A high-thermostable transglutaminase mutant and a method for expressing the same in streptomyces

Abstract

The application discloses a kind of high thermal stability glutamine transaminase mutant and its method of expression in streptomyces, belong to enzyme engineering field.The high thermal stability glutamine transaminase mutant provided by the application can efficiently catalyze casein crosslinking at high temperature, realizes the heterologous efficient expression of glutamine transaminase.Rational mutation is carried out to key amino acid site of glutamine transaminase, and the mutant strain with significantly improved thermal stability is obtained.Among them, the optimal mutant strain FRAPD-TGm2A3 has a half-life of up to 537.91 min at 60 DEG C.The mutant can still efficiently catalyze the crosslinking reaction of casein at high temperature of 85 DEG C.The present application not only lays a solid research foundation for the modification of thermal stability of glutamine transaminase, but also provides a generalizable strategy framework for the rational modification of other thermostable enzymes, and has important industrial application value and academic reference significance.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering, specifically relating to a highly thermostable glutamine transaminase mutant and its expression method in Streptomyces. Background Technology

[0002] Transglutaminase (TGase, EC 2.3.2.13) catalyzes the acyl transfer reaction of glutamine residues in proteins or peptide chains, thereby mediating intermolecular cross-linking of proteins. Currently, TGase is widely used in the food industry to improve the texture of protein-based foods. Furthermore, this enzyme shows significant potential in non-food fields, such as cross-linking of antibody-drug conjugates, quality improvement of fabrics and leather, and functional enhancement of bioscaffold materials. Compared to TGase from animal and plant sources, microbial TGase is calcium-independent. 2+ Activation offers enhanced biosafety and cost-effectiveness, thus dominating industrial applications. Among these, those derived from *Streptomyces mobara* (…) Streptomyces mobaraensis TGase (smTG) is the most widely used, but its thermal stability is poor, with a half-life of ( ) at 60℃. t 1 / 2 (60℃) less than 2 min, which severely limits its application in high-temperature processing conditions (such as tofu preparation which requires heating at 75℃ for 10-30 min).

[0003] To improve the thermal stability of smTG, researchers have employed various strategies, including directed evolution, semi-rational design, and rational design, to obtain a series of mutants with significantly improved stability. Current reports on improving the thermal stability of TGase include: (1) obtaining the mutant S2P through random mutagenesis. t 1 / 2 (60℃) 1.71 times higher than wild type (DOI10.1016 / j.jbiotec.2008.06.005); (2) S23V obtained by saturation mutation and DNA shuffling screening Y24N K294L, its t 1 / 2 (60℃) increased by 11.2 times (DOI10.1007 / s00726-011-1015-y); (3) Based on the mutation points screened in (1) and (2), multiple beneficial mutations were further integrated to construct a fivefold mutant S2P-S23V-Y24N-S199A-K294L (TGm1), which t 1 / 2(60℃) is 12.4 times that of wild type (DOI10.1080 / 09168451.2017.1403881); (4) Based on the mutation points screened in (3), FRAPD was obtained by proline scanning and substrate binding pocket modification. TGm1 E28T A265P A287P (FRAPD TGm2), with thermal stability improved by 3 times compared to TGm1 (DOI10.1021 / acs.jafc.1c05256); (5) Based on the mutation points screened in (4), the flexible region was modified to obtain FRAPD-TGm2-S116A-S179L (FRAPD-TGm2A), which t 1 / 2 (60℃) extended to 132.28 min, an improvement of 84% compared to FRAPD-TGm2 (DOI10.1021 / acs.jafc.3c00260); (6) based on the mutation points screened in (4), further dynamic cross-correlation analysis (Y34W) (DOI10.13995 / j.cnki.11-1802 / ts.038976) and disulfide bond design (D3C-G283C / A160C-G228C) were combined to obtain the mutant FRAPD-TGm2C, whose t 1 / 2 The time to reach 424.7 min (60℃) (DOI: 10.1021 / acs.jafc.5c10863) was 2.2 times higher than that of FRAPD-TGm2A, which was the highest level reported at the time.

[0004] Although the aforementioned glutamine transaminase mutant (FRAPD-TGm2C) exhibits high thermostability, its disulfide bonds prevent its expression in its natural host, *Streptomyces morihara*, thus limiting its application in the food industry. Based on the shortcomings of the prior art, the purpose of this invention is to develop a highly thermostable glutamine transaminase for expression in *Streptomyces*. Summary of the Invention

[0005] This invention provides a highly stable glutamine transaminase mutant. Through structural analysis and fold free energy prediction, specific amino acid sites in glutamine transaminase were rationally mutated to obtain an enzyme mutant with significantly enhanced thermostability. This mutant can efficiently catalyze casein crosslinking at high temperatures, promoting the application of TGase in high-temperature food processing. To obtain a TGase mutant with high crosslinking efficiency, firstly, the identified mutation site Y34W was introduced into the parent TGase, and structural analysis was performed to determine its helical corner sites. Then, a mutant with a significantly reduced fold free energy was obtained based on the RosettaCartesian_ddg script. Next, mutants with improved thermostability and catalytic activity were screened and combined from the above mutants, and the crosslinking activity of the optimal mutant on casein was evaluated.

[0006] This invention provides a thermostable glutamine transaminase mutant, which is obtained by modifying the parent enzyme, glutamine transaminase with the amino acid sequence shown in SEQ ID NO.5, as follows: The lysine residue at position 325 of the parent enzyme was mutated to glutamic acid, and the resulting enzyme was named FRAPD-TGm2A2-K325E. Alternatively, the leucine at position 294 of the parent enzyme can be mutated to isoleucine, and the resulting enzyme can be named FRAPD-TGm2A2-L294I. Alternatively, the valine at position 311 of the parent enzyme could be mutated to cysteine, and the resulting enzyme could be named FRAPD-TGm2A2-V311C. Alternatively, the threonine at position 313 of the parent enzyme could be mutated to glutamic acid, and the resulting enzyme could be named FRAPD-TGm2A2-T313E. Alternatively, the phenylalanine at position 314 of the parent enzyme could be mutated to tyrosine, and the resulting enzyme could be named FRAPD-TGm2A2-F314Y. Alternatively, the proline at position 324 of the parent enzyme could be mutated to glutamate, and the resulting product could be named FRAPD-TGm2A2-P324E. Alternatively, the lysine at position 325 of the parent enzyme can be mutated to glutamic acid, and the valine at position 311 of the parent enzyme can be mutated to cysteine, named FRAPD-TGm2A2-K325E-V311C. Alternatively, lysine at position 325 of the parent enzyme can be mutated to glutamic acid, valine at position 311 of the parent enzyme can be mutated to cysteine, and proline at position 324 of the parent enzyme can be mutated to glutamic acid, named FRAPD-TGm2A2-K325E-V311C-P324E. Alternatively, lysine at position 325 of the parent enzyme can be mutated to glutamic acid, valine at position 311 of the parent enzyme can be mutated to cysteine, and leucine at position 294 of the parent enzyme can be mutated to isoleucine, named FRAPD-TGm2A2-K325E-V311C-L294I. Alternatively, lysine at position 325 of the parent enzyme can be mutated to glutamic acid, valine at position 311 of the parent enzyme can be mutated to cysteine, and isoleucine at position 312 of the parent enzyme can be mutated to valine, named FRAPD-TGm2A2-K325E-V311C-I312V. Alternatively, the lysine at position 325 of the parent enzyme can be mutated to glutamic acid, the valine at position 311 of the parent enzyme can be mutated to cysteine, and the phenylalanine at position 314 of the parent enzyme can be mutated to tyrosine, named FRAPD-TGm2A2-K325E-V311C-F314Y.

