JP2023512130A - Transglutaminase mutants with improved thermostability - Google Patents

Abstract

The present invention discloses transglutaminase variants with improved thermostability belonging to the biological and food fields. The transglutaminase mutants of the present invention are enzymes with improved thermostability and are substitutions of the zymogen region corresponding to positions 1-45 of the sequence transglutaminase shown in SEQ ID NO: 1 and 189 and 189 of the mature region sequences. Including substitution at position 287. The transglutaminase mutants of the present invention have applications in aspects such as food processing, processing and conversion, and can be used to retain or improve food quality, consistency, elasticity, moisture or viscosity. Transglutaminase variants with improved thermostability are useful for stable applications in more harsh industrial environments, such as minced meat processing and meatball processing of meat substitutes. [Selection drawing] Fig. 1

Description

The present invention relates to transglutaminase mutants with improved thermostability, and belongs to the fields of biology and food.

Transglutaminase (TGase, EC 2.3.2.13) from Streptomyces mobaraensis AAT65817 is the mainstream enzyme preparation widely sold in China and international markets. It is a commonly used transaminase that facilitates the formation of covalent bonds between the γ-carboxamide groups of glutamine residues in proteins and small molecules by catalyzing the reaction with amino groups in acyl acceptors. . TGase has a very wide application in the food processing field, adding TGase in the processing of meats, legumes and dairy products may change the appearance, mouthfeel and stability of the food.

Along with research and applications on TGase, we find that its low thermal stability is one major problem that limits its practical application. TGase is used to process food processing processes, such as minced meat, meatballs, dairy and bean products, etc. High temperature treatment is required to achieve TGase-catalyzed cross-linking, and TGase has low thermal stability. , the temperature needs to be controlled at 40 ° C during normal operation, and the amount of addition required is large due to fast wear (see patent publication numbers: CN109907111A, CN201810480335.6, CN110771811A, CN106901205B, CN201811599451.6 ).

Because thermostability is so important, currently the methods of remodeling enzyme thermostability are mainly rational design and directed evolution. Rational design is to determine the unstable regions in the enzyme structure and introduce amino acid mutations to improve the stability of the enzyme by means of machine simulation. Directed evolution includes directed evolution based on host cells and directed evolution based on enzyme genes. However, the latter achieves directed evolution mainly by performing random mutation and genetic recombination of homologues on the enzyme gene. Currently, related reports that improve thermostability for TGase are (1) Obtaining a highly stable TGase mutation point by random mutation (doi: 10.1016/j.jbiotec.2008.06.005) , (2), and (1) were subjected to site saturation mutagenesis and DNA recombination to obtain highly stable TGase combination mutants (DOI 10.1007 /s 00726-011-1015-y), (3), (1) and (2) make further mutations based on the screened mutation points (DOI: 10.1080/09168451.2017.1403881) . However, the currently reported highly stable TGase mutants incubated at 60°C under water bath conditions are less stable than those incubated at 60°C under metal bath conditions, leaving much room for improvement. . In the actual application process, such as minced meat, corn flour, bean product processing, etc., after mixing TGase, the processing temperature is generally 40-70 ℃, but TGase is stable under the condition of 60 ℃. is extremely low, which brings about the problem of low utilization even though the addition amount is high. Therefore, obtaining a highly stable TGase by design modification is particularly important for industrial applications.

To solve the above problems, the present invention provides transglutaminase mutants with improved thermostability.

Transglutaminase variants of the present invention contain substitutions corresponding to positions 1-45 of the polypeptide set forth in SEQ ID NO: 1, substituted with SEQ ID NO: 3, and 234 of the polypeptide set forth in SEQ ID NO: 1. containing substitutions corresponding to positions 189 and 287 of the transglutaminase mature region of SEQ ID NO: 1, respectively, at positions 189 and 287;
i) the mature region of said transglutaminase mutant is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92% of the polypeptide at positions 46-376 shown in SEQ ID NO: 1 , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and/or
ii) the mature region of said transglutaminase variant is at least 60%, at least 70%, at least 80%, at least 90% of the mature polypeptide coding sequence from nucleotide positions 139-1128 shown in SEQ ID NO:2; a polypeptide encoded by a polynucleotide having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;
After the transglutaminase mutant is treated with a neutral protease, an active enzyme is obtained.

In one embodiment, the substitution at position 234 corresponds to position 189 of the mature region of the transglutaminase of SEQ ID NO: 1 and is substituted with a histidine (H) and the substitution at position 332 corresponds to the transglutaminase of SEQ ID NO: 1. corresponding to position 287 of the mature region of , and is substituted by proline (P).

In one embodiment, said transglutaminase mutant is designated D189H/A287P.

The invention further relates to polynucleotides encoding said variants, nucleic acid constructs, vectors and host cells comprising said polynucleotides, and methods of producing said variants or mature enzymes thereof. The present invention also relates to compositions comprising the transglutaminase mutants of the invention or mature enzymes thereof.

The invention further relates to methods of producing the transglutaminase mutants of the invention or the mature enzyme thereof, which methods comprise:
a) culturing a host cell of the invention under conditions compatible with expressing said variant;
b) optionally recovering said mutant.

The present invention further relates to a method of altering food appearance, mouthfeel and/or stability in raw meat processing, sausage-type products, fish balls, minced meat processing, legume products and/or dairy products, said method comprising: and adding a transglutaminase variant according to the invention or said composition according to the invention to the treatment.

The invention further relates to the use of said transglutaminase variants or mature enzymes thereof or compositions according to the invention in food treatment, processing and conversion.

In one embodiment, it is used to retain or improve food quality, consistency, elasticity, moisture or viscosity.

In one embodiment, the food is selected from cheese, yogurt, ice cream, mayonnaise and meat.

In one embodiment, wherein said food is fish.

In one embodiment, different densities of gelatin are formed and used to produce low-fat precooked food products.

Definitions or Terms:
Transglutaminase: The terms "Transglutaminase (TGase)", "R-glutaminyl-peptide amidase-γ-glutamine-transferase", "glutamyltransferase" are defined by the Enzyme Nomenclature EC 2.3.2.13 is an enzyme in the genus To achieve the object of the present invention, transglutaminase activity is determined based on the procedures described in the Examples. In one aspect, a variant of the invention is SEQ ID NO. at least 20%, such as at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% of the transglutaminase activity of one polypeptide , at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

Coding sequence: The term "coding sequence" means a polynucleotide, which directly defines the amino acid sequence of a transglutaminase variant. The boundaries of a 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). . A coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Regulatory sequence: The term “regulatory sequence” refers to nucleic acid sequences that are required for the expression of a polynucleotide encoding a transglutaminase variant of the invention. Each regulatory sequence may be native (i.e., derived from the same gene) or exogenous (i.e., derived from a different gene) with respect to the polynucleotide encoding said transglutaminase variant, or They may be natural or exogenous to each other. Such regulatory sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. Regulatory sequences include at least promoters, and transcription and translation termination signals. The regulatory sequences may be provided with linkers to introduce specific restriction sites facilitating ligation of the regulatory sequences and the coding regions of the polynucleotides encoding the transglutaminase variants of the invention.

Expression: The term "expression" includes any step relating to the production of a transglutaminase variant, 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, said molecule comprising a polynucleotide encoding a transglutaminase variant of the invention and operable with control sequences used for its expression. connected to

Fragment: The term “fragment” refers to a polypeptide that lacks one or more (eg, several) amino acids at the amino and/or carboxyl terminus of the polypeptide, wherein said fragment lacks transglutaminase activity. have In one aspect, the fragment is at least 50%, at least 55%, at least 60%, at least 65%, at least 70% of the number of amino acids 46-376 of SEQ ID NO: 1 (ie not including the length of the proenzyme region sequence). , at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%.

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

Improved Thermostability: The term "improved thermostability" refers to a characteristic of a transglutaminase variant that is improved relative to the parent transglutaminase.

