Mutants of high-thermostable thaumatin-like protein neoculin and genes and applications thereof
By performing structural analysis and site-directed mutagenesis on the saccharin neoculin, a high-thermal-stability neoculin mutant FM was constructed, solving the problem of poor thermal stability of natural saccharin and achieving high thermal stability and sweetness retention of the mutant.
Patent Information
- Application Number
- CN202411291491.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-14
- Publication Date
- 2025-11-11
- Estimated Expiration
- 2044-09-14
AI Technical Summary
The poor thermal stability of the natural sweet protein neoculin limits its commercial production and application.
By performing structural analysis and protein stability calculations on the saccharin neoculin, novel disulfide bonds and site-directed mutagenesis were designed to construct a highly thermally stable neoculin mutant, specifically involving amino acid mutations and disulfide bond formation in the NAS and NBS chains.
The denaturation temperature (Tm) of the sweet protein neoculin was increased to at least 20.3 °C while maintaining its sweetness, and a mutant FM with high thermal stability was obtained through purification.
Smart Images
Figure CN119080899B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering and protein modification technology, specifically relating to mutants, genes, and recombinant bacteria of the highly thermally stable sweet protein neoculin, and their applications. Background Technology
[0002] Sweetness is one of the most beloved flavors for humans, activating the brain's reward system and evoking feelings of happiness. As living standards improve, consumers' preference for sweet substances is further enhanced. However, excessive sugar intake can lead to diseases such as tooth decay, diabetes, obesity, and metabolic syndrome. Therefore, finding a safe and effective sweetener is of great significance to the food and pharmaceutical industries.
[0003] Neoculin, a natural sweet protein, was originally isolated from the fruit of a species called Sphaerella. It is a small-molecule plant protein with advantages such as safety, health benefits, low calories, high sweetness, high nutritional value, and high absorbability. Furthermore, it is the only sweet protein that simultaneously possesses both sweetness and taste-modifying activities, consisting of two chains (NAS and NBS, composed of 113 and 114 amino acids respectively) linked by two disulfide bonds. However, the poor thermal stability of this natural sweet protein, neoculin, limits its commercial production and application.
[0004] The thermal stability of proteins can be characterized as a visual indicator, namely the denaturation temperature T. m A higher value indicates better protein stability. The thermal shift assay technique can be used to quickly and efficiently compare protein stability: proteins usually exist in a native state, and heating causes them to transform into a denatured state, exposing hydrophobic groups. These exposed hydrophobic groups can bind to the fluorescent dye SYPRO Orange, thereby increasing the emission of the fluorescent dye. Therefore, the stability of the protein can be reflected by detecting the fluorescence intensity.
[0005] Currently, there is an urgent need for sweet proteins that are stable under high-temperature conditions. Summary of the Invention
[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a mutant of the sweet protein neoculin with high thermal stability.
[0007] A second objective of this invention is to provide the construction of mutants of the aforementioned highly thermally stable sweet protein neoculin.
[0008] A third objective of this invention is to provide the application of mutants of the aforementioned highly thermally stable sweet protein neoculin in the preparation of food sweeteners.
[0009] The technical solution of this invention is summarized as follows:
[0010] A mutant of the highly thermally stable sweet protein neoculin, comprising a NAS chain and an NBS chain linked by two disulfide bonds; the amino acid sequence of the NAS chain is shown in SEQ ID NO.1; the amino acid sequence of the NBS chain is shown in SEQ ID NO.2; the NAS chain is modified by mutating asparagine (N) at position 27 to glutamic acid (E), glutamine (Q) at position 51 to asparagine (N), methionine (M) at position 70 to serine (S), leucine (L) at position 93 to lysine (K), and valine (V) at position 95 to cysteine (C) to obtain chain A, the amino acid sequence of which is shown in SEQ ID NO.1. As shown in NO.3; the histidine (H) at position 11 of the NBS chain is mutated to alanine (A) + the glycine (G) at position 74 is mutated to alanine (A) + the tryptophan (W) at position 78 is mutated to serine (S) + the serine (S) at position 103 is mutated to cysteine (C) + the proline (P) at position 106 is mutated to aspartic acid (D), to obtain chain B, the amino acid sequence of which is shown in SEQ ID NO.4; the cysteine at position 95 of the A chain and the cysteine at position 103 of the B chain form a disulfide bond, to obtain a mutant of the highly thermally stable sweet protein neoculin, which is abbreviated as FM.
