Epimerase mutant 214d and use thereof

Abstract

This invention relates to an epimerase mutant 214D and its applications, belonging to the field of enzyme engineering technology. This invention provides a D-allulose-3-epimerase mutant, which involves mutating asparagine at position 214 of the D-allulose-3-epimerase DPEase derived from *Agrobacterium tumefaciens* to aspartic acid. Compared with the original enzyme DPEase, the D-allulose-3-epimerase mutant DPEase-214D provided by this invention shows a 2.5% increase in D-allulose conversion rate, significantly improving conversion capacity. The D-allulose-3-epimerase mutant DPEase-214D provided by this invention broadens the application range of D-allulose-3-epimerase DPEase, and has broad application prospects in the food, pharmaceutical, and other fields.

Description

Technical Field

[0001] This invention relates to an epimerase mutant 214D and its applications, belonging to the field of enzyme engineering technology. Background Technology

[0002] D-allulose (D-psicose) is a rare hexose found in nature. It is a low-energy, indigestible sugar substitute with blood sugar-lowering effects and is currently used as a sweetener by many pharmaceutical companies abroad. Furthermore, D-allulose can improve the water-holding capacity of food through the Maillard reaction, making it widely used in the food industry. D-allulose also has the following properties: (1) lowering blood sugar, making it suitable as an adjunct therapy, dietary supplement, and sweetener for type II diabetics; (2) lowering blood lipids, reducing lipase activity, and inhibiting intra-abdominal fat accumulation; (3) antioxidant activity, possessing strong reactive oxygen species (ROS) scavenging ability and glutathione reduction ability; and (4) neuroprotective and anti-inflammatory effects.

[0003] D-Allulose is the C-3 epimer of fructose, and D-allulose-3-epimerase (DAEase) is a class of enzymes that can epimerize fructose at the C-3 position to produce D-allulose. Currently, there are approximately 20 different DAEases. Most current research focuses on specific enzyme properties (such as heat resistance or alkali resistance), while studies on conversion rates are scarce.

[0004] Chinese patent document CN 106148311 A (application number: 201610818847.X) discloses a mutant of D-allulose-3-epimerase, using D-allulose-3-epimerase derived from Burkholderia sp. MR1 as a template, and containing at least one of the following mutations: amino acid residue 86 changes from glycine to aspartic acid, amino acid residue 164 changes from aspartic acid to glutamic acid, and amino acid residue 262 changes from tryptophan to serine. The D-allulose-3-epimerase mutant provided by this invention contains one or more mutation sites from G86D, D164E, and W262S, resulting in significantly improved catalytic activity, more than 1.4 times that of the wild type. This greatly reduces the amount of enzyme used in the synthesis of D-allulose and has significant industrial application value.

[0005] Chinese patent document CN 108239633 A (application number: 201611217802.3) discloses a mutant of D-allulose-3-epimerase with enhanced catalytic activity. The wild-type D-allulose-3-epimerase is derived from Paenibacillus senegalensis, and its mutation includes mutations at at least three sites: aspartic acid at position 73 (D), tyrosine at position 111 (Y), asparagine at position 188 (N), and glycine at position 250 (G). The mutant D-allulose-3-epimerase of this invention exhibits very high catalytic activity. Furthermore, the D-allulose-3-epimerase of this invention also has a high catalytic efficiency (up to 29.6%).

[0006] Chinese patent document CN 110438112 A (application number: 201910757830.1) discloses a mutant of D-allulose-3-epimerase. This D-allulose-3-epimerase is derived from the genus *Synechocystis*. The mutant amino acid mutation site is the 39th position, where I is mutated to A. Optional mutation sites also include: the 158th position, where Q is mutated to S, and the 186th position, where L is mutated to V. The efficiency of D-allulose-3-epimerase catalyzing the production of D-allulose with the above mutation sites is significantly improved.

[0007] Genetic engineering of D-allulose-3-epimerases from different sources to obtain mutants with superior properties remains of great research significance and practical application value. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides an epimerase mutant 214D and its applications. This invention involves performing an NNK mutation on asparagine (N) at position 214 of the D-allulose-3-epimerase amino acid sequence, screening for the optimal mutant, and obtaining the modified D-allulose-3-epimerase mutant 214D, which exhibits significantly improved conversion ability, increasing the conversion rate from 32.9% to 35.4%.

[0009] The technical solution of this invention is as follows:

[0010] A D-allulose-3-epimerase mutant, 214D, is based on the wild-type D-allulose-3-epimerase, with the asparagine (N) at position 214 of the amino acid sequence mutated to aspartic acid (D).

