Sucrose phosphorylase mutant and application thereof
By performing site-directed mutagenesis on sucrose phosphorylase and constructing genetically engineered cells, the problems of low yield and low conversion rate in the production of ascorbate glucoside were solved, and the efficient catalytic production of ascorbate glucoside was achieved.
Patent Information
- Application Number
- CN202511906625.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-17
- Publication Date
- 2026-02-10
AI Technical Summary
The production of ascorbate glucoside suffers from problems such as low yield and low conversion rate.
By performing site-directed mutagenesis on the amino acid sequence of sucrose phosphorylase, mutants such as Q281P, K303P, and K328F were designed and screened. Genetically engineered cells expressing this enzyme were constructed, and recombinant Escherichia coli was used as a whole-cell catalyst to catalyze the reaction of ascorbic acid and sucrose to produce ascorbate glucoside.
It significantly improves catalytic efficiency, achieving higher ascorbate glucoside yield and conversion rate, with conversion completed within 1-72 hours.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to sucrose phosphorylase (GtfA) mutants and their applications, and more specifically to their application in the production of ascorbate glucoside. Background Technology
[0002] Ascorbyl Glucoside (AA2G) is a stable derivative of vitamin C (ascorbic acid). It cannot be directly extracted from natural plants and animals and must be prepared by chemical or biosynthetic methods using vitamin C and glycosyl donors as raw materials. The core of its preparation is to form a stable glycoside structure by combining glucose molecules with ascorbic acid molecules, thus solving the problems of easy oxidation and instability of vitamin C while retaining its core efficacy. It is widely used in cosmetics, food, and pharmaceutical fields.
[0003] In the cosmetics industry, AA2G is a mainstream whitening and anti-aging ingredient. After application, it can be broken down into vitamin C by skin glycosidases, inhibiting melanin production and accelerating melanin metabolism to whiten and lighten spots. It also scavenges free radicals and promotes collagen synthesis for antioxidant and anti-aging effects. Furthermore, its pH is close to the skin's physiological environment, resulting in low irritation and relieving skin redness and sensitivity. It is commonly found in whitening serums and anti-aging creams. In the food industry, it can be added to fruit juices, dairy products, and baked goods as a nutritional fortifier to supplement vitamin C. As an antioxidant, it inhibits food oxidation and spoilage, protects natural pigments, and extends shelf life. In the pharmaceutical industry, it can be used as an adjunct treatment for vitamin C deficiency, supplementing vitamin C through oral preparations. It can also be added as an antioxidant excipient to drug preparations containing easily oxidized components to prevent oxidative degradation of the main drug ingredient, ensuring drug efficacy and safety. Overall, compared to traditional vitamin C, ascorbyl glucoside has the core advantages of high stability, low irritation, and high bioavailability. With the development of green biosynthesis technology, its applications in various fields will be further expanded.
[0004] However, the current production of ascorbate glucoside suffers from low yield and low conversion rate. This invention addresses these issues through research. Summary of the Invention
[0005] The purpose of this invention is to provide a mutant of sucrose phosphorylase and its application to solve the problems of low yield and low conversion rate in the production of ascorbate glucoside.
[0006] To achieve the above objectives, the present invention first provides a sucrose phosphorylase mutant, wherein the amino acid sequence of the sucrose phosphorylase mutant is based on the amino acid sequence shown in SEQ ID NO: 1 and undergoes the following amino acid mutations: Q281P, K303P, K328F, Q281P+K303P, K303P+K328F.
[0007] The mutants screened in this application have undergone extensive experimental verification, which ultimately proved that the catalytic activity of the enzyme is significantly improved.
[0008] The present invention also provides a nucleic acid encoding the above-mentioned sucrose phosphorylase.
[0009] The present invention also provides genetically engineered cells expressing the above-mentioned sucrose phosphorylase, which contain nucleic acids encoding the sucrose phosphorylase.
[0010] According to an embodiment of the present invention, the genetically engineered cell is a strain obtained by constructing an expression cassette of the nucleic acid and then introducing it into a host cell.
