CN100562580C - The application of one bacillus subtilis in preparation 3-oxobutanol - Google Patents
The application of one bacillus subtilis in preparation 3-oxobutanol Download PDFInfo
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- CN100562580C CN100562580C CNB200710013403XA CN200710013403A CN100562580C CN 100562580 C CN100562580 C CN 100562580C CN B200710013403X A CNB200710013403X A CN B200710013403XA CN 200710013403 A CN200710013403 A CN 200710013403A CN 100562580 C CN100562580 C CN 100562580C
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Abstract
The invention discloses the application of a bacillus subtilis in preparation 3-oxobutanol.The bacterial strain that wherein relates to has been deposited on November 23rd, 2006 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", preserving number are CGMCC NO.1869.Application method comprises the selection of (1) bacterial classification, the activation of (2) slant culture, and (3) seed culture, (4) fermentation culture, steps such as tunning are detected and collected in (5); Described method has simple to operate, and cost is low, purpose product 3-oxobutanol productive rate height, purity height, does not produce the characteristics of the common coproduction compounds of 3-oxobutanol such as dimethyl diketone and 2,3 butyleneglycols, has tempting application prospect.
Description
Technical field
The present invention relates to the application of a bacillus subtilis, relate in particular to the application of a bacillus subtilis in preparation 3-oxobutanol, belong to biological technical field.
Background technology
3-oxobutanol (acetoin) has another name called acetoin, acetyl methyl carbinol, naturally be present in the numerous food products such as corn, grape, cocoa, apple, banana, cheese, meat, have distinctive cream fragrance, in addition, beer flavor, cheese fragrance are all relevant with the 3-oxobutanol.
The 3-oxobutanol be a kind ofly be widely used, charming flavouring agent, be spices kind commonly used in the world.Main as spices such as cream, dairy products, sour milk and strawberry types, CNS GB2760-86 stipulates that it is the food spice that allows use.The 3-oxobutanol of 80% content is commonly called as " vinegar drone ", is important kind in the drinks blending.The 3-oxobutanol still is a kind of important chemical synthesis material and medicine intermediate.With the 3-oxobutanol is that raw material can generate di-acetyl and 2 by oxidation, reduction reaction, the 3-butyleneglycol, these two kinds of compounds all have purposes widely in chemical industry, particularly 2, the 3-butyleneglycol is except that self being a kind of high energy fuels, can also be used to synthetic aviation fuel, once becoming the research focus.In pharmaceutical industry, the 3-oxobutanol can be used for synthesizing rare medicine and pharmaceutical intermediate as having chiral compounds, can efficiently synthesize 4-chloro-4 as 3-oxobutanol and phosgene, 5-dimethyl-1,3 dioxolane-2-ketone (CDMDO), CDMDO is a kind of important medicine intermediate, be mainly used in the modification of antibiotics such as penicillin, penbritin, thereby improve drug effect largely, alleviate Side effects of pharmaceutical drugs, widelyd popularize and use in American-European countries and Japan.The 3-oxobutanol can also synthesize human relations Ampicillin Trihydrate hydrochloride, human relations Ampicillin Trihydrate hydrochloride is the prodrug of semisynthetic penicillin, penbritin, can be oral, have good stability in the stomach, absorb soon the blood middle concentration height, stronger 2～4 times than the effect of penbritin, untoward reaction is little, and toxicity is low, in American-European and Japan's listing.
3-oxobutanol production method mainly comprises chemical synthesis and biotransformation method.Two kinds of methods are mainly adopted in chemosynthesis: a kind of is to reduce by dimethyl diketone (di-acetyl) partial hydrogenation; Another kind is by 2,3-butyleneglycol selective oxidation.Biotransformation method also has two kinds of technologies, and a kind of is to utilize the production of enzymatic conversion method dimethyl diketone, and another is by the production of microbial fermentation saccharine material.The reduction of dimethyl diketone partial hydrogenation is to study and use more 3-oxobutanol production method at present both at home and abroad, and this method mainly is to be that different reductive agent, the catalyzer of raw material interpolation comes hydrogenating reduction to synthesize the 3-oxobutanol with the dimethyl diketone, and transformation efficiency does not wait at 50-80%.With 2,3-butyleneglycol selective oxidation processes can select for use copper as catalyzer, product is the mixture (1945 of dimethyl diketone and 3-oxobutanol, R.H.Blom), also have the investigator to adopt electrochemical oxidation 2 in addition, the 3-butyleneglycol prepares the 3-oxobutanol, and selectivity is good, the productive rate height, but this technology only is in conceptual phase (A.Hilmi in 1997).Enzymatic conversion method is produced the 3-oxobutanol: people such as Hummel in 1992 are lactobacillus or yeast separation and purification dimethyl diketone reductase enzyme and the coenzyme NAPH from cultivating once, at pH5, under 70 ℃ of the temperature, catalyzed conversion 2, the 3-dimethyl diketone generates the 3-oxobutanol, use this method productive rate to reach as high as 100%, and do not have other by product.Also once report was by sorbose bacterium or living film bacterium conversion 2 for Susan Budavari, and the 3-butyleneglycol generates the 3-oxobutanol.These methods are similar with chemosynthesis, all are to be raw material with dimethyl diketone or butyleneglycol, generate the 3-oxobutanol through enzyme process partial reduction or oxidation.Difference is enzyme process efficiency of pcr product height, does not have other by product, and product has specific rotation, but will obtain relatively difficulty of a large amount of specific enzymes.
Chemical synthesis and biological enzyme conversion method all are with dimethyl diketone or 2, the 3-butyleneglycol is a raw material, but dimethyl diketone and 2, the 3-butyleneglycol also is a synthetic perfume, but not large Chemicals, therefore raw material sources, product price and use range all are restricted, and this also is the one of the main reasons that two kinds of technologies do not obtain large-scale promotion application.
