Glutamine transaminase mutant and its application

Abstract

The present invention discloses a glutamine aminotransferase mutant and its application, wherein the amino acid sequence of the mutant is shown in SEQ ID NO.3. In the present invention, by replacing the 1-19 amino acid of MTG derived from Streptomyces marianii with the 1-18 amino acid of commercial MTG, the enzyme activity of the obtained mutant is significantly improved compared with the wild type, which can better meet the needs of industrial production. The SmMTG mutant of the present invention enriches the types of MTG and provides reference and guidance for its industrial application research.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and in particular, relates to a glutamine aminotransferase mutant and a coding gene and application thereof. Background Art

[0002] Transglutaminase (TG, EC 2.3.2.13), also known as transglutaminase, is a transferase that can catalyze the acyl transfer of glutamine residues in γ-carboxyamine and primary amines, or undergo deamidation reactions with water to cause covalent cross-linking of proteins. It can improve the thermal stability and water holding capacity of proteins, help form strong gels, and improve the quality of protein products. Therefore, it is widely used in the texture improvement of traditional protein products such as meat products and dairy products. It is a common food enzyme preparation.

[0003] TG enzymes are widely available and exist in animals, plants, and microorganisms. TG enzymes from animals require Ca 2+ Activation of microbial transglutaminase (MTGase) does not require Ca 2+ It can be activated and directly secreted outside the cell, making it suitable for large-scale industrial production.

[0004] Existing commercial MTG enzymes are all derived from Streptoverticillium mobaraense and have been heterologously expressed in multiple expression systems such as Escherichia coli, Pichia pastoris, and Bacillus subtilis, but the enzyme activity is low (about 120U / g), which limits its application in industry. Therefore, it is very meaningful to develop MTG enzymes with higher enzyme activity. Summary of the invention

[0005] Based on this, the object of the present invention is to provide a glutamine aminotransferase mutant and its encoding gene and application.

[0006] The technical solutions for achieving the above-mentioned invention objectives include the following.

[0007] In a first aspect of the present invention, a glutamine aminotransferase mutant is provided, wherein the amino acid sequence of the mutant is as shown in SEQ ID NO.3.

[0008] The second aspect of the present invention provides a gene encoding a glutamine aminotransferase mutant, the nucleotide sequence of the gene encoding is shown in SEQ ID NO.4.

[0009] The third aspect of the present invention provides the use of the above-mentioned transglutaminase mutant or the gene encoding the transglutaminase mutant in catalyzing acyl transfer or deamidation reaction.

[0010] The fourth aspect of the present invention provides a recombinant expression vector into which the coding gene of the above-mentioned transglutaminase mutant is inserted.

[0011] The fifth aspect of the present invention provides a recombinant engineered strain into which the above-mentioned recombinant expression vector is introduced.

[0012] The sixth aspect of the present invention provides the use of the above-mentioned recombinant expression vector or recombinant engineering strain in catalyzing acyl transfer or deamidation reaction.

[0013] The seventh aspect of the present invention provides a method for catalyzing acyl transfer or deamidation reaction, comprising the following steps: using the above-mentioned glutamine aminotransferase mutant to catalyze the acyl transfer reaction of glutamine residues in γ-carboxamine or primary amine, or to catalyze the deamidation reaction of γ-carboxamine or primary amine with water.

[0014] In the present invention, by replacing the amino acids 1-19 of MTG from Streptomyces marianii with the amino acids 1-18 of commercial MTG, the enzyme activity of the obtained mutant is significantly improved compared with the wild type, which can better meet the needs of industrial production. The SmMTG mutant of the present invention enriches the types of MTG and provides reference and guidance for its industrial application research. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is an expression diagram of each mutant detected by SDS-PAGE in BL21 in Example 2 of the present invention; wherein, 1: wild-type SmMTG; 2: mutant SmMTG-N19; 3: mutant SmMTG-N32; 4: mutant SmMTG-N61.

