CN113528478B - Method for efficiently producing transglutaminase and special engineering bacteria thereof - Google Patents

Abstract

The invention discloses a method for efficiently producing transglutaminase and a special engineering bacterium thereof. The invention provides a protein which is shown as a sequence 2 in a sequence table. The invention also relates to recombinant microorganisms obtained by introducing DNA molecules expressing said proteins into C.glutamicum. The recombinant microorganism can be used for preparing transglutaminase. The invention successfully realizes the high-efficiency expression of MTG in Corynebacterium glutamicum by expressing MTG derived from Streptomyces mobaraensis under a T7 promoter and inserting intein ssp-dnaB between a pro region and a mature region of the MTG, and the MTG with biological activity can be obtained without protease treatment or other condition changes after the thalli obtained by fermentation are subjected to ultrasonic disruption. The invention can be used for preparing transglutaminase, is further applied to the fields of tissue engineering, textile and leather processing and the like, and has application and popularization values.

Description

A method for efficiently producing transglutaminase and its special engineering bacteria

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for efficiently producing transglutaminase and special engineering bacteria thereof.

Background technique

Transglutaminase (EC 2.3.2.13, TGase) belongs to the family of transferases that catalyze the conversion of γ-carboxamide groups (donors) from glutamine residues to various primary amines (acyl acceptors). Acyl transfer. At present, TGase has been used in the field of food processing for more than 10 years and has been generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA). TGase can improve the properties of proteins through intramolecular and intermolecular cross-linking of proteins. Therefore, the enzyme shows wider application value in the fields of tissue engineering, textile and leather processing.

TGase exists widely in animals and plants, but its yield is low, and the cost of separation and purification is high, which is far from meeting people's needs. In 1989, microbial TGase (Microbial TGase, MTG) was discovered for the first time. Compared with TGase from other sources, MTG has the characteristics of non-Ca ²⁺ dependence, high temperature and high pH stability. In addition, the microbial production of MTG has the advantages of short production cycle, high yield, low cost, and easy industrial production. Therefore, people are more inclined to use microbial methods to produce MTG on a large scale.

MTG is usually secreted extracellularly in the form of an inactive zymogen (pro-MTG), and the active mature MTG can only be formed by removing the zymogen region (pro region) by proteolysis. The existence of the pro region plays an important role in the formation of the correct spatial structure and secretion of MTG.

Intein is a protein capable of protein splicing through nucleophilic substitution between amino acids without the involvement of other cofactors. Due to its unique mechanism of action, it has been widely used in biotechnology, biomedicine and protein chemistry. dnaB is a DNA helicase gene encoded by Synechocystis sp. After optimization and transformation, 106 amino acids at the C-terminal and 48 amino acids at the N-terminal constitute the smallest unit of intein . C-terminal cleavage of optimized intein ssp-dnaB is pH-dependent.

At present, more and more expression systems are used to express MTG, but there are still problems such as low expression level and secondary treatment with protease in the later stage. There are also some studies that use inteins to successfully express MTG in Escherichia coli and Bacillus subtilis, but it still needs to undergo secondary treatment of pH or temperature changes to realize the shearing of the pro region, which greatly increases the cost of industrial production and time cost.

Contents of the invention

The purpose of the present invention is to provide a method for efficiently producing transglutaminase and special engineering bacteria thereof.

The present invention provides a protein (protein A), which is the following (a1) or (a2):

(a1) the protein shown in the amino acid residues 1-532 of Sequence 2 in the sequence listing;

(a2) The protein shown in Sequence 2 of the Sequence Listing.

The nucleic acid molecules encoding the protein A also belong to the protection scope of the present invention.

The DNA molecule encoding the protein A also belongs to the protection scope of the present invention.

The DNA molecule is specifically any of the following (b1) to (b6):

(b1) a DNA molecule whose coding region is as shown in nucleotides 6389-7981 of Sequence 1 in the Sequence Listing;

(b2) a DNA molecule whose coding region is as shown in nucleotides 6386-7981 of Sequence 1 in the sequence listing;

(b3) a DNA molecule whose coding region is as shown in nucleotides 6389-7999 of Sequence 1 in the Sequence Listing;

(b4) a DNA molecule whose coding region is as shown in nucleotides 6386-7999 of Sequence 1 in the Sequence Listing;

(b5) a DNA molecule whose coding region is as shown in nucleotides 6389-8002 of Sequence 1 of the Sequence Listing;

(b6) A DNA molecule whose coding region is as shown in nucleotides 6386-8002 of Sequence 1 in the Sequence Listing.

The expression cassettes, recombinant vectors or recombinant microorganisms with the DNA molecules all belong to the protection scope of the present invention.

The recombinant vector is specifically as follows (c1) or (c2):

(c1) a recombinant vector having nucleotides 6304-8002 of Sequence 1 of the sequence listing;

(c2) A recombinant vector as shown in Sequence 1 of the Sequence Listing.

The recombinant microorganism is obtained by introducing the recombinant vector into Corynebacterium glutamicum.

The Corynebacterium glutamicum can specifically be Corynebacterium glutamicum C. glutamicum T7.

Corynebacterium glutamicum C. glutamicum T7 is a recombinant bacterium obtained by introducing T7-Plac into Corynebacterium glutamicum ATCC13032. Corynebacterium glutamicum C. glutamicum T7 is a recombinant bacterium obtained by integrating T7-Plac into the genome DNA of Corynebacterium glutamicum ATCC13032. T7-Plac is a DNA molecule composed of nucleotides 537-4951 in sequence 4 of the sequence listing.

Compared with Corynebacterium glutamicum ATCC13032, the difference of Corynebacterium glutamicum C. glutamicum T7 is only that glutamic acid is replaced by the double-stranded DNA molecule shown in the 17th-5461st nucleotide of sequence 4 of the sequence listing A double-stranded DNA molecule shown in Sequence 5 of the Sequence Listing in the genomic DNA of Corynebacterium ATCC13032.

The present invention also protects the application of the protein A or the nucleic acid molecule or the DNA molecule or the expression cassette or the recombinant vector or the recombinant microorganism in the preparation of transglutaminase.

The invention also protects a method for preparing transglutaminase, comprising the following steps: cultivating the recombinant microorganism.

The method for preparing the transglutaminase also includes the following steps: after the cultivation is completed, the bacteria are collected and the bacteria are broken.

The method for preparing the transglutaminase also includes the following step: collecting the supernatant after the destruction of the bacteria.

The method for preparing transglutaminase also includes the following steps: taking the supernatant, and collecting the protein with the His ₆ tag through nickel column affinity chromatography.

The method for preparing the transglutaminase also includes the following steps: after finishing the nickel column affinity chromatography, desalting and replacing the solvent system. The solvent system can specifically be Tris-HCl buffer solution with pH 8.0 and 50 mM.

The method for cultivating the recombinant microorganism specifically comprises the following steps:

(1) Cultivate the recombinant microorganism to an _OD600nm value=1 in a liquid medium;

(2) After completing step (1), add IPTG to the culture system so that the concentration in the system is 0.1-1 mM, and then culture.

The culture condition of step (1) can be: 30° C., 200 r/min shaking culture.

The culture condition of the step (2) may be: culture at 16-30°C.

The culture condition of step (2) can be: 25°C, 200r/min shaking culture for 48h.

