Alpha-galactosidase mutant as well as preparation method and application thereof
By mutating α-galactosidase in Bacillus megaterium at specific sites, the problem of low enzyme activity in existing enzyme preparations in tobacco and plant extracts has been solved, achieving efficient hydrolysis of glycosidic bonds, releasing latent aroma substances, and improving product aroma and quality.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING CHINA TOBACCO IND CO LTD
- Filing Date
- 2026-03-20
- Publication Date
- 2026-05-08
AI Technical Summary
Existing industrial α-galactosidase preparations have problems such as low enzyme activity, poor thermal stability and insufficient substrate affinity in tobacco and plant extraction, making it difficult to meet the needs of efficient and low-cost industrial production.
By performing single-point and double-point mutations on Bacillus megaterium α-galactosidase, combined with free energy virtual screening and topological cavity engineering, mutants K74R and L432V were obtained, which significantly improved its catalytic efficiency and substrate hydrolysis ability.
The mutant K74R/L432V has a nearly 1.8-fold increased specific enzyme activity, enabling it to rapidly degrade anti-nutritional oligosaccharides and polysaccharides in plant materials, release latent aroma substances, and enhance the aroma quantity and quality of tobacco and plant extracts.
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Figure CN121991929A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of genetic engineering and enzyme engineering, and in particular to an α-galactosidase mutant, its preparation method, and its application. Background Technology
[0002] α-Galactosidases are a class of exoglycosidases that specifically catalyze the hydrolysis of α-galactosidic bonds and are widely found in plants, animals, and microorganisms. In the tobacco industry, natural plant extraction, and food bioprocessing, these enzymes have significant application value due to their ability to modify specific carbohydrates. The processing quality and sensory characteristics of plant raw materials are largely related to their sugar components, mainly including macromolecular polysaccharides, functional oligosaccharides, and secondary metabolites in glycoside form. However, the natural state of these components often limits the utilization efficiency of raw materials or negatively impacts product quality, necessitating targeted improvement through enzyme engineering.
[0003] In plant matrices, macromolecular polysaccharides such as galactomannan are major components of cell walls or intercellular matrix. In plant extraction or tobacco sheet preparation processes, the presence of these high-molecular-weight polymers significantly increases the viscosity of the liquid, creating mass transfer resistance and thus inhibiting the dissolution and diffusion of active ingredients. More importantly, during thermal processing or combustion, macromolecular polysaccharides often produce a burnt odor due to incomplete pyrolysis, severely interfering with the sensory experience of the product. Enzymatic hydrolysis technology can not only effectively reduce system viscosity and improve processing efficiency but also reduce the content of undesirable flavor precursors at the source.
[0004] Meanwhile, tobacco and legumes commonly contain raffinose and stachyose, oligosaccharides belonging to the raffinose family (RFOs). Enzymatic hydrolysis can completely degrade these oligosaccharides, forming hydrolysis products such as galactose and sucrose, significantly increasing the reducing sugar content in the system. As core substrates for the Maillard reaction, reducing sugars promote the formation of aroma compounds such as pyrazines and furans during subsequent roasting and aging processes, altering the caramelization reaction pathway and thus imparting a richer, more natural roasted sweet aroma to the product, thereby improving its quality.
[0005] Furthermore, many precious aroma components in plants (such as monoterpenes, sesquiterpenes, and aromatic alcohols) do not exist entirely in a free state; a significant portion are bound to glycosyl groups to form non-volatile glycosides (bound aromas). These "latent aroma substances" cannot directly contribute to aroma, resulting in a waste of resources. α-Galactosidase has the potential to hydrolyze glycosidic bonds and can act as an "aroma releaser," breaking down glycosyl bonds and converting bound substances into free aroma components, thereby significantly increasing the total amount and richness of aroma in plant extracts or tobacco products.
