Alpha-1,3-glucan phosphorylase and its application in the preparation of digestion-resistant alpha-glucan

Abstract

The present invention discloses a kind of α-1,3-glucan phosphorylase and its application in the preparation of digestion-resistant α-glucan, belong to the field of genetic engineering and enzyme engineering. The present invention uses plasmid pET-24a (+) as expression vector and Escherichia coli BL21 as expression host, realizes the expression of α-1,3-glucan phosphorylase α-1,3-glucan phosphorylase derived from Paenibacillus sp.HMSSN-139. And the α-1,3-glucan phosphorylase expressed by recombinant Escherichia coli is used to produce digestion-resistant α-glucan containing α-1,3 bond, and the yield can reach 58.31% ~ 65.66%, which can be used for the industrial production of digestion-resistant α-glucan in the fields of food, medicine, etc.

Description

Technical Field

[0001] The invention relates to alpha-1,3-glucan phosphorylase and application thereof in the preparation of digestion-resistant alpha-glucan, belonging to the fields of genetic engineering and enzyme engineering. Background Art

[0002] Starch is mainly composed of glucose polymerized with α-1,4 and a small amount of α-1,6 glycosidic bonds. It is not only extremely easy to digest and high in calories, but also difficult to enter the colon, affecting intestinal microorganisms and failing to exert its prebiotic effects. Digestion-resistant α-glucans can be obtained by reconstructing the easily digestible α-1,4 bonds in starch molecules into slowly digestible α-1,6 and digestion-resistant α-1,2 or α-1,3 glycosidic bonds, and changing its sugar chain branching structure. The first digestion-resistant α-glucan product currently launched on the market is called "resistant dextrin". Its production process mainly uses high-temperature strong acid chemical methods to randomly break the α-1,4 bonds in starch and reorganize them into different glycosidic bond types. It is not only energy-intensive and polluting, but also contains safety hazards such as furfural substances in the product.

[0003] Enzymatic conversion is a greener, more efficient, and more controllable method for producing digestion-resistant α-glucans. DuPont has reported a series of enzymes using glucansucrases to produce digestion-resistant α-glucans containing α-1,6 or α-1,3 bonds using sucrose as a substrate. Recently, researchers at the University of Groningen in the Netherlands identified two new enzymes, 4,6- and 4,3-α-glucosyltransferases, which can introduce α-1,6 or α-1,3 bonds, respectively, into starch molecules. However, these enzymes belong to the glycoside hydrolase class, and the transglycosidation reactions they catalyze are thermodynamically disadvantageous, resulting in limited conversion rates. Furthermore, the digestion-resistant α-glucans obtained by catalysis have a relatively simple resistant bond pattern. When using sucrose as a substrate, fructose molecules may remain, increasing the cost of product purification. In recent years, glycoside phosphorylases, which can catalyze the phosphorylation of oligosaccharides to form glycosyl-1-phosphates and monosaccharides, as well as the reverse synthesis reaction, have gained increasing attention. Compared to glycoside hydrolases, glycoside phosphorylases catalyze synthesis reactions with higher donor bond energy and ease of activation. Dissociation of the sugar-phosphate bond provides energy for the formation of the product glycosidic bond, giving them a thermodynamic advantage and a relatively high conversion rate. Therefore, using glycoside phosphorylases to further introduce digestion-resistant glycosidic bonds into dextrins or α-glucans to prepare complex-bond, digestion-resistant α-glucans is a promising new approach. Summary of the Invention

[0004] The present invention identifies a new property of maltose phosphorylase (Genbank: GJM82185.1), which can be used as an α-1,3-glucan phosphorylase and catalyze the synthesis reaction of α-1,3 glycosidic bonds using various types of disaccharides, oligosaccharides, dextrins and dextran as receptors. A method for preparing digestion-resistant α-glucans containing α-1,3 bonds using the enzyme is also provided.