[0011] According to a preferred embodiment of the present invention, the wild-type D-allulose-3-epimerase is derived from Agrobacterium tumefaciens, and its amino acid sequence is shown in SEQ ID NO.1.

[0012] A gene encoding a D-allulose-3-epimerase mutant 214D is provided.

[0013] In this invention, the encoding gene of the above-mentioned D-allulose-3-epimerase mutant 214D is obtained by mutation based on the nucleotide sequence and its degenerate sequence shown in SEQ ID NO.2.

[0014] According to a preferred embodiment of the present invention, the nucleotide sequence of the gene encoding the D-allulose-3-epimerase mutant 214D is shown in SEQ ID NO.4.

[0015] A recombinant expression vector containing the encoding gene of the above-mentioned D-allulose-3-epimerase mutant 214D.

[0016] According to a preferred embodiment of the present invention, the vector plasmid of the recombinant expression vector is pET-20b(+).

[0017] A genetically engineered bacterium containing the encoding gene of the above-mentioned recombinant expression vector or the above-mentioned D-allulose-3-epimerase mutant 214D.

[0018] According to a preferred embodiment of the present invention, the host bacterium of the genetically engineered bacteria is Escherichia coli.

[0019] Application of the above-mentioned genetically engineered bacteria in the preparation of D-allulose-3-epimerase mutant 214D.

[0020] Application of the above-mentioned D-allulose-3-epimerase mutant 214D or the above-mentioned genetically engineered bacteria in the production of D-allulose.

[0021] Beneficial effects:

[0022] This invention provides a D-allulose-3-epimerase mutant, specifically, by mutating asparagine (N) at position 214 of the D-allulose-3-epimerase derived from *Agrobacterium tumefaciens* to aspartic acid (D). Compared with the original enzyme DPEase, the D-allulose-3-epimerase mutant DPEase-214D provided by this invention increases the D-allulose conversion rate by 2.5%, demonstrating a significant improvement in conversion capacity. The D-allulose-3-epimerase mutant DPEase-214D provided by this invention broadens the application range of D-allulose-3-epimerase and has broad application prospects in the food, pharmaceutical, and other fields. Attached Figure Description

[0023] Figure 1 Bar chart showing the D-allulose conversion rate of the D-allulose-3-epimerase mutant 214NNK.

[0024] Figure 2 The graph shows the D-allulose conversion rates of the original enzyme DPEase and the mutant DPEase-214D.

[0025] Figure 3 The figure shows the temperature stability results of the original enzyme DPEase and the mutant DPEase-214D.

[0026] Figure 4 The graph shows the D-allulose conversion rates of the original enzyme DPEase and the mutant DPEase-244NNK.

[0027] Figure 5 The graph shows the D-allulose conversion rate of the original enzyme DPEase and the mutant DPEase-150NNK. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto. All aspects not described in detail in the embodiments are based on existing technology in the art.

[0029] The source of the D-allulose-3-epimerase primitive enzyme in the examples:

[0030] D-allulose-3-epimerase DPEase is derived from Agrobacterium tumefaciens, with the NCBI accession number WP_010974125. The amino acid sequence of DPEase is SEQ ID NO.1, and the nucleotide sequence of its encoding gene is SEQ ID NO.2.

[0031] Example 1: Construction of recombinant plasmids containing mutant genes

[0032] Using the recombinant plasmid pET-20b(+)-DPEase (synthesized by GenScript) containing the gene encoding D-allulose-3-epimerase DPEase as a template, reverse PCR amplification was performed, and the codon corresponding to amino acid (N) at position 214 of D-allulose-3-epimerase DPEase was mutated by NNK.

[0033] The primer sequences for the reverse PCR amplification are as follows:

[0034] DPEase-214-F1:5'-TTTCACACCGGCGAAGCGNNKCGCCGTTGTTCCGGGTAAAGGTCG TA-3';

[0035] DPEase-214-R1:5'-CGCTTCGCCGGTGTGAAAGTGACCCAGCAGTGGGCCCGC-3'.

[0036] The reaction system for the reverse PCR amplification is shown in Table 1:

[0037] Table 1. Reaction system for reverse PCR amplification

[0038]

[0039] The reverse PCR amplification procedure is as follows:

[0040] Pre-denaturation at 95℃ for 5 min; denaturation at 94℃ for 30 sec, annealing at 55℃ for 30 sec, extension at 72℃ for 30 sec, 30 cycles; extension at 72℃ for 10 min, storage at 4℃.

