CN103409385A - Method for producing proenzyme of transglutaminase through fermentation - Google Patents

Method for producing proenzyme of transglutaminase through fermentation Download PDF

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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
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transglutaminase
mutant
proenzyme
glycine
cacl
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CN103409385B (en
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陈坚
王广圣
陈康康
刘松
堵国成
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Jiangnan University
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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

A kind of method of fermentative production Transglutaminase EC2.3.2.13 proenzyme
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,
Figure BDA00003630790900031
: 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:
F = μ ( VX 0 ) Y X / S ( S F - S ) exp ( μt ) - - - ( 1 )
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.
Embodiment 1
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:
F = μ ( VX 0 ) Y X / S ( S F - S ) exp ( μt ) - - - ( 1 )
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.
Embodiment 2
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.
Embodiment 3
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.
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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

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* Cited by examiner, † Cited by third party
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

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