A process for synthesizing AA-2G using sucrose phosphorylase whole cells

Abstract

This invention belongs to the field of ascorbic acid derivative preparation technology, specifically relating to a process for the whole-cell catalytic synthesis of AA-2G using sucrose phosphorylase. This invention effectively enhances the enzyme activity of sucrose phosphorylase by site-directed mutagenesis derived from *Bifidobacterium longum*, and obtains high yields of AA-2G through whole-cell catalytic synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid. Furthermore, the use of modified LB medium and treatment agents in this invention significantly improves the yield of AA-2G, providing a foundation for its industrial production. In addition, this invention employs a biocatalytic method to synthesize AA-2G under mild reaction conditions, without using toxic or harmful chemical reagents, reducing environmental pollution and aligning with the development concept of green chemistry.

Description

Technical Field

[0001] This invention belongs to the field of ascorbic acid derivative preparation technology, specifically relating to a process for the whole-cell catalytic synthesis of AA-2G using sucrose phosphorylase. Background Technology

[0002] Vitamin C is a natural active ingredient with excellent performance in skincare: it can whiten and lighten dark spots by inhibiting tyrosinase activity, stimulate collagen production and improve skin elasticity, and also has strong antioxidant properties. However, due to the presence of anthracene diol in its molecule, the hydroxyl group at C-2 is highly sensitive to temperature, oxygen, and metal ions, leading to reduced activity and decreased stability, thus limiting the efficacy of vitamin C. Vitamin C derivatives derived from structural modifications, such as metal salts, esters, and glycosyl derivatives, have improved the stability of vitamin C to some extent. Among them, 2-OD-glucanopyranosyl-L-ascorbic acid (AA-2G) stands out for its outstanding stability and its ability to be slowly released into vitamin C in the human body.

[0003] AA-2G is widely used in skincare products, mostly as a whitening additive. AA-2G also has good formula compatibility and is easily soluble in water, which increases the convenience of product development. Currently, it has been successfully applied in toners, lotions, and creams.

[0004] AA-2G is primarily synthesized enzymatically, where the glucosinolate from the donor is transferred to the C2 of vitamin C by glycosyltransferases. Currently reported enzymes for AA-2G synthesis include α-glucosidase, α-amylase, sucrosephosphorylase (SPase), cyclodextrin glucosyltransferase (CGTase), α-isomaltose-glucosyl synthase, and dextran sucrase. During the enzymatic preparation of AA-2G, some glycosyl donor and L-ascorbic acid remain, requiring the removal of these impurities to obtain high-purity AA-2G. Currently, there are few researched purification methods for AA-2G, mainly electrodialysis and ion exchange.

[0005] Sucrose phosphorylase was first discovered in Leuconostoc mesenteroides, and is mainly distributed in bacteria, with a few found in plants. To date, the main strains from which SPase originates include: Streptococcus mutans, Bifidobacterium sp., Pseudomonas saccharophila, and Leuconostoc mesenteroides.

[0006] Enzymes are environmentally friendly and highly efficient biocatalysts, but their efficiency often requires a "mild" temperature and pressure environment close to that within living organisms. However, actual industrial production places stringent requirements on enzyme reaction temperature, pressure, ion concentration, substrate specificity, and activity. To address this, researchers have employed in vitro evolution to modify enzymes at the DNA level, thereby altering their protein structure. The aim is to obtain mutants with activity, stability, and substrate specificity suitable for industrial applications.

[0007] Based on this, we propose a whole-cell catalytic synthesis process for AA-2G using sucrose phosphorylase, hoping to address the shortcomings of existing technologies. Summary of the Invention

[0008] The purpose of this invention is to address existing problems by providing a process for the whole-cell catalytic synthesis of AA-2G using sucrose phosphorylase.

[0009] This invention is achieved through the following technical solution:

[0010] A process for the whole-cell catalytic synthesis of AA-2G using sucrose phosphorylase, characterized by comprising the following steps:

[0011] S1. A key site mutation was performed on sucrose phosphorylase (SPase) from Bifidobacterium longum, and then the mutant SPase gene was transformed into Escherichia coli BL21 (DE3) through the PET-22b(+) expression vector to obtain a mutant strain.