The method for cultivating the recombinant microorganism can specifically be: inoculate 1 volume part of seed solution into 10 volume parts of liquid medium, and ferment for 48 hours. During the fermentation process, the temperature is controlled at 25°C, and the pH value is controlled at about 7.0 by feeding ammonia water. During the fermentation process, when the OD _600nm value of the system reaches 8-10, IPTG is added to make the concentration in the system 0.1-1mM. During the fermentation process, the glucose concentration of the system was monitored, and the glucose concentration of the system was maintained at 4-6g/L by feeding 50g/100ml of glucose aqueous solution.

Specifically, the concentration of IPTG in the system may be 0.5 mM.

The liquid medium can specifically be a fermentation medium containing 17 μg/mL chloramphenicol.

The preparation method of the seed solution is as follows: inoculate a single colony of the strain CG-pXMJ19-pro-ssp-dnaB ^155M -MTG into a test tube containing 5 mL of liquid BHI medium containing 17 μg/mL chloramphenicol, at 30 ° C, 200 r/mL Min shaking culture for 12h, that is, the seed solution.

The present invention also protects a protein (Protein B), which consists of the following elements from N-terminus to C-terminus: pro region of MTG, ssp-dnaB intein, amino acid residue M, mature region of MTG.

MTG is transglutaminase of microbial origin.

Specifically, MTG is MTG derived from Streptomyces mobaraensis.

The pro region of MTG is shown in amino acid residues 2 to 46 of Sequence 2 in the sequence listing, or as shown in amino acid residues 1 to 46 of Sequence 2 in the sequence listing.

The ssp-dnaB intein is shown as amino acid residues 47-200 in sequence 2 of the sequence listing.

The mature region of MTG is shown in amino acid residues 202-532 of Sequence 2 in the Sequence Listing.

The nucleic acid molecules encoding the protein B also belong to the protection scope of the present invention.

The DNA molecule encoding the protein B also belongs to the protection scope of the present invention.

The expression cassettes, recombinant vectors or recombinant microorganisms with the DNA molecules all belong to the protection scope of the present invention.

The recombinant microorganism can be used to produce transglutaminase.

The recombinant microorganism is obtained by introducing the recombinant vector into Corynebacterium glutamicum.

The present invention also protects a protein (Protein C), which sequentially includes the following segments from N-terminus to C-terminus: Protein B and protein label. The protein tag can specifically be a His ₆ tag.

The nucleic acid molecules encoding the protein C also belong to the protection scope of the present invention.

The DNA molecule encoding the protein C also belongs to the protection scope of the present invention.

The expression cassettes, recombinant vectors or recombinant microorganisms with the DNA molecules all belong to the protection scope of the present invention.

The recombinant microorganism is obtained by introducing the recombinant vector into Corynebacterium glutamicum.

The recombinant microorganism can be used to produce transglutaminase.

In the present invention, the MTG derived from Streptomyces mobaraensis is expressed under the T7 promoter, and the intein ssp-dnaB is inserted between the pro region and the mature region of MTG to successfully realize the MTG in the glutamic acid rod. Bacillus (Corynebacterium glutamicum) high-efficiency expression, the bacteria obtained from fermentation can be ultrasonically disrupted, and MTG with biological activity can be obtained without protease treatment or other condition changes.

The recombinant microorganism obtained in the present invention has an enzyme activity of 14 U/mL when fermented at 25° C. for 48 hours in a shake flask, and can reach 49 U/mL when fermented at a high density of 25° C. for 48 hours in a fermenter.

The invention can be used to prepare transglutaminase, and then applied to the fields of tissue engineering, textile and leather processing, etc., and has the value of application and popularization.

Description of drawings

Fig. 1 is the protein electrophoresis picture in the step 5 of embodiment 2.

Fig. 2 is the enzymatic activity comparison chart in the step 7 of embodiment 2.

Fig. 3 is the optimum reaction temperature result figure among the embodiment 4.

FIG. 4 is a graph showing the temperature stability results in Example 4.

Fig. 5 is the optimum pH result figure in embodiment 4.

FIG. 6 is a graph of pH stability results in Example 4. FIG.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged. L-glutamic acid-γ-monohydroxamic acid: sigma, product number G2253. N-Benzyloxycarbonyl-L-glutamylglycine (N-CBZ-Gln-Gly): sigma, product number C6154. Corynebacterium glutamicum ATCC13032 is Corynebacterium glutamicum of ATCC13032.

Brain Heart Extract Broth Medium (Liquid BHI Medium): Dissolve 37g of compound dry powder in water and make up to 1L. Compound dry powder: Beijing Runzekang Biotechnology Co., Ltd., brand OXOID, product number CM1135B.

Fermentation medium (pH7.0): 21g of 3-(N-morpholino)propanesulfonic acid, 5g of urea, 5g of ammonium sulfate, 1g of dipotassium hydrogenphosphate, 1g of potassium dihydrogenphosphate, 2g of yeast powder, 40g of glucose, sulfuric acid Magnesium 0.25g, calcium chloride 0.01g, biotin 0.2mg, trace element solution 1mL, make up to 1L with water. Trace element solution: FeSO ₄ ·7H ₂ O 16.4g, MnSO ₄ ·H ₂ O 100mg, CuSO ₄ 200mg, ZnSO ₄ ·7H ₂ O 1g, NiCl ₂ ·6H ₂ O 20mg, make up to 1L with water.

Embodiment 1, construct recombinant plasmid and recombinant bacterium

1. Construction of recombinant plasmids

1. Construct the recombinant plasmid pXMJ19-pro-ssp-dnaB ^155M -MTG.

The recombinant plasmid pXMJ19-pro-ssp-dnaB ^155M -MTG is a circular plasmid, as shown in sequence 1 of the sequence listing.

In sequence 1 of the sequence listing, nucleotides 6304-6322 constitute the T7 promoter, nucleotides 6323-6347 constitute the lac operator, nucleotides 6350-6385 constitute the RBS, and nucleotides 6386-8002 constitute the nucleotide Acid is the complete open reading frame (encoding fusion protein). In sequence 1 of the sequence listing, the 6386-6388th position is the start codon, the 6389-6523rd nucleotide codes the pro region, the 6524-6985th nucleotide codes the ssp-dnaB intein, and the 6986-6988th nucleotide Nucleotide 1 encodes amino acid residue M, nucleotide 6989-7981 encodes mature MTG, nucleotide 7982-7999 encodes His ₆ tag, nucleotide 8000-8002 is a stop codon.

The fusion protein is shown in sequence 2 of the sequence listing. In Sequence 2 of the sequence listing, amino acid residues 47-200 constitute the ssp-dnaB intein, amino acid residues 201 are M, amino acid residues 202-532 form mature MTG, and amino acids 533-538 Residues make up the His ₆ tag.

Due to the presence of the ssp-dnaB intein, the fusion protein undergoes self-cleavage (the cleavage site is between the 200th amino acid residue and the 201st amino acid residue of sequence 2), and the sequence 3 shown in the sequence table is obtained MTG-His ₆ protein.

2. Construction of recombinant plasmid pXMJ19-pro-MTG.

Compared with the recombinant plasmid pXMJ19-pro-ssp-dnaB ^155M -MTG, the difference of the recombinant plasmid pXMJ19-pro-MTG only lies in the lack of the part encoding the ssp-dnaB intein (that is, the lack of the first sequence in Sequence 1 of the sequence listing 6524-6985 nucleotides).

