High-enzyme-activity mutant of lgspase and application thereof

Abstract

This invention relates to a high-activity mutant of LgSPase, characterized by mutations at at least one site of amino acids 236, 249, 251, and 341, wherein the reference sequence of LgSPase is shown in SEQ ID NO:1. This invention, through targeted modification and semi-rational design of LgSPase, has obtained a mutant with higher enzyme activity than the wild type. The high-activity mutant provided by this invention not only improves enzyme catalytic efficiency but also exhibits different environmental adaptability and thermal stability compared to the wild type. This can reduce production costs for the biocatalytic synthesis process of 2-O-α-D-glucosyl-L-ascorbic acid and enrich the application scenarios of the process.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering, and more specifically, relates to high-activity mutants of LgSPase and their applications. Background Technology

[0002] L-Ascorbic acid (L-AA), also known as vitamin C, is an essential vitamin that the human body cannot synthesize. It participates in various physiological activities and plays a vital role in maintaining and promoting human health. Natural L-AA molecules are extremely unstable and readily oxidized by oxygen in the air, thus losing their biological efficacy. However, glycosylation modification of L-AA produces 2-O-α-D-glucopyranosyl L-ascorbic acid (AA-2G), which has a stable structure and better antioxidant, light-resistant, and heat-resistant properties. Furthermore, AA-2G releases L-AA upon hydrolysis in vivo, preserving the biological activity and physiological functions of L-AA. Therefore, AA-2G is recognized as the most stable and best-performing L-AA alternative.

[0003] Currently, the common enzymes reported in the literature capable of glycosylation of L-AA include cyclodextrin glycosyltransferase (CGTase), α-amylase, α-glucosidase, and sucrose phosphorylase. Among them, sucrose phosphorylase has become a research hotspot due to its one-step high efficiency, inexpensive and readily available glycosyl donor (sucrose), and high reactivity without activation. Sucrose phosphorylase (SPase) can transfer α-D-glucose-1-phosphate produced by the catalytic phosphorylation of sucrose to different acceptor compounds, synthesizing compounds such as ascorbate glucoside (…). Figure 1 It contains a variety of products such as glycerol glucoside and arbutin.

[0004] However, the current method for synthesizing AA-2G using SPase catalysis still has problems such as low enzyme activity, low reaction efficiency, low substrate conversion rate, and high cost. If the enzyme is improved to further enhance its activity and stability, it will help improve process efficiency and reduce production costs. Summary of the Invention

[0005] To address the above problems, the present invention provides a high-activity mutant of LgSPase, comprising mutations at at least one site of amino acids 236, 249, 251 and 341, and the reference sequence of the LgSPase is shown in SEQ ID NO:1.

[0006] In one specific implementation, the high-activity mutant of LgSPase includes at least one of the following mutations:

[0007] The mutation from proline to leucine at position 236;

[0008] A mutation from serine to threonine at position 249;

[0009] The mutation from histidine to arginine at position 251;

[0010] A mutation at position 341 from glutamine to alanine, serine, or glycine.

[0011] In one specific embodiment, the high-activity mutant of LgSPase includes mutations at position 341 (glutamine to serine) and position 337 (leucine to methionine).

[0012] In one specific embodiment, the high-activity mutant of LgSPase includes mutations at position 341 (glutamine to serine), position 137 (arginine to proline), position 138 (lysine to arginine), and position 139 (aspartic acid to proline).

[0013] The present invention also provides the application of the above-mentioned high enzyme activity mutant in the preparation of 2-O-α-D-glucosyl-L-ascorbic acid.

[0014] The present invention also provides a method for catalytically converting L-ascorbic acid into 2-O-α-D-glucosyl-L-ascorbic acid, comprising the step of co-incubating the high-enzyme-activity mutant of the above-mentioned high-enzyme-activity mutant with a glycosyl donor and L-ascorbic acid to induce an enzymatic reaction.

[0015] In one specific implementation, the glycosyl donor is sucrose.

[0016] In one specific embodiment, the glycosyl acceptor is L-AA. In another specific embodiment, the enzyme reaction is carried out at a temperature of 20-50°C and a pH of 5.2.

[0017] This invention, through site-directed mutagenesis of certain amino acids in LgSPase, yielded mutants with higher enzyme activity than the wild type. Furthermore, these mutants exhibit different temperature adaptability compared to the wild type. The high-activity mutants provided by this invention not only improve enzyme catalytic efficiency but also provide corresponding enzymes for different enzyme reaction environments, reducing production costs and expanding the application scenarios of the synthesis process of 2-O-α-D-glucosyl-L-ascorbic acid. Attached Figure Description

[0018] Figure 1 This is the reaction formula for the reaction of L-AA with sucrose catalyzed by LgSPase.

[0019] Figure 2 To study the enzyme activity of semi-rational design and site-directed mutagenesis dominant mutants.

