CN102994469B - Glutamine transaminase with improved heat stability and application thereof - Google Patents
Glutamine transaminase with improved heat stability and application thereof Download PDFInfo
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- CN102994469B CN102994469B CN201210581790.8A CN201210581790A CN102994469B CN 102994469 B CN102994469 B CN 102994469B CN 201210581790 A CN201210581790 A CN 201210581790A CN 102994469 B CN102994469 B CN 102994469B
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- 108060008539 Transglutaminase Proteins 0.000 claims abstract description 7
- 102000003601 transglutaminase Human genes 0.000 claims abstract description 7
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- 108090000623 proteins and genes Proteins 0.000 claims description 17
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 16
- 241000894006 Bacteria Species 0.000 claims description 12
- 150000001413 amino acids Chemical class 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 8
- 239000013604 expression vector Substances 0.000 claims description 6
- 238000010353 genetic engineering Methods 0.000 claims description 5
- 238000003259 recombinant expression Methods 0.000 claims description 4
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- 101710123874 Protein-glutamine gamma-glutamyltransferase Proteins 0.000 description 4
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- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000000968 intestinal effect Effects 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
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- 239000006228 supernatant Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 102100038095 Protein-glutamine gamma-glutamyltransferase 2 Human genes 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000187391 Streptomyces hygroscopicus Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
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- 239000002054 inoculum Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
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- PMKKIDFHWBBGDA-UHFFFAOYSA-N 2-(2,5-dioxopyrrol-1-yl)ethyl methanesulfonate Chemical compound CS(=O)(=O)OCCN1C(=O)C=CC1=O PMKKIDFHWBBGDA-UHFFFAOYSA-N 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
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- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
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- 102000003929 Transaminases Human genes 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
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- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
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- 235000019319 peptone Nutrition 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
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- 239000011780 sodium chloride Substances 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a glutamine transaminase with improved heat stability and application thereof. High-efficiency expression of microbial transglutaminase (MTG) in escherichia coli serves as a modification platform, a label facilitating improvement of heat stability is added at the C end of MTG maturase, preference is given to IGCIILT to obtain a mutant strain with good enzymatic property, and the heat stability is improved by 2.5 times and 3 times. Modified enzyme is suitable for industrial application, the production cost can be reduced, and the production efficiency can be improved.
Description
Technical field
The present invention relates to a kind of glutamine transaminage, the glutamine transaminage that particularly a kind of thermostability improves.
Background technology
Glutamine of microbe transaminase (Transglutaminase EC 2.3.2.13 full name R-glutaminyl-peptide:amine-γ-glutayle-transferase is called for short MTG) it can cause in protein molecule by forming ε-(γ-glutamyl) Methionin covalent linkage in the intermolecular or molecule of catalytic proteins, intermolecular generation is crosslinked, the hydrolysis of glutamy amido in the connection between protein and amino acid and protein molecule.Unique catalysis that TGase has, makes it in fields such as food, weaving, bio-pharmaceuticals, have a wide range of applications.But because some defects of MTG self are as factors such as thermostabilitys, limited the range of application of MTG.Therefore the expression platform of the MTG based on having obtained in intestinal bacteria, by being added with at C end the label that helps thermostability, carries out molecular modification to MTG, is more suitable for the MTG of industrial application to obtaining zymologic property.
Summary of the invention
For improving the present situation of MTG poor heat stability, this research is by adding label at the C of MTG terminal amino acid, thus the interaction between enhancing C terminal amino acid and other position amino acid of TGase, the thermostability of raising MTG.This research provides a kind of new transformation thinking, by being added with at TGase C end the label that helps thermostability, obtained the mutant strain that thermostability improves, in early-stage Study, this research department filters out the bacterial strain (Streptomyces hygroscopicus CCTCC M203062) of the new product Transglutaminase EC2.3.2.13 of a strain, pass through gene clone method, MTG gene order and upstream and downstream sequence thereof have been obtained, promotor and terminator (Genbank:EU477523) containing MTG self, and realize high efficient expression (the Liu S of LiaoMTGHe Qi proenzyme district in intestinal bacteria, Zhang D, Wang M, Cui W, Chen K, Liu Y, Du G, Chen J, Zhou Z (2011) The pro-region of Streptomyces hygroscopicustransglutaminase affects its secretion by Escherichia coli.FEMS Microbiol Lett 324 (2): 98-105).Based on intestinal bacteria construction platform, we are by adding label at MTG maturing enzyme C end, and described label aminoacid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2, and wherein preferred SEQ ID NO.1, has obtained the good mutant strain of zymologic property.
