CN104017764A - Bacillus subtilis NAD+regeneration system is used to effectively biotransform 2, 3-butanediol to produce acetoin - Google Patents
Bacillus subtilis NAD+regeneration system is used to effectively biotransform 2, 3-butanediol to produce acetoin Download PDFInfo
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Abstract
The invention discloses a method for producing acetoin by efficient bioconversion of 2,3-butanediol by using a Bacillus subtilis nicotinamide adenine dinucleotide (NAD)<+> regeneration system, and belongs to the field of genetic engineering. Acetoin reductase and educed form of nicotinamide-adenine dinucleotide (NADH) oxidase in a high-yield acetoin strain B.subtilis JNA with independent intellectual property rights, which are autonomously screened from a laboratory are cloned by the method disclosed by the invention, and excessive coexpression of the acetoin reductase and NADH oxidase in B.subtilis JNA is carried out, so that the production of acetoin by effective conversion of the 2,3-butanediol in the wild-type high-yield acetoin bacillus subtilis by virtue of the NAD<+> regeneration system is realized for the first time at home and abroad. The enzyme activity determination and intracellular coenzyme level research on the built gene engineering strain prove that the 2,3-butanediol can be lastingly and effectively converted by B.subtilis JNA/pMA5-bdhA-yodC to produce the acetoin. 120g/L of 2,3-butanediol can be finally converted into about 92.5g/L of acetoin by the B.subtilis JNA/pMA5-bdhA-yodC when the temperature is 40 DEG C and the pH is 8.0 under the optimal whole-cell conversion condition of adding 5mM of MnC12, the acetoin yield can be up to 2.31g/(L.h), and is the highest lever for producing the acetoin from the bacillus subtilis in the current report, and a foundation is provided for industrial production of the acetoin from microorganisms.
Description
Technical field
Utilize NAD
+regeneration system rapidly high-performance bio transforms the method for 2,3-butanediol production acetoin, the invention belongs to genetically engineered and bioengineering field.Relate in particular to a kind of acetoin reductase enzyme and nadh oxidase co expression engineering strain structure and utilize this bacterial strain to carry out the method for resting cell 2,3-butanediol production acetoin.
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.Production by Microorganism Fermentation acetoin receives increasing concern in recent years, and various countries scientist carries out the work of this respect in succession.Aspect at present the research of fermentative Production acetoin being focused mostly on and is optimized at screening wild strain with to fermention medium or technique, and genetic engineering modified bacterial strain report is relatively less, and in most of bacterial strain metabolic processes, acetoin is as 2, the by product of 3-butyleneglycol and dimethyl diketone metabolism exists, accumulated concentrations is lower, in addition, in fermenting process, can produce many by products, as acetic acid, lactic acid, ethanol and 2,3-butyleneglycol, thus directly cause downstream engineering to be difficult to carry out separation, finally cannot obtain the acetoin that purity is higher.
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 CN 101864381A), find under study for action the phenomenon that in fermentation of bacillus subtilis process, acetoin and 2,3-butanediol transform mutually, the metabolism that this mutual conversion process is subject to acetoin reductase enzyme regulates.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 this case, cofactor is controlled for very crucial further raising object product output.By cofactor regeneration technology bio-transformation synthetic product, not only can reduce synthetic expense, drive when having reacted and also can simplify product separation, prevention can be checked the by product accumulation of cofactor.
The present invention, by expression vector pMA5, has realized acetoin reductase enzyme in B.subtilis JNA (being called for short bdhA) and the nadh oxidase efficient homology coexpression of (being called for short .yodC) first.By the NAD building
+regeneration system rapidly, realizes and carries out repeatedly resting cell 2,3-butanediol with a collection of bacterial strain, finally realizes High-efficient Production acetoin.
