CN113528478B

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

Info

Publication number: CN113528478B
Application number: CN202010284432.5A
Authority: CN
Inventors: 董志扬; 张楠; 张山; 何永志; 张岩峰
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-04-13
Filing date: 2020-04-13
Publication date: 2023-01-20
Anticipated expiration: 2040-04-13
Also published as: CN113528478A

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

Method for efficiently producing transglutaminase and special engineering bacteria thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for efficiently producing transglutaminase and a special engineering bacterium thereof.

Background

Transglutaminases (TGases) belong to the transferase family, which catalyze the acyl transfer from the gamma-carboxamide group (donor) of a glutamine residue to various primary amines (acyl acceptor). Currently, TGase has been used in the food processing field for more than 10 years and is Generally Recognized As Safe (GRAS) by the united states Food and Drug Administration (FDA). TGase can improve the properties of proteins by intramolecular and intermolecular cross-linking of proteins. Therefore, the enzyme shows wider application value in the fields of tissue engineering, textile and leather processing and the like.

TGase is widely available in animals and plants, but the yield is low, and the cost of separation and purification is high, so that the TGase is far from meeting the requirements of people. In 1989, TGase (Microbial TGase, MTG) of Microbial origin was first discovered, and MTG was non-Ca-containing compared with TGase of other origin ²⁺ Dependence, high temperature, high pH stability and the like. In addition, the microbial method for producing MTG has the advantages of short production period, high yield, low cost, easy industrial production and the like. Therefore, there is a tendency to use microbiological methods for the large-scale production of MTG.

MTG is normally secreted extracellularly as an inactive proenzyme (pro-MTG) and the pro-domain (pro domain) is cleaved by proteolytic cleavage to form the active mature MTG. The existence of pro region plays an important role in the formation of correct spatial structure and secretion of MTG.

An intein is a protein that enables protein splicing through nucleophilic substitution between amino acids without the involvement of other cofactors. Due to its unique mechanism of action, it is now widely used in biotechnology, biomedicine and protein chemistry. dnaB is a DNA helicase gene derived from Synechocystis sp code, and 106 amino acids at C-terminal and 48 amino acids at N-terminal form the minimum action unit of intein after optimization and modification. The C-terminal cleavage of the optimized intein ssp-dnaB is pH dependent.

At present, more and more expression systems are used for expressing MTG, but the problems of low expression level, secondary treatment of protease in the later period and the like exist. Some researches successfully realize the expression of MTG in escherichia coli and bacillus subtilis by using intein, but the shearing of a pro region is still realized by secondary treatment with pH or temperature change, so that the industrial production cost and the time cost are greatly increased.

Disclosure of Invention

The invention aims to provide a method for efficiently producing transglutaminase and a special engineering bacterium thereof.

The invention provides a protein (protein A), which is (a 1) or (a 2) as follows:

(a1) Protein shown by 1-532 th amino acid residues in a sequence 2 in a sequence table;

(a2) A protein shown in a sequence 2 of a sequence table.

Nucleic acid molecules which code for said protein A also belong to the scope of protection of the present invention.

DNA molecules which code for the protein A also belong to the scope of protection of the invention.

The DNA molecule is specifically any one of the following (b 1) to (b 6):

(b1) The coding region is DNA molecule shown as 6389-7981 th nucleotide of sequence 1 in the sequence table;

(b2) The coding region is DNA molecule shown as 6386-7981 th nucleotide in sequence 1 of the sequence table;

(b3) The coding region is DNA molecule shown as 6389-7999 th bit nucleotide in sequence 1 of the sequence table;

(b4) The coding region is DNA molecule shown as 6386-7999 th nucleotide of sequence 1 in the sequence table;

(b5) The coding region is DNA molecule shown as 6389-8002 bit nucleotide of sequence 1 in the sequence table;

(b6) The coding region is DNA molecule shown as 6386-8002 bit nucleotide of sequence 1 in the sequence table.

