CN105420206A - Glutamine transaminase improving heat stability - Google Patents

Glutamine transaminase improving heat stability Download PDF

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CN105420206A
CN105420206A CN201511028185.8A CN201511028185A CN105420206A CN 105420206 A CN105420206 A CN 105420206A CN 201511028185 A CN201511028185 A CN 201511028185A CN 105420206 A CN105420206 A CN 105420206A
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glutamine transaminage
seqidno
enzyme
aminoacid sequence
heat stability
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CN105420206B (en
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刘松
童理明
陈坚
堵国成
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Jiangnan University
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Jiangnan University
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Priority to CN201910171552.1A priority patent/CN109897839B/en
Priority to CN201910174239.3A priority patent/CN109971733B/en
Priority to CN201910171525.4A priority patent/CN109897838B/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)
    • C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII

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  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention relates to glutamine transaminase, in particular to glutamine transaminase improving heat stability. By means of fusion expression of parental short peptide at the end N of maturase, the enzyme heat stability is improved.

Description

The glutamine transaminage that a kind of thermostability improves
Technical field
The present invention relates to a kind of glutamine transaminage, the glutamine transaminage of particularly a kind of thermostability raising.
Background technology
Transglutaminase EC2.3.2.13 (Transglutaminase, EC2.3.2.13, TGase) is that amide group shift reaction can occur one, finally forms the albumen of ε-(γ-glutamyl) Methionin covalent linkage.Based on the existence of above-mentioned catalyzed reaction, TGase can impel between various protein molecule, the hydrolysis of intramolecular crosslinking and glutamine residue, thus improves the various functional propertys of protein, such as emulsifying property, solvability etc.; Meanwhile, TGase by some small-molecule substances such as Methionin etc. is introduced protein, can increase the nutritive value of protein.Therefore, on market, widespread use TGase is as the processing treatment of the foodstuff additive had characteristic in food such as flour products, milk-product, a word used in place name baked goods, meat product and fishery products, and market demand is extremely huge.The catalytic capability that TGase is special makes it in the industrial production such as food, weaving, have widespread use, but its catalytic activity is lower, thermostability is poor, makes TGase receive serious restriction as the application in the industry of excellent catalyzer.Therefore, the focus major part of current research all concentrates on the thermostability how improving TGase.Therefore based on the expression platform in intestinal bacteria obtained, inserting small peptide by the N end at maturing enzyme, to carrying out molecular modification, being more suitable for the TGase of industrial application to obtaining zymologic property.
Self-assembly parents small peptide (SAPs) is alternately distributed by hydrophobic hydrophilic amino acid, spontaneously be assembled into special small peptide, the hydrogel formed to being fixed of macromole such as albumen, can have huge application potential in molectronics, cell cultures, nanometer biotechnology and biological medicine etc.SAPs and albumen terminal fusion, can improve catalytic efficiency and the thermostability of enzyme.
Summary of the invention
The technical problem to be solved in the present invention is the glutamine transaminage obtaining the raising of a kind of thermostability, realized the raising of enzyme heat stability by the short skin of amalgamation and expression parents held at the N of maturing enzyme, sequence carry out lacking as shown in SEQIDNO.1 or on SEQIDNO.1 basis, suddenly change after the aminoacid sequence that remains unchanged of glutamine transaminage enzymic activity.
The aminoacid sequence of described glutamine transaminage can also as shown in SEQIDNO.2, shown in SEQIDNO.3 or shown in SEQIDNO.4.Above-mentioned glutamine transaminage has all merged parents' short peptide sequence at the N of maturing enzyme end.
For the technical problem that solves the problem, the present invention adopts following example:
1, the acquisition of the short skin gene of parents, according to the aminoacid sequence of the short skin of parents, primer designs corresponding DNA sequence dna.