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

Mature Polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. means a polypeptide. In one aspect, in this patent, said mature polypeptide is the mature enzyme of transglutaminase, amino acids 46-376 of SEQ ID NO:1. As is well known to those of skill in the art, host cells produce a mixture of two or more polypeptides of different maturity (i.e., having different C-terminal and/or N-terminal amino acids) expressed on the same polynucleotide. can do.

Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide that has transglutaminase activity. In one aspect, the coding sequence for the mature polypeptide, ie the mature enzyme, is nucleotides 139-1128 of SEQ ID NO:2 (ie, does not include the codon sequence corresponding to the proenzyme region).

Mutant: The term "mutant" means a polynucleotide encoding a variant.

Nucleic acid construct: The term "nucleic acid construct" refers to a single- or double-stranded nucleic acid molecule, which is isolated from a naturally occurring gene, or which is a nucleic acid that is not naturally occurring in nature. Segment modified to contain or synthetic, said nucleic acid molecule comprises one or more regulatory sequences.

Parent or parental transglutaminase: The term "parental" or "parental transglutaminase" means the transglutaminase that is modified to give rise to the transglutaminase mutant of the invention. Said parent of this patent is ie the amino acid sequence corresponding to SEQ ID NO:1. Said parent transglutaminase may be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof, or may 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: Thermostability of the transglutaminase mutants of the invention can be demonstrated as residual activity or residual performance of said transglutaminase after exposure to different test conditions for a period of time or thereafter. It is indicated for the known activity or performance of the parent transglutaminase (eg, parent transglutaminase as set forth in SEQ ID NO:1).

Mutant: The term "mutant" refers to a polypeptide that has transglutaminase activity and is altered (i.e., substituted, inserted, and/or deleted) at one or more (e.g., several) sites. means. Substitution means replacing an amino acid occupying a position with a different amino acid, deletion means removing an amino acid occupying a position, insertion means adjacent And it means adding an amino acid behind the amino acid occupying a certain site. The variant of the present invention has the polypeptide amino acid sequence of SEQ ID NO: 1, and substitutions occur at positions 1-45, the substituted amino acid sequence is the sequence shown in SEQ ID NO: 3, and Substitutions occur simultaneously at positions 234 and 332 (ie, positions 189 and 287 of the transglutaminase mature enzyme shown in SEQ ID NO: 1) and the substituted amino acids are histidine and proline, respectively. A variant of the invention has at least 20%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of transglutaminase activity.

Wild-type transglutaminase: The term "wild-type" transglutaminase means a transglutaminase that is expressed in naturally occurring microorganisms (eg, bacteria, yeast, filamentous fungi) in nature.

In describing the variants of the present invention, the nomenclature set forth below has been adapted for reference. Accepted IUPAC single-letter or three-letter amino acid abbreviations are employed.

Substitutions: For amino acid substitutions, the nomenclature of original amino acid, site, substituted amino acid is used. Therefore, a substitution of alanine for threonine at position 226 is designated as "Thr226Ala" or "T226A." Multiple mutations are separated by a symbol ("-"), for example, "Gly205Arg-Ser411Phe" or "G205R-S411F" replace glycine (G) and serine (S) at positions 205 and 411 with arginines, respectively. (R) and phenylalanine (F) substituted.

The transglutaminase mutants of the present invention have proenzyme substitutions relative to the parent, ie, substitutions occur at positions 1-45 of SEQ ID NO: 1 as shown, and the proenzyme region sequence of Streptomyces caniferus transglutaminase (e.g. (as shown in SEQ ID NO: 3) and obtained after mutations have occurred at positions 234 and 332 (ie positions 189 and 287 of the mature region portion) of the transglutaminase shown in SEQ ID NO: 1. The 60° C. half-life of the mutant mature enzyme was 35.57 minutes, a 214.5% improvement over the parent mature enzyme's 11.31 minutes.

The transglutaminase mutants of the present invention have significantly improved thermal stability and can be applied in areas such as food processing, processing and conversion to improve food quality, consistency, elasticity, moisture or viscosity. Used to maintain or improve. Transglutaminase variants with improved thermostability are useful for stable applications in more harsh industrial environments, such as minced meat processing and meatball processing of meat substitutes.

Initial enzymatic activity and 60° C. half-life of parental and mutant mature enzymes. The parent and the mutant are E. coli strains. Whole cells were analyzed by SDS-PAGE electrophoresis after being expressed in E. coli BL21, M is protein abundance marker, units are kDa, 1 is E. coli E. coli. Whole-cell electrophoresis analysis after parental expression in E. coli BL21; Whole cell electrophoresis analysis after expression of mutants in E. coli BL21. Parent and mutant mature enzymes are SDS-PAGE analysis of purified samples, M is protein abundance marker, unit is Kda, 1 is parent mature enzyme, 2 is mutant It is a mature enzyme.

The present invention relates to improved transglutaminase variants compared to the parental transglutaminase. More specifically, the present invention relates to transglutaminase variants with improved thermostability compared to the parental transglutaminase (especially the transglutaminase as shown in SEQ ID NO:1).

According to the present invention, enzymatic activity, thermostability and kinetic parameters of transglutaminase are measured by the following methods.

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

Enzyme activity test stop solution B: 3 mol/L HCl, 5% FeCl _{3.6H 2} O (dissolved in 0.1 mol/L HCl) and 12% TCA (trichloroacetic acid) are mixed in equal volumes to form a solution. to complete.

Definition of enzymatic activity: One unit of enzymatic activity was defined as the amount of enzyme that catalyzes 1 μmol of substrate to form product per minute.

Preparation of a calibration curve for enzyme activity test: Add 100 ml of Tris-HCl 200 mM to 648 mg of standard L-glutamic acid-γ-monohydroxamic acid, adjust the solution to pH 6.0, and add Tris-HCl 200 mM, pH 6.0 solution. The solution and substrate solution A were each incubated at 37°C for 5 minutes, after which 60 µL of the standard solution was added to 150 µL of substrate solution A and diluted to 37°C. After bathing in water for 10 minutes, 60 μL of stop solution B is added, centrifuged at 10000 rpm for 1 minute, 200 μL of the supernatant is taken, and the absorbance value at 525 nm is measured. A straight line is drawn from the absorbance value versus the amount of hydroxamic acid, the slope of the straight line gives the conversion factor K, and K can be used to calculate the amount of hydroxamic acid produced after obtaining the absorbance in measuring the enzymatic activity of the sample.

Measurement method: Protein sample and 150 μL of substrate solution A are incubated at 37° C. for 5 minutes, then 60 μL of protein sample is added with 150 μL of substrate solution A, and after 10 minutes of water bathing at 37° C., 60 μL of test is stopped. Add solution B. The reaction solution is centrifuged at 10,000 rpm for 1 minute, 200 μL of the supernatant is taken, and the absorbance value at a wavelength of 525 nm is measured. For blank control, add 60 μL of protein sample to 60 μL of test stop solution B, add 150 μL of substrate solution A, centrifuge at 10000 rpm for 1 minute, remove 200 μL of supernatant, and measure absorbance at 525 nm. do. By subtracting the absorbance value obtained in the control group from the absorbance value obtained in the experimental group and substituting it into the enzyme activity calibration curve, the enzyme activity corresponding to the mass of protein added is obtained, and the enzyme activity is Divide the protein concentration to get the protein enzyme specific activity U/mg.

Thermal stability test The half-life was measured under water bath conditions of 60°C. The sample is continuously heated and incubated in a 60 ° C. water bath, sampled every minute within 0-10 minutes, and sampled every 2 minutes from 10-40 minutes, and any sample taken out is immediately An ice bath is applied at 20°C. By measuring the enzyme activity for each sample taken, the residual enzyme activity ratio over time was obtained, and non-linear fitting was performed on it by Exponential-ExpDec1 in Original 2018, and the enzyme calculated after obtaining the fitting formula The half-life is the time corresponding to the activity decreasing to 50% of the initial level.

Differential scanning calorimetry Take pure protein solutions of parent and mutants (solvent is Tris-HCl, pH 8.0), concentration is greater than 1.5 mg/ml, and perform dissolution temperature test by differential scanning calorimetry. . The solvent in which the protein resides is taken as an internal reference, the scanning temperature is 40-90° C., the heating rate is 1° C./min, the pressure is 3 atm, and finally the dissolution temperature of the protein is obtained.

