CN104087602A - Method for producing optical pure R-acetylmethylcarbinol - Google Patents
Method for producing optical pure R-acetylmethylcarbinol Download PDFInfo
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Abstract
The invention discloses a method for producing optical pure R-acetylmethylcarbinol. The method comprises the following steps of performing co-expression on (R,R)-2,3-butanediol dehydrogenase gene (bdh) and glucose dehydrogenase (gdh) gene in escherichia coli; performing conversion by using recombinant escherichia coli resting cells as a biological catalyst and butanedione as a precursor to produce the optical pure R-acetylmethylcarbinol. Glucose dehydrogenase can supply coenzyme NADH required by (R,R)-2,3-butanediol dehydrogenase while (R,R)-2,3-butanediol dehydrogenase has the characteristic of catalyzing butanedione to produce the optical pure R-acetylmethylcarbinol in the presence of NADH, and the yield of R-acetylmethylcarbinol can reach 26g/L, and the optical purity is 99.6 percent. The method is simple in culture medium, easy and simple in operation process, low in cost and convenient to separate products, and has the industrial application prospect; complicated chiral resolution steps are not needed; regeneration of intracellular coenzyme is realized.
Description
Technical field
The present invention relates to a kind of production method of optical purity R-acetoin, specifically by (R, R)-2,3-butanediol dehydrogenation enzyme gene (bdh) and glucose dehydrogenase gene (gdh) are at expression in escherichia coli, and to take this recombination bacillus coli resting cell be biological catalyst, take dimethyl diketone and the glucose method as substrate High-efficient Production optical purity R-acetoin.
Background technology
Acetoin (acetoin), be 3-hydroxy-2-butanone (3-hydroxy-2-butanone), there are two kinds of optically active isomers (R-acetoin and S-acetoin), be present in natively in the varieties of food items such as dairy products, butter, cheese, drinks, corn, grape, apple, strawberry, cocoa, standard GB/T 2760-86 stipulates that it is to allow to add edible food spice.Acetoin is widely used, and it has, and obvious cheese is fragrant, the fragrant feature of fat, is mainly used in the production of the spices such as cream, dairy products, coffee, Yoghourt.In addition, acetoin is a kind of important chemosynthesis intermediate and multifunctional material, and within 2004, USDOE is classified acetoin as one of hardware and software platform compound of 30 kinds of preferential developments utilizations, can be widely used in the fields such as food, pharmacy, chemical industry.For example, the acetoin of 80% content is commonly called as " vinegar drone ", is an extremely important kind in drinks blending.The mixture that contains acetoin and dimethyl diketone can also be used to treat mazoitis.In pharmaceutical industry, optical purity acetoin can be used for synthetic synthetic having optically active chiral drug as having the single-minded compound of three-dimensional arrangement, thereby improves largely drug effect, alleviates Side effects of pharmaceutical drugs.In addition, optical purity acetoin also can be used for synthesis of chiral liquid crystal composite material.
The production method of optical purity acetoin is mainly divided into two classes: chemical synthesis and biological synthesis process.The raw material sources of chemosynthesis are in non-renewable fossil resource---oil, raw material sources are restricted, and building-up process is loaded down with trivial details, finally also need to carry out complicated chiral separation by chemical method, have that cost is high, yield is low, the problem of optical purity low and high energy consumption, high pollution, be therefore difficult to realize low-cost scale operation.Biosynthetic means production acetoin has plurality of advantages, as natural in Product Green, reaction conditions is gentle, pollute less, energy consumption is low, is a kind of environmentally benign synthesis extremely with development prospect, has received in recent years increasing concern.Yet what natural microbial fermentation produced is generally all the mixture of optical isomers of R-acetoin and S-acetoin, tunning chiral separation technique is loaded down with trivial details, with high costs.1984, Ui etc. reported and have utilized Pseudomonas sp.S-4 fermentation method to synthesize single configuration R-acetoin [J.Ferment.Technol., 1984,62:151-156].There is the problem of production concentration low (1.72g/L) and fermentation period long (48h) in this method.Chinese Patent Application No. CN201310346916.8 discloses Hao Jian etc., and to utilize strain Cray Bai Shi pulmonitis strain (Klebsiella pneumoniae) mutant strain fermentative production R-acetoin, production peak be 35.7g/L in 2013.Yet, K.pneumoniae is classified as pathogenic strains (Risk group2, pathogenic microorganisms) by the World Health Organization, and the principle of sound accounting of large-scale industrial production has limited its application [Biotechnol.Adv. to a great extent, 2009,27:715-725; Appl.Environ.Microbiol., 2011,77:5467-5475; Bioresour.Technol., 2012,124:237-244].