[0041] The PCR product was examined by agarose gel electrophoresis. The length was approximately 4500 bp. The DNA was recovered using the SanPrep DNA Gel Extraction Kit (Shanghai Sangon Biotech). The recovered product was stored at -20°C for later use.

[0042] The obtained gel recovery product was purified and digested. Since the pET-20b(+) plasmid was derived from E. coli with Dam methylation modification function, it could be cleaved by DpnI enzyme. However, the plasmid containing the mutation site did not have methylation and therefore could not be cleaved by DpnI enzyme. After DpnI enzyme digestion, the recombinant plasmid library pET-20b(+)-DPEase-214NNK containing the mutant gene was obtained.

[0043] The digestion system is shown in Table 2:

[0044] Table 2. Digestion system for recycled adhesive products

[0045]

[0046] The digestion procedure is as follows:

[0047] React at 37℃ for 120 min, at 75℃ for 15 min, and store at 4℃.

[0048] The digestion product was examined by agarose gel electrophoresis. The product was approximately 4500 bp in length. The DNA was recovered using the SanPrep column DNA gel recovery kit (Shanghai Sangon Biotech). The recovered product was stored at -20°C for later use.

[0049] Example 2: Preparation of competent Escherichia coli cells

[0050] (i) Pick a single colony of Escherichia coli BL21(DE3) and inoculate it into LB liquid medium, and incubate overnight at 37°C at 200 r / min;

[0051] (ii) Transfer 0.1 mL of the cultured bacterial solution to 10 mL of LB liquid medium and incubate at 200 rpm and 37°C until OD reaches 100%. 600 Reaching 0.6–0.8;

[0052] (iii) Take 1 mL of OD 600 Transfer the bacterial culture to a concentration of 0.6–0.8 to a 1.5 mL sterile centrifuge tube, centrifuge at 12000 rpm for 2 min, and thoroughly remove the supernatant.

[0053] (iv) Add 100 μL of ice-cold SSCS (one-step rapid preparation kit for competent cells, product of Shanghai Sangon Biotech Co., Ltd.), and gently suspend the bacterial cells to prepare E. coli competent cells;

[0054] (v) Aliquot the prepared E. coli competent cells into 100 μL tubes and store at -80°C for later use.

[0055] Example 3: Preparation of recombinant Escherichia coli BL21(DE3)

[0056] First, the concentration of plasmid pET-20b(+)-DPEase-214NNK was determined using a nucleic acid micro-spectrophotometer. After reaching a concentration of 300 μg / mL, it was transformed into Escherichia coli BL21(DE3) competent cells prepared in Example 2 using a chemical transformation method. The resulting cells were revived and cultured at 37°C for 1 h using resuscitation medium. Then, 100 μL of the cells were spread on LB solid medium containing 50 μg / mL ampicillin and cultured overnight at 37°C. Positive recombinant colonies with ampicillin resistance were screened.

[0057] The components of the resuscitation medium per liter are as follows:

[0058] 10g peptone, 5g yeast powder, 10g sodium chloride, and the remainder water.

[0059] Culture and identification of positive recombinant bacteria:

[0060] The above-mentioned positive recombinant colonies were picked and inoculated into liquid LB medium containing 50 μg / mL ampicillin, and cultured overnight at 37°C. After culture, they were sent to Shanghai Bioengineering Co., Ltd. for sequencing verification. After correct sequencing, recombinant Escherichia coli was obtained.

[0061] After sequencing verification, the successfully mutated strains were preserved, including strains with mutations at position 214 to 19 other amino acids. Subsequent transformation rate screening was conducted to obtain the optimal mutant.

[0062] Example 4: Mutant Transformation Rate Test

[0063] The recombinant Escherichia coli (19 species) containing plasmid pET-20b(+)-DPEase-214NNK prepared in Example 3 were inoculated into 5 mL of liquid LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and the remainder water) and cultured at 200 rpm and 37 °C until OD. 600 The concentration was 0.7, and the inoculum was added to the fermentation medium (20 g / L peptone, 20 g / L yeast extract, 20 g / L dextrin, and the remainder water) at an inoculation ratio of 2% (v / v), and induced at 30°C for 48 h. After induction, the fermentation broth was centrifuged, and the precipitate was resuspended in an equal volume of PBS buffer. After ultrasonic disruption, a crude enzyme solution was obtained. The crude enzyme solution was added to a fructose solution with a final concentration of 300 g / L at pH 8.0, and reacted in a water bath at 60°C for 20 h. The concentrations of D-allulose and fructose were detected by high performance liquid chromatography, and the optimal mutant was screened based on the D-allulose conversion rate.

