Sweet protein mutant as well as preparation method and application thereof

Abstract

The invention provides a sweet protein mutant as well as a preparation method and application thereof. The mutant is obtained by amino acid mutation on the basis of an amino acid sequence as shown in SEQ ID NO.1 of sweet protein. The amino acid mutation type comprises at least one of K5D, V7R, D29N or E53R. The mutation is introduced into the amino acid sequence of the sweet protein, so that compared with the sweet protein corresponding to the original sequence, the sweetness of the mutant is obviously improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a sweet protein mutant and a preparation method and application thereof. Background Art

[0002] The World Health Organization believes that excessive sugar consumption is one of the causes of obesity, diabetes and other diseases. According to the standards announced by the WHO in 2019, the standard daily sugar intake for adults is 25g. With the changes in human living habits, the sugar substitute industry has gradually entered the field of vision of consumers, and suppliers focusing on "sugar-free products" have gradually emerged.

[0003] As a substitute for sugar, sweeteners have received much attention. In recent years, some companies have set their sights on sweet proteins, which are highly sweet, pure in taste, natural, and free of any side effects. They are expected to become 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 peculiar smell, and can maintain 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 sucrose of the same mass. Compared with other sweet proteins, Brazzein has the smallest molecular weight and the best water solubility. Its aqueous solution still retains its sweetness after being heat treated at 80°C for 4 hours, and has good thermal stability and pH stability.

[0004] However, the yield of sweet protein from natural plants is low and cannot meet market demand. In addition, sweet protein often cannot be properly folded or easily forms inclusion bodies and the expression level is too low in heterologous recombinant expression, which limits its commercial production and application. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a mutant of thaumatin and its preparation method and application. The present invention uses genetic engineering technology to artificially transform the thaumatin gene, which significantly improves the sweetness of thaumatin.

[0006] To achieve this object, the present invention adopts the following technical solutions:

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

[0008] In the present invention, by introducing a mutation into the amino acid sequence of sweet protein, the relative sweetness of the mutant is significantly improved compared with 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 the present invention, the amino acid sequence shown in SEQ ID NO.1 is as follows:

[0011] QDKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQC ICDYCEY.

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

[0013] SEQ ID NO.2:

[0014] CAGGACAAGTGCAAGAAGGTCTACGAGAACTACCCAGTTTCCAAGTGCCAGTTGGCTAACCAGTGTAACTACGACTGCAAGTTGGACAAGCACGCTAGATCTGGTGAGTGTTTCTACGACGAGAAGAGAAAACCTGCAGTGCATCTGTGACTACTGCGAGTACTAA.

[0015] In the present invention, the non-mutated sweet protein originates from the West African tropical plant Pentadiplandra brazzeana Baillon.

[0016] In a second aspect, the present invention provides a nucleic acid molecule encoding the sweet protein mutant described in the first aspect.

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

[0018] In a fourth aspect, the present invention provides a sweet protein mutant transformant, which is a genetically engineered strain that expresses the sweet protein mutant described in the first aspect.

[0019] In a fifth aspect, the present invention provides a method for preparing the thaumatin mutant according to the first aspect, the preparation method comprising the following steps:

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

[0021] Preferably, the method for preparing the thaumatin mutant comprises the following steps:

[0022] The sweet protein nucleic acid fragment derived from the West African tropical plant Pentadiplandra brazzeana Baillon is synthesized by whole gene, as shown in SEQ ID NO.2, and is recombined into an expression vector through enzyme digestion to obtain a recombinant plasmid. The recombinant plasmid is used as a template for site-directed mutagenesis, and the mutant is transformed into a host cell for cultivation; the sweet protein mutant is obtained.

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

[0024] In a sixth aspect, the present invention provides a use of the sweet protein mutant described in the first aspect in fermentation culture to synthesize sweet protein.

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

[0026] After culturing the sweet protein mutant transformant, the culture is collected, and the impurity protein is separated and filtered. The filtered solution is subjected to cation exchange resin to adsorb the sweet protein mutant, eluted, and the eluate is collected. After nanofiltration, salt removal and concentration, the lyophilization is performed to obtain the sweet protein mutant purified product.

[0027] Preferably, the separation and removal of impurities is carried out by centrifugation.

