Application of D-mannose 2-epimerase in preparation of D-mannose

Abstract

The invention discloses application of D-mannose 2-epimerase in preparation of D-mannose, and belongs to the field of gene engineering and enzyme engineering. According to the present invention, the gene of the D-mannose 2-epimerase BbME derived from Bacteroidetes bacteria is expressed in E.coli BL21 (DE3), the D-mannose 2-epimerase is applied to a reaction system using D-glucose as a substrate, an enzyme reaction is performed at a pH value of 8.0 and a temperature of 50 DEG C, and the yield of the D-mannose after the reaction is performed for 48 h can achieve 141.05 g / L. The recombinant strain is subjected to high-density fermentation, crude enzyme liquid obtained after wall breaking of thallus cells is added into a 1.5 L large reaction system, the yield of D-mannose can reach 125.35 g / L after reaction is conducted for 48 h, and industrial production of D-mannose can be achieved.

Description

Technical Field

[0001] The invention relates to application of D-mannose 2-epimerase in the preparation of D-mannose, and belongs to the fields of genetic engineering and enzyme engineering. Background Art

[0002] D-mannose is a stereoisomer of D-glucose at the C-2 position and is also a component of some polysaccharides and glycoproteins in nature. To date, D-mannose has attracted much attention for its many beneficial health effects on human health, including stimulation of insulin secretion, treatment or prevention of urinary tract infections, blocking colonization and promoting the growth of intestinal probiotics. It plays an important role in the synthesis of immunostimulants, antitumor agents, vitamins and D-mannitol. In addition, D-mannose has been used as an additive to effectively inhibit the intestinal proliferation of Salmonella in the poultry industry. D-mannose exhibits excellent physiological properties in the food, pharmaceutical and feed industries.

[0003] At present, there are three main ways to prepare D-mannose: plant extraction, chemical synthesis, and bioenzymatic method. Among them, bioenzymatic method has become the research trend of preparing D-mannose due to its advantages such as mild reaction conditions, environmental friendliness and low cost. Most of the current research focuses on the preparation of D-mannose using D-fructose as a substrate, and D-mannose isomerase (Mannoseisomerase MIase, EC 5.3.1.7) is used to produce D-mannose. However, the reaction conversion rate is low and the cost of producing D-mannose using D-fructose as a substrate is high. Compared with D-fructose substrate, D-glucose has unique advantages in terms of source and price, which requires us to explore new reaction pathways or enzyme resources that can prepare D-mannose using D-glucose as a substrate.

[0004] In 2014, Park et al. found that cellobiose 2-epimerase (CEase; EC 5.1.3.11) from Caldicellulosiruptor saccharolyticus has the activity of catalyzing the reversible epimerization between D-glucose and D-mannose, and its equilibrium conversion rate is about 15.67%. However, this reaction is a side reaction of CEase, and about 5% of D-fructose byproducts accumulate in the reaction system, and the conversion rate is too low, which is not suitable for large-scale system preparation in industry. In 2019, Saburi et al. reported two D-mannose 2-epimerase (MEase, EC 5.1.3.-) from Runella slithyiformis and Dyadobacterfermentans, named Runse_4512 and Dfer_5652, respectively. Similar to CEase, MEase can also directly convert D-glucose into D-mannose. At 50°C and pH 8.0, after 48 hours of reaction with 500 g / L D-glucose as substrate, Runse_4512 and Dfer_5652 produced 122 and 114 g / L D-mannose in the system, respectively, with conversion rates of 24.4% and 22.8%, respectively. Therefore, compared with cellobiose 2-epimerase, D-mannose 2-epimerase has more advantages in preparing D-mannose with D-glucose as substrate. However, there are very few D-mannose 2-epimerases reported in articles or patents so far, and the only reported enzyme conversion rates of Runse_4512 and Dfer_5652 from two different sources are low and not suitable for large-scale application in industry. Therefore, it is necessary to explore or identify enzyme resources with high catalytic efficiency. Summary of the invention

[0005] The present invention provides a gene encoding D-mannose 2-epimerase, and the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] In one embodiment, the amino acid sequence of the D-mannose 2-epimerase is shown as SEQ ID NO.2.