The 3-oxobutanol is the glycometabolic mesostate of multiple microorganism, can produce the 3-oxobutanol with the saccharic for the prepared using microbial fermentation, but, present according to the retrieval related microorganism produces the document of 3-oxobutanol, most reports are the research of related microorganism pathways metabolism theoretical side, the report of the 3-oxobutanol production that minority relates to also mainly is that the by product of 3-butyleneglycol fermentation is mentioned as dimethyl diketone and 2.Can utilize xylan to generate dimethyl diketone and 3-oxobutanol such as R.B.hespell report poly-viscosity bacillus, its ultimate production reaches as high as 11.3g/L.Braneni and Keenan utilize lactobacillus-fermented to produce dimethyl diketone and 3-oxobutanol, and this bacterial strain also produces 2 simultaneously, the 3-butyleneglycol.The 3-oxobutanol is produced in the fermentation of report such as R.M.Teixeira debaryomyces hansenii, and output reaches as high as 0.36g/L.At present, yet there are no, do not see that also using Bacillus subtilis strain fermentation transforming glucose directly generates and obtain the report of purpose product 3-oxobutanol about the research report of 3-oxobutanol as microorganism main purpose product.
Reference:
1.Susan?Budavari.The?Merck?Index[M].Twelfth?Edition.，1996.12.
2.Hummel.USP；5164314，1992
3.Blom?R?H..Am?Chem?Soc，1945，67：494
4.Hilmi A, Belgsir EM, Leger JM,, wait .J.Electro.Chem., 1997,435:69.
5.R.B.Hespell.Curr.Micro.1996，32：291
6.R.M.Teixeira D.Cavalheiro waits .Braz.J.Chem.Eng.19 (2): 181
7.A.L.Braneni?and?T.W.Keenan.Appl.Micro.，1971：517
Summary of the invention:
At the deficiencies in the prior art, the present invention will deal with problems and provide the application of a bacillus subtilis in preparation 3-oxobutanol.Utilize bacterial strain of the present invention transforming glucose generation 3-oxobutanol effectively, do not produce dimethyl diketone and 2, the common coproduction compounds of 3-oxobutanol such as 3-butyleneglycol, realizing obtaining with the 3-oxobutanol by microbe fermentation method is main purpose product hope.
The application of subtilis of the present invention in preparation 3-oxobutanol.
Wherein, the sequence of steps that relates to of described application method is as follows:
(1) bacterial classification is selected: select subtilis (Bacillus subtilis) CGMCC NO.1869 for use;
Above-mentioned bacterial strains has been deposited on November 23rd, 2006 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", bacterial strain are called subtilis (Bacillus subtilis) SFA-H31, and preserving number is CGMCCN0.1869.
Above-mentioned subtilis, its biological property is: do not move, living statospore, sporangiocyst does not expand, and does not have parasporal crystal to generate, and presents shaft-like during 37 ℃ of liquid culture childrens, single, in pairs or be catenation, began to give birth to spore in 16-18 hour, 24 ± 2 hours spore maturations come off (thalline nourishing body and gemma form referring to accompanying drawing 1, accompanying drawing 2); 37 ℃ of solid culture bacterium colony initial stages are rounded, along with the prolongation of incubation time is irregular gradually; Bacterium colony is flat, ground-glass appearance is surperficial, projection not; Began the spore of sprouting in slant culture 16-20 hour, gemma came off fully in 48 ± 2 hours; Physiological and biochemical property is: Gram-positive, and aerobic, chemoheterotrophic bacteria produces acid from glucose, fructose, seminose, maltose, sucrose, trehalose, and glucose fermentation is aerogenesis not, does not utilize Citrate trianion, the catalase positive, reduction nitrate; The result who measures the gene order of 16SrRNA shows that its gene order length is 1468bp.
(2) slant culture activation: with bacterial classification inoculation in slant medium, under 35～40 ℃ of conditions, static cultivation 24～48 hours, standby;
(3) seed culture: with the bacterial strain of step (2) cultivation, under aseptic condition, encircle in 30～40mL (250mL triangular flask) liquid seed culture medium with inoculation articulating 1～2, putting the rotation rotating speed is that 150～180 rev/mins, rotation radius are on the shaking table of 40mm, cultivated 12～18 hours for 35～40 ℃, get seed liquor;
(4) fermentation culture:
Shake flask fermentation: the inoculum size of volume ratio with 1～5%, seed liquor is inoculated in the shaking in the bottle of 80-100mL (500mL triangular flask) fermention medium is housed, 35～40 ℃, rotating speed is 150～180 rev/mins, shake flask fermentation 48-72 hour, when detection in the fermented liquid is residual less than glucose, fermentation ends;
50L ferment tank: the inoculum size of volume ratio with 1～5%, seed liquor is inoculated in the canned 50L fermentor tank that 33～35L fermention medium arranged, 35～40 ℃ of stir culture of ventilating, wherein mixing speed is 150～200r/min, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute count 1: 0.5 ± 0.1vvm, jar internal pressure 0.1 ± 0.02MPa, fermentation time is 40～70 hours; Regulate fermentor tank rotating speed and ventilation than the saturation ratio of control dissolved oxygen levels at 5-20%, the fermented liquid initial pH value is 7.0～7.5, stream adds soda acid and makes the pH of fermented liquid be maintained 6.0～7.0 in the fermenting process, the glucose in the fermenting process in the interval timing sampling mensuration fermented liquid and the concentration of 3-oxobutanol, glucose concn no longer reduces in fermented liquid, when 3-oxobutanol concentration no longer rises, finish fermentation;
(5) product detects: get above-mentioned fermented liquid with 3000～3500 rev/mins centrifugal 5～8 minutes, measure the content of glucose and 3-oxobutanol in the supernatant liquor, and calculate the transformation efficiency that conversion of glucose generates the 3-oxobutanol; Collect fermented liquid simultaneously and carry out underpressure distillation, distillate part condensation and collect the purity that liquid carries out 3-oxobutanol in the gas chromatographic analysis mensuration tunning at 80 ℃.