Kinetic Parameter Determination For Michaelis constant K _M test method, parent and mutant protein solutions are diluted to 0.05 mg/ml, test method refers to enzymatic activity assay. Substrate solution A should be arranged in 10 types according to the content of N-benzyloxycarbonyl-L-glutamylglycine, other components are unchanged, the content of N-benzyloxycarbonyl-L-glutamylglycine are 3 mM, 6 mM, 9 mM, 12 mM, 15 mM, 18 mM, 21 mM, 24 mM, 27 mM and 30 mM, respectively. In the enzymatic activity test method, the amount of substrate conversion in the substrate solution A for different N-benzyloxycarbonyl-L-glutamylglycine contents of the parent and mutants was measured, ie the substrate N- Benzyloxycarbonyl-L-glutamylglycine is catalyzed and converted to product quantities. Based on the conversion amount numerical values obtained above, non-linear fitting is performed on the numerical values obtained by the software Origin 2018, fitting is performed using Growth / Sigmadial-Hill, K _M and Vmax values are obtained, and enzyme concentration conversion is performed. to get the k _cat value. On this basis, K _M /k _cat gives the enzyme catalytic efficiency.

The transglutaminase of the present invention contains substitutions corresponding to positions 1-45 of the polypeptide shown in SEQ ID NO: 1 (the substitutions are in SEQ ID NO: 3), and positions 234, 332 of the polypeptide shown in SEQ ID NO: 1. containing substitutions corresponding to positions (corresponding to positions 189 and 287, respectively, of the transglutaminase mature region of SEQ ID NO: 1),
i) the transglutaminase mature region is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93% of the polypeptide at positions 46-376 shown in SEQ ID NO: 1; %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and/or ii) the mature region of said transglutaminase is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least A polypeptide encoded by a polynucleotide having 98%, or at least 99%, sequence identity.

Substitutions occur at positions 1 to 45, and the amino acid sequence after substitution is SEQ ID NO:3.

In one embodiment, the transglutaminase has a histidine (H) substituted at position 234 (ie position 189 of the transglutaminase mature region of SEQ ID NO:1).

In one example, the transglutaminase was substituted with a proline (P) at position 332 (ie position 287 of the transglutaminase mature region of SEQ ID NO:1).

In one embodiment, the transglutaminase variant is compared to the polypeptide set forth in SEQ ID NO: 1, positions 1-45 are substituted with SEQ ID NO: 3, and compared to the transglutaminase mature region set forth in SEQ ID NO: 1. only substitutions at positions 189 and 287 occurred, and the half-life at 60°C improved to 214.5%.

Table 1 lists the relative thermostability of transglutaminase mutants in which only substitutions at positions 189 and 287 of the mature region occurred compared to the parent mature enzyme and the mutant mature enzyme shown in SEQ ID NO:1. rice field.

Production of Mutants A transglutaminase of the invention can be produced using any mutagenesis program known in the art (e.g., site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.). Mutants can be produced.

Site-directed mutagenesis is a technique that introduces one or more (eg, several) mutations at one or more defined sites in the parental transglutaminase-encoding polynucleotide.

Site-directed mutagenesis can be accomplished in vitro via PCR using oligonucleotide primers containing the desired mutation. In vitro site-directed mutagenesis can also be performed by cassette mutagenesis, wherein the restriction enzyme is cleaved at a site in a plasmid containing the parent transglutaminase-encoding polynucleotide followed by mutation. to a polynucleotide. Generally, the digested plasmid and oligonucleotide restriction enzymes were the same, which allowed the cohesive ends of the plasmid and insert to be ligated together.

It is also possible to achieve site-directed mutagenesis in vivo by methods known in the art.

Any site-directed mutagenesis program can be used in the present invention. There are several commercially available kits that are used to make variants.

Synthetic gene construction requires in vitro synthesis of a polynucleotide molecule designed to encode a polypeptide of interest. Gene synthesis can be performed using a variety of techniques.

Single or multiple amino acid substitutions, deletions and/or insertions can be made and confirmed using known mutagenesis, recombination and/or shuffling methods followed by relevant screening programs. be able to.

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned and mutagenized polypeptides expressed in host cells. Mutagenized DNA molecules encoding active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow rapid determination of the importance of each amino acid residue in a polypeptide.

Semi-synthetic gene construction is achieved by combining synthetic gene construction and/or site-directed mutagenesis and/or random mutagenesis and/or shuffling. Semi-synthetic construction generally combines PCR techniques with processes that utilize synthetic polynucleotide fragments. Thus, limited regions of the gene can be synthesized de novo, while other regions can be amplified using site-directed mutagenized primers, while error-prone or non-error-prone PCR can be used in other regions. Amplification can be performed. The polynucleotide subsequences can then be shuffled.

Polynucleotides The invention further relates to isolated polynucleotides encoding the transglutaminase variants of the invention. In some aspects, the invention relates to nucleic acid constructs comprising a polynucleotide of the invention. In some aspects, the invention relates to expression vectors comprising the polynucleotides of the invention. In some aspects, the invention relates to host cells comprising a polynucleotide of the invention. In some aspects, the invention relates to a method of producing a transglutaminase variant, comprising: a) culturing a host cell of the invention under conditions compatible with expressing said transglutaminase variant; (b) recovering said transglutaminase mutant.

Nucleic Acid Constructs The invention further relates to nucleic acid constructs comprising a polynucleotide encoding a transglutaminase variant of the invention and operably linked to one or more regulatory sequences, wherein said one or more regulatory sequences are The control sequences direct the expression of the coding sequence in a suitable host cell under compatible conditions.

Polynucleotides can be manipulated in a variety of ways to provide for expression of the transglutaminase variant. Depending on the expression vector, it is ideal or necessary to manipulate the polynucleotide before it is inserted into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known to those of skill in the art.

A control sequence may be a promoter, ie, identifies a polynucleotide for expression of said polynucleotide by a host cell. 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 and includes mutant, truncated and hybrid promoters and encodes extracellular or intracellular polypeptides that are homologous or heterologous to the host cell. can be obtained.

Expression Vectors The invention further relates to recombinant expression vectors comprising a polynucleotide encoding the transglutaminase variant of the invention, a promoter, and transcription and translation termination signals. Various nucleotides and control sequences can be ligated together to generate a recombinant expression vector, which inserts or replaces a polynucleotide encoding a transglutaminase variant at such sites. One or more suitable restriction sites may be included to allow for Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. When creating an expression vector, the coding sequence is so positioned in the vector that it is operably linked to appropriate control sequences for expression.

A recombinant expression vector can be any vector (eg, plasmid or virus) that can be easily programmed by a recombinant DNA program to effect polynucleotide expression. The choice of vector generally depends on the compatibility of the vector with the host cell into which it will be introduced. Vectors may be linear or circular plasmids.

A vector may be an autonomously replicating vector, ie a vector that exists as an entity external to a chromosome, the replication of which is independent of chromosomal replication, and may be, for example, 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 a vector that, when introduced into a host cell, integrates into the genome and replicates with one or more chromosomes integrated therein. Also, a single vector or plasmid or more than one vector or plasmid can be used, which together contain the total DNA that is introduced into the host cell genome, or can use a transposon.

The vectors preferably contain one or more selectable markers that allow easy selection of cells, such as transformed, transfected, transduced cells. Selectable markers are genes whose products provide 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 resistance to antibiotics (e.g., ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance). is. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers used in filamentous fungal host cells are amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), Including, but not limited to, pyrG (orotidine-5'-phosphate decarboxylase), sC (adenylyl sulfate transferase), and trpC (anthranilate synthase), and equivalents thereof. Preferably used in Aspergillus cells are the Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and the Streptomyces hygroscopicus bar gene.

The vector preferably contains one or more elements that enable the vector to integrate into the genome of the host cell or that have caused the vector to replicate independently of the genome in the cell.

