Sucrose phosphorylase mutant and application thereof in preparation of L-ascorbyl glucoside

Abstract

The invention discloses a sucrose phosphorylase mutant and application thereof in preparation of L-ascorbyl glucoside, and belongs to the field of enzyme engineering modification. According to the invention, sucrose phosphorylase is semi-rationally modified, homologous modeling is used for mutant prediction, a small mutant library is constructed, and a mutant with improved thermal stability is obtained after screening. The mutants are used for synthesizing the L-ascorbyl glucoside, reaction conditions are optimized, the yield of the L-ascorbyl glucoside is improved, and a foundation is laid for improving stability and large-scale application of industrial production.

Description

Technical Field

[0001] This invention relates to a sucrose phosphorylase mutant and its application in the preparation of L-ascorbic acid glucoside, belonging to the field of enzyme engineering modification. Background Technology

[0002] Sucrose phosphorylase (SPase), with its excellent acceptor heterogeneity, can transfer glycosyl groups from sucrose to various carbohydrate and non-carbohydrate molecules, thereby efficiently synthesizing a variety of high-value-added glycosides and oligosaccharides. L-Ascorbic acid glucoside (AA-2G), a glycoside compound formed by linking L-ascorbic acid (vitamin C, L-AA) and glucose molecules with a glycosidic bond, has extremely wide applications in cosmetics (such as whitening skincare products), food (such as antioxidant food additives), and pharmaceuticals (such as vitamin supplements) due to its excellent stability, low irritation, and good biological activity.

[0003] In the synthesis of L-ascorbic acid glucoside, current chemical synthesis methods are hampered by the generation of numerous byproducts during the reaction process. This not only makes it difficult to improve the purity of the target product but also results in cumbersome and costly subsequent purification steps, making them extremely uneconomical. While biosynthesis aligns with environmentally friendly and sustainable development principles, it is limited by the difficulty in controlling the reaction system and low product yield, making it difficult to meet the needs of large-scale industrial production. Utilizing sucrose phosphorylase to transglycosylate some compounds, along with more convenient whole-cell catalysis, can significantly simplify the synthesis of L-ascorbic acid. However, the poor stability of SPases and the requirement for high-temperature, long-term reactions limit their industrial application. This necessitates further modification of SPases to improve their stability, laying the foundation for large-scale industrial production. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention semi-rationally modifies sucrose phosphorylase, uses homology modeling to predict mutants, constructs a small mutant library, and after screening, obtains a mutant with improved thermal stability.

[0005] This invention is achieved through the following technical solution: The first objective of this invention is to provide a sucrose phosphorylase mutant in which glycine at position 197 of the amino acid sequence shown in SEQ ID NO. 2 is mutated to alanine.

[0006] In one embodiment of the present invention, the amino acid sequence of the mutant is shown in SEQ ID NO.1.

[0007] A second object of the present invention is to provide a gene encoding the mutant.

[0008] A third objective of this invention is to provide a recombinant plasmid carrying the said gene.

[0009] In one embodiment of the present invention, the vector for the recombinant plasmid is a pET series plasmid, including but not limited to pET28a.

[0010] A fourth object of the present invention is to provide a recombinant microorganism expressing the mutant.

[0011] In one embodiment of the present invention, the recombinant microorganism uses Escherichia coli BL21(DE3) as the host and pET28a as the expression vector.

[0012] The fifth objective of this invention is to provide a method for improving the stability of sucrose phosphorylase, which involves mutating glycine at position 197 of the amino acid sequence shown in SEQ ID NO.2 to alanine to obtain the mutant G197A shown in SEQ ID NO.1.

[0013] A sixth object of the present invention is to provide the application of the sucrose phosphorylase mutant in the whole-cell catalytic production of L-ascorbic acid glucoside in Escherichia coli.

[0014] In one embodiment of the present invention, the application involves adding the mutant or recombinant microorganism expressing the mutant to a reaction system containing vitamin C and / or sucrose, at a temperature of 30-50 °C. o C. React at pH 5-6 for 40-100 hours.