[0007] FRAPD-TGm2A2 (SEQ ID NO.5) is: FRAPD-TGm2A-Y34W-A160P-G157Q-G250K-D324P. The first five amino acids in SEQ ID NO.5, FRAPD, are the five amino acids remaining after the zymogen is cleaved. The amino acid sites described in this invention start with the first position in the maturation region (positions 6 to 336 in the full-length sequence). For example, position 6 in the full-length sequence is the first position in the maturation region, which is described as D1. Another example is Y34W, which indicates that the Y at position 34 in the maturation region (position 39 in the full-length sequence) is mutated to W.

[0008] The present invention also provides a nucleic acid.

[0009] Specifically, the nucleic acid encodes the gene for the aforementioned glutamine transaminase mutant.

[0010] The present invention also provides a recombinant vector carrying the above-mentioned genes.

[0011] In one embodiment of the present invention, the recombinant vector used for expression of *E. coli* is pET-22b+, in which... Nde I- Blp I-site integration dissolution tag TrxA and S. caniferus (proC)-derived TGase proterminus and TGm2A.

[0012] The recombinant vector used for Streptomyces expression was pSET152, in which... Xba I- EcoR The TGm2A1 sequence, optimized with Streptomyces codons, is integrated at site I.

[0013] In one embodiment of the present invention, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors.

[0014] The present invention also provides recombinant cells that express the above-mentioned mutants, or carry the above-mentioned genes, or carry the above-mentioned recombinant vectors.

[0015] In one embodiment of the present invention, the recombinant cells use prokaryotic cells as expression hosts.

[0016] In one embodiment of the present invention, the eukaryotic microorganism is selected from one or more of Saccharomyces cerevisiae, Pichia pastoris and Kluyveromyces marx var. sacchari; In one embodiment of the present invention, the prokaryotic microorganism is selected from one or more of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Salmonella and Streptomyces.

[0017] 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.

[0018] In one embodiment of the invention, the composition contains water or other buffering systems.

[0019] The present invention also relates to methods for producing the glutamine transaminase variant of the present invention, the methods comprising: specifically, the expression cassette comprising the above-described nucleic acid and promoter.

[0020] More specifically, the promoter is a prokaryotic promoter, and the prokaryotic promoter is selected from T7.

[0021] The present invention also provides a gene engineering vector. Specifically, the gene engineering vector is a prokaryotic expression vector; the prokaryotic expression vector is selected from pET22b.

[0022] The present invention also provides a microorganism. Specifically, the microorganism comprises the above-mentioned highly thermostable glutamine transaminase mutant, nucleic acid, expression cassette, and / or gene engineering vector. More specifically, the microorganism is... E. coli BL21 (DE3).

[0023] The present invention also provides recombinant cells expressing the above-mentioned mutants or carrying the above-mentioned nucleic acids or carrying the above-mentioned recombinant vectors.

[0024] In one embodiment of the present invention, the recombinant cells are expressed using prokaryotic microorganisms as expression hosts.

[0025] In one embodiment of the present invention, the prokaryotic microorganism is selected from Escherichia coli.

[0026] The present invention also provides a recombinant enzyme catalyst containing the above-mentioned glutamine transaminase mutant, which is any one of the following forms: (1) Culture recombinant expression transformants containing the glutamine transaminase mutant and isolate transformant cells containing the recombinant glutamine transaminase mutant enzyme; (2) Cultivate recombinant expression transformants containing the glutamine transaminase mutant, isolate transformant cells containing the recombinant glutamine transaminase mutant enzyme, and break the transformant cells containing the recombinant glutamine transaminase mutant enzyme to obtain cell lysate; (3) Cultivate recombinant expression transformants containing the glutamine transaminase mutant, isolate transformant cells containing the recombinant glutamine transaminase mutant enzyme, break the transformant cells containing the recombinant glutamine transaminase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant glutamine transaminase mutant enzyme to obtain lyophilized powder. (4) Cultivate recombinant expression transformants containing the glutamine transaminase mutant, isolate transformant cells containing the recombinant glutamine transaminase mutant enzyme, break the transformant cells containing the recombinant glutamine transaminase mutant enzyme, obtain cell lysate, and purify the cell lysate of the recombinant glutamine transaminase mutant enzyme to obtain pure enzyme solution.

[0027] The present invention also provides a method for improving the thermostability of transglutaminase, the method comprising: mutating wild-type transglutaminase into the transglutaminase mutant shown in SEQ ID NO.5; Or the method described is: After mutating wild-type glutamine transaminase to the glutamine transaminase mutant shown in SEQ ID NO.5, and further mutating it by changing the following: lysine at position 325 to glutamic acid; or leucine at position 294 to isoleucine; or valine at position 311 to cysteine; or threonine at position 313 to glutamic acid; or phenylalanine at position 314 to tyrosine; or proline at position 324 to glutamic acid; or lysine at position 325 to glutamic acid while simultaneously changing valine at position 311 to cysteine; or lysine at position 325 to glutamic acid while simultaneously changing valine at position 311 to cysteine; or lysine at position 325 to glutamic acid while simultaneously changing valine at position 311 to cysteine. The valine is mutated to cysteine, and the proline at position 324 is mutated to glutamic acid; or the lysine at position 325 is mutated to glutamic acid, the valine at position 311 is mutated to cysteine, and the leucine at position 294 is mutated to isoleucine; or the lysine at position 325 is mutated to glutamic acid, the valine at position 311 is mutated to cysteine, and the isoleucine at position 312 is mutated to valine; or the lysine at position 325 is mutated to glutamic acid, the valine at position 311 is mutated to cysteine, and the phenylalanine at position 314 is mutated to tyrosine.

[0028] The present invention also provides a recombinant Streptomyces strain that expresses the above-mentioned glutamine transaminase mutant.

[0029] The present invention also provides the application of the above-mentioned glutamine transaminase mutant, or the above-mentioned nucleic acid, or the above-mentioned recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant enzyme catalyst, or the above-mentioned recombinant Streptomyces in food processing, processing or transformation, or in the preparation of food or pharmaceuticals.

[0030] Beneficial effects (1) This invention, through the analysis of materials from... Streptomyces mobaraensis Structural analysis and mutational free energy prediction of glutamine transaminase revealed a significant increase in its half-life. T The high thermal stability mutant with an increased m value, without loss of enzyme activity, breaks the inherent trade-off between activity and temperature stability, and provides a universal strategy framework for the rational modification of thermostable enzymes.

[0031] (2) This invention uses casein as a substrate and glutamine transaminase catalyzes it to achieve efficient cross-linking at high temperatures of 75℃, 78℃ and 85℃, thereby improving the industrial application range of glutamine transaminase. Attached Figure Description

[0032] Figure 1A , Figure 1B: SDS-PAGE image of heterologous expression of transglutaminase; where M is the protein marker, and the rest are pure enzyme bands of transglutaminase.

[0033] Figure 2 : Three-dimensional structural diagram of transglutaminase.

[0034] Figure 3A , Figure 3B Determination of enzyme activity and residual enzyme activity in glutamine transaminase mutant strains.

[0035] Figure 4 Determination of the optimal temperature for glutamine transaminase mutant strains.

[0036] Figure 5 Analysis of the effect of transglutaminase cross-linking casein.

