CN112553176A - Glutamine transaminase with improved thermal stability - Google Patents

Abstract

The invention discloses a glutamine transaminase with improved thermal stability, belonging to the fields of biology and food. The glutamine transaminase variant of the invention is an enzyme with improved thermostability comprising a substitution corresponding to positions 1 to 4 of the polypeptide shown in SEQ ID No. 1. The glutamine transaminase variants of the invention can be used in food processing, processing and transformation applications, and can be used to maintain or improve the quality, consistency, elasticity, moisture or viscosity of food. The glutamine transaminase variants with improved thermostability are more advantageous for their stable application in harsh industrial environments, such as meat analogue processing and meat ball processing.

Description

Glutamine transaminase with improved thermal stability

Technical Field

The invention relates to a glutamine transaminase with improved thermal stability, belonging to the fields of biology and food.

Background

Glutamine transaminase (TGase, EC 2.3.2.13), a class of transaminase, catalyzes the reaction of the gamma carboxamide group of glutamine residues in proteins and small molecules with the amino group of acyl acceptors to complete substrate crosslinking by forming covalent bonds. The application of TGase in the field of food processing is very wide, and the TGase can be added into fresh meat processing, sausage products, fish balls, meat paste processing, bean products and dairy products to change the food appearance, taste and stability.

With the research and application of TGase, the discovery of poor thermal stability is a major problem limiting its practical application. TGase is used in food processing, such as processing meat paste, meat balls, dairy products and bean products, and high temperature treatment is needed to complete TGase catalytic crosslinking, and the thermal stability of TGase is poor, the temperature is controlled to be within 40 ℃ in conventional operation, and the addition amount is large due to fast loss (refer to patent publication numbers: CN109907111A, CN201810480335.6, CN110771811A, CN106901205B and CN 201811599451.6).

The existing method for modifying the thermal stability of the enzyme is mainly rational design and directed evolution. The TGase from Streptomyces mobaraensis AAT65817 is a mainstream enzyme preparation widely sold in domestic and international markets. There is reported a Streptomyces mobaraensis-derived TGase with improved stability, which has a half-life of 11.31min at 60 ℃ under water bath conditions. However, in practical applications, this stability is far from desired. For example, the ambient temperature during meat emulsion processing is 30-70 ℃, and TGase is easily inactivated at this temperature, so there are certain requirements on the amount of addition and the processing time. Therefore, if high-stability TGase can be further obtained, the treatment time and the enzyme dosage can be greatly reduced and the method is more economical because the TGase can be applied at 60 ℃ or even above.

Disclosure of Invention

In order to solve the above problems, the present invention provides a transglutaminase having improved stability, a mature enzyme coding sequence of transglutaminase, and a method for obtaining a mature enzyme of transglutaminase having improved thermostability by replacing a nitrogen terminal sequence.

The mature enzyme of glutamine transaminase of the present invention comprises a substitution corresponding to positions 1 to 4 of the polypeptide represented by SEQ ID NO.1, wherein the amino acid sequence in which the substitution occurs is FRAPD.

i) A polypeptide having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the polypeptide represented by SEQ ID NO. 1; and/or

ii) the mature enzyme of glutamine transaminase is a polypeptide encoded by a polynucleotide having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the mature polypeptide coding sequence represented by SEQ ID NO. 2.

In one embodiment, the mature enzyme of glutamine transaminase is a substitution of positions 1 to 4 to FRAPD on the basis of SEQ ID NO: 1.

The present invention also relates to polynucleotides encoding the mature enzymes of glutamine transaminase; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and a method for producing the transglutaminase maturase. Furthermore, the present invention relates to a composition comprising the glutamine transaminase maturation enzyme of the present invention.

The present invention also relates to methods of producing a transglutaminase maturation enzyme of the invention, comprising:

a) culturing the host cell of the invention under conditions suitable for expression of said transglutaminase; and

b) optionally recovering said transglutaminase.

The invention also relates to a method for modifying the phase, mouthfeel and/or stability of a food product in fresh meat processing, sausage products, fish balls, meat emulsion processing, soy products and/or dairy products, said method comprising adding a transglutaminase maturase according to the invention or a composition according to the invention to the food processing for treatment.

The invention also relates to the use of said transglutaminase or of the composition according to the invention for the treatment, processing and transformation of food.

In one embodiment, for maintaining or improving the quality, consistency, elasticity, moisture or viscosity of a food product.

In one embodiment, wherein the food product is selected from the group consisting of cheese, yogurt, ice cream, mayonnaise, and meat.

In one embodiment, wherein the food product is fish.

In one embodiment, for forming gelatin of different densities and for preparing fat-reduced precooked food products.

The invention also relates to a method for improving the stability of the mature enzyme of glutamine transaminase, which is realized by zymogen replacement in practical application; in one embodiment, the pro-enzyme of glutamine transaminase (SEQ ID NO:4) of the original Streptomyces mobaraensis is replaced with Streptomyces caniferus-derived pro-glutamine transaminase (SEQ ID NO: 3).

The method for improving the stability of the mature enzyme of the transglutaminase is embodied in practical application that after a zymogen region of the transglutaminase is cut by neutral protease, the nitrogen end of the mature region of the transglutaminase has one more amino acid than the nitrogen end of the mature region of the transglutaminase which is not replaced by the zymogen, the more amino acid is asparagine (Asn), namely the nitrogen-end amino acid sequence FRAP of the sequence shown in SEQ ID NO:1 is replaced by FRAPD.

Definitions or terms:

transglutaminase: the terms "Transglutaminase (TGase)", "R-glutaminyl-peptidamidase- γ -glutamyl-transferase", "Transglutaminase" refer to enzymes in class EC 2.3.2.13 as defined by the enzyme nomenclature. Glutamine transaminase contains the sequence of the pro-nitrogen region, as well as the downstream mature region. For the purposes of the present invention, the glutamine transaminase activity is determined according to the procedure described in the examples.

Mature enzyme: the mature region of a transglutaminase encodes a transglutaminase having a transglutaminase activity.

A coding sequence: the term "coding sequence" means a polynucleotide which directly specifies the amino acid sequence of a transglutaminase. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

And (3) control sequence: the term "control sequences" means nucleic acid sequences necessary for the expression of a polynucleotide encoding a transglutaminase of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the glutamine transaminase, or native or foreign with respect to one another. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a transglutaminase variant of the invention.

Expressing: the term "expression" includes any step involved in the production of glutamine transaminase, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: the term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a glutamine transaminase of the present invention and operably linked to control sequences that provide for its expression.

Fragment (b): the term "fragment" means a polypeptide lacking one or more (e.g., several) amino acids at the amino and/or carboxy terminus of the polypeptide; wherein said fragment has glutamine transaminase activity. In one aspect, a fragment contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the number of amino acids 1 to 335 of SEQ ID NO.1 (this length being the length of the mature region after cleavage by the pro-glutamyltransferase enzyme shown in SEQ ID NO. 1).

Host cell: the term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Improved thermal stability: the term "improved thermostability" means the improved characteristics of a transglutaminase relative to a parent transglutaminase.

