A high-enzyme-activity d-allulose 3-epimerase mutant and application thereof

Abstract

The application discloses a high-enzyme-activity D-allulose 3-epimerase mutant and application thereof, and belongs to the technical field of enzyme genetic engineering. The D-allulose 3-epimerase derived from Thermoclostridium caenicola is used as a parent, a cysteine at the 258th position is mutated into a histidine by using a gene mutation technology, a single-point mutant C258H is obtained, and the enzyme activity of the mutant C258H is improved from 242.4 U / mg of a wild-type enzyme to 387.8 U / mg, with an increase of 60.0%. The finding has important research value for industrialized preparation of D-allulose.

Description

Technical Field

[0001] This invention relates to a high-activity D-allulose 3-epimerase mutant and its applications, belonging to the field of enzyme genetic engineering technology. Background Technology

[0002] D-Allulose is an important member of the rare sugar family and a novel low-energy sweetener. With a sweetness equivalent to 70% of sucrose and only 0.4 kcal / g, it is certified as "Generally Recognized as Safe" by the U.S. Food and Drug Administration (FDA) and officially approved for use in dietary supplements and foods. The labeling of D-Allulose in the "Total Sugars" and "Added Sugars" sections of nutrition labels has been removed. In recent years, D-Allulose has gained regulatory approval and widespread application in several countries, including Japan, Australia, South Korea, Canada, and New Zealand. D-Allulose's flavor, properties, and texture are similar to other sugars, making it an excellent sucrose substitute, primarily used in food, pharmaceuticals, and dietary supplements. Through the Maillard reaction, D-Allulose can enhance the flavor and quality of food during processing; furthermore, it can enhance the elasticity, viscosity, and water retention of frozen foods to increase storage stability. Therefore, D-Allulose has wide applications in the preparation of confectionery, beverages, baked goods, and health foods.

[0003] Studies on the metabolism and absorption of D-allulose show that approximately 70% of it is absorbed in the small intestine without being converted into energy, and is subsequently excreted in the urine within 24 hours, without raising blood glucose levels in diabetic patients. Furthermore, D-allulose inhibits intestinal digestive enzymes, slowing carbohydrate digestion, and competitively transports glucose across the intestinal mucosa, exhibiting excellent hypoglycemic properties. It can be used as a specific metabolic regulator of fat and glucose metabolism. In addition, D-allulose demonstrates a series of excellent physiological properties, such as enhanced antioxidant activity, anti-inflammation, inhibition of parasite growth, lowering blood lipids, prevention of diabetic nephropathy, enhanced endurance, reduced fatigue, and suppression of loss of appetite. Consumers with specific needs can purchase D-allulose as a health food or medicine. Due to many consumers' changing preferences for health foods, the application range of D-allulose is wider than that of artificial low-calorie sweeteners, resulting in significant market demand. However, since D-allulose is very rare in nature and difficult to synthesize chemically, biological conversion of D-allulose has become a research hotspot.

[0004] According to Izumoring's rare sugar conversion strategy, ketose 3-epimerases play an irreplaceable role in the biotransformation of D-allulose, catalyzing a reversible epimerization reaction of D-fructose at the C-3 position to generate D-allulose. Currently, research on ketose 3-epimerases is quite extensive, including microbial screening and identification, enzyme isolation and purification, heterologous recombination expression, enzyme immobilization, food-grade expression, molecular modification, and crystal structure determination. However, current D-allulose 3-epimerases exhibit low catalytic activity, making them unsuitable for industrial production. Obtaining D-allulose 3-epimerases with high catalytic activity is crucial to meeting the market demand for D-allulose. Summary of the Invention

[0005] This invention provides a mutant of D-allulose 3-epimerase with enhanced enzyme activity. This discovery has significant practical implications for the industrial production of D-allulose and the industrial application of D-allulose 3-epimerase. Currently, there is no research on site 258, which is located on the enzyme surface and far from the active site. This invention is the first to propose site-directed mutagenesis at this site, resulting in a mutant with significantly enhanced enzyme activity, providing more possibilities for the design of mutants to enhance the activity of D-allulose 3-epimerase.

