Thermostable transglutaminase and use thereof

Abstract

This invention relates to a thermostable transglutaminase and its applications, belonging to the fields of genetic engineering and protein engineering. The variant comprises a leader peptide and a mature polypeptide. The parental mature polypeptide of the variant corresponds to the region from position 4 to 13 of the N-terminus of the wild-type SmTG mature polypeptide, which is replaced by a target sequence consisting of n repeating units linked end-to-end. These repeating units have at least 80% identity with the original sequence and undergo substitution at position 10, with at least two repeating units having different substituted amino acids at position 10. The remaining amino acid region of the parental mature polypeptide has at least 85% sequence identity with the remaining region of the wild-type SmTG mature polypeptide. Compared to the parent, this variant maintains transglutaminase activity while significantly extending its heat-inactivation half-life at 60°C by at least 0.3 times.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and protein engineering, and in particular to a thermostable transglutaminase and its applications, especially a transglutaminase (TGase) variant with significantly improved thermostability, its encoded polynucleotide, expression vector, host cell, and its application in food processing (such as meat products, dairy products, and plant protein crosslinking). Background Technology

[0002] Glutamine transaminase (SmTG) derived from *Streptomyces mobaraensis* is a monomeric enzyme with significant industrial application value. It efficiently catalyzes the covalent binding of the γ-carboxamide group (acting as an acyl donor) of glutamine residues in protein peptide chains to various acyl acceptors (usually the ε-amino group of lysine residues) via amide transfer reactions, thereby forming strong ε-(γ-glutamyl)-lysine isopeptide bonds within or between protein molecules. This irreversible cross-linking not only enables the polymerization of large proteins but also mediates the site-specific modification of small molecules. Based on its superior protein modification capabilities, TGase is hailed as the "bio-glue" of the food industry and is currently widely used in meat products, dairy products, aquatic products, and baking. For example, it can restructure minced meat and improve its gel strength, improve the texture of yogurt and prevent whey separation, or strengthen the gluten network to improve dough elasticity, thus comprehensively improving the appearance, taste, and shelf-life stability of food products.

[0003] However, with the deepening of SmTG research and the expansion of its application scenarios, its inherent structural limitations have gradually become apparent—poor thermal stability has become the main bottleneck restricting its efficient application under extreme processing conditions (especially in specific food sectors). Taking modern beef recombination and processing technology as an example, TGase is often required to maintain its catalytic activity for several hours at relatively high temperatures of around 60°C to ensure sufficient cross-linking and gelation of the protein network. However, SmTG in its natural state is highly susceptible to structural unfolding and irreversible inactivation above 50°C. This rapid inactivation and consumption under high-temperature conditions necessitates the use of excessively high doses of enzyme to compensate for the loss of activity in actual production. This not only significantly increases industrial production costs but also brings potential risks such as enzyme residues and uneven product quality.

[0004] To overcome this challenge of thermal stability, researchers have conducted extensive research on molecular modification. Currently, a range of advanced protein engineering techniques are widely used in the modification of SmTG, including directed evolution based on error-prone PCR (used to explore random mutations across the entire sequence), semi-rational design focusing on key structural domains (such as site-directed saturation mutagenesis and DNA shuffling), and rational design based on three-dimensional structures. Although existing research has made some progress through traditional strategies such as introducing non-natural disulfide bonds, optimizing surface charge distribution, and constructing internal salt bridges, the overall modification effect still faces a ceiling. More challenging is the "activity-stability trade-off" effect prevalent in protein molecular evolution, which is particularly evident in SmTG: while attempting to improve thermal resistance by increasing the rigidity of local molecular structures, it often disrupts the conformational flexibility required for substrate binding or catalytic reactions, ultimately leading to a significant decrease in the catalytic efficiency of mutants at conventional operating temperatures. How to break this "you can't have your cake and eat it too" impasse remains a pressing challenge in the field of enzyme engineering. Summary of the Invention

[0005] To address the technical problem of insufficient thermostability of existing glutamine transaminases, this invention provides a thermostable glutamine transaminase and its applications. This invention utilizes a specific region sequence substitution strategy to obtain a highly thermostable glutamine transaminase variant. The inventors have creatively discovered that modifying a specific flexible region at the N-terminus of the TG enzyme through "direct tandem insertion of specific multiple repeat units" and "key site substitution" can significantly improve the enzyme's half-life and residual activity at high temperatures, and this modification strategy is universally applicable to the TG enzyme family with certain sequence homology.