Given this, people attempt building Recombinant organism strain and synthesize R-acetoin.1998, Ui etc. are by K.pneumoniae coding for alpha-acetolactate synthestase, alpha-acetolactate decarboxylase and meso-2, the gene cluster of 3-butanediol dehydrogenation enzyme is incorporated in intestinal bacteria, then by meso-2, the encoding gene of 3-butanediol dehydrogenation enzyme knocks out, the recombination bacillus coli obtaining can produce 17.5g/L optical purity R-acetoin [Lett.Appl.Microbiol. by glucose fermentation, 1998,26:275-278], but fermentation method, due to fermented liquid complicated component, causes tunning separation and Extraction difficulty.In addition, also there is fermentation period length and the low problem of substrate conversion efficiency in this method, has limited its commercial application.
(the R of Paenibacillus polymyxa (Paenibacillus polymyxa), R)-2, under the condition that 3-butanediol dehydrogenation enzyme exists at NADH, can be reduced to R-acetoin [Appl.Environ.Microbiol., 2011,77:4230-4233] by catalysis dimethyl diketone.The bdh gene of two strain P.polymyxa (ATCC12321 and ZJ-9) has been proved to be coding (R, R)-2,3-butanediol desaturase [Appl.Environ.Microbiol., 2011,77:4230-4233; J.Basic.Microbiol., 2012,52,1-9], but not yet have the relevant information of P.polymyxa DSM365 strain b-dh gene to report at present.Chinese Patent Application No. CN201010564858 discloses a kind of method of pure enzyme catalysis reduction dimethyl diketone of using and has prepared optical purity R-acetoin, by building (the R of expression of recombinant e. coli P.polymyxa ATCC12321 bacterial strain, R)-2,3-butanediol dehydrogenation enzyme, then smudge cells, by expression product separation and purification, obtain (R, R)-2,3-butanediol desaturase of purifying.Adding under the condition of coenzyme NAD H, (R, the R) of purifying-2,3-butanediol desaturase can transform dimethyl diketone and produce optical purity R-acetoin.Pure enzymatic process has advantages of that product separation purifying is easy, but needs smudge cells and separation and purification enzyme, and need to add expensive coenzyme NAD H, and technique is loaded down with trivial details, and cost is higher.
Resting cell catalysis refers to and utilizes complete resting cell to carry out bio-transformation as biological catalyst, its essence is to utilize intracellular various enzyme to carry out catalyzed reaction, combine the advantage of these two kinds of technology of fermentation method and enzyme law catalysis, overcome the shortcomings that fermentation method exists.So far there are two pieces of bibliographical informations to use the method for resting cell catalysis to prepare optical purity R-acetoin.2002, Yamada-Onodera etc. are at the glycerol dehydrogenase of expression in escherichia coli Hansenula polymorpha, then using recombination bacillus coli resting cell as whole-cell biocatalyst, transform optical purity (R, R)-2,3-butyleneglycol is R-acetoin, and product optical purity has reached 99.9%, but output very low (9.68g/L) [Acta Biotechnol, 2002,22:355-362].In addition Chinese Patent Application No. CN200910013902.8; PLoS ONE, 2010,5:e8860 etc. come from (the R of Bacillus subtilis by building recombination bacillus coli heterogenous expression, R)-2,3-butanediol dehydrogenation enzyme and come from the nadh oxidase of Lactobacillus brevis, acquisition can catalysis optical purity (R, R)-2,3-butyleneglycol becomes the recombination bacillus coli biological catalyst of optical purity R-acetoin, but product optical purity lower (96%).Yet above-mentioned resting cell catalyzed reaction all needs to use industry to be difficult to expensive raw material---optical purity (R, the R)-2,3-butanediol of preparation, be difficult to realize and commercially produce on a large scale.And dimethyl diketone price is well below optical purity (R, R)-2,3-butanediol, if can utilize it is the pure R-acetoin of precursor synthesizing optical, can effectively reduces the production cost of R-acetoin, thereby have more commercial applications prospect.
