CN103981141A - Strategy for producing acetoin through efficient fermentation based on AR/BDH enzymatic properties of bacillus subtilis - Google Patents
Strategy for producing acetoin through efficient fermentation based on AR/BDH enzymatic properties of bacillus subtilis Download PDFInfo
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- CN103981141A CN103981141A CN201410249909.0A CN201410249909A CN103981141A CN 103981141 A CN103981141 A CN 103981141A CN 201410249909 A CN201410249909 A CN 201410249909A CN 103981141 A CN103981141 A CN 103981141A
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Abstract
The invention relates to a strategy for producing acetoin through efficient fermentation based on AR/BDH enzymatic properties of bacillus subtilis and belongs to the field of genetic engineering and fermentation engineering. Shown by researches on the AR/BDH enzymatic properties of a strain B. subtilis JNA, which is independently screened by our laboratory, has independent intellectual property and can produce acetoin at high yield, the conditions that the optimal pH in the reduction direction of AR/BDH is 6.5 and the optimal pH of the oxidation direction of AR/BDH is 8.5 are discovered for the first time. Based on the enzymatic properties and according to the fermentation strategy, the strain is in the optimal growth pH (6.5) condition at the prophase of fermentation and in the optimal transformation pH (8.0) condition at the anaphase of fermentation, 150g/L glucose can be transformed into 56.8g/L acetoin, the yield of 2,3-butanediol is only 6.1g/L, and the production rate of acetoin reaches 0.59g/(L.h) and is increased by nearly 84%; recombinant B. subtilis which overexpresses AR/BDH consumes 180g/L glucose under the regulation and control of the same fermentation strategy, the yield of acetoin is increased to 73.6g/L, and the production rate finally reaches 0.77g/(L.h), so that the capability of producing acetoin through the anaphase reverse transformation of the target strain is enhanced, and a basis is provided for producing acetoin through microbial industrialized fermentation.
Description
Technical field
Based on subtilis AR/BDH zymologic property, improve an acetoin output fermentation strategies, the invention belongs to genetically engineered and field of fermentation engineering.Be specifically related to a kind of structure and the fermentation strategies based on this enzyme zymologic property raising acetoin output of acetoin reductase enzyme/2,3-butanediol dehydrogenase gene engineering bacteria.
Technical background
Acetoin is in the value that has a wide range of applications aspect food blending, biochemistry and pharmacology, and production synthetic to it has more deep research both at home and abroad at present.Some bacterium of occurring in nature has the ability of production acetoin, mainly comprises Klebsiella (Klebisella), enterobacter (Enterobacter), bacillus (Bacillus), serratia (Serratia) and lactococcus (Lactococcus) etc.But in most of bacterial strain metabolic processes, acetoin exists as the by product of 2,3-butanediol and dimethyl diketone metabolism, and accumulated concentrations is lower, thereby these microbial strains industrial fermentation production acetoins have directly been caused being difficult to utilize.
This laboratory preservation has a strain to take subtilis B.subtilis JNA (the preserving number CCTCC M209309 that glucose is substrate high yield acetoin; Public announcement of a patent application CN101864381A), the phenomenon that exists acetoin and 2,3-butanediol mutually to transform in its fermenting process is found in research, and this mutual conversion process is subject to the adjusting of acetoin reductase enzyme.Acetoin reductase enzyme, claims again 2,3-butanediol desaturase (being called for short AR/BDH, E.C.1.1.1.4).Be under the jurisdiction of oxydo-reductase, can in the situation that NADH exists, catalysis acetoin generate 2,3-butanediol, simultaneously at NAD
+the reversible formation acetoin of catalysis 2,3-butanediol in situation about existing.In subtilis correlative study, though determined the gene of coding acetoin reductase enzyme/2,3-butanediol desaturase, and called after bdhA, its relevant nature is failed further to study.
The present invention, by expression vector pMA5, has realized the high efficient expression of B.subtilis JNA acetoin reductase enzyme (being called for short bdhA), first this enzyme has been carried out to relevant zymologic property research.For the subtilis later stage 2, the mechanism research of the reverse conversion acetoin of 3-butyleneglycol provides the foundation, and based on acetoin reductase enzyme zymologic property, by fermentation strategies, regulate and control, reach shortening fermentation period, the object of strengthening reverse conversion capability of later stage, finally realizes B.subtilis JNA High-efficient Production acetoin.