[0012] S2. The mutant strain was inoculated into modified LB medium containing kanamycin and cultured at 37℃ and 200 r / min until OD 600 = 0.6~0.8. IPTG was added and the culture was induced at 28℃ for 14 h. After the culture was completed, the cells were centrifuged, washed three times with phosphate buffer containing 8% glycerol, and stored at -20℃ for later use.

[0013] S3. Dissolve 60-80 g / L sucrose and 40-50 g / L ascorbic acid in phosphate buffer, then add 5-6 mmol / L of the bacterial cells obtained in step S2, stir and mix well, add the treatment agent, and then place it in a shaker for a shaking reaction.

[0014] S4. Product separation and purification:

[0015] After the reaction was completed, the supernatant was centrifuged and passed through a 0.22 μm microfiltration membrane and a 10 kDa ultrafiltration membrane to remove macromolecular impurities. Then, the ultrafiltration solution was initially separated using a strong acid cation exchange resin and eluted with 0.1 mol / L hydrochloric acid solution. The eluent containing AA-2G was collected, and the eluent was concentrated and then purified by preparative high performance liquid chromatography.

[0016] More preferably, step S1 specifically includes:

[0017] 1) Using the nucleotide sequence shown in SEQ ID NO:4 as a template and L341V-F and L341V-R as primers, PCR was performed to obtain the mutant shown in SEQ ID NO:3; the amino acid sequence of the mutant is shown in SEQ ID NO:1;

[0018] 2) The mutant gene and PET-22b(+) were digested with BamHI and EcolI, respectively, purified, and ligated with T4 DNA ligase overnight at 16°C, and then sequenced.

[0019] 3) The ligation product was transformed into Escherichia coli BL21(DE3), plated on LB agar plates containing antibiotics, and cultured at 37°C for 12-16 h to obtain mutant strains.

[0020] More preferably, the sequence of L341V-F in step 1) is as shown in SEQ ID NO:5, and the sequence of L341V-R is as shown in SEQ ID NO:6.

[0021] More preferably, the antibiotic mentioned in step 3) is kanamycin, and the concentration of kanamycin is 50 μg / mL.

[0022] More preferably, the modified LB medium in step S2 comprises the following components: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 6-8 μg / L vitamin C, 6-8 μg / L vitamin E, 10-12 ng / L tyrosine, 15-19 ng / L L-arginine, 2.3-2.6 μg / L lanthanum nitrate, 1.9-2.5 μg / L cerium nitrate, and 10-12 μg / L silk fibroin.

[0023] More preferably, the concentration of IPTG in step S2 is 0.5 mmol / L;

[0024] The specific parameters for centrifugation are: centrifugation at 8000~10000 r / min for 10~15 min.

[0025] More preferably, the concentration of the phosphate buffer solution in step S3 is 10~20 mmol / L;

[0026] The components and their corresponding weight percentages in the treatment agent are as follows: Tween 80 0.1~1%, hydroxypropyl-β-cyclodextrin 4~6%, 1-ethyl-3-methylimidazolium acetate 3~4%, and the balance is deionized water.

[0027] More preferably, the temperature during the shaking culture in step S3 is controlled at 35~39℃, the shaking speed is 150~250 r / min, and the reaction time is 12~36 h.

[0028] More preferably, the centrifugation speed in step S4 is 8000~10000 r / min, and the centrifugation time is 10~15 min.

[0029] The present invention has the following advantages over the prior art:

[0030] This invention effectively enhances the activity of sucrose phosphorylase by site-directed mutagenesis of Bifidobacterium longum, and achieves high yields of AA-2G through whole-cell catalysis to synthesize 2-OD-glucanopyranosyl-L-ascorbic acid. Furthermore, the use of modified LB medium and treatment agents in this invention significantly improves the yield of AA-2G, providing a foundation for its industrial production.

