CN112553177B - Glutamine transaminase variant with improved heat stability - Google Patents

Glutamine transaminase variant with improved heat stability Download PDF

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CN112553177B
CN112553177B CN202011586610.6A CN202011586610A CN112553177B CN 112553177 B CN112553177 B CN 112553177B CN 202011586610 A CN202011586610 A CN 202011586610A CN 112553177 B CN112553177 B CN 112553177B
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variant
transglutaminase
glutamine transaminase
seq
processing
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CN112553177A (en
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刘松
王兴隆
杜建辉
钱源
周景文
陈坚
堵国成
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Taixing Dongsheng Bio Tech Co ltd
Jiangnan University
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Jiangsu Donghui Biotechnology Co ltd
Jiangnan University
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Priority to PCT/CN2020/142185 priority patent/WO2022141411A1/en
Priority to JP2021576243A priority patent/JP7395097B2/en
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Abstract

The invention discloses a glutamine transaminase variant with improved thermal stability, belonging to the fields of biology and food. The transglutaminase variants of the invention are enzymes with improved thermostability comprising a substitution of the zymogen region corresponding to positions 1-45 of the transglutaminase sequence shown in SEQ ID NO.1 and a substitution of positions 189 and 287 of the mature region sequence. 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 variant with improved heat stability
Technical Field
The invention relates to a glutamine transaminase variant with improved thermal stability, belonging to the fields of biology and food.
Background
A glutamine transaminase (TGase, EC 2.3.2.13) derived from Streptomyces mobaraensis AAT65817 is a mainstream enzyme preparation widely sold in domestic and international markets. It is a commonly used transaminase that promotes covalent cross-linking between proteins and small molecules by reacting the gamma carboxamide group of a glutamine residue with the amino group of an acyl acceptor. The application of TGase in the field of food processing is very wide, and the TGase is added in the processing of meat, bean products and dairy products to change the appearance, taste and stability of the food.
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).
Because the thermal stability is important, the current method for modifying the thermal stability of the enzyme is mainly rational design and directed evolution. The rational design is to determine the unstable region in the enzyme structure by a machine simulation mode and introduce amino acid mutation to improve the stability of the enzyme. Directed evolution includes directed evolution based on host cells mutagenized by physical, chemical, etc. means to obtain desired enzymatic properties, and directed evolution based on enzyme genes, which mainly performs random mutation, homolog gene recombination to obtain directed evolution. The following reports are related to the improvement of thermal stability of TGase: (1) obtaining a high-stability TGase mutation point through random mutation (doi: 10.1016/j.jbiotec.2008.06.005); (2) carrying out site saturation mutation and DNA recombination on the single-point mutation obtained in the step (1) to obtain a high-stability TGase combined mutant (DOI10.1007/s00726-011 and 1015-y); (3) further combinatorial mutations were performed on the basis of the selected mutation points of (1) and (2) (DOI: 10.1080/09168451.2017.1403881). However, the high-stability TGase mutant reported at present has poor stability after incubation at 60 ℃ under the water bath condition compared with the incubation at 60 ℃ under the metal bath condition, and has a larger improvement space. In practical application, such as meat paste, corn flour, bean product processing, etc., the treatment temperature of mixed TGase is usually 40-70 ℃, and the stability of TGase at 60 ℃ is very poor, resulting in high addition amount and low utilization rate. Therefore, the design and modification of the high-stability TGase are particularly important in meeting the industrial application.
Disclosure of Invention
In order to solve the above problems, the present invention provides a transglutaminase variant having improved thermostability.
The glutamine transaminase variant of the invention comprises substitutions corresponding to positions 1 to 45 of the polypeptide shown in SEQ ID NO.1, which are shown in SEQ ID NO. 3; and comprises substitutions corresponding to positions 234 and 332 of the polypeptide shown in SEQ ID NO.1 (corresponding to positions 189 and 287, respectively, of the mature region of the transglutaminase of 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 with the polypeptide of positions 46 to 376 as set forth in SEQ ID NO.1 in the mature region of said transglutaminase variant; and/or
ii) the mature region of said transglutaminase variant 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 from nucleotide 139 to 1128;
wherein the glutamine transaminase variant is treated with a neutral protease to obtain an active enzyme.
