CN104017758A - Method for producing acetoin through high-efficiency fermentation by appropriately expressing novel bacillus subtilis NADH oxidizing enzyme - Google Patents
Method for producing acetoin through high-efficiency fermentation by appropriately expressing novel bacillus subtilis NADH oxidizing enzyme Download PDFInfo
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Abstract
The invention relates to a method for producing acetoin through high-efficiency fermentation by appropriately expressing a novel bacillus subtilis NADH oxidizing enzyme and belongs to the fields of genetic engineering and fermentation engineering. According to the method, the noval bacillus subtilis NADH oxidizing enzyme is found for the first time in genome of a high-yield acetoin bacterial strain B.subtilis JNA which is autonomously screened by a laboratory and has independent intellectual property, the enzymatic property of the noval bacillus subtilis NADH oxidizing enzyme is determined while an NADH oxidizing enzyme is expressed in bdhA gene deleted bacillus subtilis by appropriately expressing a carrier pMA5-PbdhA, finally the acetoin is produced through fermentation by utilizing recombinant bacteria modified by metabolic engineering, about 56.7g/L acetoin and 1.2g/L of 2,3-butanediol are obtained, the contents of 2,3-butanediol, lactic acid and acetic acid are respectively reduced by about 92.3%, 70.1% and 75.0%, the production efficiency of the acetoin is increased to 0.68g/(L.h), and a fermentation period is shortened for 1.7 times compared with the original bacterial strain; NADH regulation in bacillus subtilis is utilized for producing the acetoin for the first time at home and abroad, and the aims of shortening the fermentation period and reducing a NADH-dependent by-product are realized.
Description
Technical field
Utilize appropriateness to express novel nadh oxidase and reduce NADH dependent form by product raising acetoin output, the invention belongs to genetically engineered and field of fermentation engineering.Be specifically related to a kind of screening of novel nadh oxidase, the structure of genetic engineering bacterium and fermentative production thereof.
Technical background
Acetoin is natural to be present in the numerous food products such as corn, grape, cocoa, apple, banana, cheese, meat.Be be a kind ofly widely used, charming flavouring agent, there is strong cream, fat, butter sample fragrance, after high dilution, have pleasant milk fragrance, so acetoin is mainly used in configuring the essence of milk flavoured type, meat odor type, strawberry odor type, or is directly used in milk preparation.China standard GB/T 2760-86 clearly stipulates that it is the food grade spices that allows use, and U.S. food and extraction association (FEMA) security number are 2008.
At present, traditional fermentation technique has become the study hotspot of China's present stage flavouring agent industry.Utilize the ultimate principle of microorganism fermentative production spices to be: the katalysis that the enzyme producing in self metabolism by microorganism and microorganism growth process carries out, biochemical reaction through series of complex, makes various organism be converted into the multiple compound with aromatic odour.Research shows that some bacterium has the ability of production acetoin both at home and abroad at present, 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.Deep research.
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), find under study for action its fermenting process meeting forming section NADH dependent form by product, as lactic acid, ethanol and 2,3-butyleneglycol, cause acetoin output further to improve, therefore consider to reduce by NADH level in reduction born of the same parents the generation of these by products.Nadh oxidase (NOX, EC1.6.99.3) can form NAD by catalyzing N ADH
+, so this enzyme is widely used above the rearrangement of NADH dependent form pathways metabolism flow.But so far, the research about NOX in subtilis have not been reported.
Reported first of the present invention come from the novel nadh oxidase from subtilis, when determining this enzyme relevant nature, the subtilis appropriateness expression vector pMA5-P building by this laboratory
bdhA(public announcement of a patent application CN102876703A) expresses nadh oxidase in bdhA Gene Deletion bacterial strain.In bdhA Gene Deletion bacterial strain, utilize appropriateness to express novel nadh oxidase, reach shortening fermentation period, reduce the object of NADH dependent form by product, finally realize the engineering strain High-efficient Production acetoin that appropriateness is expressed NOX.
Summary of the invention
Original strain used in the present invention 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 collection center, preservation address: Wuhan, China, Wuhan University; Deposit number is: CTCCM209309.