Separating: the term "isolated" means a substance in a form or environment not found in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance; (2) any substance that is at least partially removed from one or more or all of the naturally occurring components with which it is associated in nature, including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor; (3) any substance that is modified manually by man relative to that found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a stronger promoter than a promoter naturally associated with the gene encoding the substance). The isolated material may be present in a sample of fermentation broth.

Mature polypeptide: the term "mature polypeptide" means a polypeptide that is in its final form following translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and the like. In one aspect, the mature polypeptide is amino acids 5 to 335 of SEQ ID NO. 1. It is known in the art that host cells can produce a mixture of two or more different mature polypeptides (i.e., having different C-terminal and/or N-terminal amino acids) expressed from the same polynucleotide.

Mature polypeptide coding sequence: the term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having glutamine transaminase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 13 to 1005 of SEQ ID NO. 2.

Nucleic acid construct: the term "nucleic acid construct" means a nucleic acid molecule, either single-or double-stranded, that is isolated from a naturally occurring gene or that has been modified to contain segments of nucleic acids in a manner not otherwise found in nature, or that is synthetic, that contains one or more control sequences.

Maternal or glutamine transaminase maturation zone: the term "maternal" or "glutamine transaminase maturation zone" means a glutamine transaminase maturation enzyme that is altered to produce a glutamine transaminase maturation enzyme variant of the present invention. The parent transglutaminase can be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof or can be synthetically produced.

Sequence identity: the degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

Stability: the thermostability of a variant of a transglutaminase according to the invention can be expressed as the residual activity or residual performance of the transglutaminase during or after exposure to the different test conditions. May be compared to the known activity or performance of a maternal transglutaminase (e.g., a maternal transglutaminase as shown in SEQ ID NO: 1).

Variants: the term "variant" means a polypeptide having glutamine transaminase activity that comprises alterations (i.e., substitutions, insertions, and/or deletions) at one or more (e.g., several) positions. In the invention, one variant is that the 1 st-4 th amino acid is substituted as shown in SEQ ID NO.1 in the maternal sequence, and the substituted amino acid is FRAPD. Substitution means the substitution of an amino acid occupying a position with a different amino acid; deletion means the removal of an amino acid occupying a position; and an insertion means that an amino acid is added next to and immediately following the amino acid occupying a certain position.

Wild-type transglutaminase: the term "wild-type" glutamine transaminase is intended to mean a glutamine transaminase expressed by a naturally occurring microorganism (e.g., a bacterium, yeast, or filamentous fungus) found in nature.

Has the advantages that:

the mature enzyme of glutamine transaminase of the present invention contains substitutions corresponding to positions 1 to 4 of the polypeptide shown in SEQ ID NO.1 (the substituted amino acid sequence is FRAPD), and the mature enzyme variant has improved thermostability. The mature enzyme variant, in practical use, can be obtained by using a zymogen derived from Streptomyces caniferus glutamine transaminase in place of the zymogen of Streptomyces mobaraensis glutamine transaminase, expressing the zymogen with glutamine transaminase, and cleaving the zymogen region with a neutral protease. After the sample is purified by affinity chromatography, the result shows that the half-life period of the mature enzyme variant is improved by 94.33 percent compared with the half-life period of the female parent at 60 ℃. The melting point of the mature enzyme variant was increased by 0.95 ℃ as determined by differential scanning calorimetry.

The glutamine transaminase maturation enzyme variant of the invention has significantly improved thermostability, can be applied in food processing, transformation and the like, and can be used for maintaining or improving the quality, consistency, elasticity, moisture or viscosity of food. The glutamine transaminase variant with improved thermal stability is more beneficial to the stable application of the glutamine transaminase in severe industrial environments, such as meat emulsion processing and meat ball processing of artificial meat.

Drawings

FIG. 1: initial enzyme activities of the parent and the variant and half-lives at 60 ℃.

FIG. 2: the whole cell after the female parent containing zymogen and the variant (both the female parent and the variant glutamine transaminase contain zymogen region sequences) are expressed by E.coli BL21 and analyzed by SDS-PAGE electrophoresis. M is a protein mass marker with a Kda unit; 1 as female parent, expressing with E.coli BL21, and analyzing by whole cell electrophoresis; coli BL21 expression for the variant and whole cell electrophoretic analysis.

FIG. 3: SDS-PAGE analysis of samples from the purification of the parent and variants; m is a protein mass marker with the unit of kDa; 1, performing electrophoretic analysis after female parent purification; 2 electrophoretic analysis after purification of variants.

FIG. 4: the zymogen region (pro) of Streptomyces mobaraeneesis transglutaminase is aligned with the zymogen region (proC) of Streptomyces caniferus transglutaminase in amino acid sequence.

Detailed Description

The present invention relates to variants that have been improved in thermostability as compared to the parent. More specifically, the present invention relates to variants having improved thermostability compared to the parent (in particular glutamine transaminase as shown in SEQ ID NO: 1).

The enzyme activity, thermal stability and kinetic parameters of the glutamine transaminase are determined according to the following method.

Enzyme activity assay

Enzyme activity test substrate solution a: 200mM Tris-HCl, 100mM hydroxylamine, 10mM reduced glutathione, 30mM N-benzyloxycarbonyl-L-glutamylglycine, adjusted to pH 6.0.

Enzyme activity test stop solution B: 3mol/L HCl, 5% FeCl₃·6H₂The solution preparation is completed by mixing O (dissolved in 0.1mol/L HCl) and 12% TCA (trichloroacetic acid) in equal volume.

Definition of enzyme activity: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of a product from 1. mu. mol of substrate per minute.

Preparing a standard curve for enzyme activity test: weighing 648mg of standard L-glutamic acid-gamma-monohydroxamic acid, adding 100ml of Tris-HCl 200mM solution and pH 6.0 solution, sequentially diluting with Tris-HCl 200mM solution and pH 6.0 solution by a 2-fold dilution method for 5 gradients, respectively carrying out heat preservation on the solution and the substrate solution A at 37 ℃ for 5min, then taking 60 mu L of standard solution to 150 mu L of substrate solution A, adding 60 mu L of stop solution B after carrying out water bath at 37 ℃ for 10min, centrifuging for 1min at 10000rpm, and taking 200 mu L of supernatant to determine the light absorption value at 525 nm. And (3) taking the quantity of the light absorption value to the hydroxamic acid as a straight line, obtaining a conversion coefficient K from the slope of the straight line, and calculating the production quantity of the hydroxamic acid through K after the absorbance is obtained in the determination of the enzyme activity of the sample.

The determination method comprises the following steps: the protein sample and 150. mu.L of the substrate solution A were incubated at 37 ℃ for 5min, then 60. mu.L of the protein sample was added to 150. mu.L of the substrate solution A, and 60. mu.L of the test stop solution B was added after water bath at 37 ℃ for 10 min. The reaction solution was centrifuged at 10000rpm for 1min, and 200. mu.L of the supernatant was taken to measure the absorbance at 525 nm. The blank control is 60 μ L of test stop solution B, 60 μ L of protein sample, 150 μ L of substrate solution A, and 200 μ L of supernatant after centrifugation at 10000rpm for 1min to determine the light absorption value at 525 nm. And subtracting the light absorption value obtained by the control group from the light absorption value obtained by the experimental group, and bringing the light absorption value into an enzyme activity standard curve to obtain the enzyme activity corresponding to the added mass of the protein, and dividing the enzyme activity by the protein concentration to obtain the specific enzyme activity U/mg of the protein.