[0007] The present invention provides a vector, which carries a gene encoding D-mannose 2-epimerase.

[0008] In one embodiment, the starting vector of the vector is the expression vector pET-24a(+).

[0009] In one embodiment, the vector includes but is not limited to pPIC9K, pPIC3K, pET series vectors, Duet series, and pGEX series.

[0010] The present invention also provides a microbial cell carrying the gene or the vector.

[0011] In one embodiment, the microbial cells include but are not limited to bacteria and fungal cells.

[0012] In one embodiment, the microbial cell includes, but is not limited to, Escherichia coli, Pichia pastoris, or Bacillus subtilis.

[0013] The present invention provides a recombinant bacterium, wherein the recombinant bacterium uses Escherichia coli as an expression host and expresses D-mannose 2-epimerase having an amino acid sequence as shown in SEQ ID NO.2.

[0014] In one embodiment, the Escherichia coli is E. coli BL21 (DE3).

[0015] The present invention provides a method for producing D-mannose. The method comprises reacting D-mannose 2-epimerase shown in SEQ ID NO.2 in a system with D-glucose as a substrate.

[0016] In one embodiment, the method is to use the crude enzyme solution of D-mannose 2-isomerase prepared by fermentation of the recombinant bacteria or the purified pure enzyme for catalytic reaction.

[0017] In one embodiment, the crude enzyme solution is the enzyme-containing supernatant collected by centrifugation after breaking the bacterial cells of the recombinant bacteria.

[0018] In one embodiment, the method comprises using the fermentation broth obtained by fermenting the recombinant bacteria for catalytic reaction after cell disruption.

[0019] In one embodiment, the reaction solution is purified after the reaction to obtain D-mannose.

[0020] In one embodiment, the reaction is carried out in an aqueous medium, which includes but is not limited to water or a buffer.

[0021] In one embodiment, the reaction is carried out in a phosphate buffer; the concentration of the phosphate buffer used is 40-60 mM and the pH is 6.0-7.5.

[0022] In one embodiment, the reaction is carried out in a Tris-HCl buffer; the concentration of the Tris-HCl buffer used is 40-60 mM, and the pH is 7.0-8.5.

[0023] In one embodiment, the reaction is carried out in water and the pH is adjusted to 7.5-8.5 using NaOH.

[0024] In one embodiment, the amount of D-mannose 2-epimerase added is 0.2-2 mg / mL, or 0.25-1 mg / mL, or 0.5-1 mg / mL.

[0025] In one embodiment, the D-glucose concentration is 500-600 g / L.

[0026] In one embodiment, the method comprises reacting at 30-45° C. for at least 24 h, or 24-48 h.

[0027] The present invention also protects the use of the gene or the expression vector in preparing D-mannose or products containing D-mannose in the fields of food and cosmetics.

[0028] The present invention also protects the use of the recombinant bacteria in preparing D-mannose or products containing D-mannose in the fields of food and cosmetics.

[0029] Beneficial effects:

[0030] (1) The present invention screened out D-mannose 2-epimerase BbME from Bacteroidetes bacterium. The nucleotide sequence encoding BbME was synthesized by chemical method, and the plasmid pET-24a(+) was used as the expression vector and E.coli BL21(DE3) was used as the expression host to achieve the expression of bbme gene in E. coli.

[0031] (2) The present invention provides a method for producing mannose using D-mannose 2-dimerase BbME, and studies the suitable reaction conditions of the enzyme. The enzyme has a relatively ideal catalytic activity at 40°C and pH 8.0.

[0032] (3) The present invention conducts high-density fermentation on the constructed recombinant Escherichia coli, and directly uses the crude enzyme solution after the bacterial cell wall is broken for conversion and production of D-mannose. Under the condition of 500 g / L glucose concentration, D-glucose can be used to generate D-mannose, and the yield can reach 141.05 g / L, and the conversion rate can reach 28.21%, which is the highest yield of D-mannose synthesized by D-mannose 2-epimerase method so far. The enzyme is suitable for the needs of industrial applications such as food and medicine, and can be used for the industrial production of D-mannose. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the electrophoresis diagram after the expression of D-mannose 2-epimerase; M is a marker.

