CN103409385A - Method for producing proenzyme of transglutaminase through fermentation - Google Patents
Method for producing proenzyme of transglutaminase through fermentation Download PDFInfo
- Publication number
- CN103409385A CN103409385A CN2013103406898A CN201310340689A CN103409385A CN 103409385 A CN103409385 A CN 103409385A CN 2013103406898 A CN2013103406898 A CN 2013103406898A CN 201310340689 A CN201310340689 A CN 201310340689A CN 103409385 A CN103409385 A CN 103409385A
- Authority
- CN
- China
- Prior art keywords
- transglutaminase
- mutant
- proenzyme
- glycine
- cacl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108060008539 Transglutaminase Proteins 0.000 title claims abstract description 26
- 102000003601 transglutaminase Human genes 0.000 title claims abstract description 26
- 102000010911 Enzyme Precursors Human genes 0.000 title claims abstract description 12
- 108010062466 Enzyme Precursors Proteins 0.000 title claims abstract description 12
- 238000000855 fermentation Methods 0.000 title abstract description 16
- 230000004151 fermentation Effects 0.000 title abstract description 16
- 238000004519 manufacturing process Methods 0.000 title abstract description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 22
- 239000004471 Glycine Substances 0.000 claims abstract description 20
- 239000007788 liquid Substances 0.000 claims abstract description 12
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 10
- 239000001301 oxygen Substances 0.000 claims abstract description 10
- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 6
- 241000894006 Bacteria Species 0.000 claims description 30
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 8
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 102200037914 rs2985684 Human genes 0.000 claims description 6
- 238000012262 fermentative production Methods 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 239000013604 expression vector Substances 0.000 claims description 4
- 235000011187 glycerol Nutrition 0.000 claims description 4
- 238000011144 upstream manufacturing Methods 0.000 claims description 4
- 241000187391 Streptomyces hygroscopicus Species 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 238000010276 construction Methods 0.000 claims description 3
- 239000006052 feed supplement Substances 0.000 claims description 3
- 230000001939 inductive effect Effects 0.000 claims description 3
- 230000000968 intestinal effect Effects 0.000 claims description 3
- 101150025032 13 gene Proteins 0.000 claims description 2
- 239000001888 Peptone Substances 0.000 claims description 2
- 108010080698 Peptones Proteins 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 238000012217 deletion Methods 0.000 claims description 2
- 230000037430 deletion Effects 0.000 claims description 2
- 238000002703 mutagenesis Methods 0.000 claims description 2
- 231100000350 mutagenesis Toxicity 0.000 claims description 2
- 235000019319 peptone Nutrition 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 238000001308 synthesis method Methods 0.000 claims description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 abstract 1
- 230000035772 mutation Effects 0.000 abstract 1
- 239000013589 supplement Substances 0.000 abstract 1
- 101710123874 Protein-glutamine gamma-glutamyltransferase Proteins 0.000 description 30
- 102000004190 Enzymes Human genes 0.000 description 21
- 108090000790 Enzymes Proteins 0.000 description 21
- 108090000623 proteins and genes Proteins 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 10
- 241000588724 Escherichia coli Species 0.000 description 7
- 230000006698 induction Effects 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- 241001052560 Thallis Species 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000009825 accumulation Methods 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 4
- 229910001424 calcium ion Inorganic materials 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000006228 supernatant 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
- 239000003513 alkali Substances 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 230000029142 excretion Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 230000001133 acceleration Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000037358 bacterial metabolism Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
- 108010010779 glutamine-pyruvate aminotransferase Proteins 0.000 description 1
- 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
- 235000003969 glutathione Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000004845 protein aggregation Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006257 total synthesis reaction Methods 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000009941 weaving Methods 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for producing proenzyme of transglutaminase through fermentation; mutation E62D-tag 1 of the proenzyme of the transglutaminase is taken as an original strain, aqua ammonia is used for controlling the pH value to be not less than 7, and in case of dissolved oxygen bounce, feeding is carried out through an index fed-batch manner; glycine and CaC12 are added and constant-speed flow feed liquid supplement is started until induced concentration is reached. After the adoption of the method, the yield of the proenzyme of the transglutaminase is greatly improved.