Expression cassettes, recombinant vectors or recombinant microorganisms having the DNA molecules are within the scope of the invention.

The recombinant vector is specifically (c 1) or (c 2) as follows:

(c1) A recombinant vector with 6304-8002 nucleotides of sequence 1 of the sequence table;

(c2) The recombinant vector is shown as a sequence 1 in a sequence table.

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

The corynebacterium glutamicum may be corynebacterium glutamicum c.glutamicum T7.

Corynebacterium glutamicum C.glutamicum T7 is a recombinant strain obtained by introducing T7-Plac into Corynebacterium glutamicum ATCC 13032. Glutamicum T7 is a recombinant strain obtained by integrating T7-Plac into the genomic DNA of Corynebacterium glutamicum ATCC 13032. T7-Plac is a DNA molecule consisting of 537 th to 4951 th nucleotides of the sequence 4 in the sequence table.

Glutamicum T7 differs from corynebacterium glutamicum ATCC13032 only in that: the double-stranded DNA molecule shown in the sequence 5 in the sequence table in the genome DNA of Corynebacterium glutamicum ATCC13032 is replaced by the double-stranded DNA molecule shown in the 17 th to 5461 th nucleotides in the sequence table.

The 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 provides a method for preparing transglutaminase, which comprises the following steps: culturing the recombinant microorganism.

The process for preparing transglutaminase further comprises the steps of: after the completion of the culture, the cells were collected and disrupted.

The process for preparing transglutaminase further comprises the steps of: and collecting supernatant after the thalli are crushed.

The process for preparing transglutaminase further comprises the steps of: collecting the supernatant, and collecting the supernatant with His by nickel column affinity chromatography ₆ A tagged protein.

The process for preparing transglutaminase further comprises the steps of: after completion of the nickel column affinity chromatography, desalting and replacing the solvent system. The solvent system may be specifically Tris-HCl buffer solution with pH8.0 and 50 mM.

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

(1) Culturing the recombinant microorganism to OD using a liquid medium _600nm A value =1;

(2) After completion of step (1), IPTG is added to the culture system so that the concentration thereof in the system becomes 0.1 to 1mM, and then the culture is carried out.

The culture conditions of step (1) may be: shaking and culturing at 30 deg.C and 200 r/min.

The culture conditions of step (2) may be: culturing at 16-30 deg.C.

The culture conditions of step (2) may be: culturing at 25 deg.C under shaking at 200r/min for 48h.

The method for culturing the recombinant microorganism specifically may be: 1 volume part of the seed solution is inoculated into 10 volume parts of the liquid culture medium and fermented for 48 hours. During the fermentation process, the temperature is controlled at 25 ℃, and the pH value is controlled at about 7.0 by feeding ammonia water. During the fermentation process, when the system OD _600nm When the value reached 8 to 10, IPTG was added so that its concentration in the system became 0.1 to 1mM. During the fermentation, the glucose concentration of the system was monitored and maintained at 4-6g/L by feeding 50g/100ml of an aqueous glucose solution.

The concentration of IPTG in the system may be in particular 0.5mM.

The liquid culture medium can be specifically a fermentation culture medium containing 17 mu g/mL chloramphenicol.

The preparation method of the seed liquid comprises the following steps: the strain CG-pXMJ19-pro-ssp-dnaB ^155M And (3) inoculating the MTG single colony to a test tube filled with 5mL of liquid BHI culture medium containing 17 mu g/mL of chloramphenicol, and performing shake culture at 30 ℃ at 200r/min for 12h to obtain the seed solution.

The invention also provides a protein (protein B) which sequentially consists of the following components from the N end to the C end: the pro region of MTG, the ssp-dnaB intein, amino acid residue M, the mature region of MTG.

MTG is transglutaminase derived from microorganisms.

The MTG is specifically MTG derived from Streptomyces mobaraensis (Streptomyces mobaraensis).