[0037] Figure 6 Expression and properties of transglutaminase FRAPD-TGm2A1 in Streptomyces; Figure A shows the changes in enzyme activity and biomass with fermentation time, Figure B shows the SDS-PAGE analysis of crude enzyme solution at different fermentation times, Figure C shows the residual enzyme activity at different incubation times at 60℃, and Figure D shows the specific enzyme activity at different temperatures.

[0038] Figure 7 Expression and properties of glutamine transaminase FRAPD-TGm2A2 in Streptomyces.

[0039] Figure 8 Expression and properties of glutamine transaminase FRAPD-TGm2A3 in Streptomyces. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0041] The methods involved in the following embodiments are as follows: SDS-PAGE gel electrophoresis Prepare the SDS-PAGE preform, loading buffer, and protein samples.

[0042] Electrophoresis: Protein gel loading volume: 15 μL sample, 5 μL marker; Electrophoresis conditions: 120 V, 60 min.

[0043] Staining: Soak the protein gel in rapid staining solution for 10 min, then wash twice with water. Destaining: Soak in destaining solution (10% ethanol, 10% acetic acid, 80% water) and shake gently, replacing with fresh destaining solution until the bands are clearly visible.

[0044] The following examples illustrate the culture and expression methods of recombinant Escherichia coli. (1) Preparation of seed liquid A single colony was picked from an LB plate and inoculated into LB medium supplemented with 50 μg / mL ampicillin. The culture was then incubated at 37°C and 200 rpm for 8–10 h to obtain a seed culture.

[0045] (2) Fermentation culture of recombinant Escherichia coli The seed culture was inoculated at a 1% (v / v) inoculation rate into shake flasks containing TB liquid medium (50 mL / 250 mL volume) of ampicillin (concentration: 50 mg / L), and incubated at 37 °C and 200 rpm until OD... 600 When the concentration reaches 1.0, add isopropyl- to a final concentration of 0.1 mM. β -D-thiogalactoside (IPTG) was then cultured at 20°C for 36 h to induce protein expression.

[0046] The following examples illustrate the culture and expression methods of recombinant Streptomyces. (1) Preparation of seed liquid Spores were scraped from GYM plates (10 g / L glucose, 4 g / L yeast extract, 3 g / L malt extract, 20 g / L agar) and inoculated into seed culture medium containing 50 μg / mL apramycin (20 g / L glycerol, 20 g / L peptone, 5 g / L yeast extract, 4 g / L K2HPO4, 2 g / L MgSO4, pH=7.2). Seed culture was obtained by incubation at 30℃ and 220 rpm for 24 h.

[0047] (2) Fermentation culture of recombinant Streptomyces Inoculate 2 mL of seed culture into 30 mL of fermentation medium II (20 g / L glycerol, 25 g / L fish meal peptone, 5 g / L yeast extract, 5.5 g / L corn syrup (powder), 5.5 g / L (NH4)2SO4, 2 g / L MgSO4, 2 g / L K2HPO4, pH=7.2-7.4). Incubate at 30℃ and 220 rpm for 4 days.

[0048] The following examples illustrate the preparation methods of crude enzyme solutions. (1) Escherichia coli fermentation The obtained fermentation broth was centrifuged at low temperature at 8,000 × 10⁻⁶.g Centrifuge at 4°C for 10 min, discard the supernatant, and resuspend the precipitate in Tris-HCl (50 mmol / L, pH 8.0) buffer. The resulting bacterial cells are homogenized by high-pressure homogenization at 800 Pa and 4°C for 3 min, and the supernatant is collected by centrifugation at 8,000 × 10⁻⁶. g， After 10 min at 4℃, add 200 mg / mL of neutral protease (50 mmol / L Tris-HCl, 100 mmol / L NaCl, 2 mmol / L CaCl2, 1 mmol / L reduced glutathione, 200 g / L neutral protease, pH 7.0) to the supernatant until the final concentration of neutral protease is 20 mg / mL. Incubate at 37℃ for 20 min, centrifuge, and collect the supernatant, which is the crude enzyme solution described in this embodiment of the invention. The centrifugation conditions are 10,000 × 10⁻⁶. g， 20 min, 4℃.

[0049] (2) Streptomyces fermentation After centrifuging the fermentation broth and retaining the supernatant, add pre-cooled ethanol at -20℃ until the final ethanol concentration reaches 70%. After standing at 4℃ for 30 min, collect the protein precipitate and dissolve it in acetate buffer (50 mM, pH=5.5) to obtain the crude enzyme solution.

[0050] The purification of glutamine transaminase involved in the following examples (1) Escherichia coli fermentation All recombinant proteins had a 6 × His tag at the N-terminus, and the proteins were purified using a nickel affinity chromatography column (GE Healthcare, Chicago, USA). The crude enzyme solution was filtered through a 0.22 μm filter to obtain the supernatant. The supernatant was loaded onto a His-Trap HP column pre-equilibrated with purification buffer (50 mmol / L Tris-HCl, 200 mmol / L NaCl, 25 mmol / L imidazole, pH 8.0). The target protein was eluted with 150 mM imidazole in the same buffer. Finally, the imidazole was removed using a Superdex 75 gel column (GE Healthcare, Chicago, USA) and Tris-HCl buffer.

[0051] (2) Streptomyces fermentation Cation exchange chromatography was performed using a PrePack SP Purose 6 Fast Flow cation exchange column, eluted with acetate buffer containing 150 mM NaCl. Desalting was then performed using a Superdex 75 gel column with Tris-HCl buffer.

[0052] The enzyme activity assay and thermal stability assay of transglutaminase involved in the following examples (1) Determination of glutamine transaminase activity: Add 60 μL of TGase sample to 150 μL of TGase enzyme activity assay substrate (Tris-HCl 200 mmol / L, hydroxylamine 100 mmol / L, reduced glutathione 10 mmol / L, CBZ-Gln-Gly 30 mmol / L, pH 6.0), and incubate at 37℃ in a metal bath for 10 min. Then add 60 μL of TGase enzyme activity assay stop agent (equal volumes of 120 g / L trichloroacetic acid, 3 mmol / L HCl, and 50 g / L FeCl3·6H2O mixed thoroughly). After thoroughly mixing the mixture using a pipette, measure the absorbance at 525 nm using 200 μL of the mixture. For the blank control, replace the enzyme solution with Tris-HCl buffer, keeping all other conditions unchanged, and measure the absorbance at 525 nm using 200 μL of the mixture. The enzyme activity corresponding to the mass of protein added can be obtained by subtracting the absorbance value obtained from the control group from the absorbance value obtained from the experimental group and subtracting it into the enzyme activity standard curve. The specific enzyme activity (U / mg) can be obtained by dividing the enzyme activity by the protein concentration.

[0053] Enzyme activity definition: One unit (U) of activity is defined as the production of 1 μmol of L-glutamate-γ-monohydroxyxamic acid per minute under standard assay conditions. All experiments were repeated three times.

[0054] Enzyme activity definition (U / mg): The enzyme activity per milligram of enzyme.

