were stored at-20 ℃.
Example 2
Preparation of 2' -fucosyllactose
1. In vitro multienzyme catalytic conversion of glucose to synthesize mannose-6-phosphate
10.00g/L glucose, 28.50g/L ATP, 0.004 g/L6-phosphoglucokinase, 0.005g/L phosphoglucose isomerase, 0.006g/L phosphomannose isomerase and 4.00g/L phosphate buffer solution (pH 7.0) are mixed, stirred uniformly and reacted at 25 ℃ for 24h to obtain mannose-6-phosphate. The volume of the reaction system in this reaction was 50mL.
2. 2' -fucosyllactose synthesized by converting mannose-6-phosphate under catalysis of multienzyme
The concentration of mannose-6-phosphate was adjusted to 8.00g/L, and 8.00g/L mannose-6-phosphate, 17g/L lactose, 23.00g/L NADPH, 16.00g/L GTP, 2.00g/L phosphomannose mutase, 2.00g/L mannose-1-phosphoguanyltransferase, 2.00g/L GDP-mannose-6-dehydrogenase, 2.00g/L GDP-fucose synthase, and 2.00g/L α -1, 2-fucosyltransferase were put in 6.00g/L phosphate buffer (pH 8.0), stirred uniformly, and reacted at 25 ℃ for 24 hours to obtain 2' -fucosyllactose. The volume of the reaction system in this reaction was 50mL.
Example 3
1. In vitro multi-enzyme catalytic conversion of glucose to synthesize mannose-6-phosphate
Mixing 12.00g/L glucose, 30.00g/L ATP, 5 mg/L6-phosphoglucokinase, 8mg/L phosphoglucose isomerase, 8mg/L phosphomannose isomerase and 8.00g/L phosphate buffer solution, stirring uniformly, and reacting at 30 ℃ for 20h to obtain mannose-6-phosphate. The volume of the reaction system in this reaction was 50mL.
2. Synthesis of 2' -fucosyllactose by multi-enzyme catalytic conversion of mannose-6-phosphate
Adjusting the concentration of mannose-6-phosphate to 10.00g/L, placing 10.00g/L mannose-6-phosphate, 20g/L lactose, 25.00g/L NADPH, 18.00g/L GTP, 3.00g/L phosphomannose mutase, 3.00g/L mannose-1-phosphoguanyltransferase, 3.00g/L GDP-mannose-6-dehydrogenase, 3.00g/L GDP-fucose synthetase, 3.00g/L alpha-1, 2-fucosyltransferase in 3.00g/L phosphate buffer, stirring well, and reacting at 30 deg.C for 20h to obtain 2' -fucosyllactose. The volume of the reaction system in this reaction was 50mL.
Example 4
1. In vitro multienzyme catalytic conversion of glucose to synthesize mannose-6-phosphate
Mixing 8.00g/L glucose, 25.00g/L ATP, 3 mg/L6-phosphoglucokinase, 3mg/L phosphoglucose isomerase, 3mg/L phosphomannose isomerase and 5.00g/L phosphate buffer solution, stirring uniformly, and reacting at 20 ℃ for 30h to obtain mannose-6-phosphate. The volume of the reaction system in this reaction was 50mL.
2. Synthesis of 2' -fucosyllactose by multi-enzyme catalytic conversion of mannose-6-phosphate
The concentration of mannose-6-phosphate was adjusted to 6.00g/L, and 6.00g/L of mannose-6-phosphate, 15g/L of lactose, 20.00g/L of NADPH, 13.00g/L of GTP, 1.00g/L of phosphomannomutase, 1.00g/L of mannose-1-phosphoguanyltransferase, 1.00g/L of GDP-mannose-6-dehydrogenase, 1.00g/L of GDP-fucose synthetase, 1.00g/L of alpha-1, 2-fucosyltransferase were placed in 8.00g/L of phosphate buffer, stirred uniformly, and reacted at 20 ℃ for 30 hours to obtain 2' -fucosyllactose. The volume of the reaction system in this reaction was 50mL.
In examples 2 to 4, phosphomannomutase, mannose-1-phosphoguanyltransferase, GDP-mannose-6-dehydrogenase, GDP-fucose synthase and α -1, 2-fucosyltransferase were all prepared in example 1.
Experimental example 1
HPLC determination of mannose-6-phosphate, 2' -fucosyllactose production
HPLC high performance liquid chromatograph is SCIEX Triple quad 5500; and (3) chromatographic column: ACQUITY UPLC BEN C18.7 μm 2.1mm × 50mm; mobile phase: solution A (ammonia 10% (v/v)), solution B (acetonitrile); flow rate: 0.4 mL/min, detector: a differential refractive detector.
In the 0h, 2.5h, 4.5h, 8h, 12h, 18h and 24h of the reaction in step 1 or step 2 of example 3, taking a proper amount of reaction liquid, centrifuging at 12000rpm at 4 ℃ for 10min, collecting supernatant, preparing mannose-6-phosphate and 2'-fucosyllactose standard solutions, filtering the sample supernatant and the standard solutions after appropriate dilution by a 0.22 mu m microporous filter membrane, and respectively determining the contents of mannose-6-phosphate and 2' -fucosyllactose by HPLC high performance liquid chromatography and differential refraction, wherein specific results are shown in FIG. 4-5.