In one embodiment, the substitution at position 234 corresponds to position 189 with the mature region of the transglutaminase sequence of SEQ ID No. 1; substitution to histidine (H); a substitution at position 332, corresponding to position 287 of the mature region of the transglutaminase sequence of SEQ ID No. 1; substitution to proline (P).
In one embodiment, the variant glutamine transaminase variant is designated as D189H/a 287P.
The invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of producing the variants or mature enzymes thereof. Furthermore, the present invention relates to a composition comprising a glutamine transaminase variant of the present invention or a mature enzyme thereof.
The present invention also relates to methods of producing a transglutaminase variant of the invention or a mature enzyme thereof, comprising:
a) culturing a host cell of the invention under conditions suitable for expression of the variant; and
b) optionally recovering the variant.
The invention also relates to a method for modifying the appearance, 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 glutamine transaminase variant according to the invention or a composition according to the invention to the food processing.
The invention also relates to the use of said transglutaminase variant or mature enzyme thereof 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.
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. For the purposes of the present invention, the glutamine transaminase activity is determined according to the procedure described in the examples. In one aspect, a variant of the invention has at least 20%, e.g., 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%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the glutamine transaminase activity of the polypeptide of SEQ ID No. 1.
A coding sequence: the term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a variant of glutamine transaminase. 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 glutamine transaminase variant of the 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 variant, 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 a glutamine transaminase 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 comprising a polynucleotide encoding a glutamine transaminase variant 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, the 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%, but less than 100% of the number of amino acids 46 to 376 of SEQ ID NO.1 (i.e., not including the zymogen region sequence length).
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 characteristic of a variant of transglutaminase that is improved 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 of the present invention is transglutaminase mature enzyme, and is amino acids 46 through 376 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, i.e., the mature enzyme coding sequence, is nucleotides 139 to 1128 of SEQ ID NO.2 (i.e., does not include the codon sequence corresponding to the pro-enzyme region).
Mutant: the term "mutant" means a polynucleotide encoding a variant.
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 maternal transglutaminase: the term "maternal" or "maternal transglutaminase" means a transglutaminase enzyme that is altered to produce a transglutaminase variant of the invention. The parent of the patent is the amino acid sequence corresponding to SEQ ID NO. 1. 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. 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. The variant of the present invention has the polypeptide amino acid sequence of SEQ ID NO.1 and has substitution at positions 1-45, the substituted amino acid sequence is the sequence shown in SEQ ID NO.3, and on the basis, substitution at positions 234 and 332 (i.e., positions 189 and 287 of the transglutaminase maturase shown in SEQ ID NO. 1) occurs simultaneously, and the substituted amino acids are histidine and proline, respectively. A variant of the invention has at least 20%, e.g., 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 glutamine transaminase activity.
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.
In describing variations of the invention, the nomenclature described below is adapted for ease of reference. Accepted IUPAC single letter or three letter amino acid abbreviations are used.
And (3) substitution: for amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, substitution of threonine at position 226 with alanine is denoted as "Thr 226 Ala" or "T226A". Multiple mutations are separated by a symbol ("-"), e.g., "Gly 205Arg-Ser411 Phe" or "G205R-S411F" representing the substitution of glycine (G) and serine (S) at positions 205 and 411 with arginine (R) and phenylalanine (F), respectively.
Has the advantages that:
the variant glutamine transaminase according to the invention is obtained by a zymogen substitution on the maternal basis, i.e. a substitution of the indicated sequence SEQ ID NO:1 from position 1 to position 45, a substitution of the sequence into the zymogen region sequence of Streptomyces caniferus glutamine transaminase (as indicated in SEQ ID NO: 3), and a mutation at positions 234 and 332 of the glutamine transaminase of the indicated sequence SEQ ID NO:1 (i.e. positions 189 and 287 of the mature region portion). The half-life at 60 ℃ of the variant maturase was 35.57min, which was 214.5% higher than that of the parent maturase at 11.31 min.
