CN115094103A - Novel process for producing galactose by combined enzyme method by taking sucrose, starch or glycogen as raw materials - Google Patents
Novel process for producing galactose by combined enzyme method by taking sucrose, starch or glycogen as raw materials Download PDFInfo
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- 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
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- 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
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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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
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Abstract
The invention discloses a novel process for producing galactose by a combined enzyme method by using sucrose, starch or glycogen as raw materials, belonging to the field of enzyme engineering. The galactose is produced by taking sucrose, starch or glycogen as raw materials, the chemical reaction is catalyzed by combining a plurality of enzymes to generate reaction intermediate UDP-glucose through enzyme catalysis, and the UDP-glucose is catalyzed by the enzymes to generate UDP-galactose; enzyme hydrolysis of UDP-galactose produces UDP and galactose. Compared with the existing method for producing galactose by using lactose as a raw material, the method has the advantages of low cost, simple material acquisition and the like.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a novel process for producing galactose by a combined enzyme method by using sucrose, starch or glycogen as raw materials.
Background
Galactose has important application in food industry and the like. Galactose can be added into milk powder to regulate intestinal flora of infants and adults, and improve immunity. Galactose can also form galactooligosaccharide, stimulate the metabolism of probiotics and promote the absorption of nutrients by intestines and stomach of human body. Galactose is also an important constituent group of many biological macromolecules in the human body. In addition, galactose can be converted to other more expensive sugars, such as tagatose, etc., by chemical or enzymatic catalysis.
The most common method for the preparation of galactose is by chemical or enzymatic hydrolysis of lactose. However, lactose is limited in its source and can generally only be extracted from milk. This results in a less than high yield of lactose and thus galactose. Finally, the price of galactose is higher. How to reduce the production cost of galactose becomes a new research trend.
Disclosure of Invention
In order to solve the problem of reducing the production cost of galactose, the invention provides a novel process for producing galactose by a combined enzyme method by using sucrose, starch or glycogen as raw materials.
The technical scheme adopted by the invention for solving the technical problem is as follows:
the new process for producing galactose by using sucrose, starch or glycogen as raw materials and combining enzyme method, the galactose is produced by using sucrose, starch or glycogen as raw materials, and the chemical reaction is completed by combining and catalyzing a plurality of enzymes.
Further, key intermediates in the process from sucrose, starch or glycogen to galactose include UDP-glucose and UDP-galactose.
Further, the catalytic process from sucrose to galactose comprises catalyzing sucrose and UDP with sucrose synthase to produce UDP-glucose and fructose; catalyzing UDP-glucose to generate UDP-galactose by uridine diphosphate glucose-4-epimerase; hydrolysis of UDP-galactose with galactosyltransferase produces UDP and galactose.
Further, the resulting UDP can be used in a reaction catalyzed by sucrose synthase.
Further, the catalytic process from starch or glycogen to galactose comprises catalyzing glycogen or starch and phosphate with glycogen phosphorylase to produce glucose-1-phosphate; catalyzing glucose-1-phosphate and UTP with UTP-glucose-1-phosphate uridyltransferase to produce UDP-glucose and pyrophosphate; using uridine diphosphate glucose-4-epimerase to catalyze UDP-glucose to generate UDP-galactose; hydrolysis of UDP-galactose with a galactosyltransferase produces UDP and galactose.
Further, the EC number of sucrose synthase is EC 2.4.1.13; EC number of glycogen phosphorylase is EC 2.4.1.1; the EC number of UTP-glucose-1-phosphate uridyltransferase is EC 2.7.7.9; the EC number of the uridine diphosphate glucose-4-epimerase is EC 5.1.3.2; the EC number of the galactosyltransferase is EC 2.4.1.87.
Further, the novel process for producing galactose by using sucrose, starch or glycogen as raw materials and combining with an enzyme method combines the prepared tlr1047, D3A 95-07430 and P50127 and catalytically synthesizes galactose by using sucrose as a raw material under the reaction conditions of: under the condition of 35-65 deg.C, three recombinant enzymes of tlr1047, D3A 95-07430 and P50127 with concentration of 0.0005-0.1mg/ml and MnCl with concentration of 0.001-0.1M are added 2 Reacting sucrose with concentration of 0.004-0.1M and UDP with concentration of 0.004-0.1M in a reaction solution with pH of 4.0-10.0 to catalyze the generation of galactose.