[0005] The present invention provides a recombinant microorganism expressing α-1,3-glucan phosphorylase derived from Paenibacillus sp. HMSSN-139.

[0006] In one embodiment, the amino acid sequence of the α-1,3-glucan phosphorylase is shown as SEQ ID NO.2.

[0007] In one embodiment, the nucleotide sequence encoding the α-1,3-glucan phosphorylase is shown in SEQ ID NO. 1. In one embodiment, Escherichia coli is used as the starting strain.

[0008] In one embodiment, the Escherichia coli is Escherichia coli BL21 (DE3).

[0009] In one embodiment, pET-24a(+) is used as an expression vector to express the α-1,3-glucan phosphorylase gene shown in SEQ ID NO.1.

[0010] The present invention also provides a method for preparing digestion-resistant α-glucans. The method uses maltose as a substrate and utilizes the α-1,3-glucan phosphorylase and maltose phosphorylase to co-catalyze the production of digestion-resistant α-glucans containing α-1,3 bonds. In one embodiment, the amount of maltose phosphorylase added is 15 U / g substrate, and the amount of α-1,3-glucan phosphorylase added is 30 U / g substrate.

[0011] In one embodiment, the phosphate concentration in the reaction system is 20-50 mM.

[0012] In one embodiment, when maltose is used as the substrate, the concentration of maltose is 150-250 g / L.

[0013] In one embodiment, when maltose is used as a substrate and dextran is added, the concentration of maltose is 150-250 g / L and the concentration of dextran is 10-20 g / L.

[0014] In one embodiment, the reaction is carried out at pH 7.0 and 37° C., and the reaction time is not less than 24 hours.

[0015] In one embodiment, the method can be to use soluble starch as a substrate, and use glycogen phosphorylase, α-glucose 1-phosphate mutase, β-glucose 1-phosphate mutase and the α-1,3-glucan phosphorylase to jointly catalyze the conversion of maltose to produce digestion-resistant α-glucan containing α-1,3 bonds.

[0016] In one embodiment, the amount of glycogen phosphorylase added is 20 U / g substrate, the amount of α-glucose 1-phosphomutase and β-glucose 1-phosphomutase added is 10 U / g substrate, the amount of α-1,3-glucan phosphorylase added is 30 U / g substrate, the phosphate concentration in the reaction system is 20 mM, the soluble starch substrate is 100-200 g / L, and the reaction is carried out at pH 7.0 and 37°C for 72 hours.

[0017] In one embodiment, the maltose phosphorylase is derived from Lactobacillus brevis, and its amino acid sequence is shown in Uniprot ID: Q7SIE1; the glycogen phosphorylase is derived from Thermotoga maritima MSB8, and its amino acid sequence is shown in GenBank ID: BAD85297.1; the α-glucose 1-phosphate mutase is derived from Thermococcus kodakarensis KOD1, and its amino acid sequence is shown in GenBank ID: BAD85297.1; and the β-glucose 1-phosphate mutase is derived from Lactococcus sp., and its amino acid sequence is shown in GenBank ID: BAD85297.1. The present invention also provides the use of the recombinant microorganism or the method in producing digestion-resistant α-glucans.

[0018] Beneficial effects:

[0019] (1) The present invention uses the nucleotide sequence of α-1,3-glucan phosphorylase Psp13GP derived from Paenibacillus sp. HMSSN-139, uses plasmid pET-24a(+) as an expression vector, and uses Escherichia coli BL21(DE3) as an expression host to achieve efficient expression of the α-1,3-glucan phosphorylase Psp13GP gene in Escherichia coli.

[0020] (2) The present invention uses a one-pot method to catalyze the conversion of maltose into α-1,3-glucan phosphorylase Psp13GP and maltose phosphorylase LbMP to produce digestion-resistant α-glucan containing α-1,3 bonds, with a maximum yield of 65.66%. The α-1,3-glucan phosphorylase Psp13GP is combined with glycogen phosphorylase, α-glucose 1-phosphate mutase, and β-glucose 1-phosphate mutase to catalyze the conversion of soluble starch into digestion-resistant α-glucan containing α-1,3 bonds, with a maximum yield of 52.43%.

