A glutamine transaminase variant having improved thermal stability

Abstract

The application discloses a glutamine transaminase variant with improved thermal stability, and belongs to the fields of biology and food. The mutant TGase provided by the application is obtained by site-directed mutagenesis based on a parent TGase, wherein the parent TGase is composed of a zymogen region of Streptomyces caniferus TGase and a mature region of Streptomyces mobaraenesis TGase. The glutamine transaminase variant S116A / S179L of the application has a 60 DEG C half-life (132.28 min) which is 84% higher than that (71.9 min) of the glutamine transaminase shown in SEQ ID NO. 1. The TGase variant with improved thermal stability is more beneficial to stable application of the TGase in a harsh industrial environment, such as processing of artificial meat paste and beef.

Description

Technical Field

[0001] This invention relates to a thermostable transglutaminase variant, belonging to the fields of biology and food. Background Technology

[0002] Transglutaminase (TGase, EC 2.3.2.13) derived from *Streptomyces mobaraensis* AAT65817 can catalyze the formation of ε-(γ-glutamyl) isopeptide bonds between the γ-carboxamide group (donor) of glutamine residues in protein peptide chains and various acyl acceptors via amide transfer reactions, thereby cross-linking proteins or allowing them to be modified by small molecules. Currently, TGase is widely used in the food processing industries, including meat processing, dairy processing, and baking, to alter the appearance, texture, and stability of food products.

[0003] Research and application of TGase have revealed that its poor thermal stability is a major limitation to its practical applications, especially in the food industry. For example, beef processing requires TGase to maintain its catalytic activity at 60°C for several hours to complete TGase-catalyzed cross-linking. However, due to the poor stability of TGase, it is rapidly lost and consumed at high temperatures, thus requiring a large amount to be added.

[0004] To improve the thermostability of glutamine transaminase, a series of methods, including directed evolution (random mutation of the whole sequence), semi-rational design (saturation mutation, gene shuffling), and rational design (construction of disulfide bonds), have been used for its molecular modification. The following are some of the reports on improving the thermal stability of TGase: (1) obtaining highly stable TGase mutation points through random mutation (doi:10.1016 / j.jbiotec.2008.06.005); (2) performing site saturation mutation and DNA recombination on the single-point mutation obtained in (1) to obtain highly stable TGase combinatorial mutants (DOI 10.1007 / s00726-011-1015-y); (3) performing further combinatorial mutations based on the mutation points screened in (1) and (2) (DOI:10.1080 / 09168451.2017.1403881); (4) based on (3), proline scanning, active site modification and construction, and surface electrostatic charge design to obtain the most stable TGase combinatorial mutant reported to date (DOI:10.1021 / acs.jafc.1c05256). The mutant exhibited a specific enzyme activity of 83.74 U / mg and a half-life of 122.91 min at 60°C, which were the highest levels reported at the time.

[0005] Although the above-mentioned glutamine transaminase mutants have high thermostability and specific enzyme activity, their cross-linking efficiency with casein is poor. Figure 3 This is likely because the higher surface electrostatic charge increases the net electrostatic repulsion between the mutant and casein, thereby reducing its affinity for the latter. Therefore, a glutamine transaminase mutant with improved thermal stability and significantly enhanced casein cross-linking efficiency has broad industrial application prospects. Summary of the Invention

[0006] To obtain TGase mutants with high stability and high cross-linking efficiency, we first used molecular dynamics simulations of FRAPD-TGm2 to determine its flexible region. Then, we developed a Rosetta Cartesian_ddg-based script to obtain FRAPD-TGm2 mutants with significantly reduced folding free energy. Next, we screened and combined these mutants to obtain mutants with improved thermal stability and catalytic activity, and evaluated the cross-linking activity of the optimal mutants with β-casein.

[0007] The mutant TGase provided by this invention is obtained by site-directed mutagenesis based on the parent TGase. The parent TGase consists of the proenzyme region of Streptomyces caniferus TGase and the mature region of Streptomyces mobaraenesis TGase. The mature region sequence contains the mutation point S2P-S23V-Y24N-E28T-S199A-A265P-A287P-K294L compared with the wild type sequence.

[0008] The modified TGase had a half-life of 132.38 min at 60 °C, which was 84% ​​higher than the parent mutant's 71.9 min. In addition, it had a 7.6% higher half-life at 60 °C than the mutant with the highest reported stability, and also had a significantly higher casein crosslinking efficiency.

[0009] This invention provides a glutamine transaminase variant comprising a substitution at position 116 corresponding to the polypeptide shown in SEQ ID NO.1, wherein,

[0010] i) The variant is a polypeptide with glutamine transaminase activity; and

[0011] ii) The variant is a polypeptide having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the polypeptide shown in SEQ ID NO. 1; and / or

[0012] iii) The variant is a polypeptide encoded by a polynucleotide having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity with the mature polypeptide coding sequence shown in SEQ ID NO. 2; and / or

[0013] iv) The variant is a fragment of the polypeptide of ii) or iii) having glutamine transaminase activity;

[0014] The amino acid at position 116 is serine (abbreviated as S).

[0015] In one embodiment of the invention, the substitution at position 116 is replaced with alanine (A).

[0016] In one embodiment of the present invention, the glutamine transaminase, compared with the glutamine transaminase of SEQ ID NO.1, further includes a substitution of the amino acid at position 179 (serine, S).

[0017] In one embodiment of the present invention, the amino acid at position 179 is replaced with leucine (L).

[0018] The present invention provides a glutamine transaminase mutant, which is obtained by mutating the amino acid at position 116 and / or position 179 of the glutamine transaminase as shown in SEQ ID NO.1.

