A sucrose phosphorylase mutant and its application in the synthesis of glycerol glucoside

Abstract

The present invention relates to the field of genetic engineering, and in particular to a sucrose phosphorylase mutant and its application in the synthesis of glycerol glucoside. Granulicatella adiacens The sucrose phosphorylase mutant was obtained by simultaneously mutating the 36th, 133rd, 333rd, 335th and 435th amino acid positions of the wild-type sucrose phosphorylase of glycerol. The efficiency of producing 2-glycerol glucoside using sucrose and glycerol as raw materials was significantly improved. It only takes 12 hours to completely catalyze 1 M sucrose to produce glycerol glucoside. At the same time, the yield of the byproduct 1-glycerol glucoside was reduced. Therefore, it has great application value in the production of 2-glycerol glucoside.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a sucrose phosphorylase mutant and application thereof in synthesizing glycerol glucoside. Background Art

[0002] Glycerol glucoside, also known as glycerol glucoside or Glycoin, is a natural small molecule derived from organisms such as desert selaginella or salt-tolerant cyanobacteria. It is composed of a molecule of glucose and glycerol connected by an α-1,2-glycosidic bond. The molecular weight of glycerol glucoside is relatively small, only one thirty-thousandth of the hyaluronic acid molecule, so it can penetrate deep into the skin and provide a deep moisturizing effect. In addition to its moisturizing ability, glycerol glucoside can also significantly promote the synthesis of AQP3 water channel protein, activate the AQP3 synthesis pathway of skin cells themselves, thereby improving the skin's own moisturizing ability and enhancing the skin's barrier strength. It can also help inhibit the production of cell melanin, clean up the black layer by layer, even out the skin color, and make the skin bright and translucent from the inside out. In addition, glycerol glucoside is also used to prepare oral care products, such as oral sprays, toothpastes, etc., which have anti-inflammatory, antibacterial, and analgesic effects. In the food field, it can be used to prepare beverages, candies and other foods to improve the quality and shelf life of food. Therefore, glycerol glucoside has great application value and market prospects.

[0003] Using sucrose and glycerol as raw materials, 2-glycerol glucoside can be produced under the action of sucrose phosphorylase. However, the current sucrose phosphorylase also produces a large amount of byproduct 1-α-glycerol glucoside during the catalytic process. At the same time, natural sucrose phosphorylase is often difficult to meet production requirements. Most natural enzymes have problems such as low enzyme activity, instability, and poor substrate promiscuity. Summary of the invention

[0004] In order to overcome the above problems, the present invention provides a sucrose phosphorylase mutant and its application in synthesizing glycerol glucoside.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a sucrose phosphorylase mutant, which is obtained by simultaneously mutating the 36th serine, the 133rd lysine, the 333rd asparagine, the 335th aspartic acid and the 435th phenylalanine of the wild-type sucrose phosphorylase as shown in the amino acid sequence of SEQ ID NO: 2 through multiple sites.

[0007] In one or more embodiments, the wild-type sucrose phosphorylase whose amino acid sequence is shown in SEQ ID NO:2 has its serine at position 36 mutated to glycine; its lysine at position 133 mutated to arginine; its asparagine at position 333 mutated to glycine; its aspartic acid at position 335 mutated to leucine; and its phenylalanine at position 435 mutated to isoleucine.

[0008] The second aspect of the present invention provides a gene encoding the sucrose phosphorylase mutant described in the first aspect.

[0009] The third aspect of the present invention provides an expression cassette comprising the gene described in the second aspect.

[0010] The fourth aspect of the present invention provides a recombinant expression vector comprising the gene described in the second aspect.

[0011] The fifth aspect of the present invention provides a recombinant bacterium comprising the gene described in the second aspect.

[0012] The sixth aspect of the present invention provides the use of the recombinant bacteria described in the fifth aspect in catalytic synthesis of 2-glycerol glucoside.