[0034] Figure 2 This is the electrophoresis diagram of purified D-mannose 2-epimerase; M is a marker.

[0035] Figure 3 It is the enzyme activity of D-mannose 2-epimerase at different temperatures.

[0036] Figure 4 It is the activity of D-mannose 2-epimerase at different pH and temperature.

[0037] Figure 5 The yield (yellow) and conversion rate (green) of D-mannose with different addition amounts of D-mannose 2-isomerase at 40°C.

[0038] Figure 6 The yield (yellow) and conversion rate (green) of D-mannose with different addition amounts of D-mannose 2-isomerase at 50°C.

[0039] Figure 7 This is the growth curve of high-density fermentation in a 3L tank of D-mannose 2-epimerase engineered bacteria.

[0040] Figure 8 The yield (yellow) and conversion rate (green) of D-mannose when different percentages of broken cell fermentation broth were added.

[0041] Fig. 9 The yield (yellow) and conversion rate (green) of D-mannose prepared in a 1.5 L reaction system with 2.5% cell-broken fermentation broth added and water replacing the buffer. DETAILED DESCRIPTION

[0042] The culture medium involved in the following examples is as follows:

[0043] LB liquid medium: yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L.

[0044] LB solid medium: Add agar powder to LB liquid medium: 20g / L.

[0045] TB medium: yeast powder 24 g / L, glycerol 5 g / L, trypsin 12 g / L, K2HPO4·3H2O 16.43 g / L, KH2PO4 2.31 g / L.

[0046] The detection methods involved in the following embodiments are as follows:

[0047] Mannose 2-epimerase enzyme activity analysis:

[0048] Enzyme activity unit definition: Under standard conditions (pH 8.0, 40°C and 20 minutes), one unit (U) of BbME activity is defined as the amount of enzyme required to release 1 μmol of D-mannose from D-glucose per minute.

[0049] Determination of D-mannose 2-epimerase activity: The reaction system is 1 mL, add 100 μL of moderately diluted enzyme solution (final concentration 1.0 mg / mL) to 900 μL of 50 mM pH 8.0 Tris-HCl buffer containing 100 g / L D-glucose, react in a 40°C constant temperature water bath for 20 minutes, and immediately place in a boiling water bath for 10 minutes to terminate the reaction. After appropriate dilution, the corresponding product concentration is detected in the liquid phase. The enzyme solution that is inactivated by heating is treated in the same way as a blank.

[0050] Detection method of substrate and product content:

[0051] Each product component was detected by HPLC, as follows:

[0052] Detection of D-glucose and D-mannose: Agilent 1200HPLC chromatograph, Agilent autosampler, chromatographic column Agilent Hi-Plex Ca (8μm, 300×7.7mm), differential detector G1362A; mobile phase is pure water, flow rate and column temperature are 0.5mL·min-1 and 80℃ respectively. The product yield is determined according to the peak area and retention time.

[0053] Calculation of product conversion rate: Yield (%) = mass of D-mannose in the product / mass of all sugars in the product × 100%.

[0054] Example 1: Expression of D-mannose 2-epimerase gene

[0055] According to the amino acid sequence of D-mannose 2-epimerase bbme from Bacteroidetes bacterium in the Genbank database, a gene encoding D-mannose 2-epimerase with a nucleotide sequence as shown in SEQ ID NO.1 is synthesized into the multiple cloning site of the vector pET-24a(+) by a chemical synthesis method after codon optimization, and a recombinant plasmid is directly obtained, and the recombinant plasmid is transformed into Escherichia coli (Escherichia coli) JM109 to obtain a transformation product; the transformation product is spread on an LB solid culture medium (containing 40 μg / mL kanamycin), and inverted culture is carried out in a 37°C constant temperature incubator for 8 to 12 hours to obtain a transformant; the transformant is picked and inoculated into an LB liquid culture medium, and a flask is shaken for 8 to 12 hours under the conditions of 37°C and 120 to 180 rpm, and then the plasmid is extracted for sequencing verification, and the recombinant plasmid pET-24a(+)-bbme is obtained if the verification is correct.