For integration into the host cell genome, the vector can rely on the polynucleotide sequence encoding the transglutaminase variant or any other vector element that integrates into the genome via homologous or non-homologous recombination. Alternatively, the vector may contain another polynucleotide that guides it to integrate into the correct location within the chromosome of the host cell genome by homologous recombination. In order to increase the likelihood of incorporation at the correct location, the incorporation element may contain a sufficient number of nucleic acids, such as 100-10,000 base pairs, 400-10,000 base pairs and 800-10,000 base pairs. The nucleic acids should contain about 10 base pairs and have a high sequence identity with the corresponding target sequence to improve the probability of homologous recombination. The integration element may be any sequence homologous to the target sequence within the host cell genome. Incorporating elements may also be non-encoded or encoded polynucleotides. In one aspect, the vector can integrate into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication, said origin of replication enabling the vector to replicate autonomously in the host cell under consideration. An origin of replication can be any plasmid replicon that functions in a cell and allows for self-replication. The terms "origin of replication" or "plasmid replicon" refer to a polynucleotide from which a plasmid or vector can be copied into the body.

Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in E. coli; It is the origin of replication.

Examples of origins of replication in yeast host cells include the 2 μm origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

Examples of replication origins that are useful in filamentous fungal cells include AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res. [Nucleic Acid Studies] 15:9163). -9175, WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors containing said gene can be completed by the methods disclosed in WO 00/24883.

The production of transglutaminase mutants can be increased by inserting one or more copies of the polynucleotide of the invention into a host cell. Increased copy number of said polynucleotide can 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 said polynucleotide, where appropriate Culturing the cells in the presence of a selective agent can select for amplified copied cells containing the selectable marker gene and other copies from this said polynucleotide.

Programs for ligating such elements to construct the recombinant expression vectors of the present invention are well known to those skilled in the art.

Host Cells The invention further relates to a recombinant host cell comprising a polynucleotide encoding a transglutaminase variant of the invention and operably linked to one or more regulatory sequences. , said one or more regulatory sequences guide the production of the transglutaminase variants of the invention. By introducing a construct or vector containing the polynucleotide into a host cell, the construct or vector is maintained as a chromosomal integrant or a self-replicating extrachromosomal vector, as described above. The term "host cell" is intended to include the progeny of any parent cell that are not identical to the parent cell due to mutations that occur during replication. The choice of host cell is highly dependent on the gene encoding the transglutaminase variant and its origin.

The host cell may be any cell useful for the recombinant production of transglutaminase variants, including prokaryotic or eukaryotic cells.

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

Host cells may be eukaryotic, for example mammalian, insect, plant or fungal cells.

Methods of Production The invention further relates to a method of producing a transglutaminase variant of the invention, said method comprising: a) culturing a host cell of the invention under conditions compatible with expressing said transglutaminase variant; (b) recovering said transglutaminase mutant.

Host cells are cultured in a suitable nutrient medium to produce the transglutaminase mutant by using methods known in the art. For example, culturing in shake flasks, or small-scale or large-scale fermentation in laboratory or industrial fermentation tanks (fermenters) under conditions that allow transglutaminase or mutant expression and/or isolation in an appropriate medium. Cells can be cultured by conducting fermentations (including continuous fermentation, batch fermentation, batch fed fermentation or solid state fermentation). Cultivate in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts using procedures known in the art. Suitable media can be obtained from commercial sources or prepared according to the compositions disclosed. Once the transglutaminase variant is secreted into the nutrient medium, said transglutaminase variant can be recovered directly from the medium. If the transglutaminase mutant is not secreted, it can be recovered from cell lysates.

Transglutaminase mutants can be detected using specific methods for transglutaminase mutants known in the art. These detection methods include, but are not limited to, the use of specific antibodies, formation of enzymatic products or disappearance of enzymatic substrates. For example, enzymatic assays can be used to determine the activity of transglutaminase variants (eg, as described in the Examples).

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

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

In an alternative embodiment, rather than harvesting the transglutaminase mutant, host cells of the invention that express the transglutaminase mutant are used as a source of said transglutaminase mutant.

Fermentation broth formulations or cell compositions The present invention further relates to fermentation broth formulations or cell compositions comprising the polypeptides of the invention. Said fermentation broth product further comprises other components used in the fermentation process, e.g., cells (host cells containing genes encoding the polypeptides of the invention, these host cells are ), cell fragments, biomass, fermentation media and/or fermentation products. In some embodiments, the composition is one or more organic acids, dead cells and/or cell debris, and cell-killed whole culture media.

As used herein, the term "fermentate" means a preparation produced by cell fermentation that has not undergone recovery or purification or has undergone minimal recovery or purification. For example, when a microbial culture is grown by incubating to saturation under carbon-limiting conditions that allow protein synthesis (e.g., host cell-expressed enzymes) and secrete proteins into the cell medium, a fermentation broth is produced. Said fermentation broth may comprise the unfractionated or fractionated content of fermentation material obtained at the end of fermentation. Generally, the fermentation broth is unfractionated and contains the medium used and cell debris present after removal of microbial cells (eg, fungal cells), eg, by centrifugation. In some embodiments, the fermentation broth contains spent cell culture media, extracellular enzymes, and viable and/or inactive microbial cells.

In one embodiment, the fermentation broth formulation and cell composition comprise a first organic acid component (at least one 1-5 carbon organic acid and/or salt thereof) and a second organic acid component (at least one containing 6 or more carbons and/or salts thereof). In specific embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, salts thereof, or a mixture of two or more of the foregoing, and said second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4- Methylpentanoic 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 includes dead cells and/or cell debris. In one embodiment, the dead cells and/or cell debris are removed from the whole culture medium in which the cells die to provide a composition free of these components.

These fermentation broth formulations or cell compositions may further include preservatives and/or antimicrobials (e.g., bacteriostatic agents), such as sorbitol, sodium chloride, potassium sorbate, and others known in the art. including, but not limited to, reagents from

The cell-killed whole broth or composition may contain the unfractionated content of fermentation material obtained at the end of fermentation. Generally, the cell-killed whole culture or composition is combined with the medium used and the microbial cells (e.g., filamentous fungal cells) to grow to saturation and synthesize proteins under carbon-limited conditions. Contains cell debris present after incubation. In some embodiments, the cell-killed whole culture medium or composition contains spent cell culture medium, extracellular enzymes and killed filamentous fungal cells. In some embodiments, microbial cells present in whole cultures or compositions that kill cells can be permeabilized and/or lysed using methods known in the art.

All cultures or cell compositions described herein are generally liquid but contain insoluble components such as dead cells, cell debris, media components and/or one or more insoluble enzymes. be able to. In some examples, insoluble components can be removed to provide a clarified liquid composition.

Compositions The present invention also relates to compositions comprising the mutant transglutaminase of the invention.

These compositions can include the mutant transglutaminase of the invention as the main enzymatic component, eg, are single component compositions. Alternatively, the composition may comprise multiple enzymatic activities, for example one or more (eg several) enzymes selected from the group of protease, glucoamylase, β-amylase, pullulanase.

Example 1
The production and thermostability of partial transglutaminase mutants will now be described with reference to one specific embodiment thereof.

E. coli JM109 and E. coli E. E. coli BL21(DE3) was purchased from takara-Takara Bio (Beijing) Co., Ltd., pET-22b(+) plasmid was purchased from Novagen (the above strain E. coli BL21(DE3) is commercially available. and there is no need to make a patent deposit), the neutral protease was purchased from Beijing solarbio (part number Z8032), the Blunting Kination Ligation (BKL) Kit and PrimeSTAR® HS DNA Polymerase were purchased from Takara Bio (Beijing) Co., Ltd., and the Bradford protein concentration determination kit (surfactant compatible) was purchased from Shanghai beyotime.

Regarding such media:
LB liquid medium: yeast powder 5.0 g/L, tryptone 10.0 g/L, NaCl 10.0 g/L, ampicillin 100 μg/L.