Thermal stability test

Measuring the half-life period of the enzyme under the water bath condition of 60 ℃, specifically: diluting the female parent and a variant pure protein solution (with a solvent of Tris-HCl and pH 8.0) after replacing the zymogen to 0.5mg/ml, taking a certain amount of the sample for continuous thermal incubation at 60 ℃ in a water bath, sampling every minute within 0-10 min, sampling every 2min for 10-30 min, and immediately placing the taken sample at 20 ℃ for cold compress. The enzyme activity of the sampled samples is measured respectively to obtain the residual enzyme activity of the female parent and the variant which change with time. The method for obtaining the specific half-life values of the parent and the variant comprises the following steps: obtaining a residual enzyme activity percentage value which changes along with time, combining the residual enzyme activity percentage value obtained by Exponental-ExpDec 1 in origin 2018 to perform nonlinear fitting, and calculating the time corresponding to the enzyme activity reduced to the initial 50 percent after obtaining a fitting formula, namely the half-life.

Differential scanning calorimetry

The dissolution temperature test was performed by differential scanning calorimetry using the parent and variant pure protein solutions (solvent Tris-HCl, pH 8.0) at concentrations greater than 1.5 mg/ml. Taking a protein solvent as an internal reference, scanning the temperature from 40-90 ℃, heating up at a rate of 1 ℃/min and under a pressure of 3atm, and finally obtaining the dissolution temperature of the protein.

Determination of kinetic parameters

Michaelis constant K_MThe test method comprises the following steps: the parent and variant protein solutions were diluted to 0.05mg/ml and the assay was referenced to the enzyme activity assay. The substrate solution A was required to be configured into 10 types depending on the N-benzyloxycarbonyl-L-glutamylglycine content, and the other components were not changed, and the N-benzyloxycarbonyl-L-glutamylglycine contents were 3mM, 6mM, 9mM, 12mM, 15mM, 18mM, 21mM, 24mM, 27mM, and 30mM, respectively. According to the enzyme activity test method, the conversion amount of the substrate in the substrate solution A with different N-benzyloxycarbonyl-L-glutamyl glycine contents of the parent and the variant is tested, namely the amount of the substrate N-benzyloxycarbonyl-L-glutamyl glycine which is catalyzed to be converted into the final product within 10min of reaction time. Based on the obtained conversion value, nonlinear fitting is carried out on the obtained value through Origin 2018 software, and growing/sigmoidal-Hill is adopted for fitting, and K is obtained_MAnd Vmax value, k is obtained by conversion of enzyme concentration_catThe value is obtained. On the basis of the above, with K_M/k_catObtaining the catalytic efficiency of the enzyme.

The female parent of the present invention comprises a nucleotide sequence corresponding to SEQ ID NO:1, wherein,

i) a polypeptide having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide set forth in SEQ ID NO.1 in the mature region of the transglutaminase; and/or

ii) the mature region of said transglutaminase is a polypeptide encoded by a polynucleotide having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence as set forth in SEQ ID NO. 2;

the 1-4 position is substituted, and the amino acid sequence after the substitution is FRAPD.

Table 1 lists sequences similar to SEQ ID NO:1, the variant is relatively thermostable.

TABLE 1

Polynucleotide

The present invention also relates to isolated polynucleotides encoding the glutamine transaminases of the present invention. In certain aspects, the invention relates to nucleic acid constructs comprising a polynucleotide of the invention. In certain aspects, the invention relates to expression vectors comprising a polynucleotide of the invention. In certain aspects, the invention relates to host cells comprising a polynucleotide of the invention. In certain aspects, the invention relates to a method of producing a transglutaminase, comprising: (a) culturing the host cell of the invention under conditions suitable for expression of said transglutaminase; and (b) recovering said transglutaminase.

Nucleic acid constructs

The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a glutamine transaminase of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

The polynucleotide can be manipulated in a variety of ways to provide for expression of transglutaminase. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to its insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide recognized by a host cell for expression of the polynucleotide. The promoter contains transcriptional control sequences that mediate the expression of the transglutaminase variant. The promoter may be any polynucleotide that shows transcriptional activity in the host cell, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Expression vector

The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a glutamine transaminase of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of a polynucleotide encoding a glutamine transaminase variant at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector such that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for ensuring self-replication. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome or chromosomes into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell may be used, or a transposon may be used.

The vector preferably contains one or more selectable markers that allow for convenient selection of transformed cells, transfected cells, transduced cells, and the like. A selectable marker is a gene the product of which provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance (e.g., ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance). Suitable markers for yeast host cells include, but are not limited to: ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA 3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5' -phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and the Streptomyces hygroscopicus (Streptomyces hygroscopicus) bar gene.

The vector preferably contains one or more elements that allow the vector to integrate into the genome of the host cell or the vector to replicate autonomously in the cell, independently of the genome.

For integration into the host cell genome, the vector may rely on the polynucleotide sequence encoding transglutaminase or any other vector element for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the host cell genome at a precise location in the chromosome. To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, e.g., 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. Alternatively, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may additionally comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicon mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in E.coli, and the origins of replication of plasmids pUB110, pE194, pTA1060, and pAM β 1, which allow replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN 6.

Examples of origins of replication useful in filamentous fungal cells are AMA1 and ANS1(Gems et al, 1991, Gene [ Gene ]98: 61-67; Cullen et al, 1987, Nucleic Acids Res. [ Nucleic Acids research ]15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of a plasmid or vector containing the gene can be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the invention may be inserted into a host cell to increase production of a transglutaminase variant. The increased copy number of the polynucleotide may be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide, wherein cells containing amplified copies of the selectable marker gene, and thus additional copies of the polynucleotide, may be selected for by culturing the cells in the presence of the appropriate selectable agent.

Procedures for ligating the elements described above to construct the recombinant expression vectors of the invention are well known to those of ordinary skill in the art.

Host cell

The host cell may be any cell useful in the recombinant production of transglutaminase, e.g. a prokaryotic cell or a eukaryotic cell.

The prokaryotic host cell may be any gram-positive or gram-negative bacterium. Gram-positive bacteria include, but are not limited to: bacillus, Clostridium, enterococcus, Geobacillus (Geobacillus), Lactobacillus, lactococcus, Paenibacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia, Flavobacterium, Clostridium, helicobacter, Citrobacter, Neisseria, Pseudomonas, Salmonella, and Urethania.

The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.

Generation method

The present invention also relates to a method of producing a transglutaminase of the invention, comprising: (a) culturing the host cell of the invention under conditions suitable for expression of said transglutaminase; and (b) recovering said transglutaminase.

