Amylosucrase mutant and application thereof
By mutating the amino acid sequence of starch sucrase, its catalytic activity and thermal stability were improved, solving the problem of insufficient activity and stability of natural enzymes in the production of α-arbutin, and achieving a significant improvement in enzyme activity and enhanced thermal stability.
Patent Information
- Application Number
- CN202511416065.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-30
- Publication Date
- 2025-11-21
AI Technical Summary
Natural starch sucrase has insufficient catalytic activity and thermal stability in the production of α-arbutin, resulting in low reusability and limiting its application in industrial production.
By performing multiple amino acid sequence mutations on wild-type starch sucrase, mutant starch sucrase with significantly improved catalytic activity and thermal stability were obtained, including single-site mutations such as L330P and multi-site mutants such as A284V/D386P/L330P/V92E/A323R.
The mutant showed significantly increased enzyme activity, reaching up to 6.5 times that of the wild type, and its thermal stability was also increased to 1.37 times that of the wild type, thus improving the synthesis efficiency and reusability of α-arbutin.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to a starch sucrolase mutant with improved enzyme activity and its application in the production of alpha-arbutin, belonging to the field of bioengineering technology. BACKGROUND
[0002] Arbutin is a glucoside derivative of hydroquinone, which naturally exists in plants of the families Asteraceae, Ericaceae, Meliaceae and Rosaceae. Arbutin has antioxidant, anti-inflammatory, antibacterial, antitumor, expectorant and asthma-relieving biological activities. As a stable, safe and highly compatible natural whitening active substance, arbutin is widely used in the field of cosmetics. Arbutin exerts a whitening effect by inhibiting tyrosinase. Among the two isomers of arbutin, the inhibitory strength of alpha-arbutin on tyrosinase is 10 times that of beta-arbutin, and the anti-melanin ability is 15 times that of beta-arbutin. Therefore, the demand for alpha-arbutin is increasing.
[0003] Currently, there are several methods for producing arbutin, including natural plant extraction, chemical organic synthesis and biosynthesis. Plant extraction mainly obtains beta-arbutin, and non-natural alpha-arbutin cannot be obtained by plant extraction or plant transformation. Chemical synthesis is a mixture of alpha and beta glycosides, and has the disadvantages of unstable intermediates, harsh reaction conditions, complicated synthesis steps and high toxicity of reagents. The production of alpha-arbutin mainly relies on biosynthesis. Biosynthesis uses enzymes or whole cells as catalysts to convert hydroquinone into arbutin, which is environmentally friendly, has mild conditions and high efficiency, and can produce stereospecific alpha-arbutin. Currently, at least seven enzymes from microorganisms can be used for the biosynthesis of alpha-arbutin, which are alpha-amylase, sucrose phosphorylase, cyclodextrin glycosyltransferase, alpha-glucosidase, dextran sucrose, starch sucrose and sucrose isomerase.
[0004] Starch sucrose belongs to a multifunctional glucosyltransferase in the glycoside hydrolase (GH) family 13, which can catalyze the hydrolysis of sucrose and then transfer the glucose part to acceptors such as fructose and hydroquinone, thereby producing various types of glycosylation products. The conversion rate of starch sucrose for synthesizing alpha-arbutin can be more than 90%, and it has the significant advantage of low substrate price. The main challenges of using natural starch sucrose for alpha-arbutin are the insufficient catalytic activity and thermal stability of the natural enzyme, which is easily inactivated in the reaction system, has low reuse rate, and limits its application in industrial production. SUMMARY
[0005] In view of the problems existing in the prior art, the present application provides a mutant of starch sucrose and its application in the synthesis of alpha-arbutin. By performing multiple mutations on the wild-type starch sucrose, a starch sucrose mutant with significantly improved catalytic activity and thermal stability is obtained.