Description
Technical field
The present invention relates to a kind of method of fermentative production Transglutaminase EC2.3.2.13 proenzyme, belong to the fermentation engineering field.
Background technology
Glutamine of microbe transaminase (EC2.3.2.13; Microbial Transglutaminase; EC2.3.2.13 is called for short MTG) can the catalytic proteins peptide chain in the γ-carboxamide groups of glutamine residue with Methionin ε-acyl group or other acyl groups, react, form ε-(γ-glutamyl) Methionin covalent linkage.Special catalytic capability makes TGase be widely used in the fields such as food engineering, weaving and leather processing, material engineering, biological medicine.But, due to defects such as MTG heterogenous expression secretory volume are low, limited the range of application of MTG.We will transform prior art.
Summary of the invention
The invention provides a kind of method of fermentative production Transglutaminase EC2.3.2.13 proenzyme, technical scheme is as follows: a use ammoniacal liquor is controlled pH and is not less than 7, start index fed-batch mode feed supplement when dissolved oxygen rebounds; During to induced concentration, add glycine and CaCl
2, and start constant speed flow feeding liquid.
The present invention is with mutant E62D-tag1 fermentative production Transglutaminase EC2.3.2.13 proenzyme.
Described mutant E62D-tag1 construction process is as follows:
1) by PCR or the complete synthesis method of chemistry, obtain Streptomyces hygroscopicus CCTCC M203062, obtain Transglutaminase EC2.3.2.13 gene order and upstream and downstream sequence thereof, sequence is as shown in Genbank:EU477523; 2) by deletion mutantion, obtain Del1-4 Transglutaminase EC2.3.2.13 mutant; 3) with in step 2) on the mutant basis that obtains, by rite-directed mutagenesis, E62 is suddenlyd change, obtain mutant Del1-4/E62D Transglutaminase EC2.3.2.13 mutant; 4), on the mutant basis that step 3) obtains, merge tag1 and obtain the Transglutaminase EC2.3.2.13 mutant as shown in SEQ ID NO.1.
Wherein the E62D construction process is as follows;
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, by gene clone or chemical total synthesis method, MTG gene order and upstream and downstream sequence thereof have been obtained, the promotor and the terminator (Genbank:EU477523) that contain MTG self, be cloned into the template of expression plasmid pBB1-1011 in the present invention's transformation using it.
A) take above-mentioned template is material, by SEQ ID NO.3, it is upstream primer, SEQ ID NO.4 is downstream primer, PCR obtains the gene order of TG mutant, the TG mutant gene of acquisition is connected on expression vector pET-22b (+), be transformed into E.coli JM109 and select positive transformant, the mutant plasmid that order-checking is correct transforms expressive host E.coli BL21, obtains mutant Del1-4;
SEQ ID NO.2 is GAGAGGGTGACCCCTCCTGCCGAG
SEQ ID NO.3 is GGGGGCCCGGAAGAGCGCACTG
The PCR reaction conditions is: 72 ℃ of 10min of 95 ℃ 5min95 ℃ 5min65 ℃ 30s72 ℃ 1min40s24 circulation
B) the Del1-4 gene is template, and E62 is suddenlyd change, and primer is as follows.
SEQ ID NO.4 is
GACAGGGTGACCCCTCCTGCCGAG
SEQ ID NO.5 is
GGGGGCCCGGAAGAGCGCACTG
The PCR reaction conditions is: 72 ℃ of 10min of 95 ℃ 5min95 ℃ 5min65 ℃ 30s72 ℃ 7min24 circulation
The TG mutant gene of acquisition is connected to expression vector pET-22b (+) upper, is transformed into E.coli JM109 and selects positive transformant, extract the plasmid order-checking.Sequencing reaction is completed by Shanghai Sheng Gong bio-engineering corporation.The mutant plasmid that order-checking is correct transforms expressive host E.coli BL21, obtains mutant Del1-4/E62D.