3. Construction of recombinant plasmid pXMJ19-pro-ssp- ^dnaB 155K -MTG.

Compared with the recombinant plasmid pXMJ19-pro-ssp-dnaB ^155M -MTG, the difference of the recombinant plasmid pXMJ19-pro-ssp- ^dnaB 155K-MTG is only that the 6986-6988th nucleotide in the sequence 2 of the sequence table is replaced by "ATG" was mutated to "AAG".

2. Construction of Corynebacterium glutamicum C.glutamicum T7

1. The pK18mobsacB plasmid was digested with the restriction endonuclease BamHI, and the linearized plasmid and the T7 homologous recombination fragment were connected using the Gibson Assembly kit (NEB). The obtained recombinant plasmid was named recombinant plasmid pK18-T7.

The T7 homologous recombination fragment is a double-stranded DNA molecule shown in sequence 4 of the sequence list. In sequence 4 of the sequence listing, nucleotides 17-502 constitute the upstream homology arm, nucleotides 537-4951 constitute T7-Plac, and nucleotides 4955-5461 constitute the downstream homology arm.

2. The recombinant plasmid pK18-T7 was electrotransformed into Corynebacterium glutamicum ATCC13032 (electric shock condition: voltage 2.5KV, resistance 200Ω, capacitance 25 μ F), and the recombinant bacteria were obtained by two screenings (in the BHI containing 25mg/L kanamycin Carry out the first screening on the plate to obtain the single-exchange recombinant bacteria, then cultivate it overnight in the liquid BHI medium, and then carry out the second screening on the BHI plate containing 200g/L sucrose to obtain the homologous recombination double-exchange bacterial strain), which is the valley Corynebacterium acidicum C. glutamicum T7.

3. Perform sequencing verification.

Compared with Corynebacterium glutamicum ATCC13032, the difference of Corynebacterium glutamicum C. glutamicum T7 is only that glutamic acid is replaced by the double-stranded DNA molecule shown in the 17th-5461st nucleotide of sequence 4 of the sequence listing A double-stranded DNA molecule shown in Sequence 5 of the Sequence Listing in the genomic DNA of Corynebacterium ATCC13032. The genomic DNA of Corynebacterium glutamicum ATCC13032 is shown in GenBank: NC_003450.

3. Construction of recombinant bacteria

The recombinant plasmid pXMJ19-pro-ssp-dnaB ^155M -MTG was introduced into Corynebacterium glutamicum C. glutamicumT7 to obtain the recombinant strain, which was named strain CG-pXMJ19-pro-ssp-dnaB ^155M -MTG.

The recombinant plasmid pXMJ19-pro-MTG was introduced into Corynebacterium glutamicum C. glutamicum T7 to obtain the recombinant strain, which was named strain CG-pXMJ19-pro-MTG.

The recombinant plasmid pXMJ19-pro-ssp- ^{dnaB 155K} -MTG was introduced into Corynebacterium glutamicum C. glutamicumT7 to obtain the recombinant strain, which was named strain CG-pXMJ19-pro-ssp- ^dnaB 155K -MTG.

Embodiment 2, shake flask fermentation of recombinant bacteria

1. Induced fermentation culture of strain CG-pXMJ19-pro-ssp-dnaB ^155M -MTG

1. Inoculate a single colony of the strain CG-pXMJ19-pro-ssp-dnaB ^155M -MTG into a test tube containing 5 mL of liquid BHI medium containing 17 μg/mL chloramphenicol, and culture with shaking at 30°C and 200 r/min for 12 hours, that is for the seed liquid.

2. Inoculate 1 mL of seed solution into a 250 mL Erlenmeyer flask filled with 50 mL of fermentation medium containing 17 μg/mL chloramphenicol, and culture at 30°C with shaking at 200 r/min until OD _600nm value = 1.

3. After completing step 2, add IPTG to the culture system so that the concentration in the system is 0.5mM, and then shake and culture at 25°C and 200r/min for 48h. At this time, the OD _600nm value of the system is about 20, and the system at this time Named as a fermentation product.

4. Take the fermentation product (the amount of bacteria is: OD _600nm value×volume=20; the volume unit is ml), centrifuge at 12000r/min for 5min, and collect the bacterial pellet; resuspend the bacterial pellet in 1mL Tris-HCl buffer (pH8 .0, 50mM), and then perform ultrasonic crushing (ultrasonic crushing parameters: 200W, work for 5s and stop for 5s, and the total time is 20min). supernatant.

2. Non-induced fermentation culture of strain CG-pXMJ19-pro-ssp-dnaB ^155M -MTG

Do not join IPTG, other steps are the same as step 1.

3. Induced fermentation culture of strain CG-pXMJ19-pro-MTG

Replace the strain CG-pXMJ19-pro-ssp-dnaB ^155M -MTG with the strain CG-pXMJ19-pro-MTG, and the others are the same as step 1.

4. Induced fermentation culture of strain CG-pXMJ19-pro-ssp- ^dnaB 155K-MTG

Replace the strain CG-pXMJ19-pro-ssp- ^{dnaB 155M} -MTG with the strain CG-pXMJ19-pro-ssp-dnaB 155K -MTG, and the others are the same as step 1.

5. Protein electrophoresis

Take the whole bacterial solution and supernatant obtained in step 1, the whole bacterial solution and supernatant obtained in step 2, and the whole bacterial solution and supernatant obtained in step 3, and carry out SDS-PAGE.

The electropherogram is shown in Figure 1. In Figure 1: Swimming lane 1: the whole bacterial solution obtained in step 2; Swimming lane 2: the supernatant obtained in step 2; Swimming lane 3: The whole bacterial solution obtained in step 3; Swimming lane 4: The supernatant obtained in step 3; Swimming lane 5 : whole bacterial solution obtained in step 1; lane 6: supernatant obtained in step 1; lane M: protein marker.

The results showed that: without IPTG induction, the strain CG- ^pXMJ19 -pro-ssp-dnaB ^155M -MTG could not produce the target protein; , cannot produce mature MTG.

6. Method for detecting MTG enzyme activity

Definition of MTG enzyme activity: The amount of enzyme required to catalyze the production of 1 μmol L-glutamic acid-γ-monohydroxamic acid per minute at 37°C is defined as 1U.

MTG enzyme activity assay method (colorimetric method): ①Take 200 μL of the sample to be tested or the dilution of the sample to be tested after preheating at 37°C (unless otherwise specified, the solvent used for dilution is Tris at pH 6.0, 0.2mol/L -HCl buffer solution), add 500 μL of substrate solution preheated at 37°C, react at 37°C for 10 minutes, then add 200 μL of terminator, then centrifuge at 10000×g for 5 minutes, collect the supernatant, and measure the absorbance at 525 nm wavelength; ② with L-glutamic acid-γ-monohydroxamic acid is a solute, and water is a solvent to prepare standard solutions of different concentrations, measure the absorbance at 525nm wavelength respectively, and make a standard curve equation (in the standard curve equation, x is L- Glutamic acid-γ-monohydroxamic acid concentration, y is the absorbance value); ③ Substitute the absorbance value obtained in step ① into the standard curve equation obtained in step ②, and calculate the MTG enzyme activity of the sample to be tested.

Preparation method of substrate solution: 100mg N-CBZ-Gln-Gly was dissolved in 2mL 0.2mol/L NaOH aqueous solution, then added 4mL Tris-HCl buffer (pH6.0, 0.2mol/L), 2mL 0.1mol/L Hydroxylamine aqueous solution and 2mL 0.01mol/L reduced glutathione aqueous solution to adjust the pH to 6.0.