The pro region of MTG is shown as amino acid residues from 2 nd to 46 th in the sequence 2 of the sequence table, or is shown as amino acid residues from 1 st to 46 th in the sequence 2 of the sequence table.

The ssp-dnaB intein is shown as amino acid residues at 47 th to 200 th positions in a sequence 2 of a sequence table.

The mature region of MTG is shown as amino acid residues 202-532 of the sequence 2 in the sequence table.

Nucleic acid molecules which code for the protein B are also within the scope of the invention.

The DNA molecule for coding the protein B also belongs to the protection scope of the invention.

The recombinant microorganism can be used for producing transglutaminase.

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

The invention also provides a protein (protein C), which sequentially comprises the following segments from the N end to the C end: protein B and protein labels. The protein tag may specifically be His ₆ And (4) a label.

Nucleic acid molecules which code for the protein C also belong to the scope of protection of the invention.

DNA molecules which code for the protein C also belong to the scope of protection of the invention.

The recombinant microorganism can be used for producing transglutaminase.

The MTG derived from Streptomyces mobaraensis is expressed under a T7 promoter, and the intein ssp-dnaB is inserted between a pro region and a mature region of the MTG, so that the high-efficiency expression of the MTG in Corynebacterium glutamicum (Corynebacterium glutamicum) is successfully realized, and after the thalli obtained by fermentation are subjected to ultrasonic disruption, protease treatment or other condition change is not needed, and the MTG with biological activity can be obtained.

The recombinant microorganism obtained by the invention can reach 14U/mL by adopting a shake flask to ferment for 48h at 25 ℃, and can reach 49U/mL by adopting a fermentation tank to ferment for 48h at 25 ℃ in a high-density manner.

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.

Drawings

FIG. 1 is the protein electrophoretogram in step five of example 2.

FIG. 2 is a graph showing a comparison of enzyme activities in step seven of example 2.

FIG. 3 is a graph showing the results of the optimum reaction temperature in example 4.

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

FIG. 5 is a graph showing the results of optimum pH in example 4.

FIG. 6 is a graph showing the results of pH stability in example 4.

Detailed Description

The following examples are intended to facilitate a better understanding of the invention, but are not intended to limit the invention thereto. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. L-glutamic acid- γ -monohydroxyhydroxamic acid: sigma, product number G2253. N-benzyloxycarbonyl-L-glutamylglycine (N-CBZ-Gln-Gly): sigma, product number C6154. Corynebacterium glutamicum ATCC13032 is the Corynebacterium glutamicum of ATCC 13032.

Brain heart leachate broth medium (liquid BHI medium): 37g of the composite dry powder is dissolved in water and the volume is up to 1L. Compounding dry powder: beijing Runzukang Biotech, inc., brand OXOID, cat # CM1135B.

Fermentation medium (ph 7.0): 21g of 3- (N-morpholinyl) propanesulfonic acid, 5g of urea, 5g of ammonium sulfate, 1g of dipotassium hydrogen phosphate, 1g of monopotassium phosphate, 2g of yeast powder, 40g of glucose, 0.25g of magnesium sulfate, 0.01g of calcium chloride, 0.2mg of biotin and 1mL of trace element solution, and the balance is made up to 1L by using water. Solution of trace elements: 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 O20 mg, made up to 1L with water.

Example 1 construction of recombinant plasmid and recombinant bacterium

1. Construction of recombinant plasmids

1. Construction of recombinant plasmid pXMJ19-pro-ssp-dnaB ^155M -MTG。

Recombinant plasmid pXMJ19-pro-ssp-dnaB ^155M MTG is circular plasmid and is shown as sequence 1 in the sequence table.