[0011] The present invention also provides a method for constructing recombinant (e.g., Escherichia coli) engineered bacteria that express sucrose phosphorylase.
[0012] This invention provides a method for producing ascorbate glucoside, wherein the method utilizes the above-mentioned recombinant Escherichia coli strain to express sucrose phosphorylase, uses cells expressing sucrose phosphorylase as whole-cell catalysts, and uses ascorbic acid and sucrose as substrates to catalyze the production of ascorbate glucoside; wherein the recombinant Escherichia coli strain expresses the sucrose phosphorylase gene.
[0013] More specifically, the steps include: (1) linking the sucrose phosphorylase gene gtfA to the plasmid pET-30a(+) to obtain the pET30-gtfA plasmid. Transforming the pET30-gtfA plasmid into Escherichia coli B21(DE3) to obtain the transformed strain BL21-gtfA.
[0014] (2) The above-mentioned BL21-gtfA strain was inoculated into LB medium supplemented with 50 mg / L kanamycin sulfate and cultured overnight at 20-42℃ and 150-250 rpm. Then, it was inoculated at a ratio of 0.05%-15% (V / V) into LB medium supplemented with 50 mg / L kanamycin sulfate and 0.1-1 mM IPTG and induced to culture overnight at 20-42℃ and 150-250 rpm. The resulting cells were collected to obtain the whole-cell catalyst.
[0015] The culture temperature is 20-42℃, for example, 20℃, 25℃, 30℃, 37℃, 40℃, or 42℃.
[0016] The pH of the fermentation system is 6.0-8.0, for example, 6.0, 7.0, or 8.0;
[0017] The fermentation time is 6 hours to 96 hours, for example, 12 hours, 24 hours, 36 hours, or 48 hours;
[0018] The volume percentage of the inoculum for fermentation is 0.05%-15%, for example, 0.05%, 2%, 5%, 10%, or 15%.
[0019] Preferably, the culture is carried out under stirring or shaking conditions, more preferably at a shaking speed of 250 rpm.
[0020] The LB medium contains: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride.
[0021] The expression medium contains: 30 g / L peptone, 50 g / L yeast extract, and 6 g / L K2HPO4.
[0022] In one embodiment, the whole-cell conversion production system includes cells with a wet weight of 0.1-200 g / L, ascorbic acid of 1-400 g / L, and sucrose of 1-400 g / L.
[0023] In one embodiment, the pH of the ascorbic acid and sucrose solution is adjusted to 4.0-7.0, and 0.1-200 g / L of whole-cell catalyst (wet weight) is added. After stirring evenly, the mixture is converted at 15-40°C for 1-72 hours. After the conversion is completed, an ascorbic acid glucoside solution is obtained.
[0024] The conversion temperature is 35℃-60℃, for example 35℃ or 40℃ or 45℃ or 50℃ or 55℃ or 60℃;
[0025] The pH value of the conversion system is 4.0-7.0, for example, 4.0, 5.0, 6.0, or 7.0;
[0026] The conversion time is 1 hour to 72 hours, for example, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, or 72 hours.
[0027] The ascorbic acid concentration of the conversion system is 10-400 g / L, for example, 10 g / L, 50 g / L, 100 g / L, 200 g / L, 300 g / L, or 400 g / L.
[0028] The sucrose concentration of the conversion system is 10-400 g / L, for example, 10 g / L, 50 g / L, 100 g / L, 200 g / L, 300 g / L, or 400 g / L.
[0029] The total amount of catalyst added to the conversion system is 0.1-200 g / L wet weight, for example, 0.1 g / L, 1 g / L, 2 g / L, 5 g / L, 10 g / L, 20 g / L, 50 g / L, 100 g / L, or 200 g / L.
[0030] In one embodiment, the recombinant Escherichia coli is obtained by transforming a plasmid expressing the sucrose phosphorylase gene into competent Escherichia coli B21(DE3) host cells.