[0011] The construction of mutants of the highly thermally stable sweet protein neoculin includes the following steps:
[0012] 1) The gene encoding the A strand is gene A, and the gene encoding the B strand is gene B;
[0013] 2) Construct an A recombinant expression vector containing the A gene and a B recombinant expression vector containing the B gene;
[0014] 3) Construct engineered bacteria A containing the recombinant expression vector A and engineered bacteria B containing the recombinant expression vector B;
[0015] 4) The A-engineered bacteria were cultured and expressed to obtain inclusion body A-FM; the B-engineered bacteria were cultured and expressed to obtain inclusion body B-FM. The inclusion bodies were purified and renatured. The renatured protein was purified to obtain a mutant of the highly thermostable sweet protein neoculin.
[0016] The application of the aforementioned thermostable neoculin mutant in the preparation of food sweeteners.
[0017] Advantages of the present invention
[0018] This invention, based on structural analysis and computationally assisted protein stability design, employs novel disulfide bond design and site-directed mutagenesis to obtain a thermostable mutant of neoculin. Compared to wild-type neoculin, the thermostable mutant of neoculin exhibits significantly higher TT. m It increased the temperature by at least 20.3°C while maintaining its sweetness. Attached Figure Description
[0019] Figure 1 This is an SDS-PAGE (polyacrylamide gel electrophoresis) image of neoculin (WT) and a mutant of the highly thermally stable neoculin (FM).
[0020] Figure 2 The results are for the thermal stability measurements of WT and FM.
[0021] Figure 3 SDS-PAGE (polyacrylamide gel electrophoresis) images of WT after heat treatment at 80℃ for 1 hour and FM after heat treatment at 80℃ for 1 hour, 2 hours, 3 hours and 4 hours. Detailed Implementation
[0022] The following experimental materials and reagents can be used in the embodiments of the present invention:
[0023] Strains: Escherichia coli DH5α and Escherichia coli BL21(DE3), both commercially available.
[0024] Vector: pET28a.
[0025] Enzymes and kits: PCR reagents, DpnI enzyme, etc., were purchased from Takara; plasmid extraction kit and gel purification and recovery kit were purchased from TIANGEN; Protein Thermal Shift kit... TM The Dye Kit is from Thermo Fisher Scientific (China) Co., Ltd.
[0026] Culture medium formula: (each ingredient is a percentage by weight)
[0027] Escherichia coli culture medium (LB liquid medium): 0.5% yeast extract, 1% peptone, 1% sodium chloride, balance water.
[0028] LB-KANA medium: 0.5% yeast extract, 1% peptone, 1% sodium chloride, 50 μg / ml kanamycin sulfate, balance water.
[0029] LB-KANA plate: 0.5% yeast extract, 1% peptone, 1% sodium chloride, 1.5% agar, 50 μg / ml kanamycin sulfate, balance water.
[0030] The present invention will be further described below through specific embodiments.
[0031] Example 1
[0032] The construction of recombinant expression vectors and engineered bacteria includes the following steps:
[0033] The three-dimensional structure of the target saccharin neoculin (PDB: 2D04) was obtained by searching the Protein Database (PDB). The NAS chain and NBS chain of the saccharin neoculin are connected by two disulfide bonds. The amino acid sequence of the NAS chain is shown in SEQ ID NO.1, and the amino acid sequence of the NBS chain is shown in SEQ ID NO.2.
[0034] The above NAS chain
[0035] The 27th position asparagine (N) is mutated to glutamate (E)+
[0036] The glutamine (Q) at position 51 mutates to asparagine (N)+
[0037] The methionine (M) at position 70 is mutated to serine (S)+
[0038] The 93rd leucine (L) is mutated to lysine (K)+
[0039] The valine (V) at position 95 is mutated to cysteine (C), resulting in chain A, the amino acid sequence of which is shown in SEQ ID NO.3.
[0040] The above NBS chain
[0041] The histidine (H) at position 11 is mutated to alanine (A)+
[0042] The glycine (G) at position 74 is mutated to alanine (A)+.
[0043] The 78th tryptophan (W) is mutated to serine (S)+.
