A mutant of D-psicose-3-epimerase, recombinant Escherichia coli expressing the mutant and application thereof

Abstract

The present invention provides a mutant of D-psicose-3-epimerase, a recombinant Escherichia coli expressing the mutant and an application thereof, belonging to the technical field of isomerases, and the amino acid sequence of the mutant of the D-psicose-3-epimerase is shown in SEQ ID NO.10 in the sequence table. The mutant of the D-psicose-3-epimerase of the present invention contains S146T, G177A, and Y205H mutation sites, and the enzyme activity is significantly improved relative to the wild-type D-psicose-3-epimerase. The enzyme activity of the mutant of the D-psicose-3-epimerase of the present invention is 2.04 times that of the wild-type D-psicose-3-epimerase under the same conditions.

Description

Technical Field

[0001] The invention relates to a mutant of D-psicose-3-epimerase, a recombinant Escherichia coli expressing the mutant and application thereof, belonging to the technical field of isomerase. Background Art

[0002] Allulose is a functional rare sugar with 70% of the sweetness of sucrose. Its taste is similar to or close to sucrose, but its calories are much lower than sucrose. Therefore, domestic and foreign experts predict that allulose will become a new generation of healthy and functional sweeteners. In addition to being a sucrose substitute, allulose has unique physiological functions. Allulose has the health care properties of inhibiting blood sugar increase, lowering blood lipids, and inhibiting abdominal fat accumulation. Its potential health care value is gradually being explored.

[0003] However, D-psicose is extremely rare in nature and is only found in some plants (such as wheat and Acanthus plants). Direct extraction leads to resource waste and environmental damage. The chemical synthesis of D-psicose is difficult and has some disadvantages, such as complex purification process, chemical waste pollution, generation of by-products and high price. The biotransformation method has the advantages of good selectivity and mild reaction conditions, fast synthesis speed, strong reaction specificity, environmental friendliness, simple product purification process, and cheap and easy-to-obtain substrates. Therefore, the biosynthesis of D-psicose has become a hot topic of research.

[0004] D-psicose-3-epimerase (DPE enzyme for short) can catalyze the mutual conversion between D-fructose and D-psicose. When DPE enzyme catalyzes the mutual conversion between D-fructose and D-psicose, no other substances need to be added during the reaction. Since it only has one step reaction, there are few by-products after the reaction, which is conducive to reducing production costs and making the subsequent separation of D-psicose more convenient. It is currently the most ideal method for large-scale industrial production of D-psicose.

[0005] However, the DPE enzyme derived from unmodified wild strains has low enzyme activity and catalytic activity. When used for the conversion of D-fructose and D-psicose, the conversion rate of fructose is low. There are certain limitations in its industrial application, which restricts the large-scale industrial production of D-psicose. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a mutant of D-psicose-3-epimerase, a recombinant Escherichia coli expressing the mutant, and applications thereof, to achieve the following objectives of the invention: mutating D-psicose-3-epimerase to obtain a mutant of D-psicose-3-epimerase, and improving the enzymatic activity and catalytic activity of D-psicose-3-epimerase.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A mutant of D-psicose-3-epimerase, wherein the amino acid sequence of the mutant of D-psicose-3-epimerase is shown as SEQ ID NO.10 in the sequence listing.

[0009] Compared with the wild-type D-psicose-3-epimerase, the mutant of the D-psicose-3-epimerase has three amino acid mutation sites, namely: the 146th serine mutates to the threonine, the 177th glycine mutates to the alanine, and the 205th tyrosine mutates to the histidine.

[0010] The nucleotide sequence of the mutant of D-psicose-3-epimerase is shown in SEQ ID NO.9 in the sequence listing.

[0011] A recombinant Escherichia coli expressing the mutant.

[0012] Application of the mutant of D-psicose-3-epimerase in synthesizing D-psicose.