Main research of the present invention: the present invention utilizes molecular engineering to clone the nadh oxidase (being called for short NOX) from 4 supposition of subtilis, build recombinant expression vector and be converted into B.subtilis JNA, by enzyme activity determination and relevant zymetology Property Identification, determine the nadh oxidase that the yodC gene of source subtilis is coding formation water.Build appropriate expression vector pMA5-P
bdhA-yodC, and be converted into bdhA Gene Deletion B.subtilis JNA, successfully built the engineering strain of appropriate expression NOX.Finally, utilization appropriateness in bdhA Gene Deletion bacterial strain is expressed nadh oxidase fermentative production acetoin, 150g/L conversion of glucose is about to 2 of the acetoin of 56.7g/L and 1.2g/L, 3-butyleneglycol, 2,3-butyleneglycol, lactic acid and acetic acid reduce respectively 92.3%, 70.1% and 75.0%, and acetoin production efficiency rises to 0.68g/ (Lh), compared with original strain B.subtilis JNA fermentation period, shorten 1.7 times.
Advantage of the present invention and positively effect are:
(1) reported first of the present invention from the novel nadh oxidase of subtilis, and determined this enzyme zymetology relevant nature, for fermentation of bacillus subtilis production acetoin reduces NADH dependent form by product, provide certain theory may.
(2) the present invention expresses nadh oxidase by appropriateness in bdhA Gene Deletion bacterial strain, realize the method for High-efficient Production acetoin, 2 of the acetoin that the engineering strain that final appropriateness is expressed nadh oxidase is about 56.7g/L by 150g/L conversion of glucose and 1.2g/L, 3-butyleneglycol, 2,3-butyleneglycol, lactic acid and acetic acid reduce respectively approximately 92.3%, 70.1% and 75.0%, acetoin production efficiency rises to about 0.68g/ (Lh), compared with original strain B.subtilis JNA fermentation period, shortens 1.7 times.
Accompanying drawing explanation
Fig. 1 plasmid T-bdhA::cat builds schematic diagram.
Fig. 2 SDS-PAGE checking NADH enzyme purifying in B.subtilis JNA.
M, albumen Marker; 1, B.subtilis JNA; 2, B.subtilis JNA/pMA5-yodC crude enzyme liquid; 3, B.subtilis JNA/pMA5 crude enzyme liquid; 4-8, the pure enzyme of the NOX of wash-out under different imidazole concentrations, wherein imidazole concentration is followed successively by 0mmol/L, 20mmol/L, 50mmol/L, 100mmol/L, 300mmol/L
The expression of Fig. 3 NOX is to NADH in born of the same parents and NAD
+the impact of content and ratio thereof.
BM, the B.subtilis JNA of bdhA genetically deficient; BMN
q, BM/pMA5-P
bdh4-yodC; BMN; BM/pMA5-yodC
Embodiment
Embodiment 1: utilize genetic engineering means to build bdhA Gene Deletion bacterial strain
Build the bdhA gene fragment that reporter gene inserts inactivation, utilize primer P1 and P2 to clone from pBGSC6 plasmid the expressed intact gene fragment that obtains cat gene, then utilize EcoR47III to process the fragment that glue reclaims, finally by this gene fragment of alkaline phosphatase treatment and with the T-bdhA after same processing, be connected, transform e. coli jm109, screening positive clone in penbritin and the two resistant panel of paraxin.The T-bdhA::cat building is transformed to B.subtilis JNA, and on paraxin flat board, screening obtains the recombinant bacterial strain of bdhA genetically deficient, through bacterium colony PCR and enzyme activity confirmatory experiment, confirms whether bdhA gene is successfully inserted inactivation.
P1：5’-CGCAGCGCTAAAAAAGGATTGATTCTAATG-3’(Eco47III)
P2：5’-CGCAGCGCTTAGTGACATTAGAAAACCGAC-3’(Eco47III)
Result shows, bacterium colony PCR and enzyme activity checking are correct, and downstream by product 2,3-butanediol output significantly reduces, and successfully obtains bdhA Gene Deletion bacterial strain.
Embodiment 2:NADH oxydase design of primers
First, the chromosomal DNA of bacterial strain B.subtilis JNA of take is template, utilizes primer P3, P4, P5, P6, P7, P8, P9 and P10, by round pcr, is increased respectively and is obtained the ydfN of a section, ydgI, yodC and yfhC gene, by the ydfN after purifying, ydgI, yodC and yfhC gene are 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, connecting fluid is converted in intestinal bacteria competence E.coli JM109, picking positive transformant, extract the plasmid in transformant, through enzyme, cut and verify and confirm recombinant plasmid pMA5-ydfN, pMA5-ydgI, pMA5-yodC and pMA5-yfhC successfully construct.After double digestion checking, show this construction of recombinant plasmid success.By recombinant plasmid A5-ydfN, pMA5-ydgI, pMA5-yodC and pMA5-yfhC are converted into B.subtilis JNA with the method for chemical conversion, picking positive transformant, obtains recombined bacillus subtilis B.subtilisJNA/pMA5-ydfN, B.subtilis JNA/pMA5-ydgI, B.subtilis JNA/pMA5-yodC and B.subtilis JNA/pMA5-yfhC.