Above-mentioned slant medium consists of: glucose 5g/L, peptone 5g/L, yeast extract paste 5g/L, sodium-chlor 3g/L, sal epsom 0.1g/L, agar 20g/L, pH 7.0-7.2, distilled water preparation;
The aforesaid liquid seed culture medium is formed: glucose 20～25g/L, yeast extract paste 20g/L, potassium primary phosphate 0.1g/L, dipotassium hydrogen phosphate 0.1g/L, pH7.0-7.2, tap water preparation;
Consisting of of above-mentioned fermention medium: glucose 80-140g/L, yeast extract paste 2g/L, corn steep liquor 10g/L, pH7.0-7.2, tap water preparation.
In the above-mentioned application, preferred 37 ± 0.2 ℃ of step (2), (3), (4) described culture temperature.
In the above-mentioned application, preferred 30 hours of the described incubation time of step (2).
In the above-mentioned application, the described incubation time of step (3) preferred 12-14 hour.
In the above-mentioned application, the preferred 100-120g/L of the described fermention medium initial glucose concentration of step (4).
In the above-mentioned application, the saturation ratio of the preferred 10-15% of the described dissolved oxygen levels of step (4).
In the above-mentioned application, the fermented liquid pH preferred 6.5 ± 0.2 in the described fermenting process of step (4).
Above-mentioned glucose is measured as follows:
The SBA-4C type membrane bioreactor that uses the Shandong Province academy sciences Biology Research Institute to produce is measured.Measuring principle is to utilize the single-minded mensuration glucose content of fixation glucose dehydrogenation enzyme membrane.
Above-mentioned 3-oxobutanol is measured as follows:
Chemical colorimetry: consult reference (W.W.Westerfeld, A COLORIMETRIC DETERMINATIONOF BLOOD ACETOIN.J.Biol.Chem., 1945; 161:495-502).
The drafting of typical curve: according to the form below adds all ingredients and solution in proper order, and 60 ℃ of colour developing 15min use spectrophotometer to survey light absorption value under 530nm, the drawing standard curve.
Fermented liquid centrifuging and taking supernatant liquor suitably is diluted to the content of 3-oxobutanol about 10 μ g, colour developing as stated above, and 530nm measures light absorption value, according to the content of 3-oxobutanol in typical curve and the extension rate calculating fermented liquid.
The vapor-phase chromatography qualitative analysis:
GC conditions: use the GEP-20M capillary column, carrier gas is nitrogen (N
2), flow velocity is 0.9mL/min; Column cap is pressed 0.10MPa; 200 ℃ of injector temperatures; Temperature programming: 100 ℃ when protecting 3 minutes, 8 ℃/min heats up and causes 180 ℃, during the guarantor 3 minutes; Sample size 1 μ L; Splitting ratio 5: 1.The retention time of 3-oxobutanol is about 8.7 minutes under these conditions, and the retention time of di-acetyl is about 4.5 minutes, 2, and the retention time of 3-butyleneglycol is about 13.5 minutes.
Subtilis CGMCC NO.1869 of the present invention can ferment, and transforming glucose directly generates and acquisition purpose product 3-oxobutanol, 3-oxobutanol productive rate height in the unit volume fermented liquid, conversion of glucose generates the transformation efficiency height of 3-oxobutanol, produce 3-oxobutanol purity height, the strain fermentation stable performance.
Subtilis CGMCC NO.1869 of the present invention can ferment, and transforming glucose directly generates and acquisition purpose product 3-oxobutanol, the product purity height, do not produce dimethyl diketone and 2, the common coproduction compounds of 3-oxobutanol such as 3 butyleneglycols are that a strain has the 3-oxobutanol production bacterial strain that research and development are worth.
The present invention adopts the method for biological fermentation, and it is simple with glucose to be that raw material production 3-oxobutanol has production method, mild condition, and characteristics such as raw material sources are abundant, and production cost is low have tempting application prospect.
The inventor does not retrieve and utilizes subtilis is the bibliographical information of purpose product to produce the 3-oxobutanol, does not also retrieve the bibliographical information of producing bacillus subtilis 3-oxobutanol high purity, high yield and the high conversion identical with CGMCC NO.1869 bacterial strain.
Utilizing subtilis CGMCC NO.1869 fermentation of the present invention, is that fermenting raw materials production 3-oxobutanol productive rate reaches as high as more than the 55g/L with glucose, and the average conversion of conversion of glucose 3-oxobutanol is 45%, is up to 50%.Distillate about 95% of part peak area average out to total area of condensation collection liquid 3-oxobutanol in gas chromatographic analysis mensuration tunning, reach as high as more than 98%.
Description of drawings
Bacterial strain subtilis provided by the invention (Bacillus subtilis) has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 23rd, 2006, preservation address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, postcode: 100080, its deposit number is CGMCC N0.1869.
Fig. 1 shows subtilis CGMCC NO.1869 thalline nourishing body form (* 1600).
Fig. 2 shows subtilis CGMCC NO.1869 gemma form (* 1600).
Fig. 3 shows that the underpressure distillation of subtilis CGMCC NO.1869 bacterial strain fermentation liquor distillates part condensation and collects the liquid gas chromatogram.This collection of illustrative plates is that the underpressure distillation of one time fermentation fermented liquid distillates part condensation and collects a liquid gas chromatogram, among the figure retention time be 8.768 minutes be the 3-oxobutanol, peak area accounts for 96.4% of total peak area.