Further, the novel process for producing galactose by using sucrose, starch or glycogen as raw materials and combining with an enzyme method combines the prepared tlr1047, D3A 95-07430 and P50127 and catalytically synthesizes galactose by using sucrose as a raw material under the reaction conditions of: three recombinant enzymes, i.e., tlr1047, D3A 95-07430 and P50127, were mixed at a concentration of 2. mu.g/ml, MnCl at a concentration of 0.002M at a temperature of 50 ℃ 2 Sucrose with a concentration of 0.01M and UDP with a concentration of 0.01M were reacted in a reaction solution at pH7.0 for 12 hours to catalyze the formation of galactose.
Further, the new process for producing galactose by using sucrose, starch or glycogen as raw materials through a combined enzyme method combines the prepared all1272, tlr0611, D3A95_07430 and P50127_80-371, and catalytically synthesizes galactose by using starch as a raw material, wherein the reaction conditions are as follows: under the condition of 35-65 deg.C, four recombinant enzymes of all1272, tlr0611, D3A95_07430 and P50127_80-371 with concentration of 0.0005-0.1mg/ml, starch or glycogen with concentration of 0.25% -20% (w/v), UTP with concentration of 0.004-0.1M M and MnCl with concentration of 0.001-0.01M are added 2 MgCl with a concentration of 0.001-0.01M 2 The reaction was carried out in PBS reaction solution at pH7.2 to catalyze the formation of galactose.
Further, the sugar caneA novel process for producing galactose by using a combined enzyme method with sugar, starch or glycogen as raw materials is characterized in that prepared all1272, tlr0611, D3A95_07430 and P50127_80-371 are combined to catalyze and synthesize the galactose by using the starch as the raw material, and the reaction conditions are as follows: four recombinant enzymes all1272, tlr0611, D3A 95-07430 and P50127-80-371 at a concentration of 2. mu.g/ml, starch at a concentration of 1% (w/v), UTP at a concentration of 0.01M, MnCl at a concentration of 0.002M were mixed at 50 ℃ 2 MgCl with a concentration of 0.002M 2 The reaction was carried out in PBS at pH7.2 for 12 hours, whereby the formation of galactose was catalyzed.
Further, PBS was composed of 8g NaCL,0.2g KCl,1.44g Na 2 HPO 4 ,0.24 g KH 2 PO 4 Prepared by dissolving in 1L water
The technical principle and the beneficial effects of the invention are as follows:
the invention relates to a method for producing galactose by taking sucrose, starch or glycogen as raw materials and combining with an enzyme method. Important intermediates for this process include UDP-glucose and UDP-galactose. Enzymes catalyzing the production of galactose from sucrose, starch or glycogen include sucrose synthase, uridine diphosphate glucose-4-epimerase, galactosyltransferase, glycogen phosphorylase, UTP-glucose-1-phosphate uridyltransferase. Prior to the present application, enzymatic synthesis of galactose using such a combined enzyme method was not known, in which hydrolysis of UDP-galactose by galactosyltransferase was a critical step.
Galactosyltransferase catalyzed reactions typically involve two substrates, one being UDP-galactose and the other being other types of molecules such as sugars, proteins, etc. Galactosyltransferases catalyze the transfer of the galactose group on UDP-galactose to another substrate. The research fact of the invention discovers that galactosyltransferase (N-acetyllactosamide 3-alpha-galactosylsystemtransferase, EC 2.4.1.87) can break the covalent bond between UDP and galactose group under the condition that UDP-galactose only exists, and hydroxyl ions split off by water molecules are used as nucleophilic groups to attack galactose groups to form complete galactose.
The invention takes cane sugar, starch or glycogen as raw materials, has simple material selection and low price, and greatly reduces the cost for producing galactose. In addition, the present invention combines a large number of enzymes, and no one has used such combination to produce galactose before the present disclosure. The enzymes can be obtained by mature technology, and the cost is very low.
