CN104745545A - Method for efficiently producing L-glutamate oxidase - Google Patents
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Abstract
The invention discloses a method for efficiently producing L-glutamate oxidase and belongs to the technical fields of fermentation engineering and enzyme engineering. In the method disclosed by the invention, the optimal induction condition for L-glutamate oxidase recombinant bacteria is inducted for 5h in the presence of 5g/L lactose at 25-30 DEG C; through researches on different feeding modes of batch feeding fermentation, a manner of combining index feeding with Do-stat is beneficial to increase for a bacteria density; through optimization for induction condition of batch fermentation, inducing lasts for 12h in the presence of 10g/L lactose under the condition of a logarithmic phase OD600 of 40, and the highest enzyme activity reaches 156.1U/mL; high-expression recombinant wet bacteria are added in buffer solution containing 110g/L of glutamic acid with a pH value of 8.0, and converted for 24h at 37 DEG C to produce 107.9g/L of alpha-ketoglutaric acid, the conversion rate is 90% or above, and the needed bacterium solution amount is only 1/50 of that of shake-flask fermentation.
Description
Technical field
The present invention relates to a kind of method of High-efficient Production L-GLOD, belong to fermentation engineering and technical field of enzyme engineering.
Background technology
α-ketoglutaric acid (α-ketoglutarate is called for short α-KG), as a kind of important high level fine chemicals (20-25 ten thousand yuan/ton), is widely used in the industrial circles such as food, medicine, chemical industry and makeup.In field of medicaments, α-KG can alleviate nephrotic kidney burden, reduce complication and promote fast quick-recovery after corrective surgery; Composite with amino acid such as arginine, sportsmen's supplementing energy can be helped fast, be widely used in functional nutrient reinforcer; In addition, due to the chemical property that α-KG is special, chemosynthesis industry is widely used in.Along with the continuous expansion of α-KG Application Areas, the demand of domestic and international market to α-KG is caused constantly to increase.From the data that customs is recognized, current world market reaches more than 50,000 tons to food grade α-KG demand breach, and food grade α-KG market value is 20-25 ten thousand yuan/ton at present, and faces the awkward situation had price but no buyers.The production method of α-KG comprises: chemical synthesis, enzyme catalysis method and microbe fermentation method.At present, industrially producing alpha-KG mainly adopts organic synthesis method, relates to the chemical reaction process of series of complex, thus causes the aspect such as raw material sources, environmental pollution series of problems.Simultaneously owing to there is serious safety problem in chemical method synthesis α-KG process, α-KG is caused to be difficult to be directly used in the fields such as food, medicine and makeup.Therefore, how adopting biotechnology method to prepare the high α-KG of security on a large scale, is the focal issue that domestic and international academia and industrial community are paid close attention to.
This research department has built a strain L-GLOD early stage and has produced bacterial strain FMME089; and utilize Production by Enzymes α-KG can reach good effect; but the acquisition amount of this enzyme is relatively less; therefore the mass-producing of high-density means is utilized efficiently to prepare L-GLOD; reduce α-KG production cost, there is good industrial prospect.
Summary of the invention
The object of the invention is to utilize the mass-producing of high-density means efficiently to prepare L-GLOD.
First object of the present invention is to provide a kind of method of high expression L-GLOD.
Described method expresses the recombination bacillus coli of L-GLOD for production bacterial strain, fermentor tank carries out Fed batch fementation and produces L-GLOD; Described Fed batch fementation refers in batch fermentation process, adopts DO-stat feed profile to carry out feed supplement or the strategy that adopts index feed supplement and DO-stat two kinds of feed profile to combine carries out feed-batch culture.
Described recombination bacillus coli, in one embodiment of the invention, for E.coliBL21, with pET28a expression vector, expresses the recombinant bacterium deriving from the L-GLOD of StreptomycesghanaensisATCC14672.
Described DO-stat feed profile, in one embodiment of the invention, refer to and start feed supplement when the DO of fermenting process raises suddenly, control DO associates feed supplement, just carries out the feed supplement in a moment whenever the parameter value of DO higher than setting.Thus fermenting process dissolved oxygen is controlled in lower scope, such as 10-30%.
In one embodiment of the invention, the relevant parameter of described DO-stat setting is any one value in 20%-30%.
