Mutant of monellin with high thermal stability, gene and engineered bacteria

Abstract

The application discloses a mutant of single-chain sweet protein with high thermal stability, a gene and an engineering bacterium, wherein the mutant is obtained by mutating isoleucine (I) at the 5th position, glutamic acid (E) at the 23rd position, isoleucine (I) at the 26th position, tyrosine (Y) at the 65th position, glycine (G) at the 83rd position and asparagine (N) at the 90th position in the amino acid sequence shown in SEQ ID NO. 1 to alanine (A), arginine (R) and glutamic acid (E) respectively; the application is based on structural analysis and protein stability calculation aided design, and a mutant of single-chain sweet protein with high thermal stability is obtained by site-directed mutagenesis of the single-chain sweet protein; compared with the single-chain sweet protein, the mutant of single-chain sweet protein with high thermal stability has a T m which is increased by more than 19.4 DEG C and keeps the sweet taste.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and protein modification technology, specifically to mutants, genes, and engineered bacteria of highly thermally stable single-chain sweet proteins. Background Technology

[0002] Sweeteners have become an important part of the human diet, but excessive sugar intake can lead to diseases such as diabetes, obesity, and metabolic syndrome. As people pay more attention to these health problems, the demand for healthy foods is also increasing. Therefore, finding a safe and effective sweetener is of great significance to the food and pharmaceutical fields.

[0003] Monellin, a natural sweet protein, is a small plant protein originally isolated from the berries of an African plant called Dioscoreophyllum umminsii. It has unique advantages such as being safe and healthy, low in calories, high in sweetness and nutrition, and easily absorbed. It consists of two chains (A and B, composed of 44 and 50 amino acids respectively) linked by covalent bonds. However, the low yield and poor thermal stability of this natural plant sweet protein monellin have limited its commercial production and application.

[0004] Single-chain sweet proteins (MNEI, SEQ ID NO.1) created through protein engineering combine two natural chains into one to improve their thermal stability. They have a similar spatial structure to natural sweet proteins, but remain unstable under extreme conditions such as high temperatures, limiting their further applications.

[0005] Protein stability can be characterized as a visual indicator using the protein's denaturation temperature, T. m The higher the fluorescence intensity, the better the protein's stability. A technique published in Curr Protoc Protein Sci in 2015—the thermal shift assay—can be used for rapid and efficient comparison of protein stability. Proteins typically exist in their native state; 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 dye's emission. Therefore, the stability of the protein can be reflected by detecting the fluorescence intensity.

[0006] Sweet proteins have been successfully expressed in various expression systems. Among these systems, the E. coli expression system is considered the simplest and lowest-cost production system.

[0007] Currently, there is an urgent need for sweet proteins that are stable under high-temperature conditions. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide mutants of single-chain sweet proteins with high thermal stability.

[0009] A second objective of this invention is to provide a gene encoding a mutant of the aforementioned highly thermally stable single-stranded sweet protein.

[0010] A third objective of this invention is to provide a recombinant expression vector containing the aforementioned genes.

[0011] A fourth objective of this invention is to provide engineered bacteria comprising the above-described recombinant expression vector.

[0012] A fifth objective of this invention is to provide the application of the mutant of the above-mentioned highly thermally stable single-chain sweet protein in the preparation of food sweeteners.

[0013] The technical solution of this invention is summarized as follows:

[0014] A mutant of a highly thermally stable single-chain sweet protein, wherein the mutant is formed by mutating isoleucine (I) at position 5 of the amino acid sequence shown in SEQ ID NO.1 to glutamic acid (E), glutamic acid (E) at position 23 to alanine (A), isoleucine (I) at position 26 to arginine (R), tyrosine (Y) at position 65 to isoleucine (I), glycine (G) at position 83 to arginine (R), and asparagine (N) at position 90 to glutamic acid (E).

[0015] The gene encoding the mutant of the aforementioned highly thermally stable single-stranded sweet protein.

[0016] Recombinant expression vectors containing the above-mentioned genes.

[0017] Engineered bacteria containing the aforementioned recombinant expression vector.

[0018] The application of the mutants of the above-mentioned highly thermally stable single-chain sweet proteins in the preparation of food sweeteners.

[0019] Advantages of this invention:

[0020] This invention, based on structural analysis and computationally assisted design (COP) of protein stability, involves site-directed mutagenesis of a single-chain saccharin, resulting in a mutant of the single-chain saccharin with high thermal stability. Compared to the original single-chain saccharin, the mutant of this invention exhibits higher Tg. m It increased by more than 19.4°C while maintaining its sweetness. Attached Figure Description

[0021] Figure 1 This image shows SDS-PAGE (polyacrylamide gel electrophoresis) images of single-chain saccharin (MNEI) and a mutant of the highly thermally stable single-chain saccharin (referred to as Mut6-2).