[0013] Compared with the prior art, the present invention achieves the following beneficial effects:

[0014] The mutant of D-psicose-3-epimerase of the present invention contains S146T, G177A, and Y205H mutation sites, and the enzyme activity is significantly improved compared with the wild-type D-psicose-3-epimerase. The enzyme activity of the mutant of D-psicose-3-epimerase of the present invention is 2.04 times that of the wild-type D-psicose-3-epimerase under the same conditions.

[0015] The mutant of D-psicose-3-epimerase of the present invention improves the conversion rate of fructose and reduces the amount of enzyme used when used for the conversion between fructose and psicose, and has important industrial application value.

[0016] The nucleotide sequence of the mutant of the D-psicose-3-epimerase of the present invention has a high expression level and stable expression in Escherichia coli, and can significantly reduce the cost of preparing the DPE enzyme. DETAILED DESCRIPTION

[0017] LB liquid culture medium: tryptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, solvent is water, pH value is natural pH, high pressure sterilization at 121°C for 20 min.

[0018] LB liquid medium containing kanamycin: LB liquid medium was supplemented with 50 μg / mL (final concentration) of kanamycin before use.

[0019] LB solid medium: Add 10 g / L agar to LB liquid medium and sterilize at 121°C for 20 min.

[0020] LB solid medium containing kanamycin: 50 μg / mL (final concentration) of kanamycin was added to LB solid medium before use.

[0021] 100 g / L fructose solution: Weigh 100 g of fructose and dissolve it in 50 mM phosphate buffer at pH 7.5, and dilute to 1 L to obtain a 100 g / L fructose solution.

[0022] The wild-type DPE enzyme has a nucleotide sequence as shown in SEQ ID NO.1 in the sequence listing, and an amino acid sequence as shown in SEQ ID NO.2 in the sequence listing.

[0023] Example 1 Construction of a recombinant plasmid containing a DPE enzyme mutant

[0024] The nucleotide sequence of the wild-type DPE enzyme (as shown in SEQ ID NO.1 in the sequence list) was inserted into the pET24a plasmid to obtain the recombinant plasmid pET24a-DPE (commercially synthesized). The whole plasmid PCR technology was used to perform site-directed mutagenesis using the recombinant plasmid pET24a-DPE as a template to obtain recombinant plasmids pET24a-DPE S146T, pET24a-DPEG177A, pET24a -DPE Y205H, and pET24a -DPE S146T / G177A / Y205H containing DPE enzyme mutant genes.

[0025] The DPE enzyme mutant contained in the recombinant plasmid pET24a-DPE S146T has a nucleotide sequence as shown in SEQ ID NO.3 in the sequence list, and an amino acid sequence as shown in SEQ ID NO.4 in the sequence list, wherein the serine (S) at position 146 is mutated into threonine (T);

[0026] The DPE enzyme mutant contained in the recombinant plasmid pET24a-DPE G177A has a nucleotide sequence as shown in SEQ ID NO.5 in the sequence list, and an amino acid sequence as shown in SEQ ID NO.6 in the sequence list, wherein the glycine (G) at position 177 is mutated into alanine (A);

[0027] The DPE enzyme mutant contained in the recombinant plasmid pET24a-DPE Y205H has a nucleotide sequence as shown in SEQ ID NO.7 in the sequence list, an amino acid sequence as shown in SEQ ID NO.8 in the sequence list, and the tyrosine (Y) at position 205 is mutated into histidine (H);

[0028] The DPE enzyme mutant contained in the recombinant plasmid pET24a-DPE S146T / G177A / Y205H has a nucleotide sequence as shown in SEQ ID NO.9 in the sequence list, and an amino acid sequence as shown in SEQ ID NO.10 in the sequence list, wherein the serine (S) at position 146 is mutated into threonine (T), the glycine (G) at position 177 is mutated into alanine (A), and the tyrosine (Y) at position 205 is mutated into histidine (H).