[0031] First, this invention involves key point mutation of sucrose phosphorylase (SPase) from Bifidobacterium longum, mutating leucine at position 341 to valine (L341V). This enhances the flexibility of the substrate binding site, increases the enzyme's affinity for L-ascorbic acid, and reduces competition from hydrolytic side reactions by optimizing the catalytic site conformation, thereby improving the selectivity of AA-2G. The resulting mutant gene is then transformed into Escherichia coli. By adjusting the culture medium and culture conditions, the lag phase and logarithmic phase of cell growth are shortened, accelerating the logarithmic phase and eliminating the stationary phase, thereby increasing cell growth rate and proliferation, promoting cell growth, and achieving efficient intracellular expression of the mutant SPase gene to obtain a highly active and highly tolerable enzyme.

[0032] Secondly, whole-cell catalysis was used for catalytic synthesis. During the synthesis process, a treatment agent was added to the reaction system. Tween 80, hydroxypropyl-β-cyclodextrin, and 1-ethyl-3-methylimidazolium acetate in the treatment agent synergistically regulated the enzyme microenvironment, enhanced the binding ability of the substrate to the enzyme active site, improved the solubility of sucrose and vitamin C, and promoted product synthesis. Microfiltration, ultrafiltration, and cation exchange resin separation techniques were used for product separation and purification, significantly improving product purity while increasing product content.

[0033] Finally, this invention uses a biocatalytic method to synthesize AA-2G. The reaction conditions are mild, and no toxic or harmful chemical reagents are used, which reduces environmental pollution and is in line with the development concept of green chemistry. Detailed Implementation

[0034] To further explain the present invention, the following specific embodiments are described.

[0035] Example 1

[0036] A process for the whole-cell catalytic synthesis of AA-2G using sucrose phosphorylase, characterized by comprising the following steps:

[0037] S1. A key site mutation was performed on sucrose phosphorylase (SPase) from Bifidobacterium longum, and then the mutant SPase gene was transformed into Escherichia coli BL21 (DE3) through the PET-22b(+) expression vector to obtain a mutant strain.

[0038] Specifically:

[0039] 1) Using the nucleotide sequence shown in SEQ ID NO:4 as a template, and L341V-F (SEQ ID NO:5) and L341V-R (SEQ ID NO:6) as primers, PCR was performed to obtain the mutant shown in SEQ ID NO:3; the amino acid sequence of the mutant is shown in SEQ ID NO:1;

[0040] 2) The mutant gene and PET-22b(+) were digested with BamHI and EcolI, respectively, purified, and ligated with T4 DNA ligase overnight at 16°C, and then sequenced.

[0041] 3) The ligation product was transformed into Escherichia coli BL21(DE3), plated on LB agar plates containing antibiotics (kanamycin 50 μg / mL), and cultured at 37°C for 12-16 h to obtain mutant strains;

[0042] S2. The mutant strain was inoculated into modified LB medium containing kanamycin and cultured at 37℃ and 200 r / min until OD 600 = 0.6. IPTG (0.5 mmol / L) was added and the culture was induced at 28℃ for 14 h. After the culture was completed, the cells were centrifuged at 8000 r / min for 10 min, and the cells were collected. The cells were washed three times with phosphate buffer containing 8% glycerol and stored at -20℃ for later use.

[0043] The modified LB medium comprises the following components: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, vitamin C 6 μg / L, vitamin E 6 μg / L, tyrosine 10 ng / L, L-arginine 15 ng / L, lanthanum nitrate 2.3 μg / L, cerium nitrate 1.9 μg / L, and silk fibroin 10 μg / L;

[0044] S3. Dissolve 60 g / L sucrose and 40 g / L ascorbic acid in phosphate buffer (10 mmol / L), then add 5 mmol / L of the bacterial cells obtained in step S2, stir and mix well, add the treatment agent, and then place in a shaker at 35°C and 150 r / min for 12 h.