[0016] Figure 2 This is the standard curve for calculating enzyme activity in Example 3 of the present invention. DETAILED DESCRIPTION

[0017] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0018] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0019] In the present invention, a sequence analysis is first performed in a gene library to obtain the gene sequence of glutamine aminotransferase (SmMTG), and then the gene (amino acid sequence is SEQ ID NO.1, nucleotide sequence is SEQ ID NO.2) is obtained by gene synthesis, and then SmMTG is replaced with the N-terminal partial sequence of commercial MTG (Dongsheng Biotechnology Co., Ltd., amino acid sequence is SEQ ID NO.9) to obtain three mutants SmMTG-N19, SmMTG-N32 and SmMTG-N61, among which SmMTG-N19 is the 1-19 amino acids of SmMTG replaced with the 1-18 positions of the commercial MTG enzyme, and its amino acid sequence is SEQ ID NO.3, and its nucleotide sequence is SEQ ID NO.4; SmMTG-N32 is the 20-32 amino acids of SmMTG replaced with the 19-32 positions of the commercial MTG enzyme, and its amino acid sequence is SEQ ID NO.5, and its nucleotide sequence is SEQ ID NO.6; SmMTG-N61 is a SmMTG enzyme in which the amino acids at positions 33-61 are replaced with those at positions 33-49 of the commercial MTG enzyme. Its amino acid sequence is SEQ ID NO.7 and its nucleotide sequence is SEQ ID NO.8.

[0020] The amino acid sequence of SmMTG (WP_138057969, SEQ ID NO.1):MANRVEGEKRESFAEEHGLTADVRHINALNERALSLGQPDKPSGASLPSAVESPRASAAASGRVTPPAEPLDRMPDAYRTYGGRATTGVSNYIRKWQQVYSHRDGQARQMTEEQREQLSYGCVGVTWVNSGPYPTNKLAFASFDENKYKNALENTTPRPGETQAELEGRIAKESFDEGKGFKRARDVASIMNKALENAH The nucleotide sequence (SEQ ID NO.2):

[0021] ATGGCTAACCGTGTTGAAGGTGAAAAACGTGAATCTTTCGCTGAAGAACA

[0022] TGGCCTGACCGCTGATGATGTTCGTCATATTAACGCGCTGAACGAACGCGC

[0023] GCTGAGCCTGGGTCAGCCGGATAAACCGTCTGGTGCTTCTCTGCCGAGCG

[0024] CGGTTGAATCTCCGCGTGCGTCTGCGGCGGCGAGCGGTCGTGTTACCCCG

[0025] CCGGCGGAACCGCTGGATCGTATGCCGGACGCGTACCGTACCTACGGCGG

[0026] CCGTGCGACTACCGGCGTTAGCAACTACATTCGTAAATGGCAGCAGGTTTA

[0027] CTCTCATCGTGATGGCCAGGCGCGTCAGATGACCGAAGAACAGCGCGAAC

[0028] AGCTGTCTTACGGTTGCGTGGGTGTTACCTGGGTTAACTCTGGTCCGTACC

[0029] CGACCAACAAACTGGCGTTCGCGTCTTTCGATGAAAACAAATACAAAAAC

[0030] GCCCTGGAAAACACCACCCCGCGTCCGGGCGAAACCCAGGCGGAACTGG

[0031] AAGGCCGCATTGCGAAAGAAAGCTTCGATGAAGGTAAAGGTTTCAAACGT

[0032] GCTCGCGATGTTGCGTCTATCATGAACAAAGCGCTGGAAAACGCGCACGA

[0033] TGAAGGCACCTACCTGTCTAACCTGAAAGCGGAACTGACCCGTAACAACG

[0034] ATGCACTGCGTAACGAAGATGCGCGTTCTAACTTCTATAGCGCGCTGCGTA

[0035] ACACTCCGAGCTTCAAAGATGGTGAAGGTGGTGATTACGATCCAAGCCGTA

[0036] TGAAAGCAGTTATTTATTCTAAACATTTCTGGTCTGGTCAGGATCCGAGATC

[0037] TAGCGCTAACAAACGCAAATACGGTGATCCGGAAGCGTTCCGTCCAAATC

[0038] CGTCTACCGGTCTGGTTGATATGAGCAAAGACCGAAACACCTCTCGTTCTC

[0039] CGGGCAACTCTGGCGAAAGCTACGTTAACTTCGATTACGGCTGGTTCGGTG

[0040] ATCAGAAAGAAGAAGATCATGATCAGACCGTTTGGACCCATGCGAACCATT

[0041] ACCATTCTCCGAACGGTGGTATGGGTCCGATGAAAGTTTACGAATCTAAAT

[0042] TCCGTAACTGGTCTGCTGGTTACGCGGATTTCGATCGTGGTACCTATATGAT

[0043] TACCTTCATTCCGAAATCTTGGAACACCGCGCCGGCGAAAGTTACCCAGG

[0044] GTTGGCCGCTCGAG

[0045] Amino acid sequence of mutant SmMTG-N19 (SEQ ID NO.3):