[0029] The specific mutation process is as follows:

[0030] (1) Mutation primers

[0031] S146T-F: Its nucleotide sequence is shown in SEQ ID NO.11 in the sequence listing;

[0032] S146T-R: Its nucleotide sequence is shown in SEQ ID NO.12 in the sequence listing;

[0033] G177A-F: The nucleotide sequence is shown in SEQ ID NO.13 in the sequence listing;

[0034] G177A-R: Its nucleotide sequence is shown in SEQ ID NO.14 in the sequence listing;

[0035] Y205H-F: Its nucleotide sequence is shown in SEQ ID NO.15 in the sequence listing;

[0036] Y205H-R: Its nucleotide sequence is shown as SEQ ID NO.16 in the sequence listing.

[0037] (2) PCR system

[0038] Forward primer: 1 μL (the concentration of the forward primer is 120 ng / μL)

[0039] Reverse primer: 1 μL (the concentration of reverse primer is 120 ng / μL)

[0040] Template DNA: 0.5 μL (the concentration of template DNA is 200 ng / μL)

[0041] High-fidelity PCR enzyme (brand: TaKaRa, model: R045A): 25 μL

[0042] Sterile water: 22.5 μL (make up to 50 μL)

[0043] The high-fidelity PCR enzyme was purchased from the market.

[0044] (3) PCR amplification

[0045] The conditions were as follows: pre-deformation at 95°C for 10 min; then entering the following cycle: denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, 30 cycles; extension at 72°C for another 5 min; and insulation at 0°C.

[0046] (4) Verification

[0047] The PCR product was detected by agarose gel electrophoresis, and an obvious bright band appeared at the target band. The product was recovered using a gel recovery kit, and the recovered mutant fragment was constructed into the vector pET24a and transferred into Escherichia coli DH5α competent cells. The transformants were selected for sequencing, and the sequencing verification results showed that no random mutations occurred except the required mutation sites, indicating that the recombinant plasmids pET24a-DPE S146T, pET24a-DPE G177A, pET24a-DPE Y205H, and pET24a-DPES146T / G177A / Y205H were successfully constructed. Each recombinant plasmid was extracted and the concentration was adjusted to 170 ng / μL.

[0048] pET24a-DPE S146T / G177A / Y205H was obtained by sequentially mutating three sites.

[0049] Example 2 Construction of recombinant Escherichia coli containing DPE enzyme mutants

[0050] To 100 μL of E. coli BL21 (DE3) competent cell suspension melted in an ice bath, 2 μL of the recombinant plasmid obtained in Example 1 was added respectively, the mixture was allowed to stand on ice for 30 min, heat-shocked at 42°C for 90 s, and quickly cooled on ice for 3 min. 600 μL of sterile LB liquid culture medium was added, and the mixture was shaken and cultured on a shaker at 37°C and 120 r / min for 1 h. Then 100 μL of the bacterial solution was spread on an LB solid culture medium plate containing kanamycin. After the bacterial solution was completely absorbed by the culture medium, the plate was inverted and cultured in a 37°C constant temperature incubator for 16 h. Clones were randomly picked from the colonies grown on the plates and the plasmids were extracted for identification by agarose gel electrophoresis to obtain genetically engineered bacteria containing DPE enzyme mutant genes: E. coli BL21 (DE3)-pET24a-DPES146T, E. coli BL21 (DE3)-pET24a-DPE G177A, E. coli BL21 (DE3)-pET24a-DPE Y205H, and E. coli BL21 (DE3)-pET24a-DPE S146T / G177A / Y205H.

[0051] The recombinant plasmid pET24a-DPE was constructed into Escherichia coli according to the above method to obtain the wild-type genetically engineered bacteria E. coli BL21 (DE3)-pET24a-DPE.

[0052] Example 3 Inducible expression of recombinant Escherichia coli containing DPE enzyme mutants

[0053] The genetically engineered bacteria prepared in Example 2 were inoculated into 5 mL of LB liquid culture medium containing kanamycin at an inoculum of 1‰ (v / v), and cultured at 37°C and 200rpm for 16 hours to prepare seed liquid. The seed liquid was inoculated into LB liquid culture medium containing kanamycin at an inoculum of 1% (v / v), and cultured at 37°C and 200rpm until OD600 reached 0.6, and IPTG inducer (the final concentration of the inducer was 0.1mmol / L) was added, and cultured at 25°C and 160rpm for 20 hours to obtain fermentation liquid.