[0006] The first objective of this invention is to provide a glutamine transaminase variant comprising an amino acid region, except for the target region at positions 4 to 13 of the parental mature polypeptide as shown in SEQ ID NO: 1, having at least 85% sequence identity with the remaining region of wild-type SmTG. And / or, one or more sequences in the N-terminal amino acid region from position 4 to position 13 of the parental mature polypeptide are replaced by a target sequence, wherein the target sequence is composed of n repeating units directly linked end-to-end, where n is an integer from 2 to 15, and the repeating units have at least 80% sequence identity with the N-terminal amino acid region from position 4 to position 13 of the wild-type mature polypeptide. The sequence of the N-terminal amino acid region from position 4 to position 13 of the parental mature polypeptide is DRVTPPAEPL.

[0007] Furthermore, the sequence identity is preferably 85-99%, more preferably 89-99%, or 89-98.75%. For example, it can be any interval value between any two values ​​from 85, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, etc.

[0008] Furthermore, in this invention, n is preferably an integer from 2 to 13; more preferably, n is an integer from 2 to 10; more preferably, n is an integer from 2 to 5.

[0009] In this invention, the parental mature polypeptide of the glutamine transaminase variant is selected from any of the following sequences: (1) Wild-type SmTG mature polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 1; (2) A homologous mature polypeptide that has approximately 98.75% sequence identity with the wild-type SmTG mature polypeptide except for the target region corresponding to positions 4 to 13 of the wild-type SmTG mature polypeptide, and its amino acid sequence is shown in SEQ ID NO: 2; (3) A homologous mature polypeptide that has approximately 92.52% sequence identity with the wild-type SmTG mature polypeptide except for the target region corresponding to positions 4 to 13 of the wild-type SmTG mature polypeptide, and its amino acid sequence is shown in SEQ ID NO: 3; (4) A homologous mature polypeptide that has approximately 90.03% sequence identity with the wild-type SmTG mature polypeptide except for the target region corresponding to positions 4 to 13 of the wild-type SmTG mature polypeptide, and its amino acid sequence is shown in SEQ ID NO: 4; (5) A homologous mature polypeptide that has approximately 89.72% sequence identity with the wild-type SmTG mature polypeptide except for the target region corresponding to positions 4 to 13 of the wild-type SmTG mature polypeptide, and its amino acid sequence is shown in SEQ ID NO: 5.

[0010] In this invention, the variant possesses glutamine transaminase activity and has a thermal inactivation half-life t at 60°C. 1 / 2 The length of the variant mature polypeptide is extended by at least 0.3 to 0.4 times compared to the parental mature polypeptide; wherein, the parental mature polypeptide refers to the mature polypeptide obtained by replacing the target sequence back to the original sequence DRVTPPAEPL at positions 4 to 13 of the wild-type SmTG mature polypeptide in the variant mature polypeptide.

[0011] In some embodiments of the present invention, the amino acid at the position corresponding to the 10th position (alanine, Ala) of each repeating unit in the wild-type SmTG mature polypeptide is independently selected from one or more of alanine (Ala), histidine (His), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), asparagine (Asn), leucine (Leu), proline (Pro), glutamine (Gln), tryptophan (Trp), serine (Ser), arginine (Arg), valine (Val), glycine (Gly), tyrosine (Tyr), and cysteine ​​(Cys). Further, it is preferably one or more of glutamic acid (Glu), alanine (Ala), valine (Val), aspartic acid (Asp), phenylalanine (Phe), proline (Pro), and glutamine (Gln).

[0012] In some embodiments of the present invention, among the n repeating units, at least two repeating units have different types of amino acids substituted at the 10th position.

[0013] In some embodiments of the present invention, the combined sequence formed by the substitution of the n repeating units at the 10th amino acid position, arranged sequentially from the N-terminus to the C-terminus, satisfies any of the following conditions: (1) When the parental mature polypeptide is a wild-type SmTG mature polypeptide with the sequence shown in SEQ ID NO: 1, the combined sequence is selected from: SA, n=2; DHA, n=3; CNCP, n=4; CVVLE, n=5; EEWFE, n=5; (2) When the parental mature polypeptide is the homologous mature polypeptide shown in SEQ ID NO:2, the combined sequence is selected from: CPQG, n=4; VVA, n=3; (3) When the parental mature polypeptide is the homologous mature polypeptide shown in SEQ ID NO:3, the combined sequence is selected from: DA, n=2; QLG, n=3; RH, n=2; DEVALF, n=6; VVEFA, n=5; IPVDFEFGMV, n=10; (4) When the parental mature polypeptide is the homologous mature polypeptide shown in SEQ ID NO:4, the combined sequence is selected from: WQSRRDPEFA, n=10; NQVLE, n=5; LAV, n=3; VE, n=2; LEVA, n=4; RIDEW, n=5; GYE, n=3; ALPA, n=4; PIQMRHDEFD, n=10; RDFWPY, n=6; GE, n=2; (5) When the parental mature polypeptide is the homologous mature polypeptide shown in SEQ ID NO:5, the combined sequence is selected from: QP, n=2; AHDEFNNLDPQFE, n=13.