Because the C terminal amino acid has vital role to TGase catalytic activity and thermostability, therefore be chosen at TGase enzyme C end and add and stablize small peptide, by increasing it, hold or other regional interactions the raising protein stability with maturing enzyme N.At the PROTEIN C end, add suitable small peptide and can improve protein stability, 7 amino acid are inserted into to Del1-4/E62D glutamine transaminage mutant C end as stablizing small peptide, obtaining corresponding mutant enzyme is Del1-4/E62D-tag1.
The mutant plasmid Del1-4/E62D of take is template, carries out full plasmid PCR.Primer is as follows.
SEQ ID NO.6 is:
ATCGGTTGCATCATCCTGACGCTCGAGCACCACCACCACCACCACTGAG
SEQ ID NO.7 is CGACCAGCCCTGCTTCACCTCG
The PCR reaction conditions is: 72 ℃ of 10min of 95 ℃ 5min95 ℃ 5min65 ℃ 30s72 ℃ 7min24 circulation
The TG mutant gene of acquisition is connected to expression vector pET-22b (+) upper, is transformed into E.coli JM109 and selects positive transformant, extract the plasmid order-checking.Sequencing reaction is completed by Shanghai Sheng Gong bio-engineering corporation.The mutant plasmid that order-checking is correct transforms expressive host E.coli BL21, obtains mutant.
Above-mentioned batch fermentation condition is identical with seed culture, inoculation and initial incubation.
Above-mentionedly with ammoniacal liquor, control pH and be not less than 7, only control acid and do not control alkali.
Above-mentioned adjustment rotating speed 400-800r/min is to maintain dissolved oxygen not higher than 30%.
In aforesaid method, induced concentration is OD
600For 25-80.
In the present invention, induced concentration is OD
600During for 50-80, add stage by stage glycine and CaCl
2.
At the dense OD of bacterium
600Be 50 o'clock, adding final concentration is 75mmol/L glycine and 10mmol/L CaCl
2, as the dense OD of bacterium
600Reaching at 80 o'clock, is 75mmol/L glycine and 10mmol/L CaCl to again adding final concentration in fermented liquid
2.
The present invention adopts the index fed-batch mode to carry out high density fermentation to mutant.
The present invention effectively raises the output of Transglutaminase EC2.3.2.13, for the application of producing two-story valley glutamine transaminase provides convenience.
The accompanying drawing explanation
Fig. 1 SDS-PAGE analyzes the dense impact on protein excretion of not isogeneous induction bacterium;
(M: protein standard molecular weight; 1 and 2: induce the dense OD of bacterium
60025 full cells and fermentation supernatant; 3 and 4: induce the dense OD of bacterium
60050 full cells and fermentation supernatant; 5 and 6: induce the dense OD of bacterium
60075 full cells and fermentation supernatant).
Fig. 2 induces cell concentration impact of output in outer and born of the same parents on thalli growth, pro-TGase born of the same parents;
((a) thalli growth curve, (b) the outer output of pro-TGase born of the same parents, (c) output in pro-TGase born of the same parents, ●: induce OD
600Be 25, zero: induce OD
600Be 50,
: induce OD
600Be 75).
The impact on the pro-TGase fermenting process of Fig. 3 portion-wise addition glycine and calcium ion;
(
: OD; ●: extracellular enzyme is lived; Zero: with the outer total enzyme of born of the same parents, live in born of the same parents).
Embodiment
Substratum
Fermention medium (g/L): glycerine 8; (NH
4)
2HPO
46; KH
2PO
410.5; Citric acid 1.7; MgSO
47H
2O3.4; Trace element 10mL, pH7.0.
Liquid microelement (g/L): FeSO
47H
2O10; ZnSO
47H
2O5.25; CuSO
45H
2O3; MnSO
44H
2O0.5; Na
2B
4O
710H
2O0.23; CaCl
22; (NH
4)
6Mo
7O
240.1.
Fed-batch medium (g/L): glycerine 500; Peptone 15; Yeast powder 30; MgSO
47H
2O30.
The calculation formula of stream rate of acceleration F:
X and S are respectively cell and concentration of substrate, g/L; μ is specific growth rate, h
-1V is fermentating liquid volume, L; S
FFor adding the concentration of substrate, g/L; Y
X/SFor the yield coefficients of cell to substrate, g/g; (VX
0) be the initial cell amount of culture system, g; T is that stream adds the time, h; Wherein μ is set as 0.2h
-1.