Terminator: Contains 1mol/L HCl, 4g/100ml trichloroacetic acid, 5g/100ml FeCl ₃ ·6H ₂ O, and the balance is water.

7. Detection of MTG enzyme activity

Take the supernatant obtained in step 1, the supernatant obtained in step 3 and the supernatant obtained in step 4 respectively as samples to be tested, detect the MTG enzyme activity, and calculate the enzyme activity of the fermentation product.

The enzyme activity of the fermentation product obtained in step 1 was 14 U/mL.

The enzyme activity of the fermentation product obtained in step 3 is 0 U/mL.

The enzyme activity of the fermentation product obtained in step 4 is 0.2 U/mL.

The enzyme activity comparison of the fermentation product obtained in step 1 and the fermentation product obtained in step 4 is shown in Figure 2.

8. Preparation of MTG-His ₆ solution and sequencing identification and detection of enzyme activity

1. Take the supernatant obtained in step 1, filter it with a 0.22 μm filter membrane, and then perform nickel column affinity chromatography to collect the post-column solution containing the target protein (the target protein is MTG-His ₆ protein).

2, get the post-column solution that step 1 obtains, adopt the column type ultrafiltration membrane of 3KD pore size and the Tris-HCl buffer solution of pH8.0, 50mM to carry out desalination, obtain the Tris-HCl buffer solution with pH8.0, 50mM as The protein solution of the solvent system is named as MTG-His ₆ solution.

3. Perform N-terminal sequencing

Take the MTG-His ₆ solution obtained in step 2, perform 12.5% SDS-PAGE, transfer it to a PVDF membrane, and check the transfer effect with Ponceau red staining. Send it to the Protein Sequencing Laboratory of Peking University Life Center to determine the N-terminal 5 amino acid sequence as MDSDD by Edman method. The results show that the strain CG-pXMJ19-pro-ssp-dnaB ^155M -MTG expresses the fusion protein in the cell, and due to the presence of the ssp-dnaB intein, the fusion protein undergoes self-cleavage (the cleavage site is the 200th part of sequence 2 Between the amino acid residue at position 201 and the amino acid residue at position 201), the MTG-His ₆ protein shown in sequence 3 of the sequence listing was obtained. The above steps can obtain active MTG-His ₆ protein without secondary treatment.

4. Detection of MTG enzyme activity

Take the MTG-His ₆ solution obtained in step 2 as the sample to be tested, and detect the MTG enzyme activity.

Take the MTG-His ₆ solution obtained in step 2, and use the Bradford method to detect the protein concentration.

The enzyme activity of MTG-His ₆ solution was divided by the protein concentration of MTG-His ₆ solution to obtain the specific activity of MTG-His ₆ protein, which was 33 U/mg.

Embodiment 3, the fermenter cultivation of recombinant bacterium

1. Preparation of seed solution

Inoculate a single colony of the strain CG-pXMJ19-pro-ssp-dnaB ^155M -MTG into a test tube filled with 5 mL of liquid BHI medium containing 17 μg/mL chloramphenicol, and culture it with shaking at 30°C and 200 r/min for 12 hours to form the seeds liquid. Multiple seed solutions were prepared using multiple tubes.

2. Inoculate 100mL of seed liquid into a 2.5L fermenter equipped with 1L fermentation medium containing 17μg/mL chloramphenicol, and ferment for 48h. During the fermentation process, the temperature is controlled at 25°C, and the pH value is controlled at about 7.0 by feeding ammonia water. During the fermentation process, when the OD _600nm value of the system reaches 8-10, IPTG is added to make its concentration in the system 0.5mM. During the fermentation process, monitor the glucose concentration of the system, and maintain the glucose concentration of the system at 4-6g/L by feeding 50g/100ml of glucose aqueous solution. After the fermentation is completed, the OD _600nm value of the system is about 70, and the system at this time is named as a fermentation product.

3. Take the fermentation product (the amount of bacteria is: OD _600nm value×volume=20; the volume unit is ml), centrifuge at 12000r/min for 5min, and collect the bacterial pellet; resuspend the bacterial pellet in 1mL Tris-HCl buffer (pH8 .0, 50mM), and then perform ultrasonic crushing (ultrasonic crushing parameters: 200W, work for 5s and stop for 5s, the total time is 20min), and then centrifuge (10000×g, 5min) to collect the supernatant.

4. Take the supernatant obtained in step 3 as the sample to be tested, detect the MTG enzyme activity, and calculate the enzyme activity of the fermentation product.

The enzyme activity of the fermentation product was 49U/mL.

Embodiment 4, the research of MTG enzymatic properties

The MTG-His ₆ solution is the MTG-His ₆ solution prepared in Step 8 of Example 2.

1. Optimum reaction temperature and temperature stability

1. Optimum reaction temperature

Take the MTG-His ₆ solution as the sample to be tested to detect the MTG enzyme activity. For the method of detecting MTG enzyme activity, refer to Step 6 of Example 2. The reaction temperature was set at 30°C, 37°C, 45°C, 50°C, 55°C or 60°C, respectively.

The highest point of enzyme activity was regarded as 100% enzyme activity, and the relative enzyme activity at other temperatures was calculated. The results are shown in Figure 3.

The highest point of enzyme activity was taken as the optimum reaction temperature. The optimum reaction temperature is 55°C.

2. Temperature stability

The MTG-His ₆ solution was taken and placed at three temperatures (40°C, 50°C or 60°C), and samples were taken every 20 minutes as samples to be tested to detect MTG enzyme activity.

Take the MTG-His ₆ solution as the sample to be tested, and detect the MTG enzyme activity as the reference enzyme activity.

For the method of detecting MTG enzyme activity, refer to Step 6 of Example 2.

Taking the reference enzyme activity as 100%, draw the thermostability curve. The results are shown in Figure 4. 90% of the activity was retained after 100 minutes of treatment at 40°C, 30% of the activity was retained after 100 minutes of treatment at 50°C, and the activity was basically lost after 20 minutes of treatment at 60°C.

2. Optimum pH and pH stability

The substrate buffers are: pH 4.0, 50mM acetic acid-sodium acetate buffer, pH 5.0, 50mM acetic acid-sodium acetate buffer, pH 6.0, 50mM Tris-HCl buffer, pH 7.0, 50mM Tris-HCl buffer, pH8.0, 50mM Tris-HCl buffer, pH9.0, 50mM Tris-HCl buffer.

1. Optimal pH

Take the MTG-His ₆ solution, dilute it to 10 times volume with substrate buffer, and use it as the test sample to detect the MTG enzyme activity.

For the method of detecting MTG enzyme activity, refer to Step 6 of Example 2.

Preparation method of substrate solution: 100mg N-CBZ-Gln-Gly was dissolved in 2mL 0.2mol/L NaOH aqueous solution, then added 4mL substrate buffer, 2mL 0.1mol/L hydroxylamine aqueous solution and 2mL 0.01mol/L reduced gluten Glutathione aqueous solution, adjust the pH to the pH of the substrate buffer.

The highest enzyme activity was taken as 100% enzyme activity, and the relative enzyme activity at other pHs was calculated. The results are shown in Figure 5.

The highest point of enzyme activity is the optimum reaction pH. The optimum reaction pH is 7.0.

2. pH stability

Take the MTG-His ₆ solution, dilute it with substrate buffer to 10 times the volume, and place it at room temperature for 1 hour as the sample to be tested to detect the MTG enzyme activity.