[0055] (2) Determination of the thermal stability of transglutaminase: To assess the thermostability of the enzyme, the purified enzyme was incubated at 60℃, 65℃, or 70℃ for a certain period of time, and then its residual activity was measured at 37℃ according to the method for determining glutamine transaminase activity, with the initial activity set at 100%. Each experiment was performed in triplicate. Specifically, the parent protein and different mutant protein solutions were first diluted to 0.5 mg / ml. A certain amount of this sample was taken and continuously incubated in a water bath at 60℃, 65℃, or 70℃, with samples taken at intervals. All samples were immediately placed on ice for cooling. Enzyme activity was measured on each sample to obtain the percentage of residual TGase activity compared to the initial activity over time. This was determined using the Exponential method in Original 2018. ExpDec1 was used to perform a nonlinear fit, and after obtaining the fitting formula, the time corresponding to the enzyme activity decreasing to 50% of the initial value was calculated, which is the half-life.

[0056] Dynamic parameter determination The Michaelis constant (KM) is determined by diluting the parent and mutant protein solutions to 0.05 mg / ml, and following the enzyme activity assay method. According to N... benzyloxycarbonyl L Ten substrate solutions A with varying glutamylglycine content were prepared, while other components remained constant. benzyloxycarbonyl L The gamma-glutamyl glycine contents were 3 mM, 6 mM, 9 mM, 12 mM, 15 mM, 18 mM, 21 mM, 24 mM, 27 mM, and 30 mM, respectively. According to the enzyme activity assay method, the parent plant and mutants were tested for their effects on different N... benzyloxycarbonyl L The substrate conversion in substrate solution A with glutamylglycine content, i.e., the catalytic conversion of substrate N within a 10-minute reaction time. benzyloxycarbonyl L The amount of glutamyl glycine converted into the final product. Based on the conversion amounts obtained above, nonlinear fitting was performed on the obtained values ​​using Origin 2018 software, employing Growth / Sigmodial... Hill performed a fit and obtained Km and Vmax values, then converted the enzyme concentration to obtain the kcat value. Based on this, the enzyme catalytic efficiency was obtained as Km / kcat.

[0057] The following examples involve transglutaminase. T Detection method of m Circular dichroism spectroscopy was used to study transglutaminase. T The m-value was determined. Using TA NanoDSC, the protein sample was dissolved in buffer and adjusted to a concentration of 1.5 mg / mL. The protein's solvent (Tris-HCl, pH 8.0) was used as an internal control. The scanning temperature ranged from 30 to 90 °C, with a heating rate of 1 °C / min, and a pressure of 3 atm. The enthalpy change rate with increasing temperature was obtained and compared with the internal control to infer the protein's m-value. T m .

[0058] Example 1: Expression of glutamine transaminase The specific steps are as follows: (1) Construction of recombinant vector TrxA (SEQ ID NO.2) and proC (SEQ ID NO.3) were ligated into the pET-22b vector. Nde I- Blp pET-22b-TrxA-proC was constructed at site I.

[0059] Will come from Streptomyces mobaraensis The amino acid sequence of transglutaminase (SEQ ID NO.1) was obtained. Based on the codon preference of E. coli, the gene was codon optimized, and a biotechnology company was commissioned to chemically synthesize the gene. The optimized sequence was then constructed into the vector pET-22b-TrxA-proC to prepare the recombinant vector pET-22b-TrxA-proC-TGm2A. TGm2A (SEQ ID NO.1): DPDDRVTTPPAEPLDRMPDPYRPVNGRATTVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKEAFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNED ARSPFYSALRNTPLFKERNGGNHDPSRMKAVIYAKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEPDADKTVWTHGNHYHAPNGSLGPMHVYESLFRNWSEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP TrxA (SEQ ID NO.2) MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLA proC (SEQ ID NO.3) MASGGDEEWEGSYAATHGLTAEDVKNINALNKRALTAGQPGNFPAELPPSATALFRAPD (2) The recombinant vector pET-22b-TrxA-proC-TGm2A obtained in step (1) is further converted to E. coli Recombinant bacteria were obtained from BL21 (DE3) competent cells. E. coliBL21 (DE3) / pET-22b-TrxA-proC-TGm2A; (3) The prepared recombinant Escherichia coli E. coli BL21 (DE3) / pET-22b-TrxA-proC-TGm2A was inoculated into LB medium and cultured at 37 ℃ for 8 h to prepare seed culture. The prepared seed culture was inoculated into TB liquid medium at an inoculation rate of 1.0% (v / v) and cultured at 20℃ and 200 rpm for 36 h to obtain the fermentation broth. (4) Obtaining pure enzyme solution After centrifuging the prepared fermentation broth, the collected recombinant bacterial cells were resuspended in Tris-HCl (50 mmol / L, pH 8.0) buffer, homogenized using a high-pressure homogenizer, and centrifuged at 8,000 × g for 10 min at 4 °C. 200 mg / mL of neutral protease (50 mmol / L Tris-HCl, 100 mmol / L NaCl, 2 mmol / L CaCl2, 1 mmol / L reduced glutathione, 200 g / L neutral protease, pH 7.0) was added to the supernatant until the final concentration of neutral protease was 20 mg / mL. The mixture was incubated at 37 °C for 20 min, centrifuged at 10,000 × g for 20 min at 4 °C, and the supernatant was collected and filtered through a 0.22 μm aqueous filter membrane. Purification was performed using an AKTA purification system, and the supernatant was loaded into a pre-purification buffer (50 mmol / L Tris-HCl, 200 mmol / L NaCl, 25 mmol / L imidazole, pH 7.0). 8.0) Pre-equilibrated nickel column. Elute with 150 mM imidazole in the same buffer to obtain the target protein. Finally, remove imidazole using a Superdex 75 gel column (GE Healthcare, Chicago, USA) and Tris-HCl buffer. Collect the target protein. All operations were performed at 4 °C; protein purity was determined by SDS-PAGE, and the results are shown below. Figure 1A and Figure 1B As shown.

[0060] The results showed that TGm2A was efficiently expressed heterologously in Escherichia coli, and enzyme solution with high purity of TGm2A was obtained.

[0061] Example 2: Preparation of glutamine transaminase mutant The specific steps are as follows: 1. Identify the mutation site (1) Structural analysis of FRAPD-TGm2A-Y34W The protein structure of FRAPD-TGm2A-Y34W was obtained through online modeling with AlphaFold 3. Figure 2 The secondary structure was visualized using PyMOL, and the amino acids located at the junction of the α-helix, β-sheet and loop region were identified (named helix corner points).

[0062] (2) Screening of FRAPD-TGm2A-Y34W mutation hotspots Virtual saturation mutations were performed on the helical corner points, and the folding free energy was calculated using Rosetta Cartesian ddG. The top 20 sites with the largest decrease in folding free energy were selected for experimental verification.

[0063] 2. Construction of FRAPD-TGm2A-Y34W and its mutants The specific steps are as follows: Construction of single mutants (1) Construction of recombinant FRAPD-TGm2A expression vector The FRAPD-TGm2A (amino acid sequence as shown in SEQ ID NO.4) was codon-optimized and then linked to the dissolution tag TrxA (SEQ ID NO.2) from Example 1. S. caniferus The TGase prototerminus (proC, SEQ ID NO. 3) is integrated into pET-22b+. Nde I- Blp The recombinant vector pET-22b-TrxA-proC-FRAPD-TGm2A was prepared at site I.