[0020] Figure 3 The values ​​represent the enzyme activity of the combined mutants. Here, 2M indicates R137P / D139P, and 3M indicates R137P / K138R / D139P. Detailed Implementation

[0021] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0022] Sucrose phosphorylases catalyze the phosphorylation of sucrose to glucose-1-phosphate and fructose in the presence of phosphate, and also catalyze the transfer of glucose groups from sucrose to different glycosyl acceptor compounds. LgSPase (reference amino acid sequence shown in SEQ ID NO:1) is a sucrose phosphorylase derived from Lactobacillus gastricus. This application describes protein engineering modifications based on this enzyme, revealing that mutations at some key amino acid sites significantly enhance its activity in synthesizing AA-2G using L-AA as a glycosyl acceptor. Further research screened several mutants with high enzyme activity.

[0023] 1. Construction and screening of LgSPase random mutation libraries

[0024] Using plasmid pET28b-LgSPase as a template, the LgSPase gene sequence was amplified by error-prone PCR using the Diversify Random Mutagenesis Kit (Clonetech). The resulting recombinant plasmid library was transformed into E. coli BL21(DE3), and transformants of the recombinant expression strain were obtained, successfully constructing a mutant library. The mutant library was induced for expression and centrifuged for collection using commonly used methods in the field.

[0025] Based on the mutant bacterial cells collected in the previous step, an enzymatic reaction was performed. A 200 μL reaction system contained 0.3 M sucrose, 0.45 M L-AA, and 10 OD mutant bacterial cells. The reaction buffer was 50 mM sodium citrate buffer (pH 5.2). After reacting at 40℃ and 500 rpm for 4 h, 20 μL of 0.1 M HCl was added to terminate the reaction. Then, the mixture was centrifuged at 4℃ and 3700 rpm for 20 min. 5-10 μL of the supernatant was taken and diluted according to the instructions of the D-fructose / D-glucose assay kit (Megazyme), and the fructose and glucose contents were measured. Using the above method, more than 2000 transformants from the library were screened, and four strains with significantly increased enzyme activity were obtained. Further HPLC verification showed that the activity increased by 1.7-2.8 times after 4 h of reaction (Table 1).

[0026] Table 1. Dominant mutants identified through random mutation screening.

[0027]

[0028] 2. Semi-rational design modification of LgSPase

[0029] A three-dimensional structural model of LgSPase was constructed using molecular simulation. Based on this model, the amino acids related to substrate binding and mutation hotspots in its loop region were analyzed. At the same time, non-conserved amino acid residues affecting substrate binding were analyzed based on homologous sequence alignment. Subsequently, site-directed and combinatorial mutations were performed on these key residue sites.

[0030] Based on the target sites to be modified, eight amino acid sites, namely L337, I339, Q341, Y135, R137, K138, D139 and Y200, were selected for alanine scanning mutation.

[0031] The results showed that mutations at the R137, L337, and Q341 sites significantly affected enzyme activity, especially Q341A, which showed a 1.7-fold increase in activity. Therefore, Q341A was selected for further mutation.

[0032] Based on the activity detection results of alanine scanning mutations, R137, L337, and Q341 were modified to construct an NDT half-saturated mutant library. Using plasmid pET28a-LgSPase as a template, NDT primers were designed to perform site-directed half-saturated mutations on the amino acids at R137, L337, and Q341. The R137 site was mutated to F, L, I, V, H, Y, C, S, G, D, N, M, P; the L337 site was mutated to F, L, M, V, H, Y, C, S, G, D, N, R; and the Q341 site was mutated to F, L, I, V, H, Y, C, S, G, D, N, R. In addition, four site-directed combination mutants were constructed for the hot amino acids in the loop region: R137P / D139P, K136R / R137P / D139P, R137P / K138R / D139P, and K136R / R137P / K138R / D139P.

[0033] The results are as follows Figure 2 and Figure 3As shown, among the 13 mutants at the R137 site, the total amount of AA-2G produced by C, S, P, V, and H was significantly higher than that of the wild type, with an activity increase of 1.4-3.4 times. Among them, L337M produced 1.55 times more AA-2G than the wild type; Q341 S and Q341 G produced 2.3 times and 1.9 times more AA-2G than the wild type, respectively; R137P / D139P and K136R / R137P / K138R / D139P produced 4.3 times and 2.7 times more AA-2G than the wild type, respectively; and R137P / K138R / D139P produced 6 times more AA-2G than the wild type, with an AA-2G yield of 40 g / L.

[0034] 3. A combined mutant with LgSPase mutations at sites 137-139 as its core.

[0035] In further research, we constructed combinatorial mutants centered on mutations at sites 137-139, obtaining L337M+R137P / D139P and F198L+R137P / K138R / D139P. The results are as follows... Figure 3 As shown, the enzyme activity of L337M+R137P / D139P is 8.4 times that of the wild type, and the AA-2G yield can reach 50g / L; the enzyme activity of F198L+R137P / K138R / D139P and Q341S+R137P / K138R / D139P is 6 times that of the wild type, and the AA-2G yield is about 36-38g / L.