[0014] Further, the glutamine transaminase variants include SMTG-B1 (SEQ ID NO: 11), SMTG-B2 (SEQ ID NO: 12), SMTG-B3 (SEQ ID NO: 13), SMTG-B4 (SEQ ID NO: 14), SMTG-B5 (SEQ ID NO: 15), A1a (SEQ ID NO: 7), A1a-B1 (SEQ ID NO: 16), A1a-B2 (SEQ ID NO: 17), PD8 (SEQ ID NO: 8), PD8-B1 (SEQ ID NO: 18), PD8-B2 (SEQ ID NO: 19), PD8-B3 (SEQ ID NO: 20), PD8-B4 (SEQ ID NO: 21), and PD8-B5 (SEQ ID NO: 25). PD9-B1 (SEQ ID NO: 22), PD9-B2 (SEQ ID NO: 25), PD9-B3 (SEQ ID NO: 26), PD9-B4 (SEQ ID NO: 27), PD9-B5 (SEQ ID NO: 28), PD9-B6 (SEQ ID NO: 29), PD9-B7 (SEQ ID NO: 30), PD9-B8 (SEQ ID NO: 31), PD9-B9 (SEQ ID NO: 32), PD9-B10 (SEQ ID NO: 33), PD9-B11 (SEQ ID NO: 34), SNTG (SEQ ID NO: 10), SNTG-B1 (SEQ ID NO: 35), and SNTG-B2 (SEQ ID NO: 36).

[0015] A second object of the present invention is to provide an isolated polynucleotide encoding the glutamine transaminase variant; preferably, the nucleotide sequence of the polynucleotide is codon-optimized for a host cell expression system.

[0016] A third object of the present invention is to provide an expression vector comprising the aforementioned polynucleotide, wherein the polynucleotide is linked to at least one control sequence adapted to function in a host cell.

[0017] A fourth object of the present invention is to provide a host cell comprising the expression vector or nucleic acid construct described above.

[0018] In some embodiments of the present invention, the host cell is selected from Escherichia coli, Streptomyces sp. Bacillus subtilis, or Pichia pastoris.

[0019] A fifth object of the present invention is to provide a composition comprising the maturation enzyme of the aforementioned glutamine transaminase variant, and a food industry-acceptable carrier or excipient.

[0020] A sixth object of the present invention is to provide a food additive composition comprising the aforementioned transglutaminase variant for promoting protein cross-linking in food handling, processing and conversion to maintain or improve the quality, consistency, elasticity, moisture or viscosity of foods such as meat products, dairy products or plant protein products.

[0021] The present invention also provides a method for generating the said glutamine transaminase variant, comprising the following steps: (1) Culture host cells under conditions suitable for expressing the variant; (2) Recover the supernatant or cell lysate containing the variant from the culture; (3) The variant is separated and purified by at least one purification step of ion exchange chromatography, hydrophobic interaction chromatography or affinity chromatography.

[0022] The present invention also provides a method for altering the appearance, texture and / or stability of food, the method comprising, during food processing, treating the transglutaminase variant with a protein-containing food ingredient under the following conditions: a temperature of 40°C to 70°C, a pH of 5.5 to 8.0, and a processing time of 10 minutes to 120 minutes.

[0023] This invention also provides a method for improving the thermostability of glutamine transaminase: the method includes modifying a mature polypeptide of parental glutamine transaminase, wherein the parental mature polypeptide and the wild-type SmTG mature polypeptide (amino acid sequence as shown in SEQ ID NO: 1) have at least 85% sequence identity with wild-type SmTG except for the target region corresponding to positions 4 to 13 of the wild-type SmTG mature polypeptide; the modification includes replacing the amino acid region corresponding to positions 4 to 13 of the N-terminus of the wild-type SmTG mature polypeptide with the target sequence, wherein the target sequence is composed of n repeating units directly connected end to end, where n is an integer from 2 to 15; wherein each repeating unit has at least 80% sequence identity with the DRVTPPAEPL sequence, and each repeating unit independently undergoes amino acid substitution or remains alanine at position 10 (alanine, Ala) of the wild-type SmTG mature polypeptide; among the n repeating units, at least two repeating units have different types of amino acids substituted at position 10.

[0024] The technical solution of the present invention has the following advantages compared with the prior art: This invention breaks away from conventional single-point or multi-point random mutation strategies and proposes a novel protein engineering scaffold of "flexible region repeating unit tandem + specific site forced substitution", which has high modification efficiency and significantly improved thermal stability.