Take the MTG-His ₆ solution, dilute it to 10 times the volume with pH 8.0, 50mM Tris-HCl buffer solution, and use it as the sample to be tested, detect the MTG enzyme activity, and use it as the reference enzyme activity.

For the method of detecting MTG enzyme activity, refer to Step 6 of Example 2.

Taking the reference enzyme activity as 100%, draw the pH stability curve. The results are shown in Figure 6. After pH 5.0-7.0 treatment for 1 hour, more than 80% of the activity remained; when pH 8.0-9.0 was treated for 1 hour, more than 60% of the activity remained; when pH 4.0 was treated for 1 hour, 70% of the activity was lost.

3. Effects of metal ions, EDTA and PMSF on enzyme activity

Take the MTG-His ₆ solution as the sample to be tested, and detect the MTG enzyme activity as the reference enzyme activity.

Take the MTG-His ₆ solution, add the inhibitor to make the concentration 1mM, let it stand at room temperature for 1h, and use it as the test sample to detect the MTG enzyme activity.

For the method of detecting MTG enzyme activity, refer to Step 6 of Example 2.

Taking the reference enzyme activity as 100%, the relative enzyme activity after the action of each inhibitor was calculated.

The results are shown in Table 1. Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ had no effect on MTG enzyme activity. Cu ²⁺ , Mn ²⁺ , and Fe ²⁺ have certain inhibitory effects on the enzyme activity, but still retain more than 70% of the activity. Zn ²⁺ has a strong inhibitory effect on the activity of MTG, and the activity is basically lost after treatment. EDTA and PMSF did not inhibit the enzyme activity.

Table 1

Inhibitors contained in the test solution Relative enzyme activity (%) CuSO₄ 76.19±2.27 MnSO₄ 77.19±6.85 NaCl 113.46±3.64 KCl 113.67±3.90 CaCl₂ 108.20±3.46 ZnSO₄ 1.18±0.17 FeSO₄ 87.23±1.20 MgSO₄ 111.07±2.40 EDTA 116.33±6.11 PMSF 113.73±4.96

SEQUENCE LISTING

<110> Institute of Microbiology, Chinese Academy of Sciences

<120> A method for efficiently producing transglutaminase and its special engineering bacteria