[0022] Figure 2 The results show the thermal stability of single-chain saccharin (MNEI) and a mutant of the highly thermally stable single-chain saccharin (referred to as Mut6-2).

[0023] Figure 3 SDS-PAGE (polyacrylamide gel electrophoresis) images of single-chain saccharin (MNEI) and a mutant of the highly thermally stable single-chain saccharin (referred to as Mut6-2) after heat treatment at 100°C for 1 h. Detailed Implementation

[0024] The following experimental materials and reagents can be used in the embodiments of the present invention:

[0025] Strains and vectors: Escherichia coli DH5α, Escherichia coli BL21(DE3), and vector pET28a were all commercially available.

[0026] 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.

[0027] Culture medium formulation:

[0028] Escherichia coli culture medium (LB medium): 0.5% yeast extract, 1% peptone, 1% NaCl, balance water.

[0029] LB-KANA medium: 0.5% yeast extract, 1% peptone, 1% NaCl, 50 μg / ml kanamycin sulfate, balance water.

[0030] LB-KANA plate: 0.5% yeast extract, 1% peptone, 1% NaCl, 1.5% agar, 50 μg / ml kanamycin sulfate, balance water.

[0031] The present invention will be further described below through specific embodiments.

[0032] Example 1

[0033] Construction of engineered bacteria containing recombinant expression vectors

[0034] The three-dimensional structure of the target single-chain sweet protein (PDB: 1IV7) was obtained by searching the Protein Database (PDB) and the amino acid sequence (SEQ ID NO.1) was obtained. The 5th isoleucine (I) of the sequence was mutated to glutamic acid (E), the 23rd glutamic acid (E) was mutated to alanine (A), the 26th isoleucine (I) was mutated to arginine (R), the 65th tyrosine (Y) was mutated to isoleucine (I), the 83rd glycine (G) was mutated to arginine (R), and the 90th asparagine (N) was mutated to glutamic acid (E). The mutant names, mutation sites, and corresponding amino acid sequences are shown in Table 1.

[0035] Table 1 Recombinant expression vectors and engineered Escherichia coli strains

[0036]

[0037] The sequence SEQ ID NO.2 was synthesized by a biocommercial company. Specifically, the nucleotide sequences of the single-chain sweet protein gene and the mutant of the highly thermostable single-chain sweet protein were codon optimized according to the codon preference of E. coli. The codons were then inserted between the NdeI and BamHI restriction sites of the vector pET28a, respectively, to obtain the recombinant expression vector pET28a-MNEI and the recombinant expression vector pET28a-Mut6-2 containing the gene of the mutant of the highly thermostable single-chain sweet protein. The corresponding recombinant expression vectors are shown in Table 1.

[0038] The two recombinant expression vectors were transformed into Escherichia coli BL21(DE3) to obtain BL21(DE3)-Mut6-2 and BL21(DE3)-MNEI engineered bacteria, respectively.

[0039] Example 2

[0040] Induction and expression of single-chain sucralose protein (MNEI) and its mutant (Mut6-2) with high thermal stability.

[0041] Single colonies of the two positive engineered bacteria obtained in Example 1 were picked and placed in LB-KANA medium, incubated overnight at 37°C and 220 rpm, and then transferred to fresh LB-KANA medium at a ratio of 1:100 and cultured until OD500. 600 When the pH value is between 0.6 and 0.8, add IPTG to a final concentration of 0.5 mM and induce culture at 16°C for 16 h. Collect the cells by centrifugation at 4000 rpm for 15 min using a high-speed centrifuge and store at -80°C.

[0042] Example 3

[0043] Purification and preservation of single-chain sweet proteins and mutants of highly thermally stable single-chain sweet proteins

[0044] (1) Take the bacterial cells out of the -80℃ freezer and thaw them. Resuspend the bacterial cells in 20ml buffer A (50mM Tris-HCl, 150mM NaCl, pH=7.4) and add 200μl of serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF).

[0045] (2) Ultrasonic disruption: After the bacterial cells are disrupted, centrifuge at 18000 rpm for 30 min at 4℃, take the supernatant and add it to the Ni-NTA affinity resin gravity column, and repeat the flow-through twice.