[0045] The components and their corresponding weight percentages in the treatment agent are as follows: Tween 80 0.1%, hydroxypropyl-β-cyclodextrin 4%, 1-ethyl-3-methylimidazolium acetate 3%, and the balance is deionized water;

[0046] S4. Product separation and purification:

[0047] After the reaction was completed, the solution was centrifuged at 8000 r / min for 10 min. The supernatant was then passed through a 0.22 μm microfiltration membrane and a 10 kDa ultrafiltration membrane to remove macromolecular impurities. The ultrafiltration solution was then preliminarily separated using a strong acid cation exchange resin and eluted with 0.1 mol / L hydrochloric acid solution. The eluent containing AA-2G was collected, and the eluent was then concentrated and purified by preparative high performance liquid chromatography.

[0048] Example 2

[0049] A process for the whole-cell catalytic synthesis of AA-2G using sucrose phosphorylase, characterized by comprising the following steps:

[0050] S1. A key site mutation was performed on sucrose phosphorylase (SPase) from Bifidobacterium longum, and then the mutant SPase gene was transformed into Escherichia coli BL21 (DE3) through the PET-22b(+) expression vector to obtain a mutant strain.

[0051] Specifically:

[0052] 1) Using the nucleotide sequence shown in SEQ ID NO:4 as a template, and L341V-F (SEQ ID NO:5) and L341V-R (SEQ ID NO:6) as primers, PCR was performed to obtain the mutant shown in SEQ ID NO:3; the amino acid sequence of the mutant is shown in SEQ ID NO:1;

[0053] 2) The mutant gene and PET-22b(+) were digested with BamHI and EcolI, respectively, purified, and ligated with T4 DNA ligase overnight at 16°C, and then sequenced.

[0054] 3) The ligation product was transformed into Escherichia coli BL21(DE3), plated on LB agar plates containing antibiotics (kanamycin 50 μg / mL), and cultured at 37°C for 14 h to obtain mutant strains;

[0055] S2. The mutant strain was inoculated into modified LB medium containing kanamycin and cultured at 37℃ and 200 r / min until OD 600 = 0.7. IPTG (0.5 mmol / L) was added and the culture was induced at 28℃ for 14 h. After the culture was completed, the cells were centrifuged at 9000 r / min for 12 min, and the cells were collected. The cells were washed three times with phosphate buffer containing 8% glycerol and stored at -20℃ for later use.

[0056] The modified LB medium comprises the following components: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, vitamin C 7 μg / L, vitamin E 7 μg / L, tyrosine 11 ng / L, L-arginine 17 ng / L, lanthanum nitrate 2.4 μg / L, cerium nitrate 2.2 μg / L, and silk fibroin 11 μg / L;

[0057] S3. Dissolve 70 g / L sucrose and 45 g / L ascorbic acid in phosphate buffer (15 mmol / L), then add 5.5 mmol / L of the bacterial cells obtained in step S2, stir and mix well, add the treatment agent, and then place in a shaker at 37°C and 200 r / min for 24 h.

[0058] The components and their corresponding weight percentages in the treatment agent are as follows: Tween 80 0.5%, hydroxypropyl-β-cyclodextrin 5%, 1-ethyl-3-methylimidazolium acetate 3.5%, with the remainder being deionized water;

[0059] S4. Product separation and purification:

[0060] After the reaction was completed, the solution was centrifuged at 9000 r / min for 12 min. The supernatant was then passed through a 0.22 μm microfiltration membrane and a 10 kDa ultrafiltration membrane to remove macromolecular impurities. The ultrafiltration solution was then preliminarily separated using a strong acid cation exchange resin and eluted with 0.1 mol / L hydrochloric acid solution. The eluent containing AA-2G was collected, and the eluent was then concentrated and purified by preparative high performance liquid chromatography.

[0061] Example 3

[0062] A process for the whole-cell catalytic synthesis of AA-2G using sucrose phosphorylase, characterized by comprising the following steps:

[0063] S1. A key site mutation was performed on sucrose phosphorylase (SPase) from Bifidobacterium longum, and then the mutant SPase gene was transformed into Escherichia coli BL21 (DE3) through the PET-22b(+) expression vector to obtain a mutant strain.