While with the synthetic fermention medium of intestinal bacteria, carrying out fed-batch fermentation, seed culture, inoculation and initial incubation condition are identical with batch fermentation, with ammoniacal liquor, control pH and are not less than 7 (only control acid and do not control alkali).Start index stream adds when dissolved oxygen rebounds, and adjusts rotating speed 400-800r/min to maintain dissolved oxygen not higher than 30%, during to induced concentration, when cooling is induced, adds the glycine of 150mmol/L and the CaCl of 20mmol/L
2, and beginning constant speed stream adds.
The mensuration of Transglutaminase EC2.3.2.13 vigor in the present invention:
The colorimetric method for determining enzyme is lived: the N-α-CBZ-GLN-GLY of take is the effect substrate, and 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: in the time of 37 ℃, 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 2mL0.2moL/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%FeCL
3Pressing 1:1:1 mixes.
Pidolidone-γ of preparation 0-4 μ mol/mL-mono-hydroxamic acid standardized solution.Get the Pidolidone-γ of 1mL reagent A and 0.4mL different concns-mono-hydroxamic acid standardized solution and mix, 37 ℃ of water-baths 10 minutes.Add 0.4mL reagent B termination reaction, in the 525nm colorimetric, draw out typical curve.With 0.4mL, replace standardized solution through the enzyme liquid of suitably dilution, insulation and colorimetric, obtain enzyme from typical curve and live under the same conditions.The supernatant liquor of take after 10 minutes centrifugal of 100 ℃ of heating is blank.Enzyme activity (u/mL)=(6.8548 * OD
525-0.0164) * extension rate
Optimal reactive temperature detects: in 20mmol/L Tris damping fluid (pH6.0), measure TGase katalaze enzyme vigor between 30~70 ℃, the highest enzyme activity is defined as to 100%, calculates respectively the enzyme activity of TGase enzyme under condition of different temperatures, determine the TGase optimal reactive temperature.
For improving pro-TGase output, select the index fed-batch mode to realize the mutant built in this laboratory early-stage Study process of mutant E62D-tag1() high density fermentation production Transglutaminase EC2.3.2.13 proenzyme pro-TGase.
The calculation formula of stream rate of acceleration F:
X and S are respectively cell and concentration of substrate, g/L; μ is specific growth rate, h
-1V is fermentating liquid volume, L; S
FFor adding the concentration of substrate, g/L; Y
X/SFor the yield coefficients of cell to substrate, g/g; (VX
0) be the initial cell amount of culture system, g; T is that stream adds the time, h; Wherein μ is set as 0.2h
-1.
While with the synthetic fermention medium of intestinal bacteria, carrying out fed-batch fermentation, seed culture, inoculation and initial incubation condition are identical with batch fermentation, with ammoniacal liquor, control pH and are not less than 7 (only control acid and do not control alkali).Start index stream adds when dissolved oxygen rebounds, and adjusts rotating speed 400-800r/min to maintain dissolved oxygen not higher than 30%, during to induced concentration, when cooling is induced, adds the glycine of 150mmol/L and the CaCl of 20mmol/L
2, and beginning constant speed stream adds.
Adopt fermention medium, start index stream adds when thalli growth to dissolved oxygen rebounds, and makes the thalline Fast Growth, when cell concentration arrives induced concentration, changes constant speed stream into and adds to fermentation ends.
With batch fermentation, compare, above-mentioned feeding method significantly improves biomass, the dense OD of bacterium
600By 23.8, be increased to 123.1.