[0046] MDNGAGEETKSYAETYRLADDVRHINALNERALSLGQPDKPSGASLPSAVES

[0047] PRASAAASGRVTPPAEPLDRMPDAYRTYGGRATTGVSNYIRKWQQVYSHRD

[0048] GQARQMTEEQREQLSYGCVGVTWVNSGPYPTNKLAFASFDENKYKNALEN

[0049] TTPRPGETQAELEGRIAKESFDEGKGFKRARDVASIMNKALENAHDEGTYLS

[0050] NLKAELTRNNDALRNEDARSNFYSALRNTPSFKDGEGGDYDPSRMKAVIYSK

[0051] HFWSGQDPRSSANKRKYGDPEAFRPNPSTGLVDMSKDRNTSRSPGNSGESYV

[0052] NFDYGWFGDQKEEDHDQTVWTHANHYHSPNGGMGPMKVYESKFRNWSA

[0053] GYADFDRGTYMITFIPKSWNTAPAKVTQGWPLE

[0054] Nucleotide sequence of mutant SmMTG-N19 (SEQ ID NO.4):

[0055] ATGGACAATGGCGCGGGGGAAGAGACGAAGTCCTACGCCGAAACCTACC

[0056] GCCTCGCTGATGATGTTCGTCATATTAACGCGCTGAACGAACGCGCGCTGA

[0057] GCCTGGGTCAGCCGGATAAACCGTCTGGTGCTTCTCTGCCGAGCGCGGTT

[0058] GAATCTCCGCGTGCGTCTGCGGCGGCGAGCGGTCGTGTTACCCCGCCGGC

[0059] GGAACCGCTGGATCGTATGCCGGACGCGTACCGTACCTACGGCGGCCGTG

[0060] CGACTACCGGCGTTAGCAACTACATTCGTAAATGGCAGCAGGTTTACTCTC

[0061] ATCGTGATGGCCAGGCGCGTCAGATGACCGAAGAACAGCGCGAACAGCTG

[0062] TCTTACGGTTGCGTGGGTGTTACCTGGGTTAACTCTGGTCCGTACCCGACC

[0063] AACAAACTGGCGTTCGCGTCTTTCGATGAAAACAAATACAAAAACGCCCT

[0064] GGAAAACACCACCCCGCGTCCGGGCGAAACCCAGGCGGAACTGGAAGGC

[0065] CGCATTGCGAAAGAAAGCTTCGATGAAGGTAAAGGTTTCAAACGTGCTCG

[0066] CGATGTTGCGTCTATCATGAACAAAGCGCTGGAAAACGCGCACGATGAAG

[0067] GCACCTACCTGTCTAACCTGAAAGCGGAACTGACCCGTAACAACGATGCA

[0068] CTGCGTAACGAAGATGCGCGTTCTAACTTCTATAGCGCGCTGCGTAACACT

[0069] CCGAGCTTCAAAGATGGTGAAGGTGGTGATTACGATCCAAGCCGTATGAA

[0070] AGCAGTTATTTATTCTAAACATTTCTGGTCTGGTCAGGATCCGAGATCTAGC

[0071] GCTAACAAACGCAAATACGGTGATCCGGAAGCGTTCCGTCCAAATCCGTCT

[0072] ACCGGTCTGGTTGATATGAGCAAAGACCGAAACACCTCTCGTTCTCCGGG

[0073] CAACTCTGGCGAAAGCTACGTTAACTTCGATTACGGCTGGTTCGGTGATCA

[0074] GAAAGAAGAAGATCATGATCAGACCGTTTGGACCCATGCGAACCATTACC

[0075] ATTCTCCGAACGGTGGTATGGGTCCGATGAAAGTTTACGAATCTAAATTCC

[0076] GTAACTGGTCTGCTGGTTACGCGGATTTCGATCGTGGTACCTATATGATTAC

[0077] CTTCATTCCGAAATCTTGGAACACCGCGCCGGCGAAAGTTACCCAGGGTT

[0078] GGCCGCTCGAG

[0079] Amino acid sequence of mutant SmMTG-N32 (SEQ ID NO.5):