[0064] FRAPD-TGm2A is obtained by adding 5 amino acids (FRAPD) to the N-terminus of TGm2A. FRAPD consists of the five amino acids remaining after the zymogen is cleaved. The amino acid sequence is shown below (SEQ ID NO.4): FRAPDDPDDRVTTPPAEPLDRMPDPYRPVNGRATTVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKEAFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNED ARSPFYSALRNTPLFKERNGGNHDPSRMKAVIYAKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEPDADKTVWTHGNHYHAPNGSLGPMHVYESLFRNWSEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP (2) Introduction of site-directed mutation Using the vector pET-22b-TrxA-proC-FRAPD-TGm2A obtained in step (1) as a template, Y34W was introduced using the primers shown in Table 2 to construct a recombinant vector containing the mutant: pET-22b-TrxA-proC-FRAPD-TGm2A-Y34W; Based on pET-22b-TrxA-proC-FRAPD-TGm2A-Y34W, 20 sites with significant changes in folding free energy were introduced for mutation using the primers shown in Table 1 (Table 2). The PCR reaction mixture was as follows: 2 μL upstream primer, 2 μL downstream primer, 1 μL template plasmid, 25 μL 2 × Phanta Max Master Mix, and 20 μL ddH2O. The pre-denaturation temperature was 95℃ for 3 min, followed by denaturation at 95℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 4 min, and final extension at 72℃ for 5 min, for a total of 30 cycles.

[0065] Table 1: Mutation sites and primers

[0066] Recombinant vectors expressing different mutants were prepared separately.

[0067] (3) Construction of recombinant strains The recombinant vectors containing wild-type recombinant glutamine transaminase and the recombinant vectors containing mutants obtained in steps (1) and (2) were respectively transformed into... E. coli Recombinant expression strains were prepared from BL21 (DE3) competent cells.

[0068] (4) Screening for positive mutants The recombinant expression strains obtained in step (3) were expressed and purified according to the method in Example 1. The specific enzyme activity of the purified enzymes was measured, and the residual enzyme activity after incubation at 60°C for 1 h was also measured. The results are as follows: Figure 3A and Figure 3B As shown in Table 2: Table 2: Enzyme activity after single point mutation and residual enzyme activity after incubation at 60 ℃ for 1 h

[0069] The results show: Compared to FRAPD-TGm2A-Y34W (74.38±4.07), mutations in G157N, G157Q, G157W, and A160P, based on FRAPD-TGm2A-Y34W, increased the residual enzyme activity after incubation at 60℃ for 1 h by 5.30%, 5.60%, 4.19%, and 6.21%, respectively. Among them, the A160P mutation improved thermostability while maintaining a high specific enzyme activity.

[0070] Example 3: Construction of the FRAPD-TGm2A combinatorial mutant 1. Construction of double mutants Combination mutations were performed on the four positive mutations G157N, G157Q, G157W, and A160P to obtain mutants with higher thermal stability. The specific steps are as follows: (1) The recombinant strain containing the A160P positive mutation (FRAPD-TGm2A-Y34W-A160P) was inoculated into a test tube containing 5 mL of LB medium, and ampicillin was added to a final concentration of 50 mg / L. The tube was then incubated in a constant temperature shaker at 37℃ and 200 rpm for 8 h. Subsequently, plasmid was extracted using the SanPrep column-based plasmid DNA mini-extraction kit. The extraction steps were performed according to the instructions provided in the kit. The recombinant plasmid was prepared as pET-22b-TrxA-proC-FRAPD-TGm2A-Y34W-A160P.

[0071] (2) Using the plasmid pET-22b-TrxA-proC-FRAPD-TGm2A-Y34W-A160P as a template, PCR was performed using the primers shown in Table 2 to introduce the second mutation site. Based on the plasmid containing A160P (pET-22b-TrxA-proC-FRAPD-TGm2A-Y34W-A160P), G157N, G157Q, and G157W were introduced to obtain plasmids containing the mutants FRAPD-TGm2A-Y34W-A160P-G157N; FRAPD-TGm2A-Y34W-A160P-G157Q; and FRAPD-TGm2A-Y34W-A160P-G157W. A total of 3 double mutant plasmids were obtained. The PCR, template digestion, and product purification procedures were the same as those for the construction of the single mutant in Example 2. Subsequently, following the recombinant strain construction method described in the single mutant construction section, the corresponding recombinant strain was obtained. After expression and purification, the enzyme activity was measured, and the residual activity was measured after incubation at 65 °C for 1 h. The results are shown in Table 3.

[0072] Table 3: Temperature stability determination of double-spot mutants

[0073] The results showed that the combined mutation of A160P and G157Q resulted in the most significant improvement in thermal stability. Therefore, subsequent studies will focus on... E. coli BL21 (DE3) / pET-22b-TrxA-proC-FRAPD-TGm2A-Y34W-A160P-G157Q (hereinafter referred to as FRAPD-TGm2A1) is the optimal mutant strain for highly thermostable glutamine transaminase.

[0074] 2. Construction of the three mutations First, sequences were designed based on the deep learning model ProteinMPNN, and sequence consistency analysis was performed. The three-dimensional structure (PDB format) of FRAPD-TGm2A1 was used as input and submitted to the ProteinMPNN deep learning model. The default sampling temperature (T=0.1) was used in the runtime parameter settings, generating 1000 novel protein sequences. All generated sequences were then subjected to consistency analysis using MEGA 11 software. Amino acids with high frequency at each site were defined as high-probability amino acids.

[0075] Secondly, stability mutation prediction based on evolutionary information. The FRAPD-TGm2A1 3D structure file was submitted to the FireProt online server. Naturally occurring amino acid mutations (i.e., "consensus mutations") in highly conserved but distantly related homologous proteins during evolution were identified, and candidate beneficial mutations were obtained.

[0076] Finally, through cross-comparison of ProteinMPNN analysis and FireProt analysis, the beneficial mutation sites predicted by FireProt were mutated into high-probability amino acids recognized by ProteinMPNN for experimental verification.

[0077] Based on the above method, and following the methods in Examples 1 and 2, using pET-22b-TrxA-proC-FRAPD-TGm2A-Y34W-A160P-G157Q prepared in step 1 as a template, PCR was performed using the primers shown in Table 5 to introduce mutation sites. Single-point mutations were performed on FRAPD-TGm2A1 (FRAPD-TGm2A-Y34W-A160P-G157Q), resulting in 16 mutants (FRAPD-TGm2A1-R89Y, FRAPD-TGm2A1-R89A, FRAPD-TGm2A1-S101P, FRAPD-TGm2A1-S144A, FRAPD-TGm2A1-S144D, FRAPD-TGm2A1-S144E, FRAPD-TGm2A1-R236K, FRAPD- TGm2A1-G250T, FRAPD-TGm2A1-G250K, FRAPD-TGm2A1-G250S, FRAPD-TGm2A1-E300A, FRAPD-TGm2A1- E300K, FRAPD-TGm2A1-S303P, FRAPD-TGm2A1-S303D, FRAPD-TGm2A1-D324A, FRAPD-TGm2A1-D324P).

[0078] The primers involved are shown in Table 4: Table 4: Primers

[0079] The results showed that the specific enzyme activity and residual enzyme activity of 16 mutants after incubation at 60℃ for 60 min were measured. Among them, FRAPD-TGm2A1-R89A, FRAPD-TGm2A1-S144A, FRAPD-TGm2A1-S144E, FRAPD-TGm2A1-R236K, FRAPD-TGm2A1-G250K and FRAPD-TGm2A1-D324P showed good stability. The residual enzyme activity increased from 80.37% (residual enzyme activity of FRAPD-TGm2A1) to 81.97% (R89A), 83.36% (S144A), 86.23% (S144E), 87.22% (R236K), 88.19% (G250K) and 83.44% (D324P), respectively. The FRAPD-TGm2A1-G250K performed best (88.19%), and it was combined with five other points.

