Sweet protein mutants, their preparation methods and applications

Abstract

This invention provides a sweet protein mutant, its preparation method, and its application. The mutant is obtained by mutating the amino acid sequence of the sweet protein shown in SEQ ID NO.1; the type of amino acid mutation includes at least one of K5D, V7R, D29N, or E53R. By introducing mutations into the amino acid sequence of the sweet protein, this invention significantly improves the sweetness of the mutant compared to the sweet protein corresponding to the original sequence.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a sweet protein mutant, its preparation method, and its application. Background Technology

[0002] The World Health Organization (WHO) considers excessive sugar consumption to be one of the causes of obesity, diabetes, and other diseases. According to the WHO's 2019 standards, the recommended daily sugar intake for adults is 25g. With changing lifestyles, the sugar substitute industry has gradually come into focus, and suppliers specializing in "sugar-free products" have emerged.

[0003] As a sugar substitute, sweeteners have attracted considerable attention. In recent years, some companies have turned their attention to sweet proteins, which are high in sweetness, have a pure taste, are all-natural, and have no side effects, making them a promising candidate for the future of the high-intensity sweetener industry. Among the many sweet proteins, brazzein is considered the most promising because it tastes very much like sugar, has no off-flavor, and maintains its structure over a wide temperature range and at different pH values. Brazzein is isolated from the fruit of the West African tropical plant Pentadiplandra brazzeana Baillon, and its sweetness is 500-2000 times that of an equal mass of sucrose. Compared with other sweet proteins, brazzein has the smallest molecular weight, the best water solubility, and its aqueous solution retains its sweetness even after heat treatment at 80°C for 4 hours, exhibiting good thermal and pH stability.

[0004] However, the yield of sweet proteins from natural plant sources is low and cannot meet market demand. In addition, sweet proteins often fail to fold correctly or easily form inclusion bodies and have excessively low expression levels during heterologous recombination expression, which limits their commercial production and application. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a mutant of a sweet protein, its preparation method, and its applications. This invention employs genetic engineering techniques to artificially and directionally modify the sweet protein gene, significantly increasing the sweetness of the sweet protein.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] On one hand, the present invention provides a saccharin mutant, which is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 of the saccharin; the type of amino acid mutation includes at least one of K5D, V7R, D29N or E53R.

[0008] In this invention, by introducing mutations into the amino acid sequence of a sweet protein, the relative sweetness of the mutant is significantly increased compared to the sweet protein corresponding to the original sequence.

[0009] Preferably, the type of amino acid mutation is K5D, V7R, D29N, E53R, K5D+V7R, D29N+E53R or K5D+V7R+D29N+E53R.

[0010] In this invention, the amino acid sequence shown in SEQ ID NO.1 is as follows:

[0011] QDKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQC ICDYCEY.

[0012] In this invention, the unmutated sweet protein (i.e., the original sequence) includes the nucleic acid sequence shown in SEQ ID NO.2.

[0013] SEQ ID NO.2:

[0014] CAGGACAAGTGCAAGAAGGTCTACGAGAACTACCCAGTTTCCAAGTGCCAGTTGGCTAACCAGTGTAACTACGACTGCAAGTTGGACAAGCACGCTAGATCTGGTGAGTGTTTCTACGACGAGAAGAGAAAACCTGCAGTGCATCTGTGACTACTGCGAGTACTAA.

[0015] In this invention, the unmutated sweet protein is derived from the West African tropical plant Pentadiplandra brazzeana Baillon.

[0016] Secondly, the present invention provides a nucleic acid molecule that encodes the sweet protein mutant described in the first aspect.

[0017] Thirdly, the present invention provides an expression vector containing at least one copy of the nucleic acid molecule described in the second aspect.

[0018] Fourthly, the present invention provides a sweet protein mutant transformant, wherein the sweet protein mutant transformant is a genetically engineered strain expressing the sweet protein mutant described in the first aspect.

[0019] Fifthly, the present invention provides a method for preparing the sweet protein mutant described in the first aspect, the method comprising the following steps:

[0020] An expression vector was constructed and transformed into recipient cells to construct a sweet protein mutant transformant; the sweet protein mutant transformant was cultured, and the culture was collected to obtain the sweet protein mutant.