[0015] In one embodiment of the present invention, the concentration of vitamin C in the reaction system is 0.8~1.6 mol / L.

[0016] In one embodiment of the present invention, the cell density OD600 in the reaction system is 20~50 mol / L.

[0017] In one embodiment of the present invention, the reaction temperature is 45°C. o C.

[0018] In one embodiment of the present invention, the pH of the reaction system is 5.2.

[0019] A seventh object of the present invention is to provide the use of the sucrose phosphorylase mutant in the production of products containing L-ascorbic acid glucoside.

[0020] The beneficial effects of this invention are: This invention mutated the sucrose phosphorylase of *Bifidobacterium longum*, obtaining an excellent mutant, G197A. Compared to the wild-type strain, this mutant showed improved stability after cultivation: the half-life of G197A at 50℃ was 24.75 min longer than the wild-type. Simultaneously, optimized reaction conditions yielded higher L-ascorbic acid glucoside production. Finally, the whole-cell catalytic system was scaled up, achieving an L-ascorbic acid glucoside concentration of 383.13 g / L. This invention has broad industrial application prospects, enriches the methods and strategies for strain modification, and provides a theoretical basis for the enzyme engineering modification of related strains. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The half-life of WT, G222A, A84I, D35L and G197A; Figure 2 The Tm values ​​are for WT, G222A, A84I, D35L, and G197A. Figure 3 The transglycosylation activities of WT, G222A, A84I, D35L and G197A; Figure 4 The effect of L-AA concentration on the catalytic synthesis of AA-2G; Figure 5 The effect of cell density OD600 on the catalytic synthesis of AA-2G; Figure 6 To obtain the Escherichia coli cell growth curve for high-density fermentation; Figure 7 To increase the yield of AA-2G synthesized by whole-cell catalysis of G197A; Figure 8 The results are HPLC analysis of the components in the eluent and the AA-2G standard. Detailed Implementation

[0023] The present invention will be further illustrated below with specific examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0024] The culture medium formulations used in the following examples are: (1) LB medium (g / L): tryptone 10, yeast extract 5, sodium chloride 10.

[0025] (2) TB medium (g / L): tryptone 12, yeast extract 24, glycerol 4 ml, dipotassium hydrogen phosphate trihydrate 16.43, potassium dihydrogen phosphate 2.31.

[0026] (3) Kanamycin-resistant LB medium: Kanamycin was added to LB medium to a final concentration of 50 μg / mL.

[0027] (4) Kanamycin-resistant TB medium: Kanamycin was added to TB medium to a final concentration of 50 μg / mL.

[0028] The reagents used in the following examples: 2 × Phanta Flash Master Mix (Vazyme), QuickCut Dpn I. Enzymes and DL-10000 DNA Marker were purchased from Takara Bio Inc. (TaKaRa). Sucrose, kanamycin, isopropyl-β-D-thiogalactoside (IPTG), and 2-(N-morpholino)ethanesulfonic acid (MES) were purchased from Shanghai Maclean Biotechnology Co., Ltd. Commonly used reagents such as L-AA, phenol, fructose, KH2PO4, 3,5-dinitrosalicylic acid (DNS), potassium sodium tartrate, anhydrous glucose, sodium hydroxide, and imidazole were purchased from Sinopharm Chemical Reagent Co., Ltd. 180 kDa protein marker was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd. Tryptone and yeast extract were purchased from Oxioid.

[0029] The HPLC detection conditions for L-ascorbic acid glucoside (AA-2G) were as follows: organic acid column (4.6 mm × 250 mm, 5 μm), mobile phase: 5 mM H2SO4, 0.6 mL·min -1 The flow rate was [value missing], the injection volume was 5 μL, the column temperature was 45℃, and the detection method was ultraviolet light.