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 acetoin reductase gene (being called for short bdhA) and nadh oxidase (being called for short yodC) from B.subtilis JNA, build recombinant expression vector pMA5-bdhA, pMA5-yodC and pMA5-bdhA-yodC, and be converted into respectively B.subtilis JNA, successfully built engineering strain B.Subtilis JNA/pMA5-bdhA, B.Subtilis JNA/pMA5-yodC and B.Subtilis JNA/pMA5-bdhA-yodC.The engineering strain building is carried out to coenzyme level in enzyme activity determination and born of the same parents and studies have shown that B.Subtilis JNA/pMA5-bdhA-yodC can continuously and effectively transform 2,3-butanediol and generate acetoin.Finally at pH8.0,40 ℃ and add 5mM MnCl
2condition under, final B.subtilis JNA/pMA5-bdhA-yodC is by 120g/L2,3-butyleneglycol is converted into the about 92.5g/L of acetoin, and acetoin productive rate reaches 2.31g/ (Lh).
Advantage of the present invention and positively effect are:
(1) first passage of the present invention co expression acetoin reductase enzyme and nadh oxidase structure NAD in subtilis
+regeneration system rapidly, carries out bio-transformation production acetoin, not only reduces its synthetic expense, drives the separation of also having simplified product when having reacted, realizes the method for High-efficient Production acetoin.
(2) the present invention adopts and repeatedly transforms strategy in batches, final B.Subtilis JNA/pMA5-bdhA-yodC is by 120g/L2,3-butyleneglycol is converted into the about 92.5g/L of acetoin, and acetoin productive rate reaches 2.31g/ (Lh), this output is to report at present subtilis production acetoin highest level.
Accompanying drawing explanation
Fig. 1 recombinant plasmid pMA5-bdhA-yodC builds schematic diagram.
Fig. 2 SDS-PAGE analyzing proteins expression.
Fig. 3 bacterial strain B.subtilis JNA/pMA5-bdhA-yodC is at the horizontal resting cell curve of 5L-tank.
Fig. 4 bacterial strain B.subtilis JNA/pMA5-bdhA-yodC is at 5L-tank level resting cell curve repeatedly.
Embodiment
Embodiment 1: the amplification of goal gene and the structure of recombined bacillus subtilis
Plasmid pMA5-bdhA-yodC 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 PCR, bdhA gene after purifying, after restriction enzyme MluI and BamHI digestion, is connected to construction recombination plasmid pMA5-bdhA with the plasmid pMA5 of the above-mentioned two kinds of digestion with restriction enzyme of same process, after double digestion checking, show this construction of recombinant plasmid success.Subsequently, utilize primer P3 and P4 to obtain nucleotides sequence by pcr amplification and classify the gene yodC shown in SEQ ID NO:2 as, by the yodC 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, build another recombinant vectors pMA5-yodC; Then take recombinant vectors pMA5-yodC as template, utilize primer P5 and P6 to amplify the gene fragment HapII-yodC with promotor HapII by round pcr: last, after this gene fragment is processed by restriction enzyme SphI and HindIII, with the same carrier pMA5-bdhA processing through SphI and HindIII 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-yodC successfully constructs.Recombinant plasmid pMA5-bdhA-yodC is converted into subtilis with the method for chemical conversion, picking positive transformant, bacillus amyloliquefaciens B.subtilis JNA/pMA5-bdhA-yodC obtains recombinating.To original bacterium and recombinant bacterium intracellular protein expression analysis, can find out, gene bdhA and yodC successfully realize overexpression in recombinant bacterium.