[0006] Despite the enormous potential of α-galactosidase, currently industrially available naturally derived enzyme preparations generally suffer from problems such as low specific enzyme activity, poor thermal stability, or insufficient affinity for substrates in complex plant matrices, making it difficult to meet the demands of efficient and low-cost industrial production. Summary of the Invention
[0007] In view of this, the purpose of this invention is to provide an α-galactosidase mutant, its preparation method, and its application. The enzyme is extracted from Bacillus megaterium and modified using single-point mutagenesis and saturation mutagenesis techniques, coupled with a free energy virtual screening coupled with internal topological cavity engineering modification strategy. This yielded two-point mutants K74R and L432V with significantly enhanced enzyme activity. These mutants exhibit significantly improved catalytic efficiency, enabling them to rapidly degrade anti-nutritional oligosaccharides and polysaccharides in plant materials. They can also function as "flavor-enhancing enzymes" to efficiently hydrolyze glycosidic bonds and release latent aroma substances, making them applicable to tobacco processing, plant extraction, and the fragrance and flavor industry.
[0008] The present invention solves the above-mentioned technical problems through the following technical means:
[0009] In a first aspect, the present invention provides an α-galactosidase mutant, the amino acid sequence of which is obtained by mutating amino acid residues at one or more of the following sites based on the wild-type α-galactosidase amino acid sequence shown in SEQ ID NO.1: positions 74, 145, 432, 560, and 615; wherein the amino acid site numbers correspond to the sequence shown in SEQ ID NO.1.
[0010] Preferably, the mutation includes any one or a combination of the following: K74R, A145T, L432V, Q560I, D615E, wherein the K74R mutation is a mutation of lysine at position 74 of SEQ ID NO.1 to arginine, the A145T mutation is a mutation of alanine at position 145 of SEQ ID NO.1 to threonine, the L432V mutation is a mutation of leucine at position 432 of SEQ ID NO.1 to valine, the Q560I mutation is a mutation of glutamine at position 560 of SEQ ID NO.1 to isoleucine, and the D615E mutation is a mutation of aspartic acid at position 615 of SEQ ID NO.1 to glutamic acid.
[0011] Preferably, the mutant is a two-point mutant containing both K74R and L432V, and its amino acid sequence is shown in SEQ ID NO.2.
[0012] To enhance the catalytic activity of α-galactosidase on its substrates, this invention involves specific mutations at specific sites in wild-type α-galactosidase derived from Bacillus megaterium, resulting in significantly improved specific enzyme activity and substrate hydrolysis efficiency. These specific mutations involve replacing lysine (K) at position 74 with arginine (R) and leucine (L) at position 432 with valine (V), yielding the double-site mutants K74R and L432V.
[0013] Experimental results showed that the specific enzyme activity of the two-point mutants K74R and L432V reached 159.19 U / mg, which was 176.95% higher than that of the wild-type WT (57.48 U / mg). This mutant exhibits extremely high hydrolysis efficiency of α-galactosidic bonds, enabling rapid substrate degradation, and its substrate conversion rate is significantly higher than that of the wild-type enzyme in the same time period.
[0014] Secondly, the present invention provides a nucleic acid molecule that encodes the α-galactosidase mutant as described above.
[0015] Thirdly, the present invention provides a recombinant expression vector containing the aforementioned nucleic acid molecule; the recombinant expression vector is pET-28a(+).
[0016] Fourthly, the present invention provides a recombinant host cell containing the aforementioned nucleic acid molecule or the aforementioned recombinant expression vector.
[0017] Preferably, the recombinant host cell is Escherichia coli BL21(DE3).
[0018] Fifthly, the present invention provides a method for preparing the α-galactosidase mutant, characterized by comprising the following steps: (1) culturing the recombinant host cells in a suitable culture medium; (2) inducing the expression of the α-galactosidase mutant; and (3) isolating and purifying the α-galactosidase mutant from the culture.