The glutamine transaminase variant of the invention has significantly improved thermostability, can be applied in food processing, processing and transformation, and can be used for maintaining or improving 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.
Drawings
FIG. 1: initial enzyme activity and half-life at 60 ℃ of the parent and variant maturases.
FIG. 2: coli BL21 expression of the parent and variant, and SDS-PAGE electrophoresis analysis of the whole cells. M is a protein mass marker with the unit of kDa; 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 variant maturases; m is a protein mass marker with a Kda unit; 1 is maternal maturase; and 2 is variant maturase.
Detailed Description
The present invention relates to variants of transglutaminase having improved compared to the parent transglutaminase. More specifically, the present invention relates to variants of a glutamine transaminase having improved thermostability as compared to a parent glutamine transaminase (in particular a 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/LHCl) 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
The half-life period under the condition of water bath at the temperature of 60 ℃ is determined by the specific method that firstly, a protein solution of female parent glutamine transaminase mature enzyme and variant mature enzyme is diluted to 0.5mg/ml, a certain amount of sample is taken to carry out continuous hot incubation in the water bath at the temperature of 60 ℃, sampling is carried out every minute within 0-10 min, sampling is carried out every 2min within 10-40 min, and the taken samples are immediately placed at the temperature of 20 ℃ for cold compress. Respectively carrying out enzyme activity determination on the samples to obtain the residual enzyme activity ratio which changes along with time, carrying out nonlinear fitting on the samples through Exponental-ExpDec 1 in origin 2018, and calculating the time corresponding to the enzyme activity which is reduced to 50% of the initial value 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 KMThe 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 obtainedMAnd Vmax value, k is obtained by conversion of enzyme concentrationcatThe value is obtained. On the basis of the above, with KM/kcatObtaining the catalytic efficiency of the enzyme.
The glutamine transaminase of the invention comprises the substitution (the substitution is SEQ ID NO.3) corresponding to the 1 st to 45 th positions of the polypeptide shown as SEQ ID NO. 1; and comprises substitutions corresponding to positions 234 and 332 of the polypeptide shown in SEQ ID NO.1 (corresponding to positions 189 and 287, respectively, of the mature region of the transglutaminase of 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 of positions 46 to 376 as set forth in SEQ ID NO.1 in the mature region of said 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 represented by SEQ ID No.2 from nucleotide 139 to 1128;
the 1 st to 45 th positions are substituted, and the amino acid sequence after the substitution is SEQ ID NO. 3.
In one embodiment, the transglutaminase is substituted with histidine (H) at position 234 (i.e., position 189 of the mature region of the transglutaminase sequence of SEQ ID NO: 1).
In one embodiment, the glutamine transaminase, position 332 (i.e., position 287 of the mature region of the glutamine transaminase of SEQ ID NO: 1) is substituted with proline (P).
In one embodiment, the transglutaminase variant comprises a substitution at positions 1-45 of the polypeptide of SEQ ID NO.1 with SEQ ID NO.3, and only a substitution at positions 189 and 287 of the mature region of the transglutaminase variant of SEQ ID NO.1, wherein the half-life at 60 ℃ is increased by 214.5%.
Table 1 lists the relative thermostability of variants of glutamine transaminase in which substitutions at positions 189 and 287 of the maturation zone only occur compared to the parent and variant maturases shown in SEQ ID NO: 1.
TABLE 1
Figure BDA0002867321470000081
Preparation of variants
Any mutagenesis procedure known in the art (e.g., site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.) can be used to prepare transglutaminase variants of the invention.
Site-directed mutagenesis is a technique in which one or more (e.g., several) mutations are introduced at one or more defined sites in a polynucleotide encoding the parent transglutaminase.
Site-directed mutagenesis can be achieved in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. In vitro site-directed mutagenesis may also be performed by cassette mutagenesis involving cleavage by a restriction enzyme at a site in a plasmid comprising a polynucleotide encoding the parent transglutaminase and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Typically, the restriction enzymes that digest the plasmid and oligonucleotide are the same, allowing the sticky ends of the plasmid and insert to ligate to each other.