[0031] This invention utilizes a sucrose phosphorylase mutant obtained through rational design and enzyme evolution screening based on the amino acid sequence shown in SEQ ID NO: 1. Compared to the wild-type sucrose phosphorylase, this mutant exhibits alterations in protein structure and function. In practical applications, the catalytic efficiency of the sucrose phosphorylase mutant is significantly improved, resulting in a higher catalytic rate for ascorbate glucoside and thus a higher yield of salicylate glucoside. Using the constructed whole-cell catalyst, ascorbic acid and sucrose in the catalytic solution can be converted into ascorbate glucoside within 1-72 hours during fermentation. Simultaneously, the sucrose phosphorylase gene is introduced into *E. coli* to construct an *E. coli* strain expressing sucrose phosphorylase, creating a more efficient strain for catalyzing the conversion of ascorbic acid to ascorbate glucoside. This provides a powerful enzyme and strain for *E. coli* biosynthesis, and the invention has broad application prospects. Detailed Implementation
[0032] The following are examples to help to better understand the present invention.
[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0035] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.
[0036] The amino acid sequence of SEQ ID NO: 1 (derived from Coffea eugenioides) is as follows:
[0037] MPIQNKAMLITYSDSMAKNIKETHEVLKEYIGDAIGGVHLLPFFPSTGDRGFAPYRYDVVDSAFGNWDDVEALGEDYYLMFDFMINHISKKSVMYQDFKKNHDASKYSDFFIRWEKFWKAAGKGRPTQKDIDLIYKRKDKAPEQEIDFDDGSKEHLWNTFGEEQIDINVKSKVAQEFFKQTLTDMVKHGADLIRLDAFAYAVKKVDTNDFFVEPEIWDLLNEVRDILAPYKAEILPEIHEHYTIPQKISKHGYFIYDFALPMTTLYTLYSGKTERLANWLKMCPMKQFTTLDTHDGIGVVDAKDILTDDEIKYASNELYKVGANVKRKYSSAEYHNLDIYQINSTYYSALGDDDKAYLLARAFQVFAPGIPMIYYVGLLAGSNDLDLLEKTKEGRNINRHYYTKEEVAKEVQRPVVANLLKLLSWRNKFAAFDLDGSIKVETPSETTIKITRQDKDGANVAVLDADAAKKTFTITANGETVMEQK。
[0038] The protein sequence shown in SEQ ID NO: 1 above in this application corresponds to the specific nucleic acid sequence SEQ ID NO: 2:
[0039]
[0040] Example 1: Specific methods or steps for rationally designing screening
[0041] The specific methods or steps of rational design and evolutionary screening employed in this invention are as follows:
[0042] By analyzing the sequence and structure of sucrose phosphorylase, and by docking the substrate with the enzyme structure, we screened amino acids near the substrate for alanine mutation and functional verification, aiming to obtain the key amino acid residues for enzyme catalysis.
[0043] Using sequence SEQ ID NO:2 as a template, site-directed mutagenesis primers were designed, and the mutant gene was obtained using site-directed mutagenesis techniques;
[0044] Site-directed mutagenesis refers to the introduction of desired changes (usually changes that characterize a favorable direction) into a target DNA fragment (which can be a genome or a plasmid) using methods such as polymerase chain reaction (PCR). These changes include the addition, deletion, and point mutations of bases.
[0045] The gtfA gene was cloned into the pET-30a vector using a seamless cloning method to construct the recombinant plasmid pET-gtfA. Mutation was then performed using overlap extension PCR: complementary primers containing the target mutation site (located in the middle of the primers) and flanking sequences of 15-20 bp were designed. PCR amplification was performed using the recombinant pET-gtfA plasmid as a template. The product was then digested with DpnI enzyme to remove the methylated wild-type template before being transformed into DH5α. Competent cells are selected, and positive single colonies are extracted and verified by plasmid extraction and DNA sequencing. Once the mutation site is confirmed to be accurate, the recombinant pET-gtfA vector containing the mutated expression gene can be obtained.