[0019] In a sixth aspect, the present invention provides the use of the α-galactosidase mutant, the nucleic acid molecule, the recombinant expression vector, or the recombinant host cell in the preparation of enzyme preparations for hydrolyzing substrates containing α-galactosidic bonds.
[0020] Further preferably, the method of application includes the following steps: under an enzyme catalytic system, using sugars containing α-galactosidic bonds as substrates, catalyzing their hydrolysis to produce at least one of galactose, sucrose, monoterpene alcohols, and aromatic alcohols.
[0021] Further preferably, the substrate includes oligosaccharides such as raffinose and stachyose, or aroma precursors in the form of glycosides (such as linalool glycosides, geraniol glycosides, etc.).
[0022] Preferably, the application is as follows:
[0023] (1) Application in tobacco processing or preparation of tobacco extracts, specifically for degrading raffinose family oligosaccharides and / or galactomannan in tobacco raw materials; said application can improve the sensory quality of tobacco extracts;
[0024] (2) Application in the processing of natural plant fragrances, specifically used to hydrolyze the glycosidic bonds bonded to aroma substances, and promote the transformation of bound aroma substances into free aroma substances.
[0025] More preferably, the tobacco raw material is selected from tobacco leaves, tobacco flakes, stems, thin sheets, leaf groups of tobacco, or any combination thereof;
[0026] Utilizing the specific hydrolytic ability of enzymes, the raffinose family oligosaccharides (RFOs) and galactomannans in tobacco that cause pungent and off-flavors are degraded; at the same time, glycosides in tobacco are hydrolyzed.
[0027] The beneficial effects of this invention are:
[0028] (1) This invention utilizes a free energy virtual screening coupled with an internal topological cavity engineering strategy to successfully obtain a series of highly active mutants. Among them, the specific enzyme activity of the core mutant K74R / L432V is nearly 1.8 times higher than that of the wild type. Under the same enzyme dosage, the mutant can significantly shorten the reaction time or increase the substrate conversion rate.
[0029] (2) The mutant of the present invention can efficiently hydrolyze sugar components (such as specific oligosaccharides and polysaccharides) in plant raw materials that cause bitterness, spiciness and increased viscosity. In tobacco processing, the extract prepared by this enzyme treatment has significantly reduced impurities and irritation, and has a more mellow and delicate taste. At the same time, it promotes the conversion of flavor precursors and improves the overall harmony of tobacco products.
[0030] (3) The mutant of the present invention has a broad-spectrum ability to hydrolyze α-galactosidic bonds, and can be used as a highly efficient "flavor enhancer" in the preparation of fragrances. It can specifically cleave the glycosidic bonds of latent aroma substances (such as glycoside aroma precursors) that are in a "bound state" due to glycosylation modification, and release volatile aroma-producing aglycones, thereby significantly increasing the total aroma in tobacco, fruits and natural plant extracts, and giving the products a richer and more natural aroma characteristics. Attached Figure Description
[0031] Figure 1 It is an enzyme activity assay comparing wild-type and single-point mutant;
[0032] Figure 2 It is an enzyme activity assay comparing wild-type and double-point mutant;
[0033] Figure 3 TLC analysis of the hydrolysates of wild-type hydrolyzed raffinose;
[0034] Figure 4 This is a TLC analysis of the hydrolysate of raffinose by a two-point mutant. Detailed Implementation
[0035] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0036] The materials and reagents involved in the following examples are as follows:
[0037] LB liquid medium: yeast extract 5g / L, tryptone 10g / L, NaCl 10g / L.
[0038] LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, agar powder 20 g / L.