[0036] It is evident that specific mutations at sites 137, 138, and 139 significantly enhance the enzyme activity of LgSPase mutants. Based on this, combinations with other site mutations (such as L337M, F198L, and Q341S) can yield mutants with significantly higher enzyme activity than wild-type mutants.

[0037] 4. Enzymatic characterization of representative dominant mutants

[0038] The stability of the dominant mutant and its activity in synthesizing AA-2G under different pH and temperature conditions were assessed.

[0039] 1) Synthesis of AA-2G under different temperature conditions

[0040] Temperature gradients of 20℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, and 70℃ were selected for detection. The enzyme concentration was 0.5 g / L, the substrates were 0.3 M sucrose and 0.45 M L-AA, the buffer was 50 mM sodium citrate, and the reaction was carried out at pH 5.2 for 24 h. The results showed that wild-type LgSPase maintained good activity in the range of 35-50℃; the mutants L337M+R137P / D139P and R137P / K138R / D139P had the highest AA-2G yield at 35℃, and L337M+R137P / D139P showed significantly high activity in a relatively wide temperature range (30-40℃), with AA-2G yield exceeding 60 g / L; R137P / K138R / D139P was significantly affected by temperature, with ≤30℃ or ≥40℃ leading to a significant decrease in activity; F198L+R137P / K138R / D139P showed a preference for medium and low temperatures, exhibiting the maximum reactivity at 20℃.

[0041] 2) Synthesis of AA-2G under different pH conditions

[0042] In 50 mM sodium citrate buffer, the pH values ​​of 4.0, 4.5, 5.2, 6.0, 6.5, and 7.0 were selected for detection. 0.3 M sucrose and 0.45 M L-AA were prepared as substrates, and the reaction was carried out at 35 °C.

[0043] The results showed that the wild type and the L337M+R137P / D139P, R137P / K138R / D139P, and F198L+R137P / K138R / D139P mutants had good activity in acidic and neutral environments ranging from 4.0 to 7.0, with the best activity at pH 5.2.

[0044] 3) Thermal stability of wild-type and mutant LgSPase

[0045] The mutant and wild-type purified enzymes were pre-incubated at 40℃ for 0h, 0.5h, 4h, 12h, 24h, 36h, 48h, and 72h before enzyme activity was measured. The enzyme concentration was 0.5g / L, the final substrate concentration was 0.3M sucrose and 0.45M L-AA, the buffer was 50mM sodium citrate buffer, and the reaction was carried out at 35℃ and pH 5.2 for 24h.

[0046] The results showed that the wild type and the selected mutants maintained high activity after 72 hours of pre-incubation at 40°C, with all activities exceeding 80%. Among them, the mutant L337M+R137P / D139P maintained the highest activity and the best thermal stability, with almost no significant loss of activity.

[0047] It should be noted that the description of a mutation from a certain amino acid to another amino acid in this invention is based on the LgSPase reference sequence and is made to make the reader understand the mutation site more intuitively. The amino acid before the mutation at the site should not be used to limit this invention. As long as the amino acid at the site is the target amino acid, even if it was another amino acid in a non-reference sequence before the mutation, or if such a protein was obtained directly by synthesis or other means without mutation, it should be included within the scope of protection of this invention.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A high-activity mutant of LgSPase, characterized in that, The LgSPase contains at least one site mutation, including amino acids at positions 236, 249, 251, and 341, and its reference sequence is shown in SEQ ID NO:

1.

2. The high enzyme activity mutant according to claim 1, characterized in that, Includes at least one of the following mutations: The mutation from proline to leucine at position 236; A mutation from serine to threonine at position 249; The mutation from histidine to arginine at position 251; A mutation at position 341 from glutamine to cysteine, alanine, serine, or glycine.

3. The high enzyme activity mutant according to claim 2, characterized in that, This includes mutations at position 341 (glutamine to serine) and position 337 (leucine to methionine).

4. The high enzyme activity mutant according to claim 2, characterized in that, Mutations include glutamine at position 341 to serine, arginine at position 137 to proline, lysine at position 138 to arginine, and aspartic acid at position 139 to proline.

5. The use of the high enzyme activity mutant according to any one of claims 1-4 in the preparation of 2-O-α-D-glucosyl-L-ascorbic acid.

6. A method for catalytically converting L-ascorbic acid to 2-O-α-D-glucosyl-L-ascorbic acid, characterized in that, The method includes the step of co-incubating the high-enzyme-activity mutant of claims 1-4 or a protein containing the sequence of the high-enzyme-activity mutant, or the expression host cell or cell lysate of the high-enzyme-activity mutant or a protein containing the sequence of the high-enzyme-activity mutant, with a glycosyl donor and L-ascorbic acid to induce an enzymatic reaction.

7. The method according to claim 6, characterized in that, The glycosyl donor is sucrose.

8. The method according to claim 6, characterized in that, The enzyme reaction is carried out at a temperature of 20-50℃ and a pH of 4.0-7.0.

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