Enzyme activity improved tagatose-4-epimerase mutant and application thereof
By molecularly transforming the tagatose-4-episomerase, the site-point mutation of serine position 56 is alanine, which improves the enzyme activity, solves the problems of low enzyme activity and poor thermal stability, and achieves efficient industrial production of tagatose.
Patent Information
- Application Number
- CN202510696701.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-28
- Publication Date
- 2025-08-29
AI Technical Summary
The existing tagatose-4-episomerase has low enzyme activity, poor thermal stability and weak substrate adaptability, which leads to hindering industrial application, and traditional extraction methods have problems such as high cost and cumbersome processes.
By molecularly transforming the Taggarose-4-episomerase from Thermoprotei archaeon, the site-directed mutation of serine position 56 as alanine, mutant S56A was obtained and purified by nickel affinity chromatography column to improve enzyme activity.
The enzyme activity of the tagatose-4-epiamomerase mutant S56A was increased by 260%, and the optimal pH and reaction temperature did not change significantly, providing an excellent catalyst for the industrial production of tagatose and reducing production costs.
Smart Images
Figure CN120555413A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of enzyme genetic engineering, and particularly relates to a tagatose-4-epimerase mutant with improved enzyme activity and application thereof. Background Art
[0002] D-Tagatose, a rare, naturally occurring ketohexose, has a sweetness similar to sucrose (approximately 92%) but contains only 38% of the calories. It exhibits significant physiological benefits, including hypoglycemic, anti-caries, gut microbiome regulation, antioxidant, and probiotic effects. As a Generally Recognized as Safe (GRAS) food ingredient certified by the US FDA, it has significant potential for application in the food, pharmaceutical, and cosmetic sectors. However, D-tagatose's natural abundance is extremely low (it is only found in some fruits, dairy products, and hot spring microorganisms), making it difficult to achieve large-scale production using traditional extraction methods.
[0003] At present, the industrial production of D-tagatose mainly relies on chemical and biological methods. The chemical method uses D-galactose as raw material and produces it through alkaline isomerization reaction under high temperature and high pressure conditions, but there are problems such as harsh reaction conditions (requires 180-200°C, pH10-12), complex by-products (such as sorbose, mannose), difficulty in separation and purification, and environmental pollution; the biological method uses lactose or polysaccharides as substrates and synthesizes it through a multi-enzyme cascade reaction catalyzed by β-galactosidase and L-arabinose isomerase. Although this method has the advantage of being green and environmentally friendly, it has problems such as high cost of lactose raw materials (about 5 times that of D-fructose), low substrate utilization (30%-50%), and multi-step purification resulting in a cumbersome process.
[0004] In 2017, Lee's team first proposed using tagatose-4-epimerase (T4E) to directly catalyze the diastereoisomerization of the C4 hydroxyl group of D-fructose to produce D-tagatose. This single-step reaction offers advantages such as low substrate cost (D-fructose is only one-fifth the price of D-galactose) and mild reaction conditions (pH 7-9, 70-80°C). However, industrial application of native T4E has been hampered by its low enzymatic activity, poor thermostability, and weak substrate adaptability. Although researchers have attempted to modify T4E through directed evolution and rational design, technical bottlenecks such as low substrate conversion, insufficient industrial-grade enzyme activity, and poor long-term thermostability have not been overcome. Therefore, screening and modifying tagatose-4-epimerase (T4E) through directed evolution and rational design to obtain mutants with improved enzyme activity is of great significance for the industrial production of tagatose. Summary of the Invention
[0005] The purpose of the present invention is to overcome the defects in the prior art and provide a tagatose-4-epimerase mutant with improved enzyme activity and its application. The enzyme activity is greatly improved compared with the wild type, laying a solid foundation for the industrial application of D-tagatose and tagatose-4-epimerase.
[0006] To achieve the above object, the technical solutions adopted by the present invention are as follows:
[0007] A tagatose 4-epimerase mutant with improved enzyme activity, characterized in that the amino acid sequence of the tagatose 4-epimerase mutant is shown in SEQ ID No: 4.