Summary of the invention
Bacterial classification used herein is B.subtilis JNA, and this bacterial strain prior fermentation glucose synthesizes 2,3-butanediol, and the reverse acetoin that is converted into of later stage, has been preserved in Chinese Typical Representative culture Bao Zang center, preservation address: Wuhan, China, Wuhan University; Protecting a surname is numbered: CTCCM209309.
Main research of the present invention: the present invention utilizes molecular engineering to clone the acetoin reductase gene (being called for short bdhA) from B.subtilis JNA, build recombinant expression vector pMA5-bdhA, and be converted into B.subtilis JNA, successfully built engineering strain B.subtilis JNA/pMA5-bdhA.The engineering strain building is carried out to enzyme activity determination and zymologic property research discovery: enzyme reduction orienting response optimal pH is 6.5, and oxydasis orienting response optimal pH is 8.5.Based on this zymologic property, by fermentation strategies, regulate and control, earlier fermentation makes bacterial strain in optimum growing condition (pH6.5), and the fermentation later stage passes through to strengthen reverse conversion capability (pH8.0), final B.subtilis JNA fermentation 96h, 150g/L conversion of glucose is the about 56.8g/L of acetoin, by product 2,3-butanediol is only 6.1g/L, and acetoin productive rate reaches 0.59g/ (Lh), do not regulate and control to compare with tactful by fermentation, productive rate has improved nearly 84%.Utilize identical fermentation strategies, recombinant bacterial strain B.subtilis JNA/pMA5-bdhA controls the pH6.5 later stage in early stage and controls pH8.0 condition bottom fermentation 96h, the final 180g/L glucose that consumes, acetoin output is increased to about 73.6g/L, and productive rate finally reaches 0.77g/ (Lh).
Advantage of the present invention and positively effect are:
(1) the present invention has explored the zymologic property of the acetoin reductase enzyme in subtilis source first, for the phenomenon of subtilis later stage 2,3-butanediol reverse conversion acetoin provides certain theory value.
(2) the present invention is based on the zymologic property of acetoin reductase enzyme, by fermentation strategies, regulate and control, earlier fermentation makes bacterial strain in optimum growing condition (pH6.5), the fermentation later stage is by strengthening reverse conversion capability (pH8.0), realize the method for High-efficient Production acetoin, final B.subtilis JNA is the about 56.8g/L of acetoin by 150g/L conversion of glucose, by product 2,3-butyleneglycol is only about 6.1g/L, and acetoin productive rate reaches 0.59g/ (Lh), do not regulate and control to compare with tactful by fermentation, productive rate has improved nearly 84%; Recombinant bacterial strain B.subtilis JNA/pMA5-bdhA the most at last 180g/L conversion of glucose is that acetoin is increased to about 73.6g/L, and productive rate finally reaches 0.77g/ (Lh).
Accompanying drawing explanation
Fig. 1 recombinant plasmid pMA5-bdhA schematic diagram.
Fig. 2 SDS-PAGE analyzing proteins purifying situation.
The impact of Fig. 3 pH on enzyme activity.
Fig. 4 bacterial strain B.subtilis JNA is at 5L-tank fermentation diagram.
Fig. 5 bacterial strain B.subtilis JNA/pMA5-bdhA is at 5L-tank fermentation diagram.
Embodiment
Embodiment 1: the amplification of goal gene and the structure of recombined bacillus subtilis
Plasmid pMA5-bdhA builds schematic diagram as shown in Figure 1, and detailed process is as follows:
First, the chromosomal DNA of bacterial strain B.subtilis JNA of take is template, utilize primer P1 and P2, the bdhA gene of nucleotide sequence as shown in SEQ ID NO:1 that increases and obtain one section of 1041bp size by round pcr, by the bdhA gene after purifying after restriction enzyme MluI and BamHI digestion, be connected with the plasmid pMA5 of the above-mentioned two kinds of digestion with restriction enzyme of same process, construction recombination plasmid pMA5-bdhA, at T
4the lower 16 ℃ of connections of spending the night of effect of DNA ligase, are converted into connecting fluid in intestinal bacteria competence E.coli JM109, and picking positive transformant extracts the plasmid in transformant, through enzyme, cuts and verifies and confirm that recombinant plasmid pMA5-bdhA successfully constructs.After double digestion checking, show this construction of recombinant plasmid success.Recombinant plasmid pMA5-bdhA is converted into B.subtilis JNA with the method for chemical conversion, and picking positive transformant, obtains recombined bacillus subtilis B.subtilis JNA/pMA5-bdhA.To original bacterium and recombinant bacterium intracellular protein expression analysis, can find out, gene bdhA successfully realizes overexpression in recombinant bacterium.