[0028] Preferably, the filtration is through a 0.5-10 kDa (eg, 0.5 kDa, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa or 10 kDa) ultrafiltration membrane.

[0029] Preferably, the elution is performed using a NaCl aqueous 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 the present invention, by introducing mutations into the amino acid sequence of 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 DESCRIPTION

[0033] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0034] Example 1 Construction of Brazzein original plasmid

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

[0036] Example 2 Construction of Brazzein mutants

[0037] The forward / reverse K5D, V7R, D29N, and E53R mutation primers were used respectively, and the original plasmid Brazzein-pPICZαA was used as a template to perform site-directed mutagenesis on the above sites (for specific operations, refer to the Fast Mutagenesis System) to obtain Brazzein mutants.

[0038] in:

[0039] Lysine (K) at position 5 was mutated to aspartic acid (D)

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

[0041] 5'GAAAAGACAGGACAAGTGCGACAAGGTCTACGAGAACTAC 3'

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

[0043] 5'GTAGTTCTCGTAGACCTTGTCGCACTTGTCCTGTCTTTTC 3'

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

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

[0046] 5'GACAAGTGCAAGAAGCGCTACGAGAACTACC 3'

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

[0048] 5'GGTAGTTCTCGTAGCGCTTCTTGCACTTGTC3'

[0049] Aspartic acid (D) at position 29 mutated to asparagine (N)

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

[0051] 5'CTACGACTGCAAGTTGAACAAGCACGCTAGATC 3'

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

[0053] 5'GATCTAGCGTGCTTGTTCAACTTGCAGTCGTAG 3'

[0054] Mutation of glutamic acid (G) at position 53 to arginine (R)

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

[0056] 5'ATCTGTGACTACTGCAGATACTAAGCG 3'

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

[0058] 5'TCTGCAGTAGTCACAGATGCACTGCAG 3'

[0059] The PCR system is: FastPfu Fly PCR SuperMix 25μl, forward primer and reverse primer (10μM) 1μl each, template plasmid (50ng / μl) 1μl, ddH2O to 50μl. The PCR amplification program is: 94℃ pre-denaturation for 3 minutes, followed by 30 cycles of: 94℃ denaturation for 20 seconds, 55℃ annealing for 20 seconds, 72℃ extension for 2 minutes; and finally 72℃ extension for another 10 minutes. The PCR amplification product was digested with DpnI for 2h, and the digested product was transformed into E.coli Tran5α, and stored after verification by selecting single clones for sequencing.

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

[0061] The original plasmid Brazzein-pPICZαA in Example 1 and the mutant in Example 2 were linearized with Sac I, and the linearized products were purified with a column purification kit, and then transformed into Pichia X-33 by electroporation, and coated on YPD+Zeocin (100 mg / L) plates. The colonies grown on the YPD+Zeocin (100 mg / L) plates are Pichia engineering strains. Shake flask fermentation verification picked transformants and inoculated them into BMGY medium, respectively, and cultured at 30°C and 220rpm for 24 hours, and then transferred to BMMY medium, 30°C, 220rpm, and 0.5% methanol was added every 24 hours. After induced expression for 4 days, the bacteria were removed by centrifugation to obtain the fermentation supernatant containing sweet protein.

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

[0063] a) Pretreatment of fermentation broth

[0064] The fermentation broth was centrifuged at 8000 g for 30 min to collect the supernatant solution.

[0065] b) Rough treatment of fermentation broth

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

[0067] c) Purification of Brazzein

[0068] Protein purification resin column: Specification: 10×30 cm (1 L); Filler: 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 acetic acid in water at a flow rate of 10 mL / min.

[0070] Feed: Load 5 L of the crude sample from step b) at a flow rate of 5 mL / min.

[0071] Elution: 3 L of pH 4.0 acetic acid aqueous solution was used for elution at a flow rate of 10 mL / 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 eluate containing sweet protein.

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

[0074] The eluate obtained in the previous step was filtered through a nanofiltration membrane (1000 Da, Shanghai Langji Membrane Separation Equipment Engineering Co., Ltd.) to remove salt and obtain a concentrated sample.

[0075] e) Freeze drying of samples

[0076] The concentrated sample obtained in the previous step is used to remove excess water using a freeze dryer to finally obtain a dry sweet protein sample.