[0025] The modification strategy of this invention is not only applicable to wild-type SmTG from Streptomyces mobara, but can also be successfully transferred to SmTG variants with 98.75% or even 89.72% homology, which has extremely high universality and industrial application value.

[0026] The mutant modified in this invention has a significantly extended half-life at 60°C, making it perfectly adaptable to special processes such as high-temperature recombination of meat products and pasteurization of dairy products, greatly expanding the application scenarios of TG enzyme. Attached Figure Description

[0027] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 The heat inactivation half-life t of some glutamine transaminase mutants in this invention at 60°C is... 1 / 2 Compared to the half-life t of the parental mature polypeptide 1 / 2 .

[0028] Figure 2 This refers to the specific enzyme activity of some glutamine transaminase mutants in this invention at different temperatures.

[0029] Figure 3 This is a site map showing the sequences of the parental precursor peptide and the mature polypeptide in this invention. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0031] This invention relates to glutamine transaminase variants that have been improved compared to parental glutamine transaminases. More specifically, this invention relates to glutamine transaminase variants with improved thermostability compared to parental glutamine transaminases (particularly the glutamine transaminase shown in SEQ ID NO: 1).

[0032] I. Enzyme Activity Assay of Parental and Variant in this Invention Enzyme activity assay substrate solution A: 200 mM Tris-HCl, 100 mM hydroxylamine hydrochloride, 10 mM reduced glutathione, 30 mM N-benzyloxycarbonyl-L-glutamylglycine, pH adjusted to 6.0.

[0033] Enzyme activity test stop solution B: Mix equal volumes of 3 mol / L HCl, 5% FeCl3·6H2O (dissolved in 0.1 mol / L HCl), and 12% TCA (trichloroacetic acid) to complete the solution preparation.

[0034] Enzyme activity definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of product from 1 μmol of substrate per minute.

[0035] Preparation of the standard curve for enzyme activity assay: Weigh 648 mg of standard L-glutamic acid-γ-monoisohydroxamic acid and add 100 mL of Tris-HCl 200 mM, pH 6.0 solution. Dilute this solution five times using a 2-fold dilution method with Tris-HCl 200 mM, pH 6.0 solution. Incubate this solution and substrate solution A separately at 37°C for 5 min. Then, add 60 μL of the standard solution to 150 μL of substrate solution A, incubate at 37°C for 10 min, add 60 μL of stop solution B, centrifuge at 10000 rpm for 1 min, and measure the absorbance at 525 nm using 200 μL of the supernatant. Plot a linear relationship between absorbance and the amount of oxime acid. Obtain a conversion coefficient K from the slope of the line. After obtaining the absorbance in the sample enzyme activity assay, the amount of oxime acid produced can be calculated using K.

[0036] Assay method: Protein sample and 150 μL of substrate solution A were incubated at 37°C for 5 min. Then, 60 μL of protein sample was added to 150 μL of substrate solution A, and the mixture was incubated at 37°C for 10 min, followed by the addition of 60 μL of test stop solution B. The reaction mixture was centrifuged at 10,000 rpm for 1 min, and the absorbance of 200 μL of the supernatant was measured at 525 nm. For the blank control, 60 μL of test stop solution B was added to 60 μL of protein sample, followed by the addition of 150 μL of substrate solution A. After centrifugation at 10,000 rpm for 1 min, the absorbance of 200 μL of the supernatant was measured at 525 nm. The absorbance of the experimental group was subtracted from that of the control group, and the result was substituted into the enzyme activity standard curve to obtain the enzyme activity corresponding to the mass of protein added.

[0037] Definition of specific enzyme activity: Specific enzyme activity refers to the number of enzyme activity units per milligram of protein under specific conditions, usually expressed as U / mg protein, reflecting the catalytic ability of a unit mass of enzyme protein.

[0038] Determination of specific enzyme activity at different temperatures: Based on the method for measuring enzyme activity described above, after measuring the enzyme activity of the added enzyme at different temperatures, the specific enzyme activity (U / mg) can be obtained by dividing the enzyme activity by the protein concentration of the added enzyme.

[0039] II. Thermal Stability Determination To determine the thermostability of wild-type SmTG and its mutants at 60°C, the protein solution was diluted to 0.5 mg / mL and pre-alimited into 1.5 mL thin-walled centrifuge tubes (300 μL per tube). A multi-tube simultaneous water bath incubation method was used, with one tube removed every 30 seconds from 0–3 min, and samples taken every 2 min from 3–10 min. Samples were then taken at 15, 20, and 30 min. All samples were immediately immersed in an ice-water mixture for instantaneous cooling to terminate thermal denaturation. The remaining enzyme activity of each sample was then measured, with the 0 min group as the initial activity (100%). The Exponential-ExpDec1 model in Origin 2018 software was used to perform nonlinear fitting on the curve of the percentage of remaining enzyme activity over time. The time corresponding to the enzyme activity decreasing to 50% of the initial value was calculated, which is the half-life (t) of the protein at 60°C. 1 / 2 .