[0006] One of the purposes of the present application is to provide a mutant of amylosucrase, wherein the mutant is a single point mutant of amylosucrase having an amino acid sequence as shown in SEQ ID No. 1, wherein the single point mutation is any one of E38Y, V92E, V92M, T229Y, A284V, T313I, A323R, L330P or D386P; preferably, the single point mutant is any one of V92E, A284V, A323R, L330P or D386P; most preferably, the single point mutant is L330P.
[0007] In the present application, the single point mutant "L330P" means that the 330th amino acid of amylosucrase having an amino acid sequence as shown in SEQ ID No. 1 is mutated from leucine (L) to proline (P); the rest of the single point mutants are described in the same way.
[0008] As a specific embodiment of the present application, the present application further provides a multi-point mutant of amylosucrase having an amino acid sequence as shown in SEQ ID No. 1, wherein the multi-point mutant is any one of (a)-(e):
[0009] (a) a two-point combination mutant of amylosucrase having an amino acid sequence as shown in SEQ ID No. 1, wherein the two-point mutation is A284V / D386P;
[0010] (b) a three-point combination mutant of amylosucrase having an amino acid sequence as shown in SEQ ID No. 1, wherein the three-point mutation is A284V / D386P / L330P;
[0011] (c) a four-point combination mutant of amylosucrase having an amino acid sequence as shown in SEQ ID No. 1, wherein the four-point mutation is A284V / D386P / L330P / V92E;
[0012] (d) a five-point combination mutant of amylosucrase having an amino acid sequence as shown in SEQ ID No. 1, wherein the five-point mutation is A284V / D386P / L330P / V92E / A323R.
[0013] The multi-site mutant "A284V / D386P / L330P / V92E / A323R" in the present application means that the alanine (A) at position 284, the aspartic acid (D) at position 386, the leucine (L) at position 330, the valine (V) at position 92 and the alanine (A) at position 323 in the amylosucrase with the amino acid sequence shown in SEQ ID NO. 1 are simultaneously mutated into valine (V), proline (P), proline (P), glutamic acid (E) and arginine (R) respectively; the expressions of the other amino acid multi-site mutations in the present application are used by analogy.
[0014] In a second aspect of the present application, a coding gene encoding the above-mentioned amylosucrase unit point mutant or multi-site mutant is provided.
[0015] In a third aspect of the present application, a recombinant expression vector containing the above-mentioned mutant is provided.
[0016] In a fourth aspect of the present application, a recombinant microbial cell containing the above-mentioned expression vector is provided.
[0017] In a fourth aspect of the present application, the above-mentioned mutant is provided for use in the synthesis of alpha-arbutin. The synthesis of alpha-arbutin uses sucrose and hydroquinone as substrates.
[0018] The present application has the following beneficial effects:
[0019] The activity of the amylosucrase unit point mutant or multi-site mutant provided by the present application is obviously improved compared with the wild type. With the superposition or increase of the mutation sites, the multi-site mutant shows a synergistic effect, and the enzyme activity is gradually improved. Among them, the V5 enzyme activity is the highest, reaching 6.5 times that of the wild type amylosucrase; the thermal stability is also obviously improved, and the half-life at 45℃ is 1.37 times that of the wild type. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 The enzyme activity detection results of the wild type amylosucrase and the single mutant;
[0021] Figure 2 The enzyme activity detection results of the multi-site mutant combination mutant;
[0022] Figure 3 The high performance liquid chromatogram of the catalytic synthesis of alpha-arbutin by the best mutant V5. DETAILED DESCRIPTION
[0023] In order to make the purpose, technical scheme and advantages of the present application clearer, the following will be combined with specific embodiments, and the attached drawings will be referred to. Figures 1-3 The present application is further described in detail.
[0024] It should be understood that the terms such as "have", "contain", and "include" used herein do not exclude the presence or addition of one or more other elements.
[0025] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified. The specific conditions not specified in the following examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be obtained by commercial purchase.