Site-directed mutagenesis can also be accomplished in vivo by methods known in the art.
Any site-directed mutagenesis procedure can be used in the present invention. There are many commercially available kits that can be used to prepare variants.
Synthetic gene construction requires in vitro synthesis of designed polynucleotide molecules to encode the 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 tested using known mutagenesis, recombination and/or shuffling methods, followed by relevant screening procedures.
The mutagenesis/shuffling method can be combined with high throughput, automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells. Mutagenized DNA molecules encoding active polypeptides can be recovered from the host cells and rapidly sequenced using methods standard in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
Semi-synthetic gene construction is achieved by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi-synthetic construction typically utilizes a process of synthesizing polynucleotide fragments in conjunction with PCR techniques. Thus, defined regions of a gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenesis primers, while still other regions can be subjected to error-prone PCR or non-error-prone PCR amplification. The polynucleotide subsequences may then be shuffled.
Polynucleotide
The present invention also relates to isolated polynucleotides encoding the glutamine transaminase variants 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 methods of producing a transglutaminase variant, comprising: (a) culturing the host cell of the invention under conditions suitable for expression of said glutamine transaminase variant; and (b) recovering said transglutaminase variant.
Nucleic acid constructs
The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a glutamine transaminase variant 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 a transglutaminase variant. 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 variant 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 the transglutaminase variant 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 present invention also relates to recombinant host cells comprising a polynucleotide encoding a transglutaminase variant of the invention operably linked to one or more control sequences that direct the production of the transglutaminase variant of the invention. The construct or vector comprising the polynucleotide is introduced into a host cell such that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extra-chromosomal vector, as described earlier. 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. The choice of host cell depends to a large extent on the gene encoding the transglutaminase variant and its source.
The host cell may be any cell useful in the recombinant production of a variant glutamine transaminase, 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 glutamine transaminase variant of the present invention, the method comprising: (a) culturing the host cell of the invention under conditions suitable for expression of said glutamine transaminase variant; and (b) recovering said transglutaminase variant.
The host cell is cultured in a nutrient medium suitable for producing the transglutaminase variant using methods known in the art. For example, the cell 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 or variant 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 variant transglutaminase is secreted into the nutrient medium, the variant transglutaminase can be recovered directly from the medium. If the transglutaminase variant is not secreted, it can be recovered from the cell lysate.
Variants of transglutaminase can be detected using methods known in the art that are specific for variants of transglutaminase. 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 a glutamine transaminase variant (such as those described in the examples).
The transglutaminase variant may be recovered using methods known in the art. For example, the transglutaminase variant may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
A variant glutamine transaminase can be purified 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 to obtain a substantially pure variant glutamine transaminase.
In an alternative aspect, the transglutaminase variant is not recovered, but a host cell of the invention expressing the transglutaminase variant is used as a source of said transglutaminase variant.
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 present invention also relates to compositions comprising the variant transglutaminase of the invention.
These compositions may comprise a variant 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 thermostability of the partial transglutaminase variants are described below in connection with one of the embodiments.
Coli JM109 and E.coli BL21(DE3) were obtained from Takara-Baori physicians and technology (Beijing) Ltd, pET-22b (+) plasmid was obtained from Novagen (the above strain E.coli BL21(DE3) is commercially available without preservation for patent procedures), and neutral protease was obtained from Beijing Solebao science and technology Co., Ltd (product No. Z8032), Blunting Kit Ligation (BKL) Kit and Bling Ligation library (BKL) Kit
Figure BDA0002867321470000141
HS DNA Polymerase purchased from Taorigi medicineBiotech (beijing) inc, a Bradford protein concentration assay kit (detergent compatible type) was purchased from shanghai bi yunnan biotechnology, inc.