Expression of recombinant proteins can form certain pressure to bacterial metabolism, is not therefore affecting under the thalli growth prerequisite, selects suitable induction time to produce enzyme to recombinant bacterium and has material impact.Under high-density culturing condition, induce bacterium dense generally higher, but produce pro-TGase for recombination bacillus coli, highlyer induce the dense meeting of bacterium to make the pro-TGase protein aggregation in cell, and can not be secreted into outside born of the same parents.The pro-TGase fermentation research is found, induced the dense OD of bacterium early stage
600Surpass at 20 o'clock, pro-TGase is gathered in born of the same parents and can not be secreted into outside born of the same parents, and need when adding inductor, add final concentration is 150mmol/L glycine and 20mmol/L CaCl
2Just can promote pro-TGase by the secretion outside born of the same parents in born of the same parents.Therefore, this section is chosen and is induced the dense OD of bacterium
600Be 25,50 and 75, when adding inductor, add simultaneously glycine and CaCl
2, to promote protein excretion.
Different induction time thalli growth difference is also not obvious, induces the dense OD of bacterium
600Be 25 o'clock, the dense OD of the highest bacterium
600Be only 108.2, and when inducing the dense OD of bacterium
600Be 50 and 75 o'clock, the dense OD of the highest bacterium
600Be respectively 118.4 and 122.1.But different induction times have material impact to the outer output of pro-TGase born of the same parents, induce the dense OD of bacterium
600Be that the highest extracellular enzyme work of 25,50 and 75 correspondence is respectively 8.2,32.5 and 1.2U/mL.In addition, under different induction time culture condition, in born of the same parents, the pro-TGase accumulation volume is also different, and induction time is slower, and the intracellular protein accumulation is more, when inducing the dense OD of bacterium
600Be 75 o'clock, the interior pro-TGase accumulation of born of the same parents is the highest, reaches 6.8U/mL.Choose extracellular enzyme vertex sample alive under different culture condition, utilize SDS-PAGE to analyze pro-TGase and distribute, as shown in Figure 1, with born of the same parents, obvious pro-TGase band is arranged outward in born of the same parents, wherein induce the dense OD of bacterium
600Be that in 25 and 75 born of the same parents, the pro-TGase band is more obvious, and induce the dense OD of bacterium
600Be 50 o'clock, though the pro-TGase accumulation is arranged in born of the same parents, account for the total protein ratio lower.By the above results, can be found out, restructuring pro-TGase is synthesized different induction times and secretion has material impact, in thalline exponential growth early stage and later stage, induce and all be unfavorable for that recombinant protein is synthetic and secrete, and induce mid-term and can obtain high yield in thalline exponential growth, and most of albumen can be secreted into outside born of the same parents.
Although at the dense OD of bacterium
600Induced in 50 o'clock and can obtain higher pro-TGase output, but in born of the same parents, still have part pro-TGase accumulation, account for 15.4% of the total enzyme work of pro-TGase.For impelling pro-TGase in born of the same parents to be transported to outside born of the same parents, the interpolation strategy of glycine and calcium ion is optimized.
The too high meeting thalline normal growth that affects of glycine and calcium ion concn, concentration is too low less on the protein excretion impact, therefore glycine and calcium ion is joined in fermented liquid at twice.At the dense OD of bacterium
600Be 50 o'clock, adding inductor and final concentration is 75mmol/L glycine and 10mmol/L CaCl
2.As the dense OD of bacterium
600Reaching at 80 o'clock, is 75mmol/L glycine and 10mmol/L CaCl to again adding final concentration in fermented liquid
2.Under this culture condition, the dense OD of bacterium
600Reach as high as 130.7, born of the same parents' the highest enzyme work outward can reach 47.4U/mL, account for that total enzyme lives more than 95%, born of the same parents' high production intensity outward are 1.05U/mL/h.This result shows adds glycine and CaCl stage by stage
2Less on the thalli growth impact, and help the secretion of pro-TGase outside born of the same parents in born of the same parents.
Claims (10)
1. the method for a fermentative production Transglutaminase EC2.3.2.13 proenzyme, is characterized in that, with ammoniacal liquor, controls pH and be not less than 7, start index fed-batch mode feed supplement when dissolved oxygen rebounds; During to induced concentration, add glycine and CaCl
2, and start constant speed flow feeding liquid.