[0026] Example 1 Detection of amylomaltase enzyme activity
[0027] 1. Enzyme activity assay
[0028] The activity of amylomaltase was determined by 3,5-dinitrosalicylic acid (DNS) colorimetry. 900 μL of 0.1 M sucrose in Tris-HCl solution (50 mM, pH 8.0) was taken, then 100 μL of enzyme solution was added, and after 30 min of reaction at 30°C, 1 mL of DNS solution was added to terminate the reaction. The reaction mixture was treated in boiling water for 5 min, then immediately cooled with running water, and made up to 20 mL with distilled water, and the absorbance was determined at 540 nm using an enzyme marker. The enzyme activity was defined as: the amount of enzyme required to release 1 mg of reducing sugar from amylose per minute under optimal conditions was defined as one enzyme unit (U).
[0029] 2. Determination of half-life at different temperatures
[0030] The enzyme activity at different temperatures was determined by incubating the reaction system described above at 20-80°C (at intervals of 5°C) for 5 h to determine the optimal reaction temperature. Under the optimal temperature conditions, the residual enzyme activity was determined at 0, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 h, and the time at which the corresponding residual enzyme activity decreased to 50% of the initial enzyme activity was determined as the half-life T 1 / 2 .
[0031] Example 2 Construction of the three-dimensional structure of amylomaltase
[0032] 1. Construction of the three-dimensional structure model of amylomaltase
[0033] According to the amino acid sequence of the starch sucrolase as shown in SEQ ID NO. 1, open the SWISS-MODEL server, input the amino acid sequence, and use the protein structure with the highest sequence similarity as the template for homology modeling. The starch sucrolase AS derived from Neisseria polysaccharea (PDB ID: 1G5A) was used for the most similar modeling. The quality of the constructed homology three-dimensional model was evaluated, and the global model quality estimation (GMQE) and qualitative model energy analysis (QMEAN) scores were 0.81 and 0.83, which met the standard of a reliable homology three-dimensional model, indicating that the model constructed by homology modeling was acceptable.
[0034] 2. AlphaFold2 construction of starch sucrolase three-dimensional structure model
[0035] The three-dimensional structure model of starch sucrolase was constructed using the AlphaFold2 program, and the amino acid sequence as shown in SEQ ID NO. 1 was input into the AlphaFold2 online server for running to generate a structure file in PDB format. According to the reliability score of ranked 1-5, the highest scored model was selected as the highest reliability structure model.
[0036] 3. Starch sucrolase conformation rationality evaluation and molecular docking
[0037] The three-dimensional structure model of starch sucrolase obtained by SWISS-MODEL and AlphaFold2 was evaluated for protein conformation rationality using the SAVES program, and was scored by the Lari score. The structure model constructed by AlphaFold2 scored higher, so we selected this conformation for subsequent experiments.
[0038] The obtained structure was used with genetic algorithm AutoDock Vina 4.2 to perform molecular docking with the substrate sucrose, and the most reasonable complex structure with the lowest docking binding energy was selected for analysis. The molecular visualization software PyMol was used to view and analyze these conformations, and the interaction between the ligand and the receptor was evaluated. The residues within the substrate distance were defined as active pocket residues, and the key amino acid residues interacting with the substrate were found.
[0039] Example Three Unit point mutation of starch sucrolase and enzyme activity analysis
[0040] 1. Four strategies were used to improve the mutant of amylosucrase by semi-rational design: 1) Based on the multiple sequence alignment analysis of amylosucrase and high-activity homologous sequences, the possible high-activity sites were screened for site-directed mutagenesis; 2) According to the prediction results of PROSS and FireProt two major computational biology platforms, the possible mutation sites with improved activity and thermal stability were selected; 3) Using the molecular docking results, the amino acids in the substrate binding pocket were virtually mutated, and the interaction force between the protein and the substrate and the stability of the protein-substrate intermediate were analyzed, and the amino acids that could improve the activity and stability were selected for mutation; 4) Using FoldX energy calculation and Rosetta ddg_monomer module (ΔΔG) prediction algorithm, a double free energy screening standard was established to optimize the mutation sites with significant conformational stability.