The B.subtilis JNA genome DNA of take is template, designs eight primers, and the design of pcr amplification primer is as follows:
P3：5’-ACCG
GGATCCATGGCTGAATTTACTCAT-3’(BamHI)
P4：5’-ACCG
ACGCGTTTA
GTGGTGGTGGTGGTGGTGCATATACTCAACAAAC-3’(MluI)
P5：5’-ACCG
GGATCCATGATCAAAACAAACGAT-3’(BamHI)
P6：5’-ACCG
ACGCGTTTA
GTGGTGGTGGTGGTGGTGTTTCCATTCTGCAAT-3’(MluI)
P7：5’-ACCG
GGATCCATGACGAATACTCTGGAT-3’(BamHI)
P8：5’-ACCG
ACGCGTTTA
GTGGTGGTGGTGGTGGTGCAGCCAAGTTGATAC-3’(MluI)
P9：5’-ACCG
GGATCCATGCCTCAAACCGAACAA-3’(BamHI)
P10：5’-ACCG
ACGCGTTTA
GTGGTGGTGGTGGTGGTGCATTTCAGTGAATCG-3’(MluI)
Embodiment 3:NADH oxidase is measured
The recombinant bacterium B.subtilis JNA/pMA5-ydfN that embodiment 4 is built, B.subtilis JNA/pMA5-ydgI, B.subtilis JNA/pMA5-yodC and B.subtilis JNA/pMA5-yfhC, 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, cultivate 24h for 37 ℃, get fermented liquid in 4 ℃, the centrifugal 10min of 10000r/min, the sodium phosphate buffer of pH7.0 cleans 3 times, cell is resuspended in pH6.5 sodium phosphate buffer, subsequently this liquid is placed in and under ultrasonic disruption instrument, processes cell 20min, 15000rmin
-1centrifugal 30min, supernatant is 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.
Nadh oxidase vitality test: enzyme reaction system is 1mL, contains 50mmol/L potassiumphosphate (pH7.0), 0.3mmol/L EDTA, 50 μ mol/L FAD and 0.3mmol/L β-NADH; Enzymatic reaction starts immediately after adding a certain amount of enzyme liquid, and enzyme activity unit (IU) is defined as under 25 ℃ of conditions, and per minute is oxidized the required enzyme amount of NADH of 1 μ mol.
Result shows that the nadh oxidase of recombinant bacterium B.subtilis JNA/pMA5-yodC expression is than the enzyme 9.65U/mg of being alive, than the nadh oxidase of starting strain B.subtilis JNA, than enzyme work, 12 times have been improved, all the other recombinant bacterial strains are compared with starting strain B.subtilis JNA without obviously improving, and show that yodC gene may be the NOX encoding gene in subtilis.
Embodiment 4:NADH oxydase zymologic property is analyzed
(1) catalytic type is identified
Nadh oxidase has three kinds of catalytic type, in reaction system except meeting generates product NAD
+, also can generate water, hydrogen peroxide or super-oxide etc.By detecting and whether generate the catalytic type that water, hydrogen peroxide or super-oxide are determined this enzyme in the nadh oxidase enzymatic reaction solution in standard.Wherein hydrogen peroxide detects and utilizes H
2o
2the qualitative H that whether generates of test strip comparison superoxide Standard Colors
2o
2, super-oxide is by detecting A
550the minimizing of lower cytochrome C changes the qualitative super-oxide that whether generates.Result shows, reaction process does not detect hydrogen peroxide or super-oxide, indirectly shows that this enzyme is the nadh oxidase that forms water.
(2) 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 0.3mmol/L EDTA, 50 μ mol/L FAD and 0.3mmol/L β-NADH mixes, getting the different pH substrate buffer solutions of 2mL reacts with 20 μ L enzyme liquid, measure the enzyme relative activity under condition of different pH, and compare.Result shows, the optimal pH of this enzyme is 9.0, and the environment of neutral meta-alkali is applicable to the catalysis of this enzyme, and under acidity or strong alkali environment, enzyme activity declines rapidly.
(3) 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, in neutral pH environmental field (pH6.0～8.0), this enzyme is more stable.