<130> GNCYX200805

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 8186

<212>DNA

<213> Artificial sequence

<400> 1

tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg 60

ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga 120

gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc 180

gggcaggacg cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg 240

atggcctttttgcgtttcta caaactcttttgtttatttttctaaataca ttcaaatatg 300

tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 360

atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 420

gtttttgctc accccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 480

cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 540

gaagaacgtt ttccaatgat gagcactttt gcttcctcgc tcactgactc gctgcgctcg 600

gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 660

gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 720

cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 780

aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 840

tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 900

ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat 960

ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 1020

cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 1080

ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 1140

gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 1200

atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 1260

aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 1320

aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 1380

gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 1440

cttttggggt gggcgaagaa ctccagcatg agatccccgc gctggaggat catccagcca 1500

ttcggggtcg ttcactggtt cccctttctg atttctggca tagaagaacc cccgtgaact 1560

gtgtggttcc gggggttgct gatttttgcg agacttctcg cgcaattccc tagcttaggt 1620

gaaaacacca tgaaacacta gggaaacacc catgaaacac ccattagggc agtagggcgg 1680

cttcttcgtc tagggcttgc atttgggcgg tgatctggtc tttagcgtgt gaaagtgtgt 1740

cgtaggtggc gtgctcaatg cactcgaacg tcacgtcatt taccgggtca cggtgggcaa 1800

agagaactag tgggttagac attgttttcc tcgttgtcgg tggtggtgag cttttctagc 1860

cgctcggtaa acgcggcgat catgaactct tggaggtttt caccgttctg catgcctgcg 1920

cgcttcatgt cctcacgtag tgccaaagga acgcgtgcgg tgaccacgac gggcttagcc 1980

tttgcctgcg cttctagtgc ttcgatggtg gcttgtgcct gcgcttgctg cgcctgtagt 2040

gcctgttgag cttcttgtag ttgctgttct agctgtgcct tggttgccat gctttaagac 2100

tctagtagct ttcctgcgat atgtcatgcg catgcgtagc aaacattgtc ctgcaactca 2160

ttcatttatgt gcagtgctcc tgttactagt cgtacatact catatttacc tagtctgcat 2220

gcagtgcatg cacatgcagt catgtcgtgc taatgtgtaa aacatgtaca tgcagattgc 2280

tgggggtgca gggggcggag ccaccctgtc catgcggggt gtggggcttg ccccgccggt 2340

acagacagtg agcaccgggg cacctagtcg cggatacccc ccctaggtat cggacacgta 2400

accctcccat gtcgatgcaa atctttaaca ttgagtacgg gtaagctggc acgcatagcc 2460

aagctaggcg gccaccaaac accactaaaa attaatagtc cctagacaag acaaaccccc 2520

gtgcgagcta ccaactcata tgcacggggg ccacataacc cgaaggggtt tcaattgaca 2580

accatagcac tagctaagac aacgggcaca acacccgcac aaactcgcac tgcgcaaccc 2640

cgcacaacat cgggtctagg taacactgag taacactgaa atagaagtga acacctctaa 2700

ggaaccgcag gtcaatgagg gttctaaggt cactcgcgct agggcgtggc gtaggcaaaa 2760

cgtcatgtac aagatcacca atagtaaggc tctggcgggg tgccataggt ggcgcaggga 2820

cgaagctgtt gcggtgtcct ggtcgtctaa cggtgcttcg cagtttgagg gtctgcaaaa 2880

ctctcactct cgctgggggt cacctctggc tgaattggaa gtcatgggcg aacgccgcat 2940

tgagctggct attgctacta agaatcactt ggcggcgggt ggcgcgctca tgatgtttgt 3000

gggcactgtt cgacacaacc gctcacagtc atttgcgcag gttgaagcgg gtattaagac 3060

tgcgtactct tcgatggtga aaacatctca gtggaagaaa gaacgtgcac ggtacggggt 3120

ggagcacacc tatagtgact atgaggtcac agactcttgg gcgaacggtt ggcacttgca 3180

ccgcaacatg ctgttgttct tggatcgtcc actgtctgac gatgaactca aggcgtttga 3240

ggattccatg ttttcccgctggtctgctgg tgtggttaag gccggtatgg acgcgccact 3300

gcgtgagcac gggtcaaac ttgatcaggt gtctacctgg ggtggagacg ctgcgaaaat 3360

ggcaacctac ctcgctaagg gcatgtctca ggaactgact ggctccgcta ctaaaaccgc 3420

gtctaagggg tcgtacacgc cgtttcagat gttggatatg ttggccgatc aaagcgacgc 3480

cggcgaggat atggacgctg ttttggtggc tcggtggcgt gagtatgagg ttggttctaa 3540

aaacctgcgt tcgtcctggt cacgtggggc taagcgtgct ttgggcattg attacataga 3600

cgctgatgta cgtcgtgaaa tggaagaaga actgtacaag ctcgccggtc tggaagcacc 3660

ggaacgggtc gaatcaaccc gcgttgctgt tgctttggtg aagcccgatg attggaaact 3720

gattcagtct gatttcgcgg ttaggcagta cgttctcgat tgcgtggata aggctaagga 3780

cgtggccgct gcgcaacgtg tcgctaatga ggtgctggca agtctgggtg tggattccac 3840

cccgtgcatg atcgttatgg atgatgtgga cttggacgcg gttctgccta ctcatgggga 3900

cgctactaag cgtgatctga atgcggcggt gttcgcgggt aatgagcaga ctattcttcg 3960

cacccactaa aagcggcata aacccccgttc gatattttgt gcgatgaatt tatggtcaat 4020

gtcgcggggg caaactatga tgggtcttgt tgttggcgtc ccggaaaacg attccgaagc 4080

ccaacctttc atagaaggcg gcggtggaat cgaaatctcg tgatggcagg ttgggcgtcg 4140

cttggtcggt catttcgaag ggcaccaata actgccttaa aaaaattacg ccccgccctg 4200

ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca 4260

gacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata 4320

tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa 4380

actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt 4440

agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa 4500

ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg 4560

gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc 4620

catacggaac tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata 4680

aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt 4740

ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca 4800

ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc 4860

tcctgaaaat ctcgtcgaag ctcggcggat ttgtcctact caagctgatc cgacaaaatc 4920

cacacattat cccaggtgtc cggatcggtc aaatacgctg ccagctcata gaccgtatcc 4980

aaagcatccg gggctgatcc ccggcgccag ggtggttttt cttttcacca gtgagacggg 5040

caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgtg 5100

gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag 5160

ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac 5220

tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg 5280

ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag 5340

tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca 5400

gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg 5460

tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata 5520

atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag 5580

gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg 5640

acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct 5700

accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca 5760

atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt 5820

ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct 5880

tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg 5940

gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc 6000

accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcac 6060

cattcgatgg tgtcaacgta aatgccgctt cgccttcgcg cgcgaattgc aagctgatcc 6120

gggcttatcg actgcacggt gcaccaatgc ttctggcgtc cgctcatgag cccgaagtgg 6180

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 6240

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 6300

aattaatacg actcactata ggggaattgt gagcggataa caattcccca ataattttgt 6360

ttaactttaa gaaggagata tacatatgga caatggcgcg ggggaagaga cgaagtccta 6420

cgccgaaacc taccgcctca cggcggatga cgtcgcgaac atcaacgcgc tcaacgaaag 6480

cgctccggcc gcttcgagcg ccggcccgtc gttccgggcc cccgctatct ctggcgatag 6540

tctgatcagc ctggctagca caggaaaaag agtttctatt aaagatttgt tagatgaaaa 6600

agattttgaa atatgggcaa ttaatgaaca gacgatgaag ctagaatcag ctaaagttag 6660

tcgtgtattt tgtactggca aaaagctagt ttatattcta aaaactcgac taggtagaac 6720