[0040] III. Some of the raw materials used in this invention: 1. The BL21(DE3) competent cells involved in this invention were purchased from Sangon Biotech (Shanghai) Co., Ltd., the pET-22b(+) plasmid was purchased from Suzhou Genewiz Co., Ltd., the purified neutral protease was purchased from Sangon Biotech (Shanghai) Co., Ltd. (catalog number A002100-0050, the non-pure neutral protease was purchased from Beijing Solarbio Science & Technology Co., Ltd. (catalog number Z8031), the PrimeSTARMax DNA Polymerase (catalog number R045A) and QuickCutDpnI (catalog number 1609) were purchased from BioNTech Biotechnology (Beijing) Co., Ltd., and the PCR products were ligated using Taiji assembly. The T5 exonuclease (catalog number M0663S) was purchased from NEB, and the Bradford protein concentration assay kit (catalog number C600641-0100) was purchased from Sangon Biotech (Shanghai) Co., Ltd. Unless otherwise specified, all raw materials are commercially available reagents commonly used in the field.

[0041] 2. The culture medium involved in this invention is as follows: LB liquid medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, ampicillin 100 μg / L.

[0042] LB solid medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 15 g / L, ampicillin 100 μg / L.

[0043] Example 1 1. Construction and screening of parents and mutants (1) The polypeptide sequence of the parental gene (100% homologous enzyme SmTG is shown in SEQ ID NO:1, and the nucleic acid sequence is shown in SEQ ID NO:6; the polypeptide sequence of the 98.75% homologous enzyme A1a is shown in SEQ ID NO:2, and the nucleic acid sequence is shown in SEQ ID NO:7) As shown, the polypeptide sequence of the 92.52% homologous enzyme PD8 is shown in SEQ ID NO:3, and the nucleic acid sequence is shown in SEQ ID NO:8; the polypeptide sequence of the 90.03% homologous enzyme PD9 is shown in SEQ ID NO:4, and the nucleic acid sequence is shown in SEQ ID NO:9; the polypeptide sequence of the 89.72% homologous enzyme SNTG is shown in SEQ ID NO:5, and the nucleic acid sequence is shown in SEQ ID NO:10, along with the corresponding leader peptide sequences (already indicated in the corresponding amino acid sequences of the parent). These sequences were synthesized by Suzhou Genewiz Company and pET-22b-pro-tgase was obtained by inserting the restriction endonuclease sites NdeI and XhoI into the plasmid pET-22b(+). This was used for polymerase chain reaction (PCR). Primers for CR were synthesized by Sangon Biotech (Shanghai) Co., Ltd. When creating mutants, different pET-22b-pro-tgase enzymes were used as templates, and a saturation mutation was performed at position 10 of the mature parental polypeptide using the 22c-trick method. These upstream and downstream primers were compatible with the five enzymes mentioned above with different homologousities (100%, 98.75%, 92.52%, 90.03%, and 89.72%), as shown in Table 1. PCR yielded linear DNA with substitutions at positions 4-13 of the mature polypeptide. This DNA was then digested with the template and assembled using Taichi to obtain the recombinant plasmid. The PCR procedure followed the PrimeSTAR Max DNA Polymerase instruction manual from Baori Biotechnology (Beijing) Co., Ltd.

[0044] Table 1 Primer gene sequences

[0045] Note: The lowercase letters in the primers represent the 10th mutation site. (2) The parental plasmid and the recombinant plasmid were transformed with BL21(DE3), spread on LB solid medium, and cultured at 37℃ for 10h. The transformants were picked and inoculated into LB liquid medium (containing 100μg / mL ampicillin) and cultured at 37℃ for 10h.

[0046] (3) Screening for heat-resistant mutants: 1) High-throughput primary screening of high heat-resistant mutants using deep-well plates: Taking parental SMTG as an example, high-throughput culture screening was performed using 96-well deep-well plates. Single colonies transformed with the recombinant plasmid using SMTG as a template were picked and inoculated into each well of a 96-well deep-well plate containing liquid culture medium (containing 100 μg / mL ampicillin). Three colonies of parental SMTG were also picked as parallel controls. The deep-well plate was placed in a shaker and cultured for *E. coli* at 37°C and 220 rpm. The OD of the bacterial culture was then measured. 600 When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.01 mM to induce the expression of the recombinant protein. After adding IPTG, the culture temperature was lowered to 20°C, and the cells were cultured with shaking at 220 rpm for 24 h. After fermentation, the cells were collected by centrifugation, lysozyme was added to disrupt the cells, and the zymogen was activated using purified neutral protease to obtain the crude enzyme solution.

