Novel thaumatin mutant as well as preparation method and application thereof

Abstract

The invention relates to a novel thaumatin mutant as well as a preparation method and application thereof. The invention provides a thaumatin mutant. The thaumatin mutant comprises an amino acid sequence of SEQ ID NO: 5 and a 6His tag sequence. The invention also provides a construction method of pichia pastoris engineering bacteria for expressing the thaumatin mutant and a preparation method of the thaumatin mutant. The inventor constructs a novel thaumatin mutant through genetic engineering, and large-scale expression of the thaumatin mutant is realized by using a pichia pastoris expression system. Sensory evaluation proves that the novel thaumatin mutant has high sweetness, and his-tag has little influence on the sweetness of protein. Stable isotope labeling of the novel thaumatin mutant is realized through a stable isotope labeling technology and adjustment of culture medium conditions. A 1H-15N HSQC spectrogram is utilized to prove that the his-tag basically has no influence on the protein conformation.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a novel thaumatin mutant, a preparation method thereof and application thereof. Background Art

[0002] Thaumatin is a natural sweet protein extracted from African arrowroot. Thaumatin has the advantages of low calories, high sweetness, and no harm to human health. Even at a low concentration of 50nM, under equimolar conditions, its sweetness is 100,000 times higher than sucrose. It is one of the most studied and promising substitutes for sugars and artificial sweeteners. Thaumatin is mainly divided into Thaumatin I and Thaumatin II, which are composed of 207 amino acids. Its precursor form contains both a 22-amino acid N-terminal presequence and a 6-amino acid C-terminal presequence. The sweetness of thaumatin can be enhanced by removing the negative charge on the side where thaumatin interacts with taste receptors. By mutating the 21st amino acid of thaumatin from aspartic acid to asparagine, the thaumatin D21N mutant can be obtained, whose sweetness threshold is 31nM, and it is easier to feel sweet than natural thaumatin. The D21N mutant of thaumatin and the Y65R mutant of single-chain monellin are two of the sweetest proteins known.

[0003] The traditional plant extraction method has problems such as demanding raw material growth environment, limited planting area and low yield, which restricts the large-scale production and application of thaumatin. Thaumatin has broad application prospects. In order to meet market demand, researchers use transgenic technology to introduce thaumatin into different hosts, such as microorganisms and higher plants, in order to obtain a large amount of recombinant active thaumatin protein. However, the recombinant thaumatin protein is either low in yield or has no sweetness. So far, there is no research report on the industrial production of thaumatin using genetic engineering methods at home and abroad.

[0004] Among the more than 800 species of yeast discovered by humans, Pichia pastoris, which uses methanol as the only carbon source, is one of the most widely used heterologous protein expression systems. Compared with prokaryotic expression systems, in addition to the advantages of easy cultivation and fast growth, the use of Pichia pastoris to produce thaumatin has more advantages: (1) Compared with another commonly used eukaryotic expression system, Saccharomyces cerevisiae, it avoids the defects of unstable expression vectors and lax induction, and can express more recombinant proteins. (2) The compact genome of Pichia pastoris makes the gene identification process simpler. (3) Pichia pastoris has high genetic stability and low glycosylation, which is more suitable for the expression of genes in higher organisms, post-translational modification and disulfide bond formation. (4) Pichia pastoris has a tightly regulated methanol-inducible promoter, which can well control the production of recombinant proteins. At the same time, it secretes fewer impurities and is easy to purify. In recent years, the Pichia pastoris expression system has been used by many researchers, and more than 5,000 different types of proteins have been successfully expressed using this expression system.

[0005] Isotope labeling of proteins includes the following processes: constructing a plasmid containing the gene sequence of the target protein, introducing it into the host organism, culturing it in a culture medium containing an isotope marker, expressing the isotope-labeled protein through the host organism, and separating and purifying it using biochemical techniques to obtain high-purity isotope-labeled protein. Common protein isotope labeling methods include chemical labeling and metabolic labeling. Chemical labeling mainly refers to the use of chemical reactions to introduce isotope labels at specific sites of proteins or peptides after collecting protein samples; metabolic labeling refers to adding isotope labels to the culture medium. 15 N or 2 H or other isotopes, after several generations of culture, the protein will be completely labeled with isotopes. Chemical labeling can only replace part of the protein, while metabolic labeling can achieve uniform isotope labeling.