The B.subtilis JNA genome DNA of take is template, designs two primers, and the design of pcr amplification primer is as follows:
P1：5’-ACCG
GGATCCATGAAGGCAGCAAGATGG-3’(BamH?I)
P2：5’-ACCG
ACGCGTTTAGTGGTGGTGGTGGTGGTGGTTAGGTCTAACAAGG-3’(Mlu?I)
Embodiment 2: recombinant bacterium B.subtilis JNA/pMA5-bdhA acetoin reductase vitality is measured
(1) enzyme activity determination buffer system
Acetoin reductase enzyme: 0.05M acetoin, 50mM sodium phosphate buffer pH6.5,5mM NAD
+;
2,3-butanediol desaturase: 0.1M2,3-butyleneglycol, 50mM sodium phosphate buffer pH8.0,5mM NADH;
(2) enzyme activity determination
The recombinant bacterium B.subtilis JNA/pMA5-bdhA that embodiment 1 is built, be inoculated in respectively 10mL containing in the LB substratum of kantlex with starting strain B.subtilis JNA, 37 ℃ of shaking culture are spent the night, transfer in LB substratum next day by 4% inoculum size, cultivates 24h, get fermented liquid in 4 ℃ for 37 ℃, the centrifugal 10min of 10000r/min, the sodium phosphate buffer of pH7.0 cleans 3 times, is suspended in Z buffer, and ultrasonic disruption is processed and prepared crude enzyme liquid.20 μ l crude enzyme liquids are joined and in enzyme activity determination buffer system, detect immediately A
340the variation of light absorption value.Result shows the acetoin reductase enzyme AR direction ratio enzyme 89.85U/g of being alive that recombinant bacterium B.subtilis JNA/pMA5-bdhA expresses, than the work of starting strain B.subtilis JNA acetoin reductase enzyme AR direction ratio enzyme, 4 times have been improved, the acetoin reductase enzyme BDH direction ratio enzyme that recombinant bacterium B.subtilis JNA/pMA5-bdhA expresses is lived as 62.23U/g, than the work of starting strain B.subtilis JNA acetoin reductase enzyme BDH direction ratio enzyme, has improved 73 times.
Embodiment 3: acetoin reductase enzyme zymologic property research
Crude enzyme liquid obtains pure AR/BDH after Ni-NTA column purification, the pure enzyme liquid AR direction after purifying than enzyme work, be 140.92U/g, BDH direction ratio enzyme is lived as 112.56U/g, purification reaches 18.18 times, the rate of recovery is 41.95%.
(1) research of optimal pH
Prepare the damping fluid (pH4.5-5.5: 50mM sodium-acetate-acetate buffer of different pH gradients; PH5.5-9.0: 50mM sodium phosphate buffer; PH9.0-10.5: 50mM glycine-sodium hydrate buffer solution, every 0.5pH is a gradient), with 100mM2,3-butyleneglycol and 5mM NAD
+(or 50mM acetoin and 5mM NADH) mixes, and gets the different pH substrate buffer solutions of 2mL and reacts with 20 μ L enzyme liquid, measures the enzyme relative activity under condition of different pH, and compares.Result shows, when pH6.5, this enzyme is the strongest in the activity of reduction direction, and when pH8.5, enzyme is the strongest in the activity of oxidation direction.
(2) research of the pH stability of enzyme
Prepare the damping fluid (pH4.5-5.5: 50mM sodium-acetate-acetate buffer of different pH gradients; PH5.5-9.0: 50mM sodium phosphate buffer; PH9.0-10.5: 50mM glycine-sodium hydrate buffer solution, every 0.5pH is a gradient).Enzyme is deposited in different pH environment, sampled at regular intervals, measure the pH stability of this enzyme.Result shows, at neutral pH environmental field (pH6.0-8.0), acetoin reductase enzyme is comparatively stable.
(3) research of optimum temperuture
Temperature of reaction (25 ℃-65 ℃, every 5 ℃ is a gradient) is set and measures enzyme activity under condition of different temperatures, determine this enzyme reaction optimum temperuture.Result shows, along with the rising of temperature, the activity of enzyme also progressively strengthens, and in the time of 20-40 ℃, the relative enzyme work of reduction direction is below 20%, and the comparison of the growth within the scope of 40-55 ℃ is fast, and oxidation activity in the time of 20 ℃ just more than 40%.In the time of 50-55 ℃, enzymic activity is the highest, and along with the continuation of temperature is risen, enzyme activity sharply declines subsequently.