[0019] For the parent enzyme transglutaminase with an amino acid sequence as shown in SEQ ID NO.1, positions 1 to 59 constitute the zymogen region. In this invention, the mutation sites are calculated using the mature region, i.e., position 60, as the first position. Therefore, the position of all mutation sites in this invention should be incremented by 59. For example, the actual position of position 116 in the sequence is... 175 The actual position of the 179th position in the sequence is... 238 Bit.

[0020] In one embodiment of the present invention, the parent strain is composed of the proenzyme region of Streptomyces caniferus TGase and the mature region of Streptomyces mobaraensis TGase, wherein the mature region sequence contains the mutation point S2P-S23V-Y24N-E28T-S199A-A265P-A287P-K294L compared with the wild-type sequence. The amino acid sequence of the parent strain TGase is shown in SEQ ID NO.1, and the nucleotide sequence of the parent strain TGase is shown in SEQ ID NO.2.

[0021] For the parent TGase enzyme with a nucleotide sequence as shown in SEQ ID NO.2, positions 1-177 are codons of the proenzyme region.

[0022] In one embodiment of the present invention, the mutant is obtained by mutating serine at position 116 of glutamine transaminase, as shown in SEQ ID NO.1, to alanine, and is named S116A.

[0023] Alternatively, the mutant is obtained by mutating serine at position 179 of the glutamine transaminase, as shown in SEQ ID NO.1, to leucine, and is named S179L;

[0024] Alternatively, the mutant is obtained by mutating serine at position 116 of the glutamine transaminase as shown in SEQ ID NO.1 to alanine, and simultaneously mutating serine at position 179 to leucine, and is named S116A / S179L.

[0025] The present invention also provides genes encoding the above-mentioned mutants S116A, S179L, and S116A / S179L.

[0026] In one embodiment of the present invention, the amino acid sequence of the mutant S116A / S179L is shown in SEQ ID NO. 3. Since positions 1 to 59 constitute the zymogen region, the above-mentioned sites in the present invention are calculated with the mature region, i.e., position 60, as the first position. Therefore, the position of all mutation sites in the sequence of the present invention should be increased by 59. For example, the actual position of position 116 in the sequence is... 175 The actual position of the 179th position in the sequence is... 238 Bit.

[0027] In one embodiment of the present invention, the nucleotide sequence encoding the mutant S116A / S179L is shown in SEQ ID NO.4 (positions 1-177 are codons of the proenzyme region).

[0028] The present invention also provides a recombinant vector carrying the above-mentioned genes.

[0029] In one embodiment of the present invention, the recombinant vector pET-22b+ is an expression vector that integrates the dissolution tag TrxA, the proterminus of TGase from S. caniferus (proC), and TGm2 at its NdeI-BlpI site.

[0030] The present invention also provides recombinant cells expressing the above mutants S116A, S179L, S116A / S179L, or carrying the above genes, or carrying the above recombinant vector.

[0031] In one embodiment of the present invention, the recombinant cells use prokaryotic cells or eukaryotic cells as expression hosts.

[0032] The present invention also provides a method for improving the thermostability of a glutamine transaminase mutant, wherein the method comprises mutating serine at position 116 of the glutamine transaminase as shown in SEQ ID NO.1 to alanine;

[0033] Alternatively, the serine at position 179 of the glutamine transaminase, as shown in SEQ ID NO.1, could be mutated to leucine;

[0034] Alternatively, the serine at position 116 of the glutamine transaminase, as shown in SEQ ID NO.1, may be mutated to alanine, and the serine at position 179 may be mutated to leucine.

[0035] The glutamine transaminase variant provided by this invention is TGase-S116A / S179L.

[0036] The present invention also relates to polynucleotides encoding said variants; nucleic acid constructs, vectors, and host cells comprising said polynucleotides; and methods for generating said variants. Furthermore, the present invention relates to compositions comprising the transglutaminase variants of the present invention.

[0037] The present invention also relates to methods for generating the glutamine transaminase variants of the present invention, the methods comprising:

[0038] a) Culturing the host cells of the present invention under conditions suitable for expressing the variants; and

[0039] b) Optionally, the variants will be recycled.

[0040] The present invention also provides a method for screening the above-mentioned glutamine transaminase mutant, the method comprising:

[0041] Step 1), based on high-temperature (330K / 360K) molecular dynamics, identify the flexible regions of enzyme proteins at high temperatures;

[0042] Step 2), based on Cartesian_ddG, use a Python script (Table 1) to perform saturation-simulated mutagenesis on FRAPD-TGm2;

[0043] Step 3) The folding free energy change (ΔΔG) of the saturated simulated mutation results is calculated using a Python script (Table 2) and extracted to an Excel table for sorting of folding free energy; Step 4) The top 18 mutants with significantly reduced folding free energy are experimentally characterized, and positive mutants are further combined.

[0044] In one embodiment of the present invention, the method includes script 1 and script 2 developed based on Python; a flexible region is determined by molecular dynamics simulation, and then a saturation simulation mutation is performed on the above region based on script 1 and script 2, and a positive mutant is obtained by experimental screening based on the change of folding free energy.

[0045] The present invention also provides a method for altering the appearance, texture and / or stability of food, wherein the method comprises adding the above-mentioned transglutaminase mutants S116A, S179L or S116A / S179L during food processing.

[0046] In one embodiment of the present invention, the food includes processed fresh meat, sausage products, fish balls, minced meat, soy products and / or dairy products.

[0047] The present invention also relates to methods for altering the appearance, texture and / or stability of food products in fresh meat processing, sausage products, fish balls, minced meat processing, soy products and / or dairy products, said methods comprising adding the transglutaminase variant of the present invention or the composition of the present invention to the above-mentioned food processing process.

[0048] The present invention also relates to the use of the aforementioned glutamine transaminase variants or the compositions described herein in food processing, treatment and conversion.

[0049] In one embodiment of the present invention, it is used to maintain or improve the quality, consistency, elasticity, moisture or viscosity of food.

[0050] In one embodiment of the present invention, the food is selected from cheese, yogurt, ice cream, mayonnaise and meat.