[0006] His tag is a technology commonly used in protein purification and biological research. It involves fusing a short peptide sequence composed of histidine to the N-terminus or C-terminus of the target protein, making it easy to capture and identify during expression and purification. Since the His tag can specifically bind to affinity chromatography media chelated with metal ions (such as nickel or cobalt), this eliminates the need to use multiple purification steps to gradually separate proteins, achieving rapid purification and reducing the complexity of the operation. At the same time, the His tag affinity chromatography method is easy to scale up and is suitable for small-scale laboratory purification to industrial-scale production. Summary of the invention

[0007] As mentioned above, the traditional plant extraction method has the problems of demanding raw material growth environment, limited planting area and low yield, which limits the large-scale production and application of thaumatin. In order to meet market demand, researchers use transgenic technology to introduce thaumatin into different hosts, such as microorganisms and higher plants, in order to obtain a large number of recombinant active thaumatin proteins. However, the recombinant thaumatin proteins are either low in yield or have no sweetness. Therefore, new thaumatin mutants are urgently needed and suitable expression systems and purification methods are found to obtain recombinant thaumatin proteins with high sweetness, high yield and high purity. In order to improve the industrial production and application of proteins, the specific binding of his-tag and nickel column can be used for protein purification. Purification with nickel column has the advantages of good chemical stability, strong tolerance, not easily disturbed by other components in the fermentation supernatant, and simple sample pretreatment. Proteins carrying his-tag can achieve faster and more convenient purification.

[0008] However, due to the bitter taste of histidine, polyhistidine tags are usually not considered as purification tags for thaumatin proteins. Therefore, it is still unknown whether his-tag is suitable for the expression and purification of thaumatin and thaumatin mutant proteins and whether his-tag will affect the conformation and sweetness of thaumatin. In addition, the stable isotope labeling method can lay the foundation for the subsequent use of nuclear magnetic resonance (NMR) to study protein interactions and protein structure, but there is no report on stable isotope labeling of thaumatin mutants.

[0009] In order to overcome the above-mentioned problems of the prior art, the present invention uses genetic engineering and stable isotope labeling technology to utilize the Pichia pastoris expression system to recombinantly express a novel isotope-labeled thaumatin mutant, thereby making it have high sweetness, high purity and high yield, while simplifying the protein purification method.

[0010] In one aspect, the present invention provides a thaumatin mutant comprising an amino acid sequence of SEQ ID NO: 5 and a 6His tag sequence.

[0011] In some embodiments, the 6His tag sequence is located at the C-terminus of the thaumatin mutant.

[0012] In some embodiments, the thaumatin mutant further comprises a first presequence and / or a second presequence. In some specific embodiments, the first presequence is linked to the N-terminus of SEQ ID NO:5; preferably, the amino acid sequence of the first presequence is as shown in SEQ ID NO:1. In some specific embodiments, the second presequence is linked to the C-terminus of SEQ ID NO:5; preferably, the amino acid sequence of the second presequence is as shown in SEQ ID NO:2.

[0013] In some specific embodiments, the amino acid sequence of the thaumatin mutant is as shown in SEQ ID NO:7.

[0014] In the present invention, the 21st aspartic acid (Asp) of the natural thaumatin protein is mutated to asparagine (Asn) to obtain the thaumatin D21N protein, and its amino acid sequence is shown in SEQ ID NO:5. In some embodiments, the N-terminus and C-terminus of the thaumatin D21N protein may respectively include a 22-amino acid N-terminal presequence (for example, as shown in SEQ ID NO:1) and a 6-amino acid C-terminal presequence (for example, as shown in SEQ ID NO:2). In some specific embodiments, the novel thaumatin protein of the present invention may be a thaumatin D21N protein and a polyhistidine tag sequence (for example, a 6His tag sequence) located at its C-terminus. The novel thaumatin protein of the present invention may also be a thaumatin D21N protein comprising a presequence and further having a polyhistidine tag sequence located at the C-terminus, and its amino acid sequence may be shown in SEQ ID NO:7.

[0015] Another aspect of the present invention provides a nucleic acid molecule encoding the thaumatin mutant as described in the first aspect of the present invention. In some specific embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:9.

[0016] Another aspect of the present invention provides a recombinant plasmid, characterized in that the recombinant plasmid comprises the nucleic acid molecule described in the present invention.

[0017] Another aspect of the present invention provides a host cell, which comprises the nucleic acid molecule according to the present invention or the recombinant plasmid according to the present invention; preferably, the host cell is Pichia pastoris.