(4) THERMAL STABILITY
Enzyme liquid is left in respectively under-20 ℃, 0 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃ environment, every 1h sampling, measure the residual activity of enzyme under differing temps, thereby study the thermostability of this enzyme under differing temps.Result shows, under-20 ℃ of environment, this enzyme is the most stable, and along with the rising of temperature, the stability of enzyme progressively weakens, and during to 60 ℃, enzyme activity has been less than 10%.
(5) impact that different metal ion and EDTA live on enzyme
The reaction system that does not add metal ion of take is contrast, and in reaction system, adding respectively final concentration is the Cu of 1mM and 10mM
2+, Mn2
+, Zn
2+, Ca
2+, Mg
2+, Na
+, K
+, Fe
2+, Fe
3+ion and EDTA, study each metal ion and the EDTA impact on enzyme activity.Result shows, Zn
2+, Cu
2+ions enzyme reduction direction vigor has obvious restraining effect, Mg
2+, Mn
2+, Ca
2+ions enzyme oxidation direction vigor has obvious promoter action.
(6) kinetic parameter of enzyme
Respectively under pH6.5, pH8.5 and 40 ℃ of conditions, respectively by changing acetoin, 2,3-butanediol, NADH, NAD
+substrate reactions system final concentration, measure enzyme activity, investigate enzyme reaction speed.With the two counting backward technique mappings of Lineweaver-Burk, try to achieve the K of each substrate of acetoin reductase enzyme
mand V
max.Result shows, the K of acetoin
mfor 0.16mmol/L, V
maxbe 5.54 μ mol/Lmin, the K of 2,3-butanediol
mfor 0.26mmol/L, V
maxbe 2.34 μ mol/Lmin, the K of NADH
mfor 0.044mmol/L, V
maxbe 14.20 μ mol/Lmin, NAD
+k
mfor 0.078mmol/L, V
maxbe 14.04 μ mol/Lmin.
Embodiment 4: improve the research of acetoin output fermentation strategies
(1) seed culture
From activate dull and stereotyped picking list colony inoculation in seed culture medium, 37 ℃ of seed culture temperature, shaking speed 160r/min, incubation time is 12h left and right, seed culture medium forms: yeast extract 5g/L, Tryptones 10g/L, NaCl10g/L glucose 40g/L.
(2) fermentation culture
Initial fermentation culture volume is 2L, and the fermentation culture based component of employing is as follows:
Fermentation culture based component: beef extract 5g/L, corn steep liquor 20g/L, urea 2g/L, glucose 150g/L.Above-mentioned fermention medium is regulated to its pH to 6.5 with the NaOH of 5mol/L, high-temperature sterilization 30min at 121 ℃.
Fermentation condition: above-mentioned cultured seed liquor is inoculated in fermention medium and carries out fermentation culture by 4% inoculum size, 37 ℃ of leavening temperatures, air flow quantity is 120m
3/ hm
3substratum, mixing speed is 300r/min.Control the pH6.5 later stage and control pH8.0 condition bottom fermentation early stage, and timing sampling is measured cell concn, acetoin and 2,3-butanediol output.After fermentation ends, product 2,3-butanediol and gas Chromatographic Determination (GC-1690J gas chromatograph, Hangzhou Ke Xiao chemical industry instrument company) for acetoin in fermented liquid.Chromatographic condition is as follows: capillary column, and in 30m * 0.32mm chromatographic column, stationary liquid is AT.SE-30,, detector is FID, 150 ℃ of column temperatures, and the temperature of vaporizing chamber and detector is 250 ℃, and carrier gas is N
2, flow velocity 0.1Mpa, sample size 2 μ L, adopt external standard method quantitative.Final B.subtilis JNA is that acetoin is about 56.8g/L by 150g/L conversion of glucose, by product 2,3-butanediol is only 6.1g/L, and acetoin productive rate reaches 0.59g/ (Lh), do not regulate and control to compare with tactful by fermentation, productive rate has improved nearly 84%.
(3) feed supplement stream adds fermentation culture
Seed culture medium fermentation culture conditions is identical with embodiment 4 (1) and 4 (2), glucose final concentration is controlled between 10g/L to 15g/L, final B.subtilis JNA fermentation 96h, acetoin output is about 63.2g/L, and productive rate reaches 0.66g/ (Lh).