The invention produces galactose by combining a plurality of enzymes and taking sucrose, starch or glycogen as raw materials. Enzymes that catalyze the production of galactose from sucrose, starch or glycogen include sucrose synthase, uridine diphosphate glucose-4-epimerase, galactosyltransferase, glycogen phosphorylase, UTP-glucose-1-phosphate uridyltransferase. Wherein, the gene with the number of P50127 in the Unit Prot database of galactosyltransferase constructs an overexpression plasmid, and is transformed into E.coli BL21(DE3) to obtain recombinase through induced expression. The galactosyltransferase catalyzes the hydrolysis of UDP-galactose into UDP and galactose, and finally produces galactose.
In conclusion, the process for producing galactose is completely different from the subsequent process for producing galactose, and the final product galactose can be used in the industries of food and the like.
Drawings
FIG. 1 is a flow chart of a new process for producing galactose by a combined enzyme method using sucrose, starch or glycogen as raw materials.
FIG. 2 shows SDS-PAEG of all the proteins of interest prepared in example 2. 1. all1272, 2, tlr1047, 3, tlr0611, 4, D3a95_07430, 5, P50127_ 80-371.
FIG. 3 shows the reaction conditions explored in example 3: under the condition of 35-65 deg.C, three recombinant enzymes prepared in example 2 with the concentration of 0.0005-0.1mg/ml and MnCl with the concentration of 0.001-0.1M are added 2 Reacting sucrose with the concentration of 0.004-0.1M and UDP with the concentration of 0.004-0.1M in a reaction solution with the pH value of 4.0-10.0 for 12 hours, and catalyzing the generation of galactose by a one-pot method. The optimal reaction conditions are as follows: three recombinases prepared in example 1 at a concentration of 2. mu.g/ml, MnCl at a concentration of 0.002M, were added at a temperature of 50 deg.C 2 Sucrose with the concentration of 0.01M and UDP with the concentration of 0.01M react in a reaction solution with the pH value of 7.0 for 12 hours, and the generation of galactose is catalyzed by a 'one-pot method'.
FIG. 4 shows the reaction conditions explored in example 4: under the condition of 35-65 deg.C, four recombinant enzymes prepared in example 2 with concentration of 0.0005-0.1mg/ml, starch or glycogen with concentration of 0.25% -20% (w/v), UTP with concentration of 0.004-0.1M M, MnCl with concentration of 0.001-0.01M 2 MgCl with a concentration of 0.001-0.01M 2 PBS (8g NaCL,0.2g KCl,1.44g Na) at pH7.2 2 HPO 4 ,0.24g KH 2 PO4 in 1L water) in the reaction solution for 12 hours, and the "one-pot method" catalyzes the production of galactose. The optimal reaction conditions are as follows: four recombinant enzymes prepared in example 2 at a concentration of 2. mu.g/ml, starch or glycogen at a concentration of 1% (w/v), UTP at a concentration of 0.01M, MnCl at a concentration of 0.002M were mixed at a temperature of 50 ℃ 2 MgCl at a concentration of 0.002M 2 PBS (8g NaCL,0.2g KCl,1.44g Na) at pH7.2 2 HPO 4 ,0.24g KH 2 PO4 was dissolved in 1L water, pH 7.2) and reacted in the reaction solution for 12 hours to catalyze the formation of galactose.
FIG. 5 is the result of Thin Layer Chromatography (TLC) of galactose obtained in example 3 and example 4 in example 5. Fig. 5A, 1 (galactose standard), 2 (fructose standard), 3 (sucrose standard), 4(UDP standard, UDP standard is highly polar and difficult to develop), 5 (total product after reaction of example 3), 6 (galactose product prepared by example 3); fig. 5B 1 (galactose standard), 2 (starch), 3(UTP standard, UTP is highly polar and difficult to unfold), and 4 (galactose product prepared by example 4).
FIG. 6 shows the results of High Performance Liquid Chromatography (HPLC) for example 3 in example 6. FIG. 6A is a sucrose standard sample; FIG. 6B is a galactose standard sample; FIG. 6C is a fructose standard sample; FIG. 6D is an HPLC chromatogram of galactose synthesized by combination of enzymatic catalysis with sucrose as a raw material in example 3.