[0021] The preparation method and application method of the enzyme provided by the present invention are suitable for the needs of industrial applications such as food and medicine, and can be used for the industrial production of digestion-resistant α-glucan. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart of the one-pot enzymatic method for preparing digestion-resistant α-glucan using α-1,3-glucan phosphorylase.

[0023] Figure 2 It is the electrophoresis diagram of the recombinant enzyme solution of α-1,3-glucan phosphorylase Psp13GP and maltose phosphorylase LbMP; M is a marker, FS, PS, and PC represent the extracellular fermentation supernatant of the recombinant bacteria, the supernatant of the broken cell wall, and the broken cell wall precipitate, respectively.

[0024] Figure 3 This is the result of hydrogen nuclear magnetic resonance (1H-NMR) detection of the product of maltose synthesis catalyzed by the coupling of α-1,3-glucan phosphorylase Psp13GP and maltose phosphorylase LbMP.

[0025] Figure 4 This is the result of the detection of digestion-resistant α-glucan prepared by coupling catalysis of α-1,3-glucan phosphorylase Psp13GP and maltose phosphorylase LbMP at a concentration of 20 mM phosphate.

[0026] Figure 5 This is the result of the detection of digestion-resistant α-glucan prepared by coupling catalysis of α-1,3-glucan phosphorylase Psp13GP and maltose phosphorylase LbMP at a concentration of 50 mM phosphate.

[0027] Figure 6 The results are from the coupled catalytic production of digestion-resistant α-glucan by α-1,3-glucan phosphorylase Psp13GP and maltose phosphorylase LbMP in the presence of 20 mM phosphate and the addition of dextran. DETAILED DESCRIPTION

[0028] (1) Culture medium:

[0029] LB medium: yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L.

[0030] TB medium: yeast powder 24 g / L, glycerol 5 g / L, tryptone 12 g / L, K2HPO4·3H2O 16.43 g / L, KH2PO4 2.31 g / L.

[0031] (2) Detection and analysis methods of α-glucan products

[0032] The enzyme-catalyzed reaction samples were centrifuged at high speed, and the supernatant was collected and ultrafiltered using a 3 kDa ultrafiltration tube. The samples were diluted 10 times and then analyzed by high performance liquid chromatography.

[0033] Ordinary amino column: Waters 2695 liquid chromatography system, the chromatographic column is Thermo Aps-2 HYPERSIL (4.6 mm×250 mm); the mobile phase is 75% acetonitrile; the flow rate is 0.8 mL·min-1; the column temperature is 35°C, and the injection volume is 10 μL.

[0034] Special amino column: Waters 2695 liquid chromatography system, chromatographic column: Shodex Asahipak NH2P50-4E (4.6 mm×250 mm); mobile phase: 75% acetonitrile; flow rate: 0.8 mL·min-1; column temperature: 35°C; injection volume: 10 μL.

[0035] (III) α-1,3-glucan phosphorylase activity detection method

[0036] α-1,3-glucan phosphorylase activity is assayed by measuring the free phosphate released when the enzyme catalyzes the synthesis of a trisaccharide containing an α-1,3 linkage from the donor substrate β-glucose 1-phosphate (β-G-1-P) and the acceptor molecule maltose. The method is based on the 2012 paper "Discovery of nigerose phosphorylase from Clostridium phytofermentans."