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

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

1. Mutant construction:
The parental gene (nucleotide sequence is as shown in SEQ ID NO: 2, amino acid sequence is as shown in SEQ ID NO: 1) was synthesized by Suzhou GENEWIZ Co., Ltd., and had restriction enzyme sites NdeI and BlpI. was inserted into the plasmid pET-22b(+) by , resulting in pET-22b-tgase (SEQ ID NO: 4). The transglutaminase with the proenzyme substituted (ie, corresponding to the substitution site SEQ ID NO: 3 at positions 1-45 of SEQ ID NO: 1) contains the lysis-promoting protein tag TrxA upstream of the gene, and is synthesized by Suzhou GENEWIZ Co., Ltd. and inserted into plasmid pET-22b(+) by restriction enzyme sites NdeI and BlpI to obtain pET-22b-proC/tgase (SEQ ID NO:5).

Mutant D189H was obtained by PCR and circularization of linear DNA using plasmid pET-22b-proC/tgase as template and 189hf/189hr as primers, resulting plasmid was pET-22b-proC/tgase/D189H Met. Mutant D 189H/A287P was obtained by PCR and linear DNA circularization using plasmid pET-22b-proC/tgase/D189H as template and 287mf/287mr as primers, the resulting plasmid was pET-22b-proC/ It was tgase/D189H/A287P. All primers to be used above are as shown in Table 2. The PCR process referred to the Takara Bio (Beijing) Co., Ltd. PrimeSTAR (registered trademark) HS DNA Polymerase protocol, and the linear DNA circularization method referred to the Takara Bio (Beijing) Co., Ltd. Blunting Kination Ligation (BKL) Kit protocol.

2. Method for producing mutant enzyme The constructed plasmid was transformed into E. coli JM109 (purchased from Beijing TIANGEN Biotechnology Co., Ltd.), the transformation product was plated on LB solid medium, cultured at 37°C for 10 hours, and Transformants were picked and sequenced to identify sequence-accurate recombinant plasmid-transformed E. coli. E. coli BL21(DE3) was obtained to obtain recombinant E. coli expressing the corresponding transglutaminase mutant.

The resulting recombinant Escherichia coli was spread on LB solid medium and cultured at 37° C. for 10 hours, and transformants were picked up and inoculated into LB liquid medium (containing 100 μg/mL ampicillin). The cells were cultured at 37° C. for 10 hours and transferred to TB liquid medium (containing 100 μg/mL ampicillin) at a seeding rate of 1%. E. coli at 37°C. E. coli BL21(DE3) was grown to an OD ₆₀₀ of 1.0-1.5 and IPTG was added to a final concentration of 0.01 mM to induce recombinant protein expression. After adding IPTG, the culture temperature was changed to 20° C. and cultured continuously for 36 hours. All liquid cultures were cultured with shaking and the rotation speed was 220 rpm.

After fermentation, the sample was centrifuged at 7500 rpm for 10 minutes to collect the cells, 1/5 of the total volume of the fermentation liquid, Tris-HCl 50 mM, was added, the cells were suspended at pH 8.0, and ice bathed for 10 minutes. . The sample was placed on ice for ultrasonic rupture and centrifuged at 12,000 rpm for 15 minutes, after which the supernatant was recovered. Z8032) was added to activate the transglutaminase, and the incubation conditions were a 30 min water bath at 37°C. After centrifuging the incubated sample at 12000 rpm for 20 minutes, the supernatant is taken, and the protein is purified by a nickel ion affinity purification method. A Tris-HCl 50 mM, 20 mM imidazole, pH 7.8 solution was applied in sequence, followed by the sample and washed again with a Tris-HCl 50 mM, 20 mM imidazole, pH 7.8 solution until the conductivity was balanced, and then The protein was eluted and recovered with a solution Tris-HCl 50 mM, 180 mM imidazole, pH 7.8. The entire purification process was performed on the AKTA pure instrument and the protein harvest time and amount were determined by observing the A280 wavelength absorption peak. The collected protein sample was subjected to protein solution desalting by gel chromatography, and the specific method was to apply water and Tris-HCl 50 mM, pH 8.0 solution to the gel column until the conductivity balance was reached, then Protein samples were passed through the gel column at 280° C., followed by protein collection by continuing to pass Tris-HCl 50 mM, pH 8.0 solution until an absorption peak appeared at the A280 wavelength, with collection stopped timely when the conductivity changed. . The protein concentration adopts the BCA test method, and the specific method refers to the protocol of Bradford protein concentration measurement kit (surfactant compatible type) of Shanghai beyotime.

Enzyme specific activity, residual enzyme activity, 60°C half-life, etc. of the parent and mutants were measured, and the results are shown in Table 3, Figures 1, 2 and 3.

From Table 3 it can be seen that the mutant transglutaminase mature enzyme has significantly improved half-life and melting temperature (Tm) at 60°C (T1 _/ ^260°C ) compared to the parental transglutaminase matured enzyme.

From FIG. 1, it can be seen that the mutant transglutaminase mature enzyme has a significantly improved half-life at 60° C. (T _1/2 ^{60° C.} ) and a slightly improved enzyme specific activity compared to the parental transglutaminase mature enzyme.

From FIG. 2, it can be seen that the parent and the mutant are E. coli E. coli, respectively. It is found to achieve expression in E. coli BL21. Here, the parent has a distinct band at a protein molecular weight slightly below 49 KDa, close to the theoretical size of 43.3 KDa. A clear band is visible between the mutant D189H/A287P protein molecular weight 49-62 KDa, close to the theoretical size of 56.5 KDa.

From Figure 3, the protein has a single band after purification, indicating that the purity of the protein meets expectations.

Example 2: Application of Transglutaminase Maturation Enzyme Mutant in Rabbit Meat Jerky Processing The mutant transglutaminase maturation enzyme (TGase-D189H/A287P) prepared in Example 1 was used to process rabbit meat jerky. are as follows:
S1. The rabbit meat was ground and the chicken was cut into blocks to obtain mixed meat.

S2. After salt, complex phosphate and water were uniformly mixed, the mixed meat obtained in S1 was added, mixed uniformly, sealed with a wrap film, and pickled for 12 hours to obtain pickled meat.

S3. Mixed minced meat was obtained by homogenizing the pickled meat.

Mixed minced meat was obtained by adding transglutaminase, egg white protein, ginger powder, blended spice, etc. to the mixed ground meat obtained in S4 and S3 and uniformly stirring at a temperature of 4°C.

S5. The mixed minced meat obtained in S4 was sealed with a wrap film, allowed to stand in a water bath at a temperature of 60°C for 0.2 hours, extruded, dried, and naturally cooled to obtain a semi-finished product.

S6. The semi-finished product obtained in S5 is baked to obtain a composite rabbit jerky.

The sequence of the correlative sequence SEQ ID NO: 1 is as follows (parent, underlined is the proenzyme sequence, the rest is the mature region sequence):
ＤＮＧＡＧＥＥＴＫＳＹＡＥＴＹＲＬＴＡＤＤＶＡＮＩＮＡＬＮＥＳＡＰＡＡＳＳＡＧＰＳＦＲＡＰＤＰＤＤＲＶＴＰＰＡＥＰＬＤＲＭＰＤＰＹＲＰＶＮＧＲＡＥＴＶＶＮＮＹＩＲＫＷＱＱＶＹＳＨＲＤＧＲＫＱＱＭＴＥＥＱＲＥＷＬＳＹＧＣＶＧＶＴＷＶＮＳＧＱＹＰＴＮＲＬＡＦＡＳＦＤＥＤＲＦＫＮＥＬＫＮＧＲＰＲＳＧＥＴＲＡＥＦＥＧＲＶＡＫＥＳＦＤＥＥＫＧＦＱＲＡＲＥＶＡＳＶＭＮＲＡＬＥＮＡＨＤＥＳＡＹＬＤＮＬＫＫＥＬＡＮＧＮＤＡＬＲＮＥＤＡＲＳＰＦＹＳＡＬＲＮＴＰＳＦＫＥＲＮＧＧＮＨＤＰＳＲＭＫＡＶＩＹＡＫＨＦＷＳＧＱＤＲＳＳＳＡＤＫＲＫＹＧＤＰＤＡＦＲＰＡＰＧＴＧＬＶＤＭＳＲＤＲＮＩＰＲＳＰＴＳＰＧＥＧＦＶＮＦＤＹＧＷＦＧＡＱＴＥＡＤＡＤＫＴＶＷＴＨＧＮＨＹＨＡＰＮＧＳＬＧＡＭＨＶＹＥＳＬＦＲＮＷＳＥＧＹＳＤＦＤＲＧＡＹＶＩＴＦＩＰＫＳＷＮＴＡＰＤＫＶＫＱＧＷＰ