[0008] As a further technical solution, the tagatose-4-epimerase mutant is obtained by mutating the 56th serine of the tagatose-4-epimerase with an amino acid sequence as shown in SEQ ID No: 2 to alanine.
[0009] As a further technical solution, the tagatose-4-epimerase with an amino acid sequence as shown in SEQ ID No: 2 is derived from Thermoprotei archaeon.
[0010] A gene encoding the tagatose-4-epimerase mutant, the nucleotide sequence of which is shown in SEQ ID No: 3.
[0011] A recombinant plasmid carrying the gene.
[0012] As a further technical solution, the expression vector of the recombinant plasmid is pET-22b(+).
[0013] A recombinant cell expressing the tagatose-4-epimerase mutant or the gene is obtained by transforming the recombinant plasmid according to claim 5 into a host cell, wherein the host cell includes a prokaryotic cell or a eukaryotic cell.
[0014] As a further technical solution, the prokaryotic cell includes Escherichia coli.
[0015] A method for preparing the tagatose-4-epimerase mutant is obtained by inducing and culturing the recombinant cell.
[0016] Application of the tagatose-4-epimerase mutant in the preparation of tagatose.
[0017] As a further technical solution, D-fructose is used as a substrate, and an enzymatic reaction occurs under the action of a mutant of tagatose-4-epimerase to produce tagatose, wherein the enzymatic reaction system also includes 1mmol / LNi 2+, the pH of the enzymatic reaction system is 7.0, the temperature of the enzymatic reaction is 90 degrees Celsius, and the reaction time of the enzymatic reaction is 30 minutes.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] The present invention molecularly modifies tagatose 4-epimerase (T4Ease) from Thermoprotei archaeon by site-directed mutation of serine at position 56 in the amino acid sequence shown in SEQ ID No. 2 to alanine, resulting in mutant S56A. Pure S56A enzyme is then purified using a nickel affinity chromatography column. The optimal pH and reaction temperature of the tagatose 4-epimerase mutant S56A are 7.0 and 90°C, respectively, which are not significantly different from those of the wild type. The enzyme activity of the tagatose 4-epimerase mutant S56A is 2.84 U / mg, a 260% increase compared to the wild type. This provides an excellent catalyst for industrial tagatose production, helps reduce tagatose production costs, and lays a foundation for research on tagatose 4-epimerase and tagatose. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 The SDS-PAGE results of wild type and mutant S56A of tagatose-4-epimerase are shown;
[0021] exist Figure 1 Middle, band M: band 1: wild type; band 2: mutant S56A;
[0022] Figure 2 The figure shows the effect of the temperature of the enzyme reaction system on the enzyme activity of wild-type tagatose-4-epimerase and mutant S56A;
[0023] Figure 3 The effect of pH of the enzyme reaction system on the enzyme activity of wild-type tagatose-4-epimerase and mutant S56A
[0024] Figure 4 The results show the relative enzymatic activities of wild-type and mutant S56A tagatose-4-epimerase under the optimal enzymatic reaction conditions. DETAILED DESCRIPTION
[0025] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.
[0026] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.
[0027] In the present invention,
[0028] 1. All commercial products, including plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits, Bradford protein concentration assay kits, and DNA gel recovery kits, were used according to the kit instructions. Routine procedures, including colony PCR, agarose gel electrophoresis, heat shock transformation, electroporation, preparation of competent cells, and extraction and preservation of bacterial genomes, were performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition). Sequencing of plasmids and DNA products was performed by Suzhou Genewise Biotechnology Co., Ltd., and recombinant plasmids were performed by Nanjing GenScript Biotechnology Co., Ltd.
[0029] 2. Culture medium
[0030] (1) LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.
[0031] (2) LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar powder.
[0032] (3) TB medium: peptone 12.0 g / L, yeast powder 24.0 g / L, glycerol 5.0 g / L, KH2PO4 12.54 g / L, K2HPO4 2.31 g / L.