[0037] The specific assay method for α-1,3-glucan phosphorylase activity is as follows: a mixture (100 μL) containing 20 mM maltose, 20 mM β-glucose 1-phosphate (β-G-1-P), 25 mM MOPS-NaOH buffer (pH 7.0), and enzyme solution is incubated at 30°C for 10 minutes. An equal volume of 2 M HCl solution is added to the reaction mixture to terminate the enzymatic reaction. 50 μL of the reaction solution is pipetted into a 96-well plate, and 50 μL of distilled water is added. 100 μL of developer solution (containing 0.24% ascorbic acid and 0.25 M HCl) is added to allow color development to produce molybdenum blue. After 10 minutes of color development, 100 μL of stop solution (containing 3% acetic acid and 150 mM sodium citrate) is added to terminate the reaction. Absorbance is measured at 655 nm using a microplate reader. A sample without enzyme serves as a blank control, and a standard curve is generated under the same conditions. Under the above conditions, the amount of enzyme required to release 1 μmol of inorganic phosphate per minute in the synthesis reaction was defined as one unit of enzyme activity.

[0038] Example 1: Construction of recombinant α-1,3-glucan phosphorylase Psp13GP strain

[0039] Based on the amino acid sequence of α-1,3-glucan phosphorylase from Paenibacillus sp. HMSSN-139 in the Genbank database (GJM82185.1, which is annotated as maltose phosphorylase, and its α-1,3-glucan phosphorylase activity is unknown based on its annotation), the gene with the nucleotide sequence shown in SEQ ID NO. 1 was chemically synthesized and ligated into the pET-24a(+) vector.

[0040] Chemically transform the recombinant plasmid pET-24a(+)-Psp13GP that has been verified by enzyme digestion and sequencing into Escherichiacoli BL21(DE3) competent cells:

[0041] (1) Place Escherichia coli BL21 (DE3) competent cells on ice for 5 minutes in advance. After the competent cells are completely thawed, add 10 μL of plasmid to the cells, pipette gently to mix evenly, and place on ice for 30 minutes.

[0042] (2) Heat shock the competent cells in a 42°C water bath for 90 seconds and place on ice for 5 minutes.

[0043] (3) After the ice bath, add 1 mL of LB liquid medium to the competent medium, mix well, and culture at 37°C and 200 rpm for 60 min.

[0044] (4) Centrifuge the competent cells at 3000 rpm for 5 min, discard part of the supernatant, and retain about 200 μL of the supernatant to resuspend the bacteria by pipetting. Spread the suspension onto LB solid medium (containing 30 μg / mL kanamycin) and incubate in a 37°C incubator for 8-10 h until a single colony grows on the plate.

[0045] (5) A single colony was picked and inoculated into LB liquid medium (containing 30 μg / mL kanamycin). After shaking at 37°C and 200 rpm for 8 to 12 h, the plasmid was extracted and sequenced to verify the positive transformant containing the plasmid pET-24a(+)-Psp13GP, which was the recombinant strain of Escherichia coli BL21(DE3)-pET-24a(+)-Psp13GP.

[0046] The same strategy as described above was used to obtain recombinant strains of maltose phosphorylase LbMP from Lactobacillus brevis (amino acid sequence Uniprot ID Q7SIE1), glycogen phosphorylase TmαGP from Thermotoga maritima MSB8 (amino acid sequence GenBank ID: BAD85297.1), α-glucose 1-phosphate mutase TkαPGM from Thermococcus kodakarensis KOD1 (amino acid sequence GenBank ID: BAD85297.1), and β-glucose 1-phosphate mutase LsβPGM from Lactococcus sp. (amino acid sequence GenBank ID: BAD85297.1). (See Stephan Hüwel et al., Maltose phosphorylase from Lactobacillus brevis: Purification, characterization, and application in a biosensor for ortho-phosphate, published in 1997, and Takanori Nihira et al., One pot enzymatic production of nigerose from common sugar resources employing nigerose phosphorylase, published in 2014)

[0047] Example 2: Preparation of α-1,3-glucan phosphorylase Psp13GP recombinant enzyme