The B.subtilis JNA genome DNA of take is template, designs four primers, and the design of pcr amplification primer is as follows:
P1：5’-GAC
GGATCCATGAAAAAACAACGAATGCT-3’(BamH?I)
P2：5’ACCG
ACGCGTTTAGTGGTGGTGGTGGTGGTGGTTAGGTCTAACAAGG-3’(Mlu?1)
P3：5’-ACCG
GGATCCATGACGAATACTCTGGAT-3’(BamH?I)
P4：5’-ACCG
ACGCGTTTAGTGGTGGTGGTGGTGGTGCAGCCAAGTTGATAC-3’(Mlu?I)
Take pMA5-yodC plasmid as template, design two primers, the design of pcr amplification primer is as follows:
P5：5’ACCG
GCATGCGTAAGCTAGACAAAACGGAC-3’(Sph?I)
P6：5’-ACCG
AAGCTTTTAGTGGTGGTGGTGGTGGTGCAGCCAAGTTGATAC-3’(Hind?III)
Embodiment 2: recombinant bacterial strain enzyme activity determination
(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;
Nadh oxidase: 50mM potassium phosphate buffer pH7.0,0.3mMEDTA, 50 μ M FAD, 0.3mM β-NADH;
(2) enzyme activity determination
The recombinant bacterium B.subtilis JNA/pMA5-bdhA that embodiment 1 is built, B.subtilis JNA/pMA5-yodC and B.subtilis JNA/pMA5-bdhA-yodC, 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, be suspended in the sodium phosphate buffer of pH7.0, ultrasonic disruption is processed and is 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, acetoin reductase enzyme/2,3-butanediol desaturase (179.7/124.5mU/mg) that recombinant bacterium B.subtilis JNA/pMA5-bdhA expresses has improved respectively 4 times and 65 times than starting strain B.subtilis JNA (40.2/1.9mU/mg).The nadh oxidase (570.3mU/mg) that recombinant bacterium B.subtilis JNA/pMA5-yodC expresses has improved nearly 17 times than starting strain B.subtilis JNA (33.4mU/mg).Acetoin reductase enzyme/2 that recombinant bacterium B.subtilis JNA/pMA5-bdhA-yodC expresses, 3-butanediol dehydrogenation enzyme (171.8/152.8mU/mg) is all greatly improved than starting strain B.subtilis JNA (40.2/1.9mU/mg, 33.4mU/mg) with nadh oxidase (346.4mU/mg) alive.
Embodiment 3: in recombinant bacterial strain born of the same parents, cofactor level and full cell viability are measured
In recombinant bacterial strain born of the same parents, cofactor level detection utilizes AAT Bioquest company reagent to detect box.By fluorescence microplate reader, under Ex/Em=540/590nm, detect NADH, NAD+ concentration and NADH/NAD
+ratio.
The acetoin generating by reaction system detects resting cell vigor, reconstitution cell is suspended in and contains 40g/L2, in the 50mM phosphate buffered saline buffer (pH8.0) of 3-butyleneglycol, 37 ℃ of shaking tables react 20 minutes, reaction solution is with the rotating speed of 12000rpm, centrifugal 1min, measure the content of acetoin in supernatant liquor, mensuration system is as follows: 0.5mL creatine (0.5%w/v), 0.5mL naphthyl alcohol (5%w/v is dissolved in 95% ethanol), 0.5mL potassium hydroxide (10%w/v), the centrifugal gained supernatant of 0.01mL and 3.5mL deionized water, this is measured to system room temperature places several minutes, then utilize the absorbance under spectrophotometric determination 560nm.Full cell viability unit definition is under 37 ℃ of conditions, every OD per hour
600it is 1U that catalysis 2,3-butanediol generates 1mM acetoin.
Result shows, recombinant bacterial strain B.subtilis JNA/pMA5-bdhA-yodC compares with starting strain B.subtilis JNA, NADH concentration and NADH/NAD in born of the same parents
+ratio declines respectively 1.6 times and 2.2 times, but the transformation time that resting cell ability is about 2 times of starting strain and can continues more to grow is more conducive to resting cell 2,3-butanediol production acetoin.
Embodiment 4: resting cell 2,3-butanediol production acetoin condition optimizing
(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 1pH is a gradient), with 40g/L2, the recombinant bacterial strain thalline of 3-butyleneglycol and collection mixes, and 37 ℃ of shaking tables react 1 hour, and reaction solution is with the rotating speed of 12000rpm, centrifugal 10min, the content of acetoin in mensuration supernatant liquor.Result shows, at pH8.0, is optimal reaction pH.
(2) research of optimum temperuture
(20 ℃-60 ℃ of temperature of reaction are set, every 10 ℃ is a gradient), by the recombinant bacterial strain thalline of collecting and containing 40g/L2, the sodium phosphate buffer of 3-butyleneglycol (pH8.0) mixes, shaking table reaction 1 hour, reaction solution is with the rotating speed of 12000rpm, and centrifugal 10min measures the content of acetoin in supernatant liquor.Result shows, at 40 ℃, is optimal reactive temperature.