FIG. 7 shows the results of Nuclear Magnetic Resonance (NMR) analysis of galactose standard substance in example 7 and galactose obtained in example 3. FIG. 7A shows the results of Nuclear Magnetic Resonance (NMR) of galactose standard; FIG. 7B shows the results of Nuclear Magnetic Resonance (NMR) analysis of galactose obtained in example 3.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
In the embodiment of the invention, as shown in fig. 1, the novel process for producing galactose by using sucrose, starch or glycogen as raw materials and combining the enzymatic method produces galactose by using sucrose, starch or glycogen as raw materials, and the chemical reaction is completed by combining a plurality of enzymes for catalysis.
In an embodiment, key intermediates in the process from sucrose, starch or glycogen to galactose include UDP-glucose and UDP-galactose.
In an embodiment, the catalytic process from sucrose to galactose comprises catalyzing sucrose and UDP with a sucrose synthase to produce UDP-glucose and fructose; using uridine diphosphate glucose-4-epimerase to catalyze UDP-glucose to generate UDP-galactose; hydrolysis of UDP-galactose with galactosyltransferase produces UDP and galactose.
In the examples, the UDP produced is in turn available for reactions catalysed by sucrose synthases.
In embodiments, the catalyzed process from starch or glycogen to galactose comprises catalyzing glycogen or starch and phosphate with a glycogen phosphorylase to produce glucose-1-phosphate; catalyzing glucose-1-phosphate and UTP with UTP-glucose-1-phosphate uridyltransferase to produce UDP-glucose and pyrophosphate; using uridine diphosphate glucose-4-epimerase to catalyze UDP-glucose to generate UDP-galactose; hydrolysis of UDP-galactose with galactosyltransferase produces UDP and galactose.
In the examples, the sucrose synthase has an EC number of EC 2.4.1.13; EC number of glycogen phosphorylase is EC 2.4.1.1; the EC number of UTP-glucose-1-phosphate uridyltransferase is EC 2.7.7.9; the EC number of the uridine diphosphate glucose-4-epimerase is EC 5.1.3.2; the EC number of the galactosyltransferase is EC 2.4.1.87.
The following is a more specific example.
Example 1 preparation of expression plasmids for various enzymes
(1) Artificial gene synthesis the following genes for enzymes: sucrose synthase (UniProt gene No. tlr1047), glycogen phosphorylase (UniProt gene No. all1272), UTP-glucose-1-phosphate uridyltransferase (UniProt gene No. tlr0611), uridine diphosphate glucose-4-epimerase (UniProt gene No. D3a95_07430), and galactosyltransferase (UniProt gene No. P50127_ 80-371).
(2) Constructing an overexpression plasmid tlr1047-pET28a of tlr1047, wherein the double enzyme cutting sites of pET28a are NdeI and XhoI; constructing all1272 overexpression plasmid all1272-pET28a of all1272, wherein the double enzyme cutting sites of pET28a are NdeI and BamHI; constructing an overexpression plasmid tlr0611-pET28a of tlr0611, wherein the double enzyme cutting sites of pET28a are XhoI and BamHI; constructing an overexpression plasmid D3A95_07430-pET28a of D3A95_07430, wherein the double enzyme cutting sites of pET28a are NdeI and XhoI; constructing an overexpression plasmid P50127_80-371-pET28a of P50127_80-371, wherein the double enzyme cutting sites of pET28a are NdeI and BamHI; all the recombinant plasmids constructed (tlr1047-pET28a, all1272-pET28a, tlr0611-pET28a, D3A95_07430-pET28a, P50127_80-371-pET28a) were transformed into E.coli competent E.coli BL21(DE3) in this order, spread on LB solid medium plate containing kanamycin resistance (100. mu.g/mL), and the LB solid medium plate was inverted and cultured overnight in a 37 ℃ incubator.