The genetic engineering bacterium or the transgenic cell line that produce Transglutaminase EC2.3.2.13 are also the scope of protection of present invention.
Another technical problem that the present invention will solve is to provide a kind of construction process that builds described product Transglutaminase EC2.3.2.13 genetic engineering bacterium, it is characterized in that comprising the steps:
1) adopt the gene of glutamine transaminage after the complete synthesis or PCR method clones coding transformation of chemistry;
2) gene step 1) being obtained is connected to coli expression carrier, obtains recombinant expression vector;
3) by step 2) the recombinant expression vector Transformed E .coli E.coli BL 21 that obtains obtains genetic engineering bacterium.Described expression vector is pET-22b (+).
The present invention also provides a kind of method that said gene engineering bacterium fermentation is produced M-Zyme of applying, and take described aminotransierase gene of glutamine engineering bacteria as producing bacterial strain, and 37 ℃, cultivate 12h, be transferred in TB substratum, inoculum size is 3%; Thalline grows to OD
600be 2 o'clock, add IPTG induction, and culture temperature is dropped to 20 ℃, cultivate 48h.
The present invention's substratum used:
LB substratum: Tryptones 10g/L, yeast powder 5g/L, NaCl 10g/L, pH 7.0;
TB substratum: peptone 12g/L, yeast extract paste 24g/L, glycerine 8g/L, 17mmol/L KH
2pO
4, 72mmol/LK
2hPO
4.
The mensuration of Transglutaminase EC2.3.2.13 vigor in the present invention:
Colorimetric method for determining enzyme is lived: take N-α-CBZ-GLN-GLY as effect substrate, the mono-Hydroxylamine HCL of Pidolidone-γ is done typical curve.The Transglutaminase EC2.3.2.13 enzyme work of 1 unit is defined as: during 37 ° of C, per minute catalysis forms the enzyme amount (U/mL) of the mono-Hydroxylamine HCL of 1 μ mol Pidolidone-γ.
N α-CBZ-GLN-GLY of reagent A: 100mg is dissolved in the NaOH solution of 2mL 0.2moL/L, adds the Tris-HC damping fluid 4mL of 0.2mol/L pH6.0,0.1mol/L azanol 2mL, and the reduced glutathion 2mL of 0.01mol/L, and regulate pH to 6.0.
The HCL of reagent B:3mol/L, 12%TCA, 5%FeCL3 presses 1:1:1 and mixes.
Pidolidone-γ-mono-hydroxamic acid standardized solution of preparation 0-4 μ mol/mL.Get 1mL reagent A and mix with Pidolidone-γ-mono-hydroxamic acid standardized solution of 0.4mL different concns, 37 ° of C water-baths 10 minutes.Add 0.4mL reagent B termination reaction, in 525nm colorimetric, draw out typical curve.With 0.4mL, through the enzyme liquid of suitably dilution, replace standardized solution, insulation and colorimetric, obtain enzyme from typical curve and live under the same conditions.The supernatant liquor of take after 100 ° of C heating 10 minutes centrifugal is blank.Enzyme activity (u/mL)=(6.8548 * OD
525-0.0164) * extension rate
The present invention is efficiently expressed as transformation platform with MTG in intestinal bacteria, at MTG maturing enzyme C end, adds label, has obtained the good mutant strain of zymologic property, and thermostability improves respectively 2.5 times and 3 times.Improved enzyme is more suitable for industrial application, can reduce production costs, and enhances productivity.
Embodiment
Embodiment 1: the MTG crystalline structure simulation of streptomyces hygroscopicus source
The TGase crystalline structure of the S.mobaaensis that reported of take is template, the crystalline structure of (http://swissmodel.expasy.org/) simulation S.hygoscopicus TGase in swiss-model website.
Embodiment 2: the glutamine transaminage that thermostability improves
Be on the glutamine transaminage encoding gene basis of announcing at Genbank:EU477523, its C end adds amino acid label, and wherein label aminoacid sequence is shown in SEQ ID NO.1 or SEQ ID NO.2, wherein preferred SEQ ID NO.1.
Embodiment 3: thermostability improves the acquisition (linker9, linker13) of mutant strain
1, adopt the gene Genbank:EU477523 of the complete synthesis or PCR method clones coding glutamine transaminage of chemistry, at MTG C end, add different types of label that contributes to improve thermostability, the gene clone of improved glutamine transaminage is to carrier.
2, by the correct plasmid of order-checking, Transformed E .coli BL 21, selects transformant and is inoculated in LB liquid nutrient medium, and 37 ℃, cultivate 12h, be transferred in TB substratum, inoculum size is 3%.Thalline grows to OD
600be 2 o'clock, add IPTG induction, and culture temperature is dropped to 20 ℃, cultivate 48h.