[0018] In another aspect, the present invention provides a method for constructing an engineered Pichia pastoris expressing a thaumatin mutant, comprising the step of transforming the recombinant plasmid described in the present invention into a Pichia pastoris strain to obtain the engineered Pichia pastoris strain; preferably, the Pichia pastoris strain is X-33.

[0019] Another aspect of the present invention provides an engineered strain of Pichia pastoris obtained according to the construction method of the present invention.

[0020] Another aspect of the present invention provides a method for preparing a thaumatin mutant, which comprises fermenting and culturing the Pichia pastoris engineered bacteria as described in the present invention and purifying the thaumatin mutant using a Ni column.

[0021] Another aspect of the present invention provides a thaumatin mutant obtained according to the preparation method of the present invention.

[0022] Another aspect of the present invention provides a sweetener comprising the thaumatin mutant according to the present invention.

[0023] Another aspect of the present invention provides a use of the thaumatin mutant according to the present invention in the preparation of a sweetener, a sweet food or a sweet beverage.

[0024] In another aspect, the present invention provides a method for stable isotope labeling of a thaumatin mutant, comprising fermenting and culturing the Pichia pastoris engineered bacteria of the present invention in a culture medium containing an isotope-labeled raw material, obtaining the thaumatin mutant by column purification, and storing the thaumatin mutant in a solution containing 10% D2O, wherein the isotope-labeled raw material comprises 15 N-labeled ammonium chloride, amino-free yeast nitrogen source, 400 mM phosphate, biotin, and PTM1 Pichia trace elements.

[0025] Another aspect of the present invention provides a thaumatin mutant having an isotope-stable labeling obtained by the labeling method of the present invention.

[0026] Another aspect of the present invention provides a use of the isotope-stable thaumatin mutant as described in the present invention in studying protein interactions and / or protein structures; preferably, the study is performed using nuclear magnetic resonance.

[0027] The beneficial effects of the present invention are as follows: a novel thaumatin mutant is constructed by genetic engineering and its large-scale expression is achieved by using a Pichia pastoris expression system. Sensory evaluation confirms that the novel thaumatin mutant has a high sweetness and that the his-tag has little effect on the sweetness of the protein. The novel thaumatin mutant is labeled with stable isotopes by using stable isotope labeling technology and adjusting the culture medium conditions. 1 H- 15 N HSQC spectrum confirms that the his-tag has little effect on the protein conformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Design a flow chart for the present invention.

[0029] Figure 2 is a map of the expression vector pPICZB.

[0030] Figure 3 The figure shows the SDS-PAGE experimental results of different elution collection solutions after TH2-his purification.

[0031] Figure 4 The results of SDS-PAGE experiments of different elution collection solutions after TH2 purification.

[0032] Figure 5 TH2-His(D21N)1 H- 15 N HSQC spectrum.

[0033] Figure 6 For TH2(D21N) 1 H- 15 N HSQC spectrum. DETAILED DESCRIPTION

[0034] Exemplary embodiments of the present invention will now be described in detail, which should not be considered as limiting the present invention but rather should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred materials and methods are described below.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0038] Example 1: Thaumatin mutant D21N recombinant expression vector

[0039] The recombinant plasmids pPICZB-pre-TH2-pro and pPICZB-pre-TH2-pro-his were stored in our laboratory, and the expression vector pPICZB was Figure 2 As shown, a recombinant plasmid was constructed using a signal peptide pre-sequence. The recombinant plasmid was codon-optimized and suitable for expression in Pichia pastoris.

[0040] N-terminal pre-sequence amino acid sequence: MAATTCFFFLFPFLLLLTLSRA (SEQ ID NO: 1)

[0041] C-terminal pro-sequence amino acid sequence: LELEDE (SEQ ID NO: 2)

[0042] Example 2: Site-directed mutagenesis of the thaumatin gene

[0043] The vectors pPICZB-pre-TH2-pro and pPICZB-pre-TH2-pro-his carrying the presequence and mature thaumatin gene were used as templates for mutagenesis. Primer 1 (F) and primer 2 (R) were used to mutate the 21st aspartic acid (Asp) in the amino acid sequence corresponding to the thaumatin gene to asparagine (Asn). The mutants were named TH2 and TH2-his, respectively.