[0013] A seventh aspect of the present invention provides a method for synthesizing 2-glycerol glucoside, comprising:

[0014] The wet bacteria obtained by induction culture of genetically engineered bacteria of sucrose phosphorylase mutant or the crude enzyme solution extracted by crushing the wet bacteria is used as a catalyst, sucrose and glycerol are used as substrates, and pure water is used as a reaction medium to form a reaction system, and 2-glycerol glucoside is obtained by reaction;

[0015] Wherein, the genetically engineered bacteria is constructed by introducing the sucrose phosphorylase mutant described in the first aspect into a host bacteria.

[0016] In one or more embodiments, the amount of the catalyst is 5-15 g / L based on the total weight of the wet cells, the initial concentration of the substrate sucrose is 0.3 M-1 M, and the initial concentration of the substrate glycerol is 0.33 M-1.1 M.

[0017] In one or more embodiments, the method of extracting crude enzyme solution by disrupting wet bacteria comprises ultrasonic disruption.

[0018] The beneficial effects of the present invention are:

[0019] The present invention is derived from adjacent particle streptomyces ( Granulicatella adiacensThe sucrose phosphorylase mutant was obtained by simultaneously mutating the 36th, 133rd, 333rd, 335th and 435th amino acid positions of the wild-type sucrose phosphorylase of glycerol. The efficiency of producing 2-glycerol glucoside using sucrose and glycerol as raw materials was significantly improved. It only takes 12 hours to completely catalyze 1 M sucrose to produce glycerol glucoside. At the same time, the yield of the byproduct 1-glycerol glucoside was reduced. Therefore, it has great application value in the production of 2-glycerol glucoside. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 for E.coli The reaction formula for the synthesis of glycerol glucoside catalyzed by BL21(DE3) / pET28a-GASPase-S36G-K133R-N333G-D335L-F435I;

[0022] Figure 2 for E.coli Reaction process diagram of the synthesis of glycerol glucoside catalyzed by BL21(DE3) / pET28a-GASPase-S36G-K133R-N333G-D335L-F435I. DETAILED DESCRIPTION

[0023] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0024] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0025] The culture medium formula used in the following examples is as follows:

[0026] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, solvent is water, pH 7.4.

[0027] LB plate: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 18 g / L agar, solvent is water, pH 7.4.

[0028] The concentration of the product glycerol glucoside was detected by high performance liquid chromatography (HPLC). The analysis method was as follows: chromatographic column model: Waters XBridge Amide (4.6 mm × 250 mm, 5 μm); mobile phase: A (water): B (acetonitrile) = 15:85, injection volume 10 μL, differential detector, detection time: 20 min, flow rate: 1 mL / min. Column temperature: 40 ℃.

[0029] Sample treatment: Take 20 μL of the sample after the reaction is completed, dilute it 30 times with 85% acetonitrile solution, filter it through a 0.22 μm filter membrane, and perform HPLC detection.

[0030] Example 1

[0031] Through library mining, a strain from adjacent particle Streptomyces was screened. Granulicatella adiacens ) sucrose phosphorylase, NCBI accession number is WP_204703942.1, and Nanjing GenScript Biotechnology Co., Ltd. was commissioned to perform full gene synthesis. The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO:2.

[0032] Primers F1 and R1 were designed according to the nucleotide sequence shown in SEQ ID NO.1 (the nucleotide sequence of F1 is shown in SEQ ID NO.3, and the nucleotide sequence of R1 is shown in SEQ ID NO.4), and NdeI and NotI restriction enzyme sites were introduced into the primers, respectively.

[0033] F1: 5'-CATATGATGCAGATTAAAAACGAAGCCATG-3';

[0034] R1: 5'-GCGGCCGCTCACAGCGTAACGACGTTACC-3';

[0035] The pET-28a plasmid was used as the expression vector to construct Escherichia coli E.coli BL21(DE3) / pET28a.