[0006] This invention utilizes site-directed mutagenesis to molecularly modify D-allulose 3-epimerase (DAEase) from the microorganism Thermoclostridium caenicola.

[0007] The first objective of this invention is to provide a D-allulose 3-epimerase mutant, which is based on the amino acid sequence shown in SEQ ID No. 2, by mutating cysteine ​​at position 258 to histidine, and named C258H.

[0008] In one embodiment of the present invention, the nucleotide sequence encoding the D-allulose 3-epimerase is shown in SEQ ID No. 1.

[0009] In one embodiment of the present invention, the nucleotide sequence encoding the D-allulose 3-epimerase mutant is shown in SEQ ID No. 3; the amino acid sequence of the mutant is shown in SEQ ID No. 4.

[0010] A second objective of this invention is to provide a gene encoding the mutant.

[0011] A third objective of this invention is to provide an expression vector carrying the gene.

[0012] In one embodiment of the present invention, the expression vector is pET-22b(+).

[0013] A fourth object of the present invention is to provide recombinant cells that express the mutant or the gene encoding the mutant.

[0014] In one embodiment of the present invention, the recombinant cells are prokaryotic cells or eukaryotic cells.

[0015] In one embodiment of the present invention, the prokaryotic cells include Escherichia coli.

[0016] The fifth objective of this invention is to provide a method for increasing the activity of D-allulose 3-epimerase.

[0017] In one embodiment of the present invention, the method involves mutating the 258th position of the amino acid sequence shown in SEQ ID No. 2 from cysteine ​​to histidine.

[0018] The present invention also provides the application of the mutant or the genetically engineered bacteria in the fields of pharmaceutical production and food.

[0019] In one embodiment of the present invention, the application in the food field includes the preparation of baked goods and health foods.

[0020] Beneficial effects:

[0021] This invention involves the molecular modification of D-allulose 3-epimerase derived from *Thermoclostridium caenicola*. A site-directed mutagenesis was performed on amino acid position 258 of the amino acid sequence shown in SEQ ID NO.2 to obtain the mutant C258H. The pure enzyme C258H was obtained after purification using nickel affinity chromatography. The optimal pH for the D-allulose 3-epimerase mutant C258H is 7.5, and the optimal reaction temperature is 65℃, showing no significant changes compared to the wild type. The enzyme activity of the D-allulose 3-epimerase mutant C258H is 387.8 U / mg, which is 60% higher than that of the wild type. This provides an excellent catalyst for the industrial production of D-allulose, helps reduce the production cost of D-allulose, and lays the foundation for further research on D-allulose. Attached Figure Description

[0022] Figure 1 Agarose gel electrophoresis results of D-allulose 3-epimerase and its mutant C258H. Band 1 represents the original enzyme, and band 2 represents C258H.

[0023] Figure 2 SDS-PAGE results of D-allulose 3-epimerase and its mutant C258H. Band 1 represents the original enzyme, and band 2 represents C258H.

[0024] Figure 3 Specific enzyme activities of D-allulose 3-epimerase and its mutant C258H.

[0025] Figure 4 The optimal pH for D-allulose 3-epimerase and its mutant C258H.

[0026] Figure 5 Optimal temperature of D-allulose 3-epimerase and its mutant C258H. Detailed Implementation

[0027] Example 1: Preparation of Thermoclostridium caenicola DAEase mutant

[0028] Construction of pET22b-thca-dae plasmid: A gene fragment encoding DAEase (protein sequence accession number SHI77623.1) from the thermophilic microorganism T. caenicola was synthesized (as shown in SEQ ID No. 1). 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-thca-dae, which served as the wild-type recombinant plasmid.

[0029] Construction of the pET-22b(+)-C258H mutant plasmid: Using the pET22b-thca-dae plasmid as a template, the C258H site-directed mutation was introduced by PCR. Sequencing verification results showed that no random mutations occurred except for the required mutation site. Therefore, the mutant plasmid pET-22b(+)-C258H was successfully constructed.

[0030] The upstream mutation primer is: 5'-gatatcCACgtctggcgcgacttg-3'

[0031] The downstream mutation primer is: 3'-gtcgcgccagacGTGgatatcttttcccac-5' (underlined to indicate the mutation site).