2. method according to claim 1, it is characterized in that, the Transglutaminase EC2.3.2.13 proenzyme mutant E62D-tag1 of take is starting strain, described Transglutaminase EC2.3.2.13 proenzyme mutant E62D-tag1 construction process is as follows: 1) by PCR or the complete synthesis method of chemistry, obtain Streptomyces hygroscopicus CCTCC M203062, obtain Transglutaminase EC2.3.2.13 gene order and upstream and downstream sequence thereof, sequence, as shown in Genbank:EU477523, is cloned into expression vector; 2) by deletion mutantion, obtain Del1-4 Transglutaminase EC2.3.2.13 mutant; 3) with in step 2) on the mutant basis that obtains, by rite-directed mutagenesis, E62 is suddenlyd change, obtain mutant Del1-4/E62D Transglutaminase EC2.3.2.13 mutant; 4) on the mutant basis that step 3) obtains, merge tag1 and obtain the Transglutaminase EC2.3.2.13 mutant as shown in SEQ ID NO.1, transform intestinal bacteria and obtain the engineering bacteria that contains the Transglutaminase EC2.3.2.13 mutant.
3. method according to claim 1, is characterized in that, described adjustment rotating speed 400-800r/min is to maintain dissolved oxygen not higher than 30%.
4. method according to claim 1, is characterized in that, it is 0.2h that index stream adds specific growth rate
-1.
5. method according to claim 1, is characterized in that described induced concentration OD
600For 25-80.
6. method according to claim 5, is characterized in that described induced concentration OD
600For 50-80.
7. method according to claim 1, is characterized in that described fermention medium: glycerine 8g/L, (NH
4)
2HPO
46g/L, KH
2PO
410.5g/L, citric acid 1.7g/L, MgSO
47H
2O3.4g/L, micro-10mL/L, pH7.0.
8. method according to claim 1, is characterized in that described feed supplement liquid: glycerine 500g/L, peptone 15g/L, yeast powder 30g/L, MgSO
4GS
2O30g/L.
9. method according to claim 1, is characterized in that, in the time of described inducing, adds the glycine of 150mmol/L and the CaCl of 20mmol/L
2.
10. method according to claim 9, is characterized in that, at the dense OD of bacterium
600Be 50 o'clock, adding final concentration is 75mmol/L glycine and 10mmol/L CaCl
2, as the dense OD of bacterium
600Reaching at 80 o'clock, is 75mmol/L glycine and 10mmol/L CaCl to again adding final concentration in fermented liquid
2.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201310340689.8A CN103409385B (en) | 2013-08-07 | 2013-08-07 | Method for producing proenzyme of transglutaminase through fermentation |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201310340689.8A CN103409385B (en) | 2013-08-07 | 2013-08-07 | Method for producing proenzyme of transglutaminase through fermentation |
Publications (2)
Publication Number | Publication Date |
---|---|
CN103409385A true CN103409385A (en) | 2013-11-27 |
CN103409385B CN103409385B (en) | 2015-06-03 |
Family
ID=49602427
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201310340689.8A Expired - Fee Related CN103409385B (en) | 2013-08-07 | 2013-08-07 | Method for producing proenzyme of transglutaminase through fermentation |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN103409385B (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104830745A (en) * | 2015-04-28 | 2015-08-12 | 江南大学 | Efficient production method of gamma-aminobutyric acid |
CN107815446A (en) * | 2017-10-16 | 2018-03-20 | 浙江大学 | A kind of high cell density fermentation for recombinating nitrile hydratase Recombinant organism |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1493685A (en) * | 2003-09-05 | 2004-05-05 | 江南大学 | Glutamine transaminase high productive bacteria and its screening method and fermentation method producing glutamine transaminase using said bacterial strain |
CN101698836A (en) * | 2009-11-23 | 2010-04-28 | 江南大学 | Method for supplementing material in the fermentation process of transglutaminase |
CN102660515A (en) * | 2012-05-10 | 2012-09-12 | 江南大学 | Glutamine transaminase with improved enzymatic activity and thermal stability |
-
2013
- 2013-08-07 CN CN201310340689.8A patent/CN103409385B/en not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1493685A (en) * | 2003-09-05 | 2004-05-05 | 江南大学 | Glutamine transaminase high productive bacteria and its screening method and fermentation method producing glutamine transaminase using said bacterial strain |