[0080] MANRVEGEKRESFAEEHGLTTADDVANINALNESALSLGQPDKPSGASLPSAV

[0081] ESPRASAAASGRVTPPAEPLDRMPDAYRTYGGRATTGVSNYIRKWQQVYSHR

[0082] DGQARQMTEEQREQLSYGCVGVTWVNSGPYPTNKLAFASFDENKYKNALE

[0083] NTTPRPGETQAELEGRIAKESFDEGKGFKRARDVASIMNKALENAHDEGTYL

[0084] SNLKAELTRNNDALRNEDARSNFYSALRNTPSFKDGEGGDYDPSRMKAVIYS

[0085] KHFWSGQDPRSSANKRKYGDPEAFRPNPSTGLVDMSKDRNTSRSPGNSGESY

[0086] VNFDYGWFGDQKEEDHDQTVWTHANHYHSPNGGMGPMKVYESKFRNWS

[0087] AGYADFDRGTYMITFIPKSWNTAPAKVTQGWPLE

[0088] Nucleotide sequence of mutant SmMTG-N32 (SEQ ID NO.6):

[0089] ATGGCTAACCGTGTTGAAGGTGAAAAACGTGAATCTTTCGCTGAAGAACA

[0090] TGGCCTGACCACGGCGGATGACGTCGCGAACATCAACGCGCTCAACGAAA

[0091] GCGCGCTGAGCCTGGGTCAGCCGGATAAACCGTCTGGTGCTTCTCTGCCG

[0092] AGCGCGGTTGAATCTCCGCGTGCGTCTGCGGCGGCGAGCGGTCGTGTTAC

[0093] CCCGCCGGCGGAACCGCTGGATCGTATGCCGGACGCGTACCGTACCTACG

[0094] GCGGCCGTGCGACTACCGGCGTTAGCAACTACATTCGTAAATGGCAGCAG

[0095] GTTTACTCTCATCGTGATGGCCAGGCGCGTCAGATGACCGAAGAACAGCG

[0096] CGAACAGCTGTCTTACGGTTGCGTGGGTGTTACCTGGGTTAACTCTGGTCC

[0097] GTACCCGACCAACAAACTGGCGTTCGCGTCTTTCGATGAAAACAAATACA

[0098] AAAACGCCCTGGAAAACACCACCCCGCGTCCGGGCGAAACCCAGGCGGA

[0099] ACTGGAAGGCCGCATTGCGAAAGAAAGCTTCGATGAAGGTAAAGGTTTCA

[0100] AACGTGCTCGCGATGTTGCGTCTATCATGAACAAAGCGCTGGAAAACGCG

[0101] CACGATGAAGGCACCTACCTGTCTAACCTGAAAGCGGAACTGACCCGTAA

[0102] CAACGATGCACTGCGTAACGAAGATGCGCGTTCTAACTTCTATAGCGCGCT

[0103] GCGTAACACTCCGAGCTTCAAAGATGGTGAAGGTGGTGATTACGATCCAA

[0104] GCCGTATGAAAGCAGTTATTTATTCTAAACATTTCTGGTCTGGTCAGGATCC

[0105] GAGATCTAGCGCTAACAAACGCAAATACGGTGATCCGGAAGCGTTCCGTC

[0106] CAAATCCGTCTACCGGTCTGGTTGATATGAGCAAAGACCGAAACACCTCTC

[0107] GTTCTCCGGGCAACTCTGGCGAAAGCTACGTTAACTTCGATTACGGCTGGT

[0108] TCGGTGATCAGAAAGAAGAAGATCATGATCAGACCGTTTGGACCCATGCG

[0109] AACCATTACCATTCTCCGAACGGTGGTATGGGTCCGATGAAAGTTTACGAA

[0110] TCTAAATTCCGTAACTGGTCTGCTGGTTACGCGGATTTCGATCGTGGTACCT

[0111] ATATGATTACCTTCATTCCGAAATCTTGGAACACCGCGCCGGCGAAAGTTA

[0112] CCCAGGGTTGGCCGCTCGAG

[0113] Amino acid sequence of mutant SmMTG - N61 (SEQ ID NO.7):