[0064] Specifically:

[0065] 1) Using the nucleotide sequence shown in SEQ ID NO:4 as a template, and L341V-F (SEQ ID NO:5) and L341V-R (SEQ ID NO:6) as primers, PCR was performed to obtain the mutant shown in SEQ ID NO:3; the amino acid sequence of the mutant is shown in SEQ ID NO:1;

[0066] 2) The mutant gene and PET-22b(+) were digested with BamHI and EcolI, respectively, purified, and ligated with T4 DNA ligase overnight at 16°C, and then sequenced.

[0067] 3) The ligation product was transformed into Escherichia coli BL21(DE3), plated on LB agar plates containing antibiotics (kanamycin 50 μg / mL), and cultured at 37°C for 16 h to obtain mutant strains;

[0068] S2. The mutant strain was inoculated into modified LB medium containing kanamycin and cultured at 37℃ and 200 r / min until OD 600 = 0.8. IPTG (0.5 mmol / L) was added and the culture was induced at 28℃ for 14 h. After the culture was completed, the culture was centrifuged at 10000 r / min and stored at -20℃ for later use.

[0069] The modified LB medium comprises the following components: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, vitamin C 8 μg / L, vitamin E 8 μg / L, tyrosine 12 ng / L, L-arginine 19 ng / L, lanthanum nitrate 2.6 μg / L, cerium nitrate 2.5 μg / L, and silk fibroin 12 μg / L;

[0070] S3. Dissolve 80 g / L sucrose and 50 g / L ascorbic acid in phosphate buffer (20 mmol / L), then add 6 mmol / L of the bacterial cells obtained in step S2, stir and mix well, add the treatment agent, and then place in a shaker at 39°C and 250 r / min for 36 h.

[0071] The components and their corresponding weight percentages in the treatment agent are as follows: Tween 80 1%, hydroxypropyl-β-cyclodextrin 6%, 1-ethyl-3-methylimidazolium acetate 4%, and the balance is deionized water;

[0072] S4. Product separation and purification:

[0073] After the reaction was completed, the solution was centrifuged at 10,000 r / min for 15 min. The supernatant was then passed through a 0.22 μm microfiltration membrane and a 10 kDa ultrafiltration membrane to remove macromolecular impurities. The ultrafiltration solution was then preliminarily separated using a strong acid cation exchange resin and eluted with 0.1 mol / L hydrochloric acid solution. The eluent containing AA-2G was collected, and the eluent was then concentrated and purified by preparative high performance liquid chromatography.

[0074] Comparative Example 1

[0075] A process for the whole-cell catalytic synthesis of AA-2G using sucrose phosphorylase, characterized by comprising the following steps:

[0076] S1. The sucrose phosphorylase (SPase) gene from Bifidobacterium longum and PET-22b(+) were digested with BamHI and Ecol I, respectively, purified, and ligated with T4 DNA ligase overnight at 16°C, and then sequenced; the ligation product was transformed into Escherichia coli BL21(DE3), plated on LB agar plates containing antibiotics (kanamycin 50 μg / mL), and cultured at 37°C for 14 h to obtain the engineered strain;

[0077] S2. The engineered strain was inoculated into modified LB medium containing kanamycin and cultured at 37℃ and 200 r / min until OD 600 = 0.7. IPTG (0.5 mmol / L) was added and the culture was induced at 28℃ for 14 h. After the culture was completed, the cells were centrifuged at 9000 r / min for 12 min, and the cells were collected. The cells were washed three times with phosphate buffer containing 8% glycerol and stored at -20℃ for later use.