[0041] 2. According to the above analysis techniques, 25 single-point mutations were designed. Design mutation primers to perform single-point mutations on wild-type amylosucrase. The pET 28a(+) plasmid containing the SEQ NO. 1 sequence of amylosucrase was used as a template, and the coding gene of amylosucrase was subjected to site-directed mutagenesis by PCR method. The construction of PCR reaction system (50 μL) is shown in Table 1. After placing the various components in the system on ice and mixing, it was placed in a PCR amplifier for amplification, and the parameter settings were: 98℃ pre-denaturation for 3 min, followed by 30 cycles of 98℃ denaturation for 20 s, 55℃ annealing for 30 s, and 68℃ extension for 15 min, and 68℃ continued extension for 10 min.
[0042] Table 2 PCR reaction system
[0043]
[0044] The PCR product was digested with restriction endonuclease Dpn I, and the enzyme digestion product was introduced into E. coli DH 5α competent cells by heat shock method. The competent cells were cultured overnight in LB solid medium containing 100 μg / mL kanamycin, and single colonies were picked and cultured in LB liquid medium containing 100 μg / mL kanamycin. The plasmid was extracted, and the correct plasmid was transformed into the expression host E. coli BL21 cells.
[0045] 3. The mutants were induced to express using IPTG and isolated by purification using nickel affinity chromatography. The purified mutants were then tested for enzyme activity and optimum temperature. The results showed that all 25 of the designed mutants could be expressed in soluble form in E. coli BL21 cells. The enzyme activity of each single mutant was then tested at 40°C, and the results showed that 9 of the mutants exhibited activity superior to that of the wild-type amylosucrase, as shown in Table 1. Of these, E38Y had an activity of 121.5% of the original enzyme activity, D386P had an activity of 121.5%, T229Y had an activity of 129.4%, T313I had an activity of 145.3%, A284V had an activity of 167.12%, A323R had an activity of 172.4%, V92M had an activity of 174.3%, and V92E and L330P were optimal, with activities of 224.27% and 244.03% (as shown in Table 1). Therefore, these 9 single mutants were selected for the next round of combinatorial mutation. Figure 1
[0046] Table 1. Single-site mutants of amylosucrase SEQ ID NO Mutant site SEQ ID NO Mutant site 1 E38Y 6 T313I 2 V92E 7 A284V 3 V92M 8 A323R 4 L330P 9 D386P 5 T229Y
[0047] Example Three: Multi-site combinatorial mutation of amylosucrase and enzyme activity analysis
[0048] 1. Design of multi-site combinatorial mutants
[0049] The 9 single-site mutants with improved enzyme activity were ranked from high to low, and a series of double-site mutants, triple-site mutants, quadruple-site mutants, quintuple-site mutants and sextuple-site mutants were obtained by sequentially stacking the mutants. After successful expression in E. coli BL21, the enzyme activity of each mutant was tested at 40°C. In each round of stacking mutation, the mutants that exhibited a synergistic effect and improved enzyme activity after the previous round of mutation were preferentially selected. As the number of stacking sites increased, the activity of the multi-mutants gradually increased, but the mutant with the highest enzyme activity was not necessarily the one with the most mutation sites. Of the 20 multi-mutants constructed, 5 mutants with further improved enzyme activity were ultimately selected, indicating that there was a positive synergistic effect between these sites, as shown in Table 2. Of these, mutant V2 had an enzyme activity of 2.6 times that of the wild-type enzyme, V3 had an enzyme activity of 3.2 times that of the wild-type enzyme, V4 had an enzyme activity of 3.8 times that of the wild-type enzyme, and V5 had an enzyme activity of 6.5 times that of the wild-type enzyme (see Table 2). Figure 2
[0050] Table 2. Combinatorial multi-site mutants of amylosucrase
[0051] To further evaluate the heat resistance of the mutants, the optimum temperature, stability and half-life of the optimal mutant V5 were determined. The results showed that the optimum temperature of the optimal mutant V5 did not change significantly, and was still 40°C. The stability of the optimal mutant V5 was significantly improved, and the half-life T 1 / 2 was 1.37 times that of the wild-type enzyme.