[0021] Preferably, the method for preparing the sweet protein mutant specifically includes the following steps:

[0022] A sweet protein nucleic acid fragment derived from the West African tropical plant Pentadisplandra brazzeana Baillon was synthesized using the whole genome, as shown in SEQ ID NO.2. The fragment was then digested and recombined into an expression vector to obtain a recombinant plasmid. Using the recombinant plasmid as a template, site-directed mutagenesis was performed, and the mutant was transformed into host cells for culture to obtain the sweet protein mutant.

[0023] Preferably, the host cell is Pichia pastoris X-33.

[0024] In a sixth aspect, the present invention provides an application of the sweet protein mutant described in the first aspect in the fermentation culture for the synthesis of sweet proteins.

[0025] In a seventh aspect, the present invention provides a method for purifying the sweet protein mutant described in the first aspect, the purification method comprising the following steps:

[0026] After culturing the transformants of the sweet protein mutant, the culture was collected, and impurities were removed. The mixture was then filtered, and the filtered solution was subjected to cation exchange resin to adsorb the sweet protein mutant. The mixture was eluted, and the eluent was collected. After nanofiltration to remove salt and concentration, the eluent was lyophilized to obtain the purified sweet protein mutant.

[0027] Preferably, the separation and removal of impurity proteins is performed using a centrifugal separation method.

[0028] Preferably, the filtration is performed through an ultrafiltration membrane of 0.5 to 10 kDa (e.g., 0.5 kDa, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa or 10 kDa).

[0029] Preferably, the elution is performed using an aqueous NaCl solution.

[0030] Preferably, the NaCl concentration in the NaCl aqueous solution is 1-2M, such as 1M, 1.3M, 1.5M, 1.8M or 2M, and the pH value of the solution is 3.0-7.0, such as 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 or 7.0.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] In this invention, by introducing mutations into the amino acid sequence of the sweet protein, its sweetness is significantly improved. Compared with the sweet protein corresponding to the original sequence, the sweetness of the mutant is significantly improved, and its sweetness is 1.6-6 times that of the sweet protein corresponding to the original sequence. Detailed Implementation

[0033] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0034] Example 1: Construction of the original Brazzein plasmid

[0035] The Brazzein nucleic acid fragment (sequence shown in SEQ ID NO:2) from the West African tropical plant Pentadiplandra brazzeana Baillon was synthesized in its entirety by Sangon Biotech (Shanghai) Co., Ltd. After digestion with restriction endonucleases XhoⅠ and NotⅠ (purchased from New England Biolabs, and operated according to the instructions), it was recombined into the vector pPICZαA to obtain the original plasmid Brazzein-pPICZαA.

[0036] Example 2 Construction of Brazzein mutant

[0037] Using forward / reverse K5D, V7R, D29N, and E53R mutation primers, respectively, and with the original plasmid Brazzein-pPICZαA as a template, site-directed mutagenesis was performed at the above sites (for specific procedures, refer to the Fast Mutagenesis System) to obtain Brazzein mutants.

[0038] in:

[0039] The lysine (K) at position 5 is mutated to aspartic acid (D).

[0040] The forward primer K5D-F has the sequence shown in SEQ ID NO.3:

[0041] 5'GAAAAGACAGGACAAGTGCGACAAGGTCTACGAGAACTAC 3'

[0042] The reverse primer K5D-R has the sequence shown in SEQ ID NO.4:

[0043] 5'GTAGTTCTCGTAGACCTTGTCGCACTTGTCCTGTCTTTTC 3'

[0044] The valine (V) at position 7 is mutated to arginine (R).

[0045] The forward primer V7R-F has the sequence shown in SEQ ID NO.5:

[0046] 5'GACAAGTGCAAGAAGCGCTACGAGAACTACC 3'

[0047] The reverse primer V7R-R has the sequence shown in SEQ ID NO.6:

[0048] 5'GGTAGTTCTCGTAGCGCTTCTTGCACTTGTC3'

[0049] The aspartic acid (D) at position 29 is mutated to asparagine (N).