[0032] PCR amplification: The reaction system is shown in Table 1, with a total volume of 20 μL. The reaction program is as follows: 95℃, 2 min (pre-denaturation); 95℃, 15 s (denaturation); 56℃, 15 s (annealing); 72℃, 3 min 15 s (extension); 30 cycles; 72℃, 5 min (final extension); 4℃, ∞ (storage).

[0033] Table 1. PCR reaction system (20 μL)

[0034]

[0035] Nucleic acid electrophoresis verification and template digestion of PCR products: Take 2 μL of PCR product and perform agarose gel electrophoresis to verify whether the band size of the PCR product 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℃ for 1 h to remove wild-type recombinant plasmids in the system.

[0036] After the enzyme digestion reaction, the product was purified using a rapid PCR purification kit. Finally, 5 μL of the purified PCR product was transformed into E. coli DH5α competent cells. Positive clones were then selected for plasmid extraction and DNA sequencing. The successfully sequenced mutant plasmid was introduced into E. coli BL21(DE3) competent cells to construct a recombinant mutant gene for the induction of mutant enzyme expression.

[0037] Example 2 Expression and purification of Thermoclostridium caenicola DAEase wild-type and mutant

[0038] pET22b-thca-dae and the sequence-verified mutant plasmid pET-22b(+)-C258H were transformed into Escherichia coli BL21(DE3) cells. Positive transformants were picked and cultured overnight in LB medium at 37°C and 200 rpm. Then, they were inoculated into LB medium and cultured at 37°C for 3-4 hours until the OD value reached 0.6-0.8. The temperature was then lowered to 30°C, and IPTG was added to a final concentration of 1.0 mM for 6 hours of induction.

[0039] The DAEase wild-type and mutant were purified separately using the following method: The corresponding fermentation broth was centrifuged at 8000 rpm for 20 min at 4 °C, and the bacterial cells were collected. 20 mL of buffer (50 mM Tris, 200 mM NaCl, adjusted to pH 7.5) was added to fully resuspend the bacterial cells. The centrifuge tubes were then placed in an ice bath and placed in an ultrasonic cell disruptor. The ultrasonic disruption conditions were: working time 1 ls, stop time 2 s, for a total of 15 min. The obtained disrupted solution was centrifuged at low temperature and high speed at 8000 rpm for 10 min at 4 °C to obtain the crude enzyme solutions of DAEase wild-type and mutant. These solutions were then filtered through a 0.45 μm microporous membrane for later use.

[0040] To prepare a nickel ion affinity chromatography column, first, flush the column with deionized water (approximately 6–12 column volumes) using a constant flow pump 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 eluent at the bottom of the column matches that of the low-salt buffer pumped into the column (approximately 5 column volumes of buffer are needed), add the obtained crude enzyme solution to the column. First, wash away contaminating proteins 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) until baseline equilibration. Then, elute with an elution buffer containing a high concentration of imidazole (500 mmol / L NaCl, 500 mmol / L imidazole, 50 mM Tris, adjusted to pH 7.0). Collect the eluent with the absorption peak and measure its enzyme activity to obtain the target protein. After purification, the wild-type and mutant C258H of D-allulose 3-epimerase reached electrophoretic purity.

[0041] Example 3: Determination of the optimal pH for wild-type and mutant Thermoclostridium caenicola DAEase

[0042] Enzymatic reactions were performed using MES-NaOH (50 mM, pH buffer range 5.5–6.5), HEPES-NaOH (50 mM, pH buffer range 6.5–8.0), and Tris-HCl buffer (50 mM, pH buffer range 7.5–9.0) as buffer systems. 50 g / L D-fructose was used as the substrate, and 0.5 μmol / L purified enzyme and 1 mmol / L CoCl2 were added. The reactions were carried out at 65 °C for 5 min at different pH conditions, followed by boiling for 10 min for inactivation. After the reaction, the products were centrifuged, filtered through a membrane, diluted to a specific concentration, and detected by HPLC.