The described method being used alone DO-stat feed supplement, in one embodiment of the invention, specifically: recombination bacillus coli seed liquor accesses in fermentor tank according to the inoculum size of 3-6% by (1), control temperature 35-40 DEG C, rotating speed 300-500rpm, air flow 1-3vvm, before feed supplement, control DO is more than 30%; (2) in the feed-batch culture stage: start feed supplement when DO raises suddenly time, control DO associates feed supplement, whenever DO is higher than the feed supplement just carrying out a moment during the parameter value set; (3) the inducing culture stage: adopt final concentration to be that the lactose of 5g/L-15g/L-is induced when thalline reaches logarithmic growth middle and later periods or stationary phase.
The strategy that described index feed supplement and DO-stat two kinds of feed profile combine, in one embodiment of the invention, refers in batch fermentation process, starts to adopt specific growth rate 0.15-0.45h when DO rises suddenly
-1carry out index feed supplement, maintain DO and be not less than 20% in index feed supplement process, when reaching the restriction of fermentor tank dissolved oxygen, feed profile changes DO-stat into and controls dissolved oxygen not higher than the parameter value of setting, as thalline OD
600lactose is adopted to induce when reaching 30-60.
Described induction, in one embodiment of the invention, inducing temperature is 25-30 DEG C.
Described induction, in one embodiment of the invention, induction time is 8-16h.
Described dissolved oxygen restriction is exactly the maximum dissolved oxygen amount that fermenter equipment can reach.In one embodiment of the invention, the maximum dissolved oxygen that can reach under maximum speed 800-900rpm, air flow 3vvm is referred to.
The method that described two kinds of feed profile combine, in one embodiment of the invention, specifically: by recombination bacillus coli seed liquor according in the inoculum size access fermentor tank of 3-6%, control temperature 35-40 DEG C, rotating speed 300-500rpm, air flow 1-3vvm, before feed supplement, control DO is more than 30%; (2) the index feed-batch culture stage: start when DO rises suddenly to adopt specific growth rate 0.15-0.45h
-1carry out index feed supplement, in index feed supplement process, maintain DO higher than 20%; (3) DO-stat feed phase: when reaching the restriction of fermentor tank dissolved oxygen, feed profile changes DO-stat into and controls dissolved oxygen not higher than the parameter value of setting; (3) the inducing culture stage: as thalline OD
600when reaching 30-60, employing final concentration is that the lactose of 5g/L-15g/L is induced, and inducing temperature is 25-30 DEG C.
The parameter value of described DO-stat setting in one embodiment of the invention, is any one value in 20%-30%.
The method that described two kinds of feed profile combine, in one embodiment of the invention, specifically: the inoculum size with 4% is transferred to containing in fermentor tank, initial fermentation condition is rotating speed 300rpm, temperature 37 DEG C, air flow 1vvm, is start to adopt specific growth rate 0.25h when DO rises suddenly
-1carry out feed supplement, maintain DO higher than reaching the restriction of fermentor tank dissolved oxygen after 30%, 4h by adjusting rotary speed in feed supplement process, feed profile changes DO-stat into and controls dissolved oxygen not higher than 30%, when cell concentration is OD
600adopt the lactose of final concentration 10g/L to induce when=40, at 25-30 DEG C, induce 12h.
In described method, for the fermention medium in fermentor tank, those skilled in the art according to existing Escherichia coli culture medium, can select the substratum of applicable product enzyme or are optimized substratum further completely.
Second object of the present invention is to provide a kind of L-GLOD of obtaining by described method or recombination bacillus coli thalline.
3rd object of the present invention is to provide L-GLOD or recombination bacillus coli thalline is transforming the application of producing in α-ketoglutaric acid.
Described application, in one embodiment of the invention, is with Pidolidone or Sodium Glutamate for substrate, at pH7.0-9.0, at temperature 35-42 DEG C, transforms 18-24h, catalytic production α-ketoglutaric acid.
In one embodiment of the invention, its concentration is 110-135g/L to described substrate.
Beneficial effect of the present invention: the L-GLOD enzyme that 1, application the inventive method obtains is lived and reached more than 35U/mL, reaches as high as 156U/mL, achieves the high expression of L-GLOD; 2, the fermented liquid that obtains of fermenting can be directly used in transform produces α-KG, and substrate conversion efficiency can reach 98.7% and the bacteria liquid that resting cell drops into amasss and is only 1/50 of shaking flask level.