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 mutants
The parent gene (nucleotide sequence shown in SEQ ID NO.2 and amino acid sequence shown in SEQ ID NO. 1) was synthesized by Kinzyme, Suzhou, and inserted into plasmid pET-22b (+) via restriction enzyme sites NdeI and BlpI to obtain pET-22b-tgase (SEQ ID NO: 4). The pro-enzyme-substituted transglutaminase (i.e., the substitution at position 1-45 of SEQ ID NO. 1: SEQ ID NO.3) was synthesized by Kinzyme GmbH, Suzhou, including the upstream lysin tag TrxA, and inserted into plasmid pET-22b (+) via restriction enzyme sites NdeI and BlpI to obtain pET-22b-proC/tgase (SEQ ID NO: 5).
Mutant D189H was obtained by PCR and linear DNA circularization using plasmid pET-22b-proC/tgase as template and 189hf/189hr as primer, and the obtained plasmid was pET-22 b-proC/tgase/D189H. Variant D189H/A287P was obtained by PCR and linear DNA circularization using plasmid pET-22b-proC/tgase/D189H as template and 287mf/287mr as primer, and the resulting plasmid was pET-22 b-proC/tgase/D189H/A287P. All the primers used above are shown in Table 2. PCR Process reference Baori doctor Tech technology (Beijing) Ltd
Figure BDA0002867321470000151
HS DNA Polymerase instruction book, linear DNA cyclization method refer to Baozi doctor technology (Beijing) Limited company Blunting Kit Ligation (BKL) Kit instruction book.
TABLE 2 primer Gene sequences
Figure BDA0002867321470000152
2. Process for preparing mutant enzyme
And (3) transforming the constructed plasmid into Escherichia coli JM109 (whether the Escherichia coli JM109 can be stored in the early stage of a laboratory, if not, the Escherichia coli JM109 can be purchased from Beijing Tiangen Biochemical technology Co., Ltd.), 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 the corresponding glutamine transaminase variant.
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 AKTApure machine, and the protein collection time and amount were determined by observing the A280 wavelength absorbance 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.
The specific enzyme activity, percentage of residual enzyme activity, half-life at 60 ℃ and the like of the parent and the variant were tested, and the results are shown in table 3, fig. 1, fig. 2 and fig. 3.
TABLE 3 kinetic parameters of Streptomyces mobaraensis-derived glutamine transaminase and mutant enzyme
Figure BDA0002867321470000161
From table 3, it can be seen that: variant transglutaminase maturases at 60 ℃ (T) compared to the parent transglutaminase maturase1/2 60℃) The lower half-life period and the dissolving temperature (Tm) are both obviously improved.
From fig. 1, it can be seen that: variant transglutaminase maturases at 60 ℃ (T) compared to the parent transglutaminase maturase1/2 60℃) The half-life period is obviously improved, and the specific enzyme activity is also slightly improved.
From fig. 2, it can be seen that: the parental and variant achieved expression in e.coli BL21, respectively. 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. A clear band can be seen between the molecular weight of 49-62 KDa of the variant D189H/A287P protein, which is similar to the theoretical size of 56.5 KDa.
From fig. 3, it can be seen that: the purified protein has a single band, which indicates that the purity of the protein is consistent with the expectation.