[0036] Construction of expression plasmid: Under the priming of primers F1 and R1, the target gene was used as a template and amplified using high-fidelity PfuDNA polymerase to obtain the sucrose phosphorylase gene sequence. After sequencing, the amplified fragment was treated with NdeI and NotI restriction endonucleases (TaKaRa), and the fragment was ligated with the vector pET-28a treated with the same restriction endonuclease using T4 DNA ligase (TaKaRa) to construct plasmid pET28a-GASPase.

[0037] Preparation of competent cells: Obtain glycerol tubes stored in a -80 ℃ freezer. E.coli BL21(DE3) strain was streaked on an antibiotic-free LB plate and cultured at 37°C for 10 h to obtain a single colony; a single colony on the LB plate was picked and inoculated into a test tube containing 5 mL of LB medium and cultured at 37°C and 180 rpm for 9 h; 200 μL of bacterial solution was taken from the test tube and inoculated into 50 mL of LB medium and cultured at 37°C and 180 rpm to obtain an OD 600 to 0.4-0.6; precool the bacterial solution on ice, transfer the bacterial solution to a sterilized centrifuge tube, place on ice for 10 min, and centrifuge at 4°C and 5000 rpm for 10 min; pour out the supernatant, paying attention to prevent contamination, and replace with precooled 0.1 mol / L CaCl 2 The precipitated cells were resuspended in water and placed on ice for 30 min; centrifuged at 4 °C and 5000 rpm for 10 min, the supernatant was discarded, and the supernatant was washed with pre-cooled 0.1 mol / L CaCl containing 15% glycerol. 2 Resuspend the precipitated cells in aqueous solution, take 100 μL of the resuspended cells and dispense them into sterile 1.5 mL centrifuge tubes, store them in a -80 ℃ refrigerator, and take them out when needed.

[0038] Construction of recombinant E. coli: First, store at -80 ℃ E.coli BL21 (DE3) (Invitrogen) competent cells were placed on ice at 0 °C for 10 min, then 5 µL of plasmid pET28a-GASPase was added in a clean bench, placed on ice at 0 °C for 30 min, heat-shocked in a 42 °C water bath for 90 s, placed on ice at 0 °C for 2 min, and 600 µL of LB medium was added. The cells were cultured at 37 °C and 200 rpm for 1 h. The cells were spread on LB plates containing 50 μg / mL kanamycin resistance and cultured at 37 °C for 8-12 h. Clones were randomly selected to extract plasmids for sequencing and identification, and recombinant Escherichia coli containing the recombinant plasmid were screened. E.coli BL21(DE3) / pET28a-GASPase.

[0039] Example 2

[0040] Wet cells containing sucrose phosphorylase gene: the recombinant Escherichia coli obtained in Example 1 E.coli BL21(DE3) / pET28a-GASPase was inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37°C, 200 rpm for 12 h. Then, it was inoculated into fresh LB liquid medium containing 50 μg / mL kanamycin resistance at a 1% (v / v) inoculum and cultured at 37°C, 200 rpm until the bacterial OD 600 When the p-value reaches 0.6-0.8, IPTG with a final concentration of 0.1 mM is added, and the mixture is induced and cultured at 25°C for 16 h. The mixture is centrifuged at 4°C and 8000 rpm for 20 min, the supernatant is discarded, and the precipitate is collected to obtain the wet bacterial cells of the recombinant strain pET28a-GASPase containing sucrose phosphorylase.

[0041] Example 3

[0042] Constructed with Example 2 E.coli BL21(DE3) / pET28a-GASPase was the starting strain.

[0043] The complex model was obtained by docking the crystal structure of sucrose phosphorylase obtained by homology modeling through modification through directed evolution theory. The amino acid residues around the substrates sucrose and glycerol in the structure were analyzed, and sites 133, 160, 333, 335, 293, and 393 were selected for site-directed saturation mutation. The primer design is shown in Table 1. At the same time, based on the calculation of protein folding free energy, the stability-enhancing sites S36G, F435I, T428A, and D71E were selected for site-directed mutation.