[0051] In one embodiment of the present invention, the food is fish.

[0052] In one embodiment of the present invention, gelatin of different densities is used to prepare low-fat pre-cooked foods.

[0053] Beneficial effects

[0054] (1) The glutamine transaminase variant of the present invention has significantly improved thermal stability and can be used in food processing, manufacturing and conversion, and can be used to maintain or improve the quality, consistency, elasticity, moisture or viscosity of food. The thermally stable TGase variant is more conducive to the stable application of TGase in harsh industrial environments, such as the processing of artificial meat paste and beef processing.

[0055] (2) The glutamine transaminase variant S116A of the present invention has improved thermal stability compared with the glutamine transaminase shown in SEQ ID NO.1, with a 6.2% increase in residual enzyme activity at 60°C for 20 min.

[0056] (3) The glutamine transaminase variant S179L of the present invention has improved thermal stability compared with the glutamine transaminase shown in SEQ ID NO.1, with a 6.3% increase in residual enzyme activity at 60°C for 20 min.

[0057] (4) The glutamine transaminase variant S116A / S179L of the present invention has an 84% longer half-life (132.28 min) at 60 °C compared with the wild type (71.9 min) compared with the glutamine transaminase shown in SEQ ID NO.1. Attached Figure Description

[0058] Figure 1 SDS-PAGE analysis of 16 successfully expressed and purified TGase mutants.

[0059] Figure 2 Determination of specific enzyme activity and residual enzyme activity in different mutants.

[0060] Figure 3 Casein crosslinking assay; where A: SDS-PAGE analysis; B: SEC analysis.

[0061] Figure 4 The enzyme activity of the parent and modified TGase and its half-life at 60°C.

[0062] Figure 5 : The parental TGase and the modified TGase were expressed in E. coli BL21 and analyzed by SDS-PAGE; where M: protein quantitative marker; 1: intracellular electrophoresis analysis of parental TGase after expression in E. coli BL21; 2: intracellular electrophoresis analysis of modified TGase after expression in E. coli BL21.

[0063] Figure 6SDS-PAGE analysis of purified samples of parent TGase and modified TGase; where M: protein mass marker; 1: electrophoretic analysis of purified parent TGase; 2: electrophoretic analysis of purified modified TGase. Detailed Implementation

[0064] 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).

[0065] Enzyme activity assay

[0066] Enzyme activity assay substrate solution A: 200mM Tris-HCl, 100mM hydroxylamine, 10mM reduced glutathione, 30mM N-benzyloxycarbonyl-L-glutamylglycine, adjusted to pH 6.0.

[0067] 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.

[0068] 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.

[0069] Preparation of the standard curve for enzyme activity assay: Weigh 648 mg of standard L-glutamic acid-γ-monohydroxamic acid and add 100 ml of Tris-HCl 200 mM, pH 6.0 solution. Dilute this solution five times using the Tris-HCl 200 mM, pH 6.0 solution in a 2-fold dilution method. 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, and then add 60 μL of stop solution B. Centrifuge at 10000 rpm for 1 min, and then 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.

[0070] Assay method: Protein sample and 150 μL of substrate solution A were incubated at 37℃ 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℃ for 10 min, followed by the addition of 60 μL of test stop solution B. The reaction mixture was centrifuged at 10000 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 10000 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. This enzyme activity was then divided by the protein concentration to obtain the specific enzyme activity (U / mg).

[0071] Thermal stability determination

[0072] The half-life was determined under 60℃ water bath conditions. The specific method was as follows: First, TGase and different mutant protein solutions were diluted to 0.5 mg / ml. A certain amount of this sample was continuously incubated in a 60℃ water bath. Samples were taken every minute from 0 to 10 minutes, and every 2 minutes from 10 to 40 minutes. All samples were immediately placed at 20℃ for cooling. Enzyme activity was measured on each sample to obtain the percentage of residual TGase activity compared to the initial activity over time. The half-life was calculated by nonlinearly fitting the formula using Exponential-ExpDec1 in Original 2018 to obtain the fitted formula, and the time corresponding to the enzyme activity decreasing to 50% of the initial value was then determined.

[0073] Dynamic parameter determination

[0074] The Michaelis constant (KM) is determined as follows: Dilute the TGase and mutant protein solutions to 0.05 mg / ml, and follow the enzyme activity assay method. Substrate solution A needs to be prepared into 10 different solutions with varying N-benzyloxycarbonyl-L-glutamylglycine contents, keeping other components constant. The N-benzyloxycarbonyl-L-glutamylglycine contents are 3 mM, 6 mM, 9 mM, 12 mM, 15 mM, 18 mM, 21 mM, 24 mM, 27 mM, and 30 mM. According to the enzyme activity assay method, the substrate conversion amount in substrate solution A with different N-benzyloxycarbonyl-L-glutamylglycine contents, i.e., the amount of N-benzyloxycarbonyl-L-glutamylglycine catalyzed to be converted into the final product within a 10-minute reaction time, is measured by TGase and the mutant. Based on the conversion values ​​obtained above, nonlinear fitting was performed on the values ​​using Origin 2018 software, employing the Growth / Sigmodial-Hill method to obtain KM and Vmax values. The kcat value was then calculated using enzyme concentration conversion. Finally, the enzyme catalytic efficiency was obtained as KM / kcat.

[0075] Protein analysis:

[0076] Protein concentration was determined using the Bradford Protein Assay Kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 12% Tris-glycine gel (Thermo Fisher, Shanghai, China). The melting temperature (Tm) of the protease was determined using a differential scanning calorimeter (Nano DSC, TA Instruments, USA), with the system temperature gradually increased from 45°C to 90°C at 1°C per minute.