[0044] Primer 1 (F): gcagccagcaaggggaatgctgccttggac (SEQ ID NO: 3) Primer 2 (R): gtccaaggcagcattccccttgctggctgc (SEQ ID NO: 4) Amino acid sequence of thaumatin D21N:

[0045] ATFEIVNRCSYTVWAAASKGNAALDAGGRQLNSGESWTINVEPGTKGGKIWARTDCYFDDSGRGICRTGDCGGLLQCKRFGRPPTTLAEFSLNQYGKDYIDISNIKGFNVPMDFSPTTRGCRGVRCAADIVGQCPAKLKAPGGGCNDACTVFQTSEYCCTTGKCGPTEYSRFFKRLCPDAFSYVLDKPTTVTCPGSSNYRVTFCPTA (SEQ ID NO: 5) The amino acid sequence of thaumatin mutant TH2 is:

[0046] MAATTCFFFLFPFLLLLTLSRA ATFEIVNRCSYTVWAAASKGNAALDAGGRQLNSGESWTINVEPGTKGGKIWARTDCYFDDSGRGICRTGDCGGLLQCKRFGRPPTTLAEFSLNQYGKDYIDISNIKGFNVPMDFSPPTTRGCRGVRCAADIVGQCPAKLKAPGGGCNDACTVFQTSEYCCTTGKCGPTEYSRFFKRLCPDAFSYVLDKPTTVTCPGSSNYRVTFCPTA LELEDE (SEQ ID NO:6)

[0047] The nucleotide sequence of the thaumatin mutant TH2 is:

[0048] 5’-ATGGCAGCCACAACATGTTTTTTTTTCCTTTTTCCTTTCCTTTTGCTACTGACGCTAAGTAGGGCTGCAACATTCGAAATAGTCAACCGCTGTAGCTACACTGTATGGGCAGCAGCCAGCAAGGGGAATGCTGCCTTGGACGCGGGAGGCCGGCAGTTAAATTCAGGTGAATCCTGGACTATCAACGTGGAACCAGGTACCAAAGGTGGCAAGATCTGGGCGCGAACGGACTGTTATTTCGATGATAGCGGCCGGGGTATATGCCGAACTGGGGATTGTGGAGGCCTACTTCAGTGCAAACGATTTGGTAGACCGCCTACCACCCTCGCTGAGTTTTCGTTAAATCAATATGGAAAAGACTATATCGACATTTCGAATATTAAGGGGTTCAACGTTCCGATGGACTTCTCACCCACGACGAGGGGCTGCCGTGGAGTGCGGTGCGCAGCGGATATTGTTGGTCAGTGTCCGGCCAAGCTCAAAGCTCCTGGCGGGGGATGCAACGACGCGTGCACTGTCTTTCAAACATCTGAATACTGCTGTACCACGGGGAAATGCGGACCGACTGAGTATAGTCGCTTCTTCAAAAGATTATGCCCCGATGCTTTTTCCTACGTACTGGATAAGCCAACCACGGTGACCTGTCCCGGGTCTTCTAATTACCGTGTAACTTTTTGTCCAACAGCCCTGGAGTTGGAGGACGAA-3’(SEQ ID NO:8)

[0049] The amino acid sequence of thaumatin mutant TH2-his is as follows:

[0050] MAATTCFFFLFPFLLLLTLSRAATFEIVNRCSYTVWAAASKGNAALDAGGRQLNSGESWTINVEPGTKGGKIWARTDCYFDDSGRGICRTGDCGGLLQCKRFGRPPTTLAEFSLNQYGKDYIDISNIKGFNVPMDFSPPTTRGCRGVRCAADIVGQCPAKLKAPGGGCNDACTVFQTSEYCCTTGKCGPTEYSRFFKRLCPDAFSYVLDKPTTVTCPGSSNYRVTFCPTA LELEDE HHHHHH (SEQ ID NO: 7)

[0051] The nucleotide sequence of the thaumatin mutant TH2-his is:

[0052] 5'-ATGGCAGCCACAACATGTTTTTTTTTCCTTTTTCCTTTCCT -3'(SEQ ID NO:9)

[0053] Example 3: Construction of recombinant expression of supersweet thaumatin

[0054] 1. Preparation of recombinant plasmid

[0055] According to the instructions of the plasmid mini-extraction kit of Biyuntian Biotechnology, the plasmid obtained in Example 2 was extracted in a medium amount, and the concentration was determined using Nanodrop. Samples were taken for linearization treatment, and SacI was digested at 37°C for 1 hour, and the linearized vector was recovered by gel. 0.8% DNA agarose gel electrophoresis was used to determine whether the linearization was successful. The concentration was determined and lyophilized.