The calculation method of the HPLC detection substance concentration comprises the following steps: and (3) diluting the mannose-6-phosphate and 2'-fucosyllactose standard substances to different concentrations from high to low, detecting the mannose-6-phosphate and 2' -fucosyllactose standard substances with different concentrations by using HPLC (high performance liquid chromatography), obtaining the retention time of corresponding peaks of the two substances and peak areas corresponding to different concentrations, and making a standard curve according to the peak areas and the concentrations. And (3) carrying out HPLC (high performance liquid chromatography) detection on the pretreated sample supernatant, finding peaks in a corresponding retention time range, namely peaks of mannose-6-phosphate and 2'-fucosyllactose in the sample, and substituting the peak areas into a standard curve equation to calculate the concentrations of the mannose-6-phosphate and 2' -fucosyllactose in the sample.
As can be seen from FIGS. 4 to 5, in step 1 of example 3, the accumulation of the product was fast within 0 to 8 hours, the accumulation of the product was gradually slow within 8 to 24 hours, and the amount of mannose-6-phosphate produced was 13.84g/L at 24 hours of the reaction.
If all of the reactants are converted to products, the mass concentration of the product mannose-6-phosphate is the same as the mass concentration of the reactant glucose, about 0.0555mol/L. Converting the mass concentration of the substance into mass concentration to obtain the mannose-6-phosphate with the theoretical yield of 14.44g/L, wherein the ratio of the actual yield to the theoretical yield is the yield, and the calculated yield of the mannose-6-phosphate is 95.85 percent; in the step 2, the product accumulation is rapid after 0-8h, the product concentration tends to be gentle after 8h, and the yield of the obtained 2' -fucosyllactose is 14.56g/L when the reaction is carried out for 24 h. If the reactants are all converted into the product, the mass concentration of the product 2' -fucosyllactose is the same as the mass concentration of the reactant mannose-6-phosphate, which is about 0.0308mol/L. The theoretical yield of 2'-fucosyllactose obtained by converting the mass concentration of the substances into the mass concentration is 15.05g/L, the ratio of the actual yield to the theoretical yield is the yield, and the calculated yield of 2' -fucosyllactose is 96.74%. Drying the reaction liquid to constant weight, weighing 50mg of solid, dissolving the solid in 10ml of ultrapure water, performing HPLC detection on the obtained solution, and calculating the concentration of 2' -fucosyllactose in the solution to be 4.763g/L, namely that 50mg of solid product contains 47.63mg of 2' -fucosyllactose, the purity of the 2' -fucosyllactose is 95.26%, and the yield is 96.75%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A method of synthesizing 2' -fucosyllactose, comprising:
(1) Mixing glucose, ATP, 6-phosphoglucokinase, phosphoglucose isomerase and phosphomannose isomerase, and reacting to obtain mannose-6-phosphate;
(2) Mixing mannose-6-phosphate, lactose, NADPH, GTP, phosphomannose mutase, mannose-1-phosphate guanyltransferase, GDP-mannose-6-dehydrogenase, GDP-fucose synthetase and alpha-1, 2-fucosyltransferase, and reacting to obtain 2' -fucosyllactose.
2. The method according to claim 1, wherein the glucose concentration in step (1) is 8.00-12.00g/L; the ATP concentration is 25.00-30.00g/L; the concentration of 6-phosphoglucokinase is 3-5mg/L; the concentration of the phosphoglucose isomerase is 3-8mg/L; the concentration of phosphomannose isomerase is 3-8mg/L.
3. The method according to claim 2, wherein the reaction in step (1) is carried out at a temperature of 20-30 ℃, a pH of 6-8 and a time of 20-30h.
4. The method according to claim 3, wherein the pH of the reaction of step (1) is adjusted by a phosphate buffer solution having a concentration of 2.00 to 5.00g/L.
5. The method according to claim 1, wherein the concentration of mannose-6-phosphate in the step (2) is 6.00 to 10.00g/L; the lactose concentration is 15-20g/L; NADPH concentration is 20.00-25.00g/L; the GTP concentration is 13.00-18.00g/L; the concentration of the phosphomannose mutase is 1.00-3.00g/L; the concentration of mannose-1-phosphate guanyltransferase is 1.00-3.00g/L; the concentration of GDP-mannose-6-dehydrogenase is 1.00-3.00g/L; the concentration of the GDP-fucose synthetase is 1.00-3.00g/L; the concentration of alpha-1, 2-fucosyltransferase is 1.00-3.00g/L.
6. The method of claim 5, wherein the reaction in step (2) is carried out at a temperature of 20-30 ℃, a pH of 7-9, and a time of 20-30 hours.
7. The method according to claim 6, wherein the pH of the reaction of step (2) is adjusted by a phosphate buffer solution having a concentration of 3.00 to 8.00g/L.
8. The method of claim 1, wherein the alpha-1, 2-fucosyltransferase, phosphomannomutase, mannose-1-phosphate guanyltransferase, GDP-mannose-6-dehydrogenase, and GDP-fucose synthetase are purified by expression from a recombinant engineered bacterium.
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