[0114] MANRVEGEKRESFAEEHGLTADDVRHINALNERAPAASSAGPSFRAPDSDDR

[0115] VTPPAEPLDRMPDAYRTYGGRATTGVSNYIRKWQQVYSHRDGQARQMTEEQ

[0116] REQLSYGCVGVTWVNSGPYPTNKLAFASFDENKYKNALENTTPRPGETQAE

[0117] LEGRIAKESFDEGKGFKRARDVASIMNKALENAHDEGTYLSNLKAELTRNND

[0118] ALRNEDARSNFYSALRNTPSFKDGEGGDYDPSRMKAVIYSKHFWSGQDPRSS

[0119] ANKRKYGDPEAFRPNPSTGLVDMSKDRNTSRSPGNSGESYVNFDYGWFGDQ

[0120] KEEDHDQTVWTHANHYHSPNGGMGPMKVYESKFRNWSAGYADFDRGTYM

[0121] ITFIPKSWNTAPAKVTQGWPLE

[0122] Nucleotide sequence of mutant SmMTG-N61 (SEQ ID NO.8):

[0123] ATGGCTAACCGTGTTGAAGGTGAAAAACGTGAATCTTTCGCTGAAGAACA

[0124] TGGCCTGACCGCTGATGATGTTCGTCATATTAACGCGCTGAACGAACGCGC

[0125] TCCGGCCGCTTCGAGCGCCGGCCCGTCGTTCCGGGCCCCCGACTCCGACG

[0126] ACCGTGTTACCCCGCCGGCGGAACCGCTGGATCGTATGCCGGACGCGTAC

[0127] CGTACCTACGGCGGCCGTGCGACTACCGGCGTTAGCAACTACATTCGTAAA

[0128] TGGCAGCAGGTTTACTCTCATCGTGATGGCCAGGCGCGTCAGATGACCGA

[0129] AGAACAGCGCGAACAGCTGTCTTACGGTTGCGTGGGTGTTACCTGGGTTA

[0130] ACTCTGGTCCGTACCCGACCAACAAACTGGCGTTCGCGTCTTTCGATGAA

[0131] AACAAATACAAAAACGCCCTGGAAAACACCACCCCGCGTCCGGGCGAAA

[0132] CCCAGGCGGAACTGGAAGGCCGCATTGCGAAAGAAAGCTTCGATGAAGG

[0133] TAAAGGTTTCAAACGTGCTCGCGATGTTGCGTCTATCATGAACAAAGCGCT

[0134] GGAAAACGCGCACGATGAAGGCACCTACCTGTCTAACCTGAAAGCGGAAC

[0135] TGACCCGTAACAACGATGCACTGCGTAACGAAGATGCGCGTTCTAACTTCT

[0136] ATAGCGCGCTGCGTAACACTCCGAGCTTCAAAGATGGTGAAGGTGGTGATT

[0137] ACGATCCAAGCCGTATGAAAGCAGTTATTTATTCTAAACATTTCTGGTCTGG

[0138] TCAGGATCCGAGATCTAGCGCTAACAAACGCAAATACGGTGATCCGGAAG

[0139] CGTTCCGTCCAAATCCGTCTACCGGTCTGGTTGATATGAGCAAAGACCGAA

[0140] ACACCTCTCGTTCTCCGGGCAACTCTGGCGAAAGCTACGTTAACTTCGATT

[0141] ACGGCTGGTTCGGTGATCAGAAAGAAGAAGATCATGATCAGACCGTTTGG

[0142] ACCCATGCGAACCATTACCATTCTCCGAACGGTGGTATGGGTCCGATGAAA

[0143] GTTTACGAATCTAAATTCCGTAACTGGTCTGCTGGTTACGCGGATTTCGATC

[0144] GTGGTACCTATATGATTACCTTCATTCCGAAATCTTGGAACACCGCGCCGGC

[0145] GAAAGTTACCCAGGGTTGGCCGCTCGAG

[0146] Amino acid sequence of product MTG (SEQ ID NO.9):

[0147] DNGAGEETKSYAETYRLTADDVANINALNESAPAASSAGPSFRAPDSDDRVTP

[0148] PAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQRE

[0149] WLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFE

[0150] GRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDA

[0151] LRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSA

[0152] DKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTE

[0153] ADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSEGYSDFDRGAYVITF

[0154] IPKSWNTAPDKVKQGWP

[0155] In some embodiments of the present invention, a glutamine aminotransferase mutant is disclosed, and the amino acid sequence of the mutant is shown in SEQ ID NO.3.