[0064] See results Figure 1 Compared to the original enzyme DPEase (with a maximum conversion rate of 32.9% at 60℃ and pH 8.0), the mutant D-allulose-3-epimerase DPEase-214D exhibited the highest conversion rate, reaching 35.4%, a 2.5% improvement over the original enzyme DPEase. Meanwhile, the D-allulose-3-epimerase mutants DPEase-214C (22.9%), DPEase-214M (28.9%), DPEase-214S (25.6%), and DPEase-214E (22.9%) showed conversion rates of 0-5%, all lower than the conversion rate of the original enzyme DPEase.

[0065] The amino acid sequence of the D-allulose-3-epimerase mutant DPEase-214D screened above is shown in SEQ ID NO.3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4.

[0066] Example 5: Determination of the optimal temperature and temperature stability of mutant DPEase-214D

[0067] (1) Determination of the optimal reaction temperature

[0068] Take 5 mL of the crude DPEase-214D enzyme solution prepared in Example 4 and add it to 5 mL of a 200 g / L fructose solution (final fructose concentration 100 g / L, pH adjusted to 8 using KOH). Calibrate the water bath at 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C using a thermometer, with three parallel samples at each temperature. After reacting in the water bath for 1 h, immediately boil in a water bath for 10 min to ensure enzyme inactivation and prevent further reaction. Detect the D-allulose and fructose concentrations by high-performance liquid chromatography (HPLC) and calculate the D-allulose conversion rate. Use the conversion rate at the optimal reaction temperature as the relative conversion rate of 100%.

[0069] See results Figure 2 The optimal reaction temperature for both the original enzyme DPEase and the mutant DPEase-214D is 60℃.

[0070] (2) Temperature stability determination

[0071] Five mL of the crude DPEase-214D enzyme solution prepared in Example 4 was incubated at 50°C, 60°C, 70°C, and 80°C, with 21 sample tubes placed simultaneously at each temperature. At 20 min, 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min, three sample tubes were removed from each incubation at different temperatures. Each tube was then added to 5 mL of a 200 g / L fructose solution (final fructose concentration 100 g / L, pH adjusted to 8 with KOH). The mixture was reacted in a 60°C water bath for 1 h, followed immediately by a 10 min boiling water bath to ensure enzyme inactivation and prevent further reaction. The concentrations of D-allulose and fructose were determined by high-performance liquid chromatography (HPLC), and residual enzyme activity was calculated. One enzyme activity unit was defined as the amount of D-allulose-3-epimerase that generates 1 μmol of D-allulose per minute at 60°C and pH 8. The residual enzyme activity was calculated by taking the enzyme activity obtained by reacting crude enzyme solution directly with fructose solution without incubation as 100% of the relative enzyme activity.

[0072] See results Figure 3 The temperature stability of the original enzyme DPEase and the mutant DPEase-214D is essentially the same.

[0073] Comparative Example 1

[0074] An NNK mutation was performed on the codon corresponding to amino acid E at position 244 of D-allulose-3-epimerase (DPEase):

[0075] Using the recombinant plasmid pET-20b(+)-DPEase as a template, reverse PCR amplification was performed, and the recombinant plasmid library pET-20b(+)-DPEase-244NNK containing the mutant gene was prepared according to the method in Example 1.

[0076] The primer sequences for the reverse PCR amplification are as follows:

[0077] F1-1:5'-CGCAGTGATTATGNNKCCGTTTGTGAAGACTGGTGG-3';

[0078] R1-1:5'-TAGTTAATATCACGCAGGGCCAGACCG-3'.

[0079] Following the method in Example 3, recombinant Escherichia coli containing plasmid pET-20b(+)-DPEase-244NNK was prepared, and following the method in Example 4, the recombinant Escherichia coli was inoculated into 5 mL of liquid LB medium and cultured at 200 rpm and 37°C until OD500. 600 The inoculum was 0.7, and the inoculum was added to the fermentation medium at a 2% (v / v) inoculation ratio. Induction was performed at 30℃ for 48 h. After induction, the fermentation broth was centrifuged, and the precipitate was resuspended in an equal volume of PBS buffer. The precipitate was then sonicated to obtain a crude enzyme solution. This crude enzyme solution was added to a fructose solution at pH 8.0 with a final concentration of 300 g / L. The reaction was carried out in a water bath at 60℃, with samples taken every 4 h for a total of 5 times. The concentrations of D-allulose and fructose were determined by high-performance liquid chromatography (HPLC). The optimal mutant was screened based on the D-allulose conversion rate.