Embodiment
Embodiment 1 produces the seed selection of 3-oxobutanol subtilis
Starting strain SFA-W15 (bacillus subtilis strain of a strain small amount of accumulation 3-oxobutanol) is inoculated in liquid seed culture medium, cultivate logarithmic phase mid-term for 37 ℃, centrifugal collection thalline, the stroke-physiological saline solution washed twice, add stroke-physiological saline solution then and make bacteria suspension, make cell concn 1 * 10
8-10
9Individual/ml.
Get above-mentioned bacteria suspension 10ml and put into plate, put irradiation under the 30W ultraviolet lamp, irradiation distance 15cm, irradiation time is 3 minutes, 5 minutes, 7 minutes, 10 minutes, the screening flat board that the back coating contains 0.5%LiCl is suitably diluted in sampling at interval, lucifuge was cultivated 1-2 days for 37 ℃, picking list bacterium colony moves and connects the inclined-plane, cultivated 1-2 days for 37 ℃, the bottle that shakes of fermention medium is equipped with in inoculation after the inclined-plane maturation, 37 ℃ of shake flask fermentations 60 hours, and it is centrifugal 5 minutes to get 3000 rev/mins of fermented liquids, get supernatant liquor and measure concentration of residual glucose and 3-oxobutanol content in the fermented liquid, it is low to choose concentration of residual glucose, the bacterial strain that 3-oxobutanol content is high is the purpose bacterial strain.
The purpose bacterial strain is sieved and further separation screening again through shake flask fermentation again, obtain a strain and stablize the mutant strain SFA-U97 that sugar consumption rate and 3-oxobutanol output all have raising.
SFA-U97 inoculation initial glucose concentration is the fermention medium of 78.2g/L, and 37 ℃ of shake-flask culture finished fermentation in 60 hours, detected less than glucose in the fermented liquid, and 3-oxobutanol content is 30.6g/L, and transformation efficiency is 39.1%.
The seed selection of embodiment 2 high yield 3-oxobutanol subtilis CGMCC NO.1869 bacterial strains
In the mutant strain SFA-U97 inoculation liquid seed culture medium that embodiment 1 is screened, cultivate logarithmic phase mid-term, centrifugal collection thalline, stroke-physiological saline solution washed twice for 37 ℃, add stroke-physiological saline solution then and make bacteria suspension, make cell concn 1 * 10
8-10
9Individual/ml, standby.
Nitrosoguanidine treatment solution preparation: take by weighing nitrosoguanidine 20mg, be positioned in the aseptic triangular flask of 100ml, add acetone 2ml and make its dissolving, add again 18ml Tris damping fluid (pH6.0,0.5mol/L) mixing, standby.
Get above-mentioned nitrosoguanidine treatment solution 10ml, add the 10ml bacteria suspension, 30 ℃ of insulations were vibrated 50-60 minute, every sampling in 10 minutes once, at first dilute 1000 times of termination reactions after the sampling, and then appropriateness dilution, spread plate, cultivated 1-2 days for 37 ℃, picking list bacterium colony moves and connects the inclined-plane, and the bottle that shakes of fermention medium is equipped with in inoculation after the inclined-plane maturation, 37 ℃ of shake flask fermentations 60 hours, fermented liquid 3000 left the heart 5 minutes, got supernatant liquor and measured concentration of residual glucose and 3-oxobutanol content in the fermented liquid, and it is low to choose concentration of residual glucose, the bacterial strain that 3-oxobutanol content is high is the purpose bacterial strain.
The purpose bacterial strain is sieved and further separation screening again through shake flask fermentation again, and seed selection obtains a strain stable hereditary property, 3-oxobutanol output is up to the above mutant strain SFA-H31 of 55g/L.
Above-mentioned bacterial strains subtilis (Bacillus subtilis) SFA-H31, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 23rd, 2006, preservation address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, postcode: 100080, its deposit number is CGMCC N0.1869.
The aforesaid liquid seed culture medium is formed (g/L): glucose 20, and yeast extract paste 20, potassium primary phosphate 0.1, dipotassium hydrogen phosphate 0.1 is adjusted pH7.0-7.2, the tap water preparation.
Above-mentioned screening consists of (g/L) with plate culture medium: glucose 5, and peptone 5, yeast extract paste 5, sodium-chlor 3, sal epsom 0.1, agar 20 is adjusted pH7.0-7.2, the distilled water preparation.
Above-mentioned slant medium consists of (g/L): glucose 5, and peptone 5, yeast extract paste 5, sodium-chlor 3, sal epsom 0.1, agar 20 is adjusted pH7.0-7.2, the distilled water preparation.
The composition of above-mentioned fermention medium (g/L): glucose 80-140, yeast extract paste 2, corn steep liquor 10 is adjusted pH7.0-7.2, the tap water preparation.
The application of embodiment 3 subtilises in preparation 3-oxobutanol
The sequence of steps that application method relates to is as follows:
(1) bacterial classification is selected: select subtilis (Bacillus subtilis) CGMCC NO.1869 for use;
(2) slant culture activation: with bacterial classification inoculation in slant medium, under 37 ℃ of conditions, static cultivation 30 hours, standby;
(3) seed culture: with the bacterial strain of step (2) cultivation, under aseptic condition, encircle in the 30mL liquid seed culture medium (250mL triangular flask) with inoculation articulating 2, putting the rotation rotating speed is that 160 rev/mins, rotation radius are on the shaking table of 40mm, cultivated 12 hours for 37 ℃, seed liquor;
(4) fermentation culture:
Shake flask fermentation: the inoculum size of volume ratio with 3%, with seed liquor be inoculated in be equipped with the 80mL fermention medium shake the bottle (500mL triangular flask) in, 37 ℃, rotating speed is 160 rev/mins, shake flask fermentation 60 hours, when detection in the fermented liquid is residual less than glucose, fermentation ends;
(5) product detects: get above-mentioned fermented liquid with 3000 rev/mins centrifugal 8 minutes, measure the content of glucose and 3-oxobutanol in the supernatant liquor, and calculate the transformation efficiency that conversion of glucose generates the 3-oxobutanol; Collect fermented liquid simultaneously and carry out underpressure distillation, distillate part condensation and collect the purity that liquid carries out 3-oxobutanol in the gas chromatographic analysis mensuration tunning at 80 ℃.