[0050] The forward primer D29N-F has the sequence shown in SEQ ID NO.7:

[0051] 5'CTACGACTGCAAGTTGAACAAGCACGCTAGATC 3'

[0052] The reverse primer D29N-R has the sequence shown in SEQ ID NO.8:

[0053] 5'GATCTAGCGTGCTTGTTCAACTTGCAGTCGTAG 3'

[0054] The glutamic acid (G) at position 53 is mutated to arginine (R).

[0055] The forward primer E53R-F has the sequence shown in SEQ ID NO.9:

[0056] 5'ATCTGTGACTACTGCAGATACTAAGCG 3'

[0057] The reverse primer E53R-R has the sequence shown in SEQ ID NO.10:

[0058] 5'TCTGCAGTAGTCACAGATGCACTGCAG 3'

[0059] The PCR system is as follows: 25 μl of FastPfu Fly PCR SuperMix, 1 μl each of forward and reverse primers (10 μM), 1 μl of template plasmid (50 ng / μl), and ddH2O to a final volume of 50 μl. The PCR amplification program was as follows: 30 cycles of pre-denaturation at 94℃ for 3 minutes, followed by 20 seconds of denaturation at 94℃, 20 seconds of annealing at 55℃, and 2 minutes of extension at 72℃; and a final extension at 72℃ for 10 minutes. The PCR amplification product was digested with DpnI for 2 hours. The digested product was then transformed into E. coli Tran5α, and after single-clone selection and sequencing verification, it was stored.

[0060] Example 3: Expression of the original Brazzein plasmid and its mutants in Pichia pastoris

[0061] The original plasmid Brazzein-pPICZαA from Example 1 and the mutant from Example 2 were linearized using Sac I. The linearized products were purified using a column purification kit and then transformed into Pichia pastoris X-33 via electroporation. The transformed cells were then plated on YPD+Zeocin (100 mg / L) plates. Colonies growing on YPD+Zeocin (100 mg / L) plates were identified as the engineered Pichia pastoris strain. Transformants were picked and inoculated into BMGY medium, cultured at 30°C and 220 rpm for 24 hours with shaking, and then transferred to BMMY medium and cultured at 30°C and 220 rpm with shaking, adding 0.5% methanol every 24 hours. After induced expression for 4 days, the cells were removed by centrifugation, yielding the fermentation supernatant containing the sweet protein.

[0062] Example 4: Purification of Brazzein and its mutant proteins

[0063] a) Pretreatment of fermentation broth

[0064] The supernatant was collected by centrifuging the fermentation broth at 8000g for 30 minutes.

[0065] b) Coarse treatment of fermentation broth

[0066] The pretreated supernatant solution was concentrated and desalted by ultrafiltration through a 1 kDa ultrafiltration membrane. Pure water was continuously added during the concentration process to dilute the salt concentration in the concentrate until its conductivity was <0.1 mS / m.

[0067] c) Purification of Brazzein

[0068] Protein purification resin column: Specification: 10×30cm (1L); Packing material: CM cationic resin (6% cross-linked agarose, purchased from Suzhou Zhongke Senhui Microsphere Technology).

[0069] Equilibration: Equilibrate the resin column with 3 L of pH 4.0 aqueous acetic acid solution at a flow rate of 10 mL / min.

[0070] Feed: Load 5L of the sample after coarse treatment in step b) at a flow rate of 5mL / min.

[0071] Rinsing: Rinse with 3L of pH 4.0 acetic acid aqueous solution at a flow rate of 10mL / min.

[0072] Elution: Wash with 3 L of 1 M NaCl acetic acid solution at a flow rate of 10 mL / min to obtain an eluent containing sweet protein.

[0073] d) Concentration and desalting of the eluent.

[0074] The eluent obtained in the previous step was subjected to nanofiltration (1000Da, Shanghai Langji Membrane Separation Equipment Engineering Co., Ltd.) to remove salt, resulting in a concentrated sample.

[0075] e) Freeze-drying of samples

[0076] The concentrated sample obtained in the previous step was freeze-dried to remove excess moisture, and finally a dried sweet protein sample was obtained.