[0047] A portion of the crude enzyme solution was used to determine the initial enzyme activity; another equal volume of crude enzyme solution was placed in a PCR instrument for heat stress treatment. The specific heat stress temperature and time were determined based on the thermostability parameters of the parent lines. The residual enzyme activity after heat stress treatment was measured, and the ratio of residual enzyme activity to initial enzyme activity in each well was calculated. Clones corresponding to wells with a ratio significantly higher than that of the wild-type control were screened as candidate mutants. Plasmids were extracted from the candidate mutants for sequencing verification to obtain the mutant sequence, and further screening was performed by shake-flask fermentation to verify their true thermostability.

[0048] Verification of thermal stability through shake-flask culture: Candidate mutants SMTG-B1, SMTG-B2, SMTG-B3, SMTG-B4, and SMTG-B5, verified by sequencing, and the wild-type control strain were inoculated into test tubes containing liquid culture medium (containing 100 μg / mL ampicillin) and cultured overnight at 37°C with shaking at 220 rpm to prepare seed culture. The seed culture was then transferred to shake flasks containing fresh fermentation medium at an inoculation rate of 1%–5% (v / v). The cultures were incubated at 37°C with shaking at 220 rpm until OD reached... 600When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.1 mM, and the temperature was then lowered to 20°C. The culture was continued with shaking induction for 24 h. After fermentation, the sample was centrifuged at 7500 rpm for 10 min, and the cells were collected. One-fifth of the total fermentation broth volume of 50 mM Tris-HCl was added to resuspend the cells at pH 7.5. The sample was placed on ice for ultrasonic cell disruption, and the supernatant was collected after centrifugation at 12000 rpm for 15 min. 200 mg / mL of a neutral protease non-pure enzyme solution (neutral protease purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number Z8031) was added to the supernatant to activate transglutaminase. The incubation conditions were 37°C for 30 min. After incubation, the samples were centrifuged at 12,000 rpm for 20 min, and the supernatant was collected. Protein purification was then performed using nickel ion affinity purification. Specifically, the nickel ion affinity purification column was equilibrated with water and Tris-HCl 50 mM, pH 7.5 solution, respectively. The sample was then passed through the column, and the column was washed with Tris-HCl 50 mM and 20 mM imidazole, pH 7.5 solution until conductivity was re-equilibrated. The protein was then eluted with Tris-HCl 50 mM and 180 mM imidazole, pH 7.5 solution, and the protein was recovered. The entire purification process was completed using a nickel column. The protein collection time and amount were determined by observing the A280 wavelength absorption peak. Protein concentration was determined using the Bradford assay, specifically according to the Bradford Protein Assay Kit instructions from Sangon Biotech (Shanghai) Co., Ltd. The half-life of SMTG, SMTG-B1, SMTG-B2, SMTG-B3, SMTG-B4 and SMTG-B5 at 60℃ was determined through thermal stability experiments.

[0049] Example 2: Modification and thermal stability evaluation of wild-type SmTG (100% homology benchmark) The wild-type SmTG has the same sequence as the commercially available TG enzyme. The specific strain is DSM40847, GenBank:HF968462.1, and its nucleic acid sequence is shown in SEQ ID NO:6.

[0050] Following the method in Example 1, the sequence corresponding to positions 4 to 13 of the N-terminus of the mature polypeptide in the 100% homologous enzyme was replaced with a tandem repeat of DRVTPPAEPL, wherein the alanine corresponding to position 10 was forcibly mutated, such as DRVTPPSEPLDRVTPPVEPLDRVTPPEEPLDRVTPPFEPL. After thermostability screening, five mutants with high thermostability were obtained: SMTG-B1 (SEQ ID NO: 11), SMTG-B2 (SEQ ID NO: 12), SMTG-B3 (SEQ ID NO: 13), SMTG-B4 (SEQ ID NO: 14), and SMTG-B5 (SEQ ID NO: 15). The results are shown in Table 2 below. Table 2. Thermal stability results of some excellent mutants with wild-type SmTG as parent.

[0051] The results show that the direct tandem substitution strategy of the present invention can greatly improve the thermal stability of the SmTG variant.