[0077] Casein cross-linking experiment

[0078] We used β-casein (Macklin, Shanghai, China) as a substrate to detect the catalytic activity of FRAPD-TGm2 and its mutants on proteins. β-casein was diluted to 3 mg / ml with Tris-acetic acid buffer (100 mM, pH = 7). Cross-linking reactions were performed at 40 °C with FRAPD-TGm2 and its mutants added to the same enzyme activity concentration of 1 U / ml. Samples reacted at 0, 5, 10, 20, and 40 min with 8 M urea at a 1:3 ratio were then analyzed by SDS-PAGE. Samples at 5 min were analyzed by size exclusion chromatography (SEC) using a HiLoad 26 / 600 Superdex 200 pg column.

[0079] Definitions or terms:

[0080] Transglutaminase: The terms "transglutaminase (TGase)," "R-glutamine-acyl-peptidase-γ-glutamyl-transferase," and "transglutaminase" refer to enzymes in class EC2.3.2.13 as defined by enzyme nomenclature. For the purposes of this invention, transglutaminase activity is determined according to the procedure described in the examples. In one aspect, variants of the invention have at least 20% of the transglutaminase activity of the polypeptide of SEQ ID NO:1, for example, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0081] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly defines the amino acid sequence of a glutamine transaminase variant. The boundaries of a coding sequence are typically defined by a read frame, which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0082] Control Sequence: The term "control sequence" refers to the nucleic acid sequence necessary for the expression of a polynucleotide encoding a glutamine transaminase variant of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) for the polynucleotide encoding the glutamine transaminase variant, or native or exogenous relative to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. Control sequences may be provided with adapters for the purpose of introducing specific restriction sites that facilitate the connection of control sequences to the coding region of the polynucleotide encoding the glutamine transaminase variant of the present invention.

[0083] Expression: The term “expression” includes any step involved in the production of glutamine transaminase variants, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0084] Fragment: The term "fragment" means a polypeptide that has one or more (e.g., several) amino acids deleted from its amino and / or carboxyl terminus; wherein said fragment has glutamine transaminase activity. In one aspect, the fragment contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%, of the number of amino acids 1 to 331 of SEQ ID NO: 1 (i.e., excluding the length of the zymogen region).

[0085] Improved thermal stability: The term “improved thermal stability” refers to the characteristics of glutamine transaminase variants that are improved relative to their parental counterparts.

[0086] Isolated: The term “isolated” means a substance in a form or environment that does not exist in nature. Non-limiting examples of isolated substances include (1) any substance that is not naturally occurring; (2) any substance removed at least partially from one or more naturally occurring components associated with it in nature, including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide, or cofactor; (3) any substance that has been artificially modified relative to such a substance found in nature; or (4) any substance modified by increasing the amount of said substance relative to other components naturally associated with it (e.g., multiple copies of the gene encoding said substance; use of a promoter stronger than the promoter naturally associated with the gene encoding said substance). Isolated substances may be present in fermentation broth samples.

[0087] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is amino acids 1 to 331 of SEQ ID NO:1. It is known in the art that host cells can produce mixtures of two or more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide.

[0088] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide encoding a mature polypeptide having glutamine transaminase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 1 to 993 of SEQ ID NO:2 (i.e., excluding the codon sequence corresponding to the zymogen region).

[0089] Mutant: The term “mutant” refers to a polynucleotide that encodes a variant.

[0090] Nucleic acid constructs: The term “nucleic acid constructs” refers to single-stranded or double-stranded nucleic acid molecules that are isolated from naturally occurring genes, modified to contain nucleic acid segments in a manner not normally found in nature, or synthesized, and that contain one or more control sequences.

[0091] Parental or parental glutamine transaminase: The term "parental" or "parental glutamine transaminase" refers to a glutamine transaminase modified to produce a variant of the glutamine transaminase of the present invention. The parental glutamine transaminase may be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof, or it may be synthetically produced.

[0092] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0093] Stability: The thermostability of the glutamine transaminase variant of the present invention can be expressed as the residual activity or residual performance of the glutamine transaminase during or after exposure to different test conditions. This can be relative to the known activity or performance of a parent glutamine transaminase (e.g., the parent glutamine transaminase shown in SEQ ID NO:1).

[0094] Variant: The term "variant" refers to a polypeptide having glutamine transaminase activity that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. The variants of the present invention have at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the glutamine transaminase activity of the polypeptide of SEQ ID NO:1.

[0095] Wild-type transglutaminase: The term "wild-type" transglutaminase refers to transglutaminase expressed by naturally occurring microorganisms (such as bacteria, yeast, or filamentous fungi) found in nature.

[0096] In the description of variations of the invention, the nomenclature described below has been adapted for ease of reference. Accepted IUPAC single-letter or three-letter amino acid abbreviations are used. Substitution: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, the substitution of threonine at position 226 with alanine is represented as "Thr226Ala" or "T226A". Multiple mutations are separated by a symbol (" / "), for example, "Gly205Arg / Ser411Phe" or "G205R / S411F" represents the substitution of glycine (G) and serine (S) at positions 205 and 411 with arginine (R) and phenylalanine (F), respectively.

[0097] Preparation of variants

[0098] The glutamine transaminase variants of the present invention can be prepared using any mutagenesis procedure known in the art (e.g., site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.).

[0099] Site-directed mutagenesis is a technique for introducing one or more (e.g., several) mutations at one or more defined sites in a polynucleotide encoding the parental glutamine transaminase.

[0100] Site-directed mutagenesis can be achieved in vitro via PCR involving primers containing oligonucleotides with the desired mutation. In vitro site-directed mutagenesis can also be performed via cassette mutagenesis, which involves cleavage by a restriction enzyme at a site in a plasmid containing a polynucleotide encoding the parental glutamine transaminase, followed by ligation of the mutated oligonucleotide into the polynucleotide. Typically, the restriction enzymes digesting the plasmid and the oligonucleotide are the same, allowing the sticky ends of the plasmid and the insert to ligate together.