[0054] The prepared fermentation broth was centrifuged at 8000 rpm and 4°C for 10 min to obtain bacterial cells, and an equal volume of PBS buffer (pH=7.5) was added to the fermentation broth to resuspend the bacterial cells. The resuspended bacterial cells were treated with an ultrasonic disruptor in an ice bath, and then the obtained disrupted liquid was centrifuged (8000 rpm, 4°C) for 20 min, and the supernatant was taken as the crude enzyme solution.

[0055] Example 4 Determination of enzyme activity of DPE enzyme mutants

[0056] Enzyme activity detection method:

[0057] 1 mL of Co was added to 8 mL of fructose solution (100 g / L, 50 mM PBS buffer as solvent, pH 7.5). 2+ The mother solution (1 mmol / L) was preheated in a water bath at 55°C for 10 min.

[0058] The crude enzyme solution obtained in Example 3 was diluted 40 times (volume), 1 mL of the diluted crude enzyme solution was added to the above mixed solution, reacted at 55° C. for 10 min, and boiled for 10 min to terminate the reaction; after being diluted 10 times with ultrapure water, the solution was filtered through a 0.22 μm needle filter and the allulose content was detected by HPLC.

[0059] The Co 2+ The mother liquor is an aqueous solution of cobalt chloride hexahydrate;

[0060] Definition of enzyme activity unit: Under the conditions of 55°C and pH=7.5, one enzyme activity unit is the amount of 1 μmol of allulose produced per minute per mL of crude enzyme solution, expressed as U / mL.

[0061] The enzyme activities of the crude enzyme solutions obtained by fermentation of various genetically engineered bacteria are shown in Table 1.

[0062] Table 1

[0063]

[0064] As can be seen from the above table, the enzymatic activity of DPE enzyme in the crude enzyme liquid obtained by fermentation of genetically engineered bacteria containing DPE enzyme mutant gene is significantly improved compared with that of wild-type genetically engineered bacteria; compared with genetically engineered bacteria with single-site mutation, the enzymatic activity of DPE enzyme in the crude enzyme liquid obtained by fermentation of genetically engineered bacteria with simultaneous three-site mutation is significantly improved, achieving unexpected technical effects.

[0065] Example 5 Synthesis of D-psicose using crude enzyme solution containing DPE enzyme mutant

[0066] In a 250 mL Erlenmeyer flask, add 100 mL of fructose solution (300 g / L, 50 mM PBS buffer, pH 7.5) and 1 mL of Co 2+ The mother solution (1 mmol / L) was preheated in a water bath at 55°C for 20 min, 1 mL of the crude enzyme solution dilution (the crude enzyme solution of Example 3 was diluted 40 times) was added, mixed well, and placed in a water bath at 55°C for 1 h. The sample was treated with a 0.22 μm needle filter and the D-psicose content was detected by liquid phase.

[0067] The Co 2+ The mother liquor is an aqueous solution of cobalt chloride hexahydrate.

[0068] The results showed that the conversion rate of fructose in the reaction with crude enzyme solution of E.coli BL21 (DE3)-pET24a-DPE S146T / G177A / Y205H was 29.4%, while the conversion rate of fructose in the reaction with crude enzyme solution of wild-type genetically engineered bacteria E.coli BL21 (DE3)-pET24a-DPE was only 14.73%. This shows that in the reaction of catalyzing the conversion of fructose to psicose, the catalytic activity of the DPE enzyme with three mutations is significantly higher than that of the wild-type DPE enzyme, which can significantly improve the conversion rate of fructose.

Claims

1. A mutant of D-psicose-3-epimerase, characterized in that: The amino acid sequence of the mutant of D-psicose-3-epimerase is shown in SEQ ID NO.10 in the sequence listing.

2. A recombinant Escherichia coli expressing the mutant according to claim 1.

3. Use of the mutant of D-psicose-3-epimerase according to claim 1 in synthesizing D-psicose.

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