[0078] The modified LB medium comprises the following components: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, vitamin C 7 μg / L, vitamin E 7 μg / L, tyrosine 11 ng / L, L-arginine 17 ng / L, lanthanum nitrate 2.4 μg / L, cerium nitrate 2.2 μg / L, and silk fibroin 11 μg / L;

[0079] S3. Dissolve 70 g / L sucrose and 45 g / L ascorbic acid in phosphate buffer (15 mmol / L), then add 5.5 mmol / L of the bacterial cells obtained in step S2, stir and mix well, add the treatment agent, and then place in a shaker at 37°C and 200 r / min for 24 h.

[0080] The components and their corresponding weight percentages in the treatment agent are as follows: Tween 80 0.5%, hydroxypropyl-β-cyclodextrin 5%, 1-ethyl-3-methylimidazolium acetate 3.5%, with the remainder being deionized water;

[0081] S4. Product separation and purification:

[0082] After the reaction was completed, the solution was centrifuged at 9000 r / min for 12 min. The supernatant was then passed through a 0.22 μm microfiltration membrane and a 10 kDa ultrafiltration membrane to remove macromolecular impurities. The ultrafiltration solution was then preliminarily separated using a strong acid cation exchange resin and eluted with 0.1 mol / L hydrochloric acid solution. The eluent containing AA-2G was collected, and the eluent was then concentrated and purified by preparative high performance liquid chromatography.

[0083] Comparative Example 2

[0084] A process for the whole-cell catalytic synthesis of AA-2G using sucrose phosphorylase, characterized by comprising the following steps:

[0085] S1. A key site mutation was performed on sucrose phosphorylase (SPase) from Bifidobacterium longum, and then the mutant SPase gene was transformed into Escherichia coli BL21 (DE3) through the PET-22b(+) expression vector to obtain a mutant strain.

[0086] Specifically:

[0087] 1) Using the nucleotide sequence shown in SEQ ID NO:4 as a template, and L341V-F (SEQ ID NO:5) and L341V-R (SEQ ID NO:6) as primers, PCR was performed to obtain the mutant shown in SEQ ID NO:3; the amino acid sequence of the mutant is shown in SEQ ID NO:1;

[0088] 2) The mutant gene and PET-22b(+) were digested with BamHI and EcolI, respectively, purified, and ligated with T4 DNA ligase overnight at 16°C, and then sequenced.

[0089] 3) The ligation product was transformed into Escherichia coli BL21(DE3), plated on LB agar plates containing antibiotics (kanamycin 50 μg / mL), and cultured at 37°C for 14 h to obtain mutant strains;

[0090] S2. The mutant strain was inoculated into LB medium containing kanamycin and cultured at 37°C and 200 r / min until OD 600 = 0.7. IPTG (0.5 mmol / L) was added and the culture was induced at 28°C for 14 h. After the culture was completed, the cells were centrifuged at 9000 r / min for 12 min, and the cells were collected. The cells were washed three times with phosphate buffer containing 8% glycerol and stored at -20°C for later use.

[0091] S3. Dissolve 70 g / L sucrose and 45 g / L ascorbic acid in phosphate buffer (15 mmol / L), then add 5.5 mmol / L of the bacterial cells obtained in step S2, stir and mix well, add the treatment agent, and then place in a shaker at 37°C and 200 r / min for 24 h.

[0092] The components and their corresponding weight percentages in the treatment agent are as follows: Tween 80 0.5%, hydroxypropyl-β-cyclodextrin 5%, 1-ethyl-3-methylimidazolium acetate 3.5%, with the remainder being deionized water;

[0093] S4. Product separation and purification:

[0094] After the reaction was completed, the solution was centrifuged at 9000 r / min for 12 min. The supernatant was then passed through a 0.22 μm microfiltration membrane and a 10 kDa ultrafiltration membrane to remove macromolecular impurities. The ultrafiltration solution was then preliminarily separated using a strong acid cation exchange resin and eluted with 0.1 mol / L hydrochloric acid solution. The eluent containing AA-2G was collected, and the eluent was then concentrated and purified by preparative high performance liquid chromatography.