[0101] Site-directed mutagenesis can also be achieved in vivo using methods known in the art.

[0102] Any site-directed mutagenesis procedure can be used in this invention. Many commercially available kits are available for preparing variants.

[0103] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode polypeptides of interest. Gene synthesis can be performed using a variety of techniques.

[0104] Using known mutagenesis, recombination, and / or tampering methods, followed by relevant screening procedures, single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested.

[0105] Mutagenesis / reorganization methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenesis-encoded peptides expressed by host cells. Mutagenesis-encoded DNA molecules encoding the active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0106] Semi-synthetic gene construction can be achieved through a combination of synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction typically utilizes the process of synthesizing polynucleotide fragments combined with PCR technology. Therefore, defined regions of the gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenesis primers, and still others can be amplified using error-prone or non-error-prone PCR. The polynucleotide subsequence can then be shuffled.

[0107] Polynucleotides

[0108] This invention also relates to isolated polynucleotides encoding the glutamine transaminase variants of the present invention. In some aspects, the invention relates to nucleic acid constructs comprising the polynucleotides of the present invention. In some aspects, the invention relates to expression vectors comprising the polynucleotides of the present invention. In some aspects, the invention relates to host cells comprising the polynucleotides of the present invention. In some aspects, the invention relates to a method for generating glutamine transaminase variants, the method comprising: (a) culturing host cells of the present invention under conditions suitable for expressing the glutamine transaminase variants; and (b) recovering the glutamine transaminase variants.

[0109] Nucleic acid constructs

[0110] The present invention also relates to a nucleic acid construct comprising a polynucleotide operatively linked to one or more control sequences encoding a variant of the present invention, wherein the one or more control sequences, under conditions compatible with the control sequences, guide the expression of the coding sequence in a suitable host cell.

[0111] Polynucleotides can be manipulated in a variety of ways to provide expression of glutamine transaminase variants. Depending on the expression vector, manipulating the polynucleotide prior to insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0112] The control sequence can be a promoter, i.e., a polynucleotide recognized by the host cell for the expression of the polynucleotide. The promoter contains a transcriptional control sequence that mediates the expression of a glutamine transaminase variant. The promoter can be any polynucleotide exhibiting transcriptional activity in the host cell, including mutant, truncated, and heterozygous promoters, and can be a gene that encodes an extracellular or intracellular polypeptide homologous or heterologous to the host cell.

[0113] expression carrier

[0114] The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a glutamine transaminase variant of the present invention, a promoter, and transcription and translation termination signals. Various nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more suitable restriction sites to allow insertion or substitution of the polynucleotide encoding the glutamine transaminase variant at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is located in the vector such that the coding sequence is operatively linked to a suitable control sequence for expression.

[0115] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that can readily undergo recombinant DNA procedures and induce polynucleotide expression. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed circular plasmids.

[0116] The vector can be a self-replicating vector, that is, a vector that exists as an extrachromosomal entity and replicates independently of chromosome replication, such as plasmids, extrachromosomal elements, microchromosomes, or artificial chromosomes. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that, when introduced into a host cell, integrates into the genome and replicates along with one or more chromosomes to which it is integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids collectively containing the total DNA of the host cell genome to be introduced, or transposons can be used.

[0117] The vector preferably contains one or more selective markers that allow for convenient selection of cells such as transformed cells, transfected cells, and transduced cells. A selective marker is a gene whose product provides resistance to biocides or viruses, resistance to heavy metals, or prototrophic auxotrophic traits, etc.

[0118] Examples of selective bacterial markers include the dal gene in Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance (such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance). Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selective markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamipase), argB (ornithine carbamoyltransferase), bar (glufosinate-amylase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (adenosyl sulfate transtransferase), and trpC (o-aminobenzoic acid synthase), along with their equivalents. Preferred markers for use in Aspergillus cells are the amdS and pyrG genes of Aspergillus nidus or Aspergillus oryzae, and the bar gene of Streptomyces hygroscopicus.

[0119] The vector preferably contains one or more elements that allow the vector to integrate into the host cell's genome or to replicate autonomously in the cell independently of the genome.

[0120] To integrate into the host cell genome, the vector can rely on a polynucleotide sequence encoding a glutamine transaminase variant or any other vector element for integration into the genome via homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to guide integration into the host cell genome at a precise location on the chromosome via homologous recombination. To increase the likelihood of integration at a precise location, the integrative element should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, that have high sequence identity with the corresponding target sequence to enhance the probability of homologous recombination. The integrative element can be any sequence homologous to the target sequence within the host cell genome. Furthermore, the integrative element can be a non-coding or coding polynucleotide. On the other hand, the vector can integrate into the host cell genome via non-homologous recombination.

[0121] For autonomous replication, the vector may also additionally include an origin of replication, which enables the vector to replicate autonomously within the host cell discussed in this discussion. The origin of replication can be any plasmid replicon that mediates autonomous replication and functions within the cell. The terms "origin of replication" or "plasmid replicon" refer to the polynucleotide that enables a plasmid or vector to replicate in vivo.

[0122] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in Escherichia coli, and the origins of replication of plasmids pUB110, pE194, pTA1060, and pAMβ1, which allow replication in Bacillus.

[0123] Examples of replication origins used in yeast host cells include the 2-micron replication origin, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6.

[0124] Examples of useful origins of replication in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res. 15:9163-9175; WO00 / 24883). The AMA1 gene can be isolated and plasmids or vectors containing the gene can be constructed according to the methods disclosed in WO 00 / 24883.

[0125] More than one copy of the polynucleotide of the present invention can be inserted into host cells to increase the production of glutamine transaminase variants. An increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selective marker gene along with the polynucleotide, wherein cells containing amplified copies of the selective marker gene, and thus additional copies of the polynucleotide, can be selected by culturing cells in the presence of a suitable selective reagent.