[0046] (3) Wash the column with 8 column volumes of buffer A, and then wash the column with 8 column volumes of buffer B (50mM Tris-HCl, 150mM NaCl, 50mM imidazole, pH=7.4) to remove unbound protein impurities.

[0047] (4) Finally, the target protein was eluted with 8 column volumes of buffer C (50mM Tris-HCl, 150mM NaCl, 300mM imidazole, pH=7.4).

[0048] (5) The target protein was concentrated to 1 ml and subjected to molecular sieve chromatography using an AKTA rapid protein liquid chromatograph and a Superdex 75 10 / 300 column. According to A... 280 The ultraviolet absorption detection value is collected from the sample at the peak position corresponding to the size of the target protein.

[0049] (8) The purity of the protein was identified by SDS-PAGE (polyacrylamide gel electrophoresis), the protein concentration was measured by NanoDrop I spectrophotometer, and the protein was flash-frozen in liquid nitrogen to -80°C.

[0050] Example 4

[0051] Thermal stability testing of single-chain sweet proteins and mutants of highly thermally stable single-chain sweet proteins

[0052] To obtain the best experimental results, the entire experiment was conducted on ice.

[0053] The Applied Biosystems 7500 rapid real-time quantitative PCR system allows for rapid and efficient comparison of the thermal stability differences between MNEI and Mut6-2.

[0054] (1) Add Protein Thermal Shift TM Dye(1000x) diluted to 8x.

[0055] The reaction system is shown in Table 2:

[0056] Table 2. Thermal stability test reaction systems for MNEI and Mut6-2r

[0057]

[0058] (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.

[0059] The reaction program settings are shown in Table 3:

[0060] Table 3. Reaction procedures for detecting the thermal stability of proteins.

[0061]

[0062] (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 By calculating the melting point temperatures of MNEI and Mut6-2, the T0 of MNEI is... m The temperature was 76.6℃. Since the maximum temperature setting of the quantitative PCR system used was 99.9℃, fluorescence values ​​above this temperature could not be detected. Based on the curve, we can determine the T value of Mut6-2. m When the temperature is increased to above 96℃, the thermal stability is significantly improved.

[0063] To further characterize the thermostability of the mutant, MNEI and Mut6-2 were diluted to 0.5 mg / ml and heated at 100 °C for 1 h. The sample was 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, MNEI was no longer detectable in the supernatant, while the Mut6-2 band was still very obvious, indicating that it can withstand high temperatures and has good thermal stability.

[0064] Example 6

[0065] Sensory evaluation of single-chain sweet proteins and single-chain sweet proteins with high thermal stability

[0066] The sweetness activity of MNEI and Mut6-2 was evaluated using a taste test. To avoid protein aggregation, the proteins were centrifuged before determining their concentration and dilution. Evaluators tasted 1 ml of samples at concentrations of 1 μg / ml, 2.5 μg / ml, 5 μg / ml, 7.5 μg / ml, and 10 μg / ml, respectively, and rinsed their mouths with distilled water after each test.

[0067] Experimental results showed that MNEI and Mut6-2 still retain their sweetness characteristics, with a sweetness threshold of 2.5 μg / ml.

[0068] SEQ NO.1(MNEI)

[0069] GEWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTFNKVIRPCMKKTIYENEGFREIKGYEYQLYVYASDKLFRADISEDYKTRGRKLLRFNGPVPPP

[0070] SEQ NO.2Mut6-2(Six mutation sites I5E / E23A / I26R / Y65I / G83R / N90E)

[0071] GEWEEIDIGPFTQNLGKFAVDEANKRGQYGRLTFNKVIRPCMKKTIYENEGFREIKGYEYQLYVIASDKLFRADISEDYKTRRRKLLRFEGPVPPP

Claims

1. A mutant of a highly thermally stable single-chain sweet protein, characterized in that... The mutant is formed by the following mutations in the amino acid sequence shown in SEQ ID NO.1: isoleucine (I) at position 5 is mutated to glutamic acid (E), glutamic acid (E) at position 23 is mutated to alanine (A), isoleucine (I) at position 26 is mutated to arginine (R), tyrosine (Y) at position 65 is mutated to isoleucine (I), glycine (G) at position 83 is mutated to arginine (R), and asparagine (N) at position 90 is mutated to glutamic acid (E).

2. A gene encoding a mutant of the thermally stable single-stranded sweet protein of claim 1.

3. A recombinant expression vector comprising the gene of claim 2.

4. Engineered bacteria comprising the recombinant expression vector of claim 3.

5. The application of the mutant of the highly thermally stable single-chain sweet protein of claim 1 in the preparation of food sweeteners.

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