[0077] Example 5 Sensory evaluation of Brazzein and its mutant proteins

[0078] The sweetness of sweet proteins was determined using a blind test. The control group in the blind test used a 2% sucrose aqueous solution. The samples to be tested for the sweet proteins were prepared into different concentration gradients and randomly numbered. A panel of 10 people tasted the samples and determined which samples in each concentration gradient had the same or similar sweetness to the 2% sucrose aqueous solution, thus calculating the relative sweetness of the samples.

[0079] First, weigh 1.0g of the sweet protein sample and dilute it with distilled water to 100ml to prepare a 1.0% sample solution. Then, measure the 1.0% sample solution and further dilute it with distilled water to prepare test solutions with different dilution factors, such as 0.5%, 0.25%, 0.2%, 0.125%, 0.1%, 0.05%, 0.025%, and 0.0125%. After preparing the sweet protein samples at different concentration gradients, they were randomly numbered. A panel of 10 people tasted the samples and determined which samples at different concentration gradients had the same or similar sweetness to the 2% sucrose aqueous solution, thus calculating the relative sweetness of the sample. The formula is: Relative sweetness = 2% sucrose aqueous solution ÷ (1.0% sample solution × dilution factor).

[0080] The test results are shown in Table 1.

[0081] Table 1

[0082] protein Relative sweetness (the number of times sweetness is relative to 2% sucrose) WT 500 K5D 800 V7R 1200 D29N 1500 E53R 1000 K5D+V7R 1500 D29N+E53R 2000 K5D+V7R+D29N+E53R 3000

[0083] As can be seen above, the sweetness of the sweet protein mutant described in this invention is 1.6-6 times that of the sweet protein corresponding to the original sequence, and its sweetness is significantly improved.

[0084] The applicant declares that this invention illustrates the sweet protein mutant, its preparation method, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

Claims

1. A sweet protein mutant, characterized in that, The mutant was obtained by mutating the amino acid sequence shown in SEQ ID NO.1 of the sweet protein; the type of the amino acid mutation is K5D+ V7R+D29N+ E53R.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the sweet protein mutant of claim 1.

3. An expression carrier, characterized in that, The expression vector contains at least one copy of the nucleic acid molecule of claim 2.

4. A sweet protein mutant transformant, characterized in that, The sweet protein mutant transformant is a genetically engineered strain expressing the sweet protein mutant of claim 1.

5. A method for preparing the sweet protein mutant according to claim 1, characterized in that, The preparation method includes the following steps: An expression vector was constructed and transformed into recipient cells to construct a sweet protein mutant transformant; the sweet protein mutant transformant was cultured, and the culture was collected to obtain the sweet protein mutant.

6. The preparation method according to claim 5, characterized in that, The method for preparing the sweet protein mutant specifically includes the following steps: Using whole-genome synthesis derived from tropical plants in West Africa Pentadipandra brazzeana Baillon The sweet protein nucleic acid fragment, as shown in SEQ ID NO.2, was digested and recombined into an expression vector to obtain a recombinant plasmid. Using the recombinant plasmid as a template, site-directed mutagenesis was performed, and the mutant was transformed into host cells for culture to obtain the sweet protein mutant protein.

7. The application of the sweet protein mutant according to claim 1 in the fermentation culture for the synthesis of sweet proteins.

8. A method for purifying the sweet protein mutant according to claim 1, characterized in that, The purification method includes the following steps: After culturing the transformants of the sweet protein mutant, the culture was collected, and impurities were removed. The mixture was then filtered, and the filtered solution was subjected to cation exchange resin to adsorb the sweet protein mutant. The mixture was eluted, and the eluent was collected. After nanofiltration to remove salt and concentration, the eluent was lyophilized to obtain the purified sweet protein mutant.

9. The purification method for the sweet protein mutant according to claim 8, characterized in that, The separation and removal of impurities from proteins is achieved using centrifugation.

10. The purification method for the sweet protein mutant according to claim 8, characterized in that, The filtration is performed using a 0.5~10kDa ultrafiltration membrane.

11. The purification method for the sweet protein mutant according to claim 8, characterized in that, The elution was performed using an aqueous NaCl solution.

12. The purification method for the sweet protein mutant according to claim 11, characterized in that, The NaCl aqueous solution has a NaCl concentration of 1-2M and a pH value of 3.0-7.0.