Through document and patent retrieval widely, we find to derive from the glucose dehydrogenase gene gdh of subtilis 168, as coenzyme at document Engineering of cofactor regeneration enhances (2S, 3S)-2, [SCIENTIFIC REPORTS. is in the news in 3-butanediol production from diacetyl., 2013,3:1-6].But the relevant information report that not yet has at present P.polymyxa DSM365 bacterial strain (R, R)-2,3-butanediol dehydrogenase gene (bdh); And this gene and glucose dehydrogenase gene (gdh) are carried out to coexpression in intestinal bacteria, and prepare with this recombinant bacterial strain the R-acetoin production method that resting cell biological catalyst is realized the reduction of coupling dimethyl diketone and coenzyme NAD H regeneration, both at home and abroad also without open source literature patent report.
Summary of the invention
For the R-acetoin existing in above-mentioned existing method yield poorly, optical purity is low, product extracts difficulty, cost is high, be difficult to the deficiency that large-scale low-cost is produced, and the invention provides a kind of production method of optical purity R-acetoin.
The present invention utilizes (R, R)-2,3-butanediol dehydrogenation enzyme can generate optical purity R-acetoin by the single-minded catalysis dimethyl diketone of solid, and Hexose phosphate dehydrogenase can be realized the character of coenzyme NAD H regeneration in born of the same parents, by (R, R)-2,3-butanediol dehydrogenation enzyme gene (bdh) and glucose dehydrogenase gene (gdh) import in intestinal bacteria and carry out coexpression, take this recombination bacillus coli resting cell as biological catalyst conversion dimethyl diketone, and the high efficiency, low cost of realizing optical purity R-acetoin is synthetic.
The method of production optical purity R-acetoin of the present invention, by (R, R)-2,3-butanediol dehydrogenation enzyme gene (bdh) and glucose dehydrogenase gene (gdh) import in intestinal bacteria and carry out coexpression, take this recombination bacillus coli resting cell is biological catalyst, take dimethyl diketone as precursor, and glucose is regenerating coenzyme raw material, and the high efficiency, low cost of realizing optical purity R-acetoin is synthetic.
Wherein: described (R, R)-2, 3-butanediol dehydrogenation enzyme gene can derive from Paenibacillus polymyxa (Paenibacillus polymyxa), subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Cray Bai Shi pulmonitis strain (Klebsiella pneumoniae), Klebsiella oxytoca (Klebsiella oxytoca), enteroaerogen (Enterobacter aerogenes), serratia marcesens (Serratia marcescens), Aeromonas hydrophila (Aeromonas hydrophila) and Bacillus licheniformis (Bacillus licheniformis).
Further, described (R, R)-2,3-butanediol dehydrogenase gene source bacterial strain Paenibacillus polymyxa DSM365 preferably.This bacterial strain is purchased from German microbial strains preservation center (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), and strain number is DSM365.
Above-mentioned recombination bacillus coli is to adopt conventional method first to clone (R, R)-2,3-butanediol dehydrogenation enzyme gene (bdh) and glucose dehydrogenase gene (gdh), build again pETDuet-bdh-gdh recombinant plasmid, be transformed into intestinal bacteria, preferably, in e. coli bl21, then through screening, obtain; It contains bdh and gdh gene, is Gram-negative bacteria, aerobic or amphimicrobian growth, and better culture temperature is 37 ℃, its resting cell can be used for catalysis dimethyl diketone and produces optical purity R-acetoin.