The content that fermented liquid detects the 3-oxobutanol is 47.5g/L, and the transformation efficiency that conversion of glucose generates the 3-oxobutanol is 43.2%.
Fermented liquid distillates part condensation collection liquid and accounts for 94.7% of the total area through the peak shape area of gas chromatographic analysis 3-oxobutanol as stated above through underpressure distillation.
Above-mentioned slant medium consists of: glucose 5g/L, peptone 5g/L, yeast extract paste 5g/L, sodium-chlor 3g/L, sal epsom 0.1g/L, agar 20g/L, pH 7.0-7.2, distilled water preparation;
The aforesaid liquid seed culture medium is formed: glucose 22g/L, yeast extract paste 20g/L, potassium primary phosphate 0.1g/L, dipotassium hydrogen phosphate 0.1g/L, pH7.0-7.2, tap water preparation;
Consisting of of above-mentioned fermention medium: glucose 110g/L, yeast extract paste 2g/L, corn steep liquor 10g/L, pH7.0-7.2, tap water preparation.
The application of embodiment 4 subtilises in preparation 3-oxobutanol
The sequence of steps that application method relates to is as follows:
(1) bacterial classification is selected: select subtilis (Bacillus subtilis) CGMCC NO.1869 for use;
(2) slant culture activation: with bacterial classification inoculation in slant medium, under 37 ℃ of conditions, static cultivation 28 hours, standby;
(3) seed culture: with the bacterial strain of step (2) cultivation, under aseptic condition, encircle in the 60mL liquid seed culture medium (500mL triangular flask) with inoculation articulating 2,20 bottles of repeated inoculations, putting the rotation rotating speed is that 150 rev/mins, rotation radius are on the shaking table of 40mm, cultivated 14 hours for 37 ℃, get seed liquor;
(4) fermentation culture:
The 50L ferment tank: the inoculum size of volume ratio with 4%, seed liquor is inoculated in the 50L fermentor tank of the canned 33L of having fermention medium, 37 ℃ of stir culture of ventilating, wherein mixing speed is 200r/min, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute count 1: 0.5vvm, jar internal pressure 0.1MPa; Regulate fermentor tank rotating speed and ventilation than the saturation ratio of control dissolved oxygen levels at 10-15%, the fermented liquid initial pH value is 7.0～7.2, stream adds soda acid and makes the pH of fermented liquid be maintained 6.5 in the fermenting process, the glucose in the fermenting process in the timing sampling mensuration fermented liquid and the concentration of 3-oxobutanol, when 3-oxobutanol concentration no longer rises in the fermented liquid, finish fermentation, fermentation time is 52 hours;
(5) product detects: get above-mentioned fermented liquid with 3500 rev/mins centrifugal 5 minutes, measure the content of glucose and 3-oxobutanol in the supernatant liquor, and calculate the transformation efficiency that conversion of glucose generates the 3-oxobutanol; Collect fermented liquid simultaneously and carry out underpressure distillation, distillate part condensation and collect the purity that liquid carries out 3-oxobutanol in the gas chromatographic analysis mensuration tunning at 80 ℃.
The content that fermented liquid detects the 3-oxobutanol is 55.5g/L, and the transformation efficiency that conversion of glucose generates the 3-oxobutanol is 48.26%.
Fermented liquid distillates part condensation and collects liquid accounts for the total area as stated above through the peak shape area of gas chromatographic analysis 3-oxobutanol 96.4% (referring to accompanying drawing 3) through underpressure distillation.
Above-mentioned slant medium consists of: glucose 5g/L, peptone 5g/L, yeast extract paste 5g/L, sodium-chlor 3g/L, sal epsom 0.1g/L, agar 20g/L, pH 7.0-7.2, distilled water preparation;
The aforesaid liquid seed culture medium is formed: glucose 20g/L, yeast extract paste 20g/L, potassium primary phosphate 0.1g/L, dipotassium hydrogen phosphate 0.1g/L, pH7.0-7.2, tap water preparation;
Consisting of of above-mentioned fermention medium: glucose 115g/L, yeast extract paste 2g/L, corn steep liquor 10g/L, pH7.0-7.2, tap water preparation.
The application of embodiment 5 subtilises in preparation 3-oxobutanol
The sequence of steps that application method relates to is as follows:
(1) bacterial classification is selected: select subtilis (Bacillus subtilis) CGMCC NO.1869 for use;
(2) slant culture activation: with bacterial classification inoculation in slant medium, under 35 ℃ of conditions, static cultivation 40 hours, standby;
(3) seed culture: with the bacterial strain of step (2) cultivation, under aseptic condition, encircle in the 30mL liquid seed culture medium (250mL triangular flask) with inoculation articulating 1, putting the rotation rotating speed is that 150 rev/mins, rotation radius are on the shaking table of 40mm, cultivated 18 hours for 35 ℃, seed liquor;
(4) fermentation culture:
The 50L ferment tank: the inoculum size of volume ratio with 2%, seed liquor is inoculated in the 50L fermentor tank of the canned 33L of having fermention medium, 35 ℃ of stir culture of ventilating, wherein mixing speed is 150r/min, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute count 1: 0.5 ± 0.1vvm, jar internal pressure 0.1 ± 0.02MPa; Regulate fermentor tank rotating speed and ventilation than the saturation ratio of control dissolved oxygen levels at 5-10%, the fermented liquid initial pH value is 7.0～7.1, stream adds soda acid and makes the pH of fermented liquid be maintained 6.1～6.2 in the fermenting process, the glucose in the fermenting process in the timing sampling mensuration fermented liquid and the concentration of 3-oxobutanol, when 3-oxobutanol concentration no longer rises in the fermented liquid, finish fermentation, fermentation time is 48 hours;
(5) product detects: get above-mentioned fermented liquid with 3200 rev/mins centrifugal 7 minutes, measure the content of glucose and 3-oxobutanol in the supernatant liquor, and calculate the transformation efficiency that conversion of glucose generates the 3-oxobutanol; Collect fermented liquid simultaneously and carry out underpressure distillation, distillate part condensation and collect the purity that liquid carries out 3-oxobutanol in the gas chromatographic analysis mensuration tunning at 80 ℃.