[0039] Method for α-galactosidase activity assay: Take 20 μL of 5 mM p-nitrophenyl-α-D-glucopyranoside solution to bring the final concentration to 0.5 mM. Add 170 μL of 20 mM Tris-HCl buffer (pH 6.8) and incubate at 37°C. Then add 10 μL of enzyme solution and react for 5 min. Finally, add 50 μL of 0.5 M Na₂CO₃ solution to terminate the reaction. Measure the absorbance of the reaction solution at 405 nm. Calculate the target protease activity and specific enzyme activity based on the standard curve. Specific enzyme activity (U / mg) is defined as the amount of enzyme released per mg of protein per hour equal to 1 μmol of p-nitrophenol.
[0040] Example 1: Identification and Construction of Mutants
[0041] 1) The present invention adopts a free energy virtual screening coupling internal topology cavity engineering modification strategy to determine the method.
[0042] 2) Using the pET-28a(+) plasmid containing the α-galactosidase gene sequence as a template, a site-directed mutant sequence was amplified from the whole plasmid using a pair of mutation primers. The PCR product was digested with DpnI, and after template digestion, it was purified using a purification kit. The purified product was transformed into E. coli BL21(DE3) competent cells using the heat shock method and plated on LB agar plates containing 50 μg / mL kanamycin sulfate, and incubated overnight at 37°C. Successful transformation was verified by agarose gel electrophoresis, and the mutation results were sequenced by Sangon Biotech (Shanghai) Co., Ltd.
[0043] The PCR reaction system consisted of 50 μL of the following components: 1 μL plasmid template; 25 μL Phanta Max Buffer; 1 μL dNTP Mix; 1 μL each of forward and reverse primers; 1 μL Phanta Max Super-Fidelity DNA Polymerase; and 20 μL ddH2O.
[0044] PCR reaction conditions: 95℃ for 3 min (pre-denaturation); 95℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min 30 s, 33 cycles; 72℃ for 5 min; store at 22℃.
[0045] Primer sequences are shown in Table 1.
[0046] Table 1. Design of Mutant Primers
[0047]
[0048] Example 2: Culture, purification, and desalting of wild-type and mutant enzymes
[0049] 1) Inoculate correctly sequenced colonies and wild-type strains from glycerol tubes into LB liquid medium containing kanamycin resistance and incubate at 37°C for 12-16 h for activation. After activation, transfer 2% of the inoculum to 50 mL of LB liquid medium containing kanamycin resistance and incubate at 37°C, 220 r / min until OD600 reaches 0.6-0.8. Then, add 1 M IPTG to a final concentration of 0.1 mM and incubate at 18°C, 220 r / min for 24 h. Centrifuge the induced bacterial culture at 8000 r / min at 4°C to collect the bacterial cells. Wash and resuspend the precipitate with 25 mM, pH 6.8 Tris-HCl buffer and then sonicate (300 W, 3 s for 3 s, 3 s for 3 min). Centrifuge the sonicated bacterial culture at 4°C, 12000 r / min for 10 min and collect the supernatant for subsequent purification.
[0050] 2) The supernatant was purified using a Ni-NTA nickel ion affinity chromatography column. The purification steps are as follows:
[0051] Add 6 column volumes of Tris-HCl buffer containing 10 mM imidazole at pH 6.8 to the nickel column to equilibrate the column;
[0052] Add the supernatant after membrane transfer to the column, and collect all the flow-through liquid for analysis;
[0053] Elution was performed using buffers containing 50, 100, 150, and 300 mM imidazole, respectively, with each eluent eluting for 6 column volumes. The eluted products were collected and analyzed by 10% SDS-PAGE gel electrophoresis. The gels were stained with Coomassie Brilliant Blue R250 for observation.
[0054] 3) Collect the desired purified product based on the gel electrophoresis pattern and desalt it using a desalting column. The steps are as follows:
[0055] The column was equilibrated using 3 column volumes of Tris-HCl buffer at pH 6.8 containing 10 mM imidazole;
[0056] The collected purified product was added to the column, and the column was eluted again with Tris-HCl buffer (pH 6.8) containing 10 mM imidazole. The eluted product was collected and detected by SDS-PAGE electrophoresis.
[0057] 4) Result determination:
[0058] First, the purified wild-type and 16 single-point mutants were subjected to enzyme activity assays, and the results are as follows: Figure 1 As shown, five single-point mutants with improved activity were obtained through screening. These mutants included K74R, A145T, Q560I, and L432V, with the mutation at position 432 increasing enzyme activity by approximately 55.60%. Based on these results, a two-point combination mutation was designed to further explore the potential synergistic effect of these five mutations in enhancing enzyme activity. The results were presented in... Figure 2 As demonstrated, most mutants in the two-point combination mutants exhibited higher enzyme activity compared to the wild type, with K74R / L432V showing a specific activity of 159.19 U / mg (wild type 57.48 U / mg), representing a 176.95% increase. Therefore, these three K74R / L432V combination mutants were selected for further application and research. These results also confirm that this invention provides an effective method to significantly enhance enzyme activity. The enhancement of 4GT's catalytic activity is based on existing protein engineering techniques, laying the foundation for wider industrial applications.
[0059] Example 3: Application of wild-type and mutant enzymes in the degradation of simulated plant sugar substrates (raffinose)
[0060] 1) The mutant obtained in Example 2 and the crude enzyme solution of the wild type were applied to the hydrolysis of raffinose. The mixture was incubated with raffinose in 25 mM Tris-HCl buffer (pH 6.8) at 40 °C for different times (0.5, 1, and 2 hours) at a 1:1 ratio. The reaction mixture was analyzed by thin-layer chromatography (TLC) at each time point to determine the changes in the hydrolysis products.
[0061] 2) TLC detection: The developing solvent was mixed with n-butanol:methanol:water in a ratio of 5:2:1. The mixed standard (10 mg / mL raffinose and galactose) and the reaction mixture were spotted separately and then placed in a chromatography tank for development. After drying, the colorimetric reagent (methanol:sulfuric acid = 95:5) was used for color development.
[0062] 3) Experimental results: Thin-layer chromatography (TLC) analysis showed ( Figure 3 and 4 For the mutant K74R / L432V, raffinose was completely hydrolyzed within 30 min of the reaction. In contrast, the wild-type enzyme still had a large amount of raffinose unhydrolyzed after 30 min, and a small amount of raffinose remained unhydrolyzed after 1 h, until complete hydrolysis was achieved after 2 h. This demonstrates that the mutant is more efficient at hydrolyzing raffinose and shows greater promise for removing RFOs from soy products.
[0063] Example 4: Application of mutants in the preparation of low-irritation, high-quality tobacco extracts
[0064] 1) Fragments of tobacco waste, low-grade tobacco leaves, and industrial dust were pulverized through a 40-mesh sieve. 100 g of tobacco dust was weighed and added to purified water at a material-to-liquid ratio of 1:10 (w / v). The mixture was stirred in a constant-temperature water bath at 50°C, and the K74R / L432V mutant enzyme solution was added at a concentration of 20 U / g of tobacco dust. The mixture was placed in a constant-temperature water bath at 45°C and stirred at 100 r / min for 4 hours. After the reaction, the temperature was rapidly increased to 95°C and maintained for 10 min to inactivate the enzyme. After cooling to room temperature, the mixture was centrifuged at 5000 r / min for 15 min, and the supernatant was collected. The supernatant was concentrated under reduced pressure to obtain the tobacco extract.
[0065] A control group and a wild-type group were set up. The control group was sprayed with an equal volume of buffer solution. The wild-type group was sprayed with wild-type α-galactosidase.
[0066] Tobacco extract is added to the tobacco processing tubes and rolled using the same method.
[0067] 2) Evaluation of Results:
[0068] The sensory quality of the tobacco leaves was evaluated by professional tobacco tasters with reference to the sensory evaluation method for tobacco and tobacco products YC / T 138-1998. The results are shown in Table 2.
[0069] Table 2 Sensory Quality Evaluation
[0070]
[0071] Experiments have shown that this extract can reduce impurities and irritation, promote the upward movement of smoke, and significantly improve the quality of smoking.
[0072] Example 5: Application of mutants in natural plant extracts
[0073] 1) Select raisins, wash and crush them, add purified water at a material-to-liquid ratio of 1:10 (w / v), stir in a constant temperature water bath at 45℃, add mutant K74R / L432V enzyme solution, and add enzyme at a rate of 20 U / g tobacco powder. Place in a constant temperature water bath at 45℃ and stir at 100 r / min for 2 hours. After the reaction is complete, rapidly heat to 95℃ and hold for 15 min to inactivate the enzyme preparation. After cooling to room temperature, centrifuge at 4500 r / min for 10 min and collect the supernatant. Concentrate the supernatant under reduced pressure to obtain the natural plant extract.
[0074] Natural extracts are added to tobacco for smoking.
[0075] 2) Results Analysis
[0076] Experiments have shown that the extract has a sweet aroma, makes the smoke richer and more harmonious, and significantly improves the quality of smoking.
[0077] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.
Claims
1. An α-galactosidase mutant, characterized in that, The amino acid sequence of the mutant was obtained by mutating amino acid residues at one or more of the following sites based on the wild-type α-galactosidase amino acid sequence shown in SEQ ID NO.1: positions 74, 145, 432, 560, and 615; wherein the amino acid site numbers correspond to the sequence shown in SEQ ID NO.
1.
2. The α-galactosidase mutant according to claim 1, characterized in that: The mutations include any one or a combination of the following: K74R, A145T, L432V, Q560I, and D615E, wherein the K74R mutation is a mutation of lysine at position 74 of SEQ ID NO.1 to arginine, the A145T mutation is a mutation of alanine at position 145 of SEQ ID NO.1 to threonine, the L432V mutation is a mutation of leucine at position 432 of SEQ ID NO.1 to valine, the Q560I mutation is a mutation of glutamine at position 560 of SEQ ID NO.1 to isoleucine, and the D615E mutation is a mutation of aspartic acid at position 615 of SEQ ID NO.1 to glutamic acid.
3. The α-galactosidase mutant according to claim 2, characterized in that: The mutant is a two-point mutant containing both K74R and L432V, and its amino acid sequence is shown in SEQ ID NO.
2.
4. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the α-galactosidase mutant as described in any one of claims 1-3.
5. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule as described in claim 4; the recombinant expression vector is pET-28a(+).
6. A recombinant host cell, characterized in that, The recombinant host cell contains the nucleic acid molecule as described in claim 4 or the recombinant expression vector as described in claim 5.
7. The recombinant host cell according to claim 6, characterized in that: The recombinant host cell was Escherichia coli BL21(DE3).
8. A method for preparing the α-galactosidase mutant as described in any one of claims 1-3, characterized in that, The method includes the following steps: (1) culturing the recombinant host cell of claim 6 in a suitable culture medium; (2) inducing the expression of the α-galactosidase mutant; and (3) isolating and purifying the α-galactosidase mutant from the culture.
9. The use of the α-galactosidase mutant as described in any one of claims 1-3, the nucleic acid molecule as described in claim 4, the recombinant expression vector as described in claim 5, or the recombinant host cell as described in claim 6 or 7 in the preparation of enzyme preparations for hydrolyzing substrates containing α-galactosidic bonds.
10. The application according to claim 9, characterized in that, The application is as follows: (1) Application in tobacco processing or preparation of tobacco extracts, specifically for degrading raffinose family oligosaccharides and / or galactomannan in tobacco raw materials; said application can improve the sensory quality of tobacco extracts; (2) Application in the processing of natural plant fragrances, specifically used to hydrolyze the glycosidic bonds bonded to aroma substances, promoting the transformation of bound aroma substances into free aroma substances.