[0056] Example 2: Expression of D-mannose 2-epimerase

[0057] The correctly sequenced recombinant plasmid pET-24a(+)-bbme was transformed into E. coli BL21(DE3) competent cells to obtain positive transformants containing the plasmid pET-24a(+)-bbme.

[0058] The positive transformant containing the recombinant plasmid pET-24a(+)-bbme was inoculated into LB liquid medium (containing 10 μg / mL kanamycin) and cultured for 8-10 hours. Then, 2.5 mL of the culture solution was transferred to 50 mL of TB medium and cultured at 37°C for 2 hours. IPTG inducer with a final concentration of 0.4 mM was added, and then the culture was continued in a 25°C shaker for 24 hours. The fermentation broth was centrifuged at 4°C and 8000 rpm for 10 minutes, the supernatant was discarded, the bacteria were collected, 50 mL of 50 mM pH 8.0 Tris-HCl buffer was added to the bacteria, the bacteria were fully resuspended, and the cell wall was broken using a high-pressure homogenizer. After centrifugation at 12000 rpm for 15 minutes, the broken cell supernatant was collected as the crude enzyme solution, and the protein expression was analyzed by SDS-PAGE. The electrophoresis diagram is shown in Figure 1 .

[0059] Example 3: Purification of D-mannose 2-epimerase

[0060] Buffer A: 0.5M sodium chloride, 50mM pH 7.5 Tris-HCl buffer. The specific steps are as follows:

[0061] (1) After the Ni affinity column is equilibrated with buffer A, the sample is sucked into the Ni column to be completely adsorbed, and then 100 mL of buffer A, 100 mL of buffer A containing 10 mM imidazole, 100 mL of buffer A containing 20 mM imidazole, 100 mL of buffer A containing 40 mM imidazole, and 100 mL of buffer A containing 500 mM imidazole are used in sequence at a flow rate of 1 mL / min. The target protein D-mannose 2-epimerase is eluted with buffer A containing 20 mM imidazole, and the eluate is collected;

[0062] (2) The eluate was dialyzed against a pH 8.0, 50 mM Tris-HCl buffer for 10 h to obtain purified mannose 2-isomerase. The specific activity of the pure enzyme was 1.87 U / mg.

[0063] (3) After purification, the recombinant mannose 2-epimerase reached electrophoretic purity with an apparent molecular weight of about 44,000 Daltons. The purified electrophoretogram is shown in Figure 2 .

[0064] Example 4: Determination of application conditions of D-mannose 2-epimerase BbME

[0065] (1) Optimal temperature of D-mannose 2-isomerase BbME

[0066] The optimal reaction temperature of the enzyme was determined by referring to the method described in "Assay of Mannose 2-Dimerase Enzyme Activity". The reaction system was 1 mL. 100 μL of moderately diluted enzyme solution (final concentration 1.0 mg / mL) was added to 900 μL of 50 mM pH 6.5 phosphate buffer (50 mM pH 7.5 phosphate buffer or 50 mM pH 8.5 Tris-HCl buffer) containing 100 g / L D-glucose. The reaction was carried out in a constant temperature water bath at 30-80°C for 20 minutes. After 20 minutes, the reaction was immediately incubated in a boiling water bath for 10 minutes to terminate the reaction. The optimal reaction temperature of the enzyme at three different pH values ​​(6.5, 7.5 and 8.5) was determined. The enzyme specific activity data are shown in Figure 2. Figure 3 As shown, the optimal reaction temperature of the enzyme is independent of the pH value and is always 40°C.