[0056] 2. Conversion and screening

[0057] The obtained linearized product was transformed into Pichia pastoris X-33 by electroporation, and multi-copy transformants were screened on YPD plates containing different gradient concentrations of the antibiotic Zeocin (100 mg / mL, 1000 mg / mL, 2000 mg / mL). The positive transformants on the 2000 mg / mL YPD resistance plate were picked, and colony PCR verification was performed using 5'AOX1 primer: 5'-GACTGGTTCCAATTGACAAGC-3' (SEQ ID NO: 10) and 3'AOX1 primer: 5'-GCAAATGGCATTCTGACATCC-3' (SEQ ID NO: 11) as upstream and downstream primers. Among the positive copies, those with good growth status were selected and inoculated into 3 mL YPD medium, and cultured on a shaking table at 30°C and 220 rpm for 12 h. 500 μL of the bacterial solution was taken and injected into a freezing tube, and 50% glycerol was added according to a volume ratio of 1:1 between the bacterial solution and glycerol to obtain a recombinant expression strain, which was stored in a -80°C refrigerator.

[0058] Example 4: Expression and preparation of thaumatin mutant TH2-his

[0059] 1. Activation culture

[0060] The recombinant expression strain TH2-his obtained in Example 3 was streaked onto a YPD solid plate and cultured at 30°C for 72 h. A single clone was picked and transferred to 20 mL of BMGY medium and cultured overnight at 30°C and 220 rpm in a shaker. 600 After OD 2-6, transfer the bacterial suspension to 100 mL BMGY and 600 When OD was 0.5, the incubation was carried out in a shaking incubator at 30°C and 220 rpm. 600 When the cell density reaches 4-6, centrifuge at 5000×g, 4°C, 10 min and discard the supernatant.

[0061] 2. Fermentation culture

[0062] The cells obtained in step 1 were resuspended in 500 mL BMMY to an OD of 600 The cell culture temperature was 1, and the culture was shaken at 30°C and 220 rpm, and 2% (v / v) methanol was added every 12 hours to induce protein expression. After 120 hours of culture, the cells were centrifuged at 14000 rpm, 4°C, and 30 minutes, and the supernatant was collected and stored at 4°C.

[0063] 3. Purification of TH2-his

[0064] The TH2-his fermentation broth supernatant obtained in step 2 was adjusted to pH 7.5 with 500mM Tris-HCl, centrifuged at 14000rpm, 4°C, 10min, and filtered with a 0.45μm filter membrane. The Ni-NTA column was balanced with buffer 2 (25mM phosphate, 1M NaCl, pH=7.4) (column volume was 8mL), the filtered fermentation broth supernatant was added to the Ni-NTA column, placed in low temperature oscillation binding for 1.5h, and after binding, 5 column volumes of buffer 3 (25mM phosphate, 1M NaCl, 40mM imidazole, pH=7.4), 5 column volumes of buffer 4 (25mM phosphate, 1M NaCl, 75mM imidazole, pH=7.4), and 8 column volumes of buffer 5 (25mM phosphate, 1M NaCl, 1000mM imidazole, pH=7.4) were used in sequence for elution. The first two column volumes of eluate from buffer 4 were collected, and the collected eluate containing the target protein was subjected to 12% SDS-PAGE detection, and the relatively pure target protein collection solution was combined ( Figure 3 ).

[0065] 4. Freeze drying

[0066] After obtaining the high-purity protein collection solution in step 3, dialyze with ultrapure water to remove salt, take out the protein collection solution after 1 day, measure the absorbance value of the solution at 280nm by NanoDrop, and obtain powdered thaumatin protein after freeze-drying. The final yield of TH2-his is 12 mg / L fermentation broth supernatant.

[0067] Example 5: Expression and preparation of thaumatin mutant TH2

[0068] 1. Activation culture

[0069] The recombinant expression strain TH2 obtained in Example 3 was streaked onto a YPD solid plate and cultured at 30°C for 72 h. A single clone was picked and transferred to 20 mL of BMGY medium and cultured overnight at 30°C and 220 rpm in a shaker. 600 After OD 2-6, transfer the bacterial suspension to 100 mL BMGY and 600 When OD was 0.5, the incubation was carried out in a shaking incubator at 30°C and 220 rpm. 600 When the cell density reaches 4-6, centrifuge at 5000×g, 4°C, 10 min and discard the supernatant.

[0070] 2. Fermentation culture

[0071] The cells obtained in step 1 were resuspended in 500 mL BMMY to an OD of 600The cell culture temperature was 1, and the culture was shaken at 30°C and 220 rpm, and 2% (v / v) methanol was added every 12 hours to induce protein expression. After 120 hours of culture, the cells were centrifuged at 14000 rpm, 4°C, and 30 minutes, and the supernatant was collected and stored at 4°C.