The host cell is cultured in a nutrient medium suitable for the production of transglutaminase using methods known in the art. For example, the cells may be cultured by shake flask culture, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the transglutaminase to be expressed and/or isolated. Culturing occurs in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions. If the transglutaminase variant is secreted into the nutrient medium, the transglutaminase can be recovered directly from the medium. If the transglutaminase is not secreted, it can be recovered from the cell lysate.

Glutamine transaminase can be detected using methods known in the art that are specific for glutamine transaminase. These detection methods include, but are not limited to: the use of specific antibodies, the formation of enzyme products or the disappearance of enzyme substrates. For example, enzymatic assays can be used to determine the activity of glutamine transaminase (such as those described in the examples).

The transglutaminase can be recovered using methods known in the art. For example, the transglutaminase can be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

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

In an alternative aspect, no transglutaminase is recovered, but the host cell of the invention expressing a transglutaminase is used as a source of said transglutaminase.

Fermentation broth formulations or cell compositions

The invention also relates to fermentation broth formulations or cell compositions comprising the polypeptides of the invention. The fermentation broth product further comprises additional components used in the fermentation process, such as, for example, cells (including host cells containing a gene encoding a polypeptide of the invention, which host cells are used to produce the polypeptide of interest), cell debris, biomass, fermentation medium, and/or fermentation product. In some embodiments, the composition is a cell-killed whole broth comprising one or more organic acids, killed cells and/or cell debris, and culture medium.

The term "fermentation broth" as used herein refers to a preparation produced by fermentation of a cell that has not undergone or has undergone minimal recovery and/or purification. For example, a fermentation broth is produced when a microbial culture is grown to saturation by incubation under carbon-limited conditions that allow protein synthesis (e.g., expression of an enzyme by a host cell) and secretion of the protein into the cell culture medium. The fermentation broth may contain an unfractionated or fractionated content of the fermented material obtained at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises spent culture medium and cell debris present after removal of microbial cells (e.g., filamentous fungal cells), e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or non-viable microbial cells.

In one embodiment, the fermentation broth formulation and cell composition comprises a first organic acid component (comprising at least one organic acid of 1-5 carbons and/or salt thereof) and a second organic acid component (comprising at least one organic acid of 6 or more carbons and/or salt thereof). In a particular embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing; and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, salts thereof, or mixtures of two or more of the foregoing.

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

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

The cell-killed whole broth or composition may contain unfractionated contents of the fermented material obtained at the end of fermentation. Typically, the cell-killing whole broth or composition contains spent culture medium and cell debris present after microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon limiting conditions to allow protein synthesis. In some embodiments, the cell-killing whole broth or composition contains spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, methods known in the art may be used to permeabilize and/or lyse microbial cells present in a cell-killed whole broth or composition.

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

Composition comprising a metal oxide and a metal oxide

The invention also relates to compositions comprising the glutamine transaminase of the invention.

These compositions may comprise a transglutaminase of the invention as a major enzyme component, e.g., a one-component composition. Alternatively, the composition may comprise a plurality of enzyme activities, such as one or more (e.g., several) enzymes selected from the group consisting of: protease, glucoamylase, beta-amylase, pullulanase.

Example 1

The preparation and the heat stability of a partial transglutaminase are described below with reference to one of the embodiments.

Coli JM109 and E.coli BL21(DE3) were obtained from Takara-Baori physicians & materials technology (Beijing) Co., Ltd., pET-22b (+) plasmid was obtained from Novagen (the above strain E.coli BL21(DE3) was commercially available and no preservation for patent program was required), neutral protease was obtained from Beijing Solebao technologies Co., Ltd. (product No. Z8032), and Bradford protein concentration measurement kit (detergent compatible type) was obtained from Shanghai Biyutian biotechnology Co., Ltd.

The culture medium is as follows:

LB liquid medium: 5.0g/L yeast powder, 10.0g/L, NaCl 10.0.0 g/L tryptone and 100 mu g/L ampicillin.

LB solid medium: 5.0g/L yeast powder, 10.0g/L, NaCl 10.0.0 g/L tryptone, 15g/L agar powder and 100 mu g/L ampicillin.

TB culture medium: 24g/L yeast extract, 12g/L tryptone, 12.84g/L dipotassium phosphate trihydrate, 2.31g/L potassium dihydrogen phosphate, 4mL/L glycerol and 100 mu g/L ampicillin.

1. Construction of Glutamine transaminase mutants

The parent gene (nucleotide sequence shown in SEQ ID NO: 5) containing the pro-region of Streptomyces mobaraensis glutamine transaminase was synthesized by Kinzhi Kogyo, Suzhou, and inserted into plasmid pET-22b (+) through restriction enzyme sites NdeI and BlpI to obtain pET-22b-tgase (SEQ ID NO: 6). Glutamine transaminase after zymogen replacement (the zymogen of Streptomyces mobaraesesis glutamine transaminase was replaced with the zymogen of Streptomyces caniferus glutamine transaminase), contains the pro-soluble protein tag TrxA upstream of the gene synthesized by Kinzhi Navigilans GmbH, and was inserted into plasmid pET-22b (+) via restriction enzyme sites NdeI and BlpI to give pET-22b-proC/tgase (SEQ ID NO: 7).

2. Process for preparing mutant enzyme

Respectively transforming the constructed plasmids pET-22b-tgase and pET-22b-proC/tgase into Escherichia coli JM109, coating the transformation product on an LB solid culture medium, culturing at 37 ℃ for 10h, selecting a transformant for sequence determination, and obtaining the recombinant plasmid with correct sequencing to transform Escherichia coli E.coli BL21(DE3) to obtain the recombinant Escherichia coli capable of expressing corresponding glutamine transaminase variants.

The obtained recombinant E.coli was plated on LB solid medium, and transformants were picked up and inoculated into LB liquid medium (containing 100. mu.g/mL ampicillin) by culturing at 37 ℃ for 10 hours, and transferred to TB liquid medium (containing 100. mu.g/mL ampicillin) at 1% transfer amount by culturing at 37 ℃ for 10 hours. Coli BL21(DE3) to OD by cultivation at 37 ℃₆₀₀And (3) adding IPTG to a final concentration of 0.01mM to induce the expression of the recombinant protein within a range of 1.0-1.5. After the IPTG was inoculated, the culture temperature was changed to 20 ℃ and the culture was continued for 36 hours. All liquid cultures were performed by shaking at 220 rpm.