3, collect fermentation supernatant, detect fermentation supernatant enzyme and live, and sample is carried out to His-ni-sepharose purification.
4, and Km value alive to the ratio enzyme of MTG after purifying measured, and result is as shown in table 1.Experimental result shows, when the ratio enzyme of MTG when MTG maturing enzyme C end adds linker6, lives without obviously changing, and its thermostability improves 2.5 times.When C end adds linker8, the ratio enzyme of MTG is lived and is obviously declined, and its thermostability improves 3 times.Explanation is added with at MTG maturing enzyme C end the thermostability that the label that helps to improve thermostability contributes to improve MTG.
Table 1MTG mutant strain zymologic property.
Claims (3)
1. produce the genetic engineering bacterium of glutamine transaminage, it is characterized in that, described glutamine transaminage is on the amino acid basis of the aminotransierase gene of glutamine coding announced of Genbank:EU477523, its C end adds amino acid label, and the sequence of described amino acid label is as shown in SEQ ID NO.1 or SEQ ID NO.2.
2. described in claim 1, produce the construction process of aminotransierase gene of glutamine engineering bacteria, it is characterized in that comprising the steps:
1) adopt the gene of the complete synthesis or PCR method clones coding glutamine transaminage of chemistry, described Transglutaminase EC2.3.2.13 is on the amino acid basis of the aminotransierase gene of glutamine coding announced of Genbank:EU477523, its C end adds amino acid label, and the sequence of described amino acid label is as shown in SEQ ID NO.1 or SEQ ID NO.2;
2) by step 1) gene that obtains is connected to coli expression carrier, obtains recombinant expression vector;
3) by step 2) the recombinant expression vector Transformed E .coli BL 21 that obtains obtains genetic engineering bacterium.
3. method claimed in claim 2, is characterized in that described expression vector is pET-22b (+).
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103275882B (en) * | 2013-06-07 | 2015-04-15 | 江南大学 | Gene engineering bacteria highly expressing transglutaminase, and applications thereof |
| CN109897838B (en) * | 2015-12-31 | 2020-07-03 | 江南大学 | Glutamine transaminase with improved thermal stability |
| CN107574159B (en) * | 2017-10-26 | 2020-05-08 | 江南大学 | Mutant of glutamine transaminase expressed in active form |
| 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 |
| CN113699129B (en) * | 2021-08-25 | 2023-12-01 | 泰兴市东圣生物科技有限公司 | Glutamine transaminase variants with improved thermostability and catalytic activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62155081A (en) * | 1985-12-27 | 1987-07-10 | Shiseido Co Ltd | Novel microorganism and production of biotin by fermentation with said microorganism |
| EP1409689A2 (en) * | 2001-07-19 | 2004-04-21 | Instytut Biochemii I Biofizyki | A lactococcus gene, the amylolytic enzyme it encodes and its application |
| CN101544969A (en) * | 2008-03-25 | 2009-09-30 | 中国科学院上海生命科学研究院 | Mutant of D-carbamyl hydrolysis enzyme and application thereof |
| CN102719418A (en) * | 2012-03-23 | 2012-10-10 | 广西科学院 | Alpha-amylase truncated body and application thereof |
| CN102746371A (en) * | 2009-05-15 | 2012-10-24 | 善笙生物科技股份有限公司 | Method and system for protein purification |
-
2012
- 2012-12-27 CN CN201210581790.8A patent/CN102994469B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62155081A (en) * | 1985-12-27 | 1987-07-10 | Shiseido Co Ltd | Novel microorganism and production of biotin by fermentation with said microorganism |
| EP1409689A2 (en) * | 2001-07-19 | 2004-04-21 | Instytut Biochemii I Biofizyki | A lactococcus gene, the amylolytic enzyme it encodes and its application |
| CN101544969A (en) * | 2008-03-25 | 2009-09-30 | 中国科学院上海生命科学研究院 | Mutant of D-carbamyl hydrolysis enzyme and application thereof |
| CN102746371A (en) * | 2009-05-15 | 2012-10-24 | 善笙生物科技股份有限公司 | Method and system for protein purification |
| CN102719418A (en) * | 2012-03-23 | 2012-10-10 | 广西科学院 | Alpha-amylase truncated body and application thereof |
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Denomination of invention: A glutamine transaminase with improved thermal stability and its application Granted publication date: 20141203 Pledgee: Jiangsu Changjiang Commercial Bank Co.,Ltd. Taixing Branch Pledgor: JIANGSU YIMING BIOLOGICAL CO.,LTD. Registration number: Y2024980022801 |