Accompanying drawing explanation
Fig. 1: the optimization of lactose optimal conditions;
Fig. 2: batch fermentation fermentation diagram;
Fig. 3: DO-stat fermentation diagram;
Fig. 4: index fermentation and DO-stat combine fermentation diagram;
Fig. 5: the fermentation diagram finally determining high-density strategy.
Embodiment
The lactose-induced condition optimizing of embodiment 1
Recombinant bacterium FMME089 (the PanqingNiu built with this laboratory, Enzymaticproductionof α-ketoglutaricacidfromL-glutamicacidviaL-glutamateoxidase, JournalofBiotechnology, 2014, be 56-62) bacterial classification that sets out, optimize lactose-induced condition.
1) recombinant bacterial strain is inoculated in LB substratum, OD
600for between 0.5-0.6 time, add 1 respectively, 3,5, the lactose 37 DEG C induction 4h of 7g/L, compare the expression amount of LGOX.Find that the best enzyme of the lactose-induced effect of 5g/L is lived and reach 4.53U/mL (Figure 1A).
2) on the basis of the lactose-induced concentration of optimum, to measure after induction 3 respectively, 4,5, the expression amount of the LGOX of 6h.Result display induction 5h effect is best, and enzyme is lived and reached 4.73U/mL (Figure 1B).
Embodiment 2 batch fermentation
First be inoculated in by recombinant bacterium on LB seed culture medium after 10-12h, the inoculum size with 4% is transferred in the 5L fermentor tank containing 2LLB substratum.Initial fermentation condition is: rotating speed 300rpm, temperature 37 DEG C, air flow 1vvm.As OD600=0.5-0.7, adopt the lactose of final concentration 5g/L to induce, measure cell concentration and the enzyme expression amount alive of different time points, after result display induction, 4h enzyme is lived and is reached maximum value 9.43U/mL.
Embodiment 3 is based on the fed-batch fermentation of DO-stat
Fermentor tank starts that condition is identical with batch fermentation, and before feed supplement, control DO is more than 30%, when DO raises suddenly time, start feed supplement, and control DO associates feed supplement, whenever DO higher than 30% time just carry out the feed supplement in a moment.Mending material liquid component is the glycerine of 500g/L, adopts the lactose of 5g/L to induce when cell concentration no longer increases time.Fermentation 16h, cell concentration OD=47.7, enzyme 35.55U/mL (Fig. 3) alive.
The fed-batch fermentation that embodiment 4 index feed supplement and DO-stat combine
Earlier fermentation condition is identical with batch fermentation, is start to adopt specific growth rate 0.25h when DO rises suddenly
-1carry out feed supplement, in feed supplement process, maintain DO higher than 30% by adjusting rotary speed.Reach the restriction of fermentor tank dissolved oxygen after 4h, feed profile changes DO-stat into, adopts the lactose of total concn 5g/L to induce when cell concentration no longer increases time, and moment monitoring cell concentration and enzyme are lived.The cell concentration OD that fermentation 18h can obtain
600for 92-100, L-GLOD enzyme is lived and is reached 59U/mL (Fig. 4).Therefore determine that feeding strategy is that index feed supplement and DO-stat combine.
The fed-batch fermentation optimization that embodiment 5 index feed supplement and DO-stat combine
On the basis of embodiment 5, induce at logarithmic phase (OD about 40) and logarithmic growth later stage (OD about 60) respectively, fermentation same time finds that logarithmic phase inducible enzyme is lived and reaches 97.77U/mL, higher than the 73.73U/mL in logarithmic phase later stage.
On the basis of logarithmic phase induction, adopt the lactose of different concns to induce, adopt the lactose of 5g/L, 10g/L, 15g/L final concentration to induce respectively, discovery 10g/L higher than other concentration, reaches 156.1U/mL at identical fermentation time inducing effect.Therefore determine optimum high density fermentation strategy, fermentation diagram is shown in Fig. 5.
The application of embodiment 6 enzyme
By the thalline that above-mentioned fermentation obtains, transform for high-density thalline and produce α-KG.