Example 2 purification of various enzymes
The 5 enzymes of example 1 were purified in accordance with the following procedure:
(1) colonies were picked and inoculated into 5mL LB liquid medium containing kanamycin resistance (final concentration 100. mu.g/mL), cultured overnight at 37 ℃ at 200rpm, overnight-cultured bacterial liquid was expanded to 1L LB liquid medium containing kanamycin resistance (final concentration 100. mu.g/mL), cultured at 37 ℃ for 10h at 180rpm, added with 0.5mL of 1M IPTG as an inducer, and expressed protein was induced overnight at 20 ℃ at 180 rpm.
(2) The cells were harvested by centrifugation, the culture supernatant discarded and bacterial lysate (50mM Tris, pH 8.0, 200mM NaCl, 20mM imidazole) added; crushing thalli by an ultrasonic cell crusher; collecting lysate, centrifuging at 12000rpm for 30min, taking out supernatant, passing through Ni-NTA chromatographic column (column height 2cm), allowing the supernatant to flow out of the Ni-NTA chromatographic column under gravity action, allowing histidine-tagged protein to bind with Ni-NTA, and washing Ni-NTA with washing buffer (50mM Tris, pH 8.0, 200mM NaCl, 20mM imidazole) for three times; the target protein was eluted from Ni-NTA with an eluent (50mM Tris, pH 8.0, 200mM NaCl, 300mM imidazole), and the target protein was collected. A10 kDa dialysis bag was selected, and the purified 5 proteins were put into the dialysis bag and dialyzed twice at 4 ℃ in 1L of a dialysate (10mM Tris, pH 7.5, 200mM NaCl).
(3) Detecting target protein by a polyacrylamide gel electrophoresis (SDS-PAGE) method and purifying the protein: adding 3.3 μ L of 4 xSDS-PAGE Loading buffer into 10 μ L of protein sample, heating at 100 ℃ for 5min, Loading and running gel (120-220V), after electrophoresis, completely immersing the SDS-PAGE gel into Coomassie brilliant blue R-250 staining solution, and staining overnight; after the completion of SDS-PAGE decolorization, photographs were taken. The SDS-PAGE detection result is shown in figure 2, and the purity of all target proteins reaches more than 80%.
(4) After SDS-PAGE detection, an ultrafiltration tube with the specification of 10.0kDa is selected for protein concentration until the concentration reaches about 5-10mg/mL, and a small amount of 100 mu L is subpackaged and stored at-80 ℃ for later use.
Example 3 combination enzymatic Synthesis of galactose Using sucrose as raw Material
The combination of tlr1047, D3a95 — 07430 and P50127 prepared in example 2 catalyzed the synthesis of galactose from sucrose. The reaction conditions were explored as follows: at the temperature of 35-65 ℃, three recombinant enzymes prepared in the embodiment 2 with the concentration of 0.0005-0.1mg/ml and MnCl with the concentration of 0.001-0.1M are added 2 Sucrose with concentration of 0.004-0.1M and UDP with concentration of 0.004-0.1M react in a reaction solution with pH of 4.0-10.0 for 12 hours, and the generation of galactose is catalyzed by a one-pot method (figure 3).
Through comparison and determination, the optimal reaction conditions are as follows: three recombinant enzymes prepared in example 2 at a concentration of 2. mu.g/ml, Mn Cl at a concentration of 0.002M, were added at 50 ℃ 2 Sucrose at a concentration of 0.01M, UDP at a concentration of 0.01M at pH7.0The reaction is carried out in the liquid for 12 hours, and the generation of galactose is catalyzed by a one-pot method.
Example 4 Combined enzymatic Synthesis of galactose Using starch as raw Material
All1272, tlr0611, D3A95_07430 and P50127_80-371 prepared in example 2 were combined to catalyze and synthesize galactose by using starch as a raw material. The reaction conditions were explored as follows: under the condition of 35-65 deg.C, four recombinant enzymes prepared in example 2 with the concentration of 0.0005-0.1mg/ml, starch or glycogen with the concentration of 0.25% -20% (w/v), UTP with the concentration of 0.004-0.1M M and MnCl with the concentration of 0.001-0.01M are added 2 MgCl with a concentration of 0.001-0.01M 2 PBS (8g NaCL,0.2g KCl,1.44g Na) at pH7.2 2 HPO 4 ,0.24g KH 2 PO4 in 1L water) in the reaction solution for 12 hours, and the "one-pot" method catalyzes the production of galactose (fig. 4).