Example 2: application of glutamine transaminase mature enzyme variant in processing of dried rabbit meat slices
The variant transglutaminase maturase (TGase-D189H/A287P) prepared in example 1 was used to process dried rabbit meat slices, 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 0.2h 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 sequences
The sequence of SEQ ID NO 1 is as follows: (maternal, underlined is the zymogen sequence, remainder mature region sequence)
DNGAGEETKSYAETYRLTADDVANINALNESAPAASSAGPSFRAPDPDDRVTPPAEPLDRMPDPYRPVNGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYAKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESLFRNWSEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP
The sequence of SEQ ID NO 2 is as follows: (maternal, underlined is the zymogen sequence, remainder mature region sequence)
GACAATGGCGCGGGGGAAGAGACGAAGTCCTACGCCGAAACCTACCGCCTCACGGCGGATGACGTCGCG AACATCAACGCGCTCAACGAAAGCGCTCCGGCCGCTTCGAGCGCCGGCCCGTCGTTCCGGGCCCCCGATCCTGATGATAGAGTGACCCCACCTGCAGAACCACTGGATAGAATGCCTGATCCATATAGACCTGTGAATGGCAGAGCAGAAACTGTGGTGAACAACTATATTAGAAAATGGCAGCAAGTGTATAGCCATAGAGATGGCAGAAAACAGCAGATGACTGAAGAACAGAGAGAATGGTTAAGCTATGGCTGTGTGGGTGTGACCTGGGTGAACAGTGGTCAGTATCCAACCAACAGACTGGCCTTTGCAAGCTTTGATGAAGATAGATTTAAAAATGAACTGAAAAATGGCAGACCAAGAAGTGGTGAAACTAGAGCAGAATTTGAAGGCAGAGTGGCCAAAGAATCTTTTGATGAAGAGAAGGGCTTTCAGAGAGCAAGAGAAGTGGCAAGTGTGATGAACAGAGCCCTGGAAAATGCCCATGATGAAAGTGCCTATCTGGATAACCTGAAAAAAGAACTGGCCAATGGCAATGATGCCTTAAGAAATGAAGATGCAAGAAGCCCATTTTATAGTGCCCTGAGAAACACCCCAAGCTTTAAAGAAAGAAATGGTGGCAACCATGATCCAAGCAGAATGAAAGCAGTGATTTATGCCAAACATTTTTGGAGTGGCCAAGATAGAAGCAGCAGTGCAGATAAAAGAAAATATGGTGATCCTGATGCCTTTAGACCTGCCCCTGGCACTGGCCTGGTGGATATGAGCAGAGATAGAAACATTCCAAGAAGCCCAACTAGCCCTGGTGAAGGCTTTGTGAACTTTGATTATGGCTGGTTTGGTGCACAGACTGAAGCAGATGCAGATAAAACTGTGTGGACTCATGGCAACCATTATCATGCCCCAAATGGCAGCCTGGGTGCCATGCATGTGTATGAAAGCCTGTTTAGAAACTGGAGTGAAGGCTATAGTGATTTTGATAGAGGTGCCTATGTGATTACCTTTATTCCAAAAAGCTGGAACACTGCCCCTGATAAAGTGAAACAAGGCTGGCCA
The sequence of SEQ ID NO 3 is as follows:
MASGGDEEWEGSYAATHGLTAEDVKNINALNKRALTAGQPGNFPAELPPSATALFRAPD
the sequence of SEQ ID NO 4 is as follows: (the double underlined part is a gene corresponding to the proenzyme region, and the wavy line part is a gene corresponding to the transglutaminase maturase)
Figure BDA0002867321470000181
Figure BDA0002867321470000191
Figure BDA0002867321470000201
The sequence of SEQ ID NO.5 is as follows: (TrxA sequence is underlined in single underline, sequence of proenzyme region after proenzyme substitution is underlined in double underline, sequence of mature region of transglutaminase is underlined in wavy line)
Figure BDA0002867321470000202
Figure BDA0002867321470000211
Figure BDA0002867321470000221
Figure BDA0002867321470000231
SEQUENCE LISTING
<110> university of south of the Yangtze river
Jiangsu Donghui Biotechnology Co.,Ltd.