In the sequence 1 of the sequence table, nucleotides 6304-6322 form a T7 promoter, nucleotides 6323-6347 form a lac operator, nucleotides 6350-6385 form an RBS, and nucleotides 6386-8002 are complete open reading frames (encoding fusion proteins). In sequence 1 of the sequence table, the 6386-6388 th bits are initiation codons, the 6389-6523 th bits encode pro region, the 6524-6985 th bits encode ssp-dnaB intein, the 69886-6985 th bits encode amino acid residue M, the 69889-7981 th bits encode mature MTG, the 7982-7999 th bits encode His ₆ The tag is nucleotide 8000-8002 as a stop codon.

The fusion protein is shown as a sequence 2 in a sequence table. In the sequence 2 of the sequence table, the 47 th to 200 th amino acid residues form ssp-dnaB intein, the 201 th amino acid residue is M, the 202 th to 532 th amino acid residues form mature MTG, the 533 th to 538 th amino acid residues form His ₆ And (4) a label.

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

2. The recombinant plasmid pXMJ19-pro-MTG was constructed.

And recombinant plasmid pXMJ19-pro-ssp-dnaB ^155M -MTG the recombinant plasmid pXMJ19-pro-MTG differs from MTG only in the absence of the part encoding the ssp-dnaB intein (i.e.in the absence of nucleotides 6524 to 6985 of sequence 1 of the sequence listing).

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

And recombinant plasmid pXMJ19-pro-ssp-dnaB ^155M Recombinant plasmid pXMJ19-pro-ssp-dnaB compared to MTG ^155K The MTG only differs in that the nucleotides from position 6986 to position 6988 in the sequence 2 of the sequence listing are mutated from "ATG" to "AAG".

2. Construction of C.Glutamicum T7 C.Glutamicum

1. The plasmid pK18mobsacB is digested by restriction enzyme BamHI, and the linearized plasmid and the homologous recombinant fragment of T7 are connected by utilizing a Gibson Assembly kit (NEB), and the obtained recombinant plasmid is named as recombinant plasmid pK18-T7.

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

2. The recombinant plasmid pK18-T7 is electrically transformed into Corynebacterium glutamicum ATCC13032 (under the electric shock condition: voltage 2.5KV, resistance 200 omega, capacitance 25 muF), recombinant bacteria are obtained through two-time screening (single-exchange recombinant bacteria are obtained through the first screening on a BHI plate containing 25mg/L kanamycin, then the single-exchange recombinant bacteria are cultured in a liquid BHI culture medium overnight, and then the homologous recombination double-exchange strain is obtained through the second screening on a BHI plate containing 200g/L sucrose), namely the Corynebacterium glutamicum C.glutamicum T7.

3. And performing sequencing verification.

Glutamicum T7 differs from corynebacterium glutamicum ATCC13032 only in that: the double-stranded DNA molecule shown in the sequence 5 in the sequence table in the genomic DNA of Corynebacterium glutamicum ATCC13032 was replaced by a double-stranded DNA molecule shown in the 17 th to 5461 th nucleotides of the sequence table in the sequence table. 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 Introducing MTG into Corynebacterium glutamicum C.glutamicum T7 to obtain recombinant strain named as CG-pXMJ19-pro-ssp-dnaB ^155M -MTG。

And introducing the recombinant plasmid pXMJ19-pro-MTG into corynebacterium glutamicum C.glutamicum T7 to obtain a recombinant strain named as the strain CG-pXMJ19-pro-MTG.

The recombinant plasmid pXMJ19-pro-ssp-dnaB ^155K Introduction of MTG into C.glutamicum T7 to obtain recombinantThe strain is named as CG-pXMJ19-pro-ssp-dnaB ^155K -MTG。

Example 2 Shake flask fermentation of recombinant bacteria

1. Strain CG-pXMJ19-pro-ssp-dnaB ^155M -induced fermentation culture of MTG

1. The strain CG-pXMJ19-pro-ssp-dnaB ^155M And (3) inoculating the MTG single colony to a test tube filled with 5mL of liquid BHI culture medium containing 17 mu g/mL of chloramphenicol, and performing shake culture at 30 ℃ at 200r/min for 12h to obtain the seed solution.