CN101698836A (en) * | 2009-11-23 | 2010-04-28 | 江南大学 | Method for supplementing material in the fermentation process of transglutaminase |
CN102660515A (en) * | 2012-05-10 | 2012-09-12 | 江南大学 | Glutamine transaminase with improved enzymatic activity and thermal stability |
Non-Patent Citations (2)
Title |
---|
LIU S等: "The pro-region of Streptomyces hygroscopicus transglutaminase affects its secretion by Escherichia coli.", 《FEMS MICROBIOL LETT》, vol. 324, no. 2, 31 December 2011 (2011-12-31), pages 98 - 105 * |
刘松等: "微生物谷氨酰胺转胺酶的表达及分子改造研究进展", 《生物工程学报》, vol. 27, no. 12, 31 December 2011 (2011-12-31), pages 1681 - 1689 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104830745A (en) * | 2015-04-28 | 2015-08-12 | 江南大学 | Efficient production method of gamma-aminobutyric acid |
CN104830745B (en) * | 2015-04-28 | 2018-06-05 | 江南大学 | A kind of method for producing γ-aminobutyric acid |
CN107815446A (en) * | 2017-10-16 | 2018-03-20 | 浙江大学 | A kind of high cell density fermentation for recombinating nitrile hydratase Recombinant organism |
CN107815446B (en) * | 2017-10-16 | 2018-09-28 | 浙江大学 | A kind of fermentation process in high density of recombination nitrile hydratase Recombinant organism |
Also Published As
Publication number | Publication date |
---|---|
CN103409385B (en) | 2015-06-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN101605886B (en) | A microorganism of corynebacterium genus having enhanced l-lysine productivity and a method of producing l-lysine using the same | |
CN107574159A (en) | A kind of mutant for the glutamine transaminage expressed in an active | |
CN106929462B (en) | N-acetylneuraminic acid accumulated recombinant bacillus subtilis and application thereof | |
CN101600796A (en) | Have the coryneform bacteria microorganism belonging to genus of enhanced L-lysine productivity and the method for using described microorganisms producing L-Methionin | |
CN111548979B (en) | Recombinant escherichia coli for synthesizing lactoyl N-neotetraose and construction method and application thereof | |
CN107739734A (en) | The glutamine transaminage mutant that a kind of enzyme activity improves | |
CN105658793A (en) | Microorganism with enhanced l-lysine productivity and method for producing l-lysine by using same | |
CN103555779A (en) | Method for producing gamma-aminobutyric acid through fermentation | |
CN105695383A (en) | Recombinant strain and application thereof | |
CN101831414A (en) | Process for extracellularly producing recombinant alpha-cyclodextrin glucosyltransferase | |
CN105505969A (en) | Method for improving conversion rate of L-threonine and application of method | |
CN112522173A (en) | Engineering bacterium for producing heterologous alkaline protease and construction method thereof | |
CN101531988A (en) | Alkaline pectinase genetic engineering bacteria and construction method thereof | |
KR20130105433A (en) | Method of culturing e. coli for high density | |
CN103409385B (en) | Method for producing proenzyme of transglutaminase through fermentation | |
CN111218437A (en) | High-yield alkaline lipase, gene, strain and application | |
CN105316306A (en) | Fermentation method for efficiently producing keratinase through recombinant escherichia coli | |
CN103421753B (en) | Transglutaminase mutant with improved enzyme activity and thermal stability and construction method of transglutaminase mutant | |
CN116144571B (en) | Bacillus pumilus independent of antibiotics and capable of stabilizing high-yield alpha-amylase, and construction method and application thereof | |
CN102234668B (en) | Preparation of glutamic acid through three stages of fermentation | |
CN111424048A (en) | Gene for expressing acidic β -mannase, vector and application thereof | |
CN102660616A (en) | Method for producing bioactive peptides by utilizing solid state fermentation of soybean meal | |
CN102234667B (en) | Three-stage fermentation preparation of lysine | |
CN116200360B (en) | FutCB mutant and method for biosynthesis of 2' -fucosyllactose | |
CN110484521A (en) | The phytic acid enzyme mutant KsPHY9 and its gene of high thermal stability and application |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150603 |