After fermentation, the sample is centrifuged at 7500rpm for 10min and thalli are collected, Tris-HCl 50mM with one fifth of the total volume of the fermentation liquor is added, pH is 8.0, thalli are resuspended, and ice is applied for 10 min. The sample is placed on ice for ultrasonic wall breaking, centrifuged at 12000rpm for 15min, the supernatant is recovered, 200mg/ml neutral protease solution (neutral protease is purchased from Beijing Solebao scientific and technology Co., Ltd., product number Z8032) is added into the supernatant for activating glutamine transaminase, and the incubation condition is water bath at 37 ℃ for 30 min. Centrifuging the incubated sample at 12000rpm for 20min, taking the supernatant, and purifying the protein by a nickel ion affinity purification method, wherein the specific method comprises the following steps: taking a nickel ion affinity purification column, sequentially washing water and Tris-HCl 50mM, 20mM imidazole, pH 7.8 solution to balance the conductance, passing the sample, washing with Tris-HCl 50mM, 20mM imidazole, pH 7.8 solution to balance the conductance again, eluting protein by using Tris-HCl 50mM, 180mM imidazole, pH 7.8 solution, and recovering the protein. The whole purification process was carried out on an AKTA pure machine, and the protein collection time and amount were determined by observing the A280 wavelength absorption peak. Desalting the collected protein sample by using a gel chromatography method for a protein solution, wherein the specific method comprises the following steps: taking a gel column, sequentially and respectively passing water and Tris-HCl 50mM solution with pH 8.0 until the conductance is balanced, then enabling the gel column to pass through a protein sample, continuously passing through Tris-HCl 50mM solution with pH 8.0 until the wavelength of A280 appears an absorption peak, collecting protein, and stopping collecting in time when the conductance changes. Protein concentration the BCA assay was used, specifically described with reference to the Bradford protein concentration assay kit (detergent compatible type) instructions from Shanghai Bin Yuntian Biotechnology Co., Ltd.

From fig. 1, it can be seen that: variants compared to the maternal 60 ℃ (T)_1/2 ^60℃) The lower half-life period is obviously improved.

From fig. 2, it can be seen that: the female parent containing the Streptomyces mobaraensis glutamine transaminase zymogen region and the variants containing the solubilizing tag TrxA and the Streptomyces caniferus glutamine transaminase zymogen region respectively realize the expression in the Escherichia coli E.coli BL 21. Among them, the female parent can see a clear band at a protein molecular weight slightly lower than 49kDa, which is close to the theoretical size of 43.3 kDa. While the variant can see a clear band between 49 and 62KDa of protein molecular weight, which is similar to the theoretical size of 56.5 KDa.

From fig. 3, it can be seen that: the bands of the mother and variant proteins are single after purification, which indicates that the purity of the protein is consistent with the expectation.

From fig. 4, it can be seen that: compared to Streptomyces caniferus transglutaminase proenzyme (proC) and Streptomyces mobaraensis transglutaminase proenzyme (pro), the region linked to the mature region has an additional amino acid asparagine (D, Asp). It was confirmed that the nitrogen terminal of the mature region of glutamine transaminase, which is expressed by glutamine transaminase, is more amino acid asparagine (Asn) than the nitrogen terminal of glutamine transaminase, which is not substituted by the zymogen, after the zymogen region of glutamine transaminase is cleaved by neutral protease.

The specific enzyme activity, percentage of residual enzyme activity, half-life at 60 ℃ and the like of the parent (maturase) having a sequence as shown in SEQ ID NO:1 and the variant (maturase) having a sequence in which the substitution of FRAPD at positions 1 to 4 occurs as compared with SEQ ID NO:1 were tested, and the results are shown in Table 1, FIG. 1, Table 2.

TABLE 2 maternal and variant kinetic parameters

Example 2: application of glutamine transaminase mature enzyme variant in processing of minced fillet product

The glutamine transaminase mature enzyme variant (proC-TGase) prepared in example 1 was used for the processing of high calcium surimi products, specifically:

1. subcritical water combined with acetic acid for treating fishbone

(1) Preparing an acetic acid solution: diluting anhydrous acetic acid with distilled water to obtain 1.0M acetic acid solution; (2) mixing the fishbone with acetic acid solution: mixing fishbone and acetic acid solution according to the solid-to-liquid ratio of 1: 2; (3) subcritical water treatment: performing subcritical water treatment on the mixed solution of the fishbone and the acetic acid, wherein the subcritical water temperature is set to be 170 ℃, and the treatment time is 1.0 h.

2. Dynamic high-pressure micro-jet superfine fish bone

(1) Neutralizing with acid liquor: neutralizing the pH value of the fishbone solution to 7.0 by adopting 1.0M sodium carbonate solution; (2) dynamic high-pressure micro-jet treatment: treating the fishbone mixed solution by using a colloid mill for 3 times, then treating the fishbone mixed solution by using a 30MPa homogenizer for 2 times, and further performing ultramicro-refining treatment by using high-pressure microjet, wherein the microjet pressure is set to be 180MPa, and the treatment times are 2 times.

3. Mixing refined fishbone with defrozen minced fillet

Mixing the ultra-refined fishbone and the unfrozen minced fillet according to the ratio of 1:10, adjusting the pH value to 5.5, and kneading for 40 min.

4. Glutamine transaminase crosslinking

Crosslinking the mixture of fishbone and minced fillet by using glutamine transaminase, adding glutamine transaminase according to the concentration of 0.8 percent to prepare a spherical minced fillet product with the diameter of 4.0cm, setting the crosslinking temperature at 60 ℃ and the crosslinking time at 0.3 h.

5. Microwave sectional heating

(1) High-power microwave heating: carrying out microwave heating on the crosslinked minced fillet product, wherein the microwave power is set to be 1200W, and the microwave time is3 min; (2) low-power microwave heating: setting the microwave power at 500W, setting the microwave time at 12min, and processing the high-calcium minced fillet product after low-power microwave heating.

Example 3: application of glutamine transaminase mature enzyme variant in processing of dried rabbit meat slices

The variant transglutaminase prepared in example 1 (proC-TGase) was used for processing dried rabbit meat, specifically:

s1, mincing rabbit meat, and dicing chicken to obtain mixed meat;

s2, mixing the salt, the composite phosphate and the water uniformly, adding the mixed meat obtained in the S1, mixing uniformly, sealing with a preservative film, and pickling for 12 hours to obtain pickled meat;

s3, homogenizing the pickled meat to obtain mixed meat paste;

s4, adding glutamine transaminase, ovalbumin, ginger powder, thirteen spices and the like into the mixed meat paste obtained in the S3, and uniformly stirring at the temperature of 4 ℃ to obtain mixed meat paste;

s5, sealing the mixed minced meat obtained in the S4 by using a preservative film, placing for 0.3h under the water bath condition at the temperature of 60 ℃, carrying out extrusion forming, drying and naturally cooling to obtain a semi-finished product;

s6, baking the semi-finished product obtained in the S5 to obtain the composite dried rabbit meat.