[0067] (2) Optimal pH of mannose 2-isomerase BbME

[0068] The optimal reaction pH of the enzyme was determined by referring to the method described in "Assay of Mannose 2-Diamisomerase Enzyme Activity". The reaction system was 1 mL. 100 μL of moderately diluted enzyme solution (final concentration 1.0 mg / mL) was added to 900 μL of 50 mM pH 6.0-7.5 phosphate buffer or 50 mM pH 8.0-8.5 Tris-HCl buffer containing 100 g / L D-glucose. The reaction was carried out in a constant temperature water bath for 20 minutes. After 20 minutes, it was immediately placed in a boiling water bath and incubated for 10 minutes to terminate the reaction. When determining the optimal reaction temperature of the enzyme, the temperature span is large. In order to further accurately determine the optimal reaction temperature of the enzyme, multiple reaction systems at the same pH were placed at 30, 35, 40, 45, 50 and 55°C for reaction. While determining the optimal pH of the enzyme, its optimal reaction temperature was verified. The enzyme specific activity data are shown in Figure 2. Figure 4 As shown, it was further clarified that the optimal reaction temperature of the enzyme was 40°C and the optimal reaction pH was 8.0.

[0069] Example 5: Application of D-mannose 2-epimerase BbME in the preparation of mannose

[0070] D-glucose dissolved in 50mM Tris-HCl buffer, pH 8.0, with a final concentration of 500g / L was used as the substrate. The reaction was carried out at pH8.0 and 40℃ for 48h. Different enzyme addition amounts of 0.10mg / mL, 0.25mg / mL, 0.50mg / mL, and 1.00mg / mL were set to explore the enzyme amount when D-glucose was used as the substrate to synthesize D-mannose using D-mannose 2-isomerase in a 1mL reaction system.

[0071] The results are as follows Figure 5 As shown in the figure, within a certain range, the conversion rate of the substrate increases with the increase of the enzyme dosage. When the enzyme dosage reaches 0.50 mg / mL, the substrate conversion rate can reach 27.52%; further increase in the enzyme dosage, the conversion rate remains almost unchanged. Taking all factors into consideration, the enzyme dosage of 0.50 mg / mL is selected, at which time the yield of D-mannose can reach 137.60 g / L.

[0072] Example 6: Application of D-mannose 2-epimerase BbME in the preparation of mannose at 50°C

[0073] For a specific implementation, see Example 5, except that the reaction temperature is 50° C. In industrial large-scale production, high temperature is beneficial to reduce the risk of bacterial contamination of the reaction solution. Since D-mannose 2-epimerase can also retain a high activity at 50° C., its enzyme conversion efficiency at 50° C. was explored.

[0074] The results are as follows Figure 6 As shown, when the reaction was carried out at low enzyme dosage (0.10 and 0.25 mg / mL) for 48 h, the conversion rate was lower than that at 40 °C. However, when the enzyme dosage was increased to 0.50 mg / mL, the D-mannose production could reach 141.05 g / L, and the conversion rate was 28.21%.

[0075] Example 7: 3L tank high-density fermentation of engineered bacteria containing D-mannose 2-epimerase BbME and verification of enzyme conversion of fermentation broth

[0076] The specific fermentation conditions are as follows: after inoculation at 10% inoculum, ferment at 37°C, start feeding after dissolved oxygen rebounds and take samples every 4-6 hours to measure the concentration of bacteria. During the fermentation, the feeding rate is adjusted according to the bacterial concentration and dissolved oxygen curve measured by sampling; the OD of the fermented liquid is 600 After reaching 50, the fermentation temperature was adjusted to 25°C and IPTG was added at a final concentration of 0.4 mM to induce protein expression. 60 After reaching 120, continue to add IPTG with a final concentration of 0.4mM. 600 When the highest point is reached, the tank is put down. The specific fermentation data is as follows: Figure 7 After the fermentation liquid was diluted 4 times with buffer solution, it was pre-cooled on ice and broken by high-pressure homogenizer (with external 4°C condensed water circulation for cooling). After the breaking, it was centrifuged at 8000rpm for 20min, and the supernatant was the crude enzyme solution.

[0077] D-glucose dissolved in 50mM Tris-HCl buffer, pH 8.0, with a final concentration of 500g / L was used as a substrate. The reactions were carried out at pH 8.0 and 40°C for 24h and 48h, respectively. Crude enzyme solution was added at a ratio of 8.0%, 10.0% and 12.0% of the total reaction system (by volume ratio) for enzyme conversion analysis. The amount of crude enzyme solution added when D-mannose 2-epimerase was used to synthesize D-mannose using D-glucose as a substrate in a 20mL reaction system was investigated.