(4) 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, the optimal reactive temperature of this enzyme is 35 ℃.
(5) 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%.
(6) impact that different metal ion, EDTA and quinine are lived 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, EDTA and quinine, study each metal ion and the EDTA impact on enzyme activity.Result shows, these ions and EDTA significantly do not suppress or promoter action this enzyme, show that the catalytic center of this enzyme does not need metal ion, and in the situation that quinine exists, this enzyme complete deactivation, indirectly show that this enzyme belongs to flavoprotein family.
(7) kinetic parameter of enzyme
Under pH9.0 and 35 ℃ of conditions, by changing the substrate reactions system final concentration of NADH, measure enzyme activity respectively respectively, investigate enzyme reaction speed.By nonlinear least square regression method, obtain the K of NADH
mand V
max.Result shows, the K of NADH
mfor 0.36mmol/L, V
maxbe 19.8 μ mol/Lmin.
Embodiment 5: build the bdhA Gene Deletion recombinant bacterial strain that appropriateness is expressed nadh oxidase
Utilize primer P11/P12 primer clone from subtilis JNA to obtain yodC gene, utilize appropriate promotor P
bdhA(public announcement of a patent application CN102876703A) expresses NOX in bdhA Gene Deletion bacterial strain, the final recombinant bacterial strain that obtains recombinant bacterial strain appropriateness expression nadh oxidase, result shows, appropriateness is expressed NOX makes the NADH level in born of the same parents decline approximately 21.5%, NADH/NAD
+ratio declines and surpasses 37.9%, but less on the growth of cell and the impact of sugar consumption speed, and this strategy has improved the glucose specific consumption rate of earlier fermentation greatly, finally make this bacterium at 84h, just consume the glucose of 150g/L, so this strategy is suitable for for regulating the metabolism stream of NADH dependent form tunning.
P11：5’-ACCG
GGTACCATGACGAATACTCTGGAT-3’(KpNI)
P12：5’-ACCG
AAGCTTTTACAGCCAAGTTGATAC-3’(Hind?III)
Embodiment 6: appropriateness is expressed engineering strain fermentative production acetoin, 2,3-butanediol and the by product Performance Detection of NOX
The engineering strain that appropriateness is expressed NOX is inoculated in 10mL LB substratum, after 37 ℃ of shaking culture 7-9h, respectively gets 1ml and transfers in 3 bottles of 50ml containing 37 ℃ of shaking culture in the LB substratum of 40g/L glucose, when being cultured to OD
600during=5.0-6.0, be added to containing in the 5L fermentation reactor of 1.85L fermention medium and ferment, the initial pH of controlled fermentation is respectively 6.0,6.5 and 7.0, after determining that the most suitable growth pH is 6.5, after growth optimal conditions pH6.5 condition bottom fermentation 48h, improve pH to 7.5,8.0 and 8.5 continue fermentation, every 12h sampling once, detects fermentation growing state, sugar consumption speed, acetoin, 2,3-butanediol and by product output.
Fermentation of bacillus subtilis substratum: beef extract 5g/L, corn steep liquor 6g/L, urea 2g/L, glucose 150g/L.Regulate pH6.5.
Result shows, appropriateness is expressed nadh oxidase and not only promoted the glucolytic output of NADH dependent form by product 2,3-butanediol, lactic acid and ethanol in fermenting process that also reduced, and promoted the synthetic of acetoin.The engineering strain that final appropriateness is expressed NOX utilizes 150g/L glucose fermentation 84h to obtain 2 of the acetoin of 56.7g/L and 1.2g/L, 3-butyleneglycol, 2,3-butyleneglycol, lactic acid and acetic acid reduce respectively 92.3%, 70.1% and 75.0%, acetoin production efficiency rises to 0.68g/ (Lh), compared with original strain recombinant bacterium B.subtilis JNA fermentation period, shortens 1.7 times.
Claims (4)
1. a bdhA Gene Deletion subtilis (B.subtilis), it is characterized in that: from pBGSC6 plasmid, clone the expressed intact gene fragment that obtains cat gene, utilize this gene fragment of alkaline phosphatase treatment and be connected with the T-bdhA after same processing, build the bdhA gene fragment because of reporter gene insertion inactivation shown in SEQ ID NO:1, then the T-bdhA::cat building is converted in the subtilis B.subtilis JNA that deposit number is CCTCC NO:M209309, screening obtains a kind of bdhA Gene Deletion subtilis.