[0052] Example 3: Validation of the modification strategy of this invention on 98.75% homologous TG enzymes To verify whether the region substitution strategy of the present invention has sequence universality, a homologous TG enzyme (sequence shown in SEQ ID NO:2) with 98.75% sequence identity with the wild-type SmTG mature polypeptide except for the target region corresponding to the 4th to 13th positions of the wild-type SmTG mature polypeptide was selected as the parent and the same modification was performed.

[0053] Following the method in Example 1, the sequence corresponding to positions 4 to 13 of the N-terminus of the mature polypeptide in 98.75% of the homologous enzymes was replaced with the tandem repeat unit of the present invention, and a forced mutation was performed at position 10. Two mutants with high thermal stability, A1a-B1 (SEQ ID NO: 16) and A1a-B2 (SEQ ID NO: 17), were obtained by screening. The experimental results are shown in Table 2 below: Table 3. Thermostability results of mutants with 98.75% homologous TG enzymes as parents.

[0054] The results confirmed that this design approach is fully applicable to TG enzymes with 98.75% homology.

[0055] Example 4: Extended application of the modification strategy of this invention on 92.52% homologous TG enzymes Further, homologous TG enzymes (sequences such as SEQ ID NO:3) that differed significantly from the wild-type SmTG mature polypeptide in all amino acid regions except for the target region corresponding to positions 4 to 13 of the wild-type SmTG mature polypeptide (sequence identity of 92.52%) were selected as parents. Parental PD8 and parental PD9 were obtained by redesigning A1a based on variant A1a using PROSS.

[0056] Following the same modifications, six mutants with significantly improved thermal stability were successfully screened: PD8-B1 (SEQ ID NO: 18), PD8-B2 (SEQ ID NO: 19), PD8-B3 (SEQ ID NO: 20), PD8-B4 (SEQ ID NO: 21), PD8-B5 (SEQ ID NO: 22), and PD8-B6 (SEQ ID NO: 23). Specific data are shown in Table 4. Table 4. Thermostability results of mutants with 92.52% homologous TG enzymes as parents.

[0057] The results confirmed that this design approach is applicable to TG enzymes with 92.57% homology.

[0058] Example 5: Extended application of the modification strategy of this invention on 90.03% homologous TG enzymes Further, homologous TG enzymes (sequences such as SEQ ID NO:4) that differ significantly from the wild-type SmTG mature polypeptide in the remaining amino acid regions (sequence identity of 90.03%), except for the target region corresponding to positions 4 to 13 of the wild-type SmTG mature polypeptide, were selected as parents.

[0059] Following the same modifications, eleven mutants with significantly improved thermal stability were successfully screened: PD9-B1 (SEQ ID NO: 24), PD9-B2 (SEQ ID NO: 25), PD9-B3 (SEQ ID NO: 26), PD9-B4 (SEQ ID NO: 27), PD9-B5 (SEQ ID NO: 28), PD9-B6 (SEQ ID NO: 29), PD9-B7 (SEQ ID NO: 30), PD9-B8 (SEQ ID NO: 31), PD9-B9 (SEQ ID NO: 32), PD9-B10 (SEQ ID NO: 33), and PD9-B11 (SEQ ID NO: 34). Specific data are shown in Table 5. Table 5. Thermostability results of mutants with 90.03% homologous TG enzymes as parents.

[0060] The results confirmed that this design approach is applicable to TG enzymes with 90.03% homology.

[0061] In this study, the variant PD9-B1 had a half-life of 673.05±2.41 min at 70 °C, which was nearly 900% higher than that of the parent PD9. Moreover, with such a significant improvement in thermal stability, the specific enzyme activity at its respective optimal temperature was also nearly 200% higher than that of the parent PD9.

[0062] It should be noted that in the PD9-B1 sequence, based on the forced mutation of the 10th amino acid in the repeating segment, the strategy still works after the original V (valine) is mutated to I (isoleucine) at position 6 of the 7th repeating segment. This shows that it is reasonable that "the repeating unit has at least 80% sequence identity with the amino acid region from position 4 to position 13 of the N-terminus of the wild-type mature polypeptide".

[0063] Example 6: Extended application of the modification strategy of this invention on 89.72% homologous TG enzymes Further, homologous TG enzymes (sequences such as SEQ ID NO:5) that differ significantly from wild-type SmTG mature peptides except for the target amino acid regions corresponding to positions 4 to 13 of wild-type SmTG mature peptides (sequence identity approximately 89.72%) were selected as parents.

[0064] The source of SNTG is NCBI Reference Sequence: WP_398270749.1. Following the same modifications, mutants SNTG-B1 (SEQ ID NO: 35) and SNTG-B2 (SEQ ID NO: 36) with significantly improved thermal stability were successfully screened. Specific data are shown in Table 6. Table 6. Thermostability results of mutants with 89.72% homologous TG enzymes as parents.