[0072] 3. Purification of TH2

[0073] (1) Dialysis

[0074] Take the fermentation broth stored at 4°C in step 2 and dialyze it with buffer 1 (5 mM phosphate, pH = 8.60). Take out the fermentation broth after 1 day, measure the pH, centrifuge at 14000 rpm, 4°C, 10 min, collect the supernatant, filter it with a 0.45 μm filter membrane to remove insoluble matter, and store it at 4°C.

[0075] (2) Target protein collection

[0076] The Foresight Nuvia S column (column volume 5 mL) was balanced with buffer 1, and the filtered fermentation broth was added to the Foresight Nuvia S column for ion exchange column chromatography, and gradient elution was performed with a salt-containing buffer. A gradient of 50-250 mM NaCl was used for 8 column volumes, and the collected eluate containing the target protein was subjected to 12% SDS-PAGE detection, and the relatively pure target protein collection solution was combined ( Figure 4 ).

[0077] 4. Freeze drying

[0078] After obtaining a high-purity protein collection solution in step 3, dialyze with ultrapure water to remove salt, take out the protein collection solution after 1 day, measure the absorbance of the solution at 280nm by NanoDrop, and obtain powdered thaumatin protein after freeze-drying. The final yield of TH2 is 17.4 mg / L fermentation broth supernatant.

[0079] Example 6: Sweetness detection of thaumatin mutants TH2 and TH2-his

[0080] 1. Sweetness detection principles

[0081] The sweetness of sweet protein is identified by blind test. The standard sample of the blind test is 2% (2g / 100mL) sucrose aqueous solution. The samples of sweet protein to be tested are randomly numbered after being configured into different concentration gradients. An evaluation team composed of 8 people tastes the samples and judges the samples with the same or similar sweetness as the 2% sucrose aqueous solution in the samples with different concentration gradients. When more than 5 (60%) of the 8 sensory evaluators believe that the sweetness intensity and taste of the sample are no different from those of the standard sample, the ratio of the concentration of the standard solution to the concentration of the sample is the sweetness multiple of the sample.

[0082] 2. Preparation of different concentration gradient samples of sweet protein: The concentration of the sample mother solution is 1 / 10 of the standard sucrose aqueous solution, and it is further diluted with distilled water to form test solutions with different dilution multiples such as 2 times, 4 times, 6 times, 8 times, 10 times, 20 times, 50 times, 100 times, 200 times, and 300 times.

[0083] 3. The sensory evaluation results showed that at the shake flask level, the sweetness of the recombinant thaumatin mutant TH2 reached 2250 times that of standard sucrose, while the sweetness of TH2-his reached 1800 times that of standard sucrose.

[0084] Example 7: Stable isotope labeling of the thaumatin mutant TH2-his

[0085] 1. Activation culture

[0086] The activated strain TH2-his obtained in Example 3 was streaked onto a YPD solid plate and cultured at 30°C for 72 h. A single clone was picked out and transferred to 20 mL of BMGY medium and cultured overnight in a shaker at 30°C and 220 rpm.

[0087] 2. Stable isotope labeling culture

[0088] When the bacterial solution OD in step 1 is 600 When the OD is 2-6, centrifuge at 5000×g, 4°C, 10 min, and discard the supernatant. Resuspend the cells in BMG medium containing isotope-labeled raw materials to a value of OD 600 is 0.5, and the formula raw materials of BMG medium are 15 N-labeled ammonium chloride, amino-free yeast nitrogen source (denoted as YNB, without amino acids and without ammonium sulfate), 400 mM potassium phosphate buffer (pH = 6.0), glycerol, biotin and PTM1 Pichia pastoris trace elements; then cultured at 30°C, 220 rpm in a shaking incubator for 10 h and then tested the OD of the bacterial solution. 600 The cells were collected by centrifugation at 5000 × g, 4 °C, 10 min.

[0089] 3. Fermentation culture

[0090] The cells obtained in step 2 were resuspended in BMM medium containing isotope-labeled raw materials to a concentration of OD 600 The value is 1 (to ensure that the OD of BMM-bacterial solution 600 The value is 1, based on the volume of BMM medium and the OD of the bacterial solution 600 The value determines the volume of collected bacteria), the raw materials of BMM medium formula are 15N-labeled ammonium chloride, amino-free yeast nitrogen source (denoted as YNB, without amino acids and without ammonium sulfate), 400mM phosphate, biotin and PTM1 Pichia trace elements), cultured in a shaking incubator at 30°C and 220rpm, adding 2% (v / v) methanol every 24h to induce strain expression, and controlling the pH of the culture medium to 4-6 to prevent the pH of the culture medium from being too low and causing the expression level to decrease. After 96h of induction expression, the culture was centrifuged at 14000rpm, 4°C, and 30min, and the supernatant was collected and stored at 4°C.