Through comparison and determination, the optimal reaction conditions are as follows: four recombinant enzymes prepared in example 2 at a concentration of 2. mu.g/ml, starch at a concentration of 1% (w/v), UTP at a concentration of 0.01M, MnCl at a concentration of 0.002M were mixed at a temperature of 50 ℃ 2 MgCl at a concentration of 0.002M 2 PBS (8g NaCL,0.2g KCl,1.44g Na) at pH7.2 2 HPO 4 ,0.24g KH 2 PO 4 Dissolving in 1L water, pH 7.2) and reacting in the reaction solution for 12 hours to catalyze the generation of galactose.
Example 5 Thin Layer Chromatography (TLC)
The formula of the spreading agent comprises: n-butanol, acetone and water in a volume ratio of 4:3: 1. The color developing agent formula comprises: dissolving 1mL aniline in 24mL acetone to obtain solution A, dissolving 1g diphenylamine in 24mL acetone to obtain solution B, mixing 17mL phosphoric acid and 3mL water to obtain solution C, mixing solution A, B, C, placing into brown bottle, and storing in dark place. Thin Layer Chromatography (TLC): spotting the products obtained in the examples 3 and 4 at a position close to one end of a silica gel plate, and enabling the sample to rise along the plate under the action of a spreading agent; after the solvent is evaporated and the position of the separated component is determined, adding the color developing agent, heating in an oven for color development until the strip is clear, and photographing for storage.
TLC results are shown in figure 4: the combination of enzymes prepared in example 4 can catalyze the synthesis of galactose by sucrose or starch/glycogen, respectively (FIG. 5). The galactose products prepared in application examples 3 and 4 were in accordance with those of standard galactose.
EXAMPLE 6 High Performance Liquid Chromatography (HPLC)
The peak positions of galactose, fructose, sucrose and the enzyme reaction product of example 2 were determined using the Shimadzu LC-16HPLC analysis system of Japan. The differential detector model of the HPLC analytical system is RI-20A. NH 2P-504E amino column as chromatographic column. 65% acetonitrile as mobile phase, the flow rate in the HPLC system was set to 1 mL/min. The peak positions of galactose, fructose, sucrose and the enzyme reaction product of example 2 were finally obtained (fig. 6).
Example 7 Nuclear magnetic resonance analysis (NMR)
Galactose is purified from the enzyme-catalyzed reaction. Dissolving dried galactose in 550. mu.L D 2 O, NMR measurements were performed on a Bruker 500MHz NMR spectrometer with a 5mm broadband reverse probe and a shielded z-gradient. The results of nuclear magnetic resonance analysis are shown in FIG. 7, and the NMR spectra (reference) of galactose were completely consistent with those of the product galactose obtained in example 3.
The invention discloses a novel process for producing galactose by a combined enzyme method by using sucrose, starch or glycogen as raw materials, and a person skilled in the art can use the contents in the text for reference and realize the process by properly improving process parameters. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the technology can be practiced and applied by modifying or appropriately combining the products described herein without departing from the spirit and scope of the invention.
Claims (10)
1. A process for preparing galactose from cane sugar, starch or glycogen by enzyme method features that the cane sugar, starch or glycogen is used as raw material to prepare galactose, and the chemical reaction is catalyzed by the combination of more enzymes.
2. The novel process for producing galactose from sucrose, starch or glycogen by the combined enzymatic method as set forth in claim 1, wherein the key intermediates in the process from sucrose, starch or glycogen to galactose include UDP-glucose and UDP-galactose.
3. The novel process for producing galactose by the combined enzymatic method using sucrose, starch or glycogen as raw materials according to claim 1, wherein the catalytic process from sucrose to galactose comprises catalyzing sucrose and UDP with sucrose synthase to produce UDP-glucose and fructose; catalyzing UDP-glucose to generate UDP-galactose by uridine diphosphate glucose-4-epimerase; hydrolysis of UDP-galactose with a galactosyltransferase produces UDP and galactose.