[0048] The Escherichia coli BL21 (DE3) recombinant strain containing the recombinant plasmid pET-24a (+) -Psp13GP constructed in Example 1 was inoculated into LB liquid medium (containing 30 μg / mL kanamycin) and cultured for 8-10 h. 5 mL of culture solution was then transferred to 100 mL of TB medium and cultured at 37 ° C for 2 h. The inducing agent IPTG was added to a final concentration of 0.4 mM and cultured at 25 ° C for 24 h. After fermentation, the cells were collected by centrifugation at 8000 rpm for 20 min. 50 mL of 25 mM pH 7.0 3-morpholinepropanesulfonic acid-sodium hydroxide buffer was added to the cells. After fully resuspending the cells, the cells were broken using a high-pressure homogenizer and centrifuged at 8000 rpm for 20 min. The broken cell supernatant was collected as a crude enzyme solution with an enzyme activity of 157.2 U / mL. The collected crude enzyme solution of α-1,3-glucan phosphorylase (Psp13GP) was subjected to SDS-PAGE gel electrophoresis analysis. The electrophoresis diagram is shown in Figure 2 .

[0049] The same method and steps as described above were used to obtain crude enzyme solutions of maltose phosphorylase LbMP (137.8 U / mL) from Lactobacillus brevis, glycogen phosphorylase TmαGP (431.3 U / mL) from Thermotoga maritima MSB8, α-glucose 1-phosphomutase TkαPGM (67.2 U / mL) from Thermococcus kodakarensis KOD1, and β-glucose 1-phosphomutase LsβPGM (82.6 U / mL) from Lactococcus sp. (for the enzyme activity detection method, refer to the paper "One pot enzymatic production of nigerose from common sugar resources employing nigerose phosphorylase" published in 2014).

[0050] Example 3: Preparation of digestion-resistant α-glucan by coupling catalysis of α-1,3-glucan phosphorylase Psp13GP and maltose phosphorylase LbMP

[0051] Using 150-250 g / L maltose as substrate, the reaction was carried out at 37°C and pH 7.0 for 336 h. The LbMP enzyme dosage was 15 U / g substrate and the Psp13GP enzyme dosage was 30 U / g substrate. The phosphate concentrations were set to 20 mM and 50 mM respectively. The products of the enzyme-catalyzed reaction were analyzed by H NMR ( 1 H-NMR) detection, such as Figure 3 As shown, the glycosidic bond types in the products are mainly α-1,4 and α-1,3, indicating that α-1,3-glucan phosphorylase Psp13GP mainly catalyzes the synthesis of products containing α-1,3 bonds.

[0052] The content of the enzyme conversion product components was further detected by high performance liquid chromatography. The reaction results under the conditions of 200 g / L maltose substrate and 20 mM phosphate concentration are as follows Figure 4As shown, the ordinary amino column can detect a series of synthetic products with different polymerization degrees. In the early stage of the reaction, low-polymerization products with a polymerization degree of 2-4, such as disaccharides, trisaccharides and tetrasaccharides, mainly accumulate. The conversion rate continues to increase with time. After 24 hours of reaction, the content of products with a polymerization degree of 2-4 no longer increases, and high-polymerization products (5-7) begin to accumulate. The results of the special amino column test showed that no Aspergillus niger disaccharide (α-1,3 bond disaccharide) was generated during the enzymatic conversion process, that is, Psp13GP could not synthesize α-1,3 bond disaccharides using glucose monosaccharide as an acceptor, and could only use sugars with DP≥2 as an acceptor to perform reverse phosphorolysis synthesis reactions to generate digestion-resistant α-glucans containing α-1,3 bonds. The total conversion rate of digestion-resistant α-glucan products reached 54.87% at 72 hours, 60.32% at 144 hours, and 62.02% at 336 hours. The reaction results under 50mM phosphate concentration conditions are shown as follows. Figure 5 As shown, the product composition and accumulation pattern were similar to those under the reaction conditions of 20 mM phosphate concentration, but the total conversion rate of digestion-resistant α-glucan product was 51.64% at 72 h, 56.51% at 144 h, and 59.91% at 336 h, and the total conversion rate was lower than that under the conditions of 20 mM phosphate concentration.