[0080] 3. Construction of the four mutations Using the plasmid pET-22b-TrxA-proC-FRAPD-TGm2A1-G250K as a template, PCR reactions were performed using the primers shown in Table 5. R89A, S144A, S144E, R236K, and D324P were introduced to obtain plasmids containing the mutants FRAPD-TGm2A1-G250K-R89A, FRAPD-TGm2A1-G250K-S144A, FRAPD-TGm2A1-G250K-S144E, FRAPD-TGm2A1-G250K-R236K, and FRAPD-TGm2A1-G250K-D324P. A total of five double-mutant (relative to the FRAPD-TGm2A1 mutant) plasmids were obtained. The PCR, template digestion, and product purification procedures followed the single-mutant construction procedure in Example 2. Subsequently, the recombinant strain was obtained according to the method described in the construction of single mutants. After expression and purification, the enzyme activity was measured, and the residual activity was measured after incubation at 65 °C for 20 min.

[0081] The results show: FRAPD-TGm2A1-G250K-R236 and FRAPD-TGm2A1-G250K-D324P exhibited good stability, with residual enzyme activity increasing from 63.55% of FRAPD-TGm2A1-G250K to 67.97% and 69.06%, respectively. Comparing the specific enzyme activity after incubation, only FRAPD-TGm2A1-G250K-D324P showed an improvement. Therefore, further analysis was conducted on FRAPD-TGm2A1-G250K-D324P (named FRAPD-TGm2A2).

[0082] FRAPD-TGm2A2 is obtained by adding 5 amino acids (FRAPD) to the N-terminus of the TGm2A2 sequence; the underlined FRAPD in the sequence represents the five amino acids remaining after the zymogen region is cleaved. The amino acid sequence of FRAPD-TGm2A2 is as follows (SEQ ID NO.5): FRAPD DPDDRVTTPPAEPLDRMPDPYRPVNGRATTVVNNWIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKEAFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANQNDPLRNED ARSPFYSALRNTPLFKERNGGNHDPSRMKAVIYAKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEKFVNFDYGWFGAQTEPDADKTVWTHGNHYHAPNGSLGPMHVYESLFRNWSEGYSDFDRGAYVITFIPKSWNTAPPKVKQGWP The ununderlined portion represents the maturation region of transglutaminase. The amino acid sites described in this invention start with the first position of the maturation region. For example, the 6th position in the full-length sequence is the 1st position of the maturation region, which is described as D1.

[0083] 4. Construction of the five mutations Following the methods in Examples 1 and 2, using the recombinant vector pET-22b-TrxA-proC-FRAPD-TGm2A2 constructed in step 3 as a template, PCR was performed using the primers shown in Table 6 to introduce mutation sites. Single-point mutations were performed on FRAPD-TGm2A2, resulting in 20 mutants: FRAPD-TGm2A2-V311C, FRAPD-TGm2A2-T313Q, FRAPD-TGm2A2-F314V, FRAPD-TGm2A2-V311F, FRAPD-TGm2A2-K325E, FRAPD-TGm2A2-T313E, FRAPD-TGm2A2-L294I, FRAPD-TGm2A2-I312A, FRAPD-TGm2A2-F314Y, FR... APD-TGm2A2-P324E, FRAPD-TGm2A2-V311G, FRAPD-TGm2A2-L294Q, FRAPD-TGm2A2-I312V, FRAPD-TGm2A2-S318K, FRAPD-TGm2 A2-S318A, FRAPD-TGm2A2-F314I, FRAPD-TGm2A2-T313V, FRAPD-TGm2A2-V311I, FRAPD-TGm2A2-F314V, FRAPD-TGm2A2-P324M.

[0084] The primers involved are shown in Table 5.

[0085] Table 5: Primer Sequences

[0086] Following the method in Example 2, the specific enzyme activity and the residual enzyme activity after incubation at 65°C for 30 min were determined.

[0087] The results showed that six mutants exhibited significantly increased stability. Compared to FRAPD-TGm2A2, FRAPD-TGm2A2-K325E showed the most significant improvement, with a 20.49% increase in residual enzyme activity after incubation at 65℃ for 30 min. The other five mutants, FRAPD-TGm2A2-L294I, FRAPD-TGm2A2-V311C, FRAPD-TGm2A2-T313E, FRAPD-TGm2A2-F314Y, and FRAPD-TGm2A2-P324E, showed increases of 6.30%, 16.63%, 10.90%, 8.63%, and 5.56%, respectively.

[0088] 5. Construction of the six mutation Using the plasmid pET-22b-TrxA-proC-FRAPD-TGm2A2-K325E as a template, PCR reactions were performed using the primers shown in Table 6. L294I, V311C, T313E, F314Y, and P324E were introduced to obtain plasmids containing the mutants FRAPD-TGm2A2-K325E-L294I, FRAPD-TGm2A2-K325E-V311C, FRAPD-TGm2A2-K325E-T313E, FRAPD-TGm2A2-K325E-F314Y, and FRAPD-TGm2A2-K325E-P324E. A total of five double-mutant plasmids were obtained. The PCR, template digestion, and product purification procedures followed the single-mutant construction procedure in Example 2. Subsequently, following the recombinant strain construction method described in the single mutant construction section, the corresponding recombinant strains were obtained. After expression and purification, the enzyme activity was measured, and the residual activity was measured after incubation at 65 °C for 1 h. Furthermore, triple mutants were constructed according to the above method (Table 6), and the results are as follows: Figure 3A and Figure 3B As shown. Meanwhile, using FRAPD-TGm2C (described in the text of Chinese invention patent application number 202410823426.0); the results are shown in Table 7.

[0089] Table 6: Enzyme activity data of different mutants

[0090] The results showed that the residual specific enzyme activity of the mutant enzyme was improved after the FRAPD-TGm2A2 technology was improved, and FRAPD-TGm2A2-K325E-V311C was named FRAPD-TGm2A3.

[0091] The sequence of FRAPD-TGm2A3 is as follows: FRAPDDPDDRVTTPPAEPLDRMPDPYRPVNGRATTVVNNWIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKEAFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANQNDPLRNED ARSPFYSALRNTPLFKERNGGNHDPSRMKAVIYAKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEKFVNFDYGWFGAQTEPDADKTVWTHGNHYHAPNGSLGPMHVYESLFRNWSEGYSDFDRGAYCITFIPKSWNTAPPEVKQGWP The optimal temperature showed that the optimal temperature of FRAPD-TGm2A3 was not higher than that of FRAPD-TGm2A2, remaining at 65℃, but the enzyme activity was improved at the same temperature.

[0092] Example 4: Property determination of FRAPD-TGm2A, FRAPD-TGm2A1, FRAPD-TGm2A2, and FRAPD-TGm2A3 1. The half-lives of glutamine transaminases FRAPD-TGm2A, FRAPD-TGm2A1, FRAPD-TGm2A2, and FRAPD-TGm2A3 were determined at 60, 65, 70, and 75°C. The specific steps are as follows: The FRAPD-TGm2A, FRAPD-TGm2A1, FRAPD-TGm2A2, and FRAPD-TGm2A3 prepared in Example 3 were incubated in water baths at 60℃, 65℃, 70℃, and 75℃ for certain time intervals, and the residual enzyme activity was measured. The half-life was calculated by nonlinear fitting using Original 2021-Exponential-ExpDec1. The results are shown in Table 7.