tatcaaggca acagcaaatc atagattttt aactattgat ggttggaaaa gattagatga 6780

gctatcttta aaagagcata ttgctctacc ccgtaaacta gaaagctcct ctttacaatt 6840

gtcaccagaa atagaaaagt tgtctcagag tgatatttac tgggactcca tcgtttctat 6900

tacggagact ggagtcgaag aggtttttga tttgactgtg ccaggacac ataactttgt 6960

cgcgaatgac atcattgtac acaacatgga ctccgacgac agggtcaccc ctcccgccga 7020

gccgctcgac aggatgcccg acccgtaccg tccctcgtac ggcagggccg agacggtcgt 7080

caacaactac atacgcaagt ggcagcaggt ctacagccac cgcgacggca ggaagcagca 7140

gatgaccgag gagcagcggg agtggctgtc ctacggctgc gtcggtgtca cctgggtcaa 7200

ttcgggtcag tacccgacga acagactggc cttcgcgtcc ttcgacgagg acaggttcaa 7260

gaacgagctg aagaacggca ggccccggtc cggcgagacg cgggcggagt tcgagggccg 7320

cgtcgcgaag gagagcttcg acgaggagaa gggcttccag cgggcgcgtg aggtggcgtc 7380

cgtcatgaac agggccctgg agaacgccca cgacgagagc gcttacctcg acaacctcaa 7440

gaaggaactg gcgaacggca acgacgccct gcgcaacgag gacgcccgtt ccccgttcta 7500

ctcggcgctg cggaacacgc cgtccttcaa ggagcggaac ggaggcaatc acgacccgtc 7560

caggatgaag gccgtcatct actcgaagca cttctggagc ggccaggacc ggtcgagttc 7620

ggccgacaag aggaagtacg gcgacccgga cgccttccgc cccgccccgg gcaccggcct 7680

ggtcgacatg tcgagggaca ggaacattcc gcgcagcccc accagccccg gtgagggat 7740

cgtcaatttc gactacggct ggttcggcgc ccagacggaa gcggacgccg acaagaccgt 7800

ctggacccac ggaaatcact atcacgcgcc caatggcagc ctgggtgcca tgcatgtcta 7860

cgagagcaag ttccgcaact ggtccgaggg ttactcggac ttcgaccgcg gagcctatgt 7920

gatcaccttc atccccaaga gctggaacac cgcccccgac aaggtaaagc agggctggcc 7980

gcaccaccac caccaccact gaccgggtac cgagctcgaa ttcagcttgg ctgttttggc 8040

ggatgagaga agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata 8100

aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca 8160

gaagtgaaac gccgtagcgc cgatgg 8186

<210> 2

<211> 538

<212> PRT

<213> Artificial sequence

<400> 2

Met Asp Asn Gly Ala Gly Glu Glu Thr Lys Ser Tyr Ala Glu Thr Tyr

1 5 10 15

Arg Leu Thr Ala Asp Asp Val Ala Asn Ile Asn Ala Leu Asn Glu Ser

20 25 30

Ala Pro Ala Ala Ser Ser Ala Gly Pro Ser Phe Arg Ala Pro Ala Ile

35 40 45

Ser Gly Asp Ser Leu Ile Ser Leu Ala Ser Thr Gly Lys Arg Val Ser

50 55 60

Ile Lys Asp Leu Leu Asp Glu Lys Asp Phe Glu Ile Trp Ala Ile Asn

65 70 75 80

Glu Gln Thr Met Lys Leu Glu Ser Ala Lys Val Ser Arg Val Phe Cys

85 90 95

Thr Gly Lys Lys Leu Val Tyr Ile Leu Lys Thr Arg Leu Gly Arg Thr

100 105 110

Ile Lys Ala Thr Ala Asn His Arg Phe Leu Thr Ile Asp Gly Trp Lys

115 120 125

Arg Leu Asp Glu Leu Ser Leu Lys Glu His Ile Ala Leu Pro Arg Lys

130 135 140

Leu Glu Ser Ser Ser Leu Gln Leu Ser Pro Glu Ile Glu Lys Leu Ser

145 150 155 160

Gln Ser Asp Ile Tyr Trp Asp Ser Ile Val Ser Ile Thr Glu Thr Gly

165 170 175

Val Glu Glu Val Phe Asp Leu Thr Val Pro Gly Pro His Asn Phe Val

180 185 190

Ala Asn Asp Ile Ile Val His Asn Met Asp Ser Asp Asp Arg Val Thr

195 200 205

Pro Pro Ala Glu Pro Leu Asp Arg Met Pro Asp Pro Tyr Arg Pro Ser

210 215 220

Tyr Gly Arg Ala Glu Thr Val Val Asn Asn Tyr Ile Arg Lys Trp Gln

225 230 235 240

Gln Val Tyr Ser His Arg Asp Gly Arg Lys Gln Gln Met Thr Glu Glu

245 250 255

Gln Arg Glu Trp Leu Ser Tyr Gly Cys Val Gly Val Thr Trp Val Asn

260 265 270

Ser Gly Gln Tyr Pro Thr Asn Arg Leu Ala Phe Ala Ser Phe Asp Glu

275 280 285

Asp Arg Phe Lys Asn Glu Leu Lys Asn Gly Arg Pro Arg Ser Gly Glu

290 295 300

Thr Arg Ala Glu Phe Glu Gly Arg Val Ala Lys Glu Ser Phe Asp Glu

305 310 315 320

Glu Lys Gly Phe Gln Arg Ala Arg Glu Val Ala Ser Val Met Asn Arg

325 330 335

Ala Leu Glu Asn Ala His Asp Glu Ser Ala Tyr Leu Asp Asn Leu Lys

340 345 350

Lys Glu Leu Ala Asn Gly Asn Asp Ala Leu Arg Asn Glu Asp Ala Arg

355 360 365

Ser Pro Phe Tyr Ser Ala Leu Arg Asn Thr Pro Ser Phe Lys Glu Arg

370 375 380

Asn Gly Gly Asn His Asp Pro Ser Arg Met Lys Ala Val Ile Tyr Ser

385 390 395 400

Lys His Phe Trp Ser Gly Gln Asp Arg Ser Ser Ser Ala Asp Lys Arg

405 410 415

Lys Tyr Gly Asp Pro Asp Ala Phe Arg Pro Ala Pro Gly Thr Gly Leu

420 425 430

Val Asp Met Ser Arg Asp Arg Asn Ile Pro Arg Ser Pro Thr Ser Pro

435 440 445

Gly Glu Gly Phe Val Asn Phe Asp Tyr Gly Trp Phe Gly Ala Gln Thr

450 455 460

Glu Ala Asp Ala Asp Lys Thr Val Trp Thr His Gly Asn His Tyr His

465 470 475 480

Ala Pro Asn Gly Ser Leu Gly Ala Met His Val Tyr Glu Ser Lys Phe

485 490 495

Arg Asn Trp Ser Glu Gly Tyr Ser Asp Phe Asp Arg Gly Ala Tyr Val

500 505 510

Ile Thr Phe Ile Pro Lys Ser Trp Asn Thr Ala Pro Asp Lys Val Lys

515 520 525

Gln Gly Trp Pro His His His His His His His His

530 535

<210> 3

<211> 338

<212> PRT

<213> Artificial sequence

<400> 3

Met Asp Ser Asp Asp Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg

1 5 10 15

Met Pro Asp Pro Tyr Arg Pro Ser Tyr Gly Arg Ala Glu Thr Val Val

20 25 30

Asn Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly

35 40 45

Arg Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly

50 55 60

Cys Val Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg

65 70 75 80

Leu Ala Phe Ala Ser Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys

85 90 95

Asn Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg

100 105 110

Val Ala Lys Glu Ser Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg

115 120 125

Glu Val Ala Ser Val Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu

130 135 140

Ser Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp

145 150 155 160

Ala Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg

165 170 175

Asn Thr Pro Ser Phe Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser

180 185 190

Arg Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp

195 200 205

Arg Ser Ser Ser Ala Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe

210 215 220

Arg Pro Ala Pro Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn

225 230 235 240

Ile Pro Arg Ser Pro Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp

245 250 255

Tyr Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val

260 265 270

Trp Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala

275 280 285

Met His Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Glu Gly Tyr Ser

290 295 300

Asp Phe Asp Arg Gly Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp

305 310 315 320

Asn Thr Ala Pro Asp Lys Val Lys Gln Gly Trp Pro His His His His His

325 330 335

His His

<210> 4

<211> 5482

<212>DNA

<213> Artificial sequence

<400> 4

gagctcggta cccgggacaa ctctttcacg taagttcttc gtttctgcta ccacagccct 60

ggcggcagtc gcactggttg cgtgttcccc taatgagatt gattctgaac tgaaggtgcc 120

aacggcaact ggcgtttctt taccttcgaa gaacgtttcc gcgacctcaa ctgctactac 180

agatgaggat gcgcctggct aattgattg cgtagccgca ccaactcagc aacctgctga 240

aatctcacta aactgtgcaa tggatattga tcggctcacg gatatttctt ggagcgaatg 300

ggatactgat tccgcaactg gaaccggtac ccgcatcgta accgctgcaa atggtcaaga 360

gaccgaaacc gaagatattg aggtgaagct ttccttcccc accgagtctt cccaaggcct 420

agtgttcact caggtcaccg tcgatggaca ggttctcttc ctctaatcct ccataattag 480

agagcgtaag gcccctactt ccgcaactcg tgaaaggtag gcggatccag atcccggaca 540

ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat 600

tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct 660

cgtttcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg 720

aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac 780

tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc 840

cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg 900

tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg 960

cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg 1020

tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca 1080

tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg 1140

cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc 1200

tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac 1260

gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg 1320

gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg 1380

ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata 1440

ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc 1500

tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca 1560

atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa 1620

ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 1680

tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccc 1740

caggctttac actttatgct tccggctcgt ataatgtgtg gaattgtgag cggataacaa 1800

tttcacacag gaaacagcta tgaccatgat tacggattca ctggccgtcg ttttacaacg 1860

tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt 1920

cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 1980

cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc cggaaagctg 2040

gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact ggcagatgca 2100

cggttacgat gcgcccatct acaccaacgt gacctatccc attacggtca atccgccgtt 2160

tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg atgaaagctg 2220

gctacaggaa ggccagacgc gaattatttt tgatggcgtc gggatctgat ccggatttac 2280

taactggaag aggcactaaa tgaacacgat taacatcgct aagaacgact tctctgacat 2340

cgaactggct gctatcccgt tcaacactct ggctgaccat tacggtgagc gtttagctcg 2400

cgaacagttg gcccttgagc atgagtctta cgagatgggt gaagcacgct tccgcaagat 2460

gtttgagcgt caacttaaag ctggtgaggt tgcggataac gctgccgcca agcctctcat 2520

cactacccta ctccctaaga tgattgcacg catcaacgac tggtttgagg aagtgaaagc 2580

taagcgcggc aagcgcccga cagccttcca gttcctgcaa gaaatcaagc cggaagccgt 2640

agcgtacatc accattaaga ccactctggc ttgcctaacc agtgctgaca atacaaccgt 2700

tcaggctgta gcaagcgcaa tcggtcgggc cattgaggac gaggctcgct tcggtcgtat 2760

ccgtgacctt gaagctaagc acttcaagaa aaacgttgag gaacaactca acaagcgcgt 2820

agggcacgtc tacaagaaag catttatgca agttgtcgag gctgacatgc tctctaaggg 2880

tctactcggt ggcgaggcgt ggtcttcgtg gcataaggaa gactctattc atgtagggagt 2940

acgctgcatc gagatgctca ttgagtcaac cggaatggtt agcttacacc gccaaaatgc 3000

tggcgtagta ggtcaagact ctgagactat cgaactcgca cctgaatacg ctgaggctat 3060

cgcaacccgt gcaggtgcgc tggctggcat ctctccgatg ttccaacctt gcgtagttcc 3120

tcctaagccg tggactggca ttactggtgg tggctattgg gctaacggtc gtcgtcctct 3180

ggcgctggtg cgtactcaca gtaagaaagc actgatgcgc tacgaagacg tttacatgcc 3240

tgaggtgtac aaagcgatta acattgcgca aaacaccgca tggaaaatca acaagaaagt 3300

cctagcggtc gccaacgtaa tcaccaagtg gaagcattgt ccggtcgagg acatccctgc 3360

gattgagcgt gaagaactcc cgatgaaacc ggaagacatc gacatgaatc ctgaggctct 3420

caccgcgtgg aaacgtgctg ccgctgctgt gtaccgcaag gacaaggctc gcaagtctcg 3480

ccgtatcagc cttgagttca tgcttgagca agccaataag tttgctaacc ataaggccat 3540

ctggttccct tacaacatgg actggcgcgg tcgtgtttac gctgtgtcaa tgttcaaccc 3600

gcaaggtaac gatatgacca aaggactgct tacgctggcg aaaggtaaac caatcggtaa 3660

ggaaggttac tactggctga aaatccacgg tgcaaactgt gcgggtgtcg ataaggttcc 3720

gttccctgag cgcatcaagt tcattgagga aaaccacgag aacatcatgg cttgcgctaa 3780

gtctccactg gagaacactt ggtgggctga gcaagattct ccgttctgct tccttgcgtt 3840

ctgctttgag tacgctgggg tacagcacca cggcctgagc tataactgct cccttccgct 3900

ggcgtttgac gggtcttgct ctggcatcca gcacttctcc gcgatgctcc gagatgaggt 3960

aggtggtcgc gcggttaact tgcttcctag tgaaaccgtt caggacatct acggattgt 4020

tgctaagaaa gtcaacgaga ttctacaagc agacgcaatc aatgggaccg ataacgaagt 4080

agttaccgtg accgatgaga acactggtga aatctctgag aaagtcaagc tgggcactaa 4140

ggcactggct ggtcaatggc tggcttacgg tgttactcgc agtgtgacta agcgttcagt 4200

catgacgctg gcttacgggt ccaaagagtt cggcttccgt caacaagtgc tggaagatac 4260

cattcagcca gctattgatt ccggcaaggg tctgatgttc actcagccga atcaggctgc 4320

tggatacatg gctaagctga tttgggaatc tgtgagcgtg acggtggtag ctgcggttga 4380

agcaatgaac tggcttaagt ctgctgctaa gctgctggct gctgaggtca aagataagaa 4440

gactggagag attcttcgca agcgttgcgc tgtgcattgg gtaactcctg atggtttccc 4500

tgtgtggcag gaatacaaga agcctattca gacgcgcttg aacctgatgt tcctcggtca 4560

gttccgctta cagcctacca ttaacaccaa caaagatagc gagattgatg cacacaaaca 4620

ggagtctggt atcgctccta actttgtaca cagccaagac ggtagccacc ttcgtaagac 4680

tgtagtgtgg gcacacgaga agtacggaat cgaatctttt gcactgattc acgactcctt 4740

cggtaccatt ccggctgacg ctgcgaacct gttcaaagca gtgcgcgaaa ctatggttga 4800

cacatatgag tcttgtgatg tactggctga tttctacgac cagttcgctg accacgttgca 4860

cgagtctcaa ttggacaaaa tgccagcact tccggctaaa ggtaacttga acctccgtga 4920

catcttagag tcggacttcg cgttcgcgta acgcggaaat aggggccttt tgttgtcttc 4980

tcctggaggc tatttaagaa gtttaaattg tgtccatgag ttcgcgtatg gcaatgacag 5040

tttgagacgg ccacaggcga ttctgagaag ccattttctt tgggcgccgt ggcagttttt 5100

attgggtccc accgccgaac tgcatattcg aaccaaggag cctcaaaaat cgagctcgct 5160

ttggtctcaa acgcacattt atcgcgcgtt gaagtgtgcg tttgagacca aagagccctc 5220

cacaacgcac gtctttggtt tggatatgac aggtgcccaa gaactcaccc cgccccatgc 5280

tcacagagcc cccatcagaa gccaaaagac cccttccctg cccaagaaga acaggatgaa 5340

ggggtcttgt gctgcgtaaa ctagcggttt tggaagtagc taagcagacg taggattcg 5400

gtgtagagcc agaccaaggt cactgcaaga ccaagcgcaa cgccccatgc catcttggaa 5460

gggaaatagg ggccttttgttg 5482

<210> 5

<211> 1000

<212>DNA

<213> Artificial sequence

<400> 5

acaactcttt cacgtaagtt cttcgtttct gctaccacag ccctggcggc agtcgcactg 60

gttgcgtgtt cccctaatga gattgattct gaactgaagg tgccaacggc aactggcgtt 120

tctttaccctt cgaagaacgt ttccgcgacc tcaactgcta ctacagatga ggatgcgcct 180

ggctacattg attgcgtagc cgcaccaact cagcaacctg ctgaaatctc actaaactgt 240

gcaatggata ttgatcggct cacggatatt tcttggagcg aatgggatac tgattccgca 300

actggaaccg gtacccgcat cgtaaccgct gcaaatggtc aagagaccga aaccgaagat 360

attgaggtga agctttcctt ccccaccgag tcttcccaag gcctagtgtt cactcaggtc 420

accgtcgatg gacaggttct cttcctctaa tcctccataa ttagagagcg taaggcccct 480

acttcctgtt ttaggaaata ggggcctttt gttgtcttct cctggaggct atttaagaag 540

tttaaattgt gtccatgagt tcgcgtatgg caatgacagt ttgagacggc cacaggcgat 600

tctgagaagc cattttcttt gggcgccgtg gcagttttta ttgggtccca ccgccgaact 660

gcatattcga accaaggagc ctcaaaaatc gagctcgctt tggtctcaaa cgcacattta 720

tcgcgcgttg aagtgtgcgt ttgagaccaa agagccctcc acaacgcacg tctttggttt 780

ggatatgaca ggtgcccaag aactcacccc gccccatgct cacagagccc ccatcagaag 840

ccaaaagacc ccttccctgc ccaagaagaa caggatgaag gggtcttgtg ctgcgtaaac 900

tagcggtttt ggaagtagct aagcagacgt aggatttcgg tgtagagcca gaccaaggtc 960

actgcaagac caagcgcaac gccccatgcc atcttggaag 1000

Claims (8)

1. Protein, which is the following (a1) or (a2): (a1) the protein represented by amino acid residues 1-532 of Sequence 2 in the sequence listing; (a2) The protein shown in sequence 2 of the sequence listing. 2. A nucleic acid molecule encoding the protein of claim 1. 3. A DNA molecule encoding the protein of claim 1. 4. have the expression cassette of claim 3 described DNA molecule, recombinant vector or recombinant microorganism. 5. The recombinant vector according to claim 4, characterized in that: the recombinant vector is as follows (c1) or (c2): (c1) A recombinant vector having nucleotides 6304-8002 of Sequence 1 of the sequence listing; (c2) A recombinant vector as shown in Sequence 1 of the Sequence Listing. 6. The recombinant microorganism according to claim 4, characterized in that: the recombinant microorganism is obtained by introducing the recombinant vector according to claim 5 into Corynebacterium glutamicum. 7. The protein of claim 1, or the nucleic acid molecule of claim 2, or the DNA molecule of claim 3, or the expression cassette of claim 4, or the recombinant vector of claim 4 or 5, or the claim The application of the recombinant microorganism described in 4 or 6 in the preparation of transglutaminase. 8. A method for preparing transglutaminase, comprising the steps of: cultivating the recombinant microorganism according to claim 6.

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