[0156] In other embodiments of the present invention, a gene encoding a glutamine transaminase mutant is disclosed.

[0157] In some of the embodiments, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.4.

[0158] In other embodiments of the present invention, disclosed is the use of the above-mentioned transglutaminase mutant or the gene encoding the transglutaminase mutant in catalyzing acyl transfer or deamidation reaction.

[0159] In other embodiments of the present invention, a recombinant expression vector into which the above encoding gene is inserted is disclosed.

[0160] In other embodiments of the present invention, a recombinant engineered bacterium into which the above-mentioned recombinant expression vector is introduced is disclosed.

[0161] In other embodiments of the present invention, a method for constructing the above-mentioned recombinant engineered bacteria is disclosed, comprising the following steps: cloning the coding gene of the above-mentioned glutamine aminotransferase mutant into an expression vector, and transforming Escherichia coli competent cells to obtain.

[0162] In some embodiments, the expression vector is pET22b(+), and the E. coli competent cells are E. coli BL21(DE3) competent cells.

[0163] In other embodiments of the present invention, the use of the above-mentioned recombinant expression vector or recombinant engineered bacteria in catalyzing acyl transfer or deamidation reactions is disclosed.

[0164] In other embodiments of the present invention, a method for catalyzing acyl transfer or deamidation reaction is disclosed, comprising the following steps: using the above-mentioned glutamine aminotransferase mutant to catalyze the acyl transfer reaction of glutamine residues in γ-carboxamine or primary amine, or to catalyze the deamidation reaction of γ-carboxamine or primary amine with water.

[0165] In the following examples, the materials used include: primers and plasmid pET22b(+)-SmMTG synthesized by Sangon Biotech (Shanghai) Co., Ltd.; Escherichia coli TOP10 and BL21 (DE3) competent cells purchased from Weidi Biotechnology Co., Ltd.; SanPrep column plasmid DNA mini-extraction kit, SanPrep column PCR product purification kit, and DNA polymerase (2×PrimerStar Max) were all purchased from Sangon Biotech (Shanghai) Co., Ltd.; restriction endonuclease DpnⅠ was purchased from Thermo Fisher Scientific (China) Co., Ltd.; all chemicals were purchased from Sigma-Aldrich, MacLean Reagents and Aladdin, with the highest purity, and can be used without further purification.

[0166] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0167] Example 1 Construction of SmMTG mutant

[0168] The following steps are involved:

[0169] 1. Design mutant primers as shown in Table 1.

[0170] Table 1

[0171]

[0172] The plasmid pET22b(+)-SmMTG containing SmMTG was used as a template and the primers in Table 1 were used to obtain a plasmid containing a mutant gene by PCR amplification. The PCR amplification system is shown in Table 2, and the PCR amplification program is shown in Table 3.

[0173] Table 2

[0174]

[0175] Table 3

[0176]

[0177] Where T is the annealing temperature, which is selected to be 5°C lower than the Tm value of the corresponding primer.

[0178] After the PCR amplification product was confirmed by 1% agarose gel electrophoresis, the PCR product was digested and purified using restriction endonuclease Dpn I to perform template digestion to remove the original template contained in the system. The digestion system is shown in Table 4.

[0179] Table 4

[0180]

[0181]

[0182] The above system was kept at a constant temperature of 37°C for 30-60 min.

[0183] 2. After 1 hour of enzyme digestion, the enzyme digestion products were purified by PCR and the purified products were confirmed by nucleic acid electrophoresis.

[0184] 3. Mix the purified product with competent E. coli cells TOP10, place on ice for 30 min, heat shock at 42°C for 90 s, and quickly place on ice for 2 min.