2. Inoculating 1mL of the seed solution into a 250mL conical flask containing 50mL of a fermentation medium containing 17. Mu.g/mL chloramphenicol, and culturing at 30 ℃ under shaking at 200r/min to OD _600nm The value =1.

3. After completing step 2, IPTG was added to the culture system to a concentration of 0.5mM in the system, followed by shaking culture at 25 ℃ and 200r/min for 48 hours, at which time the OD of the system was _600nm The value was about 20, and the system was named as a fermentation product.

4. Taking fermentation product (with bacterial load: OD) _600nm Value x volume =20; the volume unit is ml), centrifuging for 5min at 12000r/min, and collecting thalli sediment; resuspending the thallus precipitate in 1mL Tris-HCl buffer (pH8.0, 50 mM), then carrying out ultrasonication (ultrasonication parameters: 200W, 5s stop working for 5s, total time 20 min), sampling as a whole bacteria liquid after the ultrasonication is finished, and collecting supernatant after the rest sample is centrifuged (10000 Xg, 5 min).

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

And (5) adding no IPTG, and performing the same step one.

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

Replacement of the Strain CG-pXMJ19-pro-ssp-dnaB with the Strain CG-pXMJ19-pro-MTG ^155M MTG, other same steps one.

4. Strain CG-pXMJ19-pro-ssp-dnaB ^155K -induced fermentation culture of MTG

With the strain CG-pXMJ19-pro-ssp-dnaB ^155K -MTG instead of the strain CG-pXMJ19-pro-ssp-dnaB ^155M MTG, other same steps one.

5. Protein electrophoresis

And (4) taking the whole bacteria liquid and the supernatant obtained in the step one, the whole bacteria liquid and the supernatant obtained in the step two, and the whole bacteria liquid and the supernatant obtained in the step three, and performing SDS-PAGE.

The electrophoretogram is shown in FIG. 1. In fig. 1: lane 1: the whole fungus liquid obtained in the step two; lane 2: the supernatant obtained in the step two; lane 3: step three, obtaining a whole fungus liquid; lane 4: the supernatant obtained in the third step; lane 5: the whole fungus liquid obtained in the step one; lane 6: supernatant obtained in the first step; lane M: protein marker.

The results show that: strain CG-pXMJ19-pro-ssp-dnaB without IPTG induction ^155M -MTG is unable to produce the protein of interest; in the absence of ssp-dnaB ^155M In the case of (i.e., strain CG-pXMJ 19-pro-MTG), mature MTG could not be produced.

6. Method for detecting MTG enzyme activity

MTG enzyme activity definition: the amount of enzyme required to catalyze the production of 1. Mu. Mol of L-glutamic acid-. Gamma. -monohydroxyhydroxamic acid at 37 ℃ per minute was defined as 1U.

MTG enzyme activity determination method (colorimetric method): (1) taking 200 mu L of a sample to be detected after being preheated at 37 ℃ or a diluent of the sample to be detected (if no special description is provided, the solvent adopted for dilution is Tris-HCl buffer solution with the pH value of 6.0 and the mol/L of 0.2), adding 500 mu L of substrate solution after being preheated at 37 ℃, reacting for 10min at 37 ℃, then adding 200 mu L of terminator, centrifuging for 5min at 10000 Xg, collecting supernate, and measuring the light absorption value at the wavelength of 525 nm; (2) preparing standard solutions with different concentrations by using L-glutamic acid-gamma-monohydroxyhydroxamic acid as a solute and water as a solvent, respectively measuring light absorption values at the wavelength of 525nm, and making a standard curve equation (in the standard curve equation, x is the concentration of the L-glutamic acid-gamma-monohydroxyhydroxamic acid, and y is the light absorption value); (3) substituting the light absorption value obtained in the step (1) into the standard curve equation obtained in the step (2), and calculating to obtain the MTG enzyme activity of the sample to be detected.