Related sequence information:

the sequence of SEQ ID NO 1 is as follows: (the female parent, underlined part is the amino acid remaining after cleavage of the transglutaminase proenzyme, i.e., the substituted part)

FRAPDPDDRVTPPAEPLDRMPDPYRPVNGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYAKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESLFRNWSEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP

SEQ ID NO:2 is as follows: (the female parent, underlined part is the gene sequence corresponding to the amino acid remaining after cleavage of the transglutaminase proenzyme, i.e., the substituted part)

AGAGCCCCTGATGATCCTGATGATAGAGTGACCCCACCTGCAGAACCACTGGATAGAATGCCTGATCCATATAGACCTGTGAATGGCAGAGCAGAAACTGTGGTGAACAACTATATTAGAAAATGGCAGCAAGTGTATAGCCATAGAGATGGCAGAAAACAGCAGATGACTGAAGAACAGAGAGAATGGTTAAGCTATGGCTGTGTGGGTGTGACCTGGGTGAACAGTGGTCAGTATCCAACCAACAGACTGGCCTTTGCAAGCTTTGATGAAGATAGATTTAAAAATGAACTGAAAAATGGCAGACCAAGAAGTGGTGAAACTAGAGCAGAATTTGAAGGCAGAGTGGCCAAAGAATCTTTTGATGAAGAGAAGGGCTTTCAGAGAGCAAGAGAAGTGGCAAGTGTGATGAACAGAGCCCTGGAAAATGCCCATGATGAAAGTGCCTATCTGGATAACCTGAAAAAAGAACTGGCCAATGGCAATGATGCCTTAAGAAATGAAGATGCAAGAAGCCCATTTTATAGTGCCCTGAGAAACACCCCAAGCTTTAAAGAAAGAAATGGTGGCAACCATGATCCAAGCAGAATGAAAGCAGTGATTTATGCCAAACATTTTTGGAGTGGCCAAGATAGAAGCAGCAGTGCAGATAAAAGAAAATATGGTGATCCTGATGCCTTTAGACCTGCCCCTGGCACTGGCCTGGTGGATATGAGCAGAGATAGAAACATTCCAAGAAGCCCAACTAGCCCTGGTGAAGGCTTTGTGAACTTTGATTATGGCTGGTTTGGTGCACAGACTGAAGCAGATGCAGATAAAACTGTGTGGACTCATGGCAACCATTATCATGCCCCAAATGGCAGCCTGGGTGCCATGCATGTGTATGAAAGCCTGTTTAGAAACTGGAGTGAAGGCTATAGTGATTTTGATAGAGGTGCCTATGTGATTACCTTTATTCCAAAAAGCTGGAACACTGCCCCTGATAAAGTGAAACAAGGCTGGCCA

The sequence of SEQ ID NO 3 is as follows: (zymogen region amino acid sequence of Streptomyces caniferus transglutaminase)

MASGGDEEWEGSYAATHGLTAEDVKNINALNKRALTAGQPGNFPAELPPSATALFRAPD

The sequence of SEQ ID NO 4 is as follows: (zymogen region amino acid sequence of Streptomyces mobaraensis transglutaminase)

DNGAGEETKSYAETYRLTADDVANINALNESAPAASSAGPSFRAP

The sequence of SEQ ID NO 5 is as follows:

GACAATGGCGCGGGGGAAGAGACGAAGTCCTACGCCGAAACCTACCGCCTCACGGCGGATGACGTCGCGAACATCAACGCGCTCAACGAAAGCGCTCCGGCCGCTTCGAGCGCCGGCCCGTCGTTCCGGGCCCCCGACTCCGACGACAGGGTCACCCCTCCCGCCGAGCCGCTCGACAGGATGCCCGACCCGTACCGTCCCTCGTACGGCAGGGCCGAGACGGTCGTCAACAACTACATACGCAAGTGGCAGCAGGTCTACAGCCACCGCGACGGCAGGAAGCAGCAGATGACCGAGGAGCAGCGGGAGTGGCTGTCCTACGGCTGCGTCGGTGTCACCTGGGTCAATTCGGGTCAGTACCCGACGAACAGACTGGCCTTCGCGTCCTTCGACGAGGACAGGTTCAAGAACGAGCTGAAGAACGGCAGGCCCCGGTCCGGCGAGACGCGGGCGGAGTTCGAGGGCCGCGTCGCGAAGGAGAGCTTCGACGAGGAGAAGGGCTTCCAGCGGGCGCGTGAGGTGGCGTCCGTCATGAACAGGGCCCTGGAGAACGCCCACGACGAGAGCGCTTACCTCGACAACCTCAAGAAGGAACTGGCGAACGGCAACGACGCCCTGCGCAACGAGGACGCCCGTTCCCCGTTCTACTCGGCGCTGCGGAACACGCCGTCCTTCAAGGAGCGGAACGGAGGCAATCACGACCCGTCCAGGATGAAGGCCGTCATCTACTCGAAGCACTTCTGGAGCGGCCAGGACCGGTCGAGTTCGGCCGACAAGAGGAAGTACGGCGACCCGGACGCCTTCCGCCCCGCCCCGGGCACCGGCCTGGTCGACATGTCGAGGGACAGGAACATTCCGCGCAGCCCCACCAGCCCCGGTGAGGGATTCGTCAATTTCGACTACGGCTGGTTCGGCGCCCAGACGGAAGCGGACGCCGACAAGACCGTCTGGACCCACGGAAATCACTATCACGCGCCCAATGGCAGCCTGGGTGCCATGCATGTCTACGAGAGCAAGTTCCGCAACTGGTCCGAGGGTTACTCGGACTTCGACCGCGGAGCCTATGTGATCACCTTCATCCCCAAGAGCTGGAACACCGCCCCCGACAAGGTAAAGCAGGGCTGGCCG

the sequence of SEQ ID NO.6 is as follows: (the sequence of the proenzyme region of transglutaminase is underlined in single line and the sequence of the mature region is underlined in double line)

tggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggatcgagatctcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaaggagatatacatatgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggcgatcctcgtcgatttctgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggccatgGACAAT GGCGCGGGGGAAGAGACGAAGTCCTACGCCGAAACCTACCGCCTCACGGCGGATGACGTCGCGAACATCAACGCGC TCAACGAAAGCGCTCCGGCCGCTTCGAGCGCCGGCCCGTCGTTCCGGGCCCCC

CACCACCACCACCACCACTGAgatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggat

The sequence of SEQ ID NO.7 is as follows: (TrxA sequence is underlined in single underline, sequence of proenzyme region of transglutaminase is underlined in double underline, sequence of mature region of transglutaminase is underlined in wavy line)

tggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggatcgagatctcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaaggagatatacatatgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggcgatcctcgtcgatttctgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggcc

CACCACCACCACCACCACTGAgatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggat

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> a transglutaminase with improved thermostability

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 335

<212> PRT

<213> Artificial sequence

<400> 1

Phe Arg Ala Pro Asp Pro Asp Asp Arg Val Thr Pro Pro Ala Glu Pro

1 5 10 15

Leu Asp Arg Met Pro Asp Pro Tyr Arg Pro Val Asn Gly Arg Ala Glu

20 25 30

Thr Val Val Asn Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His

35 40 45

Arg Asp Gly Arg Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ala Leu Arg Asn Thr Pro Ser Phe Lys Glu Arg Asn Gly Gly Asn His

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Lys Thr Val Trp Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser

275 280 285

Leu Gly Ala Met His Val Tyr Glu Ser Leu Phe Arg Asn Trp Ser Glu

290 295 300

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

305 310 315 320

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

325 330 335

<210> 2

<211> 1005

<212> DNA

<213> Artificial sequence

<400> 2

agagcccctg atgatcctga tgatagagtg accccacctg cagaaccact ggatagaatg 60

cctgatccat atagacctgt gaatggcaga gcagaaactg tggtgaacaa ctatattaga 120

aaatggcagc aagtgtatag ccatagagat ggcagaaaac agcagatgac tgaagaacag 180

agagaatggt taagctatgg ctgtgtgggt gtgacctggg tgaacagtgg tcagtatcca 240

accaacagac tggcctttgc aagctttgat gaagatagat ttaaaaatga actgaaaaat 300

ggcagaccaa gaagtggtga aactagagca gaatttgaag gcagagtggc caaagaatct 360

tttgatgaag agaagggctt tcagagagca agagaagtgg caagtgtgat gaacagagcc 420

ctggaaaatg cccatgatga aagtgcctat ctggataacc tgaaaaaaga actggccaat 480

ggcaatgatg ccttaagaaa tgaagatgca agaagcccat tttatagtgc cctgagaaac 540

accccaagct ttaaagaaag aaatggtggc aaccatgatc caagcagaat gaaagcagtg 600

atttatgcca aacatttttg gagtggccaa gatagaagca gcagtgcaga taaaagaaaa 660

tatggtgatc ctgatgcctt tagacctgcc cctggcactg gcctggtgga tatgagcaga 720

gatagaaaca ttccaagaag cccaactagc cctggtgaag gctttgtgaa ctttgattat 780

ggctggtttg gtgcacagac tgaagcagat gcagataaaa ctgtgtggac tcatggcaac 840

cattatcatg ccccaaatgg cagcctgggt gccatgcatg tgtatgaaag cctgtttaga 900

aactggagtg aaggctatag tgattttgat agaggtgcct atgtgattac ctttattcca 960

aaaagctgga acactgcccc tgataaagtg aaacaaggct ggcca 1005

<210> 3

<211> 59

<212> PRT

<213> Artificial sequence

<400> 3

Met Ala Ser Gly Gly Asp Glu Glu Trp Glu Gly Ser Tyr Ala Ala Thr

1 5 10 15

His Gly Leu Thr Ala Glu Asp Val Lys Asn Ile Asn Ala Leu Asn Lys

20 25 30

Arg Ala Leu Thr Ala Gly Gln Pro Gly Asn Phe Pro Ala Glu Leu Pro

35 40 45

Pro Ser Ala Thr Ala Leu Phe Arg Ala Pro Asp

50 55

<210> 4

<211> 45

<212> PRT

<213> Artificial sequence

<400> 4

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

1 5 10 15

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

20 25 30

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

35 40 45

<210> 5

<211> 1128

<212> DNA

<213> Artificial sequence

<400> 5

gacaatggcg cgggggaaga gacgaagtcc tacgccgaaa cctaccgcct cacggcggat 60

gacgtcgcga acatcaacgc gctcaacgaa agcgctccgg ccgcttcgag cgccggcccg 120

tcgttccggg cccccgactc cgacgacagg gtcacccctc ccgccgagcc gctcgacagg 180

atgcccgacc cgtaccgtcc ctcgtacggc agggccgaga cggtcgtcaa caactacata 240

cgcaagtggc agcaggtcta cagccaccgc gacggcagga agcagcagat gaccgaggag 300

cagcgggagt ggctgtccta cggctgcgtc ggtgtcacct gggtcaattc gggtcagtac 360

ccgacgaaca gactggcctt cgcgtccttc gacgaggaca ggttcaagaa cgagctgaag 420

aacggcaggc cccggtccgg cgagacgcgg gcggagttcg agggccgcgt cgcgaaggag 480

agcttcgacg aggagaaggg cttccagcgg gcgcgtgagg tggcgtccgt catgaacagg 540

gccctggaga acgcccacga cgagagcgct tacctcgaca acctcaagaa ggaactggcg 600

aacggcaacg acgccctgcg caacgaggac gcccgttccc cgttctactc ggcgctgcgg 660

aacacgccgt ccttcaagga gcggaacgga ggcaatcacg acccgtccag gatgaaggcc 720

gtcatctact cgaagcactt ctggagcggc caggaccggt cgagttcggc cgacaagagg 780

aagtacggcg acccggacgc cttccgcccc gccccgggca ccggcctggt cgacatgtcg 840

agggacagga acattccgcg cagccccacc agccccggtg agggattcgt caatttcgac 900

tacggctggt tcggcgccca gacggaagcg gacgccgaca agaccgtctg gacccacgga 960

aatcactatc acgcgcccaa tggcagcctg ggtgccatgc atgtctacga gagcaagttc 1020

cgcaactggt ccgagggtta ctcggacttc gaccgcggag cctatgtgat caccttcatc 1080

cccaagagct ggaacaccgc ccccgacaag gtaaagcagg gctggccg 1128

<210> 6

<211> 6819

<212> DNA

<213> Artificial sequence

<400> 6

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600

gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660

ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720

agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780

agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840

tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900

tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960

cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020

aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080

tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140

tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200

ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260

ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320

cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380

gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440

actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500

aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560

caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620

aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680

accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740

aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800

ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860

agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920

accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980

gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040

tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100

cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160

cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220

cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400

gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460

tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520

cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580

gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640

gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700

catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760

tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820

ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880

tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940

ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000

aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060

gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120

tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180

acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240

cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300

cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360

gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420

cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480

gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540

tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600

atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660

tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720

gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780

tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840

cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900

tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta 3960

atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020

atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080

tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140

cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200

aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260

ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320

tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380

tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440

gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500

gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560

gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620

ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680

taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740

ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800

atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860

tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920

gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980

gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040

aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100

ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc 5160

cctctagaaa taattttgtt taactttaag aaggagatat acatatgagc gataaaatta 5220

ttcacctgac tgacgacagt tttgacacgg atgtactcaa agcggacggg gcgatcctcg 5280

tcgatttctg ggcagagtgg tgcggtccgt gcaaaatgat cgccccgatt ctggatgaaa 5340

tcgctgacga atatcagggc aaactgaccg ttgcaaaact gaacatcgat caaaaccctg 5400

gcactgcgcc gaaatatggc atccgtggta tcccgactct gctgctgttc aaaaacggtg 5460

aagtggcggc aaccaaagtg ggtgcactgt ctaaaggtca gttgaaagag ttcctcgacg 5520

ctaacctggc catggacaat ggcgcggggg aagagacgaa gtcctacgcc gaaacctacc 5580

gcctcacggc ggatgacgtc gcgaacatca acgcgctcaa cgaaagcgct ccggccgctt 5640

cgagcgccgg cccgtcgttc cgggcccccg atcctgatga tagagtgacc ccacctgcag 5700

aaccactgga tagaatgcct gatccatata gacctgtgaa tggcagagca gaaactgtgg 5760

tgaacaacta tattagaaaa tggcagcaag tgtatagcca tagagatggc agaaaacagc 5820

agatgactga agaacagaga gaatggttaa gctatggctg tgtgggtgtg acctgggtga 5880

acagtggtca gtatccaacc aacagactgg cctttgcaag ctttgatgaa gatagattta 5940

aaaatgaact gaaaaatggc agaccaagaa gtggtgaaac tagagcagaa tttgaaggca 6000

gagtggccaa agaatctttt gatgaagaga agggctttca gagagcaaga gaagtggcaa 6060

gtgtgatgaa cagagccctg gaaaatgccc atgatgaaag tgcctatctg gataacctga 6120

aaaaagaact ggccaatggc aatgatgcct taagaaatga agatgcaaga agcccatttt 6180

atagtgccct gagaaacacc ccaagcttta aagaaagaaa tggtggcaac catgatccaa 6240

gcagaatgaa agcagtgatt tatgccaaac atttttggag tggccaagat agaagcagca 6300

gtgcagataa aagaaaatat ggtgatcctg atgcctttag acctgcccct ggcactggcc 6360

tggtggatat gagcagagat agaaacattc caagaagccc aactagccct ggtgaaggct 6420