[0033] 3. In the present invention, the raw materials used are commercially available unless otherwise specified.
[0034] Example 1: Preparation of Thermoprotei archaeon T4Ease mutant
[0035] 1. Construction of pET22b-G7L4-T4E plasmid: A gene fragment encoding T4Ease (protein sequence accession number RLE69020.1) from the thermophilic microorganism Thermoproteiarchaeon was synthesized (the nucleotide sequence of the gene encoding T4Ease is shown in SEQ ID NO. 1, and the amino acid sequence of T4Ease is shown in SEQ ID NO. 2). During the synthesis process, a 6-histidine tag was added to the C-terminus of the target gene fragment. The recombinant gene fragment was introduced into the E. coli expression vector pET-22b(+) using NdeI and XhoI restriction endonucleases to obtain the recombinant plasmid pET22b-G7L4-T4E as the wild-type recombinant plasmid.
[0036] 2. Construction of the pET-22b(+)-S56A mutant plasmid: Using the pET22b-G7L4-T4E plasmid as a template, the S56A site-directed mutation was introduced by OCR. Sequencing verification results showed that no random mutations occurred except the desired mutation site. Therefore, the mutant plasmid pET-22b(+)-S56A was successfully constructed.
[0037] The upstream mutagenesis primer was: 5′-TGCTGgccTTTGGTCGTGAAACCGCACCGGAATATGTTATT-3′;
[0038] The downstream mutagenesis primer was: 5′-TTTCACGACCAAAggcCAGCATCAGACCACCGGCAACACGACGTTC-3′;
[0039] PCR amplification: The reaction system is as shown in Table 1, with a total volume of 20 μL. The reaction program is as follows: 95°C, 3 min (initial denaturation); 95°C, 30 s (denaturation); 56°C, 30 s (annealing); 72°C, 1 min 45 s (extension); 30 cycles; 72°C, 5 min (final extension); 4°C, ∞ (storage).
[0040] The PCR reaction system is shown in Table 1;
[0041] Table 1: PCR reaction system (20 μL)
[0042]
[0043] 3. Verification of PCR product nucleic acid electrophoresis and template digestion: Take 3 μL of PCR product for agarose gel electrophoresis to verify whether the PCR product band size is correct. After verification, add 1 μL of Q.cut Dpn I and 2 μL of Q.cut Buffer (10×) to the PCR product system and perform enzyme digestion reaction at 37°C for 1 hour to remove the wild-type recombinant plasmid in the system.
[0044] After the enzyme digestion reaction, the product was purified using a PCR rapid purification kit. Finally, 5 μL of the purified PCR product was transformed into E. coli DH5ɑ competent cells. Positive clones were then selected for plasmid extraction and DNA sequencing. The successfully sequenced mutant plasmid pET-22b(+)-S56A was introduced into E. coli BL21(DE3) competent cells to construct a mutant gene recombinant strain for inducible expression of the tagatose-4-epimerase mutant.
[0045] Example 2: Expression and purification of wild-type and mutant Thermoclostridium caenicola DAEase
[0046] 1. Induced expression: The plasmid pET22b-G7L4-T4E and the mutant plasmid pET-22b(+)-S56A verified by sequencing were transformed into Escherichia coli BL21 (DE3) cells, respectively. Positive transformants were picked and cultured in LB liquid medium at 37°C with a shaker at 200 rpm overnight. Then, they were inoculated with TB medium and cultured at 37°C for 2-3 h to an OD value of 0.6-0.8. The cells were cooled to 28°C and IPTG was added to a final concentration of 1.0 mM for induction for 6 h to obtain wild-type fermentation broth and mutant fermentation broth.