The preparation method of the substrate solution comprises the following steps: 100mg of N-CBZ-Gln-Gly was dissolved in 2mL of 0.2mol/L NaOH aqueous solution, and then 4mL of Tris-HCl buffer (pH 6.0, 0.2 mol/L), 2mL of 0.1mol/L hydroxylamine aqueous solution and 2mL of 0.01mol/L reduced glutathione aqueous solution were added to adjust the pH to 6.0.

A terminating agent: containing 1mol/L HCl, 4g/100ml trichloroacetic acid, 5g/100ml FeCl ₃ ·6H ₂ O and the balance of water.

7. Detection of MTG enzyme Activity

And taking the supernatant obtained in the step one, the supernatant obtained in the step three and the supernatant obtained in the step four as samples to be detected respectively, detecting the MTG enzyme activity, and calculating to obtain the enzyme activity of the fermentation product.

The enzyme activity of the fermentation product obtained in the first step is 14U/mL.

And the enzyme activity of the fermentation product obtained in the third step is 0U/mL.

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

The enzyme activities of the fermentation product obtained in the first step and the fermentation product obtained in the fourth step are compared and shown in FIG. 2.

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

1. Collecting the supernatant obtained in step one, filtering with 0.22 μm filter membrane, performing nickel column affinity chromatography, and collecting the solution containing target protein (MTG-His) ₆ Protein).

2. Taking the post-column solution obtained in the step 1, desalting by adopting a column type ultrafiltration membrane with the aperture of 3KD and a Tris-HCl buffer solution with the pH value of 8.0 and 50mM to obtain a protein solution with the Tris-HCl buffer solution with the pH value of 8.0 and 50mM as a solvent system, and naming the protein solution as MTG-His ₆ And (3) solution.

3. Performing N-terminal sequencing

Taking the MTG-His obtained in the step 2 ₆ The solution was subjected to 12.5% SDS-PAGE, transferred onto a PVDF membrane, and the effect of the transfer was examined by ponceau staining. Sending to the protein sequencing laboratory of the life center of Beijing university, and determining that the 5 amino acid sequences at the N-terminal are M-D-S-D-D by an Edman method. As a result, the strain CG-pXMJ19-pro-ssp-dnaB was found ^155M MTG expresses fusion protein in cells, and the fusion protein is self-sheared (the shearing site is between the 200 th amino acid residue and the 201 th amino acid residue of the sequence 2) due to the existence of ssp-dnaB intein to obtain the sequence 3 shown in the sequence tableMTG-His ₆ A protein. The above steps can obtain active MTG-His without secondary treatment ₆ A protein.

4. Detection of MTG enzyme Activity

Taking the MTG-His obtained in the step 2 ₆ And (3) taking the solution as a sample to be detected, and detecting the MTG enzyme activity.

Taking the MTG-His obtained in the step 2 ₆ And (4) detecting the protein concentration by using a Bradford method.

MTG-His ₆ Enzyme activity of the solution was divided by MTG-His ₆ Protein concentration of the solution to obtain MTG-His ₆ The specific activity of the protein is 33U/mg.

EXAMPLE 3 fermenter culture of recombinant bacteria

1. Preparation of seed liquid

The strain CG-pXMJ19-pro-ssp-dnaB ^155M And (3) inoculating the MTG single colony to a test tube filled with 5mL of liquid BHI culture medium containing 17 mu g/mL of chloramphenicol, and carrying out shake culture at 30 ℃ at 200r/min for 12h to obtain the seed solution. Multiple tubes were used to prepare multiple seed solutions.

2. 100mL of the seed solution was inoculated into a 2.5L fermentor containing 1L of a fermentation medium containing 17. Mu.g/mL chloramphenicol and fermented for 48 hours. During the fermentation process, the temperature is controlled at 25 ℃, and the pH value is controlled at about 7.0 by feeding ammonia water. During the fermentation process, when the system OD is _600nm When the value reached 8-10, IPTG was added to make the concentration in the system 0.5mM. During the fermentation, the glucose concentration of the system was monitored and maintained at 4-6g/L by feeding 50g/100ml of an aqueous glucose solution. After fermentation is complete, the system OD _600nm The value was about 70, and the system was named as a fermentation product.