<120> a transglutaminase variant with improved thermostability
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 376
<212> PRT
<213> Artificial sequence
<400> 1
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 Asp Pro Asp
35 40 45
Asp Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met Pro Asp Pro
50 55 60
Tyr Arg Pro Val Asn Gly Arg Ala Glu Thr Val Val Asn Asn Tyr Ile
65 70 75 80
Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg Lys Gln Gln
85 90 95
Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys Val Gly Val
100 105 110
Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu Ala Phe Ala
115 120 125
Ser Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys Asn Gly Arg Pro
130 135 140
Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val Ala Lys Glu
145 150 155 160
Ser Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg Glu Val Ala Ser
165 170 175
Val Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu Ser Ala Tyr Leu
180 185 190
Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala Leu Arg Asn
195 200 205
Glu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn Thr Pro Ser
210 215 220
Phe Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser Arg Met Lys Ala
225 230 235 240
Val Ile Tyr Ala Lys His Phe Trp Ser Gly Gln Asp Arg Ser Ser Ser
245 250 255
Ala Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg Pro Ala Pro
260 265 270
Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile Pro Arg Ser
275 280 285
Pro Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp Tyr Gly Trp Phe
290 295 300
Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val Trp Thr His Gly
305 310 315 320
Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala Met His Val Tyr
325 330 335
Glu Ser Leu Phe Arg Asn Trp Ser Glu Gly Tyr Ser Asp Phe Asp Arg
340 345 350
Gly Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn Thr Ala Pro
355 360 365
Asp Lys Val Lys Gln Gly Trp Pro
370 375
<210> 2
<211> 1128
<212> DNA
<213> Artificial sequence
<400> 2
gacaatggcg cgggggaaga gacgaagtcc tacgccgaaa cctaccgcct cacggcggat 60
gacgtcgcga acatcaacgc gctcaacgaa agcgctccgg ccgcttcgag cgccggcccg 120
tcgttccggg cccccgatcc tgatgataga gtgaccccac ctgcagaacc actggataga 180
atgcctgatc catatagacc tgtgaatggc agagcagaaa ctgtggtgaa caactatatt 240
agaaaatggc agcaagtgta tagccataga gatggcagaa aacagcagat gactgaagaa 300
cagagagaat ggttaagcta tggctgtgtg ggtgtgacct gggtgaacag tggtcagtat 360
ccaaccaaca gactggcctt tgcaagcttt gatgaagata gatttaaaaa tgaactgaaa 420
aatggcagac caagaagtgg tgaaactaga gcagaatttg aaggcagagt ggccaaagaa 480
tcttttgatg aagagaaggg ctttcagaga gcaagagaag tggcaagtgt gatgaacaga 540
gccctggaaa atgcccatga tgaaagtgcc tatctggata acctgaaaaa agaactggcc 600
aatggcaatg atgccttaag aaatgaagat gcaagaagcc cattttatag tgccctgaga 660
aacaccccaa gctttaaaga aagaaatggt ggcaaccatg atccaagcag aatgaaagca 720
gtgatttatg ccaaacattt ttggagtggc caagatagaa gcagcagtgc agataaaaga 780
aaatatggtg atcctgatgc ctttagacct gcccctggca ctggcctggt ggatatgagc 840
agagatagaa acattccaag aagcccaact agccctggtg aaggctttgt gaactttgat 900
tatggctggt ttggtgcaca gactgaagca gatgcagata aaactgtgtg gactcatggc 960
aaccattatc atgccccaaa tggcagcctg ggtgccatgc atgtgtatga aagcctgttt 1020
agaaactgga gtgaaggcta tagtgatttt gatagaggtg cctatgtgat tacctttatt 1080
ccaaaaagct ggaacactgc ccctgataaa gtgaaacaag gctggcca 1128
<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> 6492
<212> DNA
<213> Artificial sequence
<400> 4
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 acatatggac aatggcgcgg 5220
gggaagagac gaagtcctac gccgaaacct accgcctcac ggcggatgac gtcgcgaaca 5280
tcaacgcgct caacgaaagc gctccggccg cttcgagcgc cggcccgtcg ttccgggccc 5340
ccgatcctga tgatagagtg accccacctg cagaaccact ggatagaatg cctgatccat 5400
atagacctgt gaatggcaga gcagaaactg tggtgaacaa ctatattaga aaatggcagc 5460
aagtgtatag ccatagagat ggcagaaaac agcagatgac tgaagaacag agagaatggt 5520
taagctatgg ctgtgtgggt gtgacctggg tgaacagtgg tcagtatcca accaacagac 5580