[0030] Table 1 Instruments and Equipment

[0031] Table 2 Primer Sequences

[0032] Enzyme activity assay: The reaction mixture consisted of 250 μL of 5% (w / v) sucrose and 225 μL of PBS buffer (50 mmol·L⁻¹). -1After reacting 25 μL of crude enzyme solution (pH 6.5) in a metal bath at 40℃ for 10 min, 750 μL of DNS was added to the system and the mixture was boiled for 10 min. OD was then measured after cooling. 540 The absorbance value is used to calculate the amount of fructose. One unit of SPase enzyme activity (U) is defined as the amount of enzyme required to hydrolyze sucrose to produce 1 mmol of fructose per minute.

[0033]

[0034] P: fructose molar concentration (mmol·L) -1 ) n: Dilution factor t: Reaction time (unit: min) Enzyme activity assay: Enzyme activity is the enzyme activity per milligram of protein.

[0035]

[0036] c: Protein concentration (unit: mg·mL) -1 ).

[0037] The technical solution of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased commercially, prepared by conventional methods, or commonly used in the industry.

[0038] Example 1: Design and construction of thermally stable mutants 1. The crystal structure of sucrose phosphorylase and its homologous sequence were simulated using the SWISS-MODEL website, the mutation sites were calculated using the PROSS website, and suitable sites were analyzed for construction.

[0039] 2. Based on the wild-type strain BL21(DE3) / pET-28a- constructed in our laboratory spase(Carrying the sucrose phosphorylase gene shown in SEQ ID NO.2, this strain is disclosed in the paper "Production of 2-O-α-d-glucopyranosyl-l-ascorbic acid using sucrose phosphorylase by semi-rational design"), using the primers in Table 2, site-directed mutagenesis was performed using a PCR program. PCR conditions were: 98℃ (30s); 98℃ (10s), 55℃ (5s), 72℃ (35s) ​​for 35 cycles; 72℃ (10min). The reaction system consisted of: 1 μL plasmid DNA, 25 μL 2×Phanta Flash Master Mix (Dye Plus), 2 μL each of upstream and downstream mutagenesis primers, and 20 μL ddH2O, for a total volume of 100 μL. After the reaction, the mixture was... Dpn I. The enzyme digested the template DNA at 37°C for two hours. The digested PCR product was then transferred to... E. coli BL21(DE3) competent cells were transformed and the mixture was plated on LB agar plates (kanamycin 100 μg / mL). -1 The samples were cultured at 37°C for 12 h. Transformants were then cultured in LB broth, and plasmids were extracted and sent for sequencing to verify whether the mutations were correct.

[0040] Example 2: Expression of wild-type enzyme and mutant Use a sterile inoculation loop from -80°C o Bacterial culture was picked from a C-preserved glycerol culture tube and streaked in four zones on the surface of LB solid medium at 37°C. o Incubate at C for 8-12 hours. Afterward, select morphologically uniform single colonies and inoculate them into 20 mL of LB liquid medium, incubating at 37°C. o C. The seed culture was prepared by shaking at 200 rpm for 10 hours. Subsequently, the seed culture was transferred to TB medium (composition: 12 g / L tryptone, 24 g / L yeast extract, 2.31 g / L KH2PO4, 16.43 g / L K2HPO4·3H2O, 4 mL / L glycerol) at a 1% (v / v) inoculation rate and incubated at 37°C. o C. Scale-up culture was carried out at 200 rpm. The LB solid medium used contained 100 μg / mL kanamycin sulfate, and the LB and TB liquid media contained 50 μg / mL kanamycin sulfate.

[0041] Example 3: Purification of wild-type and mutant strains After bacterial culture, the cells were collected by centrifugation at 6500 rpm for 10 min (Centrifuge 5430R, Eppendorf). The cells were resuspended in pre-cooled MES buffer (50 mM, pH 7.0), and the centrifugation was repeated 2-3 times to wash away any residual culture medium. The bacterial suspension was then ultrasonically disrupted with 25% amplitude, a pulse period of 2 seconds on and 4 seconds off, for a total duration of 15 minutes, followed by centrifugation at 6500 rpm for 30 minutes (4... o C) Obtain the crude enzyme supernatant.