Wherein, (R, R)-2,3-butanediol dehydrogenase gene (bdh) sequence length of the Paenibacillus polymyxa DSM365 that above-mentioned recombination bacillus coli is contained is 1053 bases, and its nucleotide sequence is as shown in SEQ ID NO.1; Glucose dehydrogenase gene (gdh) sequence length of contained Bacillus subtilis168 is 786 bases, and its nucleotide sequence is as shown in SEQ ID NO.2.
In the method for production optical purity R-acetoin of the present invention, recombination bacillus coli/pETDuet-bdh-gdh resting cell of take is biological catalyst, and the concrete grammar that the dimethyl diketone of take is produced optical purity R-acetoin as precursor conversion is:
(1) dull and stereotyped cultivation: recombination bacillus coli is scoring to to contain mass volume ratio be on LB flat board 1.5～1.8% agar and that contain 100 μ g/mL penbritins, cultivates 12 hours for 37 ℃;
(2) seed culture: under aseptic condition, with a single bacterium colony on toothpick picking step (1) flat board, be then inoculated in the LB liquid nutrient medium that 10～100mL contains 100 μ g/mL penbritins, 37 ℃ of shaking tables concussions are cultivated;
(3) fermentor cultivation: under aseptic condition, the bacterium liquid of getting step (2) gained be take the inoculum size that volume ratio is 1～10% and is inoculated in the LB liquid nutrient medium that contains 100 μ g/mL penbritins of 0.5～10L, cultivate 2～10 hours for 37 ℃, and then to add final concentration be the IPTG of 0.1～3mM, induce 2～20 hours, obtain a kind of culture for 10～37 ℃;
Wherein, the LB culture medium prescription described in above-mentioned steps (1)～(3) is: 10g/L peptone, and 5g/L yeast powder, 10g/L NaCl, under 121 ℃ of conditions, sterilizing is 15 minutes;
(4) collect thalline: step (3) is cultivated to the culture 5,000～8 obtaining, centrifugal 5～15 minutes of 000rpm; And with physiological saline washing thalline 2～3 times, then by cell suspension in the damping fluid of pH6～8, the final concentration that makes dry cell weight is 2～20g/L, obtains recombination bacillus coli resting cell biological catalyst, the refrigerator that is placed in 4 ℃ is stand-by;
(5) optical purity R-acetoin is prepared in conversion reaction: the biological catalyst that the dry cell weight concentration of take is 2～20g/L, under 10～42 ℃, pH5～9 condition, under 50～200rpm concussion condition, substrate is transformed, the dimethyl diketone that conversion starting point concentration is 5～25g/L, and add the glucose of 10～100g/L with the consumption of the required cofactors of postreaction; Every sampling in 3 hours, sample centrifugal 5～15 minutes with 8,000～12000rpm, removed the whole-cell biocatalyst adding; Utilize gas chromatography to detect the concentration of dimethyl diketone in supernatant, dimethyl diketone is added at interval, and the diacetyl concn in reaction system is maintained between 5～25g/L; Utilize SBA bio-sensing analyser to detect glucose concn, the glucose concn in reaction system is maintained between 5～50g/L.Supernatant is carried out to concentration and the optical purity that acetoin in conversion fluid is measured in gas chromatographic analysis, the method for calculation of R-acetoin optical purity: ee=([R] – [S])/([R]+[S]) * 100%; When the concentration of R-acetoin no longer increases, stop transforming.
In the method for above-mentioned production optical purity R-acetoin:
Preferably 10～30 ℃ of the described inducing temperatures of step (3); Induction time preferably 10～20 hours.
The described conversion reaction preferred cell of step (5) dry weight concentrations is 5～20g/L, at 20～37 ℃, under the condition of pH6～9, dimethyl diketone is transformed.
The described initial diacetyl concn of step (5) is 10～20g/L preferably, and initial glucose concentration is 20～50g/L preferably.