[0091] 4. Purification of TH2-his

[0092] The TH2-his fermentation broth supernatant obtained in step 3 was adjusted to pH 7.5 with 500mM Tris-HCl, centrifuged at 14000rpm, 4°C, 10min, and filtered with a 0.45μm filter membrane. The Ni-NTA column was balanced with buffer 2 (25mM phosphate, 1M NaCl, pH=7.4) (column volume was 3mL), the filtered fermentation broth supernatant was added to the Ni-NTA column, placed in low temperature oscillation binding for 1.5h, and after binding, 5 column volumes of buffer 3 (25mM phosphate, 1M NaCl, 40mM imidazole, pH=7.4), 5 column volumes of buffer 4 (25mM phosphate, 1M NaCl, 75mM imidazole, pH=7.4), and 8 column volumes of buffer 5 (25mM phosphate, 1M NaCl, 1000mM imidazole, pH=7.4) were used in sequence for elution. The first two column volumes of eluate from buffer 4 were collected, concentrated by ultrafiltration at 1500×g, 4° C., and 15 min, and stored at 4° C. The Hiload gel filtration chromatography column was equilibrated with buffer 6 (25 mM phosphate, pH=6.56), and the concentrate was added to the chromatography column for recombinant protein high-resolution gel filtration chromatography (SEC) to obtain a high-purity thaumatin mutant TH2-his, which was then stored at 4° C. after 10% D2O was added to the sample.

[0093] The prepared stable isotope-labeled thaumatin mutant TH2-his was subjected to liquid nuclear magnetic resonance (NMR) experiments ( Figure 5 ).

[0094] Example 8: Stable isotope labeling of thaumatin mutant TH2

[0095] 1. Activation culture

[0096] The activated strain TH2 obtained in Example 3 was streaked onto a YPD solid plate and cultured at 30°C for 72 h. A single clone was picked out and transferred to 20 mL of BMGY medium and cultured overnight in a shaker at 30°C and 220 rpm.

[0097] 2. Stable isotope labeling culture

[0098] When the bacterial solution OD in step 1 is 600 When the OD is 2-6, centrifuge at 5000×g, 4°C, 10 min, and discard the supernatant. Resuspend the cells in BMG medium containing isotope-labeled raw materials to a value of OD 600 is 0.5, and the raw materials of BMG medium are 15 N-labeled ammonium chloride, amino-free yeast nitrogen source (denoted as YNB, without amino acids and without ammonium sulfate), 400 mM potassium phosphate buffer (pH = 6.0), glycerol, biotin and PTM1 Pichia pastoris trace elements; then cultured at 30°C, 220 rpm in a shaking incubator for 10 h and then tested the OD of the bacterial solution. 600 The cells were collected by centrifugation at 5000 × g, 4 °C, 10 min.

[0099] 3. Fermentation culture

[0100] The cells obtained in step 2 were resuspended in BMM medium containing isotope-labeled raw materials to a concentration of OD 600 The value is 1 (to ensure that the OD of BMM-bacterial solution 600 The value is 1, based on the volume of BMM medium and the OD of the bacterial solution 600 The value determines the volume of collected bacteria), the raw materials of BMM medium formula are 15 N-labeled ammonium chloride, amino-free yeast nitrogen source (denoted as YNB, without amino acids and without ammonium sulfate), 400mM phosphate, biotin and PTM1 Pichia trace elements), cultured in a shaking incubator at 30°C and 220rpm, adding 2% (v / v) methanol every 24h to induce strain expression, and controlling the pH of the culture medium to 4-6 to prevent the pH of the culture medium from being too low and causing the expression level to decrease. After 96h of induction expression, the culture was centrifuged at 14000rpm, 4°C, and 30min, and the supernatant was collected and stored at 4°C.

[0101] 4. Purification of TH2

[0102] (1) Dialysis: The supernatant of the TH2 fermentation broth obtained in step 3 was dialyzed against buffer 1 (5 mM phosphate, pH = 8.60). After 1 day, the fermentation broth was taken out, the pH was measured, and the broth was centrifuged at 14,000 rpm, 4°C, for 10 min. The broth was filtered through a 0.45 μm filter membrane and stored at 4°C.