The content that fermented liquid detects the 3-oxobutanol is 34.6g/L, and the transformation efficiency that conversion of glucose generates the 3-oxobutanol is 43.25%.
Fermented liquid distillates part condensation collection liquid and accounts for 96.8% of the total area through the peak shape area of gas chromatographic analysis 3-oxobutanol as stated above through underpressure distillation.
Above-mentioned slant medium consists of: glucose 5g/L, peptone 5g/L, yeast extract paste 5g/L, sodium-chlor 3g/L, sal epsom 0.1g/L, agar 20g/L, pH 7.0-7.2, distilled water preparation;
The aforesaid liquid seed culture medium is formed: glucose 20g/L, yeast extract paste 20g/L, potassium primary phosphate 0.1g/L, dipotassium hydrogen phosphate 0.1g/L, pH7.0-7.2, tap water preparation;
Consisting of of above-mentioned fermention medium: glucose 80g/L, yeast extract paste 2g/L, corn steep liquor 10g/L, pH7.0-7.2, tap water preparation.
The application of embodiment 6 subtilises in preparation 3-oxobutanol
The sequence of steps that application method relates to is as follows:
(1) bacterial classification is selected: select subtilis (Bacillus subtilis) CGMCC NO.1869 for use;
(2) slant culture activation: with bacterial classification inoculation in slant medium, under 40 ℃ of conditions, static cultivation 26 hours, standby;
(3) seed culture: with the bacterial strain of step (2) cultivation, under aseptic condition, encircle in the 40mL liquid seed culture medium (250mL triangular flask) with inoculation articulating 2, putting the rotation rotating speed is that 180 rev/mins, rotation radius are on the shaking table of 40mm, cultivated 18 hours for 40 ℃, get primary seed solution, the inoculum size of volume ratio with 5%, seed liquor is inoculated in shake (the 500mL triangular flask) in the bottle that the 100mL seed culture medium is housed, 40 ℃, rotating speed is 180 rev/mins, shake-flask culture 20 hours gets secondary seed solution;
(4) fermentation culture:
The 50L ferment tank: the inoculum size of volume ratio with 5%, secondary seed solution is inoculated in the 50L fermentor tank of the canned 35L of having fermention medium, 40 ℃ of stir culture of ventilating, wherein mixing speed is 200r/min, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute count 1: 0.5 ± 0.1vvm, jar internal pressure 0.1 ± 0.02MPa; Regulate fermentor tank rotating speed and ventilation than controlling the saturation ratio of dissolved oxygen levels 20%, the fermented liquid initial pH value is 7.4～7.5, stream adds soda acid and makes the pH of fermented liquid be maintained 6.8～7.0 in the fermenting process, the at interval glucose in 2 hours sampling and measuring fermented liquids and the concentration of 3-oxobutanol in the fermenting process, when 3-oxobutanol concentration no longer rises in the fermented liquid, finish fermentation, fermentation time is 70 hours;
(5) product detects: get above-mentioned fermented liquid with 3400 rev/mins centrifugal 6 minutes, measure the content of glucose and 3-oxobutanol in the supernatant liquor, and calculate the transformation efficiency that conversion of glucose generates the 3-oxobutanol; Collect fermented liquid simultaneously and carry out underpressure distillation, distillate part condensation and collect the purity that liquid carries out 3-oxobutanol in the gas chromatographic analysis mensuration tunning at 80 ℃.
The content that fermented liquid detects the 3-oxobutanol is 55.67g/L, and the transformation efficiency that conversion of glucose generates the 3-oxobutanol is 45.63%.
Fermented liquid distillates part condensation collection liquid and accounts for 95.8% of the total area through the peak shape area of gas chromatographic analysis 3-oxobutanol as stated above through underpressure distillation.
Above-mentioned slant medium consists of: glucose 5g/L, peptone 5g/L, yeast extract paste 5g/L, sodium-chlor 3g/L, sal epsom 0.1g/L, agar 20g/L, pH 7.0-7.2, tap water preparation;
The aforesaid liquid seed culture medium is formed: glucose 25g/L, yeast extract paste 20g/L, potassium primary phosphate 0.1g/L, dipotassium hydrogen phosphate 0.1g/L, pH7.0-7.2, tap water preparation;
Consisting of of above-mentioned fermention medium: glucose 122g/L, yeast extract paste 2g/L, corn steep liquor 10g/L, pH7.0-7.2, tap water preparation.