2, with S.hygroscopicuspro-TGase expression plasmid pET-22b (+)/pro-TG (LiuS, ZhangD, WangM, CuiW, ChenK, DuG, ChenJ, ZhouZ.Theorderofexpressionisakeyfactorintheproductionofa ctivetransglutaminaseinEscherichiacolibyco-expressionwit hitspro-peptide.MicrobCellFact, 2011,10 (112): 1-7) be template, carry out full plasmid PCR.
3, pET-22b (+)/pro-SAP-TG connected is turned into JM109, turn after order-checking is correct and enter E.coliBL21 (DE3).
Substratum:
Seed culture medium (LB): yeast powder 5g/L, Tryptones 10g/L, NaCl10g/L, (pH7.0).
Fermention medium (TB): yeast powder 24g/L, Tryptones 12g/L, glycerine 5g/L, K 2hPO 472mmol/L, KH 2pO 417mmol/L, (pH7.0).
Cultural method:
Seed culture condition: LB substratum, uses 250mL shake-flask culture, and liquid amount is 10%, and culture temperature is 37 DEG C, and rotating speed is 220rpm, and incubation time is 10h.
Conditions of flask fermentation: TB substratum, adopt 250mL shaking flask to cultivate, liquid amount is 10%, inoculum size is 3%, and culture temperature is 37 DEG C, and rotating speed is 220rpm, when OD600 reaches 2.0, final concentration is the IPTG induction of 0.4mmol/mL, 20 DEG C of inducing culture 48h.
The heat-staple detection method of object enzyme:
Object enzyme is applied the separation means such as affinity chromatography respectively, obtains electrophoretically pure object enzyme.Object enzyme is incubated at a certain temperature, measures the time that enzyme is lived when comparing just beginning and end insulation required for loss.
Enzyme activity determination method:
Colorimetric method for determining enzyme is lived.Pidolidone-γ-mono-Hydroxylamine HCL is as typical curve, and α-N-CBZ-GLN-GLY is substrate.The TGase enzyme of 1 unit is lived and is defined as: under the condition of 37 DEG C, and the above-mentioned substrate of per minute catalysis synthesizes Pidolidone-γ-mono-Hydroxylamine HCL of 1 μm of ol enzyme amount (U/mL) used.Enzyme activity determination condition: react 10min under 37 DEG C of conditions.
The present invention for platform with the high expression of STG in intestinal bacteria, inserts parents' small peptide to the N end at STG maturing enzyme, obtains the mutant strain of thermally-stabilised raising, thermally-stabilisedly improve 70%, improved enzyme is more suitable for industrial application, can reduce production cost, enhance productivity.
Embodiment
Come by the following examples to illustrate the present invention further, the experimental technique of unreceipted actual conditions in the following example, substantially all operate according to the condition described in common molecular cloning handbook.
The acquisition of embodiment 1 parents small peptide
The aminoacid sequence of the short skin of parents obtains AEAEAKAKAEAEAKAK, by the DNA sequence dna design corresponding to amino acid whose sequence on primer:
Upstream primer M-F:GCAGAAGCAGAAGCGAAAGCCAAAGCGGAGGCGGAAGCTAAGGCTAAACGGG CCCCCGACGCTGC
Downstream primer M-R:GAAGAGCGCACTGACGCTCGGC
Embodiment 2 holds the structure of the recombinant bacterial strain inserting parents' small peptide at maturing enzyme N
With S.hygroscopicuspro-TGase expression plasmid pET-22b (+)/pro-TG for template, carry out full plasmid PCR with primer in above-described embodiment 1.
Embodiment 3 recombinant bacterial strain fermentative production TGase
By plasmid correct for order-checking, called after N, Transformed E .coliBL21, select transformant and be inoculated in LB liquid nutrient medium, 37 DEG C, and cultivate 12h, be transferred in TB substratum, inoculum size is 3%, works as OD 600when reaching 2.0, final concentration is the IPTG induction of 0.4mmol/mL, 20 DEG C of inducing culture 48h.
Embodiment 4 compares the thermally-stabilised of recombinant bacterial strain and wild TGase
Collect fermentation supernatant, detect fermentation supernatant enzyme and live, and His-ni-sepharose purification is carried out to sample, to the K of the TGase after purifying mvalue and t 1/2measure, result is as shown in table 2, and it is thermally-stabilisedly compared wild TGase and improve 71% (table 1) to insert parents small peptide at the N end of maturing enzyme.
Table 1 glutamine transaminase zymologic property compares
Embodiment 5 is held on the basis of insertion parents small peptide at maturing enzyme N and is done rite-directed mutagenesis to P132 site
Utilize site-directed mutagenesis kit, hold on the basis of inserting parents' small peptide P132 site at maturing enzyme N, S150 site, rite-directed mutagenesis is done in P305 site, Y100 site, sports N-P132I, N-S150G, N-Y100M, N-P305Q (SEQIDNO.2-5) transformant respectively and is checked order by the raw work in Shanghai.By the correct plasmid of order-checking, according to described fermentation purifying above, and survey its thermostability (table 2).
Table 2 glutamine transaminase mutant zymetology Nature comparison