[0126] The procedures for connecting the above-described elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art.

[0127] host cells

[0128] This invention also relates to recombinant host cells containing a polynucleotide operably linked to one or more control sequences encoding a glutamine transaminase variant of the invention, said control sequences directing the generation of the glutamine transaminase variant of the invention. A construct or vector containing the polynucleotide is introduced into the host cell such that said construct or vector is maintained as a chromosomal integrase or as an autonomously replicating extrachromosomal vector, as previously described. The term "host cell" encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations occurring during replication. The selection of the host cell depends largely on the gene encoding the glutamine transaminase variant and its origin.

[0129] The host cell can be any cell that is useful in the recombinant production of glutamine transaminase variants, such as prokaryotic or eukaryotic cells.

[0130] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Bacillus macrocephala, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Coliform, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0131] The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0132] Generation method

[0133] The present invention also relates to a method for generating the transglutaminase variant of the present invention, the method comprising: (a) culturing the host cells of the present invention under conditions suitable for expressing the transglutaminase variant; and (b) recovering the transglutaminase variant.

[0134] Host cells are cultured in a nutrient medium suitable for producing transglutaminase variants using methods known in the art. For example, cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, feed-feed fermentation, or solid-state fermentation) in a suitable medium and under conditions that allow for the expression and / or isolation of transglutaminase or variants in a laboratory or industrial fermenter. Using procedures known in the art, the culture occurs in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to a published composition. If a transglutaminase variant is secreted into the nutrient medium, the transglutaminase variant can be recovered directly from the medium. If the transglutaminase variant is not secreted, it can be recovered from cell lysates.

[0135] Glutamine transaminase variants can be detected using methods known in the art that are specific to them. These methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, enzyme assays can be used to determine the activity of glutamine transaminase variants (as described in the examples).

[0136] Glutamine transaminase variants can be recovered using methods known in the art. For example, glutamine transaminase variants can be recovered from nutrient media through routine procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0137] Glutamine transaminase variants can be purified to obtain substantially pure glutamine transaminase variants by a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), electrophoresis procedures (e.g., preparative isoelectric point focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction.

[0138] Alternatively, instead of recycling the glutamine transaminase variant, the host cells of the present invention expressing the glutamine transaminase variant are used as the source of the glutamine transaminase variant.

[0139] Fermentation broth preparations or cell compositions

[0140] The present invention also relates to fermentation broth formulations or cell compositions comprising the polypeptides of the present invention. The fermentation broth product further comprises additional components used in the fermentation process, such as cells (including host cells containing genes encoding the polypeptides of the present invention, these host cells being used to produce the target polypeptide), cell debris, biomass, fermentation medium, and / or fermentation products. In some embodiments, the composition is a cell-killing whole culture medium containing one or more organic acids, killed cells and / or cell debris, and a culture medium.

[0141] As used herein, the term "fermentation broth" refers to a preparation produced by cell fermentation that undergoes little or no recovery and / or purification. For example, fermentation broth is produced when a microbial culture is incubated to saturation under carbon-limited conditions that allow protein synthesis (e.g., expression of enzymes by the host cell) and secretion of proteins into the cell culture medium. The fermentation broth may contain unfractionated or fractionated contents of the fermentation material obtained at the end of fermentation. Typically, the fermentation broth is unfractionated and contains used culture medium and cell debris remaining after, for example, removal of microbial cells (e.g., filamentous fungal cells) by centrifugation. In some embodiments, the fermentation broth contains used cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0142] In one embodiment, the fermentation broth formulation and cell composition comprise a first organic acid component (containing at least one organic acid with 1-5 carbons and / or its salt) and a second organic acid component (containing at least one organic acid with 6 or more carbons and / or its salt). In one specific embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, its salt, or a mixture of two or more of the foregoing; and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, its salt, or a mixture of two or more of the foregoing.

[0143] In one aspect, the composition contains one or more organic acids and optionally further contains killed cells and / or cell debris. In one embodiment, these killed cells and / or cell debris are removed from the cell-killing whole culture medium to provide a composition free of these components.

[0144] These fermentation broth formulations or cell compositions may further contain preservatives and / or antimicrobial (e.g., bacteriostatic) agents, including but not limited to sorbitol, sodium chloride, potassium sorbate, and other reagents known in the art.

[0145] The cell-killing whole culture or composition may contain ungraded contents of the fermentation material obtained at the end of fermentation. Typically, the cell-killing whole culture or composition contains used culture medium and cell debris present after microbial cells (e.g., filamentous fungal cells) have been grown to saturation and incubated under carbon-limited conditions to allow protein synthesis. In some embodiments, the cell-killing whole culture or composition contains used cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, methods known in the art can be used to permeate and / or lyse the microbial cells present in the cell-killing whole culture or composition.

[0146] The whole culture medium or cell composition described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture medium components, and / or one or more insoluble enzymes. In some embodiments, insoluble components may be removed to provide a clear liquid composition.

[0147] Composition

[0148] The present invention also relates to compositions comprising the variant glutamine transaminase of the present invention.

[0149] These compositions may contain the variant of the present invention, transglutaminase, as the main enzyme component, for example, a single-component composition. Alternatively, the composition may contain a variety of enzyme activities, such as one or more (e.g., several) enzymes selected from the group consisting of: proteases, glucosylamylases, β-amylases, and amylopectinases.

[0150] The culture media involved in the following examples are as follows:

[0151] LB liquid medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, ampicillin 100 μg / L.

[0152] 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.

[0153] TB medium: yeast extract 24 g / L, tryptone 12 g / L, dipotassium hydrogen phosphate trihydrate 12.84 g / L, potassium dihydrogen phosphate 2.31 g / L, glycerol 4 mL / L, ampicillin 100 μg / L.