The application of embodiment 7 subtilis CGMCC NO.1869 in preparation 3-oxobutanol
Subtilis CGMCC NO.1869 bacterial strain bacterial classification inoculation is activated in slant medium, under 36 ℃ of conditions, static cultivation 40 hours, standby;
Press the composition preparation seed culture medium of liquid seed culture medium, the initial glucose concentration actual measurement is 21.5g/L, 250ml triangular flask liquid amount is 30ml, 118 ℃ of steam sterilizings 25 minutes are cooled to room temperature, inoculation slant strains 2 rings, putting the rotation rotating speed is that 150 rev/mins, rotation radius are on the shaking table of 40mm, cultivated 12 hours for 37 ℃, seed liquor, get 10 times of seed liquor dilutions and put 610nm to survey light absorption value be 0.358;
Press the composition preparation fermention medium of fermention medium, the initial glucose concentration actual measurement is 115g/L, 500ml triangular flask liquid amount is 80ml, 118 ℃ of steam sterilizings 25 minutes, be cooled to room temperature, inoculation seed liquor 3ml, putting the rotation rotating speed is that 150 rev/mins, rotation radius are on the 40mm shaking table, cultivate for 37 ℃ and finished fermentation in 68 hours, fermented liquid is centrifugal 5 minutes with 3000 rev/mins, the content of getting supernatant liquor mensuration glucose concn and 3-oxobutanol is respectively 0g/L and 53.5g/L, and the transformation efficiency that conversion of glucose generates the 3-oxobutanol is 46.52%.
Fermented liquid distillates part condensation collection liquid and accounts for 94.3% of the total area through the peak shape area of gas chromatographic analysis 3-oxobutanol as stated above through underpressure distillation.
The application of embodiment 8 subtilis CGMCC NO.1869 in preparation 3-oxobutanol
Subtilis CGMCC NO.1869 bacterial strain bacterial classification inoculation is activated in slant medium, under 37 ℃ of conditions, static cultivation 38 hours, standby;
Press the composition preparation seed culture medium of liquid seed culture medium, the initial glucose concentration actual measurement is 20g/L, 250ml triangular flask liquid amount is 40ml, 118 ℃ of steam sterilizings 25 minutes, be cooled to room temperature, inoculation slant strains suspension 5ml puts the rotation rotating speed and is 150 rev/mins, rotation radius and be on the shaking table of 40mm 37 ℃ and cultivated 12 hours, seed liquor, get 10 times of seed liquor dilutions and put 610nm to survey light absorption value be 0.326.
Press 30L liquid amount preparation fermention medium with the 50L fermentor tank, adjust pH7.2,118 ℃ of real jar of sterilizations 20 minutes, recirculated water cooling good seed liquor 600ml of inoculation culture after 37 ℃, after the inoculation immediately the sampling and measuring glucose concn be 116.4g/L.
Initial fermentor tank parameter is set to: mixing speed is 200 rev/mins, and ventilation is than 1: 0.5vvm (ratio of feed liquid volume and per minute air flow (measurement basis)), jar internal pressure 0.1MPa.The control leavening temperature is 37 ± 0.2 ℃ in the fermenting process, is 5-10% by regulating mixing speed and ventilation than the control dissolved oxygen, pH6.5 ± 0.2.
Sampling in per 8 hours in the fermenting process, the content and the cell concentration of glucose and 3-oxobutanol in the mensuration fermented liquid, when glucose content is lower than 10g/L, every two hours glucose concn and 3-oxobutanol content in the sampling and measuring fermented liquid, finish fermentation when 3-oxobutanol content no longer increases to fermented liquid, fermentation period is 56 hours.
Get after the fermentation ends fermented liquid with 3000 rev/mins centrifugal 5 minutes, get supernatant liquor and measure the content of glucose and 3-oxobutanol and be respectively 0g/L and 54.5g/L, the transformation efficiency that conversion of glucose generates the 3-oxobutanol is 46.8%.
Fermented liquid distillates part condensation collection liquid and accounts for 96.2% of the total area through the peak shape area of gas chromatographic analysis 3-oxobutanol as stated above through underpressure distillation.
The described glucose of embodiment is measured as follows:
The SBA-4C type membrane bioreactor that uses the Shandong Province academy sciences Biology Research Institute to produce is measured.Measuring principle is to utilize the single-minded mensuration glucose content of fixation glucose dehydrogenation enzyme membrane.
The described 3-oxobutanol of embodiment is measured as follows:
Chemical colorimetry: consult reference (W.W.Westerfeld, A COLORIMETRIC DETERMINATIONOF BLOOD ACETOIN.J.Biol.Chem., 1945; 161:495-502), slightly change.
The drafting of typical curve: according to the form below adds all ingredients and solution in proper order, and 60 ℃ of colour developing 15min use spectrophotometer to survey light absorption value under 530nm, the drawing standard curve.
Fermented liquid centrifuging and taking supernatant liquor suitably is diluted to the content of 3-oxobutanol about 10 μ g, colour developing as stated above, and 530nm measures light absorption value, according to the content of 3-oxobutanol in typical curve and the extension rate calculating fermented liquid.
Wherein:
3-oxobutanol standardized solution: accurate 3-oxobutanol 0.1g, with dissolved in distilled water and be settled to 100ml as storing solution, 4 preserve, and accurately draw storing solution 1ml during use, are settled to 100ml.
5% creatine solution: prepare with distilled water.
5% methyl naphthol solution: prepare with 2.5mol/LNaOH.Need fresh preparation.
The vapor-phase chromatography qualitative analysis:
GC conditions: use the GEP-20M capillary column, carrier gas is nitrogen (N
2), flow velocity is 0.9mL/min; Column cap is pressed 0.10MPa; 200 ℃ of injector temperatures; Temperature programming: 100 ℃ when protecting 3 minutes, 8 ℃/min heats up and causes 180 ℃, during the guarantor 3 minutes; Sample size 1 μ L; Splitting ratio 5: 1.The retention time of 3-oxobutanol is about 8.7 minutes under these conditions, and the retention time of di-acetyl is about 4.5 minutes, 2, and the retention time of 3-butyleneglycol is about 13.5 minutes.