3. Taking fermentation product (with bacterial load: OD) _600nm Value x volume =20; the volume unit is ml), centrifuging for 5min at 12000r/min, and collecting thalli sediment; the pellet was resuspended in 1mL Tris-HCl buffer (pH 8.0, 50 mM), then sonicated (sonication parameters: 200W, 5s stop for 5s for a total time of 20 min), and then centrifuged (10000 Xg, 5 min) to collect the supernatant.

4. And (4) taking the supernatant obtained in the step (3) as a sample to be detected, detecting the MTG enzyme activity, and calculating to obtain the enzyme activity of the fermentation product.

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

Example 4 study of the enzymatic Properties of MTG

MTG-His ₆ The solution was MTG-His prepared in step eight of example 2 ₆ And (3) solution.

1. Optimum reaction temperature and temperature stability

1. Optimum reaction temperature

Taking MTG-His ₆ And (3) taking the solution as a sample to be detected, and detecting the MTG enzyme activity. The method for detecting MTG enzyme activity refers to the sixth step of example 2. The reaction temperature is set to 30 ℃,37 ℃, 45 ℃,50 ℃, 55 ℃ or 60 ℃ respectively.

And taking the highest enzyme activity point as 100% enzyme activity, and calculating the relative enzyme activity at other temperatures. The results are shown in FIG. 3.

The highest point of enzyme activity is used as the optimal reaction temperature. The optimum reaction temperature is 55 ℃.

2. Temperature stability

Taking MTG-His ₆ The solutions are respectively placed at three temperatures (40 ℃,50 ℃ or 60 ℃), sampled every 20 minutes and used as samples to be detected to detect the MTG enzyme activity.

Taking MTG-His ₆ And (3) taking the solution as a sample to be detected, detecting the MTG enzyme activity and taking the MTG enzyme activity as a reference enzyme activity.

The method for detecting MTG enzyme activity refers to the sixth step of example 2.

The thermal stability curve was plotted with the reference enzyme activity as 100%. The results are shown in FIG. 4. The activity was retained at 40 ℃ for 100 minutes at 90%, at 50 ℃ for 100 minutes at 30% and at 60 ℃ for 20 minutes at substantially lost.

2. Optimum pH and pH stability

The substrate buffers were: 50mM acetate-sodium acetate buffer, pH5.0, 50mM acetate-sodium acetate buffer, pH6.0, 50mM Tris-HCl buffer, pH7.0, 50mM Tris-HCl buffer, pH8.0, 50mM Tris-HCl buffer, pH9.0, 50mM Tris-HCl buffer.

1. Optimum pH

Taking MTG-His ₆ And (3) diluting the solution to 10 times of volume by using a substrate buffer solution, and detecting the MTG enzyme activity as a sample to be detected.

The method for detecting MTG enzyme activity refers to step six of example 2.

The preparation method of the substrate solution comprises the following steps: 100mg of N-CBZ-Gln-Gly was dissolved in 2mL of 0.2mol/L NaOH aqueous solution, and then 4mL of substrate buffer, 2mL of 0.1mol/L hydroxylamine aqueous solution and 2mL of 0.01mol/L reduced glutathione aqueous solution were added to adjust the pH to the pH of the substrate buffer.

And taking the highest enzyme activity point as 100% enzyme activity, and calculating the relative enzyme activity under other pH values. The results are shown in FIG. 5.

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

2. Stability of pH

Taking MTG-His ₆ And (3) diluting the solution to 10 times of volume by using a substrate buffer solution, standing at room temperature for 1h, and detecting the MTG enzyme activity as a sample to be detected.