The sequence of SEQ ID NO:2 is as follows (parent, underlined is the proenzyme sequence, the rest is the mature region sequence):
ＧＡＣＡＡＴＧＧＣＧＣＧＧＧＧＧＡＡＧＡＧＡＣＧＡＡＧＴＣＣＴＡＣＧＣＣＧＡＡＡＣＣＴＡＣＣＧＣＣＴＣＡＣＧＧＣＧＧＡＴＧＡＣＧＴＣＧＣＧＡＡＣＡＴＣＡＡＣＧＣＧＣＴＣＡＡＣＧＡＡＡＧＣＧＣＴＣＣＧＧＣＣＧＣＴＴＣＧＡＧＣＧＣＣＧＧＣＣＣＧＴＣＧＴＴＣＣＧＧＧＣＣＣＣＣＧＡＴＣＣＴＧＡＴＧＡＴＡＧＡＧＴＧＡＣＣＣＣＡＣＣＴＧＣＡＧＡＡＣＣＡＣＴＧＧＡＴＡＧＡＡＴＧＣＣＴＧＡＴＣＣＡＴＡＴＡＧＡＣＣＴＧＴＧＡＡＴＧＧＣＡＧＡＧＣＡＧＡＡＡＣＴＧＴＧＧＴＧＡＡＣＡＡＣＴＡＴＡＴＴＡＧＡＡＡＡＴＧＧＣＡＧＣＡＡＧＴＧＴＡＴＡＧＣＣＡＴＡＧＡＧＡＴＧＧＣＡＧＡＡＡＡＣＡＧＣＡＧＡＴＧＡＣＴＧＡＡＧＡＡＣＡＧＡＧＡＧＡＡＴＧＧＴＴＡＡＧＣＴＡＴＧＧＣＴＧＴＧＴＧＧＧＴＧＴＧＡＣＣＴＧＧＧＴＧＡＡＣＡＧＴＧＧＴＣＡＧＴＡＴＣＣＡＡＣＣＡＡＣＡＧＡＣＴＧＧＣＣＴＴＴＧＣＡＡＧＣＴＴＴＧＡＴＧＡＡＧＡＴＡＧＡＴＴＴＡＡＡＡＡＴＧＡＡＣＴＧＡＡＡＡＡＴＧＧＣＡＧＡＣＣＡＡＧＡＡＧＴＧＧＴＧＡＡＡＣＴＡＧＡＧＣＡＧＡＡＴＴＴＧＡＡＧＧＣＡＧＡＧＴＧＧＣＣＡＡＡＧＡＡＴＣＴＴＴＴＧＡＴＧＡＡＧＡＧＡＡＧＧＧＣＴＴＴＣＡＧＡＧＡＧＣＡＡＧＡＧＡＡＧＴＧＧＣＡＡＧＴＧＴＧＡＴＧＡＡＣＡＧＡＧＣＣＣＴＧＧＡＡＡＡＴＧＣＣＣＡＴＧＡＴＧＡＡＡＧＴＧＣＣＴＡＴＣＴＧＧＡＴＡＡＣＣＴＧＡＡＡＡＡＡＧＡＡＣＴＧＧＣＣＡＡＴＧＧＣＡＡＴＧＡＴＧＣＣＴＴＡＡＧＡＡＡＴＧＡＡＧＡＴＧＣＡＡＧＡＡＧＣＣＣＡＴＴＴＴＡＴＡＧＴＧＣＣＣＴＧＡＧＡＡＡＣＡＣＣＣＣＡＡＧＣＴＴＴＡＡＡＧＡＡＡＧＡＡＡＴＧＧＴＧＧＣＡＡＣＣＡＴＧＡＴＣＣＡＡＧＣＡＧＡＡＴＧＡＡＡＧＣＡＧＴＧＡＴＴＴＡＴＧＣＣＡＡＡＣＡＴＴＴＴＴＧＧＡＧＴＧＧＣＣＡＡＧＡＴＡＧＡＡＧＣＡＧＣＡＧＴＧＣＡＧＡＴＡＡＡＡＧＡＡＡＡＴＡＴＧＧＴＧＡＴＣＣＴＧＡＴＧＣＣＴＴＴＡＧＡＣＣＴＧＣＣＣＣＴＧＧＣＡＣＴＧＧＣＣＴＧＧＴＧＧＡＴＡＴＧＡＧＣＡＧＡＧＡＴＡＧＡＡＡＣＡＴＴＣＣＡＡＧＡＡＧＣＣＣＡＡＣＴＡＧＣＣＣＴＧＧＴＧＡＡＧＧＣＴＴＴＧＴＧＡＡＣＴＴＴＧＡＴＴＡＴＧＧＣＴＧＧＴＴＴＧＧＴＧＣＡＣＡＧＡＣＴＧＡＡＧＣＡＧＡＴＧＣＡＧＡＴＡＡＡＡＣＴＧＴＧＴＧＧＡＣＴＣＡＴＧＧＣＡＡＣＣＡＴＴＡＴＣＡＴＧＣＣＣＣＡＡＡＴＧＧＣＡＧＣＣＴＧＧＧＴＧＣＣＡＴＧＣ ATGTGTATGAAAGCCTGTTTAGAAAACTGGAGTGAAGGCTATAGTGATTTTGATAGAGGTGCCTATGTGATTACCTTTTATTCCAAAAGCTGGAACACTGCCCCTGATAAAGTGAAAACAAGGCTGGCCA

The sequence of SEQ ID NO:3 is as follows:
MASGGDEEWEGSYAATHGLTAEDVKNINALNKRALTAGQPGNFPAELPPSATALFRAPD