[0093] Table 7: Half-life determination of mutants

[0094] 2. Determination of the optimal temperature for FRAPD-TGm2A, FRAPD-TGm2A1, FRAPD-TGm2A2, and FRAPD-TGm2A3 transglutaminases The specific steps are as follows: The specific enzyme activities of FRAPD-TGm2A, FRAPD-TGm2A1, FRAPD-TGm2A2, and FRAPD-TGm2A3 prepared in Example 3 were measured at 37, 40, 45, 50, 55, 60, 65, 70, and 75 °C to determine their optimal temperatures. The results are as follows. Figure 4 As shown.

[0095] The results show that the optimal temperatures for FRAPD-TGm2A, FRAPD-TGm2A1, FRAPD-TGm2A2, and FRAPD-TGm2A3 are 60℃, 65℃, 65℃, and 65℃, respectively.

[0096] 3. Determination of the kinetic constants of glutamine transaminase FRAPD-TGm2A and FRAPD-TGm2A1 The specific steps are as follows: The kinetic constants of the glutamine transaminases FRAPD-TGm2A, FRAPD-TGm2A1, FRAPD-TGm2A2, and FRAPD-TGm2A3 prepared in Example 3 were determined at 37°C and 65°C, respectively. Substrate solutions with concentrations of 3-30 mM were prepared, and enzyme activities at the corresponding temperatures were measured. The results were obtained through Origin 2021-Growth / Sigmodial-Hill nonlinear fitting. K m and V max The value is obtained by converting enzyme concentration. k cat Value and K m / k cat value.

[0097] The results are shown in Table 8.

[0098] Table 8: Kinetic constants of FRAPD-TGm2A and FRAPD-TGm2A1

[0099] As shown in Table 9, at 37 and 65℃, compared to FRAPD-TGm2A, FRAPD-TGm2A1... K m The value has increased, while k cat The value decreases at 37℃ and increases at 65℃. These changes affect the overall catalytic efficiency of FRAPD-TGm2A at 37℃. k cat / K mThe catalytic efficiency decreased at 37°C, while it increased at 65°C; these results correspond to enzyme activity. At 37°C and 65°C, FRAPD-TGm2A2 showed significantly higher efficiency than FRAPD-TGm2A1. K m All decreased and k cat Both increased, and this trend led to its [value] at both temperatures. k cat / K m All received improvements.

[0100] At 37℃ and 65℃, FRAPD-TGm2A3 was superior to FRAPD-TGm2A2. K m and k cat All increased, k cat / K m It also improved, indicating k cat The increase was higher than K m .

[0101] Example 5: Casein crosslinking catalyzed by mutant FRAPD-TGm2A1 The specific steps are as follows: Using β-casein as a substrate, the cross-linking activity of TGase on proteins was detected. TGase (FRAPD-TGm2A, FRAPD-TGm2A1, FRAPD-TGm2A2, and FRAPD-TGm2A3 prepared in Examples 1-3) was added to 1 mL of β-casein solution (3 mg / mL casein, Tris-acetic acid (100 mM, pH=7)) to a final concentration of 0.01 mg / mL, and reacted at 75℃, 78℃, 80℃, and 85℃ for different times (5, 10, 20, and 40 min). The treated reaction samples were reacted with 8 M urea at a 1:3 volume ratio and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples reacted for 40 min were analyzed by size exclusion chromatography (SEC) using a HiLoad26 / 600 Superdex 200 pg column (GE Healthcare).

[0102] The results are as follows Figure 5 As shown.

[0103] The results show: (1) At 75℃The results of the casein crosslinking experiment showed that the mutant FRAPD-TGm2A1 was completely crosslinked at 5 min, while FRAPD-TGm2A lost its crosslinking activity after crosslinking most of the casein bands.

[0104] SEC analysis of the sample after 40 min of reaction showed that the elution volume of large protein molecules such as casein cross-linked complexes was around 120 ml, casein eluted at around 220 ml, and the remaining small molecules eluted at 300 ml. FRAPD-TGm2A1 had completely cross-linked casein, while FRAPD-TGm2A still had residues.

[0105] At 75°C, FRAPD-TGm2A's ability to crosslink casein is not as good as that of FRAPD-TGm2A1.

[0106] (2) At 78℃ The results of the casein crosslinking experiment showed that the mutant FRAPD-TGm2A2 was completely crosslinked at 5 min, while FRAPD-TGm2A1 lost its crosslinking activity after crosslinking most of the casein bands.

[0107] SEC analysis of the sample after 40 min of reaction showed that FRAPD-TGm2A2 had completely cross-linked casein, while FRAPD-TGm2A1 still had residues.

[0108] (3) At 85℃ The results of the casein crosslinking experiment showed that the mutant FRAPD-TGm2A3 was completely crosslinked at 5 min, while FRAPD-TGm2A2 lost its crosslinking activity after crosslinking most of the casein bands.

[0109] SEC analysis was performed on the samples after 40 min of reaction. FRAPD-TGm2A3 The casein has been completely cross-linked, but FRAPD-TGm2A2 remains.

[0110] In summary, FRAPD-TGm2A3 showed the best performance in casein crosslinking experiments at high temperatures.

[0111] Example 6: Expression and property determination of mutant TGm2A1 in Streptomyces All mutants of this invention (including but not limited to FRAPD-TGm2A2-K325E, FRAPD-TGm2A2-L294I, FRAPD-TGm2A2-V311C, FRAPD-TGm2A2-I312V, FRAPD-TGm2A2-T313E, FRAPD-TGm2A2-F314Y, FRAPD-TGm2A2-P324E, FRAPD-TGm2A2-K325E-V311C, FRAPD-TGm2A2-K325E-L294I, FRAPD-TGm2A2-K325E-I312V, FRAPD-TGm2A2-K325E-F314Y ...5E-L294I, FRAPD-TGm2A2-K325E-I312V, FRAPD-TGm2A2-K325E-F314Y, FRAPD-TGm2A2-K325E-F314Y, FRAPD-TGm2A2-K325E-F315E-L294I, F The following strains (A2-K325E-P324E, FRAPD-TGm2A2-K325E-V311C-P324E, FRAPD-TGm2A2-K325E-V311C-L294I, FRAPD-TGm2A2-K325E-V311C-I312V, FRAPD-TGm2A2-K325E-V311C-F314Y, FRAPD-TGm2A2-K325E-V311C-P324E, FRAPD-TGm2A, FRAPD-TGm2A1, FRAPD-TGm2A2, and FRAPD-TGm2A3) can all be expressed in Streptomyces strains. Taking FRAPD-TGm2A1 as an example, the specific steps are as follows: (1) Construction of recombinant Streptomyces TGm2A1 strain The specific steps are as follows: TGm2A1 (amino acid sequence as shown in SEQ ID NO.3; the proenzyme region FRAPD was directly removed during synthesis, which has no impact on enzyme performance) was integrated into pSET152 after codon optimization. Xba I- EcoRAt site I, a recombinant vector, pSET152-TGm2A1, was prepared. The pSET152-TGm2A1 plasmid was introduced into unmethylated Escherichia coli strain ET12567 / pUZ8002. After culturing at 37°C for 2 hours in LB medium (5 g / L yeast extract, 10 g / L casein, 5 g / L sodium chloride) containing antibiotics (25 μg / mL chloramphenicol, 50 μg / mL kanamycin, 50 μg / mL alpramycin), the recombinant E. coli cells were collected and mixed with a smY2019-Δtg spore suspension preheated to 50°C for 10 minutes (the preparation method is described in the Chinese invention patent text with publication number CN112126613B). The suspension was spread onto MS agar medium (20 g / L mannitol, 2.5 g / L malt extract, 20 g / L soybean flour, 20 g / L agar; 50 mM magnesium chloride) and incubated at 30°C for 16 hours. Finally, the agar medium was covered with 50 μg / mL alpramycin solution, and the transformants were cultured for 48 hours.