In the described conversion reaction process of step (5), add dimethyl diketone and glucose, diacetyl concn preferably maintains 10～20g/L, and glucose concn preferably maintains 10～50g/L.
The present invention has successfully realized first by (R, R)-2,3-butanediol dehydrogenation enzyme gene (bdh) and glucose dehydrogenase gene (gdh) are at expression in escherichia coli, and using this recombination bacillus coli resting cell as biological catalyst, take dimethyl diketone as precursor, glucose is regenerating coenzyme raw material, High-efficient Production optical purity R-acetoin.
The present invention has following characteristics:
(1) having built first and applied can coexpression (R, R)-2,3-butanediol desaturase and the recombination bacillus coli of Hexose phosphate dehydrogenase, and its resting cell can be used as biological catalyst for transforming dimethyl diketone High-efficient Production optical purity R-acetoin.
(2) (R, R)-2,3-butanediol desaturase catalysis dimethyl diketone generates optical purity R-acetoin; The regeneration of coenzyme in born of the same parents, the NADH that provide (R, R)-2,3-butanediol desaturase is required can be provided Hexose phosphate dehydrogenase.
(3) substratum of bacterial strain requirement is simple, cost is low.
(4) directly with complete recombination bacillus coli resting cell, as biological catalyst, transform, do not need smudge cells and separation and purification enzyme, operating process is simple.
(5) in reaction system, only there is the R-acetoin of single configuration, therefore do not need complicated chiral separation step.
(6) biological catalyst can be removed by filtration method or centrifuging, and it is cheap that follow-up rectifying separation is extracted expense.
Accompanying drawing explanation
The colibacillary protein electrophoresis figure of Fig. 1.M:Maker; 1: e. coli bl21; 2: e. coli bl21/pETDuet-1; 3: e. coli bl21/pETDuet-gdh; 4: e. coli bl21/pETDuet-bdh-gdh; 5: (R, the R) of purifying-2,3-butanediol desaturase.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the content described in embodiment only limits to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1
The structure of recombination bacillus coli BL21/pETDuet-bdh-gdh
The genomic dna of preparing bacterial strain Paenibacillus polymyxa DSM365 by conventional method, the genome of this bacterial strain is carried out to complete sequence order-checking and metabolism network parsing, and genes involved is annotated and Function Identification, obtained (the R of this bacterial strain, the encoding sequence of R)-2,3-butanediol dehydrogenase gene; Use synthetic primer P1 and P2, the genomic dna of P.polymyxa DSM365 of take is template, and pcr amplification obtains (R, R)-2,3-butanediol dehydrogenase gene (bdh).The genome sequence of Bacillus subtilis168 bacterial strain obtains from ncbi database, uses synthetic primer P3 and P4, and the genomic dna of B.subtilis168 of take is template, and pcr amplification obtains glucose dehydrogenase gene (gdh).
Described P1, P2, P3 and P4 sequence are respectively:
P1：5’-GATGCCATGGAAGCATTGAGATGGCATGG-3’；
P2：5’-TCGCGAGCTCTTAAGCTTGCGGAGATACCAG-3’。
P3：5’-CGAATTCCATATGTATCCGGATTTAAAAGG-3’；
P4：5’-GATCCAGACGTCTTAACCGCGGCCTGC-3’。
Above-mentioned bdh gene order length is 1053 bases, and its nucleotide sequence is as described in SEQ ID NO.1; Gdh gene order length is 786 bases, and its nucleotide sequence is as described in SEQ ID NO.2.
Two PCR products that pcr amplification is obtained are double digestion be connected to pETDuet-1 plasmid respectively, obtains recombinant plasmid pETDuet-bdh-gdh; Above-mentioned recombinant plasmid is imported to host e. coli BL21 through transforming, obtain recombination bacillus coli BL21/pETDuet-bdh-gdh.This bacterial strain is in containing the LB liquid nutrient medium of 100 μ g/mL penbritins, cultivate 3 hours for 37 ℃, adding final concentration is the IPTG of 0.5mM, induce 10 hours for 16 ℃, obtain expressing the recombinant Bacillus coli cells that has (R, R)-2,3-butanediol desaturase and Hexose phosphate dehydrogenase, through 12.5% SDS-PAGE checking, result as shown in Figure 1.