[0052] Example 4. Catalytic synthesis of a- arbutin by the best mutant V5
[0053] The catalytic synthesis of a- arbutin was performed using sucrose and hydroquinone as substrates, with ascorbic acid added, and the best mutant V5. The reaction system contained 200 μL of sucrose solution (15 mM), 100 μL of hydroquinone solution (5 mM), and 600 μL of Tris-HCl buffer (50 mM, pH 8.0). After the addition was complete, the centrifuge tube was preheated in a 40°C water bath for 10 minutes, and then 100 μL of amylomaltase was added to the centrifuge tube for catalytic reaction. The reaction was maintained at 40°C and shaken at 160 rpm to ensure uniform mixing of the reaction mixture.
[0054] After 3 hours of reaction, the reaction tube was transferred to a boiling water bath for 5 minutes for heat inactivation. After the sample cooled naturally, it was centrifuged at 13,000 x g for 20 minutes at 4°C using a high-speed refrigerated centrifuge. The final clear supernatant was filtered through a 0.22 μm filter membrane, and then subjected to quantitative analysis of the product using a high-performance liquid chromatography system (HPLC) equipped with an ultraviolet detector. The detection wavelength of the HPLC was set to 280 nm, and the chromatographic column used was an Agilent ZORBAX sb-aq column (4.6 x 250 mm). The mobile phase was a mixture of acetonitrile and water (80:20, v / v), the flow rate was 0.8 mL·min-1, and the column temperature was set to 30°C.
[0055] HPLC analysis of the reaction product showed that the best mutant could catalyze the synthesis of a- arbutin ( Figure 3 ), with a yield that was 15.6% higher than that of the wild-type enzyme, indicating that the mutant has better application prospects.
Claims
1. A single site mutant of amylosucrase characterized in that, The mutant is a single-point mutant obtained by mutating any one amino acid unit point of the amino acid sequence shown in SEQ ID NO. 1 to E38Y, V92E, V92M, T229Y, A284V, A323R, T313I, L330P or D386P.
2. The single-site mutant of claim 1, wherein, The mutant is a single-point mutant obtained by mutating any one amino acid unit point of the amino acid sequence shown in SEQ ID NO. 1 to V92E, A284V, A323R, L330P or D386P.
3. A multisite mutant of amylosucrase characterized in that, The multi-site mutant is selected from any one of the following (a)-(d): (a) a two-site combined mutant V2 obtained by simultaneously mutating two sites of A284V / D386P of the amino acid sequence shown in SEQ ID No. 1; (b) a three-site combined mutant V3 obtained by simultaneously mutating three sites of A284V / D386P / L330P of the amino acid sequence shown in SEQ ID No. 1; (c) a four-site combined mutant V4 obtained by simultaneously mutating four sites of A284V / D386P / L330P / V92E of the amino acid sequence shown in SEQ ID No. 1; (d) a five-site combined mutant V5 obtained by simultaneously mutating five sites of A284V / D386P / L330P / V92E / A323R of the amino acid sequence shown in SEQ ID No.
1.
4. A gene encoding the single-point mutant of claims 1 and 2.
5. A gene encoding the multi-site mutant of claim 3.
6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises a nucleotide sequence encoding the amino acid sequence of the starch sucrolase mutant of claims 1 and 3.
7. A recombinant host cell, characterized in that, The recombinant engineering bacteria are obtained by transforming the recombinant expression vector of claim 6 into a host microorganism.
8. Use of the single-point mutant of claim 1 or the multi-site mutant of claim 3 in the synthesis of α-arbutin; the synthesis of α-arbutin uses sucrose and hydroquinone as substrates.