[0154] The *E. coli* JM109 and *E. coli* BL21(DE3) involved in the following examples were purchased from Takara Biotech (Beijing) Co., Ltd., the pET-22b(+) plasmid was purchased from Novagen (the above-mentioned strain *E. coli* BL21(DE3) is commercially available and does not require preservation for patent procedures), the neutral protease was purchased from Beijing Solarbio Technology Co., Ltd. (catalog number Z8032), and the Blunting Kination Ligation (BKL) Kit and HS DNA Polymerase was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd., and the Bradford Protein Assay Kit (detergent compatible) was purchased from Beyotime Biotechnology Co., Ltd.

[0155] Example 1: Method for screening glutamine transaminase mutants

[0156] The specific steps are as follows:

[0157] (1) Molecular dynamics simulation:

[0158] Molecular dynamics simulations of FRAPD-TGm2 were performed using GROMACS-2020 at 330K / 360K temperatures to identify its flexible regions at high temperatures.

[0159] (2) Saturation simulation of mutation:

[0160] Saturation-simulated mutations of FRAPD-TGm2 were performed using a Python script based on Cartesian_ddG (Table 1);

[0161] Table 1: Simulation of mutation based on Cartesian ddG fold free energy saturation

[0162]

[0163]

[0164] (3) Calculation and extraction of the change in folding free energy:

[0165] The folding free energy change (ΔΔG) of the saturated simulated mutation results was calculated using a Python script (Table 2) and extracted to an Excel table for sorting.

[0166] Table 2 shows the batch calculation and extraction of folding free energy changes.

[0167]

[0168]

[0169] (4) Experimental verification:

[0170] Experimental characterization was performed on the first 18 mutants with significantly reduced folding free energy, and positive mutants were further combined.

[0171] Example 2: Construction of mutants

[0172] The specific steps are as follows:

[0173] (1) Construction of pET-22b-tgase vector:

[0174] The gene of the parent TGase is shown in SEQ ID NO.2 (amino acid sequence is shown in SEQ ID NO.1). The gene was inserted into the plasmid pET-22b(+) through the restriction endonuclease sites NdeI and BlpI, and sent to Genewiz to synthesize pET-22b-tgase.

[0175] (2) Construction of recombinant vectors containing mutants

[0176] S116A, S179L, and the remaining mutants were obtained by PCR and linear DNA circularization on the plasmid pET-22b-tgase using the corresponding primers (Table 3).

[0177] The mutant S116A / S179L was obtained from the plasmid of mutant S116A through PCR and linear DNA circularization using primers 179-Leu-F / R. All primers used are listed in Table 3. The PCR procedure was performed according to the instructions of Baori Biotechnology (Beijing) Co., Ltd. Refer to the HS DNA Polymerase instruction manual. For the linear DNA circularization method, refer to the Blunting Kination Ligation (BKL) Kit instruction manual from Baori Biotechnology (Beijing) Co., Ltd.

[0178] Table 3: Primer gene sequences

[0179]

[0180]

[0181] Recombinant vectors containing different mutants were prepared respectively: pET-22b-S116A / S179L, pET-22b-S116A, pET-22b-S179L, pET-22b-E164L, pET-22b-S116C, pET-22b-S84V, pET-22b-S84Y, pET-22b-E164I, pET-22b -R183P, pET-22b-S84F, pET-22b-E164M, pET-22b-R208M, pET-22b-R208L, pET-22b-E 164V, pET-22b-S84I, pET-22b-N176I, pET-22b-K91W, pET-22b-E93I, pET-22b-G97L.

[0182] Example 3: Preparation method of mutant enzyme

[0183] The specific steps are as follows:

[0184] (1) The recombinant vectors constructed in Example 1 were transformed into *E. coli* JM109. The transformation products were plated on LB solid medium and cultured at 37°C for 10 h. Transformants were picked and sequenced. The recombinant plasmids with correct sequencing were transformed into *E. coli* BL21(DE3). Recombinant *E. coli* strains expressing the corresponding glutamine transaminase variants, namely *E. coli* BL21(DE3) / pET-22b-S116A, *E. coli* BL21(DE3) / pET-22b-S179L, *E. coli* BL21(DE3) / pET-22b-S116A / S179L, *E. coli* BL21(DE3) / pET-22b-E164L, *E. coli* BL21(DE3) / pET-22b-S116C, and *E. coli* BL21(DE3) / pET-22b-S116C, were prepared. BL21(DE3) / pET-22b-S84V, E.coli BL21(DE3) / pET-22b-S84Y, E.coli BL21(DE3) / pET-22b-E164I, E.coli BL21(DE3) / pET-22b-R183P, E.coli BL21(DE3) / pET-22b-S84F, E.coli BL21(DE3) / pET-22b-E164M, E.coli BL21(DE3) / pET-22b-R208M, E.coli BL21(DE3) / pET-22b-R208L, E.coli BL21(DE3) / pET-22b-E164V, E.coli BL21(DE3) / pET-22b-S84I, E.coli BL21(DE3) / pET-22b-N176I, E.coli BL21(DE3) / pET-22b-K91W, E.coli BL21(DE3) / pET-22b-E93I, E. coli BL21(DE3) / pET-22b-G97L.

[0185] (2) The obtained recombinant Escherichia coli were spread on LB solid medium and cultured at 37°C for 10 h. Transformants were picked and inoculated into LB liquid medium (containing 100 μg / mL ampicillin) and cultured at 37°C for 10 h to prepare seed liquids.

[0186] The prepared seed culture was transferred to TB liquid medium (containing 100 μg / mL ampicillin) at a transfer rate of 1% (v / v), and Escherichia coli BL21(DE3) was cultured at 37°C until OD200. 600 To induce recombinant protein expression, add IPTG to a final concentration of 0.01 mM, bringing the concentration to between 1.0 and 1.5 mM (intracellular electrophoresis analysis is shown below). Figure 5 (As shown). After IPTG was introduced, the culture temperature was changed to 20℃ and cultured continuously for 32 hours. All liquid cultures were cultured on a shaker at a speed of 220 rpm.