tggcctttgc aagctttgat gaagatagat ttaaaaatga actgaaaaat ggcagaccaa 5640
gaagtggtga aactagagca gaatttgaag gcagagtggc caaagaatct tttgatgaag 5700
agaagggctt tcagagagca agagaagtgg caagtgtgat gaacagagcc ctggaaaatg 5760
cccatgatga aagtgcctat ctggataacc tgaaaaaaga actggccaat ggcaatgatg 5820
ccttaagaaa tgaagatgca agaagcccat tttatagtgc cctgagaaac accccaagct 5880
ttaaagaaag aaatggtggc aaccatgatc caagcagaat gaaagcagtg atttatgcca 5940
aacatttttg gagtggccaa gatagaagca gcagtgcaga taaaagaaaa tatggtgatc 6000
ctgatgcctt tagacctgcc cctggcactg gcctggtgga tatgagcaga gatagaaaca 6060
ttccaagaag cccaactagc cctggtgaag gctttgtgaa ctttgattat ggctggtttg 6120
gtgcacagac tgaagcagat gcagataaaa ctgtgtggac tcatggcaac cattatcatg 6180
ccccaaatgg cagcctgggt gccatgcatg tgtatgaaag cctgtttaga aactggagtg 6240
aaggctatag tgattttgat agaggtgcct atgtgattac ctttattcca aaaagctgga 6300
acactgcccc tgataaagtg aaacaaggct ggccacacca ccaccaccac cactgagatc 6360
cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac 6420
tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa 6480
ctatatccgg at 6492
<210> 5
<211> 6858
<212> DNA
<213> Artificial sequence
<400> 5
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
<210> 6
<211> 36
<212> DNA
<213> Artificial sequence
<400> 6
catccaagca gaatgaaagc agtgatttat gccaaa 36
<210> 7
<211> 33
<212> DNA
<213> Artificial sequence
<400> 7
atggttgcca ccatttcttt ctttaaagct tgg 33
<210> 8
<211> 33
<212> DNA
<213> Artificial sequence
<400> 8
cctatgcatg tgtatgaaag cctgtttaga aac 33
<210> 9
<211> 18
<212> DNA
<213> Artificial sequence
<400> 9
acccaggctg ccatttgg 18

Claims (8)

1. A glutamine transaminase variant is obtained by substituting the 1 st to 45 th positions of the polypeptide shown in SEQ ID NO.1 with the sequence shown in SEQ ID NO.3 to obtain a substituted polypeptide, and substituting the 234 th position of the substituted polypeptide with histidine and the 332 th position with proline.
2. A polynucleotide encoding the variant of claim 1.
3. A nucleic acid construct, vector or host cell comprising the polynucleotide of claim 2.
4. A method for producing the variant of claim 1, wherein the method comprises culturing the host cell of claim 3 and recovering the variant of claim 1 from the culture.
5. A composition comprising a mature polypeptide of glutamine transaminase variant of claim 1 from positions 46-376.
6. 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, comprising adding the transglutaminase variant of claim 1 or a mature polypeptide represented by positions 46-376 of the transglutaminase variant of claim 1 to the food processing.
7. 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, said method comprising adding to said food product processing the composition of claim 5.
8. Use of the transglutaminase variant of claim 1 or the composition of claim 5 for food processing, processing and transformation.
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EP2404999A4 (en) * 2009-03-06 2012-01-11 Ajinomoto Kk Thermotolerant transglutaminase originating in actinomyces
CN107574159A (en) * 2017-10-26 2018-01-12 江南大学 A kind of mutant for the glutamine transaminage expressed in an active
CN108103041A (en) * 2018-02-02 2018-06-01 泰兴市东圣生物科技有限公司 A kind of thermostabilization microbial transglutaminase and its encoding gene
CN111386346A (en) * 2017-11-30 2020-07-07 天野酶制品株式会社 Modified transglutaminase
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EP2404999A4 (en) * 2009-03-06 2012-01-11 Ajinomoto Kk Thermotolerant transglutaminase originating in actinomyces
CN107574159A (en) * 2017-10-26 2018-01-12 江南大学 A kind of mutant for the glutamine transaminage expressed in an active
CN111386346A (en) * 2017-11-30 2020-07-07 天野酶制品株式会社 Modified transglutaminase
CN108103041A (en) * 2018-02-02 2018-06-01 泰兴市东圣生物科技有限公司 A kind of thermostabilization microbial transglutaminase and its encoding gene
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