The sequence of SEQ ID NO: 4 is as follows (double underline is the gene corresponding to the proenzyme region and wavy line is the gene corresponding to the transglutaminase maturation enzyme):
ｔｇｇｃｇａａｔｇｇｇａｃｇｃｇｃｃｃｔｇｔａｇｃｇｇｃｇｃａｔｔａａｇｃｇｃｇｇｃｇｇｇｔｇｔｇｇｔｇｇｔｔａｃｇｃｇｃａｇｃｇｔｇａｃｃｇｃｔａｃａｃｔｔｇｃｃａｇｃｇｃｃｃｔａｇｃｇｃｃｃｇｃｔｃｃｔｔｔｃｇｃｔｔｔｃｔｔｃｃｃｔｔｃｃｔｔｔｃｔｃｇｃｃａｃｇｔｔｃｇｃｃｇｇｃｔｔｔｃｃｃｃｇｔｃａａｇｃｔｃｔａａａｔｃｇｇｇｇｇｃｔｃｃｃｔｔｔａｇｇｇｔｔｃｃｇａｔｔｔａｇｔｇｃｔｔｔａｃｇｇｃａｃｃｔｃｇａｃｃｃｃａａａａａａｃｔｔｇａｔｔａｇｇｇｔｇａｔｇｇｔｔｃａｃｇｔａｇｔｇｇｇｃｃａｔｃｇｃｃｃｔｇａｔａｇａｃｇｇｔｔｔｔｔｃｇｃｃｃｔｔｔｇａｃｇｔｔｇｇａｇｔｃｃａｃｇｔｔｃｔｔｔａａｔａｇｔｇｇａｃｔｃｔｔｇｔｔｃｃａａａｃｔｇｇａａｃａａｃａｃｔｃａａｃｃｃｔａｔｃｔｃｇｇｔｃｔａｔｔｃｔｔｔｔｇａｔｔｔａｔａａｇｇｇａｔｔｔｔｇｃｃｇａｔｔｔｃｇｇｃｃｔａｔｔｇｇｔｔａａａａａａｔｇａｇｃｔｇａｔｔｔａａｃａａａａａｔｔｔａａｃｇｃｇａａｔｔｔｔａａｃａａａａｔａｔｔａａｃｇｔｔｔａｃａａｔｔｔｃａｇｇｔｇｇｃａｃｔｔｔｔｃｇｇｇｇａａａｔｇｔｇｃｇｃｇｇａａｃｃｃｃｔａｔｔｔｇｔｔｔａｔｔｔｔｔｃｔａａａｔａｃａｔｔｃａａａｔａｔｇｔａｔｃｃｇｃｔｃａｔｇａｇａｃａａｔａａｃｃｃｔｇａｔａａａｔｇｃｔｔｃａａｔａａｔａｔｔｇａａａａａｇｇａａｇａｇｔａｔｇａｇｔａｔｔｃａａｃａｔｔｔｃｃｇｔｇｔｃｇｃｃｃｔｔａｔｔｃｃｃｔｔｔｔｔｔｇｃｇｇｃａｔｔｔｔｇｃｃｔｔｃｃｔｇｔｔｔｔｔｇｃｔｃａｃｃｃａｇａａａｃｇｃｔｇｇｔｇａａａｇｔａａａａｇａｔｇｃｔｇａａｇａｔｃａｇｔｔｇｇｇｔｇｃａｃｇａｇｔｇｇｇｔｔａｃａｔｃｇａａｃｔｇｇａｔｃｔｃａａｃａｇｃｇｇｔａａｇａｔｃｃｔｔｇａｇａｇｔｔｔｔｃｇｃｃｃｃｇａａｇａａｃｇｔｔｔｔｃｃａａｔｇａｔｇａｇｃａｃｔｔｔｔａａａｇｔｔｃｔｇｃｔａｔｇｔｇｇｃｇｃｇｇｔａｔｔａｔｃｃｃｇｔａｔｔｇａｃｇｃｃｇｇｇｃａａｇａｇｃａａｃｔｃｇｇｔｃｇｃｃｇｃａｔａｃａｃｔａｔｔｃｔｃａｇａａｔｇａｃｔｔｇｇｔｔｇａｇｔａｃｔｃａｃｃａｇｔｃａｃａｇａａａａｇｃａｔｃｔｔａｃｇｇａｔｇｇｃａｔｇａｃａｇｔａａｇａｇａａｔｔａｔｇｃａｇｔｇｃｔｇｃｃａｔａａｃｃａｔｇａｇｔｇａｔａａｃａｃｔｇｃｇｇｃｃａａｃ 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ａｃｇａｃｔｃａｃｔａｔａｇｇｇｇａａｔｔｇｔｇａｇｃｇｇａｔａａｃａａｔｔｃｃｃｃｔｃｔａｇａａａｔａａｔｔｔｔｇｔｔｔａａｃｔｔｔａａｇａａｇｇａｇａｔａｔａｃａｔａｔｇＧＡＣＡＡＴＧＧＣＧＣＧＧＧＧＧＡＡＧＡＧＡＣＧＡＡＧＴＣＣＴＡＣＧＣＣＧＡＡＡＣＣＴＡＣＣＧＣＣＴＣＡＣＧＧＣＧＧＡＴＧＡＣＧＴＣＧＣＧＡＡＣＡＴＣＡＡＣＧＣＧＣＴＣＡＡＣＧＡＡＡＧＣＧＣＴＣＣＧＧＣＣＧＣＴＴＣＧＡＧＣＧＣＣＧＧＣＣＣＧＴＣＧＴＴＣＣＧＧＧＣＣＣＣＣＧＡＴＣＣＴＧＡＴＧＡＴＡＧＡＧＴＧＡＣＣＣＣＡＣＣＴＧＣＡＧＡＡＣＣＡＣＴＧＧＡＴＡＧＡＡＴＧＣＣＴＧＡＴＣＣＡＴＡＴＡＧＡＣＣＴＧＴＧＡＡＴＧＧＣＡＧＡＧＣＡＧＡＡＡＣＴＧＴＧＧＴＧＡＡＣＡＡＣＴＡＴＡＴＴＡＧＡＡＡＡＴＧＧＣＡＧＣＡＡＧＴＧＴＡＴＡＧＣＣＡＴＡＧＡＧＡＴＧＧＣＡＧＡＡＡＡＣＡＧＣＡＧＡＴＧＡＣＴＧＡＡＧＡＡＣＡＧＡＧＡＧＡＡＴＧＧＴＴＡＡＧＣＴＡＴＧＧＣＴＧＴＧＴＧＧＧＴＧＴＧＡＣＣＴＧＧＧＴＧＡＡＣＡＧＴＧＧＴＣＡＧＴＡＴＣＣＡＡＣＣＡＡＣＡＧＡＣＴＧＧＣＣＴＴＴＧＣＡＡＧＣＴＴＴＧＡＴＧＡＡＧＡＴＡＧＡＴＴＴＡＡＡＡＡＴＧＡＡＣＴＧＡＡＡＡＡＴＧＧＣＡＧＡＣＣＡＡＧＡＡＧＴＧＧＴＧＡＡＡＣＴＡＧＡＧＣＡＧＡＡＴＴＴＧＡＡＧＧＣＡＧＡＧＴＧＧＣＣＡＡＡＧＡＡＴＣＴＴＴＴＧＡＴＧＡＡＧＡＧＡＡＧＧＧＣＴＴＴＣＡＧＡＧＡＧＣＡＡＧＡＧＡＡＧＴＧＧＣＡＡＧＴＧＴＧＡＴＧＡＡＣＡＧＡＧＣＣＣＴＧＧＡＡＡＡＴＧＣＣＣＡＴＧＡＴＧＡＡＡＧＴＧＣＣＴＡＴＣＴＧＧＡＴＡＡＣＣＴＧＡＡＡＡＡＡＧＡＡＣＴＧＧＣＣＡＡＴＧＧＣＡＡＴＧＡＴＧＣＣＴＴＡＡＧＡＡＡＴＧＡＡＧＡＴＧＣＡＡＧＡＡＧＣＣＣＡＴＴＴＴＡＴＡＧＴＧＣＣＣＴＧＡＧＡＡＡＣＡＣＣＣＣＡＡＧＣＴＴＴＡＡＡＧＡＡＡＧＡＡＡＴＧＧＴＧＧＣＡＡＣＣＡＴＧＡＴＣＣＡＡＧＣＡＧＡＡＴＧＡＡＡＧＣＡＧＴＧＡＴＴＴＡＴＧＣＣＡＡＡＣＡＴＴＴＴＴＧＧＡＧＴＧＧＣＣＡＡＧＡＴＡＧＡＡＧＣＡＧＣＡＧＴＧＣＡＧＡＴＡＡＡＡＧＡＡＡＡＴＡＴＧＧＴＧＡＴＣＣＴＧＡＴＧＣＣＴＴＴＡＧＡＣＣＴＧＣＣＣＣＴＧＧＣＡＣＴＧＧＣＣＴＧＧＴＧＧＡＴＡＴＧＡＧＣＡＧＡＧＡＴＡＧＡＡＡＣＡＴＴＣＣＡＡＧＡＡＧＣＣＣＡＡＣＴＡＧＣＣＣＴＧＧＴＧＡＡＧＧＣＴＴＴＧＴＧＡＡＣＴＴＴＧＡＴＴＡＴＧＧＣＴＧＧＴＴＴＧ ＧＴＧＣＡＣＡＧＡＣＴＧＡＡＧＣＡＧＡＴＧＣＡＧＡＴＡＡＡＡＣＴＧＴＧＴＧＧＡＣＴＣＡＴＧＧＣＡＡＣＣＡＴＴＡＴＣＡＴＧＣＣＣＣＡＡＡＴＧＧＣＡＧＣＣＴＧＧＧＴＧＣＣＡＴＧＣＡＴＧＴＧＴＡＴＧＡＡＡＧＣＣＴＧＴＴＴＡＧＡＡＡＣＴＧＧＡＧＴＧＡＡＧＧＣＴＡＴＡＧＴＧＡＴＴＴＴＧＡＴＡＧＡＧＧＴＧＣＣＴＡＴＧＴＧＡＴＴＡＣＣＴＴＴＡＴＴＣＣＡＡＡＡＡＧＣＴＧＧＡＡＣＡＣＴＧＣＣＣＣＴＧＡＴＡＡＡＧＴＧＡＡＡＣＡＡＧＧＣＴＧＧＣＣＡＣＡＣＣＡＣＣＡＣＣＡＣＣＡＣＣＡＣＴＧＡｇａｔｃｃｇｇｃｔｇｃｔａａｃａａａｇｃｃｃｇａａａｇｇａａｇｃｔｇａｇｔｔｇｇｃｔｇｃｔｇｃｃａｃｃｇｃｔｇａｇｃａａｔａａｃｔａｇｃａｔａａｃｃｃｃｔｔｇｇｇｇｃｃｔｃｔａａａｃｇｇｇｔｃｔｔｇａｇｇｇｇｔｔｔｔｔｔｇｃｔｇａａａｇｇａｇｇａａｃｔａｔａｔｃｃｇｇａｔ