[0043] Enzyme activity (U): The amount of enzyme required to catalyze the synthesis of 1 μmol of D-allulose per unit time (min) under standard reaction conditions. The optimal reaction pH is the pH corresponding to the highest enzyme activity. To compare the effect of different reaction pH on recombinant enzyme activity, the enzyme activity at the optimal reaction pH was set as 100% relative enzyme activity, and the relative enzyme activity at other pH values ​​was calculated. Results are shown below. Figure 4 The optimal pH for mutant C258H is the same as that for wild type, which is 7.5.

[0044] Example 4: Determination of the optimal temperature for wild-type and mutant Thermoclostridium caenicola DAEase

[0045] The enzyme reaction was carried out within a temperature range of 40-80℃ (with temperature points set every 5℃), and the system pH was set to 7.5. Except for the reaction temperature, the substrate concentration, enzyme concentration, metal ion concentration, and detection conditions were the same as described in Example 3. The optimal reaction temperature is the temperature corresponding to the highest enzyme activity. To compare the effect of different reaction temperatures on the recombinant enzyme activity, the enzyme activity at the optimal reaction temperature was set as 100% relative enzyme activity to calculate the relative enzyme activity at other temperatures. The results are shown below. Figure 5 The optimal temperature for mutant C258H is the same as that for wild type, which is 65℃.

[0046] Example 5: Enzyme activity assay of Thermoclostridium caenicola DAEase wild-type and mutant

[0047] Using 50 g / L D-fructose as a substrate, 0.5 μmol / L purified enzyme and 1 mmol / L CoCl2 were added. The enzyme reaction was carried out at 65 °C and pH 7.5 for 5 min, followed by boiling for 10 min to inactivate the enzyme. After the reaction, the product was centrifuged, filtered through a membrane, diluted to a certain concentration, and then detected by HPLC.

[0048] Enzyme activity (U): The amount of enzyme required to catalyze the synthesis of 1 μmol of D-allulose per unit time (min) under standard reaction conditions.

[0049] Enzyme activity (U / mg) definition: Under standard reaction conditions, the number of enzyme activity units per unit mass (mg) of enzyme protein.

[0050] Protein quality detection method: (1) Plotting a standard curve: Dilute bovine serum albumin sample according to a certain ratio to obtain six standard solutions with a gradient interval of 0.10 mg / mL-0.60 mg / mL and a concentration of 0.10 mg / mL. Take 1 mL of each standard solution, add 5 mL of solution A and let stand for 10 min, then add solution B and let stand for 30 min. Measure the OD at a wavelength of 650 nm using a UV spectrophotometer. 650 Values, plotted with the protein concentration of the standard solution on the x-axis, and the corresponding OD values ​​measured. 650 Plot a standard curve with the value as the ordinate. (2) Process the DAEase protein sample according to the same steps as in (1) above, and measure the OD. 650 The enzyme protein concentration was calculated by comparing the value with the standard curve.

[0051] Depend on Figure 3 It can be seen that the specific enzyme activity of the wild type is 242.4 U / mg, while the specific enzyme activity of the mutant C258H is increased to 387.8 U / mg, an increase of 60.0%.

[0052] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A D-allulose 3-epimerase mutant with enhanced enzyme activity, characterized in that, The amino acid sequence shown in SEQ ID No.2 is modified so that the amino acid at position 258 is changed from cysteine ​​to histidine.

2. The gene encoding the mutant of claim 1.

3. An expression vector carrying the gene of claim 2.

4. The expression vector according to claim 3, characterized in that, The expression vector is pET-22b(+).

5. Recombinant cells expressing the mutant of claim 1 or the gene of claim 2.

6. The recombinant cell according to claim 5, characterized in that, The recombinant cells are prokaryotic or eukaryotic cells.

7. The recombinant cell according to claim 6, characterized in that, The prokaryotic cells include Escherichia coli.

8. A method for increasing the activity of D-allulose 3-epimerase, characterized in that, The amino acid sequence shown in SEQ ID No.2 is modified so that the amino acid at position 258 is changed from cysteine ​​to histidine.

9. The use of the D-allulose 3-epimerase mutant of claim 1 or the recombinant cells of claim 5 in the preparation of D-allulose.

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