[0047] 2. Purification: Centrifuge the wild-type and mutant fermentation broths at 4°C, 8000 rpm for 20 min to obtain the cells. Add 20 mL of buffer (50 mM Tris, 200 mM NaCl, adjusted to pH 7.5) to fully resuspend the cells. Place the centrifuge tube in an ice bath and place it in an ultrasonic cell disruptor. Ultrasonic disruption conditions are: 1 s on time, 2 s off time, for a total of 15 min. Subject the resulting disrupted liquid to low-temperature high-speed centrifugation at 4°C, 8000 rpm for 10 min to obtain crude enzyme solutions of wild-type and mutant DAEase. Filter each through a 0.45 μm microporous filter membrane and set aside.
[0048] Prepare a nickel ion affinity chromatography column. First, use a constant flow pump to flush the column with deionized water (approximately 6 to 12 column volumes) at 4°C. Then, equilibrate the column with a low-salt buffer (500 mmol / L NaCl, 50 mM Tris, adjusted to pH 7.0). When the pH of the effluent at the bottom of the column matches the pH of the low-salt buffer pumped into the column (approximately 5 column volumes of buffer are required), add the resulting membrane-passed crude enzyme solution to the column. First, flush the impurities with a buffer containing a low concentration of imidazole (500 mmol / L NaCl, 50 mmol / L imidazole, 50 mM Tris, adjusted to pH 7.0) to reach baseline equilibrium. Then, elute with an eluent containing a high concentration of imidazole (500 mmol / L NaCl, 500 mmol / L imidazole, 50 mM Tris, adjusted to pH 7.0). Collect the eluate at the absorption peak and determine its enzyme activity to obtain the target protein. After purification, wild-type tagatose-4-epimerase and mutant S56A were analyzed by SDS-PAGE, and both were electrophoretically pure (see Figure 1 ).
[0049] The amino acid sequence of the tagatose-4-epimerase mutant S56A is shown in SEQ ID NO. 4, and the nucleotide sequence of the gene encoding the tagatose-4-epimerase mutant S56A is shown in SEQ ID NO. 3.
[0050] Example 3: Optimization of the optimal temperature of the wild type and mutants of Thermoprotei archaeon T4Ease
[0051] The enzyme reaction was carried out in the temperature range of 70-95°C (a temperature point was set every 5°C), and the system pH was set to 9.0. 50 g / L D-fructose was used as the substrate, and pure enzyme (wild type and mutant S56A of tagatose-4-epimerase purified in Example 2) was added to a final concentration of 0.1 mg / mL, 1 mmol / L Ni 2+ The enzyme was reacted at 90°C under different pH conditions for 30 minutes and then inactivated by boiling for 10 minutes. The optimal reaction temperature is the temperature corresponding to the highest enzyme activity. In order to compare the effects of different reaction temperatures on the activity of the recombinant enzyme, the enzyme activity at the optimal reaction temperature was set as 100% relative enzyme activity to calculate the relative enzyme activity at other temperatures. Figure 2 The optimum temperature of mutant S56A was consistent with that of the wild type, both of which were 90℃.
[0052] Example 4: Determination of the optimal pH of the wild type and mutants of Thermoprotei archaeon T4Ease
[0053] The enzyme reaction was performed using NaAc-HAc (50 mM, pH 5.5-6.0), PBS (50 mM, pH 6.5-7.0), and TrisHCl (50 mM, pH 7.5-9.0) as the buffer systems, respectively. The reaction temperature was set at 90°C. Except for the reaction temperature, the reaction conditions, including substrate concentration, enzyme concentration, and metal ion concentration, and the detection conditions were the same as those described in Example 3. After the reaction, the product was centrifuged, filtered, diluted to a certain concentration, and then detected by HPLC.
[0054] Enzyme activity definition (U): The amount of enzyme required to catalyze the synthesis of 1 μmol of D-psicose per unit time (min) under standard reaction conditions. The optimal reaction pH is the pH corresponding to the highest enzyme activity. In order to compare the effects of different reaction pH on the activity of the recombinant enzyme, the enzyme activity at the optimal reaction pH was set as 100% relative enzyme activity to calculate the relative enzyme activity at other pH values. Figure 3 The optimal pH of the wild-type tagatose-4-epimerase and mutant S56A was consistent, both at 7.0.