[0103] (2) Collection of target protein: Foresight Nuvia S column (column volume of 5 mL) was balanced with buffer 1, and the filtered fermentation broth was added to the Foresight Nuvia S column for ion exchange column chromatography, and gradient elution was performed with a salt-containing buffer. The collected target protein was detected by 12% SDS-PAGE using a gradient elution of 50-250 mM NaCl for 8 column volumes, and the relatively pure target protein collection solution was combined. Ultrafiltration and concentration were performed at 1500×g, 4°C, and 15 min, and stored at 4°C. The Hiload gel filtration chromatography column was balanced with buffer 6 (25 mM phosphate, pH=6.56), and the concentrate was added to the chromatography column for recombinant protein high-resolution gel filtration chromatography (SEC) to obtain high-purity thaumatin mutant TH2, and 10% D2O was added to the sample and stored at 4°C.

[0104] The prepared stable isotope-labeled thaumatin mutant TH2 was subjected to liquid nuclear magnetic resonance (NMR) experiments ( Figure 6 ).

[0105] according to 1 H- 15 N HSQC spectrum ( Figure 5 and Figure 6 ) It can be seen that his-tag has basically no effect on protein conformation.

Claims

1. A thaumatin mutant, characterized in that: The thaumatin mutant comprises the amino acid sequence of SEQ ID NO: 5 and a 6His tag sequence.

2. The thaumatin mutant according to claim 1, characterized in that The 6His tag sequence is located at the C-terminus of the thaumatin mutant.

3. The thaumatin mutant according to claim 2, characterized in that The thaumatin mutant further comprises a first prosequence and / or a second prosequence.

4. The thaumatin mutant according to claim 3, characterized in that The first presequence is linked to the N-terminus of SEQ ID NO: 5; Preferably, the amino acid sequence of the first pre-sequence is as shown in SEQ ID NO:

1.

5. The thaumatin mutant according to claim 3, characterized in that The second presequence is linked to the C-terminus of SEQ ID NO: 5; Preferably, the amino acid sequence of the second pre-sequence is as shown in SEQ ID NO:

2.

6. The thaumatin mutant according to claim 3, characterized in that The amino acid sequence of the thaumatin mutant is shown in SEQ ID NO:

7.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the thaumatin mutant according to any one of claims 1 to 6.

8. The nucleic acid molecule according to claim 7, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

9.

9. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the nucleic acid molecule according to claim 7 or 8.

10. A host cell, characterized in that The host cell comprises the nucleic acid molecule according to claim 7 or 8 or the recombinant plasmid according to claim 9; Preferably, the host cell is Pichia pastoris.

11. A method for constructing an engineered strain of Pichia pastoris expressing a thaumatin mutant, characterized in that: The method comprises the steps of transforming the recombinant plasmid as claimed in claim 9 into a Pichia pastoris strain to obtain the Pichia pastoris engineered strain; Preferably, the Pichia pastoris strain is X-33.

12. An engineered Pichia pastoris obtained according to the construction method of claim 11.

13. A method for preparing a thaumatin mutant, characterized in that: The method comprises fermenting and culturing the Pichia pastoris engineered bacteria as claimed in claim 12 and purifying the thaumatin mutant using a Ni column.

14. A thaumatin mutant obtained according to the preparation method of claim 13.

15. A sweetener comprising the thaumatin mutant according to any one of claims 1 to 6 or 14.

16. Use of the thaumatin mutant according to any one of claims 1 to 6 or 14 in the preparation of a sweetener, a sweet food or a sweet beverage.

17. A stable isotope labeling method for a thaumatin mutant, characterized in that: The method comprises fermenting and culturing the Pichia pastoris engineered bacteria as claimed in claim 12 in a culture medium containing isotope-labeled raw materials, obtaining the thaumatin mutant by column purification, and storing the thaumatin mutant in a solution containing 10% D2O, Wherein, the isotope-labeled raw material comprises 15 N-labeled ammonium chloride, amino-free yeast nitrogen source, 400 mM phosphate, biotin, and PTM1 Pichia trace elements.

18. A thaumatin mutant having an isotope-stable labeling obtained by the labeling method according to claim 17.

19. Use of the isotope-stable thaumatin mutant according to claim 18 in studying protein interaction and / or protein structure; Preferably, the studies are performed using nuclear magnetic resonance.

Images