Above-mentioned recombination bacillus coli is Gram-negative bacteria, and aerobic or amphimicrobian growth can be produced optical purity R-acetoin for catalysis dimethyl diketone.
Embodiment 2
The preparation of recombination bacillus coli resting cell biological catalyst
(1) dull and stereotyped cultivation: recombination bacillus coli BL21/pETDuet-bdh-gdh is scoring to to contain mass volume ratio be on LB flat board 1.8% agar and that contain 100 μ g/mL penbritins, cultivates 12 hours for 37 ℃;
(2) seed culture: under aseptic condition, a single bacterium colony with on toothpick picking step (1) flat board, is then inoculated in the LB liquid nutrient medium that contains 100 μ g/mL penbritins of 20mL, 37 ℃ of shaking table concussions are cultivated 12 hours;
(3) fermentor cultivation: under aseptic condition, the bacterium liquid of getting step (2) gained be take inoculum size that volume ratio is 1% and is inoculated in the LB liquid nutrient medium that 2L contains 100 μ g/mL penbritins, cultivate 3 hours for 37 ℃, and then to add final concentration be the IPTG of 0.5mM, induce 10 hours for 16 ℃, stop cultivating;
Wherein, the LB culture medium prescription described in above-mentioned steps (1)～(3) is: 10g/L peptone, and 5g/L yeast powder, 10g/LNaCl, under 121 ℃ of conditions, sterilizing is 15 minutes;
(4) collect thalline: step (3) is cultivated to the culture 8 obtaining, centrifugal 5 minutes of 000rpm; And with physiological saline washing thalline 2 times, then by cell suspension in the damping fluid of pH7, the final concentration that makes dry cell weight is 15g/L, obtains recombination bacillus coli resting cell biological catalyst, the refrigerator that is placed in 4 ℃ is stand-by.
Embodiment 3
Utilize recombination bacillus coli resting cell to prepare optical purity R-acetoin as biological catalyst
Select the recombination bacillus coli resting cell of above-mentioned acquisition to carry out following conversion process as catalyzer: under 30 ℃, pH7 condition, the concussion of 180rpm shaking table, the dimethyl diketone that conversion starting point concentration is 20g/L, and add the glucose of 20g/L with the consumption of the required cofactor of postreaction; Every sampling in 3 hours, sample centrifugal 10 minutes with 8,000rpm, remove the whole-cell biocatalyst adding, utilize gas chromatography to detect the concentration of dimethyl diketone in supernatant, according to diacetyl concn, add dimethyl diketone, diacetyl concn is maintained between 10～20g/L; Supernatant is carried out to concentration and the optical purity that R-acetoin in conversion fluid is measured in gas chromatographic analysis; After 16 hours, detected result shows: the concentration of R-acetoin is 26g/L, and its optical purity is 99.6%.
Claims (8)
1. (R, R)-2,3-butanediol dehydrogenase gene bdh, is characterized in that, its nucleotide sequence is as shown in SEQ ID NO.1.
2. the production method of an optical purity R-acetoin, it is characterized in that, comprise the steps: described (R, R)-2,3-butanediol dehydrogenation enzyme gene bdh and glucose dehydrogenase gene gdh are at expression in escherichia coli, and to take this recombination bacillus coli resting cell be biological catalyst, the dimethyl diketone of take is produced optical purity R-acetoin as precursor conversion.