(3) its impact of different metal ion pair
The reaction system that does not add metal ion of take is contrast, and in reaction system, adding respectively final concentration is the Mn of 0.5mM, 2.5mM, 5.0mM, 7.5mM and 10mM
2+, Zn
2+, Ca
2+, Mg
2+, Fe
2+, Fe
3+ion, by the recombinant bacterial strain thalline of collecting with containing 40g/L2, the sodium phosphate buffer of 3-butyleneglycol (pH8.0) mixes, 40 ℃ of shaking tables reactions 1 hour, reaction solution is with the rotating speed of 12000rpm, centrifugal 10min measures the content of acetoin in supernatant liquor.Result shows, 5Mm Mn
2+reaction is had to obvious promoter action.
(4) research of suitable concentration of substrate
Under pH8.0 and 40 ℃ of conditions, by changing substrate 2,3-butanediol reaction system final concentration (10-60g/L), shaking table reaction 1 hour, reaction solution is with the rotating speed of 12000rpm, and centrifugal 10min measures the content of acetoin in supernatant liquor.Result shows, 40g/L2, and 3-butyleneglycol is the suitableeest concentration of substrate.
Embodiment 5: recombinant bacterial strain B.subtilis JNA/pMA5-bdhA-yodC resting cell 2,3-butanediol production acetoin
(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, NaC110g/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: soy peptone 1g/L, glucose 4g/L, corn steep liquor 1.5g/L, urea 0.3g/L, sodium-chlor 5g/L, dipotassium hydrogen phosphate 0.17g/L, potassium primary phosphate 0.23g/L, manganous sulfate 0.075g/L, deionized water configuration, pH6.5.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/ h.m
3substratum, mixing speed is 300r/min.Cultivate 36h, the centrifugal 10min of 10000rpm collects thalline.
(3) resting cell
Reaction system: 40g/L2,3-butyleneglycol, 50mM sodium phosphate buffer (pH8.0), 5mM MnCl
2
The thalline of collection is added in the 5L reactor containing 2L reaction system, under optimum reaction conditions, transform 12h, every 2h sampling once, detects acetoin, 2,3-butanediol output.2,3-butanediol and gas Chromatographic Determination (GC-1690J gas chromatograph, Hangzhou Ke Xiao chemical industry instrument company) for acetoin.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.Result shows, after end reaction 12h, and 40g/L2,3-butyleneglycol transforms and is about 31.5g/L acetoin, and productive rate reaches 2.62g/ (Lh), compared with starting strain B.Subtilis JNA, has improved 1 times.
(4) repeatedly in batches tactful
Seed culture medium fermentation culture conditions and resting cell and embodiment 5 (1), 5 (2) and 5 (3)) identical, when substrate 2, when 3-butyleneglycol only remains about 10g/L, collect the centrifugal 10min of reaction solution 10000rpm and collect thalline, add containing transforming again in the 5L reactor of 2L fresh reactant system, until speed of reaction obviously declines.Every 2h sampling once, detects acetoin, 2,3-butanediol output.Result shows, finally with a collection of bacterial strain, through 3 resting cells, reacts after 40h, and 120g/L2,3-butyleneglycol transforms and is about 92.5g/L acetoin, and productive rate reaches 2.31g/ (Lh).
Claims (3)
1. a recombined bacillus subtilis (B.subtilis) of strengthening coenzyme cyclic regeneration, it is characterized in that: the nadh oxidase gene yodC shown in the acetoin reductase gene bdhA shown in SEQ ID NO:1 and SEQ ID NO:2 is cloned into shuttle vectors pMA5 jointly and is configured to restructuring shuttle expression plasmid pMA5-bdhA-yodC and is converted in the subtilis B.subtilis JNA that deposit number is CCTCC NO:M209309, thereby improve the ability that subtilis utilizes 2,3-butanediol resting cell production acetoin.