[0044] The serine (S) at position 103 becomes cysteine (C) +
[0045] The proline (P) at position 106 is mutated to aspartic acid (D) to obtain the B chain, the amino acid sequence of which is shown in SEQ ID NO.4.
[0046] Based on the amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, a biotechnology company was commissioned to synthesize the NAS, NBS, A, and B genes according to the codon preferences of E. coli. The four synthesized gene fragments were then inserted into the pET-28a expression vector via NcoI and XhoI restriction sites, resulting in expression vectors pET28a-NAS, pET28a-NBS, pET28a-A-FM (A recombinant expression vector), and pET28a-B-FM (B recombinant expression vector).
[0047] The above pET28a-NAS, pET28a-NBS, A recombinant expression vector and B recombinant expression vector were transformed into Escherichia coli BL21(DE3) to obtain BL21(DE3)-NAS, BL21(DE3)-NBS, BL21(DE3)-A-FM (A engineered bacteria), and BL21(DE3)-B-FM (B engineered bacteria).
[0048] Example 2
[0049] The preparation and purification of the sweet protein neoculin (WT) and its highly thermally stable mutant (FM) include the following steps:
[0050] (1) Protein expression:
[0051] The specific process is as follows: Single colonies of BL21(DE3)-NAS, BL21(DE3)-NBS, BL21(DE3)-A-FM (A engineered bacteria), and BL21(DE3)-B-FM (B engineered bacteria) obtained in Example 1 were picked and added to 10 ml of LB-KANA medium. The culture was then incubated at 37°C and 220 rpm for 12 h on a shaker. The culture was then transferred to 1 L of LB-KANA medium and incubated at 37°C and 220 rpm until the OD (outlet count) reached. 600 When the concentration is 0.6-0.8, add 500 μL of IPTG (final concentration 0.5 mM) to induce expression. After 4 h, transfer the bacterial culture to a centrifuge bottle and centrifuge at 4000 rpm and 4℃ for 15 min to collect the bacterial cells.
[0052] (2) Protein denaturation and purification:
[0053] The four types of bacterial cells collected in step (1) were resuspended in PBS buffer and then sonicated. The lysed bacterial solutions were aliquoted into centrifuge tubes and centrifuged at 16,000 rpm and 4°C for 30 min to collect inclusion bodies, which were designated as inclusion body NAS, inclusion body NBS, inclusion body A-FM, and inclusion body B-FM. The inclusion bodies were washed three times with PBS + 1% Triton X 100, and then washed twice with PBS buffer to obtain clean inclusion bodies. 5 ml of 6M guanidine hydrochloride solution was added to the collected inclusion bodies to denature them overnight. The supernatant obtained by centrifugation at 16,000 rpm and 4°C for 50 min was the denatured protein, designated as denatured protein NAS, denatured protein NBS, denatured protein A-FM, and denatured protein B-FM. Denatured proteins NAS and NBS were mixed in a 1:1 mass ratio (WT), and denatured proteins A-FM and B-FM were mixed in a 1:1 mass ratio (FM). These were then added to a refolding buffer (400 mM L-arginine, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 100 mM Tirs-HCl, 0.5 mM PMSF) and refolded overnight to obtain the refolded proteins. The two refolded proteins were then subjected to ion-exchange chromatography using an AKTA rapid protein liquid chromatography system and a Q column to obtain purified proteins WT and FM (the final thermostable mutant product of the sweet protein neoculin). The purity of the proteins was then assessed using SDS-PAGE (polyacrylamide gel electrophoresis). Figure 1 As shown, high-purity WT and FM were obtained. Finally, protein concentration was measured using a NanoDrop I spectrophotometer.
[0054] A disulfide bond is formed between cysteine at position 95 of chain A and cysteine at position 103 of chain B, resulting in a mutant of the highly thermally stable sweet protein neoculin (FM).
[0055] Example 3
[0056] To obtain the best experimental results, the entire experiment for protein thermostability testing was conducted on ice. The specific steps are as follows:
[0057] (1) Add Protein Thermal Shift TM Dye(1000x) diluted to 8x.
[0058] The reaction system is shown in Table 1:
[0059] Table 1. Reaction system for thermal stability testing
[0060]
[0061] (2) After the reaction components are mixed evenly, they are added to a 96-well detection plate and centrifuged at 1000 rpm for 1 minute.