Claims (5)

1. a glutamine transaminage for thermally-stabilised raising, it is characterized in that aminoacid sequence carries out lacking as shown in SEQIDNO.1 or on SEQIDNO.1 basis, suddenly change after the aminoacid sequence that remains unchanged of glutamine transaminage enzymic activity.
2. glutamine transaminage according to claim 1, is characterized in that aminoacid sequence is as shown in SEQIDNO.2.
3. glutamine transaminage according to claim 1, is characterized in that aminoacid sequence is as shown in SEQIDNO.3.
4. glutamine transaminage according to claim 1, is characterized in that aminoacid sequence is as shown in SEQIDNO.4.
5. prepare the method for glutamine transaminage described in claim 1, it is characterized in that the N of parents' short peptide fusion expression to glutamine transaminage maturing enzyme to hold.
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CN201511028185.8A CN105420206B (en) 2015-12-31 2015-12-31 A kind of glutamine transaminage that thermal stability improves
CN201910171552.1A CN109897839B (en) 2015-12-31 2015-12-31 Glutamine transaminase with improved thermal stability
CN201910174239.3A CN109971733B (en) 2015-12-31 2015-12-31 Glutamine transaminase with improved thermal stability
CN201910171525.4A CN109897838B (en) 2015-12-31 2015-12-31 Glutamine transaminase with improved thermal stability

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN109852602A (en) * 2019-01-11 2019-06-07 江南大学 A method of improving enzyme stability
CN111593038A (en) * 2020-06-23 2020-08-28 江南大学 Glutaminase mutant with improved stability
CN114149987A (en) * 2021-12-07 2022-03-08 安徽大学 Artificially-modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis

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Publication number Priority date Publication date Assignee Title
CN110241063B (en) * 2019-06-28 2021-01-29 江南大学 Method for enhancing salt tolerance of glutaminase

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CN102994469A (en) * 2012-12-27 2013-03-27 江南大学 Glutamine transaminase with improved heat stability and application thereof

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CN102660515B (en) * 2012-05-10 2013-12-11 江南大学 Glutamine transaminase with improved enzymatic activity and thermal stability

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN109852602A (en) * 2019-01-11 2019-06-07 江南大学 A method of improving enzyme stability
CN111593038A (en) * 2020-06-23 2020-08-28 江南大学 Glutaminase mutant with improved stability
CN111593038B (en) * 2020-06-23 2022-02-15 江南大学 Glutaminase mutant with improved stability
CN114149987A (en) * 2021-12-07 2022-03-08 安徽大学 Artificially-modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis
CN114149987B (en) * 2021-12-07 2024-02-13 安徽大学 Artificially modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis

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CN109897838A (en) 2019-06-18
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CN109897839B (en) 2021-03-02
CN109971733B (en) 2020-11-06
CN109897839A (en) 2019-06-18
CN109971733A (en) 2019-07-05

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