ttgtgaactt tgattatggc tggtttggtg cacagactga agcagatgca gataaaactg 6480

tgtggactca tggcaaccat tatcatgccc caaatggcag cctgggtgcc atgcatgtgt 6540

atgaaagcct gtttagaaac tggagtgaag gctatagtga ttttgataga ggtgcctatg 6600

tgattacctt tattccaaaa agctggaaca ctgcccctga taaagtgaaa caaggctggc 6660

cacaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt 6720

tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct 6780

tgaggggttt tttgctgaaa ggaggaacta tatccggat 6819

<210> 7

<211> 6858

<212> DNA

<213> Artificial sequence

<400> 7

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600

gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660

ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720

agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780

agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840

tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900

tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960

cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020

aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080

tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140

tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200

ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260

ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320

cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380

gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440

actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500

aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560

caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620

aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680

accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740

aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800

ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860

agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920

accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980

gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040

tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100

cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160

cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220

cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400

gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460

tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520

cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580

gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640

gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700

catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760

tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820

ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880

tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940

ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000

aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060

gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120

tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180

acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240

cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300

cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360

gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420

cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480

gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540

tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600

atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660

tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720

gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780

tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840

cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900

tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta 3960

atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020

atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080

tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140

cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200

aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260

ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320

tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380

tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440

gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500

gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560

gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620

ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680

taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740

ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800

atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860

tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920

gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980

gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040

aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100

ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc 5160

cctctagaaa taattttgtt taactttaag aaggagatat acatatgagc gataaaatta 5220

ttcacctgac tgacgacagt tttgacacgg atgtactcaa agcggacggg gcgatcctcg 5280

tcgatttctg ggcagagtgg tgcggtccgt gcaaaatgat cgccccgatt ctggatgaaa 5340

tcgctgacga atatcagggc aaactgaccg ttgcaaaact gaacatcgat caaaaccctg 5400

gcactgcgcc gaaatatggc atccgtggta tcccgactct gctgctgttc aaaaacggtg 5460

aagtggcggc aaccaaagtg ggtgcactgt ctaaaggtca gttgaaagag ttcctcgacg 5520

ctaacctggc catggcctct ggtggtgatg aagaatggga aggcagctat gcagccactc 5580

atggcctgac tgcagaagat gtgaaaaaca ttaatgccct gaataaaaga gcccttactg 5640

ctggtcagcc tggtaatttt cctgcagagc tgccccctag tgccactgcc ctgtttagag 5700

cccctgatga tcctgatgat agagtgaccc cacctgcaga accactggat agaatgcctg 5760

atccatatag acctgtgaat ggcagagcag aaactgtggt gaacaactat attagaaaat 5820

ggcagcaagt gtatagccat agagatggca gaaaacagca gatgactgaa gaacagagag 5880

aatggttaag ctatggctgt gtgggtgtga cctgggtgaa cagtggtcag tatccaacca 5940

acagactggc ctttgcaagc tttgatgaag atagatttaa aaatgaactg aaaaatggca 6000

gaccaagaag tggtgaaact agagcagaat ttgaaggcag agtggccaaa gaatcttttg 6060

atgaagagaa gggctttcag agagcaagag aagtggcaag tgtgatgaac agagccctgg 6120

aaaatgccca tgatgaaagt gcctatctgg ataacctgaa aaaagaactg gccaatggca 6180

atgatgcctt aagaaatgaa gatgcaagaa gcccatttta tagtgccctg agaaacaccc 6240

caagctttaa agaaagaaat ggtggcaacc atgatccaag cagaatgaaa gcagtgattt 6300

atgccaaaca tttttggagt ggccaagata gaagcagcag tgcagataaa agaaaatatg 6360

gtgatcctga tgcctttaga cctgcccctg gcactggcct ggtggatatg agcagagata 6420

gaaacattcc aagaagccca actagccctg gtgaaggctt tgtgaacttt gattatggct 6480

ggtttggtgc acagactgaa gcagatgcag ataaaactgt gtggactcat ggcaaccatt 6540

atcatgcccc aaatggcagc ctgggtgcca tgcatgtgta tgaaagcctg tttagaaact 6600

ggagtgaagg ctatagtgat tttgatagag gtgcctatgt gattaccttt attccaaaaa 6660

gctggaacac tgcccctgat aaagtgaaac aaggctggcc acaccaccac caccaccact 6720

gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc 6780

aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag 6840

gaggaactat atccggat 6858

Claims (10)

1. A mature enzyme of glutamine transaminase with improved thermostability, comprising a substitution corresponding to positions 1 to 4 of the polypeptide shown in SEQ ID NO.1, wherein,

i) a polypeptide having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide set forth in SEQ ID NO. 1; and/or

ii) the mature enzyme of said transglutaminase is a polypeptide encoded by a polynucleotide having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence as set forth in SEQ ID NO. 2;

said substitution in positions 1 to 4 is a substitution to the amino acid sequence FRAPD.

2. A polynucleotide encoding a mature enzyme of glutamine transaminase of claim 1.

3. Nucleic acid constructs, vectors, and host cells comprising the polynucleotide of claim 2.

4. A method of producing a mature enzyme of glutamine transaminase of claim 1.

5. A composition comprising a mature enzyme of transglutaminase defined in claim 1.

6. A method for modifying the appearance, mouthfeel and/or stability of a food product in fresh meat processing, sausage products, fish pellets, meat emulsion processing, soy products and/or dairy products, comprising adding the mature enzyme of transglutaminase according to claim 1 or the composition according to claim 5 to the food product during processing.

7. Use of the mature enzyme of transglutaminase defined in claim 1 for food processing, processing and transformation.

8. Use of the composition of claim 5 for food processing, processing and transformation.

9. A method for improving the stability of a mature glutamine transaminase, which is characterized in that the amino acids at the 1 st to 4 th positions in the sequence of SEQ ID NO.1 are replaced by a polypeptide sequence FRAPD.

10. The method according to claim 9, wherein the actual use is carried out by changing the pro-region by replacing the pro-enzyme (SEQ ID NO:4) of the original Streptomyces mobaraensis-derived transglutaminase with the pro-enzyme (SEQ ID NO:3) of Streptomyces caniferus-derived transglutaminase, cleaving the pro-enzyme with neutral protease to form an amino acid at the nitrogen terminus of the mature transglutaminase one more than the amino acid at the nitrogen terminus of the transglutaminase not substituted with the pro-enzyme, wherein the amino acid at the nitrogen terminus is asparagine (Asn), i.e., the amino acid at the first 4 th amino acid position of the nitrogen terminus of the mature region, and converting FRAP into 5 amino acids FRAPD.

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