[0095] Comparative Example 3

[0096] A process for the whole-cell catalytic synthesis of AA-2G using sucrose phosphorylase, characterized by comprising the following steps:

[0097] S1. A key site mutation was performed on sucrose phosphorylase (SPase) from Bifidobacterium longum, and then the mutant SPase gene was transformed into Escherichia coli BL21 (DE3) through the PET-22b(+) expression vector to obtain a mutant strain.

[0098] Specifically:

[0099] 1) Using the nucleotide sequence shown in SEQ ID NO:4 as a template, and L341V-F (SEQ ID NO:5) and L341V-R (SEQ ID NO:6) as primers, PCR was performed to obtain the mutant shown in SEQ ID NO:3; the amino acid sequence of the mutant is shown in SEQ ID NO:1;

[0100] 2) The mutant gene and PET-22b(+) were digested with BamHI and EcolI, respectively, purified, and ligated with T4 DNA ligase overnight at 16°C, and then sequenced.

[0101] 3) The ligation product was transformed into Escherichia coli BL21(DE3), plated on LB agar plates containing antibiotics (kanamycin 50 μg / mL), and cultured at 37°C for 14 h to obtain mutant strains;

[0102] S2. The mutant strain was inoculated into modified LB medium containing kanamycin and cultured at 37℃ and 200 r / min until OD 600 = 0.7. IPTG (0.5 mmol / L) was added and the culture was induced at 28℃ for 14 h. After the culture was completed, the cells were centrifuged at 9000 r / min for 12 min, and the cells were collected. The cells were washed three times with phosphate buffer containing 8% glycerol and stored at -20℃ for later use.

[0103] The modified LB medium comprises the following components: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, vitamin C 7 μg / L, vitamin E 7 μg / L, tyrosine 11 ng / L, L-arginine 17 ng / L, lanthanum nitrate 2.4 μg / L, cerium nitrate 2.2 μg / L, and silk fibroin 11 μg / L;

[0104] S3. Dissolve 70 g / L sucrose and 45 g / L ascorbic acid in phosphate buffer (15 mmol / L) with stirring, then add 5.5 mmol / L of the bacterial cells obtained in step S2, and place in a shaker at 37°C and 200 r / min for 24 h.

[0105] S4. Product separation and purification:

[0106] After the reaction was completed, the solution was centrifuged at 9000 r / min for 12 min. The supernatant was then passed through a 0.22 μm microfiltration membrane and a 10 kDa ultrafiltration membrane to remove macromolecular impurities. The ultrafiltration solution was then preliminarily separated using a strong acid cation exchange resin and eluted with 0.1 mol / L hydrochloric acid solution. The eluent containing AA-2G was collected, and the eluent was then concentrated and purified by preparative high performance liquid chromatography.

[0107] Tests and trials

[0108] The content of AA-2G in each example and comparative example group was detected and analyzed by HPLC.

[0109] HPLC detection conditions

[0110] Chromatographic column: Diamonsil C18 column, 5 μm (250 mm × 4.6 mm). UV detection wavelength: 238 nm. Mobile phase: 1% methanol, pH adjusted to 2.0 with phosphoric acid; flow rate: 0.8 mL / min; injection volume: 10 μL; column temperature: 25℃. The peak area of ​​AA-2G in the HPLC chromatogram is directly proportional to its concentration; a standard curve for AA-2G was plotted accordingly. The concentration of AA-2G in the sample was determined using the standard curve and the peak area of ​​AA-2G in the sample.

[0111] The experimental results are shown in Table 1 below.

[0112] Table 1

[0113] Yield (g / L) Example 1 95.8 Example 2 96.3 Example 3 95.3 Comparative Example 1 52.6 Comparative Example 2 80.6 Comparative Example 3 85.8

[0114] As shown in Table 1 above, the site-directed mutagenesis of sucrose phosphorylase derived from Bifidobacterium longum in this invention effectively increased the enzyme activity of sucrose phosphorylase, and high-yield AA-2G was obtained through whole-cell catalysis to synthesize 2-OD-glucanyl-L-ascorbic acid. Furthermore, the use of modified LB medium and treatment agents in this invention significantly improved the yield of AA-2G, providing a foundation for the industrial production of AA-2G.