2. the recombined bacillus subtilis B.subtilisJNA/pMA5-bdhA-yodC that utilizes claim 1 to build, optimizes its resting cell reaction conditions, it is characterized by: reaction optimal pH is 8.0; Reaction optimum temperuture is 40 ℃; Optimal reaction substrate 2,3-butanediol concentration is 40g/L; 5mM MnCl
2reaction is had to obvious promoter action.
3. under claim 2 resting cell reaction conditions, the recombined bacillus subtilis B.subtilis JNA/pMA5-bdhA-yodC that utilizes claim 1 to build, resting cell 2, the application of 3-butyleneglycol production acetoin, it is characterized in that: bacterial strain B.subtilis JNA/pMA5-bdhA-yodC is inoculated in containing in kantlex 10mL LB substratum, after 37 ℃ of shaking culture 7-9h, get 1ml and transfer 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, (formula of this fermention medium is: soy peptone 1g/L, glucose 4g/L to add 1.85L fermention medium, corn steep liquor 1.5g/L, urea 0.3g/L, sodium-chlor 5g/L, dipotassium hydrogen phosphate 0.17g/L, potassium primary phosphate 0.23g/L, manganous sulfate 0.075g/L, deionized water configuration, pH6.5) in 5L fermentation reactor, cultivate 36h, the centrifugal 10min of 10000rpm collects thalline, adds containing 2L reaction system (2,3-butyleneglycol 40g/L, MnCl
25mM, pH8.0,40 ℃ of temperature of reaction,) 5L reactor in, when substrate 2,3-butanediol is only during surplus about 10g/L, collect the centrifugal 10min of reaction solution 10000rpm and collect thalline, again transform, until speed of reaction obviously declines, finally with a collection of bacterial strain, after 3 resting cells, 120g/L2,3-butyleneglycol is converted into about 92.5g/L acetoin, productive rate reaches 2.31g/ (Lh), is domestic and international first passage NAD
+regeneration system rapidly utilizes 2,3-butanediol Efficient Conversion production acetoin in wild-type high yield acetoin subtilis, the final object that improves conversion rate, High-efficient Production acetoin that realizes.
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| CN104404089A (en) * | 2014-12-09 | 2015-03-11 | 江南大学 | Method for improving acetoin yield by adding gluconic acid |
| 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 |
| CN106399447A (en) * | 2016-10-27 | 2017-02-15 | 天津大学 | Method for increasing acetoin yield through mixed fermentation |
| CN106967741A (en) * | 2017-04-03 | 2017-07-21 | 天津大学 | A kind of external enzyme reaction production L（+）The method of 3-hydroxy-2-butanone |
| 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 |
| CN110713965A (en) * | 2019-10-29 | 2020-01-21 | 江南大学 | Method for producing 1, 2-aminoalcohol compound by whole cell transformation |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104404089A (en) * | 2014-12-09 | 2015-03-11 | 江南大学 | Method for improving acetoin yield by adding gluconic acid |
| 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 |
| CN104404089B (en) * | 2014-12-09 | 2018-02-23 | 江南大学 | A kind of method for improving 3-hydroxy-2-butanone yield by adding gluconic acid |
| CN105154456A (en) * | 2015-08-24 | 2015-12-16 | 江南大学 | Method for enhancing 2,3-butanediol synthesis by improving intracellular coenzyme level |
| CN106399447A (en) * | 2016-10-27 | 2017-02-15 | 天津大学 | Method for increasing acetoin yield through mixed fermentation |
| CN106399447B (en) * | 2016-10-27 | 2019-12-03 | 天津大学 | The method for improving 3-hydroxy-2-butanone yield using mixed fungus fermentation |
| CN106967741A (en) * | 2017-04-03 | 2017-07-21 | 天津大学 | A kind of external enzyme reaction production L（+）The method of 3-hydroxy-2-butanone |
| CN106967741B (en) * | 2017-04-03 | 2020-02-21 | 天津大学 | Method for producing L (+) -acetoin through in vitro enzyme reaction |
| 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 |
| CN110713965A (en) * | 2019-10-29 | 2020-01-21 | 江南大学 | Method for producing 1, 2-aminoalcohol compound by whole cell transformation |
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