[0042] After obtaining the fragmented supernatant, protein purification was performed using nickel affinity chromatography. The supernatant was loaded onto a pre-equilibrated Ni-NTA resin column at a flow rate of 1 mL / min. Non-specifically bound proteins were washed with MES buffer (pH 7.0) containing 20 mM imidazole. Finally, the target protein was eluted with a gradient of elution buffer containing 500 mM imidazole. The collected protein fraction was then subjected to ultrafiltration (30 kDa molecular weight cutoff) to buffer replacement with storage buffer (50 mM MES, pH 7.0) and concentrated to the target concentration.

[0043] Example 4: Wild-type and mutant stability test After purification, the pure enzyme was uniformly concentrated at 50. o Incubate in a water bath at temperature C for 60 min, then perform phosphorylation reaction and measure its phosphorylation activity. The residual enzyme activity (RA, %) is calculated using the formula: RA (%) = (At / A0) × 100. Where A0 is the initial enzyme activity of the untreated sample, and At is the enzyme activity measured after heat treatment.

[0044] The half-life (t1 / 2) was determined by incubating the purified enzyme at a uniform protein concentration in a 50°C water bath for 240 min. Residual enzyme activity (RA, %) was measured every 60 min, and the half-life was then obtained by fitting a half-life curve. The results are as follows: Figure 1 As shown, the half-life of G197A is 24.75 min longer than that of WT.

[0045] We typically use melting temperature (Tm) to more accurately describe the thermal stability of proteins. Tm is measured using a nanometer differential scanning calorimeter (Nano DSC, Waters), which captures the change in the heat capacity of proteins during heating. After standardizing the protein concentration of the purified enzyme, it is used as 1... o The scan rate is between 20 and 80 C / min. o Scanning within interval C yields the heat capacity curve. Subtracting the blank control and automatically fitting the curve gives the melting temperature (Tm). Results are as follows: Figure 2As shown, the melting temperature of G197A is 2.01°C higher than that of WT. o C.

[0046] The transglycosylase activities of WT, G222A, A84I, D35L, and G197A were then measured, and the results are as follows: Figure 3 As shown, G197A decreased by less than 10%. Subsequent experiments were conducted using G197A to catalyze the preparation of AA-2G.

[0047] Example 5: Preparation of AA-2G catalyzed by sucrose phosphorylase The reaction system consisted of (by final concentration) vitamin C (0.8, 0.9, 1, 1.2, 1.4, 1.6 mol / L) and 0.8 mol / L sucrose in sodium citrate buffer (0.1 mmol / L, pH 5.2), with a cell density OD600 = 40 and a total system volume of 5 mL.

[0048] The final vitamin concentrations were controlled at 0.8, 0.9, 1, 1.2, 1.4, and 1.6 mol / L, respectively, and the reaction temperature was controlled at 44-46°C. o The reaction was carried out at C for 72 hours, and the yield of AA-2G was measured. The results are as follows: Figure 4 As shown, when the vitamin C concentration is 1.4 mol / L, the AA-2G yield reaches 185 g / L.

[0049] Example 6: Preparation of AA-2G catalyzed by sucrose phosphorylase The reaction system consisted of sodium citrate buffer (0.1 mmol / L, pH 5.2) containing different cell densities OD600 (20, 30, 40, 50) and 0.8 mol / L sucrose and 1.4 mol / L vitamin C, with a total volume of 5 mL.

[0050] Cell densities (OD600) were controlled at 20, 30, 40, and 50, respectively, and the reaction temperature was controlled at 44-46°C. o The reaction was carried out at C for 72 hours, and the yield of AA-2G was measured. The results are as follows: Figure 5 As shown, when the cell density OD600 is 40, the AA-2G yield reaches 185 g / L.