To supernatant be gone after centrifugal for fermented liquid 8000rpm, 5min to obtain thalline, for bioconversion reaction, substrate be the Pidolidone of 110g/L, adopts the phosphate buffered saline buffer of pH7.0, and 37 DEG C transform the output that 24h measures α-KG.Respectively high density product is applied flexibly in transformation experiment with identical LGOX enzyme with shaking flask product employing same volume fermented liquid, identical cell concentration.Result is as shown in table 1, can reach a conclusion: the high density fermentation liquid of 1mL can substitute the changing effect of the thalline that 50mL shake flask fermentation obtains completely, and α-KG output reaches 107.9g/L, and transformation efficiency is up to 98.7%.
The effect of table 1 enzyme
Embodiment 7 is based on the optimization of specific growth rates different before induction setting
On the basis of embodiment 4 and embodiment 5, set 0.15,0.25,0.35,0.45h by different specific growth rate μ
-1carry out feed supplement, index feed supplement terminates to carry out immediately inducing and changes DO-stat mode into carries out feed supplement.Inductive condition exponential phase of growth, the lactose of 10g/L is induced, after result display fermentation 20h, and μ=0.35h
-1obtain best LGOX enzyme to live, reach 176.3U/mL.
Embodiment 8 is used alone DO-stat feed supplement
Ferment by the following method:
(1) access in fermentor tank by recombination bacillus coli seed liquor according to the inoculum size of 3-6%, control temperature 35-40 DEG C, rotating speed 300-500rpm, air flow 1-3vvm, before feed supplement, control DO is more than 30%;
(2) in the feed-batch culture stage: start feed supplement when DO raises suddenly time, control DO associates feed supplement, whenever DO is higher than the feed supplement just carrying out a moment time parameter (arbitrary value in 20-30%) that set;
(3) the inducing culture stage: adopt final concentration to be that the lactose of 5g/L-15g/L-is induced when thalline reaches logarithmic growth middle and later periods or stationary phase.
Live as 30-45U/mL according to the ferment LGOX enzyme that obtains of aforesaid method.
Embodiment 9 index feed supplement and DO-stat combine fed-batch fermentation
Ferment by the following method:
By recombination bacillus coli seed liquor according in the inoculum size access fermentor tank of 3%, control temperature 35 DEG C, rotating speed 300-500rpm, air flow 1-3vvm, before feed supplement, control DO is more than 30%; (2) the index feed-batch culture stage: start when DO rises suddenly to adopt specific growth rate 0.15h
-1carry out index feed supplement, in index feed supplement process, maintain DO higher than 20%; (3) DO-stat feed phase: when reaching the restriction of fermentor tank dissolved oxygen, feed profile changes DO-stat into and controls dissolved oxygen not higher than 20%; (3) the inducing culture stage: as thalline OD
600when reaching 30, employing final concentration is that the lactose of 5g/L is induced, and inducing temperature is 25 DEG C, inducing culture 16h.
Live as 65U/mL according to the ferment LGOX enzyme that obtains of aforesaid method, cell concentration is OD
600=87.
Embodiment 10 index feed supplement and DO-stat combine fed-batch fermentation
Ferment by the following method:
By recombination bacillus coli seed liquor according in the inoculum size access fermentor tank of 6%, control temperature 40 DEG C, rotating speed 300-500rpm, air flow 1-3vvm, before feed supplement, control DO is more than 30%; (2) the index feed-batch culture stage: start when DO rises suddenly to adopt specific growth rate 0.45h
-1carry out index feed supplement, in index feed supplement process, maintain DO higher than 30%; (3) DO-stat feed phase: when reaching the restriction of fermentor tank dissolved oxygen, feed profile changes DO-stat into and controls dissolved oxygen not higher than 25%; (3) the inducing culture stage: as thalline OD
600when reaching 60, employing final concentration is that the lactose of 15g/L is induced, and inducing temperature is 30 DEG C, inducing culture 8h.
Live as 135U/mL according to the ferment LGOX enzyme that obtains of aforesaid method, cell concentration is OD
600=109.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1. a method for high expression L-GLOD, is characterized in that, described method expresses the recombination bacillus coli of L-GLOD for production bacterial strain, fermentor tank carries out Fed batch fementation and produces L-GLOD; Described Fed batch fementation refers in batch fermentation process, adopts DO-stat feed profile to carry out feed supplement or the strategy that adopts index feed supplement and DO-stat two kinds of feed profile to combine carries out feed-batch culture.