[0065] The "direct tandem + forced mutation at position 10" modification strategy of this invention is effective not only on wild-type SmTG (100%), but also achieves consistent and predictable improvements in thermostability on other TG enzymes with a sequence difference spanning 10% (98.75%, 92.52%, 90.03%, and 89.72% homology). This fully demonstrates that the "at least 85% sequence identity" requirement defined in this invention has a solid experimental and theoretical basis.

[0066] Glutamine transaminase consists of a precursor peptide and a mature polypeptide. In the parental mature polypeptide, the first D at the N-terminus of DSDDVRTPPAEPL~ is designated as position 1. The N-terminal residual amino acid FRAP remaining after cleavage of the precursor peptide using metalloproteinase TAMEP or neutral protease is not part of the mature polypeptide but belongs to the precursor peptide. (Specific details are as follows...) Figure 3 As shown.

[0067] The protein and base sequences used in this invention are as follows: The underlined portion represents the precursor peptide, while the un-underlined portion represents the mature polypeptide.

[0068] SMTG-protein (SEQ ID NO:1)

[0069] A1a-protein (SEQ ID NO:2)

[0070] PD8-protein (SEQ ID NO:3)

[0071] PD9-protein (SEQ ID NO:4)

[0072] SNTG-protein>(SEQ ID NO:5)

[0073] SMTG> (SEQ ID NO:6)

[0074] A1a>(SEQ ID NO:7)

[0075] PD8 (SEQ ID NO:8)

[0076] PD9> (SEQ ID NO:9)

[0077] SNTG> (SEQ ID NO:10)

[0078] SMTG-B1 (SEQ ID NO:11)

[0079] SMTG-B2>(SEQ ID NO:12)

[0080] SMTG-B3>(SEQ ID NO:13)

[0081] SMTG-B4>(SEQ IDNO:14)

[0082] SMTG-B5>(SEQ ID NO:15)

[0083] A1a-B1>(SEQ ID NO:16)

[0084] A1a-B2>(SEQ ID NO:17)

[0085] PD8-B1>(SEQ ID NO:18)

[0086] PD8-B2>(SEQ ID NO:19)

[0087] PD8-B3>(SEQ ID NO:20)

[0088] PD8-B4>(SEQ ID NO:21)

[0089] PD8-B5>(SEQ ID NO:22)

[0090] PD8-B6>(SEQ ID NO:23)

[0091] PD9-B1>(SEQ ID NO:24)

[0092] PD9-B2>(SEQ ID NO:25)

[0093] PD9-B3>(SEQ ID NO:26)

[0094] PD9-B4>(SEQ ID NO:27)

[0095] PD9-B5>(SEQ ID NO:28)

[0096] PD9-B6>(SEQ ID NO:29)

[0097] PD9-B7>(SEQ ID NO:30)

[0098] PD9-B8>(SEQ ID NO:31)

[0099] PD9-B9>(SEQ ID NO:32)

[0100] PD9-B10>(SEQ ID NO:33)

[0101] PD9-B11>(SEQ ID NO:34)

[0102] SNTG-B1>(SEQ ID NO:35)

[0103] SNTG-B2>(SEQ ID NO:36)

[0104] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A variant of transglutaminase, characterized in that, The polynucleotide sequences encoding the glutamine transaminase variants include one of SMTG-B1, SMTG-B2, SMTG-B3, SMTG-B4, SMTG-B5, A1a-B1, A1a-B2, PD8-B1, PD8-B2, PD8-B3, PD8-B4, PD8-B5, PD8-B6, PD9-B1, PD9-B2, PD9-B3, PD9-B4, PD9-B5, PD9-B6, PD9-B7, PD9-B8, PD9-B9, PD9-B10, PD9-B11, SNTG-B1, and SNTG-B2, as shown in SEQ ID NO:11 to SEQ ID NO:36, respectively.

2. An isolated polynucleotide, characterized in that, The polynucleotide encodes the glutamine transaminase variant of claim 1.

3. An expression carrier, characterized in that, The expression vector comprises the polynucleotide of claim 2, and the polynucleotide is linked to at least one control sequence adapted to function in a host cell.

4. A host cell, characterized in that, The host cell comprises the expression vector of claim 3.

5. The host cell according to claim 4, characterized in that, The host cell is selected from Escherichia coli. Escherichia coli Streptomyces Streptomyces sp.、 Bacillus subtilis Bacillus subtilis or Pichia pastoris Pichia pastoris .

6. A composition, characterized in that, It includes the glutamine transaminase variant of claim 1, and a food industry-acceptable carrier or excipient.

7. A food additive composition, characterized in that, It includes the glutamine transaminase variant described in claim 1.

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