[0112] (2) Enzymatic assay of smY2019-TGm2A1 The specific steps are as follows: The transformants obtained in step (1) were cultured on GYM medium (10 g / L glucose, 3 g / L malt powder, 4 g / L yeast powder) for 4-5 days. The spores and mycelia were inoculated into seed medium containing 50 μg / mL apramycin (5 g / L yeast powder, 20 g / L glycerol, 20 g / L tryptone, 2 g / L MgSO4, 4 g / L K2HPO4, pH 7.2). After culturing at 30℃ for 24 h, 2.4 mL was inoculated into 30 mL of fermentation medium (5 g / L yeast powder, 20 g / L glycerol, 5.5 g / L corn steep liquor powder, 25 g / L fish meal peptone, 2 g / L K2HPO4, 5.5 g / L (NH4)2SO4, 2 g / L MgSO4, pH 7.2-7.4). The medium was cultured at 30℃ for 96 h, and samples were taken for analysis at 12 h intervals.

[0113] The results show: After 60 h of fermentation, the bacterial biomass and extracellular TGase activity both reached their peak values. The half-life and optimal temperature of the samples at this fermentation time were determined, and the results are as follows: Figure 6 As shown, TGm2A1's t 1 / 2 The thrombolysis time reached 160.03 min at 60℃, and its optimum temperature (65℃) was 15℃ higher than that of wild-type TGase.

[0114] Following the above method, FRAPD-TGm2A2 and FRAPD-TGm2A3 were expressed. The results of FRAPD-TGm2A2 and FRAPD-TGm2A3 expression are as follows: Figures 7-8 As shown.

[0115] 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 thermostable transglutaminase mutant, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.5, or it is obtained by modifying the glutamine transaminase with the amino acid sequence shown in SEQ ID NO.5 as the parent enzyme as follows: The lysine residue at position 325 of the parent enzyme was mutated to glutamic acid; Alternatively, the leucine at position 294 of the parent enzyme could be mutated to isoleucine; Alternatively, the valine at position 311 of the parent enzyme could be mutated to cysteine. Alternatively, the threonine at position 313 of the parent enzyme could be mutated to glutamate; Alternatively, the phenylalanine at position 314 of the parent enzyme could be mutated to tyrosine. Alternatively, the proline at position 324 of the parent enzyme could be mutated to glutamate. Alternatively, lysine at position 325 of the parent enzyme can be mutated to glutamic acid, and valine at position 311 of the parent enzyme can be mutated to cysteine. Alternatively, lysine at position 325 of the parent enzyme can be mutated to glutamic acid, valine at position 311 of the parent enzyme can be mutated to cysteine, and proline at position 324 of the parent enzyme can be mutated to glutamic acid. Alternatively, lysine at position 325 of the parent enzyme can be mutated to glutamic acid, valine at position 311 of the parent enzyme can be mutated to cysteine, and leucine at position 294 of the parent enzyme can be mutated to isoleucine. Alternatively, lysine at position 325 of the parent enzyme can be mutated to glutamic acid, valine at position 311 of the parent enzyme can be mutated to cysteine, and isoleucine at position 312 of the parent enzyme can be mutated to valine. Alternatively, lysine at position 325 of the parent enzyme can be mutated to glutamic acid, valine at position 311 of the parent enzyme can be mutated to cysteine, and phenylalanine at position 314 of the parent enzyme can be mutated to tyrosine.

2. A nucleic acid, characterized in that, The nucleic acid encodes the glutamine transaminase mutant of claim 1.

3. An expression cassette or recombinant vector carrying the nucleic acid described in claim 2.

4. The recombinant vector according to claim 3, characterized in that, The vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors.

5. Recombinant cells expressing the glutamine transaminase mutant of claim 1 or carrying the nucleic acid of claim 2 or carrying the recombinant vector of claim 3.

6. The recombinant cell according to claim 5, characterized in that, The recombinant cells use prokaryotic or eukaryotic microorganisms as expression hosts; Preferably, the eukaryotic microorganism is selected from one or more of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces martensii; Preferably, the prokaryotic microorganism is selected from one or more of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Salmonella, and Streptomyces.

7. A recombinant enzyme catalyst containing the glutamine transaminase mutant of claim 1, characterized in that, The recombinase catalyst is any one of the following forms: (1) Culture recombinant expression transformants containing the glutamine transaminase mutant and isolate transformant cells containing the recombinant glutamine transaminase mutant enzyme; (2) Cultivate recombinant expression transformants containing the glutamine transaminase mutant, isolate transformant cells containing the recombinant glutamine transaminase mutant enzyme, and break the transformant cells containing the recombinant glutamine transaminase mutant enzyme to obtain cell lysate; (3) Cultivate recombinant expression transformants containing the glutamine transaminase mutant, isolate transformant cells containing the recombinant glutamine transaminase mutant enzyme, break the transformant cells containing the recombinant glutamine transaminase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant glutamine transaminase mutant enzyme to obtain lyophilized powder. (4) Cultivate recombinant expression transformants containing the glutamine transaminase mutant, isolate transformant cells containing the recombinant glutamine transaminase mutant enzyme, break the transformant cells containing the recombinant glutamine transaminase mutant enzyme, obtain cell lysate, and purify the cell lysate of the recombinant glutamine transaminase mutant enzyme to obtain pure enzyme solution.

8. A method for improving the thermostability of wild-type glutamine transaminase, characterized in that, The method involves mutating wild-type transglutaminase into the transglutaminase mutant shown in SEQ ID NO.5; Or the method described is: After mutating wild-type glutamine transaminase to the glutamine transaminase mutant shown in SEQ ID NO.5, and further mutating it by changing the following: lysine at position 325 to glutamic acid; or leucine at position 294 to isoleucine; or valine at position 311 to cysteine; or threonine at position 313 to glutamic acid; or phenylalanine at position 314 to tyrosine; or proline at position 324 to glutamic acid; or lysine at position 325 to glutamic acid while simultaneously changing valine at position 311 to cysteine; or lysine at position 325 to glutamic acid while simultaneously changing valine at position 311 to cysteine; or lysine at position 325 to glutamic acid while simultaneously changing valine at position 311 to cysteine. The valine is mutated to cysteine, and the proline at position 324 is mutated to glutamic acid; or the lysine at position 325 is mutated to glutamic acid, the valine at position 311 is mutated to cysteine, and the leucine at position 294 is mutated to isoleucine; or the lysine at position 325 is mutated to glutamic acid, the valine at position 311 is mutated to cysteine, and the isoleucine at position 312 is mutated to valine; or the lysine at position 325 is mutated to glutamic acid, the valine at position 311 is mutated to cysteine, and the phenylalanine at position 314 is mutated to tyrosine.

9. A recombinant Streptomyces, characterized in that, The recombinant Streptomyces expressed the glutamine transaminase mutant of claim 1.

10. The use of the glutamine transaminase mutant of claim 1, or the nucleic acid of claim 2, or the recombinant vector of claim 3, or the recombinant cell of claim 5 or 6, or the recombinant enzyme catalyst of claim 7, or the recombinant Streptomyces of claim 9 in food processing, manufacturing or conversion, or in the preparation of food or pharmaceuticals.

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