[0115] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A process for the whole-cell catalytic synthesis of AA-2G using sucrose phosphorylase, characterized in that, Includes the following steps: S1. A key site mutation was performed on sucrose phosphorylase derived from Bifidobacterium longum, and then the mutant SPase gene was transformed into Escherichia coli BL21(DE3) through the PET-22b(+) expression vector to obtain a mutant strain. Specifically: 1) Using the nucleotide sequence shown in SEQ ID NO:4 as a template and L341V-F and L341V-R as primers, PCR was performed to obtain the mutant shown in SEQ ID NO:3; the amino acid sequence of the mutant is shown in SEQ ID NO:1; The sequence of L341V-F is shown in SEQ ID NO:5, and the sequence of L341V-R is shown in SEQ ID NO:6; 2) The mutant gene and PET-22b(+) were digested with BamHI and EcolI, respectively, purified, and ligated with T4 DNA ligase overnight at 16°C, and then sequenced. 3) The ligation product was transformed into Escherichia coli BL21(DE3), plated on LB agar plates containing antibiotics, and cultured at 37°C for 12-16 h to obtain mutant strains; S2. The mutant strain was inoculated into modified LB medium containing kanamycin and cultured at 37℃ and 200 r / min until OD 600 = 0.6~0.

8. IPTG was added and the culture was induced at 28℃ for 14 h. After the culture was completed, the cells were centrifuged, washed three times with phosphate buffer containing 8% glycerol, and stored at -20℃ for later use. The modified LB medium comprises the following components: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, vitamin C 6-8 μg / L, vitamin E 6-8 μg / L, tyrosine 10-12 ng / L, L-arginine 15-19 ng / L, lanthanum nitrate 2.3-2.6 μg / L, cerium nitrate 1.9-2.5 μg / L, and silk fibroin 10-12 μg / L; S3. Dissolve 60-80 g / L sucrose and 40-50 g / L ascorbic acid in phosphate buffer, then add 5-6 mmol / L of the bacterial cells obtained in step S2, stir and mix well, add the treatment agent, and then place it in a shaker for a shaking reaction. The concentration of the phosphate buffer solution is 10~20 mmol / L; The components and their corresponding weight percentages in the treatment agent are as follows: Tween 80 0.1~1%, hydroxypropyl-β-cyclodextrin 4~6%, 1-ethyl-3-methylimidazolium acetate 3~4%, and the balance is deionized water; S4. Product separation and purification: After the reaction was completed, the supernatant was centrifuged and passed through a 0.22 μm microfiltration membrane and a 10 kDa ultrafiltration membrane to remove macromolecular impurities. Then, the ultrafiltration solution was initially separated using a strong acid cation exchange resin and eluted with 0.1 mol / L hydrochloric acid solution. The eluent containing AA-2G was collected, and the eluent was concentrated and then purified by preparative high performance liquid chromatography.

2. The process for whole-cell catalytic synthesis of AA-2G using sucrose phosphorylase according to claim 1, characterized in that, The antibiotic mentioned in step 3) is kanamycin, and the concentration of kanamycin is 50 μg / mL.

3. The process for whole-cell catalytic synthesis of AA-2G using sucrose phosphorylase according to claim 1, characterized in that, The concentration of IPTG mentioned in step S2 is 0.5 mmol / L; The specific parameters for centrifugation are: centrifugation at 8000~10000 r / min for 10~15 min.

4. The process for whole-cell catalytic synthesis of AA-2G using sucrose phosphorylase according to claim 1, characterized in that, In step S3, the temperature during the shaking culture is controlled at 35~39℃, the shaking speed is 150~250 r / min, and the reaction time is 12~36 h.

5. The process for whole-cell catalytic synthesis of AA-2G using sucrose phosphorylase according to claim 1, characterized in that, The centrifugation process in step S4 is carried out at a speed of 8000~10000 r / min and a centrifugation time of 10~15 min.