[0053] Example 4: Preparation of digestion-resistant α-glucan by coupling catalysis of α-1,3-glucan phosphorylase Psp13GP and maltose phosphorylase LbMP under the condition of adding dextran

[0054] Using 150-250 g / L maltose as substrate, adding 10-20 g / L dextran, reacting at 37°C, pH 7.0 for 336 hours, adding 15 U / g substrate of LbMP and 30 U / g substrate of Psp13GP, and setting two groups with phosphate concentrations of 20 mM and 50 mM respectively. The results of enzyme conversion were detected by high performance liquid chromatography. Under the condition of adding dextran, Psp13GP can catalyze the synthesis reaction with dextran as the receptor to obtain a digestion-resistant α-glucan product containing α-1,3 bond branches. The results of high performance liquid chromatography are as follows Figure 6 As shown in the figure, within a certain range, the conversion rate continued to increase with time. Under the condition of 20 mM phosphate concentration, the conversion rate of digestion-resistant α-glucan product reached 53.57% at 72 h, 59.38% at 144 h, and reached a maximum of 65.66% at 336 h.

[0055] Example 5: One-pot catalytic preparation of digestion-resistant α-glucan using starch as substrate

[0056] Using 100-200 g / L soluble starch as the substrate, the reaction was carried out at 37°C and pH 7.0 for 336 hours. The enzyme dosages of glycogen phosphorylase TmαGP, α-glucose 1-phosphomutase TkαPGM and β-glucose 1-phosphomutase LsβPGM, and α-1,3-glucan phosphorylase Psp13GP were 20 U / g substrate, respectively. The phosphate concentration was set at 20 mM. Enzymatic conversion results were detected by high-performance liquid chromatography. Results showed that when soluble starch was converted to β-glucose 1-phosphate using glycogen phosphorylase TmαGP, α-glucose 1-phosphate mutase TkαPGM, and β-glucose 1-phosphate mutase LsβPGM, Psp13GP was able to catalyze the synthesis of β-glucose 1-phosphate using maltose and maltodextrin as acceptors, yielding a digestion-resistant α-glucan containing α-1,3-linked branches. Using 100 g / L soluble starch as the substrate, the conversion rate reached 51.27% after 72 hours, 56.38% after 144 hours, and a peak of 58.31% after 336 hours.

[0057] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing digestion-resistant α-glucan, characterized in that: The α-1,3-glucan phosphorylase shown in SEQ ID NO.2 is used to catalyze the production of digestion-resistant α-glucan containing α-1,3 bonds using a substrate; the substrate is maltose or soluble starch.

2. The method according to claim 1, characterized in that Maltose was used as a substrate, and the α-1,3-glucan phosphorylase and maltose phosphorylase were used as catalysts together; the added amount of the maltose phosphorylase was 15 U / g substrate, and the added amount of the α-1,3-glucan phosphorylase was 30 U / g substrate. The amino acid sequence of the maltose phosphorylase was shown as Uniprot ID: Q7SIE1.

3. The method according to claim 2, characterized in that The reaction system also contains dextran.

4. The method according to claim 1, wherein Soluble starch is used as a substrate, and the α-1,3-glucan phosphorylase, glycogen phosphorylase, α-glucose 1-phosphate mutase and β-glucose 1-phosphate mutase are used as catalysts together; the added enzyme amount of the glycogen phosphorylase is 20 U / g substrate, the added enzyme amount of the α-glucose 1-phosphate mutase and the β-glucose 1-phosphate mutase is 10 U / g substrate, and the added enzyme amount of the α-1,3-glucan phosphorylase is 30 U / g substrate.

5. The method according to any one of claims 1 to 4, characterized in that: Incubate at pH 7.0 and 37°C for at least 24 h.

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