[0044] The mutation PCR system (100 μL) was as follows: 25 μL 2× Phanta Max buffer, 1 μL dNTPs, 1 μL each of the mutation upper and lower primers, 1 μL template (starting strain), 0.5 μL Pfu DNA polymerase, supplemented with ddH 2 O to 50 μL. PCR conditions were: 95℃ pre-denaturation for 3 min, 30 cycles of: 95℃ 15 s, 60℃ 15 s, 72℃ 7 min20 s, and final extension at 72℃ for 10 min. PCR results were verified by DNA agarose gel electrophoresis, PCR products were digested with DpnI enzyme template, 37℃, 1 hour, 200 rpm, 65℃, 1 minute inactivation, PCR products were heat-shock transformed, and Escherichia coli were cultured. E.coli BL21(DE3) was activated and cultured at 37°C, 200 rpm for 1 hour, then spread on LB plates containing 50 μg / mL kanamycin resistance and cultured inverted at 37°C overnight.

[0045] Table 1 Primer design for site-directed mutagenesis of sucrose phosphorylase

[0046]

[0047] Example 4

[0048] Screening of sucrose phosphorylase gene mutation library:

[0049] A single clone was picked from the plate obtained in Example 3 and inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance, and cultured at 37°C, 200 rpm for 12 h. Then, it was inoculated into fresh LB liquid medium containing 50 μg / mL kanamycin resistance at a 1% (v / v) inoculation amount and cultured at 37°C, 200 rpm until the bacterial OD reached 0. 600 When the p-value reaches 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1 mM, induce and culture at 25°C for 16 h, centrifuge at 4°C and 8000 rpm for 20 min, discard the supernatant, collect the precipitate, and obtain the wet cells containing the sucrose phosphorylase gene mutation library.

[0050] 1. Initial screening:

[0051] The reaction solution (200 μL) was prepared as follows: the final concentration of the substrate sucrose was 100 mM, the final concentration of the glycerol was 110 mM, the amount of the catalyst was 5 g / L based on the total weight of the wet bacteria, and the reaction solution was composed of pure water as the reaction medium. Reaction conditions: The reaction was carried out in a reactor at 40°C and 500 rpm for 2 hours. After the reaction was completed, 20 μL of the sample after the reaction was completed was taken, diluted 5 times, filtered through a 0.22 μm filter membrane, and HPLC detection was performed. The test results are shown in Table 2.

[0052] Table 2 Results of initial screening reactions

[0053]

[0054] 2. Rescreening:

[0055] The strains obtained in the initial screening were rescreened, and the rescreening reaction solution (10 mL) was configured: the final concentration of substrate sucrose was 300 mM, the final concentration of glycerol was 330 mM, the catalyst amount was 5 g / L based on the total weight of wet bacteria, and pure water was used as the reaction medium to constitute the reaction solution. Reaction conditions: After reacting in a reactor at 40 °C and 500 rpm for 2 hours, 20 μL of the sample after the reaction was completed was taken, diluted 10 times, filtered through a 0.22 μm filter membrane, and HPLC detection was performed. The test results are shown in Table 3.

[0056] Table 3 Rescreening reaction results

[0057]

[0058] Example 5

[0059] Application of sucrose phosphorylase in catalyzing the synthesis of glycerol glucoside:

[0060] The recombinant sucrose phosphorylase mutant with the highest activity obtained in Example 4 E.coli BL21(DE3) / pET28a-GASPase-S36G-K133R-N333G-D335L-F435I was inoculated into LB liquid medium containing kanamycin at a final concentration of 50 μg / mL, and cultured at 37 °C for 9 hours. As the seed solution, it was inoculated into a 5 L fermenter containing 3 L fermentation medium at a volume concentration of 3.5%. The prepared culture medium was added, the air outlet and air inlet were sealed and sealed, the inoculation port was opened, and the inoculation port was placed in the autoclave together with the prepared lactose inducer for sterilization at 115 °C for 30 min. The sterilized fermenter was screwed on the inoculation port and installed on the operating system, condensed water and air were passed (the air inlet pipe should be installed with a sterilization film), the air outlet was inserted below the liquid level of the conical flask, and when the autoclave dropped to 37 °C, the fire ring was put on the inoculation port, and the cultured seed solution was inoculated into the fermenter. Cultivate at 37°C, 500 rpm for about 3-4 hours, and when the bacterial density OD reaches 6-8, the fermentation tank temperature is lowered to 25°C, and lactose with a final concentration of 16 g / L is added as an inducer, and then culture at 25°C, 500 rpm for another 12 hours. Centrifuge the fermented liquid at 8000 rpm for 10 minutes to obtain the mutant containing sucrose phosphorylase. E.coli Wet bacteria of BL21(DE3) / pET28a-GASPase-S36G-K133R-N333G-D335L-F435I.

[0061] The fermentation tank culture medium consists of: 45 g of tryptone, 36 g of yeast extract, 30 g of sodium chloride, 4.08 g of potassium dihydrogen phosphate, 45 g of glycerol, 6.84 g of dipotassium hydrogen phosphate trihydrate, 15 g of ammonium sulfate, 1.125 g of magnesium sulfate, and 4 g of defoaming agent, and distilled water is added to make the volume to 3 L for dissolution.

[0062] Catalyst dosage: 15 g / L of total wet bacterial cell weight, 1M substrate sucrose at a final concentration, 1.2M substrate glycerol at a final concentration, and pure water as the reaction medium, with a total volume of 1 L. Reaction conditions: 40 °C, 500 rpm for 12 hours. After the reaction, take 20 μL of the sample, dilute 50 times, filter through a 0.22 μm filter membrane, and perform HPLC detection. The reactions that occur during the catalytic process are as follows: Figure 1 The reaction progress curve is shown in Figure 2As shown, after the reaction was completed, the concentration of 2-α-glycerol glucoside was 228.3 g / L, and the by-product 1-α-glycerol glucoside was 14.6 g / L.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A sucrose phosphorylase mutant, characterized in that: The wild-type sucrose phosphorylase with an amino acid sequence as shown in SEQ ID NO: 2 has serine at position 36 mutated to glycine; lysine at position 133 mutated to arginine; asparagine at position 333 mutated to glycine; aspartic acid at position 335 mutated to leucine; and phenylalanine at position 435 mutated to isoleucine.

2. A gene encoding the sucrose phosphorylase mutant according to claim 1.

3. An expression cassette, characterized in that Comprising the gene according to claim 2.

4. A recombinant expression vector, characterized in that: Comprising the gene according to claim 2.

5. A recombinant bacterium, characterized in that: Comprising the gene according to claim 2.

6. Use of the recombinant bacteria according to claim 5 in catalyzing the synthesis of 2-glycerol glucoside.

7. A method for synthesizing 2-glycerol glucoside, characterized in that: include: The wet bacteria obtained by induction culture of genetically engineered bacteria of sucrose phosphorylase mutant or the crude enzyme solution extracted by crushing the wet bacteria is used as a catalyst, sucrose and glycerol are used as substrates, and pure water is used as a reaction medium to form a reaction system, and 2-glycerol glucoside is obtained by reaction; Wherein, the genetically engineered bacteria is constructed by introducing the sucrose phosphorylase mutant described in claim 1 into a host bacteria.

8. The method according to claim 7, characterized in that The amount of the catalyst used is 5-15 g / L based on the total weight of the wet cells, the initial concentration of the substrate sucrose is 0.3 M-1 M, and the initial concentration of the substrate glycerol is 0.33 M-1.1 M.

9. The method according to claim 7, characterized in that The method of crushing wet bacteria to extract crude enzyme solution includes ultrasonic crushing.

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