[0080] See results Figure 4 Compared with the original enzyme DPEase (which achieved a maximum conversion rate of 32.9% at 60℃ and pH 8.0), the mutant D-allulose-3-epimerase DPEase-244NNK achieved a maximum conversion rate of 20%, which is lower than that of the original enzyme DPEase.

[0081] Comparative Example 2

[0082] An NNK mutation was performed on the codon corresponding to amino acid E at position 150 of D-allulose-3-epimerase DPEase:

[0083] Using the recombinant plasmid pET-20b(+)-DPEase as a template, reverse PCR amplification was performed, and the recombinant plasmid library pET-20b(+)-DPEase-150NNK containing the mutant gene was prepared according to the method in Example 1.

[0084] The primer sequences for the reverse PCR amplification are as follows:

[0085] F1-2:5'-CAACCTGTGTATTNNKGTGCTCAATCGCTTCGAAAA-3';

[0086] R1-2:5'-AGATCATGGCGAAATCCG-3'.

[0087] Following the method in Example 3, recombinant Escherichia coli containing plasmid pET-20b(+)-DPEase-150NNK was prepared, and following the method in Example 4, the recombinant Escherichia coli was inoculated into 5 mL of liquid LB medium and cultured at 200 rpm and 37°C until OD500. 600 The inoculum was 0.7, and the inoculum was added to the fermentation medium at a 2% (v / v) inoculation ratio. Induction was performed at 30℃ for 48 h. After induction, the fermentation broth was centrifuged, and the precipitate was resuspended in an equal volume of PBS buffer. The precipitate was then sonicated to obtain a crude enzyme solution. This crude enzyme solution was added to a fructose solution at pH 8.0 with a final concentration of 300 g / L. The reaction was carried out in a water bath at 60℃, with samples taken every 4 h for a total of 5 times. The concentrations of D-allulose and fructose were determined by high-performance liquid chromatography (HPLC). The optimal mutant was screened based on the D-allulose conversion rate.

[0088] See results Figure 5 Compared with the original enzyme DPEase (which achieved a maximum conversion rate of 32.9% at 60℃ and pH 8.0), the mutant D-allulose-3-epimerase DPEase-150NNK achieved a maximum conversion rate of 25%, which is lower than that of the original enzyme DPEase.

[0089] In summary, the D-allulose-3-epimerase mutant DPEase-244NNK in Comparative Example 1 and the D-allulose-3-epimerase mutant DPEase-150NNK in Comparative Example 2 were obtained by mutating the 244th and 150th amino acids of the D-allulose-3-epimerase DPEase, respectively, into NNK. However, the conversion rates of D-allulose in the resulting mutants were lower than those of the original enzyme DPEase. The D-allulose-3-epimerase mutant DPEase-214D protected by this invention is obtained by mutating the 214th amino acid of the original enzyme DPEase, changing the original asparagine (N) to aspartic acid (D). Compared with the original enzyme DPEase, the D-allulose-3-epimerase mutant DPEase-214D increased the conversion rate of D-allulose by 2.5%.

Claims

1. A D-allulose-3-epimerase mutant 214D, characterized in that, It is based on wild-type D-allulose-3-epimerase, by mutating asparagine at position 214 of the amino acid sequence to aspartic acid; The wild-type D-allulose-3-epimerase was derived from Agrobacterium tumefaciens (… Agrobacterium tumefaciens Its amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding a D-allulose-3-epimerase mutant 214D, characterized in that, It encodes the D-allulose-3-epimerase mutant 214D as described in claim 1.

3. The encoding gene as described in claim 2, characterized in that, The encoding gene was obtained by mutation based on the nucleotide sequence shown in SEQ ID NO.

2.

4. The encoding gene as described in claim 3, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

4.

5. A recombinant expression vector containing the encoding gene of the D-allulose-3-epimerase mutant 214D as described in claim 2.

6. The recombinant expression vector as described in claim 5, characterized in that, The recombinant expression vector uses pET-20b(+) as its vector plasmid.

7. A genetically engineered bacterium containing the encoding gene of the recombinant expression vector of claim 5 or the D-allulose-3-epimerase mutant 214D of claim 2.

8. The genetically engineered bacteria as described in claim 7, characterized in that, The host bacterium of the genetically engineered bacteria is Escherichia coli.

9. The use of the genetically engineered bacteria according to claim 7 in the preparation of D-allulose-3-epimerase mutant 214D.

10. The use of the D-allulose-3-epimerase mutant 214D according to claim 1 or the genetically engineered bacteria according to claim 7 in the production of D-allulose.

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