The sequence of SEQ ID NO: 5 is as follows (single underline is the TrxA sequence, double underline is the proenzyme region sequence after proenzyme substitution, and wavy line is the transglutaminase maturation region sequence):
ｔｇｇｃｇａａｔｇｇｇａｃｇｃｇｃｃｃｔｇｔａｇｃｇｇｃｇｃａｔｔａａｇｃｇｃｇｇｃｇｇｇｔｇｔｇｇｔｇｇｔｔａｃｇｃｇｃａｇｃｇｔｇａｃｃｇｃｔａｃａｃｔｔｇｃｃａｇｃｇｃｃｃｔａｇｃｇｃｃｃｇｃｔｃｃｔｔｔｃｇｃｔｔｔｃｔｔｃｃｃｔｔｃｃｔｔｔｃｔｃｇｃｃａｃｇｔｔｃｇｃｃｇｇｃｔｔｔｃｃｃｃｇｔｃａａｇｃｔｃｔａａａｔｃｇｇｇｇｇｃｔｃｃｃｔｔｔａｇｇｇｔｔｃｃｇａｔｔｔａｇｔｇｃｔｔｔａｃｇｇｃａｃｃｔｃｇａｃｃｃｃａａａａａａｃｔｔｇａｔｔａｇｇｇｔｇａｔｇｇｔｔｃａｃｇｔａｇｔｇｇｇｃｃａｔｃｇｃｃｃｔｇａｔａｇａｃｇｇｔｔｔｔｔｃｇｃｃｃｔｔｔｇａｃｇｔｔｇｇａｇｔｃｃａｃｇｔｔｃｔｔｔａａｔａｇｔｇｇａｃｔｃｔｔｇｔｔｃｃａａａｃｔｇｇａａｃａａｃａｃｔｃａａｃｃｃｔａｔｃｔｃｇｇｔｃｔａｔｔｃｔｔｔｔｇａｔｔｔａｔａａｇｇｇａｔｔｔｔｇｃｃｇａｔｔｔｃｇｇｃｃｔａｔｔｇｇｔｔａａａａａａｔｇａｇｃｔｇａｔｔｔａａｃａａａａａｔｔｔａａｃｇｃｇａａｔｔｔｔａａｃａａａａｔａｔｔａａｃｇｔｔｔａｃａａｔｔｔｃａｇｇｔｇｇｃａｃｔｔｔｔｃｇｇｇｇａａａｔｇｔｇｃｇｃｇｇａａｃｃｃｃｔａｔｔｔｇｔｔｔａｔｔｔｔｔｃｔａａａｔａｃａｔｔｃａａａｔａｔｇｔａｔｃｃｇｃｔｃａｔｇａｇａｃａａｔａａｃｃｃｔｇａｔａａａｔｇｃｔｔｃａａｔａａｔａｔｔｇａａａａａｇｇａａｇａｇｔａｔｇａｇｔａｔｔｃａａｃａｔｔｔｃｃｇｔｇｔｃｇｃｃｃｔｔａｔｔｃｃｃｔｔｔｔｔｔｇｃｇｇｃａｔｔｔｔｇｃｃｔｔｃｃｔｇｔｔｔｔｔｇｃｔｃａｃｃｃａｇａａａｃｇｃｔｇｇｔｇａａａｇｔａａａａｇａｔｇｃｔｇａａｇａｔｃａｇｔｔｇｇｇｔｇｃａｃｇａｇｔｇｇｇｔｔａｃａｔｃｇａａｃｔｇｇａｔｃｔｃａａｃａｇｃｇｇｔａａｇａｔｃｃｔｔｇａｇａｇｔｔｔｔｃｇｃｃｃｃｇａａｇａａｃｇｔｔｔｔｃｃａａｔｇａｔｇａｇｃａｃｔｔｔｔａａａｇｔｔｃｔｇｃｔａｔｇｔｇｇｃｇｃｇｇｔａｔｔａｔｃｃｃｇｔａｔｔｇａｃｇｃｃｇｇｇｃａａｇａｇｃａａｃｔｃｇｇｔｃｇｃｃｇｃａｔａｃａｃｔａｔｔｃｔｃａｇａａｔｇａｃｔｔｇｇｔｔｇａｇｔａｃｔｃａｃｃａｇｔｃａｃａｇａａａａｇｃａｔｃｔｔａｃｇｇａｔｇｇｃａｔｇａｃａｇｔａａｇａｇａａｔｔａｔｇｃａｇｔｇｃｔｇｃｃａｔａａｃｃａｔｇａｇｔｇａｔａａｃａｃｔｇｃｇｇｃｃａａｃ 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Claims (10)

A transglutaminase mutant comprising substitutions corresponding to positions 1-45 of the polypeptide shown in SEQ ID NO: 1, substituted with the sequence shown in SEQ ID NO: 3, and further comprising the polypeptide shown in SEQ ID NO: 1 containing substitutions corresponding to positions 234 and 332 of the peptide;
i) the mature region of said transglutaminase mutant is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92% of the polypeptide at positions 46-376 shown in SEQ ID NO: 1 , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and/or ii) said transglutaminase variant is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96% with the mature polypeptide coding sequence from nucleotide positions 139 to 1128 shown in SEQ ID NO:2, a polypeptide encoded by a polynucleotide having at least 97%, at least 98%, or at least 99% sequence identity;
Transglutaminase mutant. Transglutaminase mutant according to claim 1, characterized in that it contains a substitution corresponding to position 234 of the polypeptide shown in SEQ ID NO: 1 (i.e. position 189 of the mature region sequence), replaced by histidine. . 2. The method according to claim 1, wherein said variant further comprises a substitution at position 332 of the polypeptide shown in SEQ ID NO: 1 (i.e. position 287 of the mature region sequence) with a proline (P) substitution. Transglutaminase mutants as described. 4. A polynucleotide encoding the variant of any one of claims 1-3. Nucleic acid constructs, vectors and host cells comprising the polynucleotide of claim 4. 4. A method of producing a mutant according to any one of claims 1-3. 4. A composition comprising the mature enzyme of the transglutaminase variant of any one of claims 1-3. A method for altering the appearance, mouthfeel and/or stability of food products in raw meat processing, sausage-type products, fish balls, minced meat processing, legume products and/or dairy products, wherein the A method comprising adding a transglutaminase mutant or mature enzyme thereof according to any one of claims. 8. A method of altering the appearance, mouthfeel and/or stability of foods in raw meat processing, sausage-like products, fish balls, minced meat processing, legume products and/or dairy products, wherein said food processing process comprises: A method comprising adding a composition to perform a treatment. Use of a transglutaminase variant according to any one of claims 1 to 3 or a composition according to claim 7 in food treatment, processing and conversion.

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