[0077] Example 5 Sensory Evaluation of Brazzein and Its Mutant Proteins

[0078] The sweetness of sweet protein is identified by blind test. The control group of the blind test uses 2% sucrose aqueous solution. The sweet protein samples to be tested are prepared into different concentration gradients and randomly numbered. A 10-person evaluation team tastes the samples and determines the samples with the same or similar sweetness as the 2% sucrose aqueous solution in the samples of different concentration gradients, so as to calculate the relative sweetness of the samples.

[0079] First, weigh 1.0g of the sweet protein sample and dilute it to 100ml with distilled water to form a 1.0% sample solution; then measure the 1.0% sample solution and further dilute it with distilled water to 0.5%, 0.25%, 0.2%, 0.125%, 0.1%, 0.05%, 0.025%, 0.0125% and other different dilution multiples of the test solution. The sweet protein samples to be tested are prepared into different concentration gradients and randomly numbered. A 10-person evaluation team tastes the samples and determines the samples with the same or similar sweetness as the 2% sucrose aqueous solution in the samples with different concentration gradients, so as to calculate the relative sweetness of the samples. The formula is: relative sweetness = 2% sucrose aqueous solution ÷ (1.0% sample solution × dilution multiple).

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

[0081] Table 1

[0082] protein Relative sweetness (relative to 2% sucrose sweetness multiple) 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 from the above, the sweetness of the thaumatin mutant of the present invention is 1.6-6 times that of the thaumatin corresponding to the original sequence, and its sweetness is significantly improved.

[0084] The applicant declares that the present invention illustrates the sweet protein mutant and its preparation method and application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A thaumatin mutant, characterized in that: The mutant is obtained by amino acid mutation based on the amino acid sequence of sweet protein shown in SEQ ID NO.1; the type of amino acid mutation includes at least one of K5D, V7R, D29N or E53R.

2. The thaumatin mutant according to claim 1, characterized in that The sweet protein includes the nucleic acid sequence shown in SEQ ID NO.

2.

3. The thaumatin mutant according to claim 1, characterized in that The types of amino acid mutations are K5D, V7R, D29N, E53R, K5D+V7R, D29N+E53R or K5D+V7R+D29N+E53R.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the sweet protein mutant according to any one of claims 1 to 3.

5. An expression vector, characterized in that: The expression vector contains at least one copy of the nucleic acid molecule of claim 4.

6. A thaumatin mutant transformant, characterized in that: The sweet protein mutant transformant is a genetically engineered strain expressing the sweet protein mutant according to any one of claims 1 to 3.

7. A method for preparing the thaumatin mutant according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: An expression vector is constructed and transformed into a recipient cell to construct a sweet protein mutant transformant; the sweet protein mutant transformant is cultured and the culture is collected to obtain the sweet protein mutant.

8. The preparation method according to claim 7, characterized in that: The method for preparing the thaumatin mutant specifically comprises the following steps: The whole gene is used to synthesize a thaumatin nucleic acid fragment derived from the West African tropical plant Pentadiplandra brazzeana Baillon, as shown in SEQ ID NO.2, which is digested and recombined into an expression vector to obtain a recombinant plasmid. The recombinant plasmid is used as a template to perform site-directed mutagenesis, and the mutant is transformed into a host cell for cultivation; the thaumatin mutant protein is obtained.

9. Use of the sweet protein mutant according to any one of claims 1 to 3 in fermentation culture to synthesize sweet protein.

10. A method for purifying the thaumatin mutant according to any one of claims 1 to 3, characterized in that: The purification method comprises the following steps: After culturing the thaumatin mutant transformant, the culture is collected, and impurity proteins are separated and removed, followed by filtration. The filtered solution is subjected to cation exchange resin to adsorb the thaumatin mutant, eluted, and the eluate is collected. After nanofiltration and desalination, the eluate is concentrated and freeze-dried to obtain a purified thaumatin mutant; Preferably, the separation and removal of impurities is carried out by centrifugal separation; Preferably, the filtration is performed through a 0.5-10 kDa ultrafiltration membrane; Preferably, the elution is performed using a NaCl aqueous solution; Preferably, the NaCl concentration in the NaCl aqueous solution is 1-2 M, and the pH value of the solution is 3.0-7.0.