Taking MTG-His ₆ And (3) diluting the solution to 10 times of volume by using Tris-HCl buffer solution with the pH value of 8.0 and the concentration of 50mM, and taking the diluted solution as a sample to be detected to detect the MTG enzyme activity and the activity as reference enzyme activity.

The method for detecting MTG enzyme activity refers to step six of example 2.

The pH stability curve was plotted with the reference enzyme activity as 100%. The results are shown in FIG. 6. Treating at pH5.0-7.0 for 1 hr, and retaining more than 80% activity; treating at pH8.0-9.0 for 1 hr to retain over 60% activity; treatment at pH4.0 for 1h,70% activity was lost.

3. Influence of Metal ions, EDTA, PMSF on enzymatic Activity

Taking MTG-His ₆ Adding an inhibitor into the solution, enabling the concentration of the inhibitor to be 1mM, standing the solution at room temperature for 1h, and taking the solution as a sample to be detected to detect the MTG enzyme activity.

The method for detecting MTG enzyme activity refers to step six of example 2.

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

The results are shown in Table 1.Na (Na) ⁺ 、K ⁺ 、Ca ²⁺ 、Mg ²⁺ Has no influence on the activity of MTG enzyme. Cu ²⁺ 、Mn ²⁺ 、Fe ²⁺ Has certain inhibition effect on enzyme activity, but still retains more than 70 percent of activity. Zn ²⁺ Has strong inhibition effect on MTG activity, and the activity is basically lost after treatment. EDTA and PMSF do not produce inhibition effect on enzyme activity.

TABLE 1

Inhibitor contained in test solution	Relative enzyme activity (%)
		CuSO ₄	76.19±2.27
MnSO ₄	77.19±6.85
		NaCl	113.46±3.64
KCl	113.67±3.90
		CaCl ₂	108.20±3.46
ZnSO ₄	1.18±0.17
		FeSO ₄	87.23±1.20
MgSO ₄	111.07±2.40
		EDTA	116.33±6.11
PMSF	113.73±4.96

SEQUENCE LISTING

<110> institute of microbiology of Chinese academy of sciences

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

<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

atggcctttt tgcgtttcta caaactcttt tgtttatttt tctaaataca ttcaaatatg 300

tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 360

atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 420

gtttttgctc acccagaaac 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

ttcattatgt 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 ttttcccgct ggtctgctgg tgtggttaag gccggtatgg acgcgccact 3300

gcgtgagcac ggggtcaaac 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 aaccccgttc 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 ccaggaccac 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 gtgagggatt 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

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

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 acattgattg 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

cttatcagac 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 atgtaggagt 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 acgggattgt 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 accagttgca 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 taggatttcg 5400

gtgtagagcc agaccaaggt cactgcaaga ccaagcgcaa cgccccatgc catcttggaa 5460

gggaaatagg ggccttttgt tg 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

tctttacctt 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

1. A protein which is (a 1) or (a 2) below:

(a2) A protein shown in a sequence 2 of a sequence table.

2. A nucleic acid molecule encoding the protein of claim 1.

3. A DNA molecule encoding the protein of claim 1.

4. An expression cassette, recombinant vector or recombinant microorganism having the DNA molecule of claim 3.

5. The recombinant vector of claim 4, wherein: the recombinant vector is (c 1) or (c 2) as follows:

(c2) The recombinant vector is shown as a sequence 1 in a sequence table.

6. The recombinant microorganism of claim 4, wherein: the recombinant microorganism is obtained by introducing the recombinant vector according to claim 5 into Corynebacterium glutamicum.

7. Use of a protein according to claim 1, or a nucleic acid molecule according to claim 2, or a DNA molecule according to claim 3, or an expression cassette according to claim 4, or a recombinant vector according to claim 4 or 5, or a recombinant microorganism according to claim 4 or 6 for the preparation of a transglutaminase.

8. A method of preparing transglutaminase comprising the steps of: culturing the recombinant microorganism of claim 6.