[0051] Example 7: Shake-flask fermentation culture of Escherichia coli and whole-cell catalysis of L-ascorbic acid glucoside Recombinant Escherichia coli was streaked onto LB agar plates containing 100 μg / mL, incubated overnight, and then a single colony was picked and transferred to 20 mL of liquid LB medium containing 50 μg / mL. The culture was then incubated at 37°C. oCultured at C for 10 h. Then, at a 1% inoculum, transfer to several 50 ml liquid LB medium solutions and incubate at 37°C. o After culturing at C20 until the cell density OD600 reaches 0.6-0.8, add IPTG to a final concentration of 0.5 mM and cool to 25°C. o Induction was initiated at C for 22 hours. After induction, the cells were collected by centrifugation, and the cell density (OD600) was controlled at 40. Whole-cell catalytic synthesis of AA-2G was then performed on wild-type and G197A cells. The system contained 1.4 M vitamin C, 0.8 M sucrose, and 0.1 M sodium citrate as glucose donors. A final concentration of 0.65 M sucrose solution was added after 24 hours of reaction, and the catalytic temperature was controlled at 44-46°C. o The concentration was maintained between 5.0 and 5.5, and the total catalytic time was 72 h. Results are as follows: Figure 6 As shown, the yield of AA-2G catalyzed by G197A was 375 g / L, while the yield of AA-2G catalyzed by wild-type was 337 g / L.

[0052] Example 8: High-density fermentation of Escherichia coli and scale-up production of L-ascorbic acid glucoside Recombinant Escherichia coli was streaked onto LB agar plates containing 100 μg / mL, incubated overnight, and then a single colony was picked and transferred to 50 mL of liquid LB medium containing 50 μg / mL. The culture was then incubated at 37°C. o Cultured at C for 10 h. Then, at a 1% inoculum, transfer to several 50 ml liquid LB medium containers and incubate at 37°C. o C was cultured to an OD600 of approximately 2.6 to 2.8. Then, it was transferred at a 7.5% inoculum to a 10 L fermenter containing 4 L of material. Subsequently, it was fermented at 6 vvm, 200 rpm, and 37°C. o Fermentation was carried out under C conditions, with ammonia used for pH adjustment to maintain the pH between 6.8 and 7.0. When the cell density OD600 reached 40, the temperature was lowered to 25°C. o C, and start adding IPTG at a rate of 30 ml / h, then continue incubation for 14 to 15 h. Results are as follows: Figure 5 As shown, the cell density OD600 eventually reached 142.

[0053] The cell density was adjusted to 40, and whole-cell catalytic production of AA-2G was carried out. A 100 mL system contained 1.4 M vitamin C, 0.8 M sucrose, and 0.1 M sodium citrate as glucose donors. A 0.65 M sucrose solution was added after 24 h of reaction. The catalytic temperature was controlled at 44-46 °C. oThe concentration was maintained between 5.0 and 5.5, and the total catalytic time was 72 h. Results are as follows: Figure 7 As shown, the final result was determined by HPLC ( Figure 8 The yield of AA-2G was 383.13 g / L, while the yield of AA-2G in WT showed almost no increase in the later stages of catalysis, indicating that G197A can ultimately effectively improve the yield of AA-2G (e.g., Figure 7 ).

[0054] Example 9: Isolation and purification of L-ascorbic acid glucoside To improve the downstream process for producing AA-2G, this study used anion exchange resin to separate and purify AA-2G and L-AA. Since the concentration of AA-2G in the original conversion solution was high, and the resin's adsorption capacity was limited, the conversion solution was diluted before resin adsorption to a concentration of approximately 50 g / L of AA-2G. After resin regeneration, the solution was mixed with the diluted conversion solution, with a total volume of 0.45 L. After 24 h of static adsorption, approximately 6.1 g of AA-2G remained unadsorbed. Batch elution with 0.3 M HCl was then initiated. This step also employed static elution. Due to the limited exchange capacity of each batch of eluent, four batches of elution were performed, with the eluent changed every hour. It can be seen that the amount of AA-2G that can be eluted decreased sharply during the fourth batch of elution. In order to save time and cost, no further elution was performed. The total amount of AA-2G eluted in the four batches was 14.89g, and the elution rate reached 90.79%. This shows that using anion exchange resin for the downstream separation and purification of AA-2G is an efficient and feasible method.