[0185] 4. Add the competent cells obtained in the previous step to 1ml LB medium and activate at 37℃, 200rpm for 45min. After activation, centrifuge the competent cells at 8000rpm for 2min, remove most of the medium, resuspend the cells and evenly spread the transformation solution on the LB plate containing 100μg / mL ampicillin. After static culture in a 37℃ incubator overnight, pick a single colony on the plate and inoculate it in 5ml LB liquid medium (containing 100μg / mL Amp), culture overnight at 37℃, 200rpm, and extract the plasmid for use after confirming the amplification results by gene sequencing.

[0186] Example 2 Expression and protein purification of SmMTG mutants

[0187] 1. Expression of mutants

[0188] The plasmids of the SmMTG mutants obtained in Example 1 (hereinafter named SmMTG-N19, SmMTG-N32 and SmMTG-N61) were transformed into Escherichia coli competent cells BL21 (DE3), and the transformation solution was spread on an LB plate containing 100 μg / mL ampicillin and cultured overnight at 37°C. The single colony grown was the expression strain.

[0189] A single colony of the SmMTG mutant was picked and inoculated into 5 ml LB medium and cultured overnight at 37°C and 220 rpm, and then further inoculated into 500 ml autoinduction medium (1% (w / v) peptone, 0.5% (w / v) yeast extract, 0.05% (w / v) glucose, 0.2% (w / v) lactose, 0.5% (w / v) glycerol, 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5 mM Na2SO4, 2 mM MgSO4) for expansion. Culture at 37°C until OD 600When the logarithmic phase is reached, continue culturing for 20 hours before harvesting the bacteria. The collected bacteria are made into a suspension with PBS buffer, broken by ultrasonic instrument, and the broken suspension is centrifuged at 12000rpm for 25min. The supernatant is collected and filtered through a 0.45μm filter membrane to further remove the bacteria and impurities.

[0190] 2. Purification of mutants

[0191] First, equilibrate the Ni column with 3 volumes of PBS buffer, and load the prepared crude enzyme solution; then equilibrate the purification system with PBS buffer, and after equilibration, elute the impurities with PBS buffer with 20mM imidazole. Then elute with elution buffer (300mM imidazole and 500mM NaCl, pH 7.0 PBS buffer), and collect the elution peak, which is the purified enzyme.

[0192] 3. Detection of mutant protein concentration

[0193] The mutant protein concentration was determined using the Brandford protein assay kit. A series of concentrations of bovine serum albumin were prepared as protein concentration standard solutions to prepare a standard curve. 10 μl of standard protein of different concentrations were added to a 96-well plate, followed by 200 μl of Brandford reagent. After reacting at room temperature for 5 minutes, the absorbance was measured at 595 nm. A protein concentration standard curve was obtained based on the known protein concentration and the corresponding absorbance. The absorbance of the mutant protein at 595 nm was measured under the same reaction conditions, and the test was conducted in parallel for more than three times. The mutant protein concentration was obtained by comparing the protein concentration standard curve. The results showed that the purity of all proteins was above 75%.

[0194] Example 3: Dynamic detection of enzyme activity of SmMTG mutants

[0195] The enzyme activity was determined by the hydroxamic acid colorimetric method. Enzyme activity definition: At 37°C, the amount of enzyme required to catalyze the generation of 1 μmol of L-glutamic acid-γ-monohydroxamic acid per minute is defined as one enzyme activity unit, represented by U, i.e. 1U.

[0196] The specific steps are as follows:

[0197] 1. Preparation of reagents

[0198] (1) 0.2 mol / L Tris-Hac: Dissolve 2.42 g Tris in 95 ml ultrapure water, add glacial acetic acid to adjust the dilution to 6.0, and then make up to 100 ml.

[0199] (2) Substrate reagent A solution: Z-Gln-Gly 20 mg, hydroxylamine hydrochloride 13.898 mg, glutathione (reduced form) 6.074 mg, add 0.2 mol / L Tris-HAc buffer 2 ml, store at 4°C, valid for five days.

[0200] (3) Stop reagent solution B: 3 mol / L HCl, 5% ferric chloride solution, and 12% trichloroacetic acid, prepared in a 1:1:1 ratio and stored at 4°C in the dark.