[0187] (3) Protein purification

[0188] After fermentation, the sample was centrifuged at 7500 rpm for 10 min and the cells were collected. One-fifth of the total volume of fermentation broth was added to Tris-HCl 50 mM at pH 8.0 to resuspend the cells, and the cells were then placed on ice for 10 min.

[0189] The resuspended bacterial culture was homogenized using a high-pressure homogenizer (Union-Biotech, Shanghai, China) at 1000 bar for 3 min, followed by centrifugation at 12000 rpm for 20 min. The supernatant was collected, and neutral protease (Solarbio, Beijing, China) was added to a final concentration of 20 mg / ml. The mixture was then incubated at 37°C for 25 min. After incubation, the sample was centrifuged at 12000 rpm for 20 min, and the supernatant was collected for protein purification using nickel ion affinity purification (e.g., ...). Figure 1 As shown), the specific method is as follows:

[0190] The nickel ion affinity purification column was sequentially flushed with water and Tris-HCl 50mM, Tris-HCl 20mM imidazole, pH 7.8 solution until conductivity equilibrium was reached. The sample was then passed through the column, and it was washed with Tris-HCl 50mM, Tris-HCl 20mM imidazole, pH 7.8 solution until conductivity re-equilibration. The protein was then eluted with Tris-HCl 50mM, Tris-HCl 180mM imidazole, pH 7.8 solution, and the protein was recovered. The entire purification process was performed on an AKTA Pure instrument, and the protein collection time and amount were determined by observing the A280 wavelength absorption peak.

[0191] After collection, the protein samples were desalted using gel permeation chromatography (GPC). The specific method was as follows: the gel column was first passed through water and then through 50 mM Tris-HCl solution (pH 8.0) until conductivity equilibrium was reached. The protein sample was then passed through the gel column, followed by further passage through 50 mM Tris-HCl solution (pH 8.0) until an absorption peak appeared at the A280 wavelength. The protein was then collected, and collection was stopped immediately upon observing a change in conductivity. Protein concentration was determined using the BCA assay method, following the instructions for the Bradford Protein Concentration Assay Kit (detergent compatible) from Shanghai Beyotime Biotechnology Co., Ltd.

[0192] Pure enzyme solutions containing S116A / S179L mutants, S116A mutant, S179L mutant, E164L mutant, S116C mutant, S84V mutant, S84Y mutant, E164I mutant, R183P mutant, S84F mutant, E164M mutant, R208M mutant, R208L mutant, E164V mutant, S84I mutant, K91W mutant, and N176I mutant were prepared respectively. SDS-PAGE analysis of the purified samples of the parent TGase and the modified TGase is shown below. Figure 6 (As shown).

[0193] (4) The percentage of residual enzyme activity of the mutant pure enzyme obtained in step (3) was detected respectively, and the results are as follows: Figure 2 As shown in Table 4.

[0194] Table 4: Specific enzyme activity and residual enzyme activity (TGase) of different mutants, original enzymes, and pure enzymes.

[0195]

[0196] The results showed that the residual enzyme activity (60℃) of mutants S116A, S179L, and S116A / 179L at 20 min was 6.2%, 6.3%, and 8.8% higher than that of the parent TGase, respectively.

[0197] (5) The half-life at 60℃ and the temperature range of the maternal FRAPD-TGm2 and FRAPD-TGm2-S116A-S179L combined mutants were measured respectively. m The values ​​and kinetic constants are shown in Table 5. Figure 4 As shown.

[0198] Table 5: Determination of residual enzyme activity

[0199]

[0200] The results showed that the FRAPD-TGm2-S116A-S179L combined mutant exhibited significantly enhanced stability, with an 84% increase in half-life at 60℃ compared to the parent TGase. In addition, the enzyme activity and T... m All of them have been improved to varying degrees, making them more suitable for applications under high-temperature conditions.

[0201] (5) The casein cross-linking activity of the maternal FRAPD-TGm2, FRAPD-TGm2-N96E-S144E-N163D-R183E-R208E-K325E (FRAPD-TGm3), and the FRAPD-TGm2-S116A-S179L combined mutants were tested respectively. Figure 3 ).

[0202] The results showed that the FRAPD-TGm2-S116A-S179L combined mutant and FRAPD-TGm2 had similar casein crosslinking activity, but the activity was significantly higher than that of FRAPD-TGm3, as indicated by the peak area.

[0203] In addition, our combined mutant exhibits the highest thermal stability, with an 8% improvement in the 60°C half-life compared to the currently best-stability mutant FRAPD-TGm3 (Reference: Significantly improving thethermostability and catalytic efficiency of streptomyces mobaraenesistransglutaminase through combined rational design).

[0204] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A glutamine transaminase mutant, characterized in that, The mutant was obtained by mutating serine at position 116 of the glutamine transaminase, as shown in SEQ ID NO.1, to alanine, and serine at position 179 to leucine.

2. The gene encoding the mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. Recombinant cells expressing the mutant of claim 1, or carrying the gene of claim 2, or carrying the recombinant vector of claim 3.

5. The recombinant cell according to claim 4, characterized in that, The recombinant cells use prokaryotic or eukaryotic cells as expression hosts.

6. A method for improving the thermal stability of glutamine transaminase mutants, characterized in that, The method involves mutating serine at position 116 of the glutamine transaminase, as shown in SEQ ID NO.1, to alanine, and mutating serine at position 179 to leucine.

7. A method for altering the appearance, texture, and / or stability of food, characterized in that, The method involves adding the glutamine transaminase mutant described in claim 1 during food processing.

8. The method according to claim 7, characterized in that, The food products mentioned include processed fresh meat, sausage products, fish balls, minced meat, soy products and / or dairy products.

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