Claims (7)
1. the application of a bacillus subtilis in preparation 3-oxobutanol, its sequence of steps that relates to is as follows:
(1) bacterial classification is selected: select subtilis (Bacillus subtilis) CGMCC NO.1869 for use;
(2) slant culture activation: with bacterial classification inoculation in slant medium, under 35～40 ℃ of conditions, static cultivation 24～48 hours, standby;
(3) seed culture: with the bacterial strain of step (2) cultivation, under aseptic condition, encircle in 30～40mL liquid seed culture medium with inoculation articulating 1～2, putting the rotation rotating speed is that 150～180 rev/mins, rotation radius are on the shaking table of 40mm, cultivated 12～18 hours for 35～40 ℃, seed liquor;
(4) fermentation culture:
Shake flask fermentation: the inoculum size of volume ratio with 1～5%, with seed liquor be inoculated in 80～100mL fermention medium is housed shake in the bottle 35～40 ℃, rotating speed is 150～180 rev/mins, shake flask fermentation 48-72 hour, when detection in the fermented liquid is residual less than glucose, fermentation ends;
50L ferment tank: the inoculum size of volume ratio with 1～5%, seed liquor is inoculated in the canned 50L fermentor tank that 33～35L fermention medium arranged, 35～40 ℃ of stir culture of ventilating, wherein mixing speed is 150～200r/min, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute count 1: 0.5 ± 0.1vvm, jar internal pressure 0.1 ± 0.02MPa, fermentation time is 40～70 hours; Regulate fermentor tank rotating speed and ventilation than the saturation ratio of control dissolved oxygen levels at 5-20%, the fermented liquid initial pH value is 7.0～7.5, stream adds soda acid and makes the pH of fermented liquid be maintained 6.0～7.0 in the fermenting process, the glucose in the fermenting process in the timing sampling mensuration fermented liquid and the concentration of 3-oxobutanol, glucose concn no longer reduces in fermented liquid, when 3-oxobutanol concentration no longer rises, finish fermentation;
(5) product detects: get above-mentioned fermented liquid with 3000～3500 rev/mins centrifugal 5～8 minutes, measure the content of glucose and 3-oxobutanol in the supernatant liquor, and calculate the transformation efficiency that conversion of glucose generates the 3-oxobutanol; Collect fermented liquid simultaneously and carry out underpressure distillation, distillate part condensation and collect the purity that liquid carries out 3-oxobutanol in the gas chromatographic analysis mensuration tunning at 80 ℃;
Above-mentioned slant medium consists of: glucose 5g/L, peptone 5g/L, yeast extract paste 5g/L, sodium-chlor 3g/L, sal epsom 0.1g/L, agar 20g/L, pH 7.0-7.2, distilled water preparation;
The aforesaid liquid seed culture medium is formed: glucose 20～25g/L, yeast extract paste 20g/L, potassium primary phosphate 0.1g/L, dipotassium hydrogen phosphate 0.1g/L, pH7.0-7.2, tap water preparation;
Consisting of of above-mentioned fermention medium: glucose 80-140g/L, yeast extract paste 2g/L, corn steep liquor 10g/L, pH7.0-7.2, tap water preparation.
2。Application as claimed in claim 1 is characterized in that, step (2), (3), (4) described culture temperature are 37 ± 0.2 ℃.
3. application as claimed in claim 1 is characterized in that, the described incubation time of step (2) is 30 hours.
4. application as claimed in claim 1 is characterized in that, the described incubation time of step (3) is 12-14 hour.
5. application as claimed in claim 1 is characterized in that, the described fermention medium initial glucose concentration of step (4) is 100-120g/L.
6. application as claimed in claim 1 is characterized in that, the described dissolved oxygen levels of step (4) is the saturation ratio of 10-15%.
7. application as claimed in claim 1 is characterized in that, the fermented liquid pH in the described fermenting process of step (4) is 6.5 ± 0.2.
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| CN101864381A (en) * | 2010-05-10 | 2010-10-20 | 江南大学 | Breeding of microbial strain for producing 3-hydroxy-2-butanone by fermenting substrate glucose |
| CN102391970B (en) * | 2011-11-18 | 2012-11-07 | 山东大学 | Method for producing 2, 3-butanediol and special bacillus licheniformis thereof |
| CN103320335B (en) * | 2012-03-20 | 2015-02-11 | 南京工业大学 | Clostridium acetobutylicum and application thereof |
| CN102604878A (en) * | 2012-03-27 | 2012-07-25 | 南京工业大学 | Genetically engineered bacterium for producing 3-hydroxy butanone and construction method and application thereof |
| CN102924253B (en) * | 2012-11-20 | 2014-07-30 | 南京工业大学 | Method for extracting acetoin from fermentation liquor |
| CN106399218A (en) * | 2016-12-16 | 2017-02-15 | 山东省食品发酵工业研究设计院 | Bacillus subtilis engineering bacteria and application thereof |
| CN106636251B (en) * | 2016-12-16 | 2020-03-27 | 山东省食品发酵工业研究设计院 | Method for high yield of D-ribose by using bacillus subtilis engineering bacteria |
| CN107058173B (en) * | 2017-03-01 | 2020-04-14 | 大连理工大学 | Bacillus subtilis for producing (3R) -acetoin through fermentation and application thereof |
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Assignee: Ji'nan jiuzhou-fude flavor LLC Assignor: SHANDONG FOOD FERMENT INDUSTRY RESEARCH & DESIGN INSTITUTE Contract record no.: 2010370000452 Denomination of invention: Use of Bacillus subtilis (Ehrenberg)Cohn in preparing 3-hydroxy butanone Granted publication date: 20091125 License type: Exclusive License Open date: 20070801 Record date: 20100812 |
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