[0062] The reaction program settings are shown in Table 2:
[0063] Table 2. Reaction procedures for detecting the thermal stability of proteins.
[0064]
[0065] (3) Export the data to Excel, based on its curve, such as Figure 2 As shown, the melting point temperature T of the protein is calculated by first-order differentiation. m .
[0066] To further characterize thermal stability, WT and FM were diluted to 0.5 mg / ml. WT was heated at 80℃ for 1 h, while FM was heated at 80℃ for 1 h, 2 h, 3 h, and 4 h, respectively. The samples were then centrifuged at 14000 rpm for 20 min, and the supernatant was analyzed by SDS-PAGE. The results are as follows: Figure 3 As shown, WT was no longer detectable in the supernatant, while the FM band was still very obvious, indicating that it can withstand high temperatures and has good thermal stability.
[0067] Example 4
[0068] Sensory evaluation was performed on the FM (a mutant product of neoculin, a sweet protein with high thermal stability) prepared in Example 2.
[0069] The sweetness activity of FM was evaluated using a taste test. To avoid protein aggregation, the protein was centrifuged before determining the concentration and dilution. Before the evaluation, eight evaluators tasted sucrose solutions at concentrations of 0%, 2%, 4%, 6%, and 10% (w / v) and recorded their scores: the average score for the 2% sucrose solution was 1.0 (slightly sweet), the average score for the 4% sucrose solution was 2.0 (sweet), and the average score for solutions above 4% sucrose was 3.0 (very sweet). Subsequently, the evaluators tasted samples at concentrations of 50, 70, 80, 100, 125, 150, and 200 μg / mL, tasting 1 ml at a time and scoring them. After the test, the evaluators rinsed their mouths with distilled water. The numerical scores of all evaluators are summarized in Table 3. The sweetness threshold was determined to be the average concentration reaching a score of 2. Through experimental determination and calculation, the sweetness threshold of FM was found to be 120.625 μg / mL, essentially preserving its sweetness characteristics.
[0070] Table 3. Sweetness scores of mutant products (FM) of neoculin, a highly thermally stable sweet protein.
[0071]
Claims
1. A mutant of the highly thermally stable sweet protein neoculin, said sweet protein neoculin comprising a NAS chain and an NBS chain, said NAS chain and NBS chain being linked by two disulfide bonds; the amino acid sequence of said NAS chain is shown in SEQ ID NO.1; the amino acid sequence of said NBS chain is shown in SEQ ID NO.2; characterized in that... The NAS chain undergoes a mutation at position 27 (asparagine) to glutamic acid, position 51 (glutamine) to asparagine, position 70 (methionine) to serine, position 93 (leucine) to lysine, and position 95 (valine) to cysteine, resulting in chain A. The amino acid sequence of chain A is shown in SEQ ID NO.
3. The NBS chain undergoes a mutation at position 11 (histidine) to alanine, position 74 (glycine) to alanine, position 78 (tryptophan) to serine, position 103 (serine) to cysteine, and position 106 (proline) to aspartic acid, resulting in chain B. The amino acid sequence of chain B is shown in SEQ ID NO.
4. The cysteine at position 95 of chain A and the cysteine at position 103 of chain B form a disulfide bond, resulting in a mutant of the highly thermostable sweet protein neoculin, abbreviated as FM.
2. The method for preparing the mutant of the highly thermally stable sweet protein neoculin as described in claim 1, characterized in that: Includes the following steps: 1) The gene encoding the A strand is gene A, and the gene encoding the B strand is gene B; 2) Construct an A recombinant expression vector containing the A gene and a B recombinant expression vector containing the B gene; 3) Construct engineered bacteria A containing the recombinant expression vector A and engineered bacteria B containing the recombinant expression vector B; 4) The A-engineered bacteria were cultured and expressed to obtain inclusion bodies A-FM; The B-engineered bacteria were cultured and expressed to obtain inclusion bodies B-FM. The inclusion bodies were purified and renatured. The renatured protein was purified to obtain a mutant of the highly thermostable sweet protein neoculin.
3. The application of a mutant of the highly thermally stable sweet protein neoculin according to claim 1 in the preparation of food sweeteners.