2. method according to claim 1, it is characterized in that, described DO-stat feed profile refers to and starts feed supplement when the DO of fermenting process raises suddenly, control DO associates feed supplement, whenever DO is higher than the feed supplement just carrying out a moment during the parameter value set, by feed supplement, fermenting process dissolved oxygen is controlled at 10-30%; The parameter value of described DO-stat setting is any one value in 20%-30%.
3. method according to claim 1, is characterized in that, the strategy that described index feed supplement and DO-stat two kinds of feed profile combine refers in batch fermentation process, starts to adopt specific growth rate 0.15-0.45h when DO rises suddenly
-1carry out index feed supplement, maintain DO and be not less than 20% in index feed supplement process, when reaching the restriction of fermentor tank dissolved oxygen, feed profile changes DO-stat into and controls dissolved oxygen not higher than the parameter value of setting, as thalline OD
600lactose is adopted to induce when reaching 30-60; The parameter value of described DO-stat setting is any one value in 20%-30%.
4. method according to claim 2, it is characterized in that, described method is specifically: (1) by recombination bacillus coli seed liquor according in the inoculum size access fermentor tank of 3-6%, control temperature 35-40 DEG C, rotating speed 300-500rpm, air flow 1-3vvm, before feed supplement, control DO is more than 30%; (2) in the feed-batch culture stage: start feed supplement when DO raises suddenly time, control DO associates feed supplement, whenever DO is higher than the feed supplement just carrying out a moment during the parameter value set; (3) the inducing culture stage: adopt final concentration to be that the lactose of 5g/L-15g/L-is induced when thalline reaches logarithmic growth middle and later periods or stationary phase; The parameter value of described DO-stat setting is any one value in 20%-30%.
5. method according to claim 3, is characterized in that, described method specifically: by recombination bacillus coli seed liquor according to 3-6% inoculum size access fermentor tank in, control temperature 35-40 DEG C, rotating speed 300-500rpm, air flow 1-3vvm, before feed supplement, control DO is more than 30%; (2) the index feed-batch culture stage: start when DO rises suddenly to adopt specific growth rate 0.15-0.45h
-1carry out index feed supplement, in index feed supplement process, maintain DO higher than 20%; (3) DO-stat feed phase: when reaching the restriction of fermentor tank dissolved oxygen, feed profile changes DO-stat into and controls dissolved oxygen not higher than the parameter value of setting; (3) the inducing culture stage: as thalline OD
600when reaching 30-60, employing final concentration is that the lactose of 5g/L-15g/L is induced; The parameter value of described DO-stat setting is any one value in 20%-30%.
6. method according to claim 5, it is characterized in that, described method is specifically: the inoculum size with 4% is transferred to containing in fermentor tank, and initial fermentation condition is rotating speed 300rpm, temperature 37 DEG C, air flow 1vvm, is start to adopt specific growth rate 0.25h when DO rises suddenly
-1carry out feed supplement, maintain DO higher than reaching the restriction of fermentor tank dissolved oxygen after 30%, 4h by adjusting rotary speed in feed supplement process, feed profile changes DO-stat into and controls dissolved oxygen not higher than 30%, when cell concentration is OD
600the lactose of final concentration 10g/L is adopted to induce when=40, induction 12h.
7. method according to claim 1, is characterized in that, described recombination bacillus coli is with E.coli BL21, with pET28a expression vector, expresses the L-GLOD deriving from StreptomycesghanaensisATCC14672.
8. the L-GLOD obtained according to the arbitrary described method of claim 1-7 or recombination bacillus coli thalline.
9. L-GLOD described in claim 8 or recombination bacillus coli thalline are transforming the application of producing in α-ketoglutaric acid.
10. application according to claim 9, is characterized in that, is with Pidolidone or Sodium Glutamate for substrate, at pH 7.0-9.0, temperature 35-42 DEG C, transforms 18-24h, catalytic production α-ketoglutaric acid.
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| CN111349577B (en) * | 2018-12-21 | 2022-07-08 | 中国石油化工股份有限公司 | Culture method of ammonia oxidizing bacteria |
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| CN104745545B (en) | 2018-02-23 |
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