[0055] At the same time, such as Figure 8 As shown, HPLC analysis of the components in the eluent revealed a single peak with a retention time consistent with that of the standard AA-2G.

[0056] The sequences involved in the embodiments are as follows: SEQ ID NO.1: G197A amino acid sequence MKNKVQLITYADRLGDGTLSSMADILRTRFDGVYDGVHILPFFTPFDGADAGFDPIDHTKVDERLGSWDDVAELSKTHNIMVDAIVNHMSWESKQFQDVLEKGEESEYYPMFLTMSSVFPNGATEEDLAGIYRPRPGLPFTHYKFAGKTRLVWVSFTPQQVDIDTDSDKGWEYLMSIFDQMAASHVSYIRLDAVGYAAKEAGTSCFMTPKTFKLISRLREEGVKRGLEILIEVHSYYKKQVEIASKVDRVYDFALPPLLHSLFTGHVEPVAHWTEIRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDEDVDNLVNTIHANTHGESQAATGAAASNVDLYQPNSTYYSALGCNDQHYLAARAVQFFLPGVPQVYYVGALAGRNDMELLRRTNNGRDINRHYYSTAEIDENLERPVVKALNALAKFRNELPAFDGEFSYEVDGDTSITFRWTAADGTSTAALTFEPGRGLGTDNATPVASLAWSDAAGDHETRDLLANPPIADID SEQ ID NO.2: wild type SPase amino acid sequence MKNKVQLITYADRLGDGTLSSMADILRTRFDGVYDGVHILPFFTPFDGADAGFDPIDHTKVDERLGSWDDVAELSKTHNIMVDAIVNHMSWESKQFQDVLEKGEESEYYPMFLTMSSVFPNGATEED LAGIYRPRPGLPFTHYKFAGKTRLVWVSFTPQQVDIDTDSDKGWEYLMSIFDQMAASHVSYIRLDAVGYGAKEAGTSCFMTPKTFKLISRLREEGVKRGLEILIEVHSYYKKQVEIASKVDRVYDFA LPPLLLHSLFTGHVEPVAHWTEIRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDEDVDNLVNTIHANTHGESQAATGAAASNVDLYQPNSTYYSALGCNDQHYLAARAVQFFLPGVPQVYYVG ALAGRNDMELLRRTNNGRDINRHYYSTAEIDENLERPVVKALNALAKFRNELPAFDGEFSYEVDGDTSITFRWTAADGTSTAALTFEPGRGLGTDNATPVASLAWSDAAGDHETRDLLANPPIADID The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A sucrose phosphorylase mutant, characterized in that, The mutant is a mutant G197A obtained by mutating the glycine at position 197 of the amino acid sequence shown in SEQ ID NO. 2 to alanine.

2. A gene encoding the sucrose phosphorylase mutant of claim 1.

3. A recombinant plasmid carrying the gene of claim 2.

4. A recombinant microorganism expressing the sucrose phosphorylase mutant of claim 1.

5. The recombinant microorganism of claim 4, wherein, The recombinant microorganism takes Escherichia coli BL21 (DE3) as a host and takes pET28a as an expression vector.

6. A method of improving the stability of sucrose phosphorylase, characterized in that, The mutant G197A is obtained by mutating the glycine at position 197 of the amino acid sequence shown in SEQ ID NO. 2 to alanine.

7. Use of the sucrose phosphorylase mutant of claim 1 in catalyzing production of L-ascorbyl glucoside by whole cells of Escherichia coli.

8. Use according to claim 7, characterized in that, The application is to add the mutant or the recombinant microorganism expressing the mutant into a reaction system containing vitamin C and / or sucrose, and to react at 30~50 o C, pH 5~6 for 40~100 h.

9. Use according to claim 8, characterized in that, The concentration of vitamin C in the reaction system is 0.8-1.6 mol / L.

10. Use according to claim 8, characterized in that, The cell density OD600 in the reaction system is 20-50 mol / L.

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