[0078] The results are as follows Figure 8 As shown in the figure, within a certain range, the conversion rate of the substrate increases with the increase of crude enzyme solution. When the addition amount reaches 10.0% (v / v, corresponding to 2.5% fermentation broth by volume, i.e. 0.5 mL OD 600 When the fermentation liquid was 200 mL, the substrate conversion rate was 23.72% in 24 hours and 25.66% in 48 hours. When the enzyme dosage was further increased, the conversion rate remained almost unchanged. After comprehensive consideration, the enzyme dosage of 2.5% was selected, and the yield of D-mannose in 48 hours could reach 128.30 g / L.

[0079] Example 8: Enzyme conversion system expansion and optimization of fermentation broth containing D-mannose 2-epimerase BbME

[0080] In order to further verify the catalytic performance of D-mannose 2-epimerase and reduce the production cost in industry, the entire reaction was attempted to be carried out in an aqueous solution (without buffer salt components). In order to maintain the normal activity of the enzyme, the pH of the reaction system was adjusted to 8.0 using NaOH solution. A crude enzyme solution (prepared according to the method of Example 7) was added in an amount of 10.0% (v / v). D-mannose was synthesized in a 1.5L reaction system using D-glucose as a substrate using a fermentation broth containing D-mannose 2-epimerase, and the D-glucose concentration in the reaction system was controlled to be 500 g / L.

[0081] The results are as follows Fig. 9 As shown, when the amount of crude enzyme solution added was 10.0% (v / v, corresponding to 2.5% fermentation broth by volume, i.e., 0.5 mL OD 600 When the fermentation liquid is 200, the substrate conversion rate can reach 25.07% after 48 hours of reaction, and the output of D-mannose can reach 125.35g / L.

[0082] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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 gene encoding D-mannose 2-epimerase, characterized in that The nucleotide sequence is shown in SEQ ID NO.

1.

2. A recombinant expression vector carrying the gene according to claim 1.

3. A microbial cell carrying the gene according to claim 1, or containing the recombinant expression vector according to claim 2.

4. A recombinant Escherichia coli, characterized in that The D-mannose 2-epimerase having an amino acid sequence as shown in SEQ ID NO.2 is expressed.

5. The recombinant Escherichia coli according to claim 4, characterized in that The D-mannose 2-epimerase gene shown in SEQ ID NO. 1 was expressed in E. coli BL21 (DE3) using pET-24a(+) as an expression vector.

6. A method for preparing D-mannose 2-epimerase, characterized in that: The recombinant Escherichia coli according to claim 4 or 5 is cultured in a culture medium, and the D-mannose 2-epimerase in the culture medium is collected.

7. A method for producing D-mannose, characterized in that: The D-mannose 2-epimerase shown in SEQ ID NO.2 is reacted in a reaction system with D-glucose as a substrate; the D-mannose 2-epimerase includes but is not limited to any one of (a) to (e): (a) D-mannose 2-epimerase prepared by the method of claim 6; (b) a crude enzyme solution of D-mannose 2-epimerase obtained by fermentation of the recombinant Escherichia coli according to claim 4 or 5; (c) the purified enzyme described in (b); (d) the bacterial cell of the recombinant Escherichia coli according to claim 4 or 5; (e) The supernatant of (d) after cell disruption.

8. The method according to claim 7, characterized in that The reaction is carried out in a buffer system or pure water; the buffer includes but is not limited to phosphate buffer and Tris-HCl buffer.

9. The method according to claim 7 or 8, characterized in that: The added amount of the D-mannose 2-epimerase is 0.2-2 mg / mL; the concentration of the D-glucose is 500-600 g / L.

10. Use of the microbial cell according to claim 3, or the recombinant Escherichia coli according to any one of claims 4 to 5, or the method according to any one of claims 6 to 9 in the preparation of products containing D-mannose in the field of food or cosmetics.

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