(3) recombinant bacterial strain feed supplement stream adds fermentation culture
Seed culture medium fermentation culture conditions is identical with embodiment 4 (1) and 4 (2), glucose final concentration is controlled between 10g/L to 15g/L, final B.subtilis JNA/pMA5-bdhA fermentation 96h, acetoin output is increased to and is about 73.6g/L, and productive rate reaches 0.77g/ (Lh).
Claims (5)
1. strengthen the recombined bacillus subtilis (B.subtilis) that acetoin reductase enzyme is expressed for one kind, it is characterized in that: the acetoin reductase gene bdhA shown in SEQ IDNO:1 is cloned into shuttle vectors pMA5 and is configured to restructuring shuttle expression plasmid pMA5-bdhA and is converted in the subtilis B.subtilis JNA that deposit number is CCTCC NO:M209309, thereby its zymologic property is studied.
2. Accessory Right requires to obtain acetoin reductase enzyme/2,3-butanediol desaturase in the recombinant bacterial strain described in 1, it is characterized by: this enzyme reduction orienting response optimal pH is 6.5, and oxydasis orienting response optimal pH is 8.5; This enzyme reaction optimum temperuture is 50-55 ℃; Zn
2+, Cu
2+ions enzyme reduction direction vigor has obvious restraining effect, Mg
2+, Mn
2+, Ca
2+ions enzyme oxidation direction vigor has obvious promoter action.
3. acetoin according to claim 2 reductase enzyme/2, the feature of 3-butanediol dehydrogenation enzyme, be applied to a kind of method that fermentation strategies improves acetoin output, it is characterized in that: earlier fermentation controlled fermentation liquid pH is 6.5, make bacterial strain in the most suitable growth environment, when fermentation enters, stablize the later stage, regulate fermented liquid pH to 8.0, strengthen reverse conversion acetoin ability, finally improve acetoin output.
4. the application of fermentation strategies claimed in claim 3 in fermentative production acetoin, it is characterized in that: fermentation strain B.subtilis JNA in earlier stage controls the pH6.5 later stage and controls pH8.0 condition bottom fermentation 96 hours, 150g/L conversion of glucose is the about 56.8g/L of acetoin, by product 2,3-butyleneglycol is only about 6.1g/L, and acetoin productive rate reaches 0.59g/ (Lh), do not regulate and control to compare with tactful by fermentation, productive rate has improved nearly 84%.
5. bacterial strain claimed in claim 1 application in fermentative production acetoin at fermentation strategies claimed in claim 3, it is characterized in that: bacterial strain B.subtilis JNA/pMA5-bdhA is under fermentation strategies regulation and control, ferment 96 hours, 150g/L conversion of glucose is that acetoin output reaches about 73.6g/L, and productive rate reaches 0.77g/ (Lh).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104403984A (en) * | 2014-12-09 | 2015-03-11 | 江南大学 | Yield improvement method of acetoin by strengthening expression of bacillus subtilis glucose-6-phosphate dehydrogenase |
| CN111705027A (en) * | 2020-06-05 | 2020-09-25 | 江南大学 | Method for producing acetoin by microbial fermentation |
| CN116121158A (en) * | 2022-08-10 | 2023-05-16 | 齐鲁工业大学 | Genetic engineering strain for producing (R, R) -2, 3-butanediol, acetoin and tetramethylpyrazine and application thereof |
-
2014
- 2014-06-05 CN CN201410249909.0A patent/CN103981141A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| XIAN ZHANG，ET AL: "Two-Stage pH Control Strategy Based on the pH Preference of Acetoin Reductase Regulates Acetoin and 2,3-Butanediol Distribution in Bacillus subtilis", 《PLOS ONE》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104403984A (en) * | 2014-12-09 | 2015-03-11 | 江南大学 | Yield improvement method of acetoin by strengthening expression of bacillus subtilis glucose-6-phosphate dehydrogenase |
| CN111705027A (en) * | 2020-06-05 | 2020-09-25 | 江南大学 | Method for producing acetoin by microbial fermentation |
| CN111705027B (en) * | 2020-06-05 | 2021-11-16 | 江南大学 | Method for producing acetoin by microbial fermentation |
| CN116121158A (en) * | 2022-08-10 | 2023-05-16 | 齐鲁工业大学 | Genetic engineering strain for producing (R, R) -2, 3-butanediol, acetoin and tetramethylpyrazine and application thereof |
| CN116121158B (en) * | 2022-08-10 | 2023-09-12 | 齐鲁工业大学 | Genetic engineering strain for producing (R, R) -2, 3-butanediol, acetoin and tetramethylpyrazine and application thereof |
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Application publication date: 20140813 |