3. produce as claimed in claim 2 the method for optical purity R-acetoin, it is characterized in that, describedly take recombination bacillus coli resting cell as biological catalyst, the dimethyl diketone of take is the method that precursor conversion is produced optical purity R-acetoin, comprises the steps:
(1) dull and stereotyped cultivation: recombination bacillus coli is scoring to to contain mass volume ratio be on LB flat board 1.5～1.8% agar and that contain 100 μ g/mL penbritins, 37 ℃ of incubated overnight;
(2) seed culture: under aseptic condition, with a single bacterium colony on toothpick picking step (1) flat board, be then inoculated in the LB liquid nutrient medium that 10～100mL contains 100 μ g/mL penbritins, 37 ℃ of shaking tables concussions are cultivated;
(3) fermentor cultivation: under aseptic condition, the bacterium liquid of getting step (2) gained be take the inoculum size that volume ratio is 1～10% and is inoculated in the LB liquid nutrient medium that contains 100 μ g/mL penbritins of 0.5～10L, cultivate 2～10 hours for 37 ℃, and then to add final concentration be the IPTG of 0.1～3mM, induce 2～20 hours, obtain a kind of culture for 10～37 ℃;
Wherein, the LB culture medium prescription described in above-mentioned steps (1)～(3) is: 10g/L peptone, and 5g/L yeast powder, 10g/LNaCl, under 121 ℃ of conditions, sterilizing is 15 minutes;
(4) collect thalline: step (3) is cultivated to the culture 5,000～8 obtaining, centrifugal 5～15 minutes of 000rpm; And with physiological saline washing thalline 2～3 times, then by cell suspension in the damping fluid of pH6～8, the final concentration that makes dry cell weight is 2～20g/L, obtains recombination bacillus coli resting cell biological catalyst, the refrigerator that is placed in 4 ℃ is stand-by;
(5) optical purity R-acetoin is prepared in conversion reaction: the biological catalyst that the dry cell weight concentration of take is 2～20g/L, under 10～42 ℃, pH5～9 condition, under 50～200rpm concussion condition, substrate is transformed, the dimethyl diketone that conversion starting point concentration is 5～25g/L, and add the glucose of 10～100g/L with the consumption of the required cofactors of postreaction; Dimethyl diketone and glucose are added in interval, and the diacetyl concn in reaction system is maintained between 5～25g/L, and glucose concn maintains between 5～50g/L, when the concentration of R-acetoin no longer increases, stop transforming.
4. produce as claimed in claim 3 the method for optical purity R-acetoin, it is characterized in that, preferably 10～30 ℃ of the described inducing temperatures of step (3); Induction time preferably 10～20 hours.
5. produce as claimed in claim 3 the method for optical purity R-acetoin, it is characterized in that, the described conversion reaction preferred cell of step (5) dry weight concentrations is 5～20g/L, at 20～37 ℃, under the condition of pH6～9, dimethyl diketone is transformed.
6. produce as claimed in claim 3 the method for optical purity R-acetoin, it is characterized in that, the described initial diacetyl concn of step (5) is 10～20g/L preferably, and initial glucose concentration is 20～50g/L preferably.
7. produce as claimed in claim 3 the method for optical purity R-acetoin, it is characterized in that, in the described conversion reaction process of step (5), add dimethyl diketone and glucose, diacetyl concn preferably maintains 10～20g/L, and glucose concn preferably maintains 10～50g/L.
8. the application of (R, R)-2,3-butanediol dehydrogenase gene bdh aspect the production of optical purity R-acetoin.
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| 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 |
| CN115124414A (en) * | 2022-05-20 | 2022-09-30 | 山东省食品发酵工业研究设计院 | Preparation method and application of high-optical-purity 3-hydroxy butanone |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105969812A (en) * | 2015-07-13 | 2016-09-28 | 广西科学院 | Method for preparing chiral (S)-acetoin by virtue of whole-cell biological catalysis |
| 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 |
| CN115124414A (en) * | 2022-05-20 | 2022-09-30 | 山东省食品发酵工业研究设计院 | Preparation method and application of high-optical-purity 3-hydroxy butanone |
| CN117535372A (en) * | 2023-10-23 | 2024-02-09 | 镇江市德尔生物制品研究所有限公司 | Beta-glucosidase and application thereof in preparation of ginsenoside |
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