4. The novel process for producing galactose by the combined enzymatic method using sucrose, starch or glycogen as raw materials according to claim 3, wherein the UDP produced is further used in the reaction catalyzed by sucrose synthase.
5. The novel process for producing galactose by the combined enzymatic method of sucrose, starch or glycogen as raw materials according to claim 1, wherein the catalytic process from starch or glycogen to galactose comprises catalyzing glycogen or starch and phosphate with glycogen phosphorylase to produce glucose-1-phosphate; catalyzing glucose-1-phosphate and UTP with UTP-glucose-1-phosphate uridyltransferase to produce UDP-glucose and pyrophosphate; using uridine diphosphate glucose-4-epimerase to catalyze UDP-glucose to generate UDP-galactose; hydrolysis of UDP-galactose with a galactosyltransferase produces UDP and galactose.
6. The novel process for preparing galactose by combined enzyme method using sucrose, starch or glycogen as raw materials according to claim 1, wherein the prepared tlr1047, D3A95_07430 and P50127 are combined to catalyze and synthesize galactose by using sucrose as raw material, and the reaction conditions are as follows: under the condition of 35-65 deg.C, three recombinant enzymes of tlr1047, D3A 95-07430 and P50127 with concentration of 0.0005-0.1mg/ml and MnCl with concentration of 0.001-0.1M are added 2 In a concentration of0.004-0.1M of sucrose and 0.004-0.1M of UDP are reacted in a reaction solution with pH4.0-10.0 to catalyze the generation of galactose.
7. The novel process for preparing galactose by combined enzyme method using sucrose, starch or glycogen as raw materials according to claim 6, wherein the prepared tlr1047, D3A95_07430 and P50127 are combined to catalyze and synthesize galactose by using sucrose as raw material, and the reaction conditions are as follows: three recombinant enzymes, tlr1047, D3A95_07430 and P50127, at a concentration of 2. mu.g/ml, MnCl at a concentration of 0.002M at a temperature of 50 ℃ were added 2 Sucrose with a concentration of 0.01M and UDP with a concentration of 0.01M were reacted in a reaction solution at pH7.0 for 12 hours to catalyze the formation of galactose.
8. The novel process for producing galactose by using sucrose, starch or glycogen as raw materials through combined enzymatic method as claimed in claim 1, wherein all1272, tlr0611, D3A95_07430 and P50127_80-371 are combined to catalyze and synthesize galactose by using starch as raw materials, and the reaction conditions are as follows: under the condition of 35-65 deg.C, four recombinant enzymes of all1272, tlr0611, D3A95_07430 and P50127_80-371 with concentration of 0.0005-0.1mg/ml, starch or glycogen with concentration of 0.25% -20% (w/v), UTP with concentration of 0.004-0.1MM and MnCl with concentration of 0.001-0.01M are added 2 MgCl with a concentration of 0.001-0.01M 2 The reaction was carried out in PBS reaction solution at pH7.2 to catalyze the formation of galactose.
9. The novel process for producing galactose by a combined enzyme method using sucrose, starch or glycogen as raw materials according to claim 8, wherein all1272, tlr0611, D3a95_07430 and P50127_80-371 prepared are combined to catalyze and synthesize galactose by using starch as raw material, and the reaction conditions are as follows: four recombinant enzymes all1272, tlr0611, D3A 95-07430 and P50127-80-371 at a concentration of 2. mu.g/ml, starch at a concentration of 1% (w/v), UTP at a concentration of 0.01M, MnCl at a concentration of 0.002M were mixed at 50 ℃ 2 MgCl with a concentration of 0.002M 2 Reacting in PBS (phosphate buffer solution) at pH7.2 for 12 hr to catalyze generation of galactoseAnd (4) obtaining.
10. The novel process for producing galactose by the combined enzymatic method using sucrose, starch or glycogen as raw materials according to claim 8 or 9, wherein the PBS is composed of 8g of NaCl,0.2g of KCl, and 1.44g of Na 2 HPO 4 ,0.24gKH 2 PO 4 Dissolving in 1L water.
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