[0201] 2. Drawing of standard curve

[0202] The prepared (20mmol / L) L-glutamic acid-γ-monohydroxamic acid standard solution was gradiently diluted with Tris-HAc buffer at pH 6.0 to obtain standard solutions with concentrations of 0, 0.5, 1, 2, 5, 10, and 15mmol / L. 50μL of the diluted standard solution was placed in clean centrifuge tubes, incubated in a 37℃ water bath for 30min, and then 50μLB solution was added to terminate the reaction and develop color. The color development solution was centrifuged at 12000r / min for 1min, and 100μL of the color development supernatant was taken by a pipette and placed in a 96-well ELISA plate. The absorbance value (A) was measured at a wavelength of 525nm on an ELISA instrument. The concentration of L-glutamic acid-γ-monohydroxamic acid (mmol / ) was used as the horizontal coordinate, and the absorbance value at OD525nm was used as the vertical coordinate to draw the standard curve of the content of L-glutamic acid-γ-monohydroxamic acid.

[0203] 3. Enzyme activity determination

[0204] Take 50μL enzyme solution and place it in a 1.5ml centrifuge tube, add 50μL substrate A solution and mix evenly, put it in a 37℃ water bath for 30min, then add 50μL B solution to terminate the reaction and develop color. Centrifuge at 12000r / min for 1min, pipette 100μL color supernatant and place it in a 96-well ELISA plate, and measure the absorbance of the supernatant at a wavelength of 525nm on the ELISA reader; take another 50μL supernatant and boil it for inactivation, and follow the same steps as above, as a control for zeroing when measuring the absorbance of the sample.

[0205] The results of enzyme activity comparison between wild type and mutant SmMTG are shown in Table 2.

[0206] Table 2 Comparison of enzyme activities of wild type and mutant SmMTG

[0207] SmMTG wild type Commodity MTG SmMTG-N19 SmMTG-N32 SmMTG-61 Specific enzyme activity (U / g) 121 124 196 133 116

[0208] From the results in Table 2, it can be seen that the enzyme activity of the wild type SmMTG is 121U / g, the enzyme activity of the commercial MTG is 124U / g, and the enzyme activity of the mutant SmMTG-N19 is significantly higher than the specific enzyme activities of other mutants, wild type and commercial MTG, which is about 1.6 times the specific enzyme activity of the wild type SmMTG and commercial MTG, and can better meet the needs of industrial production.

[0209] Example 4 Application of SmMTG mutants in food

[0210] 1 kg of raw beef (purchased from the supermarket) was chopped and kneaded into handmade meatballs with a diameter of 3 cm. The meatballs were divided into three groups and different amounts of the mutant SmMTG-N19 were added. The enzyme addition amounts were 0 U / g, 0.2 U / g, and 0.4 U / g, respectively (the blank group added the same volume of water as a control variable). Each meatball was then hand-kneaded for 5 minutes to ensure that the enzyme was evenly mixed. The handmade meatballs were refrigerated at 4°C for 24 hours and boiled in boiling water for 5 minutes to observe whether the meatballs were loose in the boiling water, and to compare the elasticity and chewiness of the meatballs after boiling.

[0211] The experimental results showed that compared with the blank group, the looseness of the meatballs added with 0.2U / g and 0.4U / g mutant SmMTG-N19 was significantly reduced. After boiling, the elasticity and chewiness of the meatballs were significantly improved, and the meatballs added with 0.4U / g mutant SmMTG-N19 had better effects. This shows that mutant SmMTG-N19 can be used to change the gel properties of beef meatballs and enhance the water holding capacity and texture properties of meatballs.

[0212] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0213] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A glutamine aminotransferase mutant, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.

3.

2. A gene encoding the glutamine aminotransferase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

4.

4. A recombinant expression vector into which the coding gene according to claim 2 or 3 is inserted.

5. A recombinant engineered bacterium transformed with the recombinant expression vector according to claim 4.

6. A method for constructing the recombinant engineered bacteria according to claim 5, characterized in that: The method comprises the following steps: cloning the coding gene of the glutamine aminotransferase mutant according to claim 2 or 3 into an expression vector, and transforming competent cells of Escherichia coli to obtain the obtained product.

7. The method according to claim 6, characterized in that The expression vector is pET22b, and the Escherichia coli competent cells are Escherichia coli BL21 (DE3) competent cells.

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