[0046] The functions of R118A, D123A, T153A, I192A, Q281A, F295A, K303A, and K328A were verified by alanine scanning. After obtaining mutants with significantly altered activities, further saturation mutagenesis was used on Q281, K303, and K328 to obtain more mutants with superior performance. Combined mutagenesis of the obtained superior mutants Q281P, K303P, and K328F, along with rational design and screening based on the above data and structure, yielded mutants with high enzyme activity.
[0047] The recombinant pET-gtfA vector containing the mutant expression gene was transformed into *E. coli* cells and fermented. Whole-cell catalyst was obtained from the fermented cells and added to a solution containing ascorbic acid and sucrose for catalysis. After catalysis, the catalytic solution was analyzed by HPLC to determine the ascorbyl glucoside yield, and finally, the activity of the relevant mutant was detected.
[0048] Example 2: Functional verification of sucrose phosphorylase mutant
[0049] Engineered *E. coli* strains were activated in LB broth. Seed culture was prepared in LB broth (37℃, 250 rpm, 16 h). A 10% inoculum was inoculated into 250 mL Erlenmeyer flasks containing 30 mL of fermentation broth. After incubation at 30℃ and 250 rpm for 4 hours, 0.5 mM IPTG was added, and incubation continued for 20 hours. Finally, the liquid in the Erlenmeyer flasks was transferred to 50 mL centrifuge tubes, centrifuged at 8000 rpm for 5 min, and the bacterial cells were collected. The collected cells were added to a catalytic system (containing 1 M ascorbic acid and 0.8 M sucrose, pH 5.0) and catalyzed at 45℃ for 24 hours. After catalysis, the catalytic solution was centrifuged, and the supernatant was collected. The ascorbyl glucoside yield was detected by HPLC. Quantification was performed using ascorbic acid standards at different concentrations. The results are shown in Table 1.
[0050] Table 1
[0051]
[0052] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A sucrose phosphorylase mutant, characterized in that, The amino acid sequence of the sucrose phosphorylase mutant is based on the amino acid sequence shown in SEQ ID NO: 1 and the following amino acid mutations occur: Q281P, K303P, K328F, Q281P+K303P, K303P+K328F.
2. A nucleic acid, characterized in that, Encodes the sucrose phosphorylase mutant of claim 1.
3. A recombinant expression cassette, characterized in that, It includes the nucleic acid as described in claim 2.
4. An expression carrier, characterized in that, It includes the nucleic acid as described in claim 2 or the recombinant expression cassette as described in claim 3, specifically using pET-30a(+) as the vector backbone.
5. A recombinant genetically engineered cell, characterized in that, It contains the recombinant expression cassette as described in claim 3, or the expression vector as described in claim 4.
6. The recombinant genetically engineered cell according to claim 4, characterized in that, These are prokaryotic or eukaryotic cells, such as Escherichia coli cells.
7. A method for producing ascorbic acid glucoside, characterized in that: Using the sucrose phosphorylase as described in claim 1 as a catalyst, ascorbic acid and sucrose are used as substrates for catalysis to produce ascorbate glucoside. Optionally, the method further includes the step of separating the produced ascorbate glucoside.
8. The method as described in claim 7, characterized in that, The sucrose phosphorylase mutant is a pure enzyme, with crude enzyme solution as a catalyst, or the recombinant genetically engineered cells containing the sucrose phosphorylase mutant produced by culturing the recombinant genetically engineered cells as described in claim 5 or 6 as a whole-cell catalyst.
9. The method as described in claim 8, characterized in that, The recombinant genetically engineered cells are Escherichia coli engineered cells, and their culture method is as follows: the culture temperature is 20-40℃; the culture time is 6-96h; preferably, the culture is carried out under stirring or shaking conditions, more preferably the shaking speed is 250 rpm.
10. The method as described in claim 8, characterized in that, The catalytic system contains 0.1-200 g / L of cell wet weight, 1-400 g / L of ascorbic acid, and 1-400 g / L of sucrose. The pH is 4-7 (e.g., 5.0); the temperature for catalysis is 20-60°C, preferably 40-50°C (e.g., 45°C); and the catalytic duration is 12-96 hours, e.g., 24-72 hours (e.g., 24 hours).