2. a novel subtilis nadh oxidase, it is characterized in that: the nadh oxidase gene yodC shown in SEQ ID NO:2 is cloned into expression shuttle vectors pMA5 and is configured to restructuring shuttle expression plasmid pMA5-yodC and is converted in the subtilis B.subtilis JNA that deposit number is CCTCC NO:M209309, through enzyme activity determination and relevant zymetology Property Identification, determine that yodC is the nadh oxidase gene that coding subtilis forms water.
3. the bdhA Gene Deletion recombined bacillus subtilis (B.subtilis) of an appropriateness expression nadh oxidase, is characterized in that: the nadh oxidase gene yodC shown in SEQ ID NO:2 is cloned into appropriateness and expresses shuttle vectors pMA5-P
bdhAbe configured to restructuring shuttle expression plasmid pMA5-P
bdhA-yodC is also converted into described in claim 1 in subtilis, builds the bdhA Gene Deletion bacterial strain that appropriateness is expressed nadh oxidase.
4. the application of bacterial strain claimed in claim 3 in fermentative production acetoin, it is characterized in that: 150g/L conversion of glucose is about 2 of the acetoin of 56.7g/L and 1.2g/L the most at last, 3-butyleneglycol, 2,3-butyleneglycol, lactic acid and acetic acid reduce respectively 92.3%, 70.1% and 75.0%, acetoin production efficiency rises to 0.68g/ (Lh), compared with original strain B.subtilis JNA fermentation period, shortens 1.7 times.For utilize first NADH regulation and control production acetoin both at home and abroad in subtilis, realized the object that shortens fermentation period, reduces NADH dependent form by product.
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Cited By (8)
| 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 |
| CN105154456A (en) * | 2015-08-24 | 2015-12-16 | 江南大学 | Method for enhancing 2,3-butanediol synthesis by improving intracellular coenzyme level |
| CN107955805A (en) * | 2017-12-13 | 2018-04-24 | 江南大学 | A kind of stability-enhanced nadh oxidase and its application in 3-hydroxy-2-butanone production |
| KR20190074917A (en) * | 2017-12-20 | 2019-06-28 | 건국대학교 산학협력단 | A method for producing acetoin from ethanol |
| KR20190088648A (en) * | 2018-01-19 | 2019-07-29 | 건국대학교 산학협력단 | A method for producing butandiol from ethanol |
| WO2019124782A3 (en) * | 2017-12-20 | 2019-08-15 | 건국대학교 산학협력단 | Method for producing acetoin, butanediol, or butanol from ethanol |
| US11441142B2 (en) | 2017-12-20 | 2022-09-13 | Konkuk University Industrial Cooperation Corporation | FLS variant having increased activity |
| CN115838714A (en) * | 2022-12-07 | 2023-03-24 | 福建农林大学 | Acetaldehyde lyase, acetaldehyde lyase fusion protein, and preparation method and application thereof |
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Cited By (11)
| 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 |
| CN105154456A (en) * | 2015-08-24 | 2015-12-16 | 江南大学 | Method for enhancing 2,3-butanediol synthesis by improving intracellular coenzyme level |
| CN107955805A (en) * | 2017-12-13 | 2018-04-24 | 江南大学 | A kind of stability-enhanced nadh oxidase and its application in 3-hydroxy-2-butanone production |
| CN107955805B (en) * | 2017-12-13 | 2020-03-06 | 江南大学 | NADH oxidase with improved stability and application thereof in acetoin production |
| KR20190074917A (en) * | 2017-12-20 | 2019-06-28 | 건국대학교 산학협력단 | A method for producing acetoin from ethanol |
| WO2019124782A3 (en) * | 2017-12-20 | 2019-08-15 | 건국대학교 산학협력단 | Method for producing acetoin, butanediol, or butanol from ethanol |
| KR102093546B1 (en) * | 2017-12-20 | 2020-04-23 | 건국대학교 산학협력단 | A method for producing acetoin from ethanol |
| US11441142B2 (en) | 2017-12-20 | 2022-09-13 | Konkuk University Industrial Cooperation Corporation | FLS variant having increased activity |
| KR20190088648A (en) * | 2018-01-19 | 2019-07-29 | 건국대학교 산학협력단 | A method for producing butandiol from ethanol |
| KR102013058B1 (en) * | 2018-01-19 | 2019-08-21 | 건국대학교 산학협력단 | A method for producing butandiol from ethanol |
| CN115838714A (en) * | 2022-12-07 | 2023-03-24 | 福建农林大学 | Acetaldehyde lyase, acetaldehyde lyase fusion protein, and preparation method and application thereof |
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