[0055] Example 5: Determination of Enzyme Activity of Thermoprotei archaeon T4Ease Wild Type and Mutants
[0056] 50 g / L D-fructose was used as substrate, pure enzyme (wild type and mutant S56A of tagatose-4-epimerase purified in Example 2) was added to a final concentration of 0.1 mg / mL, 1 mmol / L Ni 2+ The enzyme reaction was carried out at 90°C, pH 7.0 for 30 minutes and then inactivated by boiling for 10 minutes. After the reaction, the product was centrifuged, filtered through a membrane, and diluted to a certain concentration for detection by HPLC.
[0057] Enzyme activity definition (U): The amount of enzyme required to catalyze the synthesis of 1 μmol of D-tagatose per unit time (min) under standard reaction conditions.
[0058] Definition of specific enzyme activity (U / mg): The number of units of enzyme activity per unit mass (mg) of enzyme protein under standard reaction conditions.
[0059] Protein quality detection method: (1) Draw a standard curve: dilute the bovine serum albumin sample in a certain proportion to obtain protein standard solutions with concentrations of 0, 0.125, 0.25, 0.5, 0.75, 1, and 1.5 mg / mL. Take 5 μL of protein standard solutions of different concentrations into the sample wells of a 96-well plate, add 250 μL of G250 staining solution to each well, and measure the OD at a wavelength of 595 nm using a UV spectrophotometer. 595 The protein concentration of the standard solution is used as the horizontal axis, and the corresponding OD 595The values are plotted as the vertical axis and the standard curve is drawn. (2) Treat the T4Ease protein sample in the same manner as in (1) above and measure the OD 595 The enzyme protein concentration was calculated by comparing the values with the standard curve.
[0060] Depend on Figure 4 It can be seen that the specific enzyme activity of the wild type is 0.79 U / mg, while the specific enzyme activity of the mutant S56A is increased to 2.84 U / mg, an increase of 260.0%.
[0061] The above-described embodiments are only preferred embodiments of the present invention and are not exhaustive of all feasible implementations of the present invention. For those skilled in the art, any obvious modifications made thereto without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.
Claims
1. A tagatose-4-epimerase mutant with improved enzyme activity, characterized in that: The amino acid sequence of the tagatose-4-epimerase mutant is shown in SEQ ID No:
4.
2. The tagatose-4-epimerase mutant according to claim 1, characterized in that The tagatose-4-epimerase mutant is obtained by mutating the 56th serine of the tagatose-4-epimerase with an amino acid sequence as shown in SEQ ID No: 2 to alanine.
3. The tagatose-4-epimerase mutant according to claim 2, characterized in that The tagatose-4-epimerase with the amino acid sequence shown in SEQ ID No: 2 is derived from Thermoprotei archaeon. A gene encoding the tagatose-4-epimerase mutant according to claim 1.
5. A recombinant plasmid carrying the gene according to claim 4.
6. The recombinant plasmid according to claim 5, characterized in that The expression vector of the recombinant plasmid is pET-22b(+).
7. A recombinant cell expressing the tagatose-4-epimerase mutant according to claim 1 or the gene according to claim 2, characterized in that: The recombinant plasmid according to claim 5 is transformed into a host cell, wherein the host cell includes a prokaryotic cell or a eukaryotic cell.
8. A method for preparing the tagatose-4-epimerase mutant according to claim 1, characterized in that: The recombinant cell according to claim 7 is obtained by inducing culture.
9. Use of the tagatose-4-epimerase mutant according to claim 1 in the preparation of tagatose.
10. The use according to claim 9, characterized in that: With D-fructose as substrate, under the action of tagatose-4-epimerase mutant, an enzymatic reaction occurs to produce tagatose, wherein the enzymatic reaction system also includes 1mmol / LNi 2+ , the pH of the enzymatic reaction system is 7.0, the temperature of the enzymatic reaction